Transcriptome assembly and digital gene expression atlas of the rainbow trout

USDA-ARS?s Scientific Manuscript database

Background: Transcriptome analysis is a preferred method for gene discovery, marker development and gene expression profiling in non-model organisms. Previously, we sequenced a transcriptome reference using Sanger-based and 454-pyrosequencing, however, a transcriptome assembly is still incomplete an...

Surface analysis of Dicrocoelium dendriticum. The molecular characterization of exosomes reveals the presence of miRNAs.

PubMed

Bernal, Dolores; Trelis, Maria; Montaner, Sergio; Cantalapiedra, Fernando; Galiano, Alicia; Hackenberg, Michael; Marcilla, Antonio

2014-06-13

With the aim of characterizing the molecules involved in the interaction of Dicrocoelium dendriticum adults and the host, we have performed proteomic analyses of the external surface of the parasite using the currently available datasets including the transcriptome of the related species Echinostoma caproni. We have identified 182 parasite proteins on the outermost surface of D. dendriticum. The presence of exosome-like vesicles in the ESP of D. dendriticum and their components has also been characterized. Using proteomic approaches, we have characterized 84 proteins in these vesicles. Interestingly, we have detected miRNA in D. dendriticum exosomes, thus representing the first report of miRNA in helminth exosomes. In order to identify potential targets for intervention against parasitic helminths, we have analyzed the surface of the parasitic helminth Dicrocoelium dendriticum. Along with the proteomic analyses of the outermost layer of the parasite, our work describes the molecular characterization of the exosomes of D. dendriticum. Our proteomic data confirm the improvement of protein identification from "non-model organisms" like helminths, when using different search engines against a combination of available databases. In addition, this work represents the first report of miRNAs in parasitic helminth exosomes. These vesicles can pack specific proteins and RNAs providing stability and resistance to RNAse digestion in body fluids, and provide a way to regulate host-parasite interplay. The present data should provide a solid foundation for the development of novel methods to control this non-model organism and related parasites. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparative Transcriptomic Approaches Exploring Contamination Stress Tolerance in Salix sp. Reveal the Importance for a Metaorganismal de Novo Assembly Approach for Nonmodel Plants1[OPEN

PubMed Central

Brereton, Nicholas J. B.; Marleau, Julie; Nissim, Werther Guidi; Labrecque, Michel; Joly, Simon; Pitre, Frederic E.

2016-01-01

Metatranscriptomic study of nonmodel organisms requires strategies that retain the highly resolved genetic information generated from model organisms while allowing for identification of the unexpected. A real-world biological application of phytoremediation, the field growth of 10 Salix cultivars on polluted soils, was used as an exemplar nonmodel and multifaceted crop response well-disposed to the study of gene expression. Sequence reads were assembled de novo to create 10 independent transcriptomes, a global transcriptome, and were mapped against the Salix purpurea 94006 reference genome. Annotation of assembled contigs was performed without a priori assumption of the originating organism. Global transcriptome construction from 3.03 billion paired-end reads revealed 606,880 unique contigs annotated from 1588 species, often common in all 10 cultivars. Comparisons between transcriptomic and metatranscriptomic methodologies provide clear evidence that nonnative RNA can mistakenly map to reference genomes, especially to conserved regions of common housekeeping genes, such as actin, α/β-tubulin, and elongation factor 1-α. In Salix, Rubisco activase transcripts were down-regulated in contaminated trees across all 10 cultivars, whereas thiamine thizole synthase and CP12, a Calvin Cycle master regulator, were uniformly up-regulated. De novo assembly approaches, with unconstrained annotation, can improve data quality; care should be taken when exploring such plant genetics to reduce de facto data exclusion by mapping to a single reference genome alone. Salix gene expression patterns strongly suggest cultivar-wide alteration of specific photosynthetic apparatus and protection of the antenna complexes from oxidation damage in contaminated trees, providing an insight into common stress tolerance strategies in a real-world phytoremediation system. PMID:27002060

De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis.

PubMed

Haas, Brian J; Papanicolaou, Alexie; Yassour, Moran; Grabherr, Manfred; Blood, Philip D; Bowden, Joshua; Couger, Matthew Brian; Eccles, David; Li, Bo; Lieber, Matthias; MacManes, Matthew D; Ott, Michael; Orvis, Joshua; Pochet, Nathalie; Strozzi, Francesco; Weeks, Nathan; Westerman, Rick; William, Thomas; Dewey, Colin N; Henschel, Robert; LeDuc, Richard D; Friedman, Nir; Regev, Aviv

2013-08-01

De novo assembly of RNA-seq data enables researchers to study transcriptomes without the need for a genome sequence; this approach can be usefully applied, for instance, in research on 'non-model organisms' of ecological and evolutionary importance, cancer samples or the microbiome. In this protocol we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-seq data in non-model organisms. We also present Trinity-supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples and approaches to identify protein-coding genes. In the procedure, we provide a workflow for genome-independent transcriptome analysis leveraging the Trinity platform. The software, documentation and demonstrations are freely available from http://trinityrnaseq.sourceforge.net. The run time of this protocol is highly dependent on the size and complexity of data to be analyzed. The example data set analyzed in the procedure detailed herein can be processed in less than 5 h.

Comprehensive evaluation of non-hybrid genome assembly tools for third-generation PacBio long-read sequence data.

PubMed

Jayakumar, Vasanthan; Sakakibara, Yasubumi

2017-11-03

Long reads obtained from third-generation sequencing platforms can help overcome the long-standing challenge of the de novo assembly of sequences for the genomic analysis of non-model eukaryotic organisms. Numerous long-read-aided de novo assemblies have been published recently, which exhibited superior quality of the assembled genomes in comparison with those achieved using earlier second-generation sequencing technologies. Evaluating assemblies is important in guiding the appropriate choice for specific research needs. In this study, we evaluated 10 long-read assemblers using a variety of metrics on Pacific Biosciences (PacBio) data sets from different taxonomic categories with considerable differences in genome size. The results allowed us to narrow down the list to a few assemblers that can be effectively applied to eukaryotic assembly projects. Moreover, we highlight how best to use limited genomic resources for effectively evaluating the genome assemblies of non-model organisms. © The Author 2017. Published by Oxford University Press.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gilchrist, Michael J.; Sobral, Daniel; Khoueiry, Pierre

Genome-wide resources, such as collections of cDNA clones encoding for complete proteins (full-ORF clones), are crucial tools for studying the evolution of gene function and genetic interactions. Non-model organisms, in particular marine organisms, provide a rich source of functional diversity. Marine organism genomes are, however, frequently highly polymorphic and encode proteins that diverge significantly from those of well-annotated model genomes. The construction of full-ORF clone collections from non-model organisms is hindered by the difficulty of predicting accurately the N-terminal ends of proteins, and distinguishing recent paralogs from highly polymorphic alleles. We also report a computational strategy that overcomes these difficulties,more » and allows for accurate gene level clustering of transcript data followed by the automated identification of full-ORFs with correct 5'- and 3'-ends. It is robust to polymorphism, includes paralog calling and does not require evolutionary proximity to well annotated model organisms. Here, we developed this pipeline for the ascidian Ciona intestinalis, a highly polymorphic member of the divergent sister group of the vertebrates, emerging as a powerful model organism to study chordate gene function, Gene Regulatory Networks and molecular mechanisms underlying human pathologies. Furthermore, using this pipeline we have generated the first full-ORF collection for a highly polymorphic marine invertebrate. It contains 19,163 full-ORF cDNA clones covering 60% of Ciona coding genes, and full-ORF orthologs for approximately half of curated human disease-associated genes.« less

Chromatin immunoprecipitation (ChIP) method for non-model fruit flies (Diptera: Tephritidae) and evidence of histone modifications.

PubMed

Nagalingam, Kumaran; Lorenc, Michał T; Manoli, Sahana; Cameron, Stephen L; Clarke, Anthony R; Dudley, Kevin J

2018-01-01

Interactions between DNA and proteins located in the cell nucleus play an important role in controlling physiological processes by specifying, augmenting and regulating context-specific transcription events. Chromatin immunoprecipitation (ChIP) is a widely used methodology to study DNA-protein interactions and has been successfully used in various cell types for over three decades. More recently, by combining ChIP with genomic screening technologies and Next Generation Sequencing (e.g. ChIP-seq), it has become possible to profile DNA-protein interactions (including covalent histone modifications) across entire genomes. However, the applicability of ChIP-chip and ChIP-seq has rarely been extended to non-model species because of a number of technical challenges. Here we report a method that can be used to identify genome wide covalent histone modifications in a group of non-model fruit fly species (Diptera: Tephritidae). The method was developed by testing and refining protocols that have been used in model organisms, including Drosophila melanogaster. We demonstrate that this method is suitable for a group of economically important pest fruit fly species, viz., Bactrocera dorsalis, Ceratitis capitata, Zeugodacus cucurbitae and Bactrocera tryoni. We also report an example ChIP-seq dataset for B. tryoni, providing evidence for histone modifications in the genome of a tephritid fruit fly for the first time. Since tephritids are major agricultural pests globally, this methodology will be a valuable resource to study taxa-specific evolutionary questions and to assist with pest management. It also provides a basis for researchers working with other non-model species to undertake genome wide DNA-protein interaction studies.

Clustered regulatory interspaced short palindromic repeats (CRISPR)-mediated mutagenesis and phenotype rescue by piggyBac transgenesis in a nonmodel Drosophila species.

PubMed

Tanaka, R; Murakami, H; Ote, M; Yamamoto, D

2016-08-01

How behavioural diversity emerged in evolution is an unexplored subject in biology. To tackle this problem, genes and circuits for a behaviour need to be determined in different species for phylogenetic comparisons. The recently developed clustered regulatory interspaced short palindromic repeats/CRISPR associated protein9 (CRISPR/Cas9) system made such a challenge possible by providing the means to induce mutations in a gene of interest in any organism. Aiming at elucidating diversification in genetic and neural networks for courtship behaviour, we attempted to generate a genetic tool kit in Drosophila subobscura, a nonmodel species distantly related to the genetic model Drosophila melanogaster. Here we report the generation of yellow (y) and white mutations with the aid of the CRISPR/Cas9 system, and the rescue of the y mutant phenotype by germline transformation of the newly established y mutant fly line with a y(+) -marked piggyBac vector. This successful mutagenesis and transformation in D. subobscura open up an avenue for comprehensive genetic analyses of higher functions in this and other nonmodel Drosophila species, representing a key step toward systematic comparisons of genes and circuitries underlying behaviour amongst species. © 2016 The Royal Entomological Society.

AmphiBase: A new genomic resource for non-model amphibian species.

PubMed

Kwon, Taejoon

2017-01-01

More than five thousand genes annotated in the recently published Xenopus laevis and Xenopus tropicalis genomes do not have a candidate orthologous counterpart in other vertebrate species. To determine whether these sequences represent genuine amphibian-specific genes or annotation errors, it is necessary to analyze them alongside sequences from other amphibian species. However, due to large genome sizes and an abundance of repeat sequences, there are limited numbers of gene sequences available from amphibian species other than Xenopus. AmphiBase is a new genomic resource covering non-model amphibian species, based on public domain transcriptome data and computational methods developed during the X. laevis genome project. Here, I review the current status of AmphiBase, including amphibian species with available transcriptome data or biological samples, and describe the challenges of building a comprehensive amphibian genomic resource in the absence of genomes. This mini-review will be informative for researchers interested in functional genomic experiments using amphibian model organisms, such as Xenopus and axolotl, and will assist in interpretation of results implicating "orphan genes." Additionally, this study highlights an opportunity for researchers working on non-model amphibian species to collaborate in their future efforts and develop amphibian genomic resources as a community. © 2017 Wiley Periodicals, Inc.

Draft genome sequence of the oleaginous yeast Cryptococcus curvatus ATCC 20509

DOE Office of Scientific and Technical Information (OSTI.GOV)

Close, Dan; Ojumu, John O.

Cryptococcus curvatus ATCC 20509 is a commonly used nonmodel oleaginous yeast capable of converting a variety of carbon sources into fatty acids. In addition, we present the draft genome sequence of this popular organism to provide a means for more in-depth studies of its fatty acid production potential.

Draft genome sequence of the oleaginous yeast Cryptococcus curvatus ATCC 20509

DOE PAGES

Close, Dan; Ojumu, John O.

2016-11-03

Cryptococcus curvatus ATCC 20509 is a commonly used nonmodel oleaginous yeast capable of converting a variety of carbon sources into fatty acids. In addition, we present the draft genome sequence of this popular organism to provide a means for more in-depth studies of its fatty acid production potential.

Genome size of 14 species of fireflies (Insecta, Coleoptera, Lampyridae)

PubMed Central

Liu, Gui-Chun; Dong, Zhi-Wei; He, Jin-Wu; Zhao, Ruo-Ping; Wang, Wen; Li, Xue-Yan

2017-01-01

Eukaryotic genome size data are important both as the basis for comparative research into genome evolution and as estimators of the cost and difficulty of genome sequencing programs for non-model organisms. In this study, the genome size of 14 species of fireflies (Lampyridae) (two genera in Lampyrinae, three genera in Luciolinae, and one genus in subfamily incertae sedis) were estimated by propidium iodide (PI)-based flow cytometry. The haploid genome sizes of Lampyridae ranged from 0. 42 to 1. 31 pg, a 3. 1-fold span. Genome sizes of the fireflies varied within the tested subfamilies and genera. Lamprigera and Pyrocoelia species had large and small genome sizes, respectively. No correlation was found between genome size and morphological traits such as body length, body width, eye width, and antennal length. Our data provide additional information on genome size estimation of the firefly family Lampyridae. Furthermore, this study will help clarify the cost and difficulty of genome sequencing programs for non-model organisms and will help promote studies on firefly genome evolution. PMID:29280364

System-level insights into the cellular interactome of a non-model organism: inferring, modelling and analysing functional gene network of soybean (Glycine max).

PubMed

Xu, Yungang; Guo, Maozu; Zou, Quan; Liu, Xiaoyan; Wang, Chunyu; Liu, Yang

2014-01-01

Cellular interactome, in which genes and/or their products interact on several levels, forming transcriptional regulatory-, protein interaction-, metabolic-, signal transduction networks, etc., has attracted decades of research focuses. However, such a specific type of network alone can hardly explain the various interactive activities among genes. These networks characterize different interaction relationships, implying their unique intrinsic properties and defects, and covering different slices of biological information. Functional gene network (FGN), a consolidated interaction network that models fuzzy and more generalized notion of gene-gene relations, have been proposed to combine heterogeneous networks with the goal of identifying functional modules supported by multiple interaction types. There are yet no successful precedents of FGNs on sparsely studied non-model organisms, such as soybean (Glycine max), due to the absence of sufficient heterogeneous interaction data. We present an alternative solution for inferring the FGNs of soybean (SoyFGNs), in a pioneering study on the soybean interactome, which is also applicable to other organisms. SoyFGNs exhibit the typical characteristics of biological networks: scale-free, small-world architecture and modularization. Verified by co-expression and KEGG pathways, SoyFGNs are more extensive and accurate than an orthology network derived from Arabidopsis. As a case study, network-guided disease-resistance gene discovery indicates that SoyFGNs can provide system-level studies on gene functions and interactions. This work suggests that inferring and modelling the interactome of a non-model plant are feasible. It will speed up the discovery and definition of the functions and interactions of other genes that control important functions, such as nitrogen fixation and protein or lipid synthesis. The efforts of the study are the basis of our further comprehensive studies on the soybean functional interactome at the genome and microRNome levels. Additionally, a web tool for information retrieval and analysis of SoyFGNs can be accessed at SoyFN: http://nclab.hit.edu.cn/SoyFN.

System-Level Insights into the Cellular Interactome of a Non-Model Organism: Inferring, Modelling and Analysing Functional Gene Network of Soybean (Glycine max)

PubMed Central

Xu, Yungang; Guo, Maozu; Zou, Quan; Liu, Xiaoyan; Wang, Chunyu; Liu, Yang

2014-01-01

Cellular interactome, in which genes and/or their products interact on several levels, forming transcriptional regulatory-, protein interaction-, metabolic-, signal transduction networks, etc., has attracted decades of research focuses. However, such a specific type of network alone can hardly explain the various interactive activities among genes. These networks characterize different interaction relationships, implying their unique intrinsic properties and defects, and covering different slices of biological information. Functional gene network (FGN), a consolidated interaction network that models fuzzy and more generalized notion of gene-gene relations, have been proposed to combine heterogeneous networks with the goal of identifying functional modules supported by multiple interaction types. There are yet no successful precedents of FGNs on sparsely studied non-model organisms, such as soybean (Glycine max), due to the absence of sufficient heterogeneous interaction data. We present an alternative solution for inferring the FGNs of soybean (SoyFGNs), in a pioneering study on the soybean interactome, which is also applicable to other organisms. SoyFGNs exhibit the typical characteristics of biological networks: scale-free, small-world architecture and modularization. Verified by co-expression and KEGG pathways, SoyFGNs are more extensive and accurate than an orthology network derived from Arabidopsis. As a case study, network-guided disease-resistance gene discovery indicates that SoyFGNs can provide system-level studies on gene functions and interactions. This work suggests that inferring and modelling the interactome of a non-model plant are feasible. It will speed up the discovery and definition of the functions and interactions of other genes that control important functions, such as nitrogen fixation and protein or lipid synthesis. The efforts of the study are the basis of our further comprehensive studies on the soybean functional interactome at the genome and microRNome levels. Additionally, a web tool for information retrieval and analysis of SoyFGNs can be accessed at SoyFN: http://nclab.hit.edu.cn/SoyFN. PMID:25423109

Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae)

PubMed Central

Chouteau, Mathieu; Whibley, Annabel; Joron, Mathieu; Llaurens, Violaine

2016-01-01

Identifying the genetic basis of adaptive variation is challenging in non-model organisms and quantitative real time PCR. is a useful tool for validating predictions regarding the expression of candidate genes. However, comparing expression levels in different conditions requires rigorous experimental design and statistical analyses. Here, we focused on the neotropical passion-vine butterflies Heliconius, non-model species studied in evolutionary biology for their adaptive variation in wing color patterns involved in mimicry and in the signaling of their toxicity to predators. We aimed at selecting stable reference genes to be used for normalization of gene expression data in RT-qPCR analyses from developing wing discs according to the minimal guidelines described in Minimum Information for publication of Quantitative Real-Time PCR Experiments (MIQE). To design internal RT-qPCR controls, we studied the stability of expression of nine candidate reference genes (actin, annexin, eF1α, FK506BP, PolyABP, PolyUBQ, RpL3, RPS3A, and tubulin) at two developmental stages (prepupal and pupal) using three widely used programs (GeNorm, NormFinder and BestKeeper). Results showed that, despite differences in statistical methods, genes RpL3, eF1α, polyABP, and annexin were stably expressed in wing discs in late larval and pupal stages of Heliconius numata. This combination of genes may be used as a reference for a reliable study of differential expression in wings for instance for genes involved in important phenotypic variation, such as wing color pattern variation. Through this example, we provide general useful technical recommendations as well as relevant statistical strategies for evolutionary biologists aiming to identify candidate-genes involved adaptive variation in non-model organisms. PMID:27271971

The use of PacBio and Hi-C data in denovo assembly of the goat genome

USDA-ARS?s Scientific Manuscript database

Generating de novo reference genome assemblies for non-model organisms is a laborious task that often requires a large amount of data from several sequencing platforms and cytogenetic surveys. By using PacBio sequence data and new library creation techniques, we present a de novo, high quality refer...

Microinjection-based RNA interference knockdown of ecdysteroid biosynthetic genes in a non-model hemipteran pest, Lygus hesperus (western tarnished plant bug)

USDA-ARS?s Scientific Manuscript database

RNAi-mediated knockdown of target transcripts offers great potential, both in terms of insect functional genomics and the development of novel insect pest management strategies. Frequently, dsRNAs targeting transcripts of interest are introduced orally to the target organism via feeding. This delive...

Proteomics identifies the composition and manufacturing recipe of the 2500-year old sourdough bread from Subeixi cemetery in China.

PubMed

Shevchenko, Anna; Yang, Yimin; Knaust, Andrea; Thomas, Henrik; Jiang, Hongen; Lu, Enguo; Wang, Changsui; Shevchenko, Andrej

2014-06-13

We report on the geLC-MS/MS proteomics analysis of cereals and cereal food excavated in Subeixi cemetery (500-300BC) in Xinjiang, China. Proteomics provided direct evidence that at the Subexi sourdough bread was made from barley and broomcorn millet by leavening with a renewable starter comprising baker's yeast and lactic acid bacteria. The baking recipe and flour composition indicated that barley and millet bread belonged to the staple food already in the first millennium BC and suggested the role of Turpan basin as a major route for cultural communication between Western and Eastern Eurasia in antiquity. This article is part of a Special Issue entitled: Proteomics of non-model organisms. We demonstrate that organic residues of thousand year old foods unearthed by archeological excavations can be analyzed by geLC-MS/MS proteomics with good representation of protein source organisms and coverage of sequences of identified proteins. In-depth look into the foods proteome identifies the food type and its individual ingredients, reveals ancient food processing technologies, projects their social and economic impact and provides evidence of intercultural communication between ancient populations. Proteomics analysis of ancient organic residues is direct, quantitative and informative and therefore has the potential to develop into a valuable, generally applicable tool in archaeometry. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2013. Published by Elsevier B.V.

Using phylogenetically-informed annotation (PIA) to search for light-interacting genes in transcriptomes from non-model organisms.

PubMed

Speiser, Daniel I; Pankey, M Sabrina; Zaharoff, Alexander K; Battelle, Barbara A; Bracken-Grissom, Heather D; Breinholt, Jesse W; Bybee, Seth M; Cronin, Thomas W; Garm, Anders; Lindgren, Annie R; Patel, Nipam H; Porter, Megan L; Protas, Meredith E; Rivera, Ajna S; Serb, Jeanne M; Zigler, Kirk S; Crandall, Keith A; Oakley, Todd H

2014-11-19

Tools for high throughput sequencing and de novo assembly make the analysis of transcriptomes (i.e. the suite of genes expressed in a tissue) feasible for almost any organism. Yet a challenge for biologists is that it can be difficult to assign identities to gene sequences, especially from non-model organisms. Phylogenetic analyses are one useful method for assigning identities to these sequences, but such methods tend to be time-consuming because of the need to re-calculate trees for every gene of interest and each time a new data set is analyzed. In response, we employed existing tools for phylogenetic analysis to produce a computationally efficient, tree-based approach for annotating transcriptomes or new genomes that we term Phylogenetically-Informed Annotation (PIA), which places uncharacterized genes into pre-calculated phylogenies of gene families. We generated maximum likelihood trees for 109 genes from a Light Interaction Toolkit (LIT), a collection of genes that underlie the function or development of light-interacting structures in metazoans. To do so, we searched protein sequences predicted from 29 fully-sequenced genomes and built trees using tools for phylogenetic analysis in the Osiris package of Galaxy (an open-source workflow management system). Next, to rapidly annotate transcriptomes from organisms that lack sequenced genomes, we repurposed a maximum likelihood-based Evolutionary Placement Algorithm (implemented in RAxML) to place sequences of potential LIT genes on to our pre-calculated gene trees. Finally, we implemented PIA in Galaxy and used it to search for LIT genes in 28 newly-sequenced transcriptomes from the light-interacting tissues of a range of cephalopod mollusks, arthropods, and cubozoan cnidarians. Our new trees for LIT genes are available on the Bitbucket public repository ( http://bitbucket.org/osiris_phylogenetics/pia/ ) and we demonstrate PIA on a publicly-accessible web server ( http://galaxy-dev.cnsi.ucsb.edu/pia/ ). Our new trees for LIT genes will be a valuable resource for researchers studying the evolution of eyes or other light-interacting structures. We also introduce PIA, a high throughput method for using phylogenetic relationships to identify LIT genes in transcriptomes from non-model organisms. With simple modifications, our methods may be used to search for different sets of genes or to annotate data sets from taxa outside of Metazoa.

A pipeline for the systematic identification of non-redundant full-ORF cDNAs for polymorphic and evolutionary divergent genomes: Application to the ascidian Ciona intestinalis

DOE PAGES

Gilchrist, Michael J.; Sobral, Daniel; Khoueiry, Pierre; ...

2015-05-27

Genome-wide resources, such as collections of cDNA clones encoding for complete proteins (full-ORF clones), are crucial tools for studying the evolution of gene function and genetic interactions. Non-model organisms, in particular marine organisms, provide a rich source of functional diversity. Marine organism genomes are, however, frequently highly polymorphic and encode proteins that diverge significantly from those of well-annotated model genomes. The construction of full-ORF clone collections from non-model organisms is hindered by the difficulty of predicting accurately the N-terminal ends of proteins, and distinguishing recent paralogs from highly polymorphic alleles. We also report a computational strategy that overcomes these difficulties,more » and allows for accurate gene level clustering of transcript data followed by the automated identification of full-ORFs with correct 5'- and 3'-ends. It is robust to polymorphism, includes paralog calling and does not require evolutionary proximity to well annotated model organisms. Here, we developed this pipeline for the ascidian Ciona intestinalis, a highly polymorphic member of the divergent sister group of the vertebrates, emerging as a powerful model organism to study chordate gene function, Gene Regulatory Networks and molecular mechanisms underlying human pathologies. Furthermore, using this pipeline we have generated the first full-ORF collection for a highly polymorphic marine invertebrate. It contains 19,163 full-ORF cDNA clones covering 60% of Ciona coding genes, and full-ORF orthologs for approximately half of curated human disease-associated genes.« less

Inference of Expanded Lrp-Like Feast/Famine Transcription Factor Targets in a Non-Model Organism Using Protein Structure-Based Prediction

PubMed Central

Ashworth, Justin; Plaisier, Christopher L.; Lo, Fang Yin; Reiss, David J.; Baliga, Nitin S.

2014-01-01

Widespread microbial genome sequencing presents an opportunity to understand the gene regulatory networks of non-model organisms. This requires knowledge of the binding sites for transcription factors whose DNA-binding properties are unknown or difficult to infer. We adapted a protein structure-based method to predict the specificities and putative regulons of homologous transcription factors across diverse species. As a proof-of-concept we predicted the specificities and transcriptional target genes of divergent archaeal feast/famine regulatory proteins, several of which are encoded in the genome of Halobacterium salinarum. This was validated by comparison to experimentally determined specificities for transcription factors in distantly related extremophiles, chromatin immunoprecipitation experiments, and cis-regulatory sequence conservation across eighteen related species of halobacteria. Through this analysis we were able to infer that Halobacterium salinarum employs a divergent local trans-regulatory strategy to regulate genes (carA and carB) involved in arginine and pyrimidine metabolism, whereas Escherichia coli employs an operon. The prediction of gene regulatory binding sites using structure-based methods is useful for the inference of gene regulatory relationships in new species that are otherwise difficult to infer. PMID:25255272

Inference of expanded Lrp-like feast/famine transcription factor targets in a non-model organism using protein structure-based prediction.

PubMed

Ashworth, Justin; Plaisier, Christopher L; Lo, Fang Yin; Reiss, David J; Baliga, Nitin S

2014-01-01

Widespread microbial genome sequencing presents an opportunity to understand the gene regulatory networks of non-model organisms. This requires knowledge of the binding sites for transcription factors whose DNA-binding properties are unknown or difficult to infer. We adapted a protein structure-based method to predict the specificities and putative regulons of homologous transcription factors across diverse species. As a proof-of-concept we predicted the specificities and transcriptional target genes of divergent archaeal feast/famine regulatory proteins, several of which are encoded in the genome of Halobacterium salinarum. This was validated by comparison to experimentally determined specificities for transcription factors in distantly related extremophiles, chromatin immunoprecipitation experiments, and cis-regulatory sequence conservation across eighteen related species of halobacteria. Through this analysis we were able to infer that Halobacterium salinarum employs a divergent local trans-regulatory strategy to regulate genes (carA and carB) involved in arginine and pyrimidine metabolism, whereas Escherichia coli employs an operon. The prediction of gene regulatory binding sites using structure-based methods is useful for the inference of gene regulatory relationships in new species that are otherwise difficult to infer.

Tapping the promise of genomics in species with complex, nonmodel genomes.

PubMed

Hirsch, Candice N; Buell, C Robin

2013-01-01

Genomics is enabling a renaissance in all disciplines of plant biology. However, many plant genomes are complex and remain recalcitrant to current genomic technologies. The complexities of these nonmodel plant genomes are attributable to gene and genome duplication, heterozygosity, ploidy, and/or repetitive sequences. Methods are available to simplify the genome and reduce these barriers, including inbreeding and genome reduction, making these species amenable to current sequencing and assembly methods. Some, but not all, of the complexities in nonmodel genomes can be bypassed by sequencing the transcriptome rather than the genome. Additionally, comparative genomics approaches, which leverage phylogenetic relatedness, can aid in the interpretation of complex genomes. Although there are limitations in accessing complex nonmodel plant genomes using current sequencing technologies, genome manipulation and resourceful analyses can allow access to even the most recalcitrant plant genomes.

Phylogenetic marker development for target enrichment from transcriptome and genome skim data: the pipeline and its application in southern African Oxalis (Oxalidaceae)

Treesearch

Roswitha Schmickl; Aaron Liston; Vojtěch Zeisek; Kenneth Oberlander; Kevin Weitemier; Shannon C. K. Straub; Richard C. Cronn; Léanne L. Dreyer; Jan Suda

2016-01-01

Phylogenetics benefits from using a large number of putatively independent nuclear loci and their combination with other sources of information, such as the plastid and mitochondrial genomes. To facilitate the selection of orthologous low-copy nuclear (LCN) loci for phylogenetics in nonmodel organisms, we created an automated and interactive script to select hundreds...

Improving transcriptome construction in non-model organisms: integrating manual and automated gene definition in Emiliania huxleyi.

PubMed

Feldmesser, Ester; Rosenwasser, Shilo; Vardi, Assaf; Ben-Dor, Shifra

2014-02-22

The advent of Next Generation Sequencing technologies and corresponding bioinformatics tools allows the definition of transcriptomes in non-model organisms. Non-model organisms are of great ecological and biotechnological significance, and consequently the understanding of their unique metabolic pathways is essential. Several methods that integrate de novo assembly with genome-based assembly have been proposed. Yet, there are many open challenges in defining genes, particularly where genomes are not available or incomplete. Despite the large numbers of transcriptome assemblies that have been performed, quality control of the transcript building process, particularly on the protein level, is rarely performed if ever. To test and improve the quality of the automated transcriptome reconstruction, we used manually defined and curated genes, several of them experimentally validated. Several approaches to transcript construction were utilized, based on the available data: a draft genome, high quality RNAseq reads, and ESTs. In order to maximize the contribution of the various data, we integrated methods including de novo and genome based assembly, as well as EST clustering. After each step a set of manually curated genes was used for quality assessment of the transcripts. The interplay between the automated pipeline and the quality control indicated which additional processes were required to improve the transcriptome reconstruction. We discovered that E. huxleyi has a very high percentage of non-canonical splice junctions, and relatively high rates of intron retention, which caused unique issues with the currently available tools. While individual tools missed genes and artificially joined overlapping transcripts, combining the results of several tools improved the completeness and quality considerably. The final collection, created from the integration of several quality control and improvement rounds, was compared to the manually defined set both on the DNA and protein levels, and resulted in an improvement of 20% versus any of the read-based approaches alone. To the best of our knowledge, this is the first time that an automated transcript definition is subjected to quality control using manually defined and curated genes and thereafter the process is improved. We recommend using a set of manually curated genes to troubleshoot transcriptome reconstruction.

Building a Genome Engineering Toolbox in Non-Model Prokaryotic Microbes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eckert, Carrie A; Freed, Emily; Smolinski, Sharon

The realization of a sustainable bioeconomy requires our ability to understand and engineer complex design principles for the development of platform organisms capable of efficient conversion of cheap and sustainable feedstocks (e.g. sunlight, CO2, non-food biomass) to biofuels and bioproducts at sufficient titers and costs. For model microbes such as E. coli, advances in DNA reading and writing technologies are driving adoption of new paradigms for engineering biological systems. Unfortunately, microbes with properties of interest for the utilization of cheap and renewable feedstocks such as photosynthesis, autotrophic growth, and cellulose degradation have very few, if any, genetic tools for metabolicmore » engineering. Therefore, it is important to begin to develop 'design rules' for building a genetic toolbox for novel microbes. Here, we present an overview of our current understanding of these rules for the genetic manipulation of prokaryotic microbes and available genetic tools to expand our ability to genetically engineer non-model systems.« less

Building a genome engineering toolbox in nonmodel prokaryotic microbes.

PubMed

Freed, Emily; Fenster, Jacob; Smolinski, Sharon L; Walker, Julie; Henard, Calvin A; Gill, Ryan; Eckert, Carrie A

2018-05-11

The realization of a sustainable bioeconomy requires our ability to understand and engineer complex design principles for the development of platform organisms capable of efficient conversion of cheap and sustainable feedstocks (e.g., sunlight, CO 2 , and nonfood biomass) into biofuels and bioproducts at sufficient titers and costs. For model microbes, such as Escherichia coli, advances in DNA reading and writing technologies are driving the adoption of new paradigms for engineering biological systems. Unfortunately, microbes with properties of interest for the utilization of cheap and renewable feedstocks, such as photosynthesis, autotrophic growth, and cellulose degradation, have very few, if any, genetic tools for metabolic engineering. Therefore, it is important to develop "design rules" for building a genetic toolbox for novel microbes. Here, we present an overview of our current understanding of these rules for the genetic manipulation of prokaryotic microbes and the available genetic tools to expand our ability to genetically engineer nonmodel systems. © 2018 Wiley Periodicals, Inc.

Exploring Pandora's Box: Potential and Pitfalls of Low Coverage Genome Surveys for Evolutionary Biology

PubMed Central

Leese, Florian; Mayer, Christoph; Agrawal, Shobhit; Dambach, Johannes; Dietz, Lars; Doemel, Jana S.; Goodall-Copstake, William P.; Held, Christoph; Jackson, Jennifer A.; Lampert, Kathrin P.; Linse, Katrin; Macher, Jan N.; Nolzen, Jennifer; Raupach, Michael J.; Rivera, Nicole T.; Schubart, Christoph D.; Striewski, Sebastian; Tollrian, Ralph; Sands, Chester J.

2012-01-01

High throughput sequencing technologies are revolutionizing genetic research. With this “rise of the machines”, genomic sequences can be obtained even for unknown genomes within a short time and for reasonable costs. This has enabled evolutionary biologists studying genetically unexplored species to identify molecular markers or genomic regions of interest (e.g. micro- and minisatellites, mitochondrial and nuclear genes) by sequencing only a fraction of the genome. However, when using such datasets from non-model species, it is possible that DNA from non-target contaminant species such as bacteria, viruses, fungi, or other eukaryotic organisms may complicate the interpretation of the results. In this study we analysed 14 genomic pyrosequencing libraries of aquatic non-model taxa from four major evolutionary lineages. We quantified the amount of suitable micro- and minisatellites, mitochondrial genomes, known nuclear genes and transposable elements and searched for contamination from various sources using bioinformatic approaches. Our results show that in all sequence libraries with estimated coverage of about 0.02–25%, many appropriate micro- and minisatellites, mitochondrial gene sequences and nuclear genes from different KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways could be identified and characterized. These can serve as markers for phylogenetic and population genetic analyses. A central finding of our study is that several genomic libraries suffered from different biases owing to non-target DNA or mobile elements. In particular, viruses, bacteria or eukaryote endosymbionts contributed significantly (up to 10%) to some of the libraries analysed. If not identified as such, genetic markers developed from high-throughput sequencing data for non-model organisms may bias evolutionary studies or fail completely in experimental tests. In conclusion, our study demonstrates the enormous potential of low-coverage genome survey sequences and suggests bioinformatic analysis workflows. The results also advise a more sophisticated filtering for problematic sequences and non-target genome sequences prior to developing markers. PMID:23185309

Population genomics of C. melanopterus using target gene capture data: demographic inferences and conservation perspectives

PubMed Central

Maisano Delser, Pierpaolo; Corrigan, Shannon; Hale, Matthew; Li, Chenhong; Veuille, Michel; Planes, Serge; Naylor, Gavin; Mona, Stefano

2016-01-01

Population genetics studies on non-model organisms typically involve sampling few markers from multiple individuals. Next-generation sequencing approaches open up the possibility of sampling many more markers from fewer individuals to address the same questions. Here, we applied a target gene capture method to deep sequence ~1000 independent autosomal regions of a non-model organism, the blacktip reef shark (Carcharhinus melanopterus). We devised a sampling scheme based on the predictions of theoretical studies of metapopulations to show that sampling few individuals, but many loci, can be extremely informative to reconstruct the evolutionary history of species. We collected data from a single deme (SID) from Northern Australia and from a scattered sampling representing various locations throughout the Indian Ocean (SCD). We explored the genealogical signature of population dynamics detected from both sampling schemes using an ABC algorithm. We then contrasted these results with those obtained by fitting the data to a non-equilibrium finite island model. Both approaches supported an Nm value ~40, consistent with philopatry in this species. Finally, we demonstrate through simulation that metapopulations exhibit greater resilience to recent changes in effective size compared to unstructured populations. We propose an empirical approach to detect recent bottlenecks based on our sampling scheme. PMID:27651217

Towards a system level understanding of non-model organisms sampled from the environment: a network biology approach.

PubMed

Williams, Tim D; Turan, Nil; Diab, Amer M; Wu, Huifeng; Mackenzie, Carolynn; Bartie, Katie L; Hrydziuszko, Olga; Lyons, Brett P; Stentiford, Grant D; Herbert, John M; Abraham, Joseph K; Katsiadaki, Ioanna; Leaver, Michael J; Taggart, John B; George, Stephen G; Viant, Mark R; Chipman, Kevin J; Falciani, Francesco

2011-08-01

The acquisition and analysis of datasets including multi-level omics and physiology from non-model species, sampled from field populations, is a formidable challenge, which so far has prevented the application of systems biology approaches. If successful, these could contribute enormously to improving our understanding of how populations of living organisms adapt to environmental stressors relating to, for example, pollution and climate. Here we describe the first application of a network inference approach integrating transcriptional, metabolic and phenotypic information representative of wild populations of the European flounder fish, sampled at seven estuarine locations in northern Europe with different degrees and profiles of chemical contaminants. We identified network modules, whose activity was predictive of environmental exposure and represented a link between molecular and morphometric indices. These sub-networks represented both known and candidate novel adverse outcome pathways representative of several aspects of human liver pathophysiology such as liver hyperplasia, fibrosis, and hepatocellular carcinoma. At the molecular level these pathways were linked to TNF alpha, TGF beta, PDGF, AGT and VEGF signalling. More generally, this pioneering study has important implications as it can be applied to model molecular mechanisms of compensatory adaptation to a wide range of scenarios in wild populations.

Towards a System Level Understanding of Non-Model Organisms Sampled from the Environment: A Network Biology Approach

PubMed Central

Williams, Tim D.; Turan, Nil; Diab, Amer M.; Wu, Huifeng; Mackenzie, Carolynn; Bartie, Katie L.; Hrydziuszko, Olga; Lyons, Brett P.; Stentiford, Grant D.; Herbert, John M.; Abraham, Joseph K.; Katsiadaki, Ioanna; Leaver, Michael J.; Taggart, John B.; George, Stephen G.; Viant, Mark R.; Chipman, Kevin J.; Falciani, Francesco

2011-01-01

The acquisition and analysis of datasets including multi-level omics and physiology from non-model species, sampled from field populations, is a formidable challenge, which so far has prevented the application of systems biology approaches. If successful, these could contribute enormously to improving our understanding of how populations of living organisms adapt to environmental stressors relating to, for example, pollution and climate. Here we describe the first application of a network inference approach integrating transcriptional, metabolic and phenotypic information representative of wild populations of the European flounder fish, sampled at seven estuarine locations in northern Europe with different degrees and profiles of chemical contaminants. We identified network modules, whose activity was predictive of environmental exposure and represented a link between molecular and morphometric indices. These sub-networks represented both known and candidate novel adverse outcome pathways representative of several aspects of human liver pathophysiology such as liver hyperplasia, fibrosis, and hepatocellular carcinoma. At the molecular level these pathways were linked to TNF alpha, TGF beta, PDGF, AGT and VEGF signalling. More generally, this pioneering study has important implications as it can be applied to model molecular mechanisms of compensatory adaptation to a wide range of scenarios in wild populations. PMID:21901081

Population genomics of C. melanopterus using target gene capture data: demographic inferences and conservation perspectives.

PubMed

Maisano Delser, Pierpaolo; Corrigan, Shannon; Hale, Matthew; Li, Chenhong; Veuille, Michel; Planes, Serge; Naylor, Gavin; Mona, Stefano

2016-09-21

Population genetics studies on non-model organisms typically involve sampling few markers from multiple individuals. Next-generation sequencing approaches open up the possibility of sampling many more markers from fewer individuals to address the same questions. Here, we applied a target gene capture method to deep sequence ~1000 independent autosomal regions of a non-model organism, the blacktip reef shark (Carcharhinus melanopterus). We devised a sampling scheme based on the predictions of theoretical studies of metapopulations to show that sampling few individuals, but many loci, can be extremely informative to reconstruct the evolutionary history of species. We collected data from a single deme (SID) from Northern Australia and from a scattered sampling representing various locations throughout the Indian Ocean (SCD). We explored the genealogical signature of population dynamics detected from both sampling schemes using an ABC algorithm. We then contrasted these results with those obtained by fitting the data to a non-equilibrium finite island model. Both approaches supported an Nm value ~40, consistent with philopatry in this species. Finally, we demonstrate through simulation that metapopulations exhibit greater resilience to recent changes in effective size compared to unstructured populations. We propose an empirical approach to detect recent bottlenecks based on our sampling scheme.

Myticalins: A Novel Multigenic Family of Linear, Cationic Antimicrobial Peptides from Marine Mussels (Mytilus spp.).

PubMed

Leoni, Gabriele; De Poli, Andrea; Mardirossian, Mario; Gambato, Stefano; Florian, Fiorella; Venier, Paola; Wilson, Daniel N; Tossi, Alessandro; Pallavicini, Alberto; Gerdol, Marco

2017-08-22

The application of high-throughput sequencing technologies to non-model organisms has brought new opportunities for the identification of bioactive peptides from genomes and transcriptomes. From this point of view, marine invertebrates represent a potentially rich, yet largely unexplored resource for de novo discovery due to their adaptation to diverse challenging habitats. Bioinformatics analyses of available genomic and transcriptomic data allowed us to identify myticalins, a novel family of antimicrobial peptides (AMPs) from the mussel Mytilus galloprovincialis , and a similar family of AMPs from Modiolus spp., named modiocalins. Their coding sequence encompasses two conserved N-terminal (signal peptide) and C-terminal (propeptide) regions and a hypervariable central cationic region corresponding to the mature peptide. Myticalins are taxonomically restricted to Mytiloida and they can be classified into four subfamilies. These AMPs are subject to considerable interindividual sequence variability and possibly to presence/absence variation. Functional assays performed on selected members of this family indicate a remarkable tissue-specific expression (in gills) and broad spectrum of activity against both Gram-positive and Gram-negative bacteria. Overall, we present the first linear AMPs ever described in marine mussels and confirm the great potential of bioinformatics tools for the de novo discovery of bioactive peptides in non-model organisms.

Long Non-Coding RNAs (lncRNAs) of Sea Cucumber: Large-Scale Prediction, Expression Profiling, Non-Coding Network Construction, and lncRNA-microRNA-Gene Interaction Analysis of lncRNAs in Apostichopus japonicus and Holothuria glaberrima During LPS Challenge and Radial Organ Complex Regeneration.

PubMed

Mu, Chuang; Wang, Ruijia; Li, Tianqi; Li, Yuqiang; Tian, Meilin; Jiao, Wenqian; Huang, Xiaoting; Zhang, Lingling; Hu, Xiaoli; Wang, Shi; Bao, Zhenmin

2016-08-01

Long non-coding RNA (lncRNA) structurally resembles mRNA but cannot be translated into protein. Although the systematic identification and characterization of lncRNAs have been increasingly reported in model species, information concerning non-model species is still lacking. Here, we report the first systematic identification and characterization of lncRNAs in two sea cucumber species: (1) Apostichopus japonicus during lipopolysaccharide (LPS) challenge and in heathy tissues and (2) Holothuria glaberrima during radial organ complex regeneration, using RNA-seq datasets and bioinformatics analysis. We identified A. japonicus and H. glaberrima lncRNAs that were differentially expressed during LPS challenge and radial organ complex regeneration, respectively. Notably, the predicted lncRNA-microRNA-gene trinities revealed that, in addition to targeting protein-coding transcripts, miRNAs might also target lncRNAs, thereby participating in a potential novel layer of regulatory interactions among non-coding RNA classes in echinoderms. Furthermore, the constructed coding-non-coding network implied the potential involvement of lncRNA-gene interactions during the regulation of several important genes (e.g., Toll-like receptor 1 [TLR1] and transglutaminase-1 [TGM1]) in response to LPS challenge and radial organ complex regeneration in sea cucumbers. Overall, this pioneer systematic identification, annotation, and characterization of lncRNAs in echinoderm pave the way for similar studies and future genetic, genomic, and evolutionary research in non-model species.

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved (Non-model) Organisms

PubMed Central

Joyce, Blake L.; Haug-Baltzell, Asher K.; Hulvey, Jonathan P.; McCarthy, Fiona; Devisetty, Upendra Kumar; Lyons, Eric

2017-01-01

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative transcriptomics. It also serves as a primer for biologists to develop data scientist computational skills, e.g. executing bash commands, visualization and management of large data sets. All command line code and further explanations of each command or step can be found on the wiki (https://wiki.cyverse.org/wiki/x/dgGtAQ). The Discovery Environment and Atmosphere platforms are connected together through the CyVerse Data Store. As such, once the initial raw sequencing data has been uploaded there is no more need to transfer large data files over an Internet connection, minimizing the amount of time needed to conduct analyses. This protocol is designed to analyze only two experimental treatments or conditions. Differential gene expression analysis is conducted through pairwise comparisons, and will not be suitable to test multiple factors. This workflow is also designed to be manual rather than automated. Each step must be executed and investigated by the user, yielding a better understanding of data and analytical outputs, and therefore better results for the user. Once complete, this protocol will yield de novo assembled transcriptome(s) for underserved (non-model) organisms without the need to map to previously assembled reference genomes (which are usually not available in underserved organism). These de novo transcriptomes are further used in pairwise differential gene expression analysis to investigate genes differing between two experimental conditions. Differentially expressed genes are then functionally annotated to understand the genetic response organisms have to experimental conditions. In total, the data derived from this protocol is used to test hypotheses about biological responses of underserved organisms. PMID:28518075

Enhanced guide-RNA design and targeting analysis for precise CRISPR genome editing of single and consortia of industrially relevant and non-model organisms.

PubMed

Mendoza, Brian J; Trinh, Cong T

2018-01-01

Genetic diversity of non-model organisms offers a repertoire of unique phenotypic features for exploration and cultivation for synthetic biology and metabolic engineering applications. To realize this enormous potential, it is critical to have an efficient genome editing tool for rapid strain engineering of these organisms to perform novel programmed functions. To accommodate the use of CRISPR/Cas systems for genome editing across organisms, we have developed a novel method, named CRISPR Associated Software for Pathway Engineering and Research (CASPER), for identifying on- and off-targets with enhanced predictability coupled with an analysis of non-unique (repeated) targets to assist in editing any organism with various endonucleases. Utilizing CASPER, we demonstrated a modest 2.4% and significant 30.2% improvement (F-test, P < 0.05) over the conventional methods for predicting on- and off-target activities, respectively. Further we used CASPER to develop novel applications in genome editing: multitargeting analysis (i.e. simultaneous multiple-site modification on a target genome with a sole guide-RNA requirement) and multispecies population analysis (i.e. guide-RNA design for genome editing across a consortium of organisms). Our analysis on a selection of industrially relevant organisms revealed a number of non-unique target sites associated with genes and transposable elements that can be used as potential sites for multitargeting. The analysis also identified shared and unshared targets that enable genome editing of single or multiple genomes in a consortium of interest. We envision CASPER as a useful platform to enhance the precise CRISPR genome editing for metabolic engineering and synthetic biology applications. https://github.com/TrinhLab/CASPER. ctrinh@utk.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Managing uncertainty in metabolic network structure and improving predictions using EnsembleFBA

PubMed Central

2017-01-01

Genome-scale metabolic network reconstructions (GENREs) are repositories of knowledge about the metabolic processes that occur in an organism. GENREs have been used to discover and interpret metabolic functions, and to engineer novel network structures. A major barrier preventing more widespread use of GENREs, particularly to study non-model organisms, is the extensive time required to produce a high-quality GENRE. Many automated approaches have been developed which reduce this time requirement, but automatically-reconstructed draft GENREs still require curation before useful predictions can be made. We present a novel approach to the analysis of GENREs which improves the predictive capabilities of draft GENREs by representing many alternative network structures, all equally consistent with available data, and generating predictions from this ensemble. This ensemble approach is compatible with many reconstruction methods. We refer to this new approach as Ensemble Flux Balance Analysis (EnsembleFBA). We validate EnsembleFBA by predicting growth and gene essentiality in the model organism Pseudomonas aeruginosa UCBPP-PA14. We demonstrate how EnsembleFBA can be included in a systems biology workflow by predicting essential genes in six Streptococcus species and mapping the essential genes to small molecule ligands from DrugBank. We found that some metabolic subsystems contributed disproportionately to the set of predicted essential reactions in a way that was unique to each Streptococcus species, leading to species-specific outcomes from small molecule interactions. Through our analyses of P. aeruginosa and six Streptococci, we show that ensembles increase the quality of predictions without drastically increasing reconstruction time, thus making GENRE approaches more practical for applications which require predictions for many non-model organisms. All of our functions and accompanying example code are available in an open online repository. PMID:28263984

Managing uncertainty in metabolic network structure and improving predictions using EnsembleFBA.

PubMed

Biggs, Matthew B; Papin, Jason A

2017-03-01

Genome-scale metabolic network reconstructions (GENREs) are repositories of knowledge about the metabolic processes that occur in an organism. GENREs have been used to discover and interpret metabolic functions, and to engineer novel network structures. A major barrier preventing more widespread use of GENREs, particularly to study non-model organisms, is the extensive time required to produce a high-quality GENRE. Many automated approaches have been developed which reduce this time requirement, but automatically-reconstructed draft GENREs still require curation before useful predictions can be made. We present a novel approach to the analysis of GENREs which improves the predictive capabilities of draft GENREs by representing many alternative network structures, all equally consistent with available data, and generating predictions from this ensemble. This ensemble approach is compatible with many reconstruction methods. We refer to this new approach as Ensemble Flux Balance Analysis (EnsembleFBA). We validate EnsembleFBA by predicting growth and gene essentiality in the model organism Pseudomonas aeruginosa UCBPP-PA14. We demonstrate how EnsembleFBA can be included in a systems biology workflow by predicting essential genes in six Streptococcus species and mapping the essential genes to small molecule ligands from DrugBank. We found that some metabolic subsystems contributed disproportionately to the set of predicted essential reactions in a way that was unique to each Streptococcus species, leading to species-specific outcomes from small molecule interactions. Through our analyses of P. aeruginosa and six Streptococci, we show that ensembles increase the quality of predictions without drastically increasing reconstruction time, thus making GENRE approaches more practical for applications which require predictions for many non-model organisms. All of our functions and accompanying example code are available in an open online repository.

Diversity Arrays Technology (DArT) for Pan-Genomic Evolutionary Studies of Non-Model Organisms

PubMed Central

James, Karen E.; Schneider, Harald; Ansell, Stephen W.; Evers, Margaret; Robba, Lavinia; Uszynski, Grzegorz; Pedersen, Niklas; Newton, Angela E.; Russell, Stephen J.; Vogel, Johannes C.; Kilian, Andrzej

2008-01-01

Background High-throughput tools for pan-genomic study, especially the DNA microarray platform, have sparked a remarkable increase in data production and enabled a shift in the scale at which biological investigation is possible. The use of microarrays to examine evolutionary relationships and processes, however, is predominantly restricted to model or near-model organisms. Methodology/Principal Findings This study explores the utility of Diversity Arrays Technology (DArT) in evolutionary studies of non-model organisms. DArT is a hybridization-based genotyping method that uses microarray technology to identify and type DNA polymorphism. Theoretically applicable to any organism (even one for which no prior genetic data are available), DArT has not yet been explored in exclusively wild sample sets, nor extensively examined in a phylogenetic framework. DArT recovered 1349 markers of largely low copy-number loci in two lineages of seed-free land plants: the diploid fern Asplenium viride and the haploid moss Garovaglia elegans. Direct sequencing of 148 of these DArT markers identified 30 putative loci including four routinely sequenced for evolutionary studies in plants. Phylogenetic analyses of DArT genotypes reveal phylogeographic and substrate specificity patterns in A. viride, a lack of phylogeographic pattern in Australian G. elegans, and additive variation in hybrid or mixed samples. Conclusions/Significance These results enable methodological recommendations including procedures for detecting and analysing DArT markers tailored specifically to evolutionary investigations and practical factors informing the decision to use DArT, and raise evolutionary hypotheses concerning substrate specificity and biogeographic patterns. Thus DArT is a demonstrably valuable addition to the set of existing molecular approaches used to infer biological phenomena such as adaptive radiations, population dynamics, hybridization, introgression, ecological differentiation and phylogeography. PMID:18301759

dDocent: a RADseq, variant-calling pipeline designed for population genomics of non-model organisms.

PubMed

Puritz, Jonathan B; Hollenbeck, Christopher M; Gold, John R

2014-01-01

Restriction-site associated DNA sequencing (RADseq) has become a powerful and useful approach for population genomics. Currently, no software exists that utilizes both paired-end reads from RADseq data to efficiently produce population-informative variant calls, especially for non-model organisms with large effective population sizes and high levels of genetic polymorphism. dDocent is an analysis pipeline with a user-friendly, command-line interface designed to process individually barcoded RADseq data (with double cut sites) into informative SNPs/Indels for population-level analyses. The pipeline, written in BASH, uses data reduction techniques and other stand-alone software packages to perform quality trimming and adapter removal, de novo assembly of RAD loci, read mapping, SNP and Indel calling, and baseline data filtering. Double-digest RAD data from population pairings of three different marine fishes were used to compare dDocent with Stacks, the first generally available, widely used pipeline for analysis of RADseq data. dDocent consistently identified more SNPs shared across greater numbers of individuals and with higher levels of coverage. This is due to the fact that dDocent quality trims instead of filtering, incorporates both forward and reverse reads (including reads with INDEL polymorphisms) in assembly, mapping, and SNP calling. The pipeline and a comprehensive user guide can be found at http://dDocent.wordpress.com.

dDocent: a RADseq, variant-calling pipeline designed for population genomics of non-model organisms

PubMed Central

Hollenbeck, Christopher M.; Gold, John R.

2014-01-01

Restriction-site associated DNA sequencing (RADseq) has become a powerful and useful approach for population genomics. Currently, no software exists that utilizes both paired-end reads from RADseq data to efficiently produce population-informative variant calls, especially for non-model organisms with large effective population sizes and high levels of genetic polymorphism. dDocent is an analysis pipeline with a user-friendly, command-line interface designed to process individually barcoded RADseq data (with double cut sites) into informative SNPs/Indels for population-level analyses. The pipeline, written in BASH, uses data reduction techniques and other stand-alone software packages to perform quality trimming and adapter removal, de novo assembly of RAD loci, read mapping, SNP and Indel calling, and baseline data filtering. Double-digest RAD data from population pairings of three different marine fishes were used to compare dDocent with Stacks, the first generally available, widely used pipeline for analysis of RADseq data. dDocent consistently identified more SNPs shared across greater numbers of individuals and with higher levels of coverage. This is due to the fact that dDocent quality trims instead of filtering, incorporates both forward and reverse reads (including reads with INDEL polymorphisms) in assembly, mapping, and SNP calling. The pipeline and a comprehensive user guide can be found at http://dDocent.wordpress.com. PMID:24949246

Simulating Nonmodel-Fitting Responses in a CAT Environment. ACT Research Report Series 98-10.

ERIC Educational Resources Information Center

Yi, Qing; Nering, Michael L.

This study developed a model to simulate nonmodel-fitting responses in a computerized adaptive testing (CAT) environment, and to examine the effectiveness of the model. The underlying idea was to simulate examinees' test behaviors realistically. This study simulated a situation in which examinees are exposed to or are coached on test items before…

The Influence of Nonmodel-Fitting Examinees in Estimating Person Parameters.

ERIC Educational Resources Information Center

Nering, Michael L.

The issue of person fit has received an increasing amount of attention by researchers in the past few years. Several studies have focused on the issue of how nonmodel-fitting responses affect the accuracy of ability estimates (e.g. R. Meijer and S. Nering, in press; Reise, 1995). The purpose of this study was to examine the effects that…

Who's your daddy? Using RADseq to explore survival and paternity in the clownfish, Amphiprion clarkii.

NASA Astrophysics Data System (ADS)

Stuart, M. R.; Pinsky, M. L.

2016-02-01

The ability to use DNA to identify individuals and their offspring has begun to revolutionize marine ecology. However, genetic mark-recapture and parentage studies typically require large numbers of individuals and associated high genotyping costs. Here, we describe a rapid and relatively low-cost protocol for genotyping non-model organisms at thousands of Single Nucleotide Polymorphisms (SNPs) using massively parallel sequencing. We apply the approach to a population of yellowtail clownfish, Amphiprion clarkii, to detect genetic mark-recaptures and parent-offspring relationships. We test multiple bioinformatic approaches and describe how this method could be applied to a wide variety of marine organisms.

Fixation of CO2 and CO on a diverse range of carbohydrates using anaerobic, non-photosynthetic mixotrophy.

PubMed

Maru, Biniam T; Munasinghe, Pradeep C; Gilary, Hadar; Jones, Shawn W; Tracy, Bryan P

2018-04-01

Biological CO2 fixation is an important technology that can assist in combating climate change. Here, we show an approach called anaerobic, non-photosynthetic mixotrophy can result in net CO2 fixation when using a reduced feedstock. This approach uses microbes called acetogens that are capable of concurrent utilization of both organic and inorganic substrates. In this study, we investigated the substrate utilization of 17 different acetogens, both mesophilic and thermophilic, on a variety of different carbohydrates and gases. Compared to most model acetogen strains, several non-model mesophilic strains displayed greater substrate flexibility, including the ability to utilize disaccharides, glycerol and an oligosaccharide, and growth rates. Three of these non-model strains (Blautia producta, Clostridium scatologenes and Thermoanaerobacter kivui) were chosen for further characterization, under a variety of conditions including H2- or syngas-fed sugar fermentations and a CO2-fed glycerol fermentation. In all cases, CO2 was fixed and carbon yields approached 100%. Finally, the model acetogen C. ljungdahlii was engineered to utilize glucose, a non-preferred sugar, while maintaining mixotrophic behavior. This work demonstrates the flexibility and robustness of anaerobic, non-photosynthetic mixotrophy as a technology to help reduce CO2 emissions.

From molecules to mating: Rapid evolution and biochemical studies of reproductive proteins

PubMed Central

Wilburn, Damien B.; Swanson, Willie J.

2015-01-01

Sexual reproduction and the exchange of genetic information are essential biological processes for species across all branches of the tree of life. Over the last four decades, biochemists have continued to identify many of the factors that facilitate reproduction, but the molecular mechanisms that mediate this process continue to elude us. However, a recurring observation in this research has been the rapid evolution of reproductive proteins. In animals, the competing interests of males and females often result in arms race dynamics between pairs of interacting proteins. This phenomenon has been observed in all stages of reproduction, including pheromones, seminal fluid components, and gamete recognition proteins. In this article, we review how the integration of evolutionary theory with biochemical experiments can be used to study interacting reproductive proteins. Examples are included from both model and non-model organisms, and recent studies are highlighted for their use of state-of-the-art genomic and proteomic techniques. Significance Despite decades of research, our understanding of the molecular mechanisms that mediate fertilization remain poorly characterized. To date, molecular evolutionary studies on both model and non-model organisms have provided some of the best inferences to elucidating the molecular underpinnings of animal reproduction. This review article details how biochemical and evolutionary experiments have jointly enhanced the field for 40 years, and how recent work using high-throughput genomic and proteomic techniques have shed additional insights into this crucial biological process. PMID:26074353

De novo assembled expressed gene catalog of a fast-growing Eucalyptus tree produced by Illumina mRNA-Seq

PubMed Central

2010-01-01

Background De novo assembly of transcript sequences produced by short-read DNA sequencing technologies offers a rapid approach to obtain expressed gene catalogs for non-model organisms. A draft genome sequence will be produced in 2010 for a Eucalyptus tree species (E. grandis) representing the most important hardwood fibre crop in the world. Genome annotation of this valuable woody plant and genetic dissection of its superior growth and productivity will be greatly facilitated by the availability of a comprehensive collection of expressed gene sequences from multiple tissues and organs. Results We present an extensive expressed gene catalog for a commercially grown E. grandis × E. urophylla hybrid clone constructed using only Illumina mRNA-Seq technology and de novo assembly. A total of 18,894 transcript-derived contigs, a large proportion of which represent full-length protein coding genes were assembled and annotated. Analysis of assembly quality, length and diversity show that this dataset represent the most comprehensive expressed gene catalog for any Eucalyptus tree. mRNA-Seq analysis furthermore allowed digital expression profiling of all of the assembled transcripts across diverse xylogenic and non-xylogenic tissues, which is invaluable for ascribing putative gene functions. Conclusions De novo assembly of Illumina mRNA-Seq reads is an efficient approach for transcriptome sequencing and profiling in Eucalyptus and other non-model organisms. The transcriptome resource (Eucspresso, http://eucspresso.bi.up.ac.za/) generated by this study will be of value for genomic analysis of woody biomass production in Eucalyptus and for comparative genomic analysis of growth and development in woody and herbaceous plants. PMID:21122097

Gene expression profiling via LongSAGE in a non-model plant species: a case study in seeds of Brassica napus

PubMed Central

Obermeier, Christian; Hosseini, Bashir; Friedt, Wolfgang; Snowdon, Rod

2009-01-01

Background Serial analysis of gene expression (LongSAGE) was applied for gene expression profiling in seeds of oilseed rape (Brassica napus ssp. napus). The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana. Results Transcripts from 3,094 genes were detected at two time-points of seed development, 23 days and 35 days after pollination (DAP). Differential expression showed a shift from gene expression involved in diverse developmental processes including cell proliferation and seed coat formation at 23 DAP to more focussed metabolic processes including storage protein accumulation and lipid deposition at 35 DAP. The most abundant transcripts at 23 DAP were coding for diverse protease inhibitor proteins and proteases, including cysteine proteases involved in seed coat formation and a number of lipid transfer proteins involved in embryo pattern formation. At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant. Over both time-points, 18.6% of the detected genes were matched by Brassica ESTs identified by LongSAGE tags in antisense orientation. This suggests a strong involvement of antisense transcript expression in regulatory processes during B. napus seed development. Conclusion This study underlines the potential of transcript tagging approaches for gene expression profiling in Brassica crop species via EST matching to annotated A. thaliana genes. Limits of tag detection for low-abundance transcripts can today be overcome by ultra-high throughput sequencing approaches, so that tag-based gene expression profiling may soon become the method of choice for global expression profiling in non-model species. PMID:19575793

De novo assembly of the transcriptome of the non-model plant Streptocarpus rexii employing a novel heuristic to recover locus-specific transcript clusters.

PubMed

Chiara, Matteo; Horner, David S; Spada, Alberto

2013-01-01

De novo transcriptome characterization from Next Generation Sequencing data has become an important approach in the study of non-model plants. Despite notable advances in the assembly of short reads, the clustering of transcripts into unigene-like (locus-specific) clusters remains a somewhat neglected subject. Indeed, closely related paralogous transcripts are often merged into single clusters by current approaches. Here, a novel heuristic method for locus-specific clustering is compared to that implemented in the de novo assembler Oases, using the same initial transcript collections, derived from Arabidopsis thaliana and the developmental model Streptocarpus rexii. We show that the proposed approach improves cluster specificity in the A. thaliana dataset for which the reference genome is available. Furthermore, for the S. rexii data our filtered transcript collection matches a larger number of distinct annotated loci in reference genomes than the Oases set, while containing a reduced overall number of loci. A detailed discussion of advantages and limitations of our approach in processing de novo transcriptome reconstructions is presented. The proposed method should be widely applicable to other organisms, irrespective of the transcript assembly method employed. The S. rexii transcriptome is available as a sophisticated and augmented publicly available online database.

tropiTree: An NGS-Based EST-SSR Resource for 24 Tropical Tree Species

PubMed Central

Russell, Joanne R.; Hedley, Peter E.; Cardle, Linda; Dancey, Siobhan; Morris, Jenny; Booth, Allan; Odee, David; Mwaura, Lucy; Omondi, William; Angaine, Peter; Machua, Joseph; Muchugi, Alice; Milne, Iain; Kindt, Roeland; Jamnadass, Ramni; Dawson, Ian K.

2014-01-01

The development of genetic tools for non-model organisms has been hampered by cost, but advances in next-generation sequencing (NGS) have created new opportunities. In ecological research, this raises the prospect for developing molecular markers to simultaneously study important genetic processes such as gene flow in multiple non-model plant species within complex natural and anthropogenic landscapes. Here, we report the use of bar-coded multiplexed paired-end Illumina NGS for the de novo development of expressed sequence tag-derived simple sequence repeat (EST-SSR) markers at low cost for a range of 24 tree species. Each chosen tree species is important in complex tropical agroforestry systems where little is currently known about many genetic processes. An average of more than 5,000 EST-SSRs was identified for each of the 24 sequenced species, whereas prior to analysis 20 of the species had fewer than 100 nucleotide sequence citations. To make results available to potential users in a suitable format, we have developed an open-access, interactive online database, tropiTree (http://bioinf.hutton.ac.uk/tropiTree), which has a range of visualisation and search facilities, and which is a model for the efficient presentation and application of NGS data. PMID:25025376

Prediction models for transfer of arsenic from soil to corn grain (Zea mays L.).

PubMed

Yang, Hua; Li, Zhaojun; Long, Jian; Liang, Yongchao; Xue, Jianming; Davis, Murray; He, Wenxiang

2016-04-01

In this study, the transfer of arsenic (As) from soil to corn grain was investigated in 18 soils collected from throughout China. The soils were treated with three concentrations of As and the transfer characteristics were investigated in the corn grain cultivar Zhengdan 958 in a greenhouse experiment. Through stepwise multiple-linear regression analysis, prediction models were developed combining the As bioconcentration factor (BCF) of Zhengdan 958 and soil pH, organic matter (OM) content, and cation exchange capacity (CEC). The possibility of applying the Zhengdan 958 model to other cultivars was tested through a cross-cultivar extrapolation approach. The results showed that the As concentration in corn grain was positively correlated with soil pH. When the prediction model was applied to non-model cultivars, the ratio ranges between the predicted and measured BCF values were within a twofold interval between predicted and measured values. The ratios were close to a 1:1 relationship between predicted and measured values. It was also found that the prediction model (Log [BCF]=0.064 pH-2.297) could effectively reduce the measured BCF variability for all non-model corn cultivars. The novel model is firstly developed for As concentration in crop grain from soil, which will be very useful for understanding the As risk in soil environment.

The comet assay in Environmental Risk Assessment of marine pollutants: applications, assets and handicaps of surveying genotoxicity in non-model organisms.

PubMed

Martins, Marta; Costa, Pedro M

2015-01-01

Determining the genotoxic effects of pollutants has long been a priority in Environmental Risk Assessment (ERA) for coastal ecosystems, especially of complex areas such as estuaries and other confined waterbodies. The acknowledged link between DNA damage, mutagenicity and carcinogenicity to the exposure to certain toxicants has been responsible to the growing interest in determining the genotoxic effects of xenobiotics to wildlife as a measure of environmental risk. The comet assay, although widely employed in in vivo and in vitro toxicology, still holds many constraints in ERA, in large part owing to difficulties in obtaining conclusive cause-effect relationships from complex environments. Nevertheless, these challenges do not hinder the attempts to apply the alkaline comet assay on sentinel organisms, wild or subjected to bioassays in or ex situ (from fish to molluscs) as well to standardise protocols and establish general guidelines to the interpretation of findings. Fish have been regarded as an appealing subject due to the ease of performing the comet assay in whole blood. However, the application of the comet assay is becoming increasingly common in invertebrates (e.g. in molluscan haemocytes and solid tissues such as gills). Virtually all sorts of results have been obtained from the application of the comet assay in ERA (null, positive and inconclusive). However, it has become clear that interpreting DNA damage data from wild organisms is particularly challenging due to their ability to adapt to continuous environmental stressors, including toxicants. Also, the comet assay in non-model organisms for the purpose of ERA implies different constraints, assumptions and interpretation of findings, compared with the in vitro procedures from which most guidelines have been derived. This paper critically reviews the application of the comet assay in ERA, focusing on target organisms and tissues; protocol developments, case studies plus data handling and interpretation. © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

PARRoT- a homology-based strategy to quantify and compare RNA-sequencing from non-model organisms.

PubMed

Gan, Ruei-Chi; Chen, Ting-Wen; Wu, Timothy H; Huang, Po-Jung; Lee, Chi-Ching; Yeh, Yuan-Ming; Chiu, Cheng-Hsun; Huang, Hsien-Da; Tang, Petrus

2016-12-22

Next-generation sequencing promises the de novo genomic and transcriptomic analysis of samples of interests. However, there are only a few organisms having reference genomic sequences and even fewer having well-defined or curated annotations. For transcriptome studies focusing on organisms lacking proper reference genomes, the common strategy is de novo assembly followed by functional annotation. However, things become even more complicated when multiple transcriptomes are compared. Here, we propose a new analysis strategy and quantification methods for quantifying expression level which not only generate a virtual reference from sequencing data, but also provide comparisons between transcriptomes. First, all reads from the transcriptome datasets are pooled together for de novo assembly. The assembled contigs are searched against NCBI NR databases to find potential homolog sequences. Based on the searched result, a set of virtual transcripts are generated and served as a reference transcriptome. By using the same reference, normalized quantification values including RC (read counts), eRPKM (estimated RPKM) and eTPM (estimated TPM) can be obtained that are comparable across transcriptome datasets. In order to demonstrate the feasibility of our strategy, we implement it in the web service PARRoT. PARRoT stands for Pipeline for Analyzing RNA Reads of Transcriptomes. It analyzes gene expression profiles for two transcriptome sequencing datasets. For better understanding of the biological meaning from the comparison among transcriptomes, PARRoT further provides linkage between these virtual transcripts and their potential function through showing best hits in SwissProt, NR database, assigning GO terms. Our demo datasets showed that PARRoT can analyze two paired-end transcriptomic datasets of approximately 100 million reads within just three hours. In this study, we proposed and implemented a strategy to analyze transcriptomes from non-reference organisms which offers the opportunity to quantify and compare transcriptome profiles through a homolog based virtual transcriptome reference. By using the homolog based reference, our strategy effectively avoids the problems that may cause from inconsistencies among transcriptomes. This strategy will shed lights on the field of comparative genomics for non-model organism. We have implemented PARRoT as a web service which is freely available at http://parrot.cgu.edu.tw .

Microsatellite loci discovery from next-generation sequencing data and loci characterization in the epizoic barnacle Chelonibia testudinaria (Linnaeus, 1758)

PubMed Central

Zardus, John D.; Wares, John P.

2016-01-01

Microsatellite markers remain an important tool for ecological and evolutionary research, but are unavailable for many non-model organisms. One such organism with rare ecological and evolutionary features is the epizoic barnacle Chelonibia testudinaria (Linnaeus, 1758). Chelonibia testudinaria appears to be a host generalist, and has an unusual sexual system, androdioecy. Genetic studies on host specificity and mating behavior are impeded by the lack of fine-scale, highly variable markers, such as microsatellite markers. In the present study, we discovered thousands of new microsatellite loci from next-generation sequencing data, and characterized 12 loci thoroughly. We conclude that 11 of these loci will be useful markers in future ecological and evolutionary studies on C. testudinaria. PMID:27231653

AD-LIBS: inferring ancestry across hybrid genomes using low-coverage sequence data.

PubMed

Schaefer, Nathan K; Shapiro, Beth; Green, Richard E

2017-04-04

Inferring the ancestry of each region of admixed individuals' genomes is useful in studies ranging from disease gene mapping to speciation genetics. Current methods require high-coverage genotype data and phased reference panels, and are therefore inappropriate for many data sets. We present a software application, AD-LIBS, that uses a hidden Markov model to infer ancestry across hybrid genomes without requiring variant calling or phasing. This approach is useful for non-model organisms and in cases of low-coverage data, such as ancient DNA. We demonstrate the utility of AD-LIBS with synthetic data. We then use AD-LIBS to infer ancestry in two published data sets: European human genomes with Neanderthal ancestry and brown bear genomes with polar bear ancestry. AD-LIBS correctly infers 87-91% of ancestry in simulations and produces ancestry maps that agree with published results and global ancestry estimates in humans. In brown bears, we find more polar bear ancestry than has been published previously, using both AD-LIBS and an existing software application for local ancestry inference, HAPMIX. We validate AD-LIBS polar bear ancestry maps by recovering a geographic signal within bears that mirrors what is seen in SNP data. Finally, we demonstrate that AD-LIBS is more effective than HAPMIX at inferring ancestry when preexisting phased reference data are unavailable and genomes are sequenced to low coverage. AD-LIBS is an effective tool for ancestry inference that can be used even when few individuals are available for comparison or when genomes are sequenced to low coverage. AD-LIBS is therefore likely to be useful in studies of non-model or ancient organisms that lack large amounts of genomic DNA. AD-LIBS can therefore expand the range of studies in which admixture mapping is a viable tool.

Transcriptome discovery in non-model wild fish species for the development of quantitative transcript abundance assays

USGS Publications Warehouse

Hahn, Cassidy M.; Iwanowicz, Luke R.; Cornman, Robert S.; Mazik, Patricia M.; Blazer, Vicki S.

2016-01-01

Environmental studies increasingly identify the presence of both contaminants of emerging concern (CECs) and legacy contaminants in aquatic environments; however, the biological effects of these compounds on resident fishes remain largely unknown. High throughput methodologies were employed to establish partial transcriptomes for three wild-caught, non-model fish species; smallmouth bass (Micropterus dolomieu), white sucker (Catostomus commersonii) and brown bullhead (Ameiurus nebulosus). Sequences from these transcriptome databases were utilized in the development of a custom nCounter CodeSet that allowed for direct multiplexed measurement of 50 transcript abundance endpoints in liver tissue. Sequence information was also utilized in the development of quantitative real-time PCR (qPCR) primers. Cross-species hybridization allowed the smallmouth bass nCounter CodeSet to be used for quantitative transcript abundance analysis of an additional non-model species, largemouth bass (Micropterus salmoides). We validated the nCounter analysis data system with qPCR for a subset of genes and confirmed concordant results. Changes in transcript abundance biomarkers between sexes and seasons were evaluated to provide baseline data on transcript modulation for each species of interest.

Next Generation Sequencing Technologies: The Doorway to the Unexplored Genomics of Non-Model Plants

PubMed Central

Unamba, Chibuikem I. N.; Nag, Akshay; Sharma, Ram K.

2015-01-01

Non-model plants i.e., the species which have one or all of the characters such as long life cycle, difficulty to grow in the laboratory or poor fecundity, have been schemed out of sequencing projects earlier, due to high running cost of Sanger sequencing. Consequently, the information about their genomics and key biological processes are inadequate. However, the advent of fast and cost effective next generation sequencing (NGS) platforms in the recent past has enabled the unearthing of certain characteristic gene structures unique to these species. It has also aided in gaining insight about mechanisms underlying processes of gene expression and secondary metabolism as well as facilitated development of genomic resources for diversity characterization, evolutionary analysis and marker assisted breeding even without prior availability of genomic sequence information. In this review we explore how different Next Gen Sequencing platforms, as well as recent advances in NGS based high throughput genotyping technologies are rewarding efforts on de-novo whole genome/transcriptome sequencing, development of genome wide sequence based markers resources for improvement of non-model crops that are less costly than phenotyping. PMID:26734016

Comparative description of ten transcriptomes of newly sequenced invertebrates and efficiency estimation of genomic sampling in non-model taxa

PubMed Central

2012-01-01

Introduction Traditionally, genomic or transcriptomic data have been restricted to a few model or emerging model organisms, and to a handful of species of medical and/or environmental importance. Next-generation sequencing techniques have the capability of yielding massive amounts of gene sequence data for virtually any species at a modest cost. Here we provide a comparative analysis of de novo assembled transcriptomic data for ten non-model species of previously understudied animal taxa. Results cDNA libraries of ten species belonging to five animal phyla (2 Annelida [including Sipuncula], 2 Arthropoda, 2 Mollusca, 2 Nemertea, and 2 Porifera) were sequenced in different batches with an Illumina Genome Analyzer II (read length 100 or 150 bp), rendering between ca. 25 and 52 million reads per species. Read thinning, trimming, and de novo assembly were performed under different parameters to optimize output. Between 67,423 and 207,559 contigs were obtained across the ten species, post-optimization. Of those, 9,069 to 25,681 contigs retrieved blast hits against the NCBI non-redundant database, and approximately 50% of these were assigned with Gene Ontology terms, covering all major categories, and with similar percentages in all species. Local blasts against our datasets, using selected genes from major signaling pathways and housekeeping genes, revealed high efficiency in gene recovery compared to available genomes of closely related species. Intriguingly, our transcriptomic datasets detected multiple paralogues in all phyla and in nearly all gene pathways, including housekeeping genes that are traditionally used in phylogenetic applications for their purported single-copy nature. Conclusions We generated the first study of comparative transcriptomics across multiple animal phyla (comparing two species per phylum in most cases), established the first Illumina-based transcriptomic datasets for sponge, nemertean, and sipunculan species, and generated a tractable catalogue of annotated genes (or gene fragments) and protein families for ten newly sequenced non-model organisms, some of commercial importance (i.e., Octopus vulgaris). These comprehensive sets of genes can be readily used for phylogenetic analysis, gene expression profiling, developmental analysis, and can also be a powerful resource for gene discovery. The characterization of the transcriptomes of such a diverse array of animal species permitted the comparison of sequencing depth, functional annotation, and efficiency of genomic sampling using the same pipelines, which proved to be similar for all considered species. In addition, the datasets revealed their potential as a resource for paralogue detection, a recurrent concern in various aspects of biological inquiry, including phylogenetics, molecular evolution, development, and cellular biochemistry. PMID:23190771

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant.

PubMed

Kramer, Annemarie; Beck, Hans Christian; Kumar, Abhishek; Kristensen, Lars Peter; Imhoff, Johannes F; Labes, Antje

2015-01-01

The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum.

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

PubMed Central

Kramer, Annemarie; Beck, Hans Christian; Kumar, Abhishek; Kristensen, Lars Peter; Imhoff, Johannes F.; Labes, Antje

2015-01-01

The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum. PMID:26460745

Reproducibility and consistency of proteomic experiments on natural populations of a non-model aquatic insect.

PubMed

Hidalgo-Galiana, Amparo; Monge, Marta; Biron, David G; Canals, Francesc; Ribera, Ignacio; Cieslak, Alexandra

2014-01-01

Population proteomics has a great potential to address evolutionary and ecological questions, but its use in wild populations of non-model organisms is hampered by uncontrolled sources of variation. Here we compare the response to temperature extremes of two geographically distant populations of a diving beetle species (Agabus ramblae) using 2-D DIGE. After one week of acclimation in the laboratory under standard conditions, a third of the specimens of each population were placed at either 4 or 27°C for 12 h, with another third left as a control. We then compared the protein expression level of three replicated samples of 2-3 specimens for each treatment. Within each population, variation between replicated samples of the same treatment was always lower than variation between treatments, except for some control samples that retained a wider range of expression levels. The two populations had a similar response, without significant differences in the number of protein spots over- or under-expressed in the pairwise comparisons between treatments. We identified exemplary proteins among those differently expressed between treatments, which proved to be proteins known to be related to thermal response or stress. Overall, our results indicate that specimens collected in the wild are suitable for proteomic analyses, as the additional sources of variation were not enough to mask the consistency and reproducibility of the response to the temperature treatments.

A quantitative and qualitative comparison of illumina MiSeq and 454 amplicon sequencing for genotyping the highly polymorphic major histocompatibility complex (MHC) in a non-model species.

PubMed

Razali, Haslina; O'Connor, Emily; Drews, Anna; Burke, Terry; Westerdahl, Helena

2017-07-28

High-throughput sequencing enables high-resolution genotyping of extremely duplicated genes. 454 amplicon sequencing (454) has become the standard technique for genotyping the major histocompatibility complex (MHC) genes in non-model organisms. However, illumina MiSeq amplicon sequencing (MiSeq), which offers a much higher read depth, is now superseding 454. The aim of this study was to quantitatively and qualitatively evaluate the performance of MiSeq in relation to 454 for genotyping MHC class I alleles using a house sparrow (Passer domesticus) dataset with pedigree information. House sparrows provide a good study system for this comparison as their MHC class I genes have been studied previously and, consequently, we had prior expectations concerning the number of alleles per individual. We found that 454 and MiSeq performed equally well in genotyping amplicons with low diversity, i.e. amplicons from individuals that had fewer than 6 alleles. Although there was a higher rate of failure in the 454 dataset in resolving amplicons with higher diversity (6-9 alleles), the same genotypes were identified by both 454 and MiSeq in 98% of cases. We conclude that low diversity amplicons are equally well genotyped using either 454 or MiSeq, but the higher coverage afforded by MiSeq can lead to this approach outperforming 454 in amplicons with higher diversity.

Reproducibility and Consistency of Proteomic Experiments on Natural Populations of a Non-Model Aquatic Insect

PubMed Central

Hidalgo-Galiana, Amparo; Monge, Marta; Biron, David G.; Canals, Francesc; Ribera, Ignacio; Cieslak, Alexandra

2014-01-01

Population proteomics has a great potential to address evolutionary and ecological questions, but its use in wild populations of non-model organisms is hampered by uncontrolled sources of variation. Here we compare the response to temperature extremes of two geographically distant populations of a diving beetle species (Agabus ramblae) using 2-D DIGE. After one week of acclimation in the laboratory under standard conditions, a third of the specimens of each population were placed at either 4 or 27°C for 12 h, with another third left as a control. We then compared the protein expression level of three replicated samples of 2–3 specimens for each treatment. Within each population, variation between replicated samples of the same treatment was always lower than variation between treatments, except for some control samples that retained a wider range of expression levels. The two populations had a similar response, without significant differences in the number of protein spots over- or under-expressed in the pairwise comparisons between treatments. We identified exemplary proteins among those differently expressed between treatments, which proved to be proteins known to be related to thermal response or stress. Overall, our results indicate that specimens collected in the wild are suitable for proteomic analyses, as the additional sources of variation were not enough to mask the consistency and reproducibility of the response to the temperature treatments. PMID:25133588

Proteomics informed by transcriptomics for characterising active transposable elements and genome annotation in Aedes aegypti.

PubMed

Maringer, Kevin; Yousuf, Amjad; Heesom, Kate J; Fan, Jun; Lee, David; Fernandez-Sesma, Ana; Bessant, Conrad; Matthews, David A; Davidson, Andrew D

2017-01-19

Aedes aegypti is a vector for the (re-)emerging human pathogens dengue, chikungunya, yellow fever and Zika viruses. Almost half of the Ae. aegypti genome is comprised of transposable elements (TEs). Transposons have been linked to diverse cellular processes, including the establishment of viral persistence in insects, an essential step in the transmission of vector-borne viruses. However, up until now it has not been possible to study the overall proteome derived from an organism's mobile genetic elements, partly due to the highly divergent nature of TEs. Furthermore, as for many non-model organisms, incomplete genome annotation has hampered proteomic studies on Ae. aegypti. We analysed the Ae. aegypti proteome using our new proteomics informed by transcriptomics (PIT) technique, which bypasses the need for genome annotation by identifying proteins through matched transcriptomic (rather than genomic) data. Our data vastly increase the number of experimentally confirmed Ae. aegypti proteins. The PIT analysis also identified hotspots of incomplete genome annotation, and showed that poor sequence and assembly quality do not explain all annotation gaps. Finally, in a proof-of-principle study, we developed criteria for the characterisation of proteomically active TEs. Protein expression did not correlate with a TE's genomic abundance at different levels of classification. Most notably, long terminal repeat (LTR) retrotransposons were markedly enriched compared to other elements. PIT was superior to 'conventional' proteomic approaches in both our transposon and genome annotation analyses. We present the first proteomic characterisation of an organism's repertoire of mobile genetic elements, which will open new avenues of research into the function of transposon proteins in health and disease. Furthermore, our study provides a proof-of-concept that PIT can be used to evaluate a genome's annotation to guide annotation efforts which has the potential to improve the efficiency of annotation projects in non-model organisms. PIT therefore represents a valuable new tool to study the biology of the important vector species Ae. aegypti, including its role in transmitting emerging viruses of global public health concern.

Short read Illumina data for the de novo assembly of a non-model snail species transcriptome (Radix balthica, Basommatophora, Pulmonata), and a comparison of assembler performance

PubMed Central

2011-01-01

Background Until recently, read lengths on the Solexa/Illumina system were too short to reliably assemble transcriptomes without a reference sequence, especially for non-model organisms. However, with read lengths up to 100 nucleotides available in the current version, an assembly without reference genome should be possible. For this study we created an EST data set for the common pond snail Radix balthica by Illumina sequencing of a normalized transcriptome. Performance of three different short read assemblers was compared with respect to: the number of contigs, their length, depth of coverage, their quality in various BLAST searches and the alignment to mitochondrial genes. Results A single sequencing run of a normalized RNA pool resulted in 16,923,850 paired end reads with median read length of 61 bases. The assemblies generated by VELVET, OASES, and SeqMan NGEN differed in the total number of contigs, contig length, the number and quality of gene hits obtained by BLAST searches against various databases, and contig performance in the mt genome comparison. While VELVET produced the highest overall number of contigs, a large fraction of these were of small size (< 200bp), and gave redundant hits in BLAST searches and the mt genome alignment. The best overall contig performance resulted from the NGEN assembly. It produced the second largest number of contigs, which on average were comparable to the OASES contigs but gave the highest number of gene hits in two out of four BLAST searches against different reference databases. A subsequent meta-assembly of the four contig sets resulted in larger contigs, less redundancy and a higher number of BLAST hits. Conclusion Our results document the first de novo transcriptome assembly of a non-model species using Illumina sequencing data. We show that de novo transcriptome assembly using this approach yields results useful for downstream applications, in particular if a meta-assembly of contig sets is used to increase contig quality. These results highlight the ongoing need for improvements in assembly methodology. PMID:21679424

Pathway Inspector: a pathway based web application for RNAseq analysis of model and non-model organisms.

PubMed

Bianco, Luca; Riccadonna, Samantha; Lavezzo, Enrico; Falda, Marco; Formentin, Elide; Cavalieri, Duccio; Toppo, Stefano; Fontana, Paolo

2017-02-01

Pathway Inspector is an easy-to-use web application helping researchers to find patterns of expression in complex RNAseq experiments. The tool combines two standard approaches for RNAseq analysis: the identification of differentially expressed genes and a topology-based analysis of enriched pathways. Pathway Inspector is equipped with ad hoc interactive graphical interfaces simplifying the discovery of modulated pathways and the integration of the differentially expressed genes in the corresponding pathway topology. Pathway Inspector is available at the website http://admiral.fmach.it/PI and has been developed in Python, making use of the Django Web Framework. Contact:paolo.fontana@fmach.it

Maximizing ecological and evolutionary insight in bisulfite sequencing data sets

PubMed Central

Lea, Amanda J.; Vilgalys, Tauras P.; Durst, Paul A.P.; Tung, Jenny

2017-01-01

Preface Genome-scale bisulfite sequencing approaches have opened the door to ecological and evolutionary studies of DNA methylation in many organisms. These approaches can be powerful. However, they introduce new methodological and statistical considerations, some of which are particularly relevant to non-model systems. Here, we highlight how these considerations influence a study’s power to link methylation variation with a predictor variable of interest. Relative to current practice, we argue that sample sizes will need to increase to provide robust insights. We also provide recommendations for overcoming common challenges and an R Shiny app to aid in study design. PMID:29046582

Comparative mapping for bighead carp (Aristichthys nobilis) against model and non-model fishes provides insights into the genomic evolution of cyprinids.

PubMed

Zhu, Chuankun; Tong, Jingou; Yu, Xiaomu; Guo, Wenjie

2015-08-01

Comparative mapping provides an efficient method to connect genomes of non-model and model fishes. In this study, we used flanking sequences of the 659 microsatellites on a genetic map of bighead carp (Aristichthys nobilis) to comprehensively study syntenic relationships between bighead carp and nine model and non-model fishes. Of the five model and two food fishes with whole genome data, Cyprinus carpio showed the highest rate of positive BLAST hits (95.3 %) with bighead carp map, followed by Danio rerio (70.9 %), Oreochromis niloticus (21.7 %), Tetraodon nigroviridis (6.4 %), Gasterosteus aculeatus (5.2 %), Oryzias latipes (4.7 %) and Fugu rubripes (3.5 %). Chromosomal syntenic analyses showed that inversion was the basic chromosomal rearrangement during genomic evolution of cyprinids, and the extent of inversions and translocations was found to be positively correlated with evolutionary relationships among fishes studied. Among the five investigated cyprinids, linkage groups (LGs) of bighead carp, Hypophthalmichthys molitrix and Ctenopharyngodon idella exhibited a one-to-one relationship. Besides, LG 9 of bighead carp and homologous LGs of silver carp and grass carp all corresponded to the chromosomes 10 and 22 of zebrafish, suggesting that chromosomal fission may have occurred in the ancestor of zebrafish. On the other hand, LGs of bighead carp and common carp showed an approximate one-to-two relationship with extensive translocations, confirming the occurrence of a 4th whole genome duplication in common carp. This study provides insights into the understanding of genome evolution among cyprinids and would aid in transferring positional and functional information of genes from model fish like zebrafish to non-model fish like bighead carp.

Application of isotope labeling experiments and (13)C flux analysis to enable rational pathway engineering.

PubMed

McAtee, Allison G; Jazmin, Lara J; Young, Jamey D

2015-12-01

Isotope labeling experiments (ILEs) and (13)C flux analysis provide actionable information for metabolic engineers to identify knockout, overexpression, and/or media optimization targets. ILEs have been used in both academic and industrial labs to increase product formation, discover novel metabolic functions in previously uncharacterized organisms, and enhance the metabolic efficiency of host cell factories. This review highlights specific examples of how ILEs have been used in conjunction with enzyme or metabolic engineering to elucidate host cell metabolism and improve product titer, rate, or yield in a directed manner. We discuss recent progress and future opportunities involving the use of ILEs and (13)C flux analysis to characterize non-model host organisms and to identify and subsequently eliminate wasteful byproduct pathways or metabolic bottlenecks. Copyright © 2015 Elsevier Ltd. All rights reserved.

Parallel labeling experiments for pathway elucidation and (13)C metabolic flux analysis.

PubMed

Antoniewicz, Maciek R

2015-12-01

Metabolic pathway models provide the foundation for quantitative studies of cellular physiology through the measurement of intracellular metabolic fluxes. For model organisms metabolic models are well established, with many manually curated genome-scale model reconstructions, gene knockout studies and stable-isotope tracing studies. However, for non-model organisms a similar level of knowledge is often lacking. Compartmentation of cellular metabolism in eukaryotic systems also presents significant challenges for quantitative (13)C-metabolic flux analysis ((13)C-MFA). Recently, innovative (13)C-MFA approaches have been developed based on parallel labeling experiments, the use of multiple isotopic tracers and integrated data analysis, that allow more rigorous validation of pathway models and improved quantification of metabolic fluxes. Applications of these approaches open new research directions in metabolic engineering, biotechnology and medicine. Copyright © 2015 Elsevier Ltd. All rights reserved.

Frogs as integrative models for understanding digestive organ development and evolution

PubMed Central

Womble, Mandy; Pickett, Melissa; Nascone-Yoder, Nanette

2016-01-01

The digestive system comprises numerous cells, tissues and organs that are essential for the proper assimilation of nutrients and energy. Many aspects of digestive organ function are highly conserved among vertebrates, yet the final anatomical configuration of the gut varies widely between species, especially those with different diets. Improved understanding of the complex molecular and cellular events that orchestrate digestive organ development is pertinent to many areas of biology and medicine, including the regeneration or replacement of diseased organs, the etiology of digestive organ birth defects, and the evolution of specialized features of digestive anatomy. In this review, we highlight specific examples of how investigations using Xenopus laevis frog embryos have revealed insight into the molecular and cellular dynamics of digestive organ patterning and morphogenesis that would have been difficult to obtain in other animal models. Additionally, we discuss recent studies of gut development in non-model frog species with unique feeding strategies, such as Lepidobatrachus laev is and Eleutherodactylouscoqui, which are beginning to provide glimpses of the evolutionary mechanisms that may generate morphological variation in the digestive tract. The unparalleled experimental versatility of frog embryos make them excellent, integrative models for studying digestive organ development across multiple disciplines. PMID:26851628

Transcriptome discovery in non-model wild fish species for the development of quantitative transcript abundance assays.

PubMed

Hahn, Cassidy M; Iwanowicz, Luke R; Cornman, Robert S; Mazik, Patricia M; Blazer, Vicki S

2016-12-01

Environmental studies increasingly identify the presence of both contaminants of emerging concern (CECs) and legacy contaminants in aquatic environments; however, the biological effects of these compounds on resident fishes remain largely unknown. High throughput methodologies were employed to establish partial transcriptomes for three wild-caught, non-model fish species; smallmouth bass (Micropterus dolomieu), white sucker (Catostomus commersonii) and brown bullhead (Ameiurus nebulosus). Sequences from these transcriptome databases were utilized in the development of a custom nCounter CodeSet that allowed for direct multiplexed measurement of 50 transcript abundance endpoints in liver tissue. Sequence information was also utilized in the development of quantitative real-time PCR (qPCR) primers. Cross-species hybridization allowed the smallmouth bass nCounter CodeSet to be used for quantitative transcript abundance analysis of an additional non-model species, largemouth bass (Micropterus salmoides). We validated the nCounter analysis data system with qPCR for a subset of genes and confirmed concordant results. Changes in transcript abundance biomarkers between sexes and seasons were evaluated to provide baseline data on transcript modulation for each species of interest. Published by Elsevier Inc.

Gene editing tools: state-of-the-art and the road ahead for the model and non-model fishes.

PubMed

Barman, Hirak Kumar; Rasal, Kiran Dashrath; Chakrapani, Vemulawada; Ninawe, A S; Vengayil, Doyil T; Asrafuzzaman, Syed; Sundaray, Jitendra K; Jayasankar, Pallipuram

2017-10-01

Advancements in the DNA sequencing technologies and computational biology have revolutionized genome/transcriptome sequencing of non-model fishes at an affordable cost. This has led to a paradigm shift with regard to our heightened understandings of structure-functional relationships of genes at a global level, from model animals/fishes to non-model large animals/fishes. Whole genome/transcriptome sequencing technologies were supplemented with the series of discoveries in gene editing tools, which are being used to modify genes at pre-determined positions using programmable nucleases to explore their respective in vivo functions. For a long time, targeted gene disruption experiments were mostly restricted to embryonic stem cells, advances in gene editing technologies such as zinc finger nuclease, transcriptional activator-like effector nucleases and CRISPR (clustered regulatory interspaced short palindromic repeats)/CRISPR-associated nucleases have facilitated targeted genetic modifications beyond stem cells to a wide range of somatic cell lines across species from laboratory animals to farmed animals/fishes. In this review, we discuss use of different gene editing tools and the strategic implications in fish species for basic and applied biology research.

Trap Nesting Wasps and Bees in Agriculture: A Comparison of Sown Wildflower and Fallow Plots in Florida

PubMed Central

Smithers, Cherice; Irvin, Allyn; Stanley-Stahr, Cory; Daniels, Jaret C.; Ellis, James D.

2017-01-01

Wildflower strip plantings in intensive agricultural systems have become a widespread tool for promoting pollination services and biological conservation because of their use by wasps and bees. Many of the trap-nesting wasps are important predators of common crop pests, and cavity-nesting bees that utilize trap-nests are important pollinators for native plants and many crops. The impact of wildflower strips on the nesting frequency of trap-nesting wasps or bees within localized areas has not been thoroughly investigated. Trap-nests made of bamboo reeds (Bambusa sp.) were placed adjacent to eight 0.1 ha wildflower plots and paired fallow areas (control plots) to determine if wildflower strips encourage the nesting of wasps and bees. From August 2014 to November 2015, occupied reeds were gathered and adults were collected as they emerged from the trap-nests. Treatment (wildflower or fallow plots) did not impact the number of occupied reeds or species richness of trap-nesting wasps using the occupied reeds. The wasps Pachodynerus erynnis, Euodynerus megaera, Parancistrocerus pedestris, and Isodontia spp. were the most common trap-nesting species collected. Less than 2% of the occupied reeds contained bees, and all were from the genus Megachile. The nesting wasp and bee species demonstrated preferences for reeds with certain inside diameters (IDs). The narrow range of ID preferences exhibited by each bee/wasp may provide opportunities to take advantage of their natural histories for biological control and/or pollination purposes. PMID:28994726

A portable expression resource for engineering cross-species genetic circuits and pathways

PubMed Central

Kushwaha, Manish; Salis, Howard M.

2015-01-01

Genetic circuits and metabolic pathways can be reengineered to allow organisms to process signals and manufacture useful chemicals. However, their functions currently rely on organism-specific regulatory parts, fragmenting synthetic biology and metabolic engineering into host-specific domains. To unify efforts, here we have engineered a cross-species expression resource that enables circuits and pathways to reuse the same genetic parts, while functioning similarly across diverse organisms. Our engineered system combines mixed feedback control loops and cross-species translation signals to autonomously self-regulate expression of an orthogonal polymerase without host-specific promoters, achieving nontoxic and tuneable gene expression in diverse Gram-positive and Gram-negative bacteria. Combining 50 characterized system variants with mechanistic modelling, we show how the cross-species expression resource's dynamics, capacity and toxicity are controlled by the control loops' architecture and feedback strengths. We also demonstrate one application of the resource by reusing the same genetic parts to express a biosynthesis pathway in both model and non-model hosts. PMID:26184393

CRISPR-enabled tools for engineering microbial genomes and phenotypes.

PubMed

Tarasava, Katia; Oh, Eun Joong; Eckert, Carrie A; Gill, Ryan T

2018-06-19

In recent years CRISPR-Cas technologies have revolutionized microbial engineering approaches. Genome editing and non-editing applications of various CRISPR-Cas systems have expanded the throughput and scale of engineering efforts, as well as opened up new avenues for manipulating genomes of non-model organisms. As we expand the range of organisms used for biotechnological applications, we need to develop better, more versatile tools for manipulation of these systems. Here we summarize the current advances in microbial gene editing using CRISPR-Cas based tools, and highlight state-of-the-art methods for high-throughput, efficient genome-scale engineering in model organisms Escherichia coli and Saccharomyces cerevisiae. We also review non-editing CRISPR-Cas applications available for gene expression manipulation, epigenetic remodeling, RNA editing, labeling and synthetic gene circuit design. Finally, we point out the areas of research that need further development in order to expand the range of applications and increase the utility of these new methods. This article is protected by copyright. All rights reserved.

Methods for MHC genotyping in non-model vertebrates.

PubMed

Babik, W

2010-03-01

Genes of the major histocompatibility complex (MHC) are considered a paradigm of adaptive evolution at the molecular level and as such are frequently investigated by evolutionary biologists and ecologists. Accurate genotyping is essential for understanding of the role that MHC variation plays in natural populations, but may be extremely challenging. Here, I discuss the DNA-based methods currently used for genotyping MHC in non-model vertebrates, as well as techniques likely to find widespread use in the future. I also highlight the aspects of MHC structure that are relevant for genotyping, and detail the challenges posed by the complex genomic organization and high sequence variation of MHC loci. Special emphasis is placed on designing appropriate PCR primers, accounting for artefacts and the problem of genotyping alleles from multiple, co-amplifying loci, a strategy which is frequently necessary due to the structure of the MHC. The suitability of typing techniques is compared in various research situations, strategies for efficient genotyping are discussed and areas of likely progress in future are identified. This review addresses the well established typing methods such as the Single Strand Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Reference Strand Conformational Analysis (RSCA) and cloning of PCR products. In addition, it includes the intriguing possibility of direct amplicon sequencing followed by the computational inference of alleles and also next generation sequencing (NGS) technologies; the latter technique may, in the future, find widespread use in typing complex multilocus MHC systems. © 2009 Blackwell Publishing Ltd.

Pathway Inspector: a pathway based web application for RNAseq analysis of model and non-model organisms

PubMed Central

Bianco, Luca; Riccadonna, Samantha; Lavezzo, Enrico; Falda, Marco; Formentin, Elide; Cavalieri, Duccio; Toppo, Stefano

2017-01-01

Abstract Summary: Pathway Inspector is an easy-to-use web application helping researchers to find patterns of expression in complex RNAseq experiments. The tool combines two standard approaches for RNAseq analysis: the identification of differentially expressed genes and a topology-based analysis of enriched pathways. Pathway Inspector is equipped with ad hoc interactive graphical interfaces simplifying the discovery of modulated pathways and the integration of the differentially expressed genes in the corresponding pathway topology. Availability and Implementation: Pathway Inspector is available at the website http://admiral.fmach.it/PI and has been developed in Python, making use of the Django Web Framework. Contact: paolo.fontana@fmach.it PMID:28158604

Integrating diverse databases into an unified analysis framework: a Galaxy approach

PubMed Central

Blankenberg, Daniel; Coraor, Nathan; Von Kuster, Gregory; Taylor, James; Nekrutenko, Anton

2011-01-01

Recent technological advances have lead to the ability to generate large amounts of data for model and non-model organisms. Whereas, in the past, there have been a relatively small number of central repositories that serve genomic data, an increasing number of distinct specialized data repositories and resources have been established. Here, we describe a generic approach that provides for the integration of a diverse spectrum of data resources into a unified analysis framework, Galaxy (http://usegalaxy.org). This approach allows the simplified coupling of external data resources with the data analysis tools available to Galaxy users, while leveraging the native data mining facilities of the external data resources. Database URL: http://usegalaxy.org PMID:21531983

Synthetic Biology Expands the Industrial Potential of Yarrowia lipolytica.

PubMed

Markham, Kelly A; Alper, Hal S

2018-06-04

The oleaginous yeast Yarrowia lipolytica is quickly emerging as the most popular non-conventional (i.e., non-model organism) yeast in the bioproduction field. With a high propensity for flux through tricarboxylic acid (TCA) cycle intermediates and biological precursors such as acetyl-CoA and malonyl-CoA, this host is especially well suited to meet our industrial chemical production needs. Recent progress in synthetic biology tool development has greatly enhanced our ability to rewire this organism, with advances in genetic component design, CRISPR technologies, and modular cloning strategies. In this review we investigate recent developments in metabolic engineering and describe how the new tools being developed help to realize the full industrial potential of this host. Finally, we conclude with our vision of the developments that will be necessary to enhance future engineering efforts. Copyright © 2018 Elsevier Ltd. All rights reserved.

Frogs as integrative models for understanding digestive organ development and evolution.

PubMed

Womble, Mandy; Pickett, Melissa; Nascone-Yoder, Nanette

2016-03-01

The digestive system comprises numerous cells, tissues and organs that are essential for the proper assimilation of nutrients and energy. Many aspects of digestive organ function are highly conserved among vertebrates, yet the final anatomical configuration of the gut varies widely between species, especially those with different diets. Improved understanding of the complex molecular and cellular events that orchestrate digestive organ development is pertinent to many areas of biology and medicine, including the regeneration or replacement of diseased organs, the etiology of digestive organ birth defects, and the evolution of specialized features of digestive anatomy. In this review, we highlight specific examples of how investigations using Xenopus laevis frog embryos have revealed insight into the molecular and cellular dynamics of digestive organ patterning and morphogenesis that would have been difficult to obtain in other animal models. Additionally, we discuss recent studies of gut development in non-model frog species with unique feeding strategies, such as Lepidobatrachus laevis and Eleutherodactylous coqui, which are beginning to provide glimpses of the evolutionary mechanisms that may generate morphological variation in the digestive tract. The unparalleled experimental versatility of frog embryos make them excellent, integrative models for studying digestive organ development across multiple disciplines. Copyright © 2016 Elsevier Ltd. All rights reserved.

Polar bear encephalitis: establishment of a comprehensive next-generation pathogen analysis pipeline for captive and free-living wildlife.

PubMed

Szentiks, C A; Tsangaras, K; Abendroth, B; Scheuch, M; Stenglein, M D; Wohlsein, P; Heeger, F; Höveler, R; Chen, W; Sun, W; Damiani, A; Nikolin, V; Gruber, A D; Grobbel, M; Kalthoff, D; Höper, D; Czirják, G Á; Derisi, J; Mazzoni, C J; Schüle, A; Aue, A; East, M L; Hofer, H; Beer, M; Osterrieder, N; Greenwood, A D

2014-05-01

This report describes three possibly related incidences of encephalitis, two of them lethal, in captive polar bears (Ursus maritimus). Standard diagnostic methods failed to identify pathogens in any of these cases. A comprehensive, three-stage diagnostic 'pipeline' employing both standard serological methods and new DNA microarray and next generation sequencing-based diagnostics was developed, in part as a consequence of this initial failure. This pipeline approach illustrates the strengths, weaknesses and limitations of these tools in determining pathogen caused deaths in non-model organisms such as wildlife species and why the use of a limited number of diagnostic tools may fail to uncover important wildlife pathogens. Copyright © 2013 Elsevier Ltd. All rights reserved.

Candidate adaptive genes associated with lineage divergence: identifying SNPs via next-generation targeted resequencing in mule deer (Odocoileus hemionus).

PubMed

Powell, John H; Amish, Stephen J; Haynes, Gwilym D; Luikart, Gordon; Latch, Emily K

2016-09-01

Mule deer (Odocoileus hemionus) are an excellent nonmodel species for empirically testing hypotheses in landscape and population genomics due to their large population sizes (low genetic drift), relatively continuous distribution, diversity of occupied habitats and phenotypic variation. Because few genomic resources are currently available for this species, we used exon data from a cattle (Bos taurus) reference genome to direct targeted resequencing of 5935 genes in mule deer. We sequenced approximately 3.75 Mbp at minimum 20X coverage in each of the seven mule deer, identifying 23 204 single nucleotide polymorphisms (SNPs) within, or adjacent to, 6886 exons in 3559 genes. We found 91 SNP loci (from 69 genes) with putatively fixed allele frequency differences between the two major lineages of mule deer (mule deer and black-tailed deer), and our estimate of mean genetic divergence (genome-wide FST  = 0.123) between these lineages was consistent with previous findings using microsatellite loci. We detected an over-representation of gamete generation and amino acid transport genes among the genes with SNPs exhibiting potentially fixed allele frequency differences between lineages. This targeted resequencing approach using exon capture techniques has identified a suite of loci that can be used in future research to investigate the genomic basis of adaptation and differentiation between black-tailed deer and mule deer. This study also highlights techniques (and an exon capture array) that will facilitate population genomic research in other cervids and nonmodel organisms. © 2016 John Wiley & Sons Ltd.

De novo transcriptome assembly for a non-model species, the blood-sucking bug Triatoma brasiliensis, a vector of Chagas disease.

PubMed

Marchant, A; Mougel, F; Almeida, C; Jacquin-Joly, E; Costa, J; Harry, M

2015-04-01

High throughput sequencing (HTS) provides new research opportunities for work on non-model organisms, such as differential expression studies between populations exposed to different environmental conditions. However, such transcriptomic studies first require the production of a reference assembly. The choice of sampling procedure, sequencing strategy and assembly workflow is crucial. To develop a reliable reference transcriptome for Triatoma brasiliensis, the major Chagas disease vector in Northeastern Brazil, different de novo assembly protocols were generated using various datasets and software. Both 454 and Illumina sequencing technologies were applied on RNA extracted from antennae and mouthparts from single or pooled individuals. The 454 library yielded 278 Mb. Fifteen Illumina libraries were constructed and yielded nearly 360 million RNA-seq single reads and 46 million RNA-seq paired-end reads for nearly 45 Gb. For the 454 reads, we used three assemblers, Newbler, CAP3 and/or MIRA and for the Illumina reads, the Trinity assembler. Ten assembly workflows were compared using these programs separately or in combination. To compare the assemblies obtained, quantitative and qualitative criteria were used, including contig length, N50, contig number and the percentage of chimeric contigs. Completeness of the assemblies was estimated using the CEGMA pipeline. The best assembly (57,657 contigs, completeness of 80 %, <1 % chimeric contigs) was a hybrid assembly leading to recommend the use of (1) a single individual with large representation of biological tissues, (2) merging both long reads and short paired-end Illumina reads, (3) several assemblers in order to combine the specific advantages of each.

A comparison across non-model animals suggests an optimal sequencing depth for de novo transcriptome assembly

PubMed Central

2013-01-01

Background The lack of genomic resources can present challenges for studies of non-model organisms. Transcriptome sequencing offers an attractive method to gather information about genes and gene expression without the need for a reference genome. However, it is unclear what sequencing depth is adequate to assemble the transcriptome de novo for these purposes. Results We assembled transcriptomes of animals from six different phyla (Annelids, Arthropods, Chordates, Cnidarians, Ctenophores, and Molluscs) at regular increments of reads using Velvet/Oases and Trinity to determine how read count affects the assembly. This included an assembly of mouse heart reads because we could compare those against the reference genome that is available. We found qualitative differences in the assemblies of whole-animals versus tissues. With increasing reads, whole-animal assemblies show rapid increase of transcripts and discovery of conserved genes, while single-tissue assemblies show a slower discovery of conserved genes though the assembled transcripts were often longer. A deeper examination of the mouse assemblies shows that with more reads, assembly errors become more frequent but such errors can be mitigated with more stringent assembly parameters. Conclusions These assembly trends suggest that representative assemblies are generated with as few as 20 million reads for tissue samples and 30 million reads for whole-animals for RNA-level coverage. These depths provide a good balance between coverage and noise. Beyond 60 million reads, the discovery of new genes is low and sequencing errors of highly-expressed genes are likely to accumulate. Finally, siphonophores (polymorphic Cnidarians) are an exception and possibly require alternate assembly strategies. PMID:23496952

A comparison across non-model animals suggests an optimal sequencing depth for de novo transcriptome assembly.

PubMed

Francis, Warren R; Christianson, Lynne M; Kiko, Rainer; Powers, Meghan L; Shaner, Nathan C; Haddock, Steven H D

2013-03-12

The lack of genomic resources can present challenges for studies of non-model organisms. Transcriptome sequencing offers an attractive method to gather information about genes and gene expression without the need for a reference genome. However, it is unclear what sequencing depth is adequate to assemble the transcriptome de novo for these purposes. We assembled transcriptomes of animals from six different phyla (Annelids, Arthropods, Chordates, Cnidarians, Ctenophores, and Molluscs) at regular increments of reads using Velvet/Oases and Trinity to determine how read count affects the assembly. This included an assembly of mouse heart reads because we could compare those against the reference genome that is available. We found qualitative differences in the assemblies of whole-animals versus tissues. With increasing reads, whole-animal assemblies show rapid increase of transcripts and discovery of conserved genes, while single-tissue assemblies show a slower discovery of conserved genes though the assembled transcripts were often longer. A deeper examination of the mouse assemblies shows that with more reads, assembly errors become more frequent but such errors can be mitigated with more stringent assembly parameters. These assembly trends suggest that representative assemblies are generated with as few as 20 million reads for tissue samples and 30 million reads for whole-animals for RNA-level coverage. These depths provide a good balance between coverage and noise. Beyond 60 million reads, the discovery of new genes is low and sequencing errors of highly-expressed genes are likely to accumulate. Finally, siphonophores (polymorphic Cnidarians) are an exception and possibly require alternate assembly strategies.

Similar Ratios of Introns to Intergenic Sequence across Animal Genomes

PubMed Central

Wörheide, Gert

2017-01-01

Abstract One central goal of genome biology is to understand how the usage of the genome differs between organisms. Our knowledge of genome composition, needed for downstream inferences, is critically dependent on gene annotations, yet problems associated with gene annotation and assembly errors are usually ignored in comparative genomics. Here, we analyze the genomes of 68 species across 12 animal phyla and some single-cell eukaryotes for general trends in genome composition and transcription, taking into account problems of gene annotation. We show that, regardless of genome size, the ratio of introns to intergenic sequence is comparable across essentially all animals, with nearly all deviations dominated by increased intergenic sequence. Genomes of model organisms have ratios much closer to 1:1, suggesting that the majority of published genomes of nonmodel organisms are underannotated and consequently omit substantial numbers of genes, with likely negative impact on evolutionary interpretations. Finally, our results also indicate that most animals transcribe half or more of their genomes arguing against differences in genome usage between animal groups, and also suggesting that the transcribed portion is more dependent on genome size than previously thought. PMID:28633296

NGScloud: RNA-seq analysis of non-model species using cloud computing.

PubMed

Mora-Márquez, Fernando; Vázquez-Poletti, José Luis; López de Heredia, Unai

2018-05-03

RNA-seq analysis usually requires large computing infrastructures. NGScloud is a bioinformatic system developed to analyze RNA-seq data using the cloud computing services of Amazon that permit the access to ad hoc computing infrastructure scaled according to the complexity of the experiment, so its costs and times can be optimized. The application provides a user-friendly front-end to operate Amazon's hardware resources, and to control a workflow of RNA-seq analysis oriented to non-model species, incorporating the cluster concept, which allows parallel runs of common RNA-seq analysis programs in several virtual machines for faster analysis. NGScloud is freely available at https://github.com/GGFHF/NGScloud/. A manual detailing installation and how-to-use instructions is available with the distribution. unai.lopezdeheredia@upm.es.

Targeted Quantification of Isoforms of a Thylakoid-Bound Protein: MRM Method Development.

PubMed

Bru-Martínez, Roque; Martínez-Márquez, Ascensión; Morante-Carriel, Jaime; Sellés-Marchart, Susana; Martínez-Esteso, María José; Pineda-Lucas, José Luis; Luque, Ignacio

2018-01-01

Targeted mass spectrometric methods such as selected/multiple reaction monitoring (SRM/MRM) have found intense application in protein detection and quantification which competes with classical immunoaffinity techniques. It provides a universal procedure to develop a fast, highly specific, sensitive, accurate, and cheap methodology for targeted detection and quantification of proteins based on the direct analysis of their surrogate peptides typically generated by tryptic digestion. This methodology can be advantageously applied in the field of plant proteomics and particularly for non-model species since immunoreagents are scarcely available. Here, we describe the issues to take into consideration in order to develop a MRM method to detect and quantify isoforms of the thylakoid-bound protein polyphenol oxidase from the non-model and database underrepresented species Eriobotrya japonica Lindl.

Signal Correlations in Ecological Niches Can Shape the Organization and Evolution of Bacterial Gene Regulatory Networks

PubMed Central

Dufour, Yann S.; Donohue, Timothy J.

2015-01-01

Transcriptional regulation plays a significant role in the biological response of bacteria to changing environmental conditions. Therefore, mapping transcriptional regulatory networks is an important step not only in understanding how bacteria sense and interpret their environment but also to identify the functions involved in biological responses to specific conditions. Recent experimental and computational developments have facilitated the characterization of regulatory networks on a genome-wide scale in model organisms. In addition, the multiplication of complete genome sequences has encouraged comparative analyses to detect conserved regulatory elements and infer regulatory networks in other less well-studied organisms. However, transcription regulation appears to evolve rapidly, thus, creating challenges for the transfer of knowledge to nonmodel organisms. Nevertheless, the mechanisms and constraints driving the evolution of regulatory networks have been the subjects of numerous analyses, and several models have been proposed. Overall, the contributions of mutations, recombination, and horizontal gene transfer are complex. Finally, the rapid evolution of regulatory networks plays a significant role in the remarkable capacity of bacteria to adapt to new or changing environments. Conversely, the characteristics of environmental niches determine the selective pressures and can shape the structure of regulatory network accordingly. PMID:23046950

PLAZA 3.0: an access point for plant comparative genomics

PubMed Central

Proost, Sebastian; Van Bel, Michiel; Vaneechoutte, Dries; Van de Peer, Yves; Inzé, Dirk; Mueller-Roeber, Bernd; Vandepoele, Klaas

2015-01-01

Comparative sequence analysis has significantly altered our view on the complexity of genome organization and gene functions in different kingdoms. PLAZA 3.0 is designed to make comparative genomics data for plants available through a user-friendly web interface. Structural and functional annotation, gene families, protein domains, phylogenetic trees and detailed information about genome organization can easily be queried and visualized. Compared with the first version released in 2009, which featured nine organisms, the number of integrated genomes is more than four times higher, and now covers 37 plant species. The new species provide a wider phylogenetic range as well as a more in-depth sampling of specific clades, and genomes of additional crop species are present. The functional annotation has been expanded and now comprises data from Gene Ontology, MapMan, UniProtKB/Swiss-Prot, PlnTFDB and PlantTFDB. Furthermore, we improved the algorithms to transfer functional annotation from well-characterized plant genomes to other species. The additional data and new features make PLAZA 3.0 (http://bioinformatics.psb.ugent.be/plaza/) a versatile and comprehensible resource for users wanting to explore genome information to study different aspects of plant biology, both in model and non-model organisms. PMID:25324309

A nearest neighbor approach for automated transporter prediction and categorization from protein sequences.

PubMed

Li, Haiquan; Dai, Xinbin; Zhao, Xuechun

2008-05-01

Membrane transport proteins play a crucial role in the import and export of ions, small molecules or macromolecules across biological membranes. Currently, there are a limited number of published computational tools which enable the systematic discovery and categorization of transporters prior to costly experimental validation. To approach this problem, we utilized a nearest neighbor method which seamlessly integrates homologous search and topological analysis into a machine-learning framework. Our approach satisfactorily distinguished 484 transporter families in the Transporter Classification Database, a curated and representative database for transporters. A five-fold cross-validation on the database achieved a positive classification rate of 72.3% on average. Furthermore, this method successfully detected transporters in seven model and four non-model organisms, ranging from archaean to mammalian species. A preliminary literature-based validation has cross-validated 65.8% of our predictions on the 11 organisms, including 55.9% of our predictions overlapping with 83.6% of the predicted transporters in TransportDB.

Estimating the efficiency of fish cross-species cDNA microarray hybridization.

PubMed

Cohen, Raphael; Chalifa-Caspi, Vered; Williams, Timothy D; Auslander, Meirav; George, Stephen G; Chipman, James K; Tom, Moshe

2007-01-01

Using an available cross-species cDNA microarray is advantageous for examining multigene expression patterns in non-model organisms, saving the need for construction of species-specific arrays. The aim of the present study was to estimate relative efficiency of cross-species hybridizations across bony fishes, using bioinformatics tools. The methodology may serve also as a model for similar evaluations in other taxa. The theoretical evaluation was done by substituting comparative whole-transcriptome sequence similarity information into the thermodynamic hybridization equation. Complementary DNA sequence assemblages of nine fish species belonging to common families or suborders and distributed across the bony fish taxonomic branch were selected for transcriptome-wise comparisons. Actual cross-species hybridizations among fish of different taxonomic distances were used to validate and eventually to calibrate the theoretically computed relative efficiencies.

Introduction to MOVES2010, October 2010 Webinar Slides

EPA Pesticide Factsheets

This presentation provides a general overview of MOVES (MOtor Vehicle Emission Simulator) for non-modelers who need to understand the transition from MOBILE to MOVES, and background information on MOVES for modelers.

The life cycle of a genome project: perspectives and guidelines inspired by insect genome projects

PubMed Central

Papanicolaou, Alexie

2016-01-01

Many research programs on non-model species biology have been empowered by genomics. In turn, genomics is underpinned by a reference sequence and ancillary information created by so-called “genome projects”. The most reliable genome projects are the ones created as part of an active research program and designed to address specific questions but their life extends past publication. In this opinion paper I outline four key insights that have facilitated maintaining genomic communities: the key role of computational capability, the iterative process of building genomic resources, the value of community participation and the importance of manual curation. Taken together, these ideas can and do ensure the longevity of genome projects and the growing non-model species community can use them to focus a discussion with regards to its future genomic infrastructure. PMID:27006757

The life cycle of a genome project: perspectives and guidelines inspired by insect genome projects.

PubMed

Papanicolaou, Alexie

2016-01-01

Many research programs on non-model species biology have been empowered by genomics. In turn, genomics is underpinned by a reference sequence and ancillary information created by so-called "genome projects". The most reliable genome projects are the ones created as part of an active research program and designed to address specific questions but their life extends past publication. In this opinion paper I outline four key insights that have facilitated maintaining genomic communities: the key role of computational capability, the iterative process of building genomic resources, the value of community participation and the importance of manual curation. Taken together, these ideas can and do ensure the longevity of genome projects and the growing non-model species community can use them to focus a discussion with regards to its future genomic infrastructure.

A pipeline for the de novo assembly of the Themira biloba (Sepsidae: Diptera) transcriptome using a multiple k-mer length approach.

PubMed

Melicher, Dacotah; Torson, Alex S; Dworkin, Ian; Bowsher, Julia H

2014-03-12

The Sepsidae family of flies is a model for investigating how sexual selection shapes courtship and sexual dimorphism in a comparative framework. However, like many non-model systems, there are few molecular resources available. Large-scale sequencing and assembly have not been performed in any sepsid, and the lack of a closely related genome makes investigation of gene expression challenging. Our goal was to develop an automated pipeline for de novo transcriptome assembly, and to use that pipeline to assemble and analyze the transcriptome of the sepsid Themira biloba. Our bioinformatics pipeline uses cloud computing services to assemble and analyze the transcriptome with off-site data management, processing, and backup. It uses a multiple k-mer length approach combined with a second meta-assembly to extend transcripts and recover more bases of transcript sequences than standard single k-mer assembly. We used 454 sequencing to generate 1.48 million reads from cDNA generated from embryo, larva, and pupae of T. biloba and assembled a transcriptome consisting of 24,495 contigs. Annotation identified 16,705 transcripts, including those involved in embryogenesis and limb patterning. We assembled transcriptomes from an additional three non-model organisms to demonstrate that our pipeline assembled a higher-quality transcriptome than single k-mer approaches across multiple species. The pipeline we have developed for assembly and analysis increases contig length, recovers unique transcripts, and assembles more base pairs than other methods through the use of a meta-assembly. The T. biloba transcriptome is a critical resource for performing large-scale RNA-Seq investigations of gene expression patterns, and is the first transcriptome sequenced in this Dipteran family.

Single-nucleotide polymorphism discovery in Leptographium longiclavatum, a mountain pine beetle-associated symbiotic fungus, using whole-genome resequencing.

PubMed

Ojeda, Dario I; Dhillon, Braham; Tsui, Clement K M; Hamelin, Richard C

2014-03-01

Single-nucleotide polymorphisms (SNPs) are rapidly becoming the standard markers in population genomics studies; however, their use in nonmodel organisms is limited due to the lack of cost-effective approaches to uncover genome-wide variation, and the large number of individuals needed in the screening process to reduce ascertainment bias. To discover SNPs for population genomics studies in the fungal symbionts of the mountain pine beetle (MPB), we developed a road map to discover SNPs and to produce a genotyping platform. We undertook a whole-genome sequencing approach of Leptographium longiclavatum in combination with available genomics resources of another MPB symbiont, Grosmannia clavigera. We sequenced 71 individuals pooled into four groups using the Illumina sequencing technology. We generated between 27 and 30 million reads of 75 bp that resulted in a total of 1, 181 contigs longer than 2 kb and an assembled genome size of 28.9 Mb (N50 = 48 kb, average depth = 125x). A total of 9052 proteins were annotated, and between 9531 and 17,266 SNPs were identified in the four pools. A subset of 206 genes (containing 574 SNPs, 11% false positives) was used to develop a genotyping platform for this species. Using this roadmap, we developed a genotyping assay with a total of 147 SNPs located in 121 genes using the Illumina(®) Sequenom iPLEX Gold. Our preliminary genotyping (success rate = 85%) of 304 individuals from 36 populations supports the utility of this approach for population genomics studies in other MPB fungal symbionts and other fungal nonmodel species. © 2013 John Wiley & Sons Ltd.

Effective de novo assembly of fish genome using haploid larvae.

PubMed

Iwasaki, Yuki; Nishiki, Issei; Nakamura, Yoji; Yasuike, Motoshige; Kai, Wataru; Nomura, Kazuharu; Yoshida, Kazunori; Nomura, Yousuke; Fujiwara, Atushi; Kobayashi, Takanori; Ototake, Mitsuru

2016-02-01

Recent improvements in next-generation sequencing technology have made it possible to do whole genome sequencing, on even non-model eukaryote species with no available reference genomes. However, de novo assembly of diploid genomes is still a big challenge because of allelic variation. The aim of this study was to determine the feasibility of utilizing the genome of haploid fish larvae for de novo assembly of whole-genome sequences. We compared the efficiency of assembly using the haploid genome of yellowtail (Seriola quinqueradiata) with that using the diploid genome obtained from the dam. De novo assembly from the haploid and the diploid sequence reads (100 million reads per each datasets) generated by the Ion Proton sequencer (200 bp) was done under two different assembly algorithms, namely overlap-layout-consensus (OLC) and de Bruijn graph (DBG). This revealed that the assembly of the haploid genome significantly reduced (approximately 22% for OLC, 9% for DBG) the total number of contigs (with longer average and N50 contig lengths) when compared to the diploid genome assembly. The haploid assembly also improved the quality of the scaffolds by reducing the number of regions with unassigned nucleotides (Ns) (total length of Ns; 45,331,916 bp for haploids and 67,724,360 bp for diploids) in OLC-based assemblies. It appears clear that the haploid genome assembly is better because the allelic variation in the diploid genome disrupts the extension of contigs during the assembly process. Our results indicate that utilizing the genome of haploid larvae leads to a significant improvement in the de novo assembly process, thus providing a novel strategy for the construction of reference genomes from non-model diploid organisms such as fish. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Proteogenomic Analysis Greatly Expands the Identification of Proteins Related to Reproduction in the Apogamous Fern Dryopteris affinis ssp. affinis.

PubMed

Grossmann, Jonas; Fernández, Helena; Chaubey, Pururawa M; Valdés, Ana E; Gagliardini, Valeria; Cañal, María J; Russo, Giancarlo; Grossniklaus, Ueli

2017-01-01

Performing proteomic studies on non-model organisms with little or no genomic information is still difficult. However, many specific processes and biochemical pathways occur only in species that are poorly characterized at the genomic level. For example, many plants can reproduce both sexually and asexually, the first one allowing the generation of new genotypes and the latter their fixation. Thus, both modes of reproduction are of great agronomic value. However, the molecular basis of asexual reproduction is not well understood in any plant. In ferns, it combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells (apogamy). To set the basis to study these processes, we performed transcriptomics by next-generation sequencing (NGS) and shotgun proteomics by tandem mass spectrometry in the apogamous fern D. affinis ssp. affinis . For protein identification we used the public viridiplantae database (VPDB) to identify orthologous proteins from other plant species and new transcriptomics data to generate a "species-specific transcriptome database" (SSTDB). In total 1,397 protein clusters with 5,865 unique peptide sequences were identified (13 decoy proteins out of 1,410, protFDR 0.93% on protein cluster level). We show that using the SSTDB for protein identification increases the number of identified peptides almost four times compared to using only the publically available VPDB. We identified homologs of proteins involved in reproduction of higher plants, including proteins with a potential role in apogamy. With the increasing availability of genomic data from non-model species, similar proteogenomics approaches will improve the sensitivity in protein identification for species only distantly related to models.

ExprAlign - the identification of ESTs in non-model species by alignment of cDNA microarray expression profiles

PubMed Central

2009-01-01

Background Sequence identification of ESTs from non-model species offers distinct challenges particularly when these species have duplicated genomes and when they are phylogenetically distant from sequenced model organisms. For the common carp, an environmental model of aquacultural interest, large numbers of ESTs remained unidentified using BLAST sequence alignment. We have used the expression profiles from large-scale microarray experiments to suggest gene identities. Results Expression profiles from ~700 cDNA microarrays describing responses of 7 major tissues to multiple environmental stressors were used to define a co-expression landscape. This was based on the Pearsons correlation coefficient relating each gene with all other genes, from which a network description provided clusters of highly correlated genes as 'mountains'. We show that these contain genes with known identities and genes with unknown identities, and that the correlation constitutes evidence of identity in the latter. This procedure has suggested identities to 522 of 2701 unknown carp ESTs sequences. We also discriminate several common carp genes and gene isoforms that were not discriminated by BLAST sequence alignment alone. Precision in identification was substantially improved by use of data from multiple tissues and treatments. Conclusion The detailed analysis of co-expression landscapes is a sensitive technique for suggesting an identity for the large number of BLAST unidentified cDNAs generated in EST projects. It is capable of detecting even subtle changes in expression profiles, and thereby of distinguishing genes with a common BLAST identity into different identities. It benefits from the use of multiple treatments or contrasts, and from the large-scale microarray data. PMID:19939286

Metabolome analysis of 20 taxonomically related benzylisoquinoline alkaloid-producing plants.

PubMed

Hagel, Jillian M; Mandal, Rupasri; Han, Beomsoo; Han, Jun; Dinsmore, Donald R; Borchers, Christoph H; Wishart, David S; Facchini, Peter J

2015-09-15

Recent progress toward the elucidation of benzylisoquinoline alkaloid (BIA) metabolism has focused on a small number of model plant species. Current understanding of BIA metabolism in plants such as opium poppy, which accumulates important pharmacological agents such as codeine and morphine, has relied on a combination of genomics and metabolomics to facilitate gene discovery. Metabolomics studies provide important insight into the primary biochemical networks underpinning specialized metabolism, and serve as a key resource for metabolic engineering, gene discovery, and elucidation of governing regulatory mechanisms. Beyond model plants, few broad-scope metabolomics reports are available for the vast number of plant species known to produce an estimated 2500 structurally diverse BIAs, many of which exhibit promising medicinal properties. We applied a multi-platform approach incorporating four different analytical methods to examine 20 non-model, BIA-accumulating plant species. Plants representing four families in the Ranunculales were chosen based on reported BIA content, taxonomic distribution and importance in modern/traditional medicine. One-dimensional (1)H NMR-based profiling quantified 91 metabolites and revealed significant species- and tissue-specific variation in sugar, amino acid and organic acid content. Mono- and disaccharide sugars were generally lower in roots and rhizomes compared with stems, and a variety of metabolites distinguished callus tissue from intact plant organs. Direct flow infusion tandem mass spectrometry provided a broad survey of 110 lipid derivatives including phosphatidylcholines and acylcarnitines, and high-performance liquid chromatography coupled with UV detection quantified 15 phenolic compounds including flavonoids, benzoic acid derivatives and hydroxycinnamic acids. Ultra-performance liquid chromatography coupled with high-resolution Fourier transform mass spectrometry generated extensive mass lists for all species, which were mined for metabolites putatively corresponding to BIAs. Different alkaloids profiles, including both ubiquitous and potentially rare compounds, were observed. Extensive metabolite profiling combining multiple analytical platforms enabled a more complete picture of overall metabolism occurring in selected plant species. This study represents the first time a metabolomics approach has been applied to most of these species, despite their importance in modern and traditional medicine. Coupled with genomics data, these metabolomics resources serve as a key resource for the investigation of BIA biosynthesis in non-model plant species.

Two-year-olds use the generic/non-generic distinction to guide their inferences about novel kinds

PubMed Central

Graham, Susan A.; Nayer, Samantha L.; Gelman, Susan A.

2011-01-01

These studies investigated 24- and 30-month-olds’ sensitivity to generic versus nongeneric language when acquiring knowledge about novel kinds. Toddlers were administered an inductive inference task, during which they heard a generic noun-phrase (e.g., “Blicks drink milk”) or a non-generic noun-phrase (e.g., “This blick drinks milk”) paired with an action (e.g., drinking) modeled on an object. They were then provided with the model and a non-model exemplar and asked to imitate the action. After hearing non-generic phrases, 30-month-olds, but not 24-month-olds, imitated more often with the model than with the non-model exemplar. In contrast, after hearing generic phrases, 30-month-olds imitated equally often with both exemplars. These results suggest that 30-month-olds use the generic/non-generic distinction to guide their inferences about novel kinds. PMID:21410928

Similar Ratios of Introns to Intergenic Sequence across Animal Genomes.

PubMed

Francis, Warren R; Wörheide, Gert

2017-06-01

One central goal of genome biology is to understand how the usage of the genome differs between organisms. Our knowledge of genome composition, needed for downstream inferences, is critically dependent on gene annotations, yet problems associated with gene annotation and assembly errors are usually ignored in comparative genomics. Here, we analyze the genomes of 68 species across 12 animal phyla and some single-cell eukaryotes for general trends in genome composition and transcription, taking into account problems of gene annotation. We show that, regardless of genome size, the ratio of introns to intergenic sequence is comparable across essentially all animals, with nearly all deviations dominated by increased intergenic sequence. Genomes of model organisms have ratios much closer to 1:1, suggesting that the majority of published genomes of nonmodel organisms are underannotated and consequently omit substantial numbers of genes, with likely negative impact on evolutionary interpretations. Finally, our results also indicate that most animals transcribe half or more of their genomes arguing against differences in genome usage between animal groups, and also suggesting that the transcribed portion is more dependent on genome size than previously thought. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

PLAZA 3.0: an access point for plant comparative genomics.

PubMed

Proost, Sebastian; Van Bel, Michiel; Vaneechoutte, Dries; Van de Peer, Yves; Inzé, Dirk; Mueller-Roeber, Bernd; Vandepoele, Klaas

2015-01-01

Comparative sequence analysis has significantly altered our view on the complexity of genome organization and gene functions in different kingdoms. PLAZA 3.0 is designed to make comparative genomics data for plants available through a user-friendly web interface. Structural and functional annotation, gene families, protein domains, phylogenetic trees and detailed information about genome organization can easily be queried and visualized. Compared with the first version released in 2009, which featured nine organisms, the number of integrated genomes is more than four times higher, and now covers 37 plant species. The new species provide a wider phylogenetic range as well as a more in-depth sampling of specific clades, and genomes of additional crop species are present. The functional annotation has been expanded and now comprises data from Gene Ontology, MapMan, UniProtKB/Swiss-Prot, PlnTFDB and PlantTFDB. Furthermore, we improved the algorithms to transfer functional annotation from well-characterized plant genomes to other species. The additional data and new features make PLAZA 3.0 (http://bioinformatics.psb.ugent.be/plaza/) a versatile and comprehensible resource for users wanting to explore genome information to study different aspects of plant biology, both in model and non-model organisms. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Phenoscape: Identifying Candidate Genes for Evolutionary Phenotypes

PubMed Central

Edmunds, Richard C.; Su, Baofeng; Balhoff, James P.; Eames, B. Frank; Dahdul, Wasila M.; Lapp, Hilmar; Lundberg, John G.; Vision, Todd J.; Dunham, Rex A.; Mabee, Paula M.; Westerfield, Monte

2016-01-01

Phenotypes resulting from mutations in genetic model organisms can help reveal candidate genes for evolutionarily important phenotypic changes in related taxa. Although testing candidate gene hypotheses experimentally in nonmodel organisms is typically difficult, ontology-driven information systems can help generate testable hypotheses about developmental processes in experimentally tractable organisms. Here, we tested candidate gene hypotheses suggested by expert use of the Phenoscape Knowledgebase, specifically looking for genes that are candidates responsible for evolutionarily interesting phenotypes in the ostariophysan fishes that bear resemblance to mutant phenotypes in zebrafish. For this, we searched ZFIN for genetic perturbations that result in either loss of basihyal element or loss of scales phenotypes, because these are the ancestral phenotypes observed in catfishes (Siluriformes). We tested the identified candidate genes by examining their endogenous expression patterns in the channel catfish, Ictalurus punctatus. The experimental results were consistent with the hypotheses that these features evolved through disruption in developmental pathways at, or upstream of, brpf1 and eda/edar for the ancestral losses of basihyal element and scales, respectively. These results demonstrate that ontological annotations of the phenotypic effects of genetic alterations in model organisms, when aggregated within a knowledgebase, can be used effectively to generate testable, and useful, hypotheses about evolutionary changes in morphology. PMID:26500251

The easy road to genome-wide medium density SNP screening in a non-model species: development and application of a 10 K SNP-chip for the house sparrow (Passer domesticus).

PubMed

Hagen, Ingerid J; Billing, Anna M; Rønning, Bernt; Pedersen, Sindre A; Pärn, Henrik; Slate, Jon; Jensen, Henrik

2013-05-01

With the advent of next generation sequencing, new avenues have opened to study genomics in wild populations of non-model species. Here, we describe a successful approach to a genome-wide medium density Single Nucleotide Polymorphism (SNP) panel in a non-model species, the house sparrow (Passer domesticus), through the development of a 10 K Illumina iSelect HD BeadChip. Genomic DNA and cDNA derived from six individuals were sequenced on a 454 GS FLX system and generated a total of 1.2 million sequences, in which SNPs were detected. As no reference genome exists for the house sparrow, we used the zebra finch (Taeniopygia guttata) reference genome to determine the most likely position of each SNP. The 10 000 SNPs on the SNP-chip were selected to be distributed evenly across 31 chromosomes, giving on average one SNP per 100 000 bp. The SNP-chip was screened across 1968 individual house sparrows from four island populations. Of the original 10 000 SNPs, 7413 were found to be variable, and 99% of these SNPs were successfully called in at least 93% of all individuals. We used the SNP-chip to demonstrate the ability of such genome-wide marker data to detect population sub-division, and compared these results to similar analyses using microsatellites. The SNP-chip will be used to map Quantitative Trait Loci (QTL) for fitness-related phenotypic traits in natural populations. © 2013 Blackwell Publishing Ltd.

A Proteomic Workflow Using High-Throughput De Novo Sequencing Towards Complementation of Genome Information for Improved Comparative Crop Science.

PubMed

Turetschek, Reinhard; Lyon, David; Desalegn, Getinet; Kaul, Hans-Peter; Wienkoop, Stefanie

2016-01-01

The proteomic study of non-model organisms, such as many crop plants, is challenging due to the lack of comprehensive genome information. Changing environmental conditions require the study and selection of adapted cultivars. Mutations, inherent to cultivars, hamper protein identification and thus considerably complicate the qualitative and quantitative comparison in large-scale systems biology approaches. With this workflow, cultivar-specific mutations are detected from high-throughput comparative MS analyses, by extracting sequence polymorphisms with de novo sequencing. Stringent criteria are suggested to filter for confidential mutations. Subsequently, these polymorphisms complement the initially used database, which is ready to use with any preferred database search algorithm. In our example, we thereby identified 26 specific mutations in two cultivars of Pisum sativum and achieved an increased number (17 %) of peptide spectrum matches.

Network analysis of oyster transcriptome revealed a cascade of cellular responses during recovery after heat shock.

PubMed

Zhang, Lingling; Hou, Rui; Su, Hailin; Hu, Xiaoli; Wang, Shi; Bao, Zhenmin

2012-01-01

Oysters, as a major group of marine bivalves, can tolerate a wide range of natural and anthropogenic stressors including heat stress. Recent studies have shown that oysters pretreated with heat shock can result in induced heat tolerance. A systematic study of cellular recovery from heat shock may provide insights into the mechanism of acquired thermal tolerance. In this study, we performed the first network analysis of oyster transcriptome by reanalyzing microarray data from a previous study. Network analysis revealed a cascade of cellular responses during oyster recovery after heat shock and identified responsive gene modules and key genes. Our study demonstrates the power of network analysis in a non-model organism with poor gene annotations, which can lead to new discoveries that go beyond the focus on individual genes.

Gap Gene Regulatory Dynamics Evolve along a Genotype Network

PubMed Central

Crombach, Anton; Wotton, Karl R.; Jiménez-Guri, Eva; Jaeger, Johannes

2016-01-01

Developmental gene networks implement the dynamic regulatory mechanisms that pattern and shape the organism. Over evolutionary time, the wiring of these networks changes, yet the patterning outcome is often preserved, a phenomenon known as “system drift.” System drift is illustrated by the gap gene network—involved in segmental patterning—in dipteran insects. In the classic model organism Drosophila melanogaster and the nonmodel scuttle fly Megaselia abdita, early activation and placement of gap gene expression domains show significant quantitative differences, yet the final patterning output of the system is essentially identical in both species. In this detailed modeling analysis of system drift, we use gene circuits which are fit to quantitative gap gene expression data in M. abdita and compare them with an equivalent set of models from D. melanogaster. The results of this comparative analysis show precisely how compensatory regulatory mechanisms achieve equivalent final patterns in both species. We discuss the larger implications of the work in terms of “genotype networks” and the ways in which the structure of regulatory networks can influence patterns of evolutionary change (evolvability). PMID:26796549

RNA-Seq for gene identification and transcript profiling of three Stevia rebaudiana genotypes.

PubMed

Chen, Junwen; Hou, Kai; Qin, Peng; Liu, Hongchang; Yi, Bin; Yang, Wenting; Wu, Wei

2014-07-07

Stevia (Stevia rebaudiana) is an important medicinal plant that yields diterpenoid steviol glycosides (SGs). SGs are currently used in the preparation of medicines, food products and neutraceuticals because of its sweetening property (zero calories and about 300 times sweeter than sugar). Recently, some progress has been made in understanding the biosynthesis of SGs in Stevia, but little is known about the molecular mechanisms underlying this process. Additionally, the genomics of Stevia, a non-model species, remains uncharacterized. The recent advent of RNA-Seq, a next generation sequencing technology, provides an opportunity to expand the identification of Stevia genes through in-depth transcript profiling. We present a comprehensive landscape of the transcriptome profiles of three genotypes of Stevia with divergent SG compositions characterized using RNA-seq. 191,590,282 high-quality reads were generated and then assembled into 171,837 transcripts with an average sequence length of 969 base pairs. A total of 80,160 unigenes were annotated, and 14,211 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Gene sequences of all enzymes known to be involved in SG synthesis were examined. A total of 143 UDP-glucosyltransferase (UGT) unigenes were identified, some of which might be involved in SG biosynthesis. The expression patterns of eight of these genes were further confirmed by RT-QPCR. RNA-seq analysis identified candidate genes encoding enzymes responsible for the biosynthesis of SGs in Stevia, a non-model plant without a reference genome. The transcriptome data from this study yielded new insights into the process of SG accumulation in Stevia. Our results demonstrate that RNA-Seq can be successfully used for gene identification and transcript profiling in a non-model species.

Comparative Gut Microbiota of 59 Neotropical Bird Species

PubMed Central

Hird, Sarah M.; Sánchez, César; Carstens, Bryan C.; Brumfield, Robb T.

2015-01-01

The gut microbiota of vertebrates are essential to host health. Most non-model vertebrates, however, lack even a basic description of natural gut microbiota biodiversity. Here, we sampled 116 intestines from 59 Neotropical bird species and used the V6 region of the 16S rRNA molecule as a microbial fingerprint (average coverage per bird ~80,000 reads). A core microbiota of Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria was identified, as well as several gut-associated genera. We tested 18 categorical variables associated with each bird for significant correlation to the gut microbiota; host taxonomic categories were most frequently significant and explained the most variation. Ecological variables (e.g., diet, foraging stratum) were also frequently significant but explained less variation. Little evidence was found for a significant influence of geographic space. Finally, we suggest that microbial sampling during field collection of organisms would propel biological understanding of evolutionary history and ecological significance of host-associated microbiota. PMID:26733954

Oral delivery of dsRNA by microbes: Beyond pest control.

PubMed

Abrieux, Antoine; Chiu, Joanna C

2016-01-01

RNA interference (RNAi) by oral delivery of dsRNA in insects has great potential as a tool for integrated pest management (IPM), especially with respect to addressing the need to reduce off-target effect and slow down resistance development to chemical insecticides. Employing the natural association existing between insect and yeast, we developed a novel method to enable the knock down of vital genes in the pest insect Drosophila suzukii through oral delivery of species-specific dsRNA using genetically modified Saccharomyces cerevisae. D. suzukii that were fed with our "yeast biopesticide" showed a significant decrease in fitness. In this perspective article, we postulate that this approach could be adapted to a large number of species, given the great diversity of symbiotic interactions involving microorganisms and host species. Furthermore, we speculate that beyond its application as biopesticide, dsRNA delivery by genetically modified microbes can also serve to facilitate reverse genetic applications, specifically in non-model organisms.

Icarus: visualizer for de novo assembly evaluation.

PubMed

Mikheenko, Alla; Valin, Gleb; Prjibelski, Andrey; Saveliev, Vladislav; Gurevich, Alexey

2016-11-01

: Data visualization plays an increasingly important role in NGS data analysis. With advances in both sequencing and computational technologies, it has become a new bottleneck in genomics studies. Indeed, evaluation of de novo genome assemblies is one of the areas that can benefit from the visualization. However, even though multiple quality assessment methods are now available, existing visualization tools are hardly suitable for this purpose. Here, we present Icarus-a novel genome visualizer for accurate assessment and analysis of genomic draft assemblies, which is based on the tool QUAST. Icarus can be used in studies where a related reference genome is available, as well as for non-model organisms. The tool is available online and as a standalone application. http://cab.spbu.ru/software/icarus CONTACT: aleksey.gurevich@spbu.ruSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Evaluating imputation algorithms for low-depth genotyping-by-sequencing (GBS) data

USDA-ARS?s Scientific Manuscript database

Well-powered genomic studies require genome-wide marker coverage across many individuals. For non-model species with few genomic resources, high-throughput sequencing (HTS) methods, such as Genotyping-By-Sequencing (GBS), offer an inexpensive alternative to array-based genotyping. Although affordabl...

Toward Universal Forward Genetics: Using a Draft Genome Sequence of the Nematode Oscheius tipulae To Identify Mutations Affecting Vulva Development

PubMed Central

Besnard, Fabrice; Koutsovoulos, Georgios; Dieudonné, Sana; Blaxter, Mark; Félix, Marie-Anne

2017-01-01

Mapping-by-sequencing has become a standard method to map and identify phenotype-causing mutations in model species. Here, we show that a fragmented draft assembly is sufficient to perform mapping-by-sequencing in nonmodel species. We generated a draft assembly and annotation of the genome of the free-living nematode Oscheius tipulae, a distant relative of the model Caenorhabditis elegans. We used this draft to identify the likely causative mutations at the O. tipulae cov-3 locus, which affect vulval development. The cov-3 locus encodes the O. tipulae ortholog of C. elegans mig-13, and we further show that Cel-mig-13 mutants also have an unsuspected vulval-development phenotype. In a virtuous circle, we were able to use the linkage information collected during mutant mapping to improve the genome assembly. These results showcase the promise of genome-enabled forward genetics in nonmodel species. PMID:28630114

Toward Universal Forward Genetics: Using a Draft Genome Sequence of the Nematode Oscheius tipulae To Identify Mutations Affecting Vulva Development.

PubMed

Besnard, Fabrice; Koutsovoulos, Georgios; Dieudonné, Sana; Blaxter, Mark; Félix, Marie-Anne

2017-08-01

Mapping-by-sequencing has become a standard method to map and identify phenotype-causing mutations in model species. Here, we show that a fragmented draft assembly is sufficient to perform mapping-by-sequencing in nonmodel species. We generated a draft assembly and annotation of the genome of the free-living nematode Oscheius tipulae , a distant relative of the model Caenorhabditis elegans We used this draft to identify the likely causative mutations at the O. tipulae cov -3 locus, which affect vulval development. The cov-3 locus encodes the O. tipulae ortholog of C. elegans mig-13 , and we further show that Cel-mig-13 mutants also have an unsuspected vulval-development phenotype. In a virtuous circle, we were able to use the linkage information collected during mutant mapping to improve the genome assembly. These results showcase the promise of genome-enabled forward genetics in nonmodel species. Copyright © 2017 by the Genetics Society of America.

Construction of a robust microarray from a non-model species (largemouth bass) using pyrosequencing technology

PubMed Central

Garcia-Reyero, Natàlia; Griffitt, Robert J.; Liu, Li; Kroll, Kevin J.; Farmerie, William G.; Barber, David S.; Denslow, Nancy D.

2009-01-01

A novel custom microarray for largemouth bass (Micropterus salmoides) was designed with sequences obtained from a normalized cDNA library using the 454 Life Sciences GS-20 pyrosequencer. This approach yielded in excess of 58 million bases of high-quality sequence. The sequence information was combined with 2,616 reads obtained by traditional suppressive subtractive hybridizations to derive a total of 31,391 unique sequences. Annotation and coding sequences were predicted for these transcripts where possible. 16,350 annotated transcripts were selected as target sequences for the design of the custom largemouth bass oligonucleotide microarray. The microarray was validated by examining the transcriptomic response in male largemouth bass exposed to 17β-œstradiol. Transcriptomic responses were assessed in liver and gonad, and indicated gene expression profiles typical of exposure to œstradiol. The results demonstrate the potential to rapidly create the tools necessary to assess large scale transcriptional responses in non-model species, paving the way for expanded impact of toxicogenomics in ecotoxicology. PMID:19936325

Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway

PubMed Central

Moghe, Gaurav D; Leong, Bryan J; Hurney, Steven M; Daniel Jones, A

2017-01-01

The diversity of life on Earth is a result of continual innovations in molecular networks influencing morphology and physiology. Plant specialized metabolism produces hundreds of thousands of compounds, offering striking examples of these innovations. To understand how this novelty is generated, we investigated the evolution of the Solanaceae family-specific, trichome-localized acylsugar biosynthetic pathway using a combination of mass spectrometry, RNA-seq, enzyme assays, RNAi and phylogenomics in different non-model species. Our results reveal hundreds of acylsugars produced across the Solanaceae family and even within a single plant, built on simple sugar cores. The relatively short biosynthetic pathway experienced repeated cycles of innovation over the last 100 million years that include gene duplication and divergence, gene loss, evolution of substrate preference and promiscuity. This study provides mechanistic insights into the emergence of plant chemical novelty, and offers a template for investigating the ~300,000 non-model plant species that remain underexplored. PMID:28853706

Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway.

PubMed

Moghe, Gaurav D; Leong, Bryan J; Hurney, Steven M; Daniel Jones, A; Last, Robert L

2017-08-30

The diversity of life on Earth is a result of continual innovations in molecular networks influencing morphology and physiology. Plant specialized metabolism produces hundreds of thousands of compounds, offering striking examples of these innovations. To understand how this novelty is generated, we investigated the evolution of the Solanaceae family-specific, trichome-localized acylsugar biosynthetic pathway using a combination of mass spectrometry, RNA-seq, enzyme assays, RNAi and phylogenomics in different non-model species. Our results reveal hundreds of acylsugars produced across the Solanaceae family and even within a single plant, built on simple sugar cores. The relatively short biosynthetic pathway experienced repeated cycles of innovation over the last 100 million years that include gene duplication and divergence, gene loss, evolution of substrate preference and promiscuity. This study provides mechanistic insights into the emergence of plant chemical novelty, and offers a template for investigating the ~300,000 non-model plant species that remain underexplored.

Reconstruction of 24 Penicillium genome-scale metabolic models shows diversity based on their secondary metabolism.

PubMed

Prigent, Sylvain; Nielsen, Jens Christian; Frisvad, Jens Christian; Nielsen, Jens

2018-06-05

Modelling of metabolism at the genome-scale have proved to be an efficient method for explaining observed phenotypic traits in living organisms. Further, it can be used as a means of predicting the effect of genetic modifications e.g. for development of microbial cell factories. With the increasing amount of genome sequencing data available, a need exists to accurately and efficiently generate such genome-scale metabolic models (GEMs) of non-model organisms, for which data is sparse. In this study, we present an automatic reconstruction approach applied to 24 Penicillium species, which have potential for production of pharmaceutical secondary metabolites or used in the manufacturing of food products such as cheeses. The models were based on the MetaCyc database and a previously published Penicillium GEM, and gave rise to comprehensive genome-scale metabolic descriptions. The models proved that while central carbon metabolism is highly conserved, secondary metabolic pathways represent the main diversity among the species. The automatic reconstruction approach presented in this study can be applied to generate GEMs of other understudied organisms, and the developed GEMs are a useful resource for the study of Penicillium metabolism, for example with the scope of developing novel cell factories. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

The environmental genomics of metazoan thermal adaptation

PubMed Central

Porcelli, D; Butlin, R K; Gaston, K J; Joly, D; Snook, R R

2015-01-01

Continued and accelerating change in the thermal environment places an ever-greater priority on understanding how organisms are going to respond. The paradigm of ‘move, adapt or die', regarding ways in which organisms can respond to environmental stressors, stimulates intense efforts to predict the future of biodiversity. Assuming that extinction is an unpalatable outcome, researchers have focussed attention on how organisms can shift in their distribution to stay in the same thermal conditions or can stay in the same place by adapting to a changing thermal environment. How likely these respective outcomes might be depends on the answer to a fundamental evolutionary question, namely what genetic changes underpin adaptation to the thermal environment. The increasing access to and decreasing costs of next-generation sequencing (NGS) technologies, which can be applied to both model and non-model systems, provide a much-needed tool for understanding thermal adaptation. Here we consider broadly what is already known from non-NGS studies about thermal adaptation, then discuss the benefits and challenges of different NGS methodologies to add to this knowledge base. We then review published NGS genomics and transcriptomics studies of thermal adaptation to heat stress in metazoans and compare these results with previous non-NGS patterns. We conclude by summarising emerging patterns of genetic response and discussing future directions using these increasingly common techniques. PMID:25735594

Comparative high-throughput transcriptome sequencing and development of SiESTa, the Silene EST annotation database

PubMed Central

2011-01-01

Background The genus Silene is widely used as a model system for addressing ecological and evolutionary questions in plants, but advances in using the genus as a model system are impeded by the lack of available resources for studying its genome. Massively parallel sequencing cDNA has recently developed into an efficient method for characterizing the transcriptomes of non-model organisms, generating massive amounts of data that enable the study of multiple species in a comparative framework. The sequences generated provide an excellent resource for identifying expressed genes, characterizing functional variation and developing molecular markers, thereby laying the foundations for future studies on gene sequence and gene expression divergence. Here, we report the results of a comparative transcriptome sequencing study of eight individuals representing four Silene and one Dianthus species as outgroup. All sequences and annotations have been deposited in a newly developed and publicly available database called SiESTa, the Silene EST annotation database. Results A total of 1,041,122 EST reads were generated in two runs on a Roche GS-FLX 454 pyrosequencing platform. EST reads were analyzed separately for all eight individuals sequenced and were assembled into contigs using TGICL. These were annotated with results from BLASTX searches and Gene Ontology (GO) terms, and thousands of single-nucleotide polymorphisms (SNPs) were characterized. Unassembled reads were kept as singletons and together with the contigs contributed to the unigenes characterized in each individual. The high quality of unigenes is evidenced by the proportion (49%) that have significant hits in similarity searches with the A. thaliana proteome. The SiESTa database is accessible at http://www.siesta.ethz.ch. Conclusion The sequence collections established in the present study provide an important genomic resource for four Silene and one Dianthus species and will help to further develop Silene as a plant model system. The genes characterized will be useful for future research not only in the species included in the present study, but also in related species for which no genomic resources are yet available. Our results demonstrate the efficiency of massively parallel transcriptome sequencing in a comparative framework as an approach for developing genomic resources in diverse groups of non-model organisms. PMID:21791039

MADS-box out of the black box

USDA-ARS?s Scientific Manuscript database

The compelling elegance of using genome-wide scans to detect the signature of selection is difficult to resist, but is countered by the low demonstrated efficacy of pinpointing the actual genes and traits that are the targets of selection in non-model species. While the difficulty of going from a s...

Development and transferability of black and red raspberry microsatellite markers from short-read sequences

USDA-ARS?s Scientific Manuscript database

The advent of next-generation sequencing technologies has been a boon to the cost-effective development of molecular markers, particularly in non-model species. Here, we demonstrate the efficiency of microsatellite or simple sequence repeat (SSR) marker development from short-read sequences using th...

Detection and mapping of QTL for temperature tolerance and body size in Chinook salmon (Oncorhynchus tshawytscha) using genotyping by sequencing

PubMed Central

Everett, Meredith V; Seeb, James E

2014-01-01

Understanding how organisms interact with their environments is increasingly important for conservation efforts in many species, especially in light of highly anticipated climate changes. One method for understanding this relationship is to use genetic maps and QTL mapping to detect genomic regions linked to phenotypic traits of importance for adaptation. We used high-throughput genotyping by sequencing (GBS) to both detect and map thousands of SNPs in haploid Chinook salmon (Oncorhynchus tshawytscha). We next applied this map to detect QTL related to temperature tolerance and body size in families of diploid Chinook salmon. Using these techniques, we mapped 3534 SNPs in 34 linkage groups which is consistent with the haploid chromosome number for Chinook salmon. We successfully detected three QTL for temperature tolerance and one QTL for body size at the experiment-wide level, as well as additional QTL significant at the chromosome-wide level. The use of haploids coupled with GBS provides a robust pathway to rapidly develop genomic resources in nonmodel organisms; these QTL represent preliminary progress toward linking traits of conservation interest to regions in the Chinook salmon genome. PMID:24822082

Mind the gap; seven reasons to close fragmented genome assemblies.

PubMed

Thomma, Bart P H J; Seidl, Michael F; Shi-Kunne, Xiaoqian; Cook, David E; Bolton, Melvin D; van Kan, Jan A L; Faino, Luigi

2016-05-01

Like other domains of life, research into the biology of filamentous microbes has greatly benefited from the advent of whole-genome sequencing. Next-generation sequencing (NGS) technologies have revolutionized sequencing, making genomic sciences accessible to many academic laboratories including those that study non-model organisms. Thus, hundreds of fungal genomes have been sequenced and are publically available today, although these initiatives have typically yielded considerably fragmented genome assemblies that often lack large contiguous genomic regions. Many important genomic features are contained in intergenic DNA that is often missing in current genome assemblies, and recent studies underscore the significance of non-coding regions and repetitive elements for the life style, adaptability and evolution of many organisms. The study of particular types of genetic elements, such as telomeres, centromeres, repetitive elements, effectors, and clusters of co-regulated genes, but also of phenomena such as structural rearrangements, genome compartmentalization and epigenetics, greatly benefits from having a contiguous and high-quality, preferably even complete and gapless, genome assembly. Here we discuss a number of important reasons to produce gapless, finished, genome assemblies to help answer important biological questions. Copyright © 2015 Elsevier Inc. All rights reserved.

Biotic interactions as drivers of algal origin and evolution.

PubMed

Brodie, Juliet; Ball, Steven G; Bouget, François-Yves; Chan, Cheong Xin; De Clerck, Olivier; Cock, J Mark; Gachon, Claire; Grossman, Arthur R; Mock, Thomas; Raven, John A; Saha, Mahasweta; Smith, Alison G; Vardi, Assaf; Yoon, Hwan Su; Bhattacharya, Debashish

2017-11-01

Contents 670 I. 671 II. 671 III. 676 IV. 678 678 References 678 SUMMARY: Biotic interactions underlie life's diversity and are the lynchpin to understanding its complexity and resilience within an ecological niche. Algal biologists have embraced this paradigm, and studies building on the explosive growth in omics and cell biology methods have facilitated the in-depth analysis of nonmodel organisms and communities from a variety of ecosystems. In turn, these advances have enabled a major revision of our understanding of the origin and evolution of photosynthesis in eukaryotes, bacterial-algal interactions, control of massive algal blooms in the ocean, and the maintenance and degradation of coral reefs. Here, we review some of the most exciting developments in the field of algal biotic interactions and identify challenges for scientists in the coming years. We foresee the development of an algal knowledgebase that integrates ecosystem-wide omics data and the development of molecular tools/resources to perform functional analyses of individuals in isolation and in populations. These assets will allow us to move beyond mechanistic studies of a single species towards understanding the interactions amongst algae and other organisms in both the laboratory and the field. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Speciation genetics: current status and evolving approaches

PubMed Central

Wolf, Jochen B. W.; Lindell, Johan; Backström, Niclas

2010-01-01

The view of species as entities subjected to natural selection and amenable to change put forth by Charles Darwin and Alfred Wallace laid the conceptual foundation for understanding speciation. Initially marred by a rudimental understanding of hereditary principles, evolutionists gained appreciation of the mechanistic underpinnings of speciation following the merger of Mendelian genetic principles with Darwinian evolution. Only recently have we entered an era where deciphering the molecular basis of speciation is within reach. Much focus has been devoted to the genetic basis of intrinsic postzygotic isolation in model organisms and several hybrid incompatibility genes have been successfully identified. However, concomitant with the recent technological advancements in genome analysis and a newfound interest in the role of ecology in the differentiation process, speciation genetic research is becoming increasingly open to non-model organisms. This development will expand speciation research beyond the traditional boundaries and unveil the genetic basis of speciation from manifold perspectives and at various stages of the splitting process. This review aims at providing an extensive overview of speciation genetics. Starting from key historical developments and core concepts of speciation genetics, we focus much of our attention on evolving approaches and introduce promising methodological approaches for future research venues. PMID:20439277

Molecular Regulation of Antibiotic Biosynthesis in Streptomyces

PubMed Central

Liu, Gang; Chandra, Govind; Niu, Guoqing

2013-01-01

SUMMARY Streptomycetes are the most abundant source of antibiotics. Typically, each species produces several antibiotics, with the profile being species specific. Streptomyces coelicolor, the model species, produces at least five different antibiotics. We review the regulation of antibiotic biosynthesis in S. coelicolor and other, nonmodel streptomycetes in the light of recent studies. The biosynthesis of each antibiotic is specified by a large gene cluster, usually including regulatory genes (cluster-situated regulators [CSRs]). These are the main point of connection with a plethora of generally conserved regulatory systems that monitor the organism's physiology, developmental state, population density, and environment to determine the onset and level of production of each antibiotic. Some CSRs may also be sensitive to the levels of different kinds of ligands, including products of the pathway itself, products of other antibiotic pathways in the same organism, and specialized regulatory small molecules such as gamma-butyrolactones. These interactions can result in self-reinforcing feed-forward circuitry and complex cross talk between pathways. The physiological signals and regulatory mechanisms may be of practical importance for the activation of the many cryptic secondary metabolic gene cluster pathways revealed by recent sequencing of numerous Streptomyces genomes. PMID:23471619

Hybrid striped bass national breeding program: research towards genetic improvement of a non-model species

USDA-ARS?s Scientific Manuscript database

The hybrid striped bass (HSB) farming industry at present relies almost totally on wild broodstock for annual production of larvae and fingerlings, and industry efforts to domesticate the parent species of the HSB (white bass: WB, Morone chrysops; striped bass: SB, M. saxatilis) have been fairly lim...

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design

USDA-ARS?s Scientific Manuscript database

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive ex...

Exploring DNA variant segregation types in pooled genome sequencing enables effective mapping of weeping trait in Malus

USDA-ARS?s Scientific Manuscript database

In recent years, next generation sequencing (NGS) based bulked segregant analysis (BSA) has become a powerful approach for allele discovery in non-model plant species. However, challenges remain, particular for out-crossing species with complex genomes. Here, the genetic control of a weeping bran...

Hybrid striped bass National Breeding Program: Research towards genetic improvement of a non-model species

USDA-ARS?s Scientific Manuscript database

The hybrid striped bass (HSB) farming industry at present relies almost totally on wild broodstock for annual production of larvae and fingerlings, and industry efforts to domesticate the parent species of the HSB (white bass: WB, Morone chrysops; striped bass: SB, M. saxatilis) have been fairly lim...

Proteomic analysis of tung tree (Vernicia fordii) oilseeds during the developmental stages

USDA-ARS?s Scientific Manuscript database

Tung tree (Vernicia fordii), a non-model woody plant belonging to the Euphorbiaceae family, is a promising economic plant due to the high content of novel high-value oil in its seeds. Many metabolic pathways are active during seed development. Oil (triacylglycerols or TAGs) accumulates in oil bodies...

Cell permeability and nuclear DNA staining by propidium iodide in basidiomycetous yeasts.

PubMed

Zhang, Ning; Fan, Yuxuan; Li, Chen; Wang, Qiming; Leksawasdi, Noppol; Li, Fuli; Wang, Shi'an

2018-05-01

Non-model yeasts within basidiomycetes have considerable importance in agriculture, industry, and environment, but they are not as well studied as ascomycetous yeasts. Serving as a basic technique, nuclear DNA staining is widely used in physiology, ecology, cell biology, and genetics. However, it is unclear whether the classical nuclear DNA staining method for ascomycetous yeasts is applicable to basidiomycetous yeasts. In this study, 5 yeasts ineffectively stained by the classical propidium iodide (PI) staining method were identified from 23 representative basidiomycetous yeasts. Pretreatment of cells using dimethyl sulfoxide (DMSO) or snailase markedly improved cell penetration to PI and thus enabled DNA content determination by flow cytometry on the recalcitrant yeasts. The pretreatments are efficient, simple, and fast, avoiding tedious mutagenesis or genetic engineering used in previous reports. The heterogeneity of cell penetration to PI among basidiomycetous yeasts was attributed to the discrepancy in cell wall polysaccharides instead of capsule or plasma membrane. This study also indicated that care must be taken in attributing PI-negative staining as viable cells when studying non-model microorganisms.

Challenges to Applying a Metamodel for Groundwater Flow Beyond Underlying Numerical Model Boundaries

NASA Astrophysics Data System (ADS)

Reeves, H. W.; Fienen, M. N.; Feinstein, D.

2015-12-01

Metamodels of environmental behavior offer opportunities for decision support, adaptive management, and increased stakeholder engagement through participatory modeling and model exploration. Metamodels are derived from calibrated, computationally demanding, numerical models. They may potentially be applied to non-modeled areas to provide screening or preliminary analysis tools for areas that do not yet have the benefit of more comprehensive study. In this decision-support mode, they may be fulfilling a role often accomplished by application of analytical solutions. The major challenge to transferring a metamodel to a non-modeled area is how to quantify the spatial data in the new area of interest in such a way that it is consistent with the data used to derive the metamodel. Tests based on transferring a metamodel derived from a numerical groundwater-flow model of the Lake Michigan Basin to other glacial settings across the northern U.S. show that the spatial scale of the numerical model must be appropriately scaled to adequately represent different settings. Careful GIS analysis of the numerical model, metamodel, and new area of interest is required for successful transfer of results.

ATGC transcriptomics: a web-based application to integrate, explore and analyze de novo transcriptomic data.

PubMed

Gonzalez, Sergio; Clavijo, Bernardo; Rivarola, Máximo; Moreno, Patricio; Fernandez, Paula; Dopazo, Joaquín; Paniego, Norma

2017-02-22

In the last years, applications based on massively parallelized RNA sequencing (RNA-seq) have become valuable approaches for studying non-model species, e.g., without a fully sequenced genome. RNA-seq is a useful tool for detecting novel transcripts and genetic variations and for evaluating differential gene expression by digital measurements. The large and complex datasets resulting from functional genomic experiments represent a challenge in data processing, management, and analysis. This problem is especially significant for small research groups working with non-model species. We developed a web-based application, called ATGC transcriptomics, with a flexible and adaptable interface that allows users to work with new generation sequencing (NGS) transcriptomic analysis results using an ontology-driven database. This new application simplifies data exploration, visualization, and integration for a better comprehension of the results. ATGC transcriptomics provides access to non-expert computer users and small research groups to a scalable storage option and simple data integration, including database administration and management. The software is freely available under the terms of GNU public license at http://atgcinta.sourceforge.net .

Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods.

PubMed

Olsen, Morten Tange; Bérubé, Martine; Robbins, Jooke; Palsbøll, Per J

2012-09-06

Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay. Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays. Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments.

The plasticizer bisphenol A affects somatic and sexual development, but differently in pipid, hylid and bufonid anurans.

PubMed

Tamschick, Stephanie; Rozenblut-Kościsty, Beata; Ogielska, Maria; Kekenj, David; Gajewski, Franz; Krüger, Angela; Kloas, Werner; Stöck, Matthias

2016-09-01

Due to their terrestrial habitats and aquatic reproduction, many amphibians are both very vulnerable and highly suitable bioindicators. The plasticizer bisphenol A (BPA) is one of the most produced chemical substances worldwide, and knowledge on its impacts on humans and animals is mounting. BPA is used for the industrial production of polycarbonate plastics and epoxy resins and found in a multitude of consumer products. Studies on BPA have involved mammals, fish and the fully aquatic anuran model Xenopus laevis. However, our knowledge about the sexual development of non-model, often semi-terrestrial anuran amphibians remains poor. Using a recently developed experimental design, we simultaneously applied BPA to two non-model species (Hyla arborea, Hylidae; Bufo viridis, Bufonidae) and the model X. laevis (Pipidae), compared their genetic and phenotypic sex for detection of sex reversals, and studied sexual development, focusing on anatomical and histological features of gonads. We compared three concentrations of BPA (0.023, 2.28 and 228 μg/L) to control groups in a high-standard flow-through-system, and tested whether conclusions, drawn from the model species, can be extrapolated to non-model anurans. In contrast to previous studies on fish and Xenopus, often involving dosages much higher than most environmental pollution data, we show that BPA causes neither the development of mixed sex nor of sex-reversed individuals (few, seemingly BPA-independent sex reversals) in all focal species. However, environmentally relevant concentrations, as low as 0.023 μg/L, were sufficient to provoke species-specific anatomically and histologically detectable impairments of gonads, and affected morphological traits of metamorphs. As the intensity of these effects differed between the three species, our data imply that BPA diversely affects amphibians with different evolutionary history, sex determination systems and larval ecologies. These results highlight the role of amphibians as a sensitive group that is responsive to environmental pollution. Copyright © 2016 Elsevier Ltd. All rights reserved.

Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods

PubMed Central

2012-01-01

Background Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay. Results Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays. Conclusion Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments. PMID:22954451

[The evolution of heat shock genes and expression patterns of heat shock proteins in the species from temperature contrasting habitats].

PubMed

Garbuz, D G; Evgen’ev, M B

2017-01-01

Heat shock genes are the most evolutionarily ancient among the systems responsible for adaptation of organisms to a harsh environment. The encoded proteins (heat shock proteins, Hsps) represent the most important factors of adaptation to adverse environmental conditions. They serve as molecular chaperones, providing protein folding and preventing aggregation of damaged cellular proteins. Structural analysis of the heat shock genes in individuals from both phylogenetically close and very distant taxa made it possible to reveal the basic trends of the heat shock gene organization in the context of adaptation to extreme conditions. Using different model objects and nonmodel species from natural populations, it was demonstrated that modulation of the Hsps expression during adaptation to different environmental conditions could be achieved by changing the number and structural organization of heat shock genes in the genome, as well as the structure of their promoters. It was demonstrated that thermotolerant species were usually characterized by elevated levels of Hsps under normal temperature or by the increase in the synthesis of these proteins in response to heat shock. Analysis of the heat shock genes in phylogenetically distant organisms is of great interest because, on one hand, it contributes to the understanding of the molecular mechanisms of evolution of adaptogenes and, on the other hand, sheds the light on the role of different Hsps families in the development of thermotolerance and the resistance to other stress factors.

Ecotoxicity of two organic UV-filters to the freshwater caddisfly Sericostoma vittatum.

PubMed

Campos, Diana; Gravato, Carlos; Fedorova, Ganna; Burkina, Viktoriia; Soares, Amadeu M V M; Pestana, João L T

2017-09-01

Organic ultraviolet filters (UV-filters) used for protection against radiation in personal care products and other materials (e.g. textiles, plastic products) are considered emerging contaminants of aquatic ecosystem. Benzophenone-3 (BP3) and 3-(4-methylbenzylidene)camphor (4-MBC) are the most commonly used organic UV-filters and have been reported in freshwater environments due to contamination through discharges from wastewater treatment plants and swimming pools or by direct contamination from recreational activities. Our aim was to evaluate the ecotoxicological effects of these UV-filters using the freshwater caddisfly Sericostoma vittatum' biochemical biomarkers and energy processing related endpoints (feeding behaviour, energy reserves and cellular metabolism). In laboratory trials, both compounds induced feeding inhibition of S. vittatum at 3.55 mg/kg of BP3 and at concentrations ≥2.57 mg/kg of 4-MBC, decreased carbohydrates content at 3.55 and 6.95 mg/kg of BP3 and 4-MBC respectively, and increased total glutathione levels at concentrations ≥1.45 and 1.35 mg/kg of BP3 and 4-MBC respectively. No significant effects were observed on endpoints associated with oxidative stress, antioxidant defences, phase II biotransformation or neurotoxicity after exposure to the two UV-filters. Our results show that environmental relevant concentrations of BP3 and 4-MBC, can negatively impact freshwater insects and demonstrate the importance of monitoring the ecological effects of organic UV-filters using non-model invertebrate species. Copyright © 2017 Elsevier Ltd. All rights reserved.

The aquatic animals' transcriptome resource for comparative functional analysis.

PubMed

Chou, Chih-Hung; Huang, Hsi-Yuan; Huang, Wei-Chih; Hsu, Sheng-Da; Hsiao, Chung-Der; Liu, Chia-Yu; Chen, Yu-Hung; Liu, Yu-Chen; Huang, Wei-Yun; Lee, Meng-Lin; Chen, Yi-Chang; Huang, Hsien-Da

2018-05-09

Aquatic animals have great economic and ecological importance. Among them, non-model organisms have been studied regarding eco-toxicity, stress biology, and environmental adaptation. Due to recent advances in next-generation sequencing techniques, large amounts of RNA-seq data for aquatic animals are publicly available. However, currently there is no comprehensive resource exist for the analysis, unification, and integration of these datasets. This study utilizes computational approaches to build a new resource of transcriptomic maps for aquatic animals. This aquatic animal transcriptome map database dbATM provides de novo assembly of transcriptome, gene annotation and comparative analysis of more than twenty aquatic organisms without draft genome. To improve the assembly quality, three computational tools (Trinity, Oases and SOAPdenovo-Trans) were employed to enhance individual transcriptome assembly, and CAP3 and CD-HIT-EST software were then used to merge these three assembled transcriptomes. In addition, functional annotation analysis provides valuable clues to gene characteristics, including full-length transcript coding regions, conserved domains, gene ontology and KEGG pathways. Furthermore, all aquatic animal genes are essential for comparative genomics tasks such as constructing homologous gene groups and blast databases and phylogenetic analysis. In conclusion, we establish a resource for non model organism aquatic animals, which is great economic and ecological importance and provide transcriptomic information including functional annotation and comparative transcriptome analysis. The database is now publically accessible through the URL http://dbATM.mbc.nctu.edu.tw/ .

Selection of low-variance expressed Malus x domestica (apple) genes for use as quantitative PCR reference genes (housekeepers)

USDA-ARS?s Scientific Manuscript database

To accurately measure gene expression using PCR-based approaches, there is the need for reference genes that have low variance in expression (housekeeping genes) to normalise the data for RNA quantity and quality. For non-model species such as Malus x domestica (apples), previously, the selection of...

We can't all be supermodels: the value of comparative transcriptomics to the study of non-model insects

PubMed Central

Oppenheim, Sara J; Baker, Richard H; Simon, Sabrina; DeSalle, Rob

2015-01-01

Insects are the most diverse group of organisms on the planet. Variation in gene expression lies at the heart of this biodiversity and recent advances in sequencing technology have spawned a revolution in researchers' ability to survey tissue-specific transcriptional complexity across a wide range of insect taxa. Increasingly, studies are using a comparative approach (across species, sexes and life stages) that examines the transcriptional basis of phenotypic diversity within an evolutionary context. In the present review, we summarize much of this research, focusing in particular on three critical aspects of insect biology: morphological development and plasticity; physiological response to the environment; and sexual dimorphism. A common feature that is emerging from these investigations concerns the dynamic nature of transcriptome evolution as indicated by rapid changes in the overall pattern of gene expression, the differential expression of numerous genes with unknown function, and the incorporation of novel, lineage-specific genes into the transcriptional profile. PMID:25524309

Quality of Computationally Inferred Gene Ontology Annotations

PubMed Central

Škunca, Nives; Altenhoff, Adrian; Dessimoz, Christophe

2012-01-01

Gene Ontology (GO) has established itself as the undisputed standard for protein function annotation. Most annotations are inferred electronically, i.e. without individual curator supervision, but they are widely considered unreliable. At the same time, we crucially depend on those automated annotations, as most newly sequenced genomes are non-model organisms. Here, we introduce a methodology to systematically and quantitatively evaluate electronic annotations. By exploiting changes in successive releases of the UniProt Gene Ontology Annotation database, we assessed the quality of electronic annotations in terms of specificity, reliability, and coverage. Overall, we not only found that electronic annotations have significantly improved in recent years, but also that their reliability now rivals that of annotations inferred by curators when they use evidence other than experiments from primary literature. This work provides the means to identify the subset of electronic annotations that can be relied upon—an important outcome given that >98% of all annotations are inferred without direct curation. PMID:22693439

Synthetic biology to access and expand nature’s chemical diversity

PubMed Central

Smanski, Michael J.; Zhou, Hui; Claesen, Jan; Shen, Ben; Fischbach, Michael; Voigt, Christopher A.

2016-01-01

Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Economically accessing the potential encoded within sequenced genomes promises to reinvigorate waning drug discovery pipelines and provide novel routes to intricate chemicals. This is a tremendous undertaking, as the pathways often comprise dozens of genes spanning as much as 100+ kiliobases of DNA, are controlled by complex regulatory networks, and the most interesting molecules are made by non-model organisms. Advances in synthetic biology address these issues, including DNA construction technologies, genetic parts for precision expression control, synthetic regulatory circuits, computer aided design, and multiplexed genome engineering. Collectively, these technologies are moving towards an era when chemicals can be accessed en mass based on sequence information alone. This will enable the harnessing of metagenomic data and massive strain banks for high-throughput molecular discovery and, ultimately, the ability to forward design pathways to complex chemicals not found in nature. PMID:26876034

Gene silencing in non-model insects: Overcoming hurdles using symbiotic bacteria for trauma-free sustainable delivery of RNA interference: Sustained RNA interference in insects mediated by symbiotic bacteria: Applications as a genetic tool and as a biocide.

PubMed

Whitten, Miranda; Dyson, Paul

2017-03-01

Insight into animal biology and development provided by classical genetic analysis of the model organism Drosophila melanogaster was an incentive to develop advanced genetic tools for this insect. But genetic systems for the over one million other known insect species are largely undeveloped. With increasing information about insect genomes resulting from next generation sequencing, RNA interference is now the method of choice for reverse genetics, although it is constrained by the means of delivery of interfering RNA. A recent advance to ensure sustained delivery with minimal experimental intervention or trauma to the insect is to exploit commensal bacteria for symbiont-mediated RNA interference. This technology not only offers an efficient means for RNA interference in insects in laboratory conditions, but also has potential for use in the control of human disease vectors, agricultural pests and pathogens of beneficial insects. © 2017 WILEY Periodicals, Inc.

Re"CYC"ling molecular regulators in the evolution and development of flower symmetry.

PubMed

Spencer, Victoria; Kim, Minsung

2018-07-01

Flower forms are both highly diverse and multifaceted. As well as varying in colour, size, organ number, and much more, flowers show different types of symmetry. Floral symmetry can be grouped into three main categories: asymmetry, bilateral symmetry and radial symmetry, characterised by zero, one, and multiple planes of symmetry, respectively. This review will first explore floral symmetry from a classical morphological view, then from a modern molecular perspective. The recent molecular work on symmetry in monocots and eudicots will be discussed, followed by an in-depth discussion into the evolution of CYC genes, particularly in the capitulum of the sunflower family (Asteraceae). Whilst recent studies on non-model species are helping to bring new light to this field, more species coverage is required to understand how traits such as bilateral symmetry have evolved so many times, and whether the same molecular regulators were recruited for this function. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

No more non-model species: the promise of next generation sequencing for comparative immunology.

PubMed

Dheilly, Nolwenn M; Adema, Coen; Raftos, David A; Gourbal, Benjamin; Grunau, Christoph; Du Pasquier, Louis

2014-07-01

Next generation sequencing (NGS) allows for the rapid, comprehensive and cost effective analysis of entire genomes and transcriptomes. NGS provides approaches for immune response gene discovery, profiling gene expression over the course of parasitosis, studying mechanisms of diversification of immune receptors and investigating the role of epigenetic mechanisms in regulating immune gene expression and/or diversification. NGS will allow meaningful comparisons to be made between organisms from different taxa in an effort to understand the selection of diverse strategies for host defence under different environmental pathogen pressures. At the same time, it will reveal the shared and unique components of the immunological toolkit and basic functional aspects that are essential for immune defence throughout the living world. In this review, we argue that NGS will revolutionize our understanding of immune responses throughout the animal kingdom because the depth of information it provides will circumvent the need to concentrate on a few "model" species. Copyright © 2014 Elsevier Ltd. All rights reserved.

Adaptive and neutral markers both show continent-wide population structure of mountain pine beetle (Dendroctonus ponderosae).

PubMed

Batista, Philip D; Janes, Jasmine K; Boone, Celia K; Murray, Brent W; Sperling, Felix A H

2016-09-01

Assessments of population genetic structure and demographic history have traditionally been based on neutral markers while explicitly excluding adaptive markers. In this study, we compared the utility of putatively adaptive and neutral single-nucleotide polymorphisms (SNPs) for inferring mountain pine beetle population structure across its geographic range. Both adaptive and neutral SNPs, and their combination, allowed range-wide structure to be distinguished and delimited a population that has recently undergone range expansion across northern British Columbia and Alberta. Using an equal number of both adaptive and neutral SNPs revealed that adaptive SNPs resulted in a stronger correlation between sampled populations and inferred clustering. Our results suggest that adaptive SNPs should not be excluded prior to analysis from neutral SNPs as a combination of both marker sets resulted in better resolution of genetic differentiation between populations than either marker set alone. These results demonstrate the utility of adaptive loci for resolving population genetic structure in a nonmodel organism.

Characterization of RNase MRP RNA and novel snoRNAs from Giardia intestinalis and Trichomonas vaginalis

PubMed Central

2011-01-01

Background Eukaryotic cells possess a complex network of RNA machineries which function in RNA-processing and cellular regulation which includes transcription, translation, silencing, editing and epigenetic control. Studies of model organisms have shown that many ncRNAs of the RNA-infrastructure are highly conserved, but little is known from non-model protists. In this study we have conducted a genome-scale survey of medium-length ncRNAs from the protozoan parasites Giardia intestinalis and Trichomonas vaginalis. Results We have identified the previously 'missing' Giardia RNase MRP RNA, which is a key ribozyme involved in pre-rRNA processing. We have also uncovered 18 new H/ACA box snoRNAs, expanding our knowledge of the H/ACA family of snoRNAs. Conclusions Results indicate that Giardia intestinalis and Trichomonas vaginalis, like their distant multicellular relatives, contain a rich infrastructure of RNA-based processing. From here we can investigate the evolution of RNA processing networks in eukaryotes. PMID:22053856

Engineering xylose metabolism for production of polyhydroxybutyrate in the non-model bacterium Burkholderia sacchari.

PubMed

Guamán, Linda P; Barba-Ostria, Carlos; Zhang, Fuzhong; Oliveira-Filho, Edmar R; Gomez, José Gregório C; Silva, Luiziana F

2018-05-15

Despite its ability to grow and produce high-value molecules using renewable carbon sources, two main factors must be improved to use Burkholderia sacchari as a chassis for bioproduction at an industrial scale: first, the lack of molecular tools to engineer this organism and second, the inherently slow growth rate and poly-3-hydroxybutyrate [P(3HB)] production using xylose. In this work, we have addressed both factors. First, we adapted a set of BglBrick plasmids and showed tunable expression in B. sacchari. Finally, we assessed growth rate and P(3HB) production through overexpression of xylose transporters, catabolic or regulatory genes. Overexpression of xylR significantly improved growth rate (55.5% improvement), polymer yield (77.27% improvement), and resulted in 71% of cell dry weight as P(3HB). These values are unprecedented for P(3HB) accumulation using xylose as a sole carbon source and highlight the importance of precise expression control for improving utilization of hemicellulosic sugars in B. sacchari.

RAD sequencing yields a high success rate for westslope cutthroat and rainbow trout species-diagnostic SNP assays

USGS Publications Warehouse

Stephen J. Amish,; Paul A. Hohenlohe,; Sally Painter,; Robb F. Leary,; Muhlfeld, Clint C.; Fred W. Allendorf,; Luikart, Gordon

2012-01-01

Hybridization with introduced rainbow trout threatens most native westslope cutthroat trout populations. Understanding the genetic effects of hybridization and introgression requires a large set of high-throughput, diagnostic genetic markers to inform conservation and management. Recently, we identified several thousand candidate single-nucleotide polymorphism (SNP) markers based on RAD sequencing of 11 westslope cutthroat trout and 13 rainbow trout individuals. Here, we used flanking sequence for 56 of these candidate SNP markers to design high-throughput genotyping assays. We validated the assays on a total of 92 individuals from 22 populations and seven hatchery strains. Forty-six assays (82%) amplified consistently and allowed easy identification of westslope cutthroat and rainbow trout alleles as well as heterozygote controls. The 46 SNPs will provide high power for early detection of population admixture and improved identification of hybrid and nonhybridized individuals. This technique shows promise as a very low-cost, reliable and relatively rapid method for developing and testing SNP markers for nonmodel organisms with limited genomic resources.

Low-coverage, whole-genome sequencing of Artocarpus camansi (Moraceae) for phylogenetic marker development and gene discovery1

PubMed Central

Gardner, Elliot M.; Johnson, Matthew G.; Ragone, Diane; Wickett, Norman J.; Zerega, Nyree J. C.

2016-01-01

Premise of the study: We used moderately low-coverage (17×) whole-genome sequencing of Artocarpus camansi (Moraceae) to develop genomic resources for Artocarpus and Moraceae. Methods and Results: A de novo assembly of Illumina short reads (251,378,536 pairs, 2 × 100 bp) accounted for 93% of the predicted genome size. Predicted coding regions were used in a three-way orthology search with published genomes of Morus notabilis and Cannabis sativa. Phylogenetic markers for Moraceae were developed from 333 inferred single-copy exons. Ninety-eight putative MADS-box genes were identified. Analysis of all predicted coding regions resulted in preliminary annotation of 49,089 genes. An analysis of synonymous substitutions for pairs of orthologs (Ks analysis) in M. notabilis and A. camansi strongly suggested a lineage-specific whole-genome duplication in Artocarpus. Conclusions: This study substantially increases the genomic resources available for Artocarpus and Moraceae and demonstrates the value of low-coverage de novo assemblies for nonmodel organisms with moderately large genomes. PMID:27437173

HNK-1 immunoreactivity during early morphogenesis of the head region in a nonmodel vertebrate, crocodile embryo

NASA Astrophysics Data System (ADS)

Kundrát, Martin

2008-11-01

The present study examines HNK-1 immunoidentification of a population of the neural crest (NC) during early head morphogenesis in the nonmodel vertebrate, the crocodile ( Crocodylus niloticus) embryos. Although HNK-1 is not an exclusive NC marker among vertebrates, temporospatial immunoreactive patterns found in the crocodile are almost consistent with NC patterns derived from gene expression studies known in birds (the closest living relatives of crocodiles) and mammals. In contrast to birds, the HNK-1 epitope is immunoreactive in NC cells at the neural fold level in crocodile embryos and therefore provides sufficient base to assess early migratory events of the cephalic NC. I found that crocodile NC forms three classic migratory pathways in the head: mandibular, hyoid, and branchial. Further, I demonstrate that, besides this classic phenotype, there is also a forebrain-derived migratory population, which consolidates into a premandibular stream in the crocodile. In contrast to the closely related chick model, crocodilian premandibular and mandibular NC cells arise from the open neural tube suggesting that species-specific heterochronic behavior of NC may be involved in the formation of different vertebrate facial phenotypes.

De novo characterization of Lentinula edodes C(91-3) transcriptome by deep Solexa sequencing.

PubMed

Zhong, Mintao; Liu, Ben; Wang, Xiaoli; Liu, Lei; Lun, Yongzhi; Li, Xingyun; Ning, Anhong; Cao, Jing; Huang, Min

2013-02-01

Lentinula edodes, has been utilized as food, as well as, in popular medicine, moreover, its extract isolated from its mycelium and fruiting body have shown several therapeutic properties. Yet little is understood about its genes involved in these properties, and the absence of L.edodes genomes has been a barrier to the development of functional genomics research. However, high throughput sequencing technologies are now being widely applied to non-model species. To facilitate research on L.edodes, we leveraged Solexa sequencing technology in de novo assembly of L.edodes C(91-3) transcriptome. In a single run, we produced more than 57 million sequencing reads. These reads were assembled into 28,923 unigene sequences (mean size=689bp) including 18,120 unigenes with coding sequence (CDS). Based on similarity search with known proteins, assembled unigene sequences were annotated with gene descriptions, gene ontology (GO) and clusters of orthologous group (COG) terms. Our data provides the first comprehensive sequence resource available for functional genomics studies in L.edodes, and demonstrates the utility of Illumina/Solexa sequencing for de novo transcriptome characterization and gene discovery in a non-model mushroom. Copyright © 2012 Elsevier Inc. All rights reserved.

ICG: a wiki-driven knowledgebase of internal control genes for RT-qPCR normalization.

PubMed

Sang, Jian; Wang, Zhennan; Li, Man; Cao, Jiabao; Niu, Guangyi; Xia, Lin; Zou, Dong; Wang, Fan; Xu, Xingjian; Han, Xiaojiao; Fan, Jinqi; Yang, Ye; Zuo, Wanzhu; Zhang, Yang; Zhao, Wenming; Bao, Yiming; Xiao, Jingfa; Hu, Songnian; Hao, Lili; Zhang, Zhang

2018-01-04

Real-time quantitative PCR (RT-qPCR) has become a widely used method for accurate expression profiling of targeted mRNA and ncRNA. Selection of appropriate internal control genes for RT-qPCR normalization is an elementary prerequisite for reliable expression measurement. Here, we present ICG (http://icg.big.ac.cn), a wiki-driven knowledgebase for community curation of experimentally validated internal control genes as well as their associated experimental conditions. Unlike extant related databases that focus on qPCR primers in model organisms (mainly human and mouse), ICG features harnessing collective intelligence in community integration of internal control genes for a variety of species. Specifically, it integrates a comprehensive collection of more than 750 internal control genes for 73 animals, 115 plants, 12 fungi and 9 bacteria, and incorporates detailed information on recommended application scenarios corresponding to specific experimental conditions, which, collectively, are of great help for researchers to adopt appropriate internal control genes for their own experiments. Taken together, ICG serves as a publicly editable and open-content encyclopaedia of internal control genes and accordingly bears broad utility for reliable RT-qPCR normalization and gene expression characterization in both model and non-model organisms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

The birds and the bees and the flowers and the trees: lessons from genetic mapping of sex determination in plants and animals.

PubMed

Charlesworth, Deborah; Mank, Judith E

2010-09-01

The ability to identify genetic markers in nonmodel systems has allowed geneticists to construct linkage maps for a diversity of species, and the sex-determining locus is often among the first to be mapped. Sex determination is an important area of study in developmental and evolutionary biology, as well as ecology. Its importance for organisms might suggest that sex determination is highly conserved. However, genetic studies have shown that sex determination mechanisms, and the genes involved, are surprisingly labile. We review studies using genetic mapping and phylogenetic inferences, which can help reveal evolutionary pattern within this lability and potentially identify the changes that have occurred among different sex determination systems. We define some of the terminology, particularly where confusion arises in writing about such a diverse range of organisms, and highlight some major differences between plants and animals, and some important similarities. We stress the importance of studying taxa suitable for testing hypotheses, and the need for phylogenetic studies directed to taxa where the patterns of changes can be most reliably inferred, if the ultimate goal of testing hypotheses regarding the selective forces that have led to changes in such an essential trait is to become feasible.

Overcoming the anaerobic hurdle in phenotypic microarrays: Generation andvisualization of growth curve data for Desulfovibrio vulgaris Hildenborough

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borglin, Sharon E; Joyner, Dominique; Jacobsen, Janet

2008-10-04

Growing anaerobic microorganisms in phenotypic microarrays (PM) and 96-well microtiter plates is an emerging technology that allows high throughput survey of the growth and physiology and/or phenotype of cultivable microorganisms. For non-model bacteria, a method for phenotypic analysis is invaluable, not only to serve as a starting point for further evaluation, but also to provide a broad understanding of the physiology of an uncharacterized wild-type organism or the physiology/phenotype of a newly created mutant of that organism. Given recent advances in genetic characterization and targeted mutations to elucidate genetic networks and metabolic pathways, high-throughput methods for determining phenotypic differences aremore » essential. Here we outline challenges presented in studying the physiology and phenotype of a sulfate reducing anaerobic delta proteobacterium, Desulfovibrio vulgaris Hildenborough. Modifications of the commercially available OmniLog(TM) system (Hayward, CA) for experimental setup, and configuration, as well as considerations in PM data analysis are presented. Also highlighted here is data viewing software that enables users to view and compare multiple PM data sets. The PM method promises to be a valuable strategy in our systems biology approach to D. vulgaris studies and is readily applicable to other anaerobic and aerobic bacteria.« less

The Embryonic Transcriptome of the Red-Eared Slider Turtle (Trachemys scripta)

PubMed Central

Kaplinsky, Nicholas J.; Gilbert, Scott F.; Cebra-Thomas, Judith; Lilleväli, Kersti; Saare, Merly; Chang, Eric Y.; Edelman, Hannah E.; Frick, Melissa A.; Guan, Yin; Hammond, Rebecca M.; Hampilos, Nicholas H.; Opoku, David S. B.; Sariahmed, Karim; Sherman, Eric A.; Watson, Ray

2013-01-01

The bony shell of the turtle is an evolutionary novelty not found in any other group of animals, however, research into its formation has suggested that it has evolved through modification of conserved developmental mechanisms. Although these mechanisms have been extensively characterized in model organisms, the tools for characterizing them in non-model organisms such as turtles have been limited by a lack of genomic resources. We have used a next generation sequencing approach to generate and assemble a transcriptome from stage 14 and 17 Trachemys scripta embryos, stages during which important events in shell development are known to take place. The transcriptome consists of 231,876 sequences with an N50 of 1,166 bp. GO terms and EC codes were assigned to the 61,643 unique predicted proteins identified in the transcriptome sequences. All major GO categories and metabolic pathways are represented in the transcriptome. Transcriptome sequences were used to amplify several cDNA fragments designed for use as RNA in situ probes. One of these, BMP5, was hybridized to a T. scripta embryo and exhibits both conserved and novel expression patterns. The transcriptome sequences should be of broad use for understanding the evolution and development of the turtle shell and for annotating any future T. scripta genome sequences. PMID:23840449

Consistent prediction of GO protein localization.

PubMed

Spetale, Flavio E; Arce, Debora; Krsticevic, Flavia; Bulacio, Pilar; Tapia, Elizabeth

2018-05-17

The GO-Cellular Component (GO-CC) ontology provides a controlled vocabulary for the consistent description of the subcellular compartments or macromolecular complexes where proteins may act. Current machine learning-based methods used for the automated GO-CC annotation of proteins suffer from the inconsistency of individual GO-CC term predictions. Here, we present FGGA-CC + , a class of hierarchical graph-based classifiers for the consistent GO-CC annotation of protein coding genes at the subcellular compartment or macromolecular complex levels. Aiming to boost the accuracy of GO-CC predictions, we make use of the protein localization knowledge in the GO-Biological Process (GO-BP) annotations to boost the accuracy of GO-CC prediction. As a result, FGGA-CC + classifiers are built from annotation data in both the GO-CC and GO-BP ontologies. Due to their graph-based design, FGGA-CC + classifiers are fully interpretable and their predictions amenable to expert analysis. Promising results on protein annotation data from five model organisms were obtained. Additionally, successful validation results in the annotation of a challenging subset of tandem duplicated genes in the tomato non-model organism were accomplished. Overall, these results suggest that FGGA-CC + classifiers can indeed be useful for satisfying the huge demand of GO-CC annotation arising from ubiquitous high throughout sequencing and proteomic projects.

Mapping the expression of the sex determining factor Doublesex1 in Daphnia magna using a knock-in reporter.

PubMed

Nong, Quang Dang; Mohamad Ishak, Nur Syafiqah; Matsuura, Tomoaki; Kato, Yasuhiko; Watanabe, Hajime

2017-11-02

Sexually dimorphic traits are common and widespread among animals. The expression of the Doublesex-/Mab-3-domain (DM-domain) gene family has been widely studied in model organisms and has been proven to be essential for the development and maintenance of sex-specific traits. However, little is known about the detailed expression patterns in non-model organisms. In the present study, we demonstrated the spatiotemporal expression of the DM-domain gene, doublesex1 (dsx1), in the crustacean Daphnia magna, which parthenogenetically produces males in response to environmental cues. We developed a dsx1 reporter strain to track dsx1 activity in vivo by inserting the mCherry gene into the dsx1 locus using the TALEN-mediated knock-in approach. After confirming dsx1 expression in male-specific traits in juveniles and adults, we performed time-lapse imaging of embryogenesis. Shortly after gastrulation stage, a presumptive primary organiser, named cumulus, first showed male-specific dsx1 expression. This cell mass moved to the posterior growth zone that distributes dsx1-expressing progenitor cells across the body during axial elongation, before embryos start male-specific dsx1 expression in sexually dimorphic structures. The present study demonstrated the sex-specific dsx1 expression in cell populations involved in basal body formation.

Characterization of the transcriptome of an ecologically important avian species, the Vinous-throated Parrotbill Paradoxornis webbianus bulomachus (Paradoxornithidae; Aves)

PubMed Central

2012-01-01

Background Adaptive divergence driven by environmental heterogeneity has long been a fascinating topic in ecology and evolutionary biology. The study of the genetic basis of adaptive divergence has, however, been greatly hampered by a lack of genomic information. The recent development of transcriptome sequencing provides an unprecedented opportunity to generate large amounts of genomic data for detailed investigations of the genetics of adaptive divergence in non-model organisms. Herein, we used the Illumina sequencing platform to sequence the transcriptome of brain and liver tissues from a single individual of the Vinous-throated Parrotbill, Paradoxornis webbianus bulomachus, an ecologically important avian species in Taiwan with a wide elevational range of sea level to 3100 m. Results Our 10.1 Gbp of sequences were first assembled based on Zebra Finch (Taeniopygia guttata) and chicken (Gallus gallus) RNA references. The remaining reads were then de novo assembled. After filtering out contigs with low coverage (<10X), we retained 67,791 of 487,336 contigs, which covered approximately 5.3% of the P. w. bulomachus genome. Of 7,779 contigs retained for a top-hit species distribution analysis, the majority (about 86%) were matched to known Zebra Finch and chicken transcripts. We also annotated 6,365 contigs to gene ontology (GO) terms: in total, 122 GO-slim terms were assigned, including biological process (41%), molecular function (32%), and cellular component (27%). Many potential genetic markers for future adaptive genomic studies were also identified: 8,589 single nucleotide polymorphisms, 1,344 simple sequence repeats and 109 candidate genes that might be involved in elevational or climate adaptation. Conclusions Our study shows that transcriptome data can serve as a rich genetic resource, even for a single run of short-read sequencing from a single individual of a non-model species. This is the first study providing transcriptomic information for species in the avian superfamily Sylvioidea, which comprises more than 1,000 species. Our data can be used to study adaptive divergence in heterogeneous environments and investigate other important ecological and evolutionary questions in parrotbills from different populations and even in other species in the Sylvioidea. PMID:22530590

Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens

PubMed Central

Suchan, Tomasz; Pitteloud, Camille; Gerasimova, Nadezhda S.; Kostikova, Anna; Schmid, Sarah; Arrigo, Nils; Pajkovic, Mila; Ronikier, Michał; Alvarez, Nadir

2016-01-01

In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes loci dropout. This should enable the application of hyRAD to analyses at broader evolutionary scales. PMID:26999359

The C. elegans dauer larva as a paradigm to study metabolic suppression and desiccation tolerance.

PubMed

Erkut, Cihan; Kurzchalia, Teymuras V

2015-08-01

The hypometabolic, stress-resistant dauer larva of Caenorhabditis elegans serves as an excellent model to study the molecular mechanisms of desiccation tolerance, such as maintenance of membrane organization, protein folding, xenobiotic and ROS detoxification in the dry state. Many organisms from diverse taxa of life have the remarkable ability to survive extreme desiccation in the nature by entering an ametabolic state known as anhydrobiosis (life without water). The hallmark of the anhydrobiotic state is the achievement and maintenance of an exceedingly low metabolic rate, as well as preservation of the structural integrity of the cell. Although described more than three centuries ago, the biochemical and biophysical mechanisms underlying this phenomenon are still not fully comprehended. This is mainly due to the fact that anhydrobiosis in animals was studied using non-model organisms, which are very difficult, if not impossible, to manipulate at the molecular level. Recently, we introduced the roundworm (nematode) Caenorhabditis elegans as a model for anhydrobiosis. Taking advantage of powerful genetic, biochemical and biophysical tools, we investigated several aspects of anhydrobiosis in a particular developmental stage (the dauer larva) of this organism. First, our studies allowed confirming the previously suggested role of the disaccharide trehalose in the preservation of lipid membranes. Moreover, in addition to known pathways such as reactive oxygen species defense, heat-shock and intrinsically disordered protein expression, evidence for some novel strategies of anhydrobiosis has been obtained. These are increased glyoxalase activity, polyamine and polyunsaturated fatty acid biosynthesis. All these pathways may constitute a generic toolbox of anhydrobiosis, which is possibly conserved between animals and plants.

Strategies for Cd accumulation in Dittrichia viscosa (L.) Greuter: role of the cell wall, non-protein thiols and organic acids.

PubMed

Fernández, R; Fernández-Fuego, D; Bertrand, A; González, A

2014-05-01

Dittrichia viscosa (L.) Greuter is plant species commonly found in degraded zones of Asturias (Spain), where it accumulates high levels of Cd, but the mechanisms involved in this response in non-model plants have not been elucidated. In this way, we analysed the fraction of the total Cd bound to the cell walls, the ultrastructural localization of this metal, and non-protein thiol and organic acid concentrations of two clones of D. viscosa: DV-A (from a metal-polluted soil) and DV-W (from a non-polluted area). After 10 days of hydroponic culture with Cd, fractionation and ultrastructural localisation studies showed that most of the Cd accumulated by D. viscosa was kept in the cell wall. The non-protein thiol content rose in D. viscosa with Cd exposure, especially in the non-metallicolous DV-W clone, and in both clones we found with Cd exposure a synthesis de novo of phytochelatins PC2 and PC3 in shoots and roots and also of other phytochelatin-related compounds, particularly in roots. Regarding organic acids, their concentration in both clones decreased in shoots after Cd treatment, but increased in roots, mainly due to changes in the citric acid concentration. Thus, retention of Cd in the cell wall seems to be the first strategy in response to metal entry in D. viscosa and once inside cells non-protein thiols and organic acids might also participate in Cd tolerance. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Pb-induced responses in Zygophyllum fabago plants are organ-dependent and modulated by salicylic acid.

PubMed

López-Orenes, Antonio; Martínez-Pérez, Ascensión; Calderón, Antonio A; Ferrer, María A

2014-11-01

Zygophyllum fabago is a promising species for restoring heavy metal (HM) polluted soils, although the mechanisms involved in HM tolerance in this non-model plant remain largely unknown. This paper analyses the extent to which redox-active compounds and enzymatic antioxidants in roots, stems and leaves are responsible for Pb tolerance in a metallicolous ecotype of Z. fabago and the possible influence of salicylic acid (SA) pretreatment (24 h, 0.5 mM SA) in the response to Pb stress. SA pretreatment reduced both the accumulation of Pb in roots and even more so the concentration of Pb in aerial parts of the plants, although a similar drop in the content of chlorophylls and in the maximum quantum yield of photosystem II was observed in both Pb- and SA-Pb-treated plants. Pb increased the endogenous free SA levels in all organs and this response was enhanced in root tissues upon SA pretreatment. Generally, Pb induced a reduction in catalase, ascorbate peroxidase and glutathione reductase specific activities, whereas dehydroascorbate reductase was increased in all organs of control plants. SA pretreatment enhanced the Pb-induced H2O2 accumulation in roots by up-regulating Fe-superoxide dismutase isoenzymes. Under Pb stress, the GSH redox ratio remained highly reduced in all organs while the ascorbic acid redox ratio dropped in leaf tissues where a rise in lipid peroxidation products and electrolyte leakage was observed. Finally, an organ-dependent accumulation of proline and β-carboline alkaloids was found, suggesting these nitrogen-redox-active compounds could play a role in the adaptation strategies of this species to Pb stress. Copyright © 2014. Published by Elsevier Masson SAS.

Proteomic characterization of the hemolymph of Octopus vulgaris infected by the protozoan parasite Aggregata octopiana.

PubMed

Castellanos-Martínez, Sheila; Diz, Angel P; Álvarez-Chaver, Paula; Gestal, Camino

2014-06-13

The immune system of cephalopods is poorly known to date. The lack of genomic information makes difficult to understand vital processes like immune defense mechanisms and their interaction with pathogens at molecular level. The common octopus Octopus vulgaris has a high economic relevance and potential for aquaculture. However, disease outbreaks provoke serious reductions in production with potentially severe economic losses. In this study, a proteomic approach is used to analyze the immune response of O. vulgaris against the coccidia Aggregata octopiana, a gastrointestinal parasite which impairs the cephalopod nutritional status. The hemocytes and plasma proteomes were compared by 2-DE between sick and healthy octopus. The identities of 12 differentially expressed spots and other 27 spots without significant alteration from hemocytes, and 5 spots from plasma, were determined by mass spectrometry analysis aided by a six reading-frame translation of an octopus hemocyte RNA-seq database and also public databases. Principal component analysis pointed to 7 proteins from hemocytes as the major contributors to the overall difference between levels of infection and so could be considered as potential biomarkers. Particularly, filamin, fascin and peroxiredoxin are highlighted because of their implication in octopus immune defense activity. From the octopus plasma, hemocyanin was identified. This work represents a first step forward in order to characterize the protein profile of O. vulgaris hemolymph, providing important information for subsequent studies of the octopus immune system at molecular level and also to the understanding of the basis of octopus tolerance-resistance to A. octopiana. The immune system of cephalopods is poorly known to date. The lack of genomic information makes difficult to understand vital processes like immune defense mechanisms and their interaction with pathogens at molecular level. The study herein presented is focused to the comprehension of the octopus immune defense against a parasite infection. Particularly, it is centered in the host-parasite relationship developed between the octopus and the protozoan A. octopiana, which induces severe gastrointestinal injuries in octopus that produce a malabsorption syndrome. The common octopus is a commercially important species with a high potential for aquaculture in semi-open systems, and this pathology reduces the condition of the octopus populations on-growing in open-water systems resulting in important economical loses. This is the first proteomic approach developed on this host-parasite relationship, and therefore, the contribution of this work goes from i) ecological, since this particular relationship is tending to be established as a model of host-parasite interaction in natural populations; ii) evolutionary, due to the characterization of immune molecules that could contribute to understand the functioning of the immune defense in these highly evolved mollusks; and iii) to economical view. The results of this study provide an overview of the octopus hemolymph proteome. Furthermore, proteins influenced by the level of infection and implicated in the octopus cellular response are also showed. Consequently, a set of biomarkers for disease resistance is suggested for further research that could be valuable for the improvement of the octopus culture, taken into account their high economical value, the declining of landings and the need for the diversification of reared species in order to ensure the growth of the aquaculture activity. Although cephalopods are model species for biomedical studies and possess potential in aquaculture, their genomes have not been sequenced yet, which limits the application of genomic data to research important biological processes. Similarly, the octopus proteome, like other non-model organisms, is poorly represented in public databases. Most of the proteins were identified from an octopus' hemocyte RNA-seq database that we have performed, which will be the object of another manuscript in preparation. Therefore, the need to increase molecular data from non-model organisms is herein highlighted. Particularly, here is encouraged to expand the knowledge of the genomic of cephalopods in order to increase successful protein identifications. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2013 Elsevier B.V. All rights reserved.

Cells, walls, and endless forms.

PubMed

Monniaux, Marie; Hay, Angela

2016-12-01

A key question in biology is how the endless diversity of forms found in nature evolved. Understanding the cellular basis of this diversity has been aided by advances in non-model experimental systems, quantitative image analysis tools, and modeling approaches. Recent work in plants highlights the importance of cell wall and cuticle modifications for the emergence of diverse forms and functions. For example, explosive seed dispersal in Cardamine hirsuta depends on the asymmetric localization of lignified cell wall thickenings in the fruit valve. Similarly, the iridescence of Hibiscus trionum petals relies on regular striations formed by cuticular folds. Moreover, NAC transcription factors regulate the differentiation of lignified xylem vessels but also the water-conducting cells of moss that lack a lignified secondary cell wall, pointing to the origin of vascular systems. Other novel forms are associated with modified cell growth patterns, including oriented cell expansion or division, found in the long petal spurs of Aquilegia flowers, and the Sarracenia purpurea pitcher leaf, respectively. Another good example is the regulation of dissected leaf shape in C. hirsuta via local growth repression, controlled by the REDUCED COMPLEXITY HD-ZIP class I transcription factor. These studies in non-model species often reveal as much about fundamental processes of development as they do about the evolution of form. Copyright © 2016 Elsevier Ltd. All rights reserved.

Transcriptome analyses and differential gene expression in a non-model fish species with alternative mating tactics

PubMed Central

2014-01-01

Background Social dominance is important for the reproductive success of males in many species. In the black-faced blenny (Tripterygion delaisi) during the reproductive season, some males change color and invest in nest making and defending a territory, whereas others do not change color and ‘sneak’ reproductions when females lay their eggs. Using RNAseq, we profiled differential gene expression between the brains of territorial males, sneaker males, and females to study the molecular signatures of male dimorphism. Results We found that more genes were differentially expressed between the two male phenotypes than between males and females, suggesting that during the reproductive period phenotypic plasticity is a more important factor in differential gene expression than sexual dimorphism. The territorial male overexpresses genes related to synaptic plasticity and the sneaker male overexpresses genes involved in differentiation and development. Conclusions Previously suggested candidate genes for social dominance in the context of alternative mating strategies seem to be predominantly species-specific. We present a list of novel genes which are differentially expressed in Tripterygion delaisi. This is the first genome-wide study for a molecular non-model species in the context of alternative mating strategies and provides essential information for further studies investigating the molecular basis of social dominance. PMID:24581002

Transcriptome changes induced by arbuscular mycorrhizal fungi in sunflower (Helianthus annuus L.) roots.

PubMed

Vangelisti, Alberto; Natali, Lucia; Bernardi, Rodolfo; Sbrana, Cristiana; Turrini, Alessandra; Hassani-Pak, Keywan; Hughes, David; Cavallini, Andrea; Giovannetti, Manuela; Giordani, Tommaso

2018-01-08

Arbuscular mycorrhizal (AM) fungi are essential elements of soil fertility, plant nutrition and productivity, facilitating soil mineral nutrient uptake. Helianthus annuus is a non-model, widely cultivated species. Here we used an RNA-seq approach for evaluating gene expression variation at early and late stages of mycorrhizal establishment in sunflower roots colonized by the arbuscular fungus Rhizoglomus irregulare. mRNA was isolated from roots of plantlets at 4 and 16 days after inoculation with the fungus. cDNA libraries were built and sequenced with Illumina technology. Differential expression analysis was performed between control and inoculated plants. Overall 726 differentially expressed genes (DEGs) between inoculated and control plants were retrieved. The number of up-regulated DEGs greatly exceeded the number of down-regulated DEGs and this difference increased in later stages of colonization. Several DEGs were specifically involved in known mycorrhizal processes, such as membrane transport, cell wall shaping, and other. We also found previously unidentified mycorrhizal-induced transcripts. The most important DEGs were carefully described in order to hypothesize their roles in AM symbiosis. Our data add a valuable contribution for deciphering biological processes related to beneficial fungi and plant symbiosis, adding an Asteraceae, non-model species for future comparative functional genomics studies.

Transcriptome analyses and differential gene expression in a non-model fish species with alternative mating tactics.

PubMed

Schunter, Celia; Vollmer, Steven V; Macpherson, Enrique; Pascual, Marta

2014-02-28

Social dominance is important for the reproductive success of males in many species. In the black-faced blenny (Tripterygion delaisi) during the reproductive season, some males change color and invest in nest making and defending a territory, whereas others do not change color and 'sneak' reproductions when females lay their eggs. Using RNAseq, we profiled differential gene expression between the brains of territorial males, sneaker males, and females to study the molecular signatures of male dimorphism. We found that more genes were differentially expressed between the two male phenotypes than between males and females, suggesting that during the reproductive period phenotypic plasticity is a more important factor in differential gene expression than sexual dimorphism. The territorial male overexpresses genes related to synaptic plasticity and the sneaker male overexpresses genes involved in differentiation and development. Previously suggested candidate genes for social dominance in the context of alternative mating strategies seem to be predominantly species-specific. We present a list of novel genes which are differentially expressed in Tripterygion delaisi. This is the first genome-wide study for a molecular non-model species in the context of alternative mating strategies and provides essential information for further studies investigating the molecular basis of social dominance.

We can't all be supermodels: the value of comparative transcriptomics to the study of non-model insects.

PubMed

Oppenheim, Sara J; Baker, Richard H; Simon, Sabrina; DeSalle, Rob

2015-04-01

Insects are the most diverse group of organisms on the planet. Variation in gene expression lies at the heart of this biodiversity and recent advances in sequencing technology have spawned a revolution in researchers' ability to survey tissue-specific transcriptional complexity across a wide range of insect taxa. Increasingly, studies are using a comparative approach (across species, sexes and life stages) that examines the transcriptional basis of phenotypic diversity within an evolutionary context. In the present review, we summarize much of this research, focusing in particular on three critical aspects of insect biology: morphological development and plasticity; physiological response to the environment; and sexual dimorphism. A common feature that is emerging from these investigations concerns the dynamic nature of transcriptome evolution as indicated by rapid changes in the overall pattern of gene expression, the differential expression of numerous genes with unknown function, and the incorporation of novel, lineage-specific genes into the transcriptional profile. © 2014 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society.

A FASTQ compressor based on integer-mapped k-mer indexing for biologist.

PubMed

Zhang, Yeting; Patel, Khyati; Endrawis, Tony; Bowers, Autumn; Sun, Yazhou

2016-03-15

Next generation sequencing (NGS) technologies have gained considerable popularity among biologists. For example, RNA-seq, which provides both genomic and functional information, has been widely used by recent functional and evolutionary studies, especially in non-model organisms. However, storing and transmitting these large data sets (primarily in FASTQ format) have become genuine challenges, especially for biologists with little informatics experience. Data compression is thus a necessity. KIC, a FASTQ compressor based on a new integer-mapped k-mer indexing method, was developed (available at http://www.ysunlab.org/kic.jsp). It offers high compression ratio on sequence data, outstanding user-friendliness with graphic user interfaces, and proven reliability. Evaluated on multiple large RNA-seq data sets from both human and plants, it was found that the compression ratio of KIC had exceeded all major generic compressors, and was comparable to those of the latest dedicated compressors. KIC enables researchers with minimal informatics training to take advantage of the latest sequence compression technologies, easily manage large FASTQ data sets, and reduce storage and transmission cost. Copyright © 2015 Elsevier B.V. All rights reserved.

CBrowse: a SAM/BAM-based contig browser for transcriptome assembly visualization and analysis.

PubMed

Li, Pei; Ji, Guoli; Dong, Min; Schmidt, Emily; Lenox, Douglas; Chen, Liangliang; Liu, Qi; Liu, Lin; Zhang, Jie; Liang, Chun

2012-09-15

To address the impending need for exploring rapidly increased transcriptomics data generated for non-model organisms, we developed CBrowse, an AJAX-based web browser for visualizing and analyzing transcriptome assemblies and contigs. Designed in a standard three-tier architecture with a data pre-processing pipeline, CBrowse is essentially a Rich Internet Application that offers many seamlessly integrated web interfaces and allows users to navigate, sort, filter, search and visualize data smoothly. The pre-processing pipeline takes the contig sequence file in FASTA format and its relevant SAM/BAM file as the input; detects putative polymorphisms, simple sequence repeats and sequencing errors in contigs and generates image, JSON and database-compatible CSV text files that are directly utilized by different web interfaces. CBowse is a generic visualization and analysis tool that facilitates close examination of assembly quality, genetic polymorphisms, sequence repeats and/or sequencing errors in transcriptome sequencing projects. CBrowse is distributed under the GNU General Public License, available at http://bioinfolab.muohio.edu/CBrowse/ liangc@muohio.edu or liangc.mu@gmail.com; glji@xmu.edu.cn Supplementary data are available at Bioinformatics online.

Microsatellite DNA capture from enriched libraries.

PubMed

Gonzalez, Elena G; Zardoya, Rafael

2013-01-01

Microsatellites are DNA sequences of tandem repeats of one to six nucleotides, which are highly polymorphic, and thus the molecular markers of choice in many kinship, population genetic, and conservation studies. There have been significant technical improvements since the early methods for microsatellite isolation were developed, and today the most common procedures take advantage of the hybrid capture methods of enriched-targeted microsatellite DNA. Furthermore, recent advents in sequencing technologies (i.e., next-generation sequencing, NGS) have fostered the mining of microsatellite markers in non-model organisms, affording a cost-effective way of obtaining a large amount of sequence data potentially useful for loci characterization. The rapid improvements of NGS platforms together with the increase in available microsatellite information open new avenues to the understanding of the evolutionary forces that shape genetic structuring in wild populations. Here, we provide detailed methodological procedures for microsatellite isolation based on the screening of GT microsatellite-enriched libraries, either by cloning and Sanger sequencing of positive clones or by direct NGS. Guides for designing new species-specific primers and basic genotyping are also given.

Genomic insights into the evolution of industrial yeast species Brettanomyces bruxellensis.

PubMed

Curtin, Christopher D; Pretorius, Isak S

2014-11-01

Brettanomyces bruxellensis, like its wine yeast counterpart Saccharomyces cerevisiae, is intrinsically linked with industrial fermentations. In wine, B. bruxellensis is generally considered to contribute negative influences on wine quality, whereas for some styles of beer, it is an essential contributor. More recently, it has shown some potential for bioethanol production. Our relatively poor understanding of B. bruxellensis biology, at least when compared with S. cerevisiae, is partly due to a lack of laboratory tools. As it is a nonmodel organism, efforts to develop methods for sporulation and transformation have been sporadic and largely unsuccessful. Recent genome sequencing efforts are now providing B. bruxellensis researchers unprecedented access to gene catalogues, the possibility of performing transcriptomic studies and new insights into evolutionary drivers. This review summarises these findings, emphasises the rich data sets already available yet largely unexplored and looks over the horizon at what might be learnt soon through comprehensive population genomics of B. bruxellensis and related species. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Spontaneous Mutation Rate in the Smallest Photosynthetic Eukaryotes

PubMed Central

Krasovec, Marc; Eyre-Walker, Adam; Sanchez-Ferandin, Sophie

2017-01-01

Abstract Mutation is the ultimate source of genetic variation, and knowledge of mutation rates is fundamental for our understanding of all evolutionary processes. High throughput sequencing of mutation accumulation lines has provided genome wide spontaneous mutation rates in a dozen model species, but estimates from nonmodel organisms from much of the diversity of life are very limited. Here, we report mutation rates in four haploid marine bacterial-sized photosynthetic eukaryotic algae; Bathycoccus prasinos, Ostreococcus tauri, Ostreococcus mediterraneus, and Micromonas pusilla. The spontaneous mutation rate between species varies from μ = 4.4 × 10−10 to 9.8 × 10−10 mutations per nucleotide per generation. Within genomes, there is a two-fold increase of the mutation rate in intergenic regions, consistent with an optimization of mismatch and transcription-coupled DNA repair in coding sequences. Additionally, we show that deviation from the equilibrium GC content increases the mutation rate by ∼2% to ∼12% because of a GC bias in coding sequences. More generally, the difference between the observed and equilibrium GC content of genomes explains some of the inter-specific variation in mutation rates. PMID:28379581

The importance of living botanical collections for plant biology and the “next generation” of evo-devo research

PubMed Central

Dosmann, Michael; Groover, Andrew

2012-01-01

Living botanical collections include germplasm repositories, long-term experimental plantings, and botanical gardens. We present here a series of vignettes to illustrate the central role that living collections have played in plant biology research, including evo-devo research. Looking toward the future, living collections will become increasingly important in support of future evo-devo research. The driving force behind this trend is nucleic acid sequencing technologies, which are rapidly becoming more powerful and cost-effective, and which can be applied to virtually any species. This allows for more extensive sampling, including non-model organisms with unique biological features and plants from diverse phylogenetic positions. Importantly, a major challenge for sequencing-based evo-devo research is to identify, access, and propagate appropriate plant materials. We use a vignette of the ongoing 1,000 Transcriptomes project as an example of the challenges faced by such projects. We conclude by identifying some of the pinch points likely to be encountered by future evo-devo researchers, and how living collections can help address them. PMID:22737158

Physiological effects of polybrominated diphenyl ether (PBDE-47) on pregnant gartersnakes and resulting offspring.

PubMed

Neuman-Lee, Lorin A; Carr, James; Vaughn, Katelynn; French, Susannah S

2015-08-01

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants and are persistent contaminants found in virtually every environment and organism sampled to date, including humans. There is growing evidence that PBDEs are the source of thyroid, neurodevelopmental, and reproductive toxicity. Yet little work has focused on how this pervasive contaminant may influence the reproduction and physiology of non-traditional model species. This is especially critical because in many cases non-model species, such as reptiles, are most likely to come into contact with PBDEs in nature. We tested how short-term, repeated exposure to the PBDE congener BDE-47 during pregnancy affected physiological processes in pregnant female gartersnakes (thyroid follicular height, bactericidal ability, stress responsiveness, reproductive output, and tendency to terminate pregnancy) and their resulting offspring (levels of corticosterone, bactericidal ability, and size differences). We found potential effects of BDE-47 on both the mother, such as increased size and higher thyroid follicular height, and her offspring (increased size), suggesting the effects on physiological function of PBDEs do indeed extend beyond the traditional rodent models. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation and integration of functional annotation pipelines for newly sequenced organisms: the potato genome as a test case.

PubMed

Amar, David; Frades, Itziar; Danek, Agnieszka; Goldberg, Tatyana; Sharma, Sanjeev K; Hedley, Pete E; Proux-Wera, Estelle; Andreasson, Erik; Shamir, Ron; Tzfadia, Oren; Alexandersson, Erik

2014-12-05

For most organisms, even if their genome sequence is available, little functional information about individual genes or proteins exists. Several annotation pipelines have been developed for functional analysis based on sequence, 'omics', and literature data. However, researchers encounter little guidance on how well they perform. Here, we used the recently sequenced potato genome as a case study. The potato genome was selected since its genome is newly sequenced and it is a non-model plant even if there is relatively ample information on individual potato genes, and multiple gene expression profiles are available. We show that the automatic gene annotations of potato have low accuracy when compared to a "gold standard" based on experimentally validated potato genes. Furthermore, we evaluate six state-of-the-art annotation pipelines and show that their predictions are markedly dissimilar (Jaccard similarity coefficient of 0.27 between pipelines on average). To overcome this discrepancy, we introduce a simple GO structure-based algorithm that reconciles the predictions of the different pipelines. We show that the integrated annotation covers more genes, increases by over 50% the number of highly co-expressed GO processes, and obtains much higher agreement with the gold standard. We find that different annotation pipelines produce different results, and show how to integrate them into a unified annotation that is of higher quality than each single pipeline. We offer an improved functional annotation of both PGSC and ITAG potato gene models, as well as tools that can be applied to additional pipelines and improve annotation in other organisms. This will greatly aid future functional analysis of '-omics' datasets from potato and other organisms with newly sequenced genomes. The new potato annotations are available with this paper.

Genealogy of the nuclear beta-fibrinogen locus in a highly structured lizard species: comparison with mtDNA and evidence for intragenic recombination in the hybrid zone.

PubMed

Godinho, R; Mendonça, B; Crespo, E G; Ferrand, N

2006-06-01

The study of nuclear genealogies in natural populations of nonmodel organisms is expected to provide novel insights into the evolutionary history of populations, especially when developed in the framework of well-established mtDNA phylogeographical scenarios. In the Iberian Peninsula, the endemic Schreiber's green lizard Lacerta schreiberi exhibits two highly divergent and allopatric mtDNA lineages that started to split during the late Pliocene. In this work, we performed a fine-scale analysis of the putative mtDNA contact zone together with a global analysis of the patterns of variation observed at the nuclear beta-fibrinogen intron 7 (beta-fibint7). Using a combination of DNA sequencing with single-strand conformational polymorphism (SSCP) analysis, we show that the observed genealogy at the beta-fibint7 locus reveals extensive admixture between two formerly isolated lizard populations while the two mtDNA lineages remain essentially allopatric. In addition, a private beta-fibint7 haplotype detected in the single population where both mtDNA lineages were found in sympatry is probably the result of intragenic recombination between the two more common and divergent beta-fibint7 haplotypes. Our results suggest that the progressive incorporation of nuclear genealogies in investigating the ancient demography and admixture dynamics of divergent genomes will be necessary to obtain a more comprehensive picture of the evolutionary history of organisms.

Germline DNA methylation in reef corals: patterns and potential roles in response to environmental change.

PubMed

Dimond, James L; Roberts, Steven B

2016-04-01

DNA methylation is an epigenetic mark that plays an inadequately understood role in gene regulation, particularly in nonmodel species. Because it can be influenced by the environment, DNA methylation may contribute to the ability of organisms to acclimatize and adapt to environmental change. We evaluated the distribution of gene body methylation in reef-building corals, a group of organisms facing significant environmental threats. Gene body methylation in six species of corals was inferred from in silico transcriptome analysis of CpG O/E, an estimate of germline DNA methylation that is highly correlated with patterns of methylation enrichment. Consistent with what has been documented in most other invertebrates, all corals exhibited bimodal distributions of germline methylation suggestive of distinct fractions of genes with high and low levels of methylation. The hypermethylated fractions were enriched with genes with housekeeping functions, while genes with inducible functions were highly represented in the hypomethylated fractions. High transcript abundance was associated with intermediate levels of methylation. In three of the coral species, we found that genes differentially expressed in response to thermal stress and ocean acidification exhibited significantly lower levels of methylation. These results support a link between gene body hypomethylation and transcriptional plasticity that may point to a role of DNA methylation in the response of corals to environmental change. © 2015 John Wiley & Sons Ltd.

Galaxy tools and workflows for sequence analysis with applications in molecular plant pathology.

PubMed

Cock, Peter J A; Grüning, Björn A; Paszkiewicz, Konrad; Pritchard, Leighton

2013-01-01

The Galaxy Project offers the popular web browser-based platform Galaxy for running bioinformatics tools and constructing simple workflows. Here, we present a broad collection of additional Galaxy tools for large scale analysis of gene and protein sequences. The motivating research theme is the identification of specific genes of interest in a range of non-model organisms, and our central example is the identification and prediction of "effector" proteins produced by plant pathogens in order to manipulate their host plant. This functional annotation of a pathogen's predicted capacity for virulence is a key step in translating sequence data into potential applications in plant pathology. This collection includes novel tools, and widely-used third-party tools such as NCBI BLAST+ wrapped for use within Galaxy. Individual bioinformatics software tools are typically available separately as standalone packages, or in online browser-based form. The Galaxy framework enables the user to combine these and other tools to automate organism scale analyses as workflows, without demanding familiarity with command line tools and scripting. Workflows created using Galaxy can be saved and are reusable, so may be distributed within and between research groups, facilitating the construction of a set of standardised, reusable bioinformatic protocols. The Galaxy tools and workflows described in this manuscript are open source and freely available from the Galaxy Tool Shed (http://usegalaxy.org/toolshed or http://toolshed.g2.bx.psu.edu).

Identification of long non-coding RNAs in two anthozoan species and their possible implications for coral bleaching.

PubMed

Huang, Chen; Morlighem, Jean-Étienne R L; Cai, Jing; Liao, Qiwen; Perez, Carlos Daniel; Gomes, Paula Braga; Guo, Min; Rádis-Baptista, Gandhi; Lee, Simon Ming-Yuen

2017-07-13

Long non-coding RNAs (lncRNAs) have been shown to play regulatory roles in a diverse range of biological processes and are associated with the outcomes of various diseases. The majority of studies about lncRNAs focus on model organisms, with lessened investigation in non-model organisms to date. Herein, we have undertaken an investigation on lncRNA in two zoanthids (cnidarian): Protolpalythoa varibilis and Palythoa caribaeorum. A total of 11,206 and 13,240 lncRNAs were detected in P. variabilis and P. caribaeorum transcriptome, respectively. Comparison using NONCODE database indicated that the majority of these lncRNAs is taxonomically species-restricted with no identifiable orthologs. Even so, we found cases in which short regions of P. caribaeorum's lncRNAs were similar to vertebrate species' lncRNAs, and could be associated with lncRNA conserved regulatory functions. Consequently, some high-confidence lncRNA-mRNA interactions were predicted based on such conserved regions, therefore revealing possible involvement of lncRNAs in posttranscriptional processing and regulation in anthozoans. Moreover, investigation of differentially expressed lncRNAs, in healthy colonies and colonial individuals undergoing natural bleaching, indicated that some up-regulated lncRNAs in P. caribaeorum could posttranscriptionally regulate the mRNAs encoding proteins of Ras-mediated signal transduction pathway and components of innate immune-system, which could contribute to the molecular response of coral bleaching.

SSHscreen and SSHdb, generic software for microarray based gene discovery: application to the stress response in cowpea

PubMed Central

2010-01-01

Background Suppression subtractive hybridization is a popular technique for gene discovery from non-model organisms without an annotated genome sequence, such as cowpea (Vigna unguiculata (L.) Walp). We aimed to use this method to enrich for genes expressed during drought stress in a drought tolerant cowpea line. However, current methods were inefficient in screening libraries and management of the sequence data, and thus there was a need to develop software tools to facilitate the process. Results Forward and reverse cDNA libraries enriched for cowpea drought response genes were screened on microarrays, and the R software package SSHscreen 2.0.1 was developed (i) to normalize the data effectively using spike-in control spot normalization, and (ii) to select clones for sequencing based on the calculation of enrichment ratios with associated statistics. Enrichment ratio 3 values for each clone showed that 62% of the forward library and 34% of the reverse library clones were significantly differentially expressed by drought stress (adjusted p value < 0.05). Enrichment ratio 2 calculations showed that > 88% of the clones in both libraries were derived from rare transcripts in the original tester samples, thus supporting the notion that suppression subtractive hybridization enriches for rare transcripts. A set of 118 clones were chosen for sequencing, and drought-induced cowpea genes were identified, the most interesting encoding a late embryogenesis abundant Lea5 protein, a glutathione S-transferase, a thaumatin, a universal stress protein, and a wound induced protein. A lipid transfer protein and several components of photosynthesis were down-regulated by the drought stress. Reverse transcriptase quantitative PCR confirmed the enrichment ratio values for the selected cowpea genes. SSHdb, a web-accessible database, was developed to manage the clone sequences and combine the SSHscreen data with sequence annotations derived from BLAST and Blast2GO. The self-BLAST function within SSHdb grouped redundant clones together and illustrated that the SSHscreen plots are a useful tool for choosing anonymous clones for sequencing, since redundant clones cluster together on the enrichment ratio plots. Conclusions We developed the SSHscreen-SSHdb software pipeline, which greatly facilitates gene discovery using suppression subtractive hybridization by improving the selection of clones for sequencing after screening the library on a small number of microarrays. Annotation of the sequence information and collaboration was further enhanced through a web-based SSHdb database, and we illustrated this through identification of drought responsive genes from cowpea, which can now be investigated in gene function studies. SSH is a popular and powerful gene discovery tool, and therefore this pipeline will have application for gene discovery in any biological system, particularly non-model organisms. SSHscreen 2.0.1 and a link to SSHdb are available from http://microarray.up.ac.za/SSHscreen. PMID:20359330

A Comprehensive Structural Study of Offshore Wind Turbine Foundation and Non-Model Based Damage Detection using Effective Mass with Application to Small Components/ Cables and a Truss Wind Turbine Tower

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Scott A.

This research has two areas of focus. The first area is to investigate offshore wind turbine (OWT) designs, for use in the Maryland offshore wind area (MOWA), using intensive modeling techniques. The second focus area is to investigate a way to detect damage in wind turbine towers and small electrical components.

A rapid and cost-effective fluorescence detection in tube (FDIT) method to analyze protein phosphorylation.

PubMed

Jin, Xiao; Gou, Jin-Ying

2016-01-01

Protein phosphorylation is one of the most important post-translational modifications catalyzed by protein kinases in living organisms. The advance of genome sequencing provided the information of protein kinase families in many organisms, including both model and non-model plants. The development of proteomics technologies also enabled scientists to efficiently reveal a large number of protein phosphorylations of an organism. However, kinases and phosphorylation targets are still to be connected to illustrate the complicated network in life. Here we adapted Pro-Q ® Diamond (Pro-Q ® Diamond Phosphoprotein Gel Stain), a widely used phosphoprotein gel-staining fluorescence dye, to establish a rapid, economical and non-radioactive fluorescence detection in tube (FDIT) method to analyze phosphorylated proteins. Taking advantages of high sensitivity and specificity of Pro-Q ® diamond, the FDIT method is also demonstrated to be rapid and reliable, with a suitable linear range for in vitro protein phosphorylation. A significant and satisfactory protein kinase reaction was detected as fast as 15 min from Wheat Kinase START 1.1 (WKS1.1) on a thylakoid ascorbate peroxidase (tAPX), an established phosphorylation target in our earlier study. The FDIT method saves up to 95% of the dye consumed in a gel staining method. The FDIT method is remarkably quick, highly reproducible, unambiguous and capable to be scaled up to dozens of samples. The FDIT method could serve as a simple and sensitive alternative procedure to determine protein kinase reactions with zero radiation exposure, as a supplementation to other widely used radioactive and in-gel assays.

A comparative simulation study of AR(1) estimators in short time series.

PubMed

Krone, Tanja; Albers, Casper J; Timmerman, Marieke E

2017-01-01

Various estimators of the autoregressive model exist. We compare their performance in estimating the autocorrelation in short time series. In Study 1, under correct model specification, we compare the frequentist r 1 estimator, C-statistic, ordinary least squares estimator (OLS) and maximum likelihood estimator (MLE), and a Bayesian method, considering flat (B f ) and symmetrized reference (B sr ) priors. In a completely crossed experimental design we vary lengths of time series (i.e., T = 10, 25, 40, 50 and 100) and autocorrelation (from -0.90 to 0.90 with steps of 0.10). The results show a lowest bias for the B sr , and a lowest variability for r 1 . The power in different conditions is highest for B sr and OLS. For T = 10, the absolute performance of all measurements is poor, as expected. In Study 2, we study robustness of the methods through misspecification by generating the data according to an ARMA(1,1) model, but still analysing the data with an AR(1) model. We use the two methods with the lowest bias for this study, i.e., B sr and MLE. The bias gets larger when the non-modelled moving average parameter becomes larger. Both the variability and power show dependency on the non-modelled parameter. The differences between the two estimation methods are negligible for all measurements.

Microbial ecology-based methods to characterize the bacterial communities of non-model insects.

PubMed

Prosdocimi, Erica M; Mapelli, Francesca; Gonella, Elena; Borin, Sara; Crotti, Elena

2015-12-01

Among the animals of the Kingdom Animalia, insects are unparalleled for their widespread diffusion, diversity and number of occupied ecological niches. In recent years they have raised researcher interest not only because of their importance as human and agricultural pests, disease vectors and as useful breeding species (e.g. honeybee and silkworm), but also because of their suitability as animal models. It is now fully recognized that microorganisms form symbiotic relationships with insects, influencing their survival, fitness, development, mating habits and the immune system and other aspects of the biology and ecology of the insect host. Thus, any research aimed at deepening the knowledge of any given insect species (perhaps species of applied interest or species emerging as novel pests or vectors) must consider the characterization of the associated microbiome. The present review critically examines the microbiology and molecular ecology techniques that can be applied to the taxonomical and functional analysis of the microbiome of non-model insects. Our goal is to provide an overview of current approaches and methods addressing the ecology and functions of microorganisms and microbiomes associated with insects. Our focus is on operational details, aiming to provide a concise guide to currently available advanced techniques, in an effort to extend insect microbiome research beyond simple descriptions of microbial communities. Copyright © 2015 Elsevier B.V. All rights reserved.

Efficient and Heritable Gene Targeting in Tilapia by CRISPR/Cas9

PubMed Central

Li, Minghui; Yang, Huihui; Zhao, Jiue; Fang, Lingling; Shi, Hongjuan; Li, Mengru; Sun, Yunlv; Zhang, Xianbo; Jiang, Dongneng; Zhou, Linyan; Wang, Deshou

2014-01-01

Studies of gene function in non-model animals have been limited by the approaches available for eliminating gene function. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated) system has recently become a powerful tool for targeted genome editing. Here, we report the use of the CRISPR/Cas9 system to disrupt selected genes, including nanos2, nanos3, dmrt1, and foxl2, with efficiencies as high as 95%. In addition, mutations in dmrt1 and foxl2 induced by CRISPR/Cas9 were efficiently transmitted through the germline to F1. Obvious phenotypes were observed in the G0 generation after mutation of germ cell or somatic cell-specific genes. For example, loss of Nanos2 and Nanos3 in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, while masculinization of somatic cells in both XY and XX gonads was demonstrated by Dmrt1 and Cyp11b2 immunohistochemistry and by up-regulation of serum androgen levels. Our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing a convenient and effective approach for generating loss-of-function mutants. Furthermore, our study shows the utility of the CRISPR/Cas9 system for genetic engineering in non-model species like tilapia and potentially in many other teleost species. PMID:24709635

Effect of gastric emptying and entero-hepatic circulation on bioequivalence assessment of ranitidine.

PubMed

Chrenova, J; Durisova, M; Mircioiu, C; Dedik, L

2010-01-01

The aim of study was to compare the bioavailability of ranitidine obtained from either Ranitidine (300 mg tablet; LPH® S.C. LaborMed Pharma S.A. Romania: the test formulation) and Zantac® (300 mg tablet; GlaxoSmithKline, Austria: the reference formulation). Twelve, Romanian, healthy volunteers were enrolled in the study. An open-label, two-period, crossover, randomized design was used. Plasma levels of ranitidine were determined using the validated, high-pressure liquid chromatography (HPLC) method. The physiologically motivated time-delayed model was used for the data evaluation and a paired Student's t-test and Schuirmann's two one-sided tests were carried out to compare parameters. Nonmodeling parameters (AUC(t), AUC, C(max), T(max)) were tested by the paired Student's t-test and the 90 confidence intervals of the geometric mean ratios were determined by Schuirmann's tests. Paired Student's t-test showed no significant differences between nonmodeling and modeling parameters. The results of the Schuirmann's tests however indicated significant statistical differences with reference to AUC(t), AUC, C(max), T(max) and other modeling parameters, especially MT(c) and τ(c). Schuirmann's tests revealed significant bioequivalence between ranitidine formulations using the modeling parameters MRT and n. The presented model can be useful as an additional tool to assess drug bioequivalence, by screening for disruptive parameters. Copyright 2010 Prous Science, S.A.U. or its licensors. All rights reserved.

Contrasting the Chromosomal Organization of Repetitive DNAs in Two Gryllidae Crickets with Highly Divergent Karyotypes

PubMed Central

Palacios-Gimenez, Octavio M.; Carvalho, Carlos Roberto; Ferrari Soares, Fernanda Aparecida; Cabral-de-Mello, Diogo C.

2015-01-01

A large percentage of eukaryotic genomes consist of repetitive DNA that plays an important role in the organization, size and evolution. In the case of crickets, chromosomal variability has been found using classical cytogenetics, but almost no information concerning the organization of their repetitive DNAs is available. To better understand the chromosomal organization and diversification of repetitive DNAs in crickets, we studied the chromosomes of two Gryllidae species with highly divergent karyotypes, i.e., 2n(♂) = 29,X0 (Gryllus assimilis) and 2n = 9, neo-X1X2Y (Eneoptera surinamensis). The analyses were performed using classical cytogenetic techniques, repetitive DNA mapping and genome-size estimation. Conserved characteristics were observed, such as the occurrence of a small number of clusters of rDNAs and U snDNAs, in contrast to the multiple clusters/dispersal of the H3 histone genes. The positions of U2 snDNA and 18S rDNA are also conserved, being intermingled within the largest autosome. The distribution and base-pair composition of the heterochromatin and repetitive DNA pools of these organisms differed, suggesting reorganization. Although the microsatellite arrays had a similar distribution pattern, being dispersed along entire chromosomes, as has been observed in some grasshopper species, a band-like pattern was also observed in the E. surinamensis chromosomes, putatively due to their amplification and clustering. In addition to these differences, the genome of E. surinamensis is approximately 2.5 times larger than that of G. assimilis, which we hypothesize is due to the amplification of repetitive DNAs. Finally, we discuss the possible involvement of repetitive DNAs in the differentiation of the neo-sex chromosomes of E. surinamensis, as has been reported in other eukaryotic groups. This study provided an opportunity to explore the evolutionary dynamics of repetitive DNAs in two non-model species and will contribute to the understanding of chromosomal evolution in a group about which little chromosomal and genomic information is known. PMID:26630487

Digital Gene Expression Analysis Based on De Novo Transcriptome Assembly Reveals New Genes Associated with Floral Organ Differentiation of the Orchid Plant Cymbidium ensifolium

PubMed Central

Yang, Fengxi; Zhu, Genfa

2015-01-01

Cymbidium ensifolium belongs to the genus Cymbidium of the orchid family. Owing to its spectacular flower morphology, C. ensifolium has considerable ecological and cultural value. However, limited genetic data is available for this non-model plant, and the molecular mechanism underlying floral organ identity is still poorly understood. In this study, we characterize the floral transcriptome of C. ensifolium and present, for the first time, extensive sequence and transcript abundance data of individual floral organs. After sequencing, over 10 Gb clean sequence data were generated and assembled into 111,892 unigenes with an average length of 932.03 base pairs, including 1,227 clusters and 110,665 singletons. Assembled sequences were annotated with gene descriptions, gene ontology, clusters of orthologous group terms, the Kyoto Encyclopedia of Genes and Genomes, and the plant transcription factor database. From these annotations, 131 flowering-associated unigenes, 61 CONSTANS-LIKE (COL) unigenes and 90 floral homeotic genes were identified. In addition, four digital gene expression libraries were constructed for the sepal, petal, labellum and gynostemium, and 1,058 genes corresponding to individual floral organ development were identified. Among them, eight MADS-box genes were further investigated by full-length cDNA sequence analysis and expression validation, which revealed two APETALA1/AGL9-like MADS-box genes preferentially expressed in the sepal and petal, two AGAMOUS-like genes particularly restricted to the gynostemium, and four DEF-like genes distinctively expressed in different floral organs. The spatial expression of these genes varied distinctly in different floral mutant corresponding to different floral morphogenesis, which validated the specialized roles of them in floral patterning and further supported the effectiveness of our in silico analysis. This dataset generated in our study provides new insights into the molecular mechanisms underlying floral patterning of Cymbidium and supports a valuable resource for molecular breeding of the orchid plant. PMID:26580566

Contrasting the Chromosomal Organization of Repetitive DNAs in Two Gryllidae Crickets with Highly Divergent Karyotypes.

PubMed

Palacios-Gimenez, Octavio M; Carvalho, Carlos Roberto; Ferrari Soares, Fernanda Aparecida; Cabral-de-Mello, Diogo C

2015-01-01

A large percentage of eukaryotic genomes consist of repetitive DNA that plays an important role in the organization, size and evolution. In the case of crickets, chromosomal variability has been found using classical cytogenetics, but almost no information concerning the organization of their repetitive DNAs is available. To better understand the chromosomal organization and diversification of repetitive DNAs in crickets, we studied the chromosomes of two Gryllidae species with highly divergent karyotypes, i.e., 2n(♂) = 29,X0 (Gryllus assimilis) and 2n = 9, neo-X1X2Y (Eneoptera surinamensis). The analyses were performed using classical cytogenetic techniques, repetitive DNA mapping and genome-size estimation. Conserved characteristics were observed, such as the occurrence of a small number of clusters of rDNAs and U snDNAs, in contrast to the multiple clusters/dispersal of the H3 histone genes. The positions of U2 snDNA and 18S rDNA are also conserved, being intermingled within the largest autosome. The distribution and base-pair composition of the heterochromatin and repetitive DNA pools of these organisms differed, suggesting reorganization. Although the microsatellite arrays had a similar distribution pattern, being dispersed along entire chromosomes, as has been observed in some grasshopper species, a band-like pattern was also observed in the E. surinamensis chromosomes, putatively due to their amplification and clustering. In addition to these differences, the genome of E. surinamensis is approximately 2.5 times larger than that of G. assimilis, which we hypothesize is due to the amplification of repetitive DNAs. Finally, we discuss the possible involvement of repetitive DNAs in the differentiation of the neo-sex chromosomes of E. surinamensis, as has been reported in other eukaryotic groups. This study provided an opportunity to explore the evolutionary dynamics of repetitive DNAs in two non-model species and will contribute to the understanding of chromosomal evolution in a group about which little chromosomal and genomic information is known.

Genome sequence and genetic diversity of European ash trees.

PubMed

Sollars, Elizabeth S A; Harper, Andrea L; Kelly, Laura J; Sambles, Christine M; Ramirez-Gonzalez, Ricardo H; Swarbreck, David; Kaithakottil, Gemy; Cooper, Endymion D; Uauy, Cristobal; Havlickova, Lenka; Worswick, Gemma; Studholme, David J; Zohren, Jasmin; Salmon, Deborah L; Clavijo, Bernardo J; Li, Yi; He, Zhesi; Fellgett, Alison; McKinney, Lea Vig; Nielsen, Lene Rostgaard; Douglas, Gerry C; Kjær, Erik Dahl; Downie, J Allan; Boshier, David; Lee, Steve; Clark, Jo; Grant, Murray; Bancroft, Ian; Caccamo, Mario; Buggs, Richard J A

2017-01-12

Ash trees (genus Fraxinus, family Oleaceae) are widespread throughout the Northern Hemisphere, but are being devastated in Europe by the fungus Hymenoscyphus fraxineus, causing ash dieback, and in North America by the herbivorous beetle Agrilus planipennis. Here we sequence the genome of a low-heterozygosity Fraxinus excelsior tree from Gloucestershire, UK, annotating 38,852 protein-coding genes of which 25% appear ash specific when compared with the genomes of ten other plant species. Analyses of paralogous genes suggest a whole-genome duplication shared with olive (Olea europaea, Oleaceae). We also re-sequence 37 F. excelsior trees from Europe, finding evidence for apparent long-term decline in effective population size. Using our reference sequence, we re-analyse association transcriptomic data, yielding improved markers for reduced susceptibility to ash dieback. Surveys of these markers in British populations suggest that reduced susceptibility to ash dieback may be more widespread in Great Britain than in Denmark. We also present evidence that susceptibility of trees to H. fraxineus is associated with their iridoid glycoside levels. This rapid, integrated, multidisciplinary research response to an emerging health threat in a non-model organism opens the way for mitigation of the epidemic.

A Comprehensive Linkage Map of the Dog Genome

PubMed Central

Wong, Aaron K.; Ruhe, Alison L.; Dumont, Beth L.; Robertson, Kathryn R.; Guerrero, Giovanna; Shull, Sheila M.; Ziegle, Janet S.; Millon, Lee V.; Broman, Karl W.; Payseur, Bret A.; Neff, Mark W.

2010-01-01

We have leveraged the reference sequence of a boxer to construct the first complete linkage map for the domestic dog. The new map improves access to the dog's unique biology, from human disease counterparts to fascinating evolutionary adaptations. The map was constructed with ∼3000 microsatellite markers developed from the reference sequence. Familial resources afforded 450 mostly phase-known meioses for map assembly. The genotype data supported a framework map with ∼1500 loci. An additional ∼1500 markers served as map validators, contributing modestly to estimates of recombination rate but supporting the framework content. Data from ∼22,000 SNPs informing on a subset of meioses supported map integrity. The sex-averaged map extended 21 M and revealed marked region- and sex-specific differences in recombination rate. The map will enable empiric coverage estimates and multipoint linkage analysis. Knowledge of the variation in recombination rate will also inform on genomewide patterns of linkage disequilibrium (LD), and thus benefit association, selective sweep, and phylogenetic mapping approaches. The computational and wet-bench strategies can be applied to the reference genome of any nonmodel organism to assemble a de novo linkage map. PMID:19966068

Integration of parallel 13 C-labeling experiments and in silico pathway analysis for enhanced production of ascomycin.

PubMed

Qi, Haishan; Lv, Mengmeng; Song, Kejing; Wen, Jianping

2017-05-01

Herein, the hyper-producing strain for ascomycin was engineered based on 13 C-labeling experiments and elementary flux modes analysis (EFMA). First, the metabolism of non-model organism Streptomyces hygroscopicus var. ascomyceticus SA68 was investigated and an updated network model was reconstructed using 13 C- metabolic flux analysis. Based on the precise model, EFMA was further employed to predict genetic targets for higher ascomycin production. Chorismatase (FkbO) and pyruvate carboxylase (Pyc) were predicted as the promising overexpression and deletion targets, respectively. The corresponding mutant TD-FkbO and TD-ΔPyc exhibited the consistency effects between model prediction and experimental results. Finally, the combined genetic manipulations were performed, achieving a high-yield ascomycin engineering strain TD-ΔPyc-FkbO with production up to 610 mg/L, 84.8% improvement compared with the parent strain SA68. These results manifested that the integration of 13 C-labeling experiments and in silico pathway analysis could serve as a promising concept to enhance ascomycin production, as well as other valuable products. Biotechnol. Bioeng. 2017;114: 1036-1044. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The Global Genome Biodiversity Network (GGBN) Data Standard specification

PubMed Central

Droege, G.; Barker, K.; Seberg, O.; Coddington, J.; Benson, E.; Berendsohn, W. G.; Bunk, B.; Butler, C.; Cawsey, E. M.; Deck, J.; Döring, M.; Flemons, P.; Gemeinholzer, B.; Güntsch, A.; Hollowell, T.; Kelbert, P.; Kostadinov, I.; Kottmann, R.; Lawlor, R. T.; Lyal, C.; Mackenzie-Dodds, J.; Meyer, C.; Mulcahy, D.; Nussbeck, S. Y.; O'Tuama, É.; Orrell, T.; Petersen, G.; Robertson, T.; Söhngen, C.; Whitacre, J.; Wieczorek, J.; Yilmaz, P.; Zetzsche, H.; Zhang, Y.; Zhou, X.

2016-01-01

Genomic samples of non-model organisms are becoming increasingly important in a broad range of studies from developmental biology, biodiversity analyses, to conservation. Genomic sample definition, description, quality, voucher information and metadata all need to be digitized and disseminated across scientific communities. This information needs to be concise and consistent in today’s ever-increasing bioinformatic era, for complementary data aggregators to easily map databases to one another. In order to facilitate exchange of information on genomic samples and their derived data, the Global Genome Biodiversity Network (GGBN) Data Standard is intended to provide a platform based on a documented agreement to promote the efficient sharing and usage of genomic sample material and associated specimen information in a consistent way. The new data standard presented here build upon existing standards commonly used within the community extending them with the capability to exchange data on tissue, environmental and DNA sample as well as sequences. The GGBN Data Standard will reveal and democratize the hidden contents of biodiversity biobanks, for the convenience of everyone in the wider biobanking community. Technical tools exist for data providers to easily map their databases to the standard. Database URL: http://terms.tdwg.org/wiki/GGBN_Data_Standard PMID:27694206

Highly Efficient Genome Editing via CRISPR/Cas9 to Create Clock Gene Knockout Cells.

PubMed

Korge, Sandra; Grudziecki, Astrid; Kramer, Achim

2015-10-01

Targeted genome editing using CRISPR/Cas9 is a relatively new, revolutionary technology allowing for efficient and directed alterations of the genome. It has been widely used for loss-of-function studies in animals and cell lines but has not yet been used to study circadian rhythms. Here, we describe the application of CRISPR/Cas9 genome editing for the generation of an F-box and leucine-rich repeat protein 3 (Fbxl3) knockout in a human cell line. Genomic alterations at the Fbxl3 locus occurred with very high efficiency (70%-100%) and specificity at both alleles, resulting in insertions and deletions that led to premature stop codons and hence FBXL3 knockout. Fbxl3 knockout cells displayed low amplitude and long period oscillations of Bmal1-luciferase reporter activity as well as increased CRY1 protein stability in line with previously published phenotypes for Fbxl3 knockout in mice. Thus, CRISPR/Cas9 genome editing should be highly valuable for studying circadian rhythms not only in human cells but also in classic model systems as well as nonmodel organisms. © 2015 The Author(s).

Marine genomics: News and views.

PubMed

Ribeiro, Ângela M; Foote, Andrew D; Kupczok, Anne; Frazão, Bárbara; Limborg, Morten T; Piñeiro, Rosalía; Abalde, Samuel; Rocha, Sara; da Fonseca, Rute R

2017-02-01

Marine ecosystems occupy 71% of the surface of our planet, yet we know little about their diversity. Although the inventory of species is continually increasing, as registered by the Census of Marine Life program, only about 10% of the estimated two million marine species are known. This lag between observed and estimated diversity is in part due to the elusiveness of most aquatic species and the technical difficulties of exploring extreme environments, as for instance the abyssal plains and polar waters. In the last decade, the rapid development of affordable and flexible high-throughput sequencing approaches have been helping to improve our knowledge of marine biodiversity, from the rich microbial biota that forms the base of the tree of life to a wealth of plant and animal species. In this review, we present an overview of the applications of genomics to the study of marine life, from evolutionary biology of non-model organisms to species of commercial relevance for fishing, aquaculture and biomedicine. Instead of providing an exhaustive list of available genomic data, we rather set to present contextualized examples that best represent the current status of the field of marine genomics. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparative Transcriptomics Among Four White Pine Species

PubMed Central

Baker, Ethan A. G.; Wegrzyn, Jill L.; Sezen, Uzay U.; Falk, Taylor; Maloney, Patricia E.; Vogler, Detlev R.; Delfino-Mix, Annette; Jensen, Camille; Mitton, Jeffry; Wright, Jessica; Knaus, Brian; Rai, Hardeep; Cronn, Richard; Gonzalez-Ibeas, Daniel; Vasquez-Gross, Hans A.; Famula, Randi A.; Liu, Jun-Jun; Kueppers, Lara M.; Neale, David B.

2018-01-01

Conifers are the dominant plant species throughout the high latitude boreal forests as well as some lower latitude temperate forests of North America, Europe, and Asia. As such, they play an integral economic and ecological role across much of the world. This study focused on the characterization of needle transcriptomes from four ecologically important and understudied North American white pines within the Pinus subgenus Strobus. The populations of many Strobus species are challenged by native and introduced pathogens, native insects, and abiotic factors. RNA from the needles of western white pine (Pinus monticola), limber pine (Pinus flexilis), whitebark pine (Pinus albicaulis), and sugar pine (Pinus lambertiana) was sampled, Illumina short read sequenced, and de novo assembled. The assembled transcripts and their subsequent structural and functional annotations were processed through custom pipelines to contend with the challenges of non-model organism transcriptome validation. Orthologous gene family analysis of over 58,000 translated transcripts, implemented through Tribe-MCL, estimated the shared and unique gene space among the four species. This revealed 2025 conserved gene families, of which 408 were aligned to estimate levels of divergence and reveal patterns of selection. Specific candidate genes previously associated with drought tolerance and white pine blister rust resistance in conifers were investigated. PMID:29559535

A divide-and-conquer algorithm for large-scale de novo transcriptome assembly through combining small assemblies from existing algorithms.

PubMed

Sze, Sing-Hoi; Parrott, Jonathan J; Tarone, Aaron M

2017-12-06

While the continued development of high-throughput sequencing has facilitated studies of entire transcriptomes in non-model organisms, the incorporation of an increasing amount of RNA-Seq libraries has made de novo transcriptome assembly difficult. Although algorithms that can assemble a large amount of RNA-Seq data are available, they are generally very memory-intensive and can only be used to construct small assemblies. We develop a divide-and-conquer strategy that allows these algorithms to be utilized, by subdividing a large RNA-Seq data set into small libraries. Each individual library is assembled independently by an existing algorithm, and a merging algorithm is developed to combine these assemblies by picking a subset of high quality transcripts to form a large transcriptome. When compared to existing algorithms that return a single assembly directly, this strategy achieves comparable or increased accuracy as memory-efficient algorithms that can be used to process a large amount of RNA-Seq data, and comparable or decreased accuracy as memory-intensive algorithms that can only be used to construct small assemblies. Our divide-and-conquer strategy allows memory-intensive de novo transcriptome assembly algorithms to be utilized to construct large assemblies.

Optimization of the genotyping-by-sequencing strategy for population genomic analysis in conifers.

PubMed

Pan, Jin; Wang, Baosheng; Pei, Zhi-Yong; Zhao, Wei; Gao, Jie; Mao, Jian-Feng; Wang, Xiao-Ru

2015-07-01

Flexibility and low cost make genotyping-by-sequencing (GBS) an ideal tool for population genomic studies of nonmodel species. However, to utilize the potential of the method fully, many parameters affecting library quality and single nucleotide polymorphism (SNP) discovery require optimization, especially for conifer genomes with a high repetitive DNA content. In this study, we explored strategies for effective GBS analysis in pine species. We constructed GBS libraries using HpaII, PstI and EcoRI-MseI digestions with different multiplexing levels and examined the effect of restriction enzymes on library complexity and the impact of sequencing depth and size selection of restriction fragments on sequence coverage bias. We tested and compared UNEAK, Stacks and GATK pipelines for the GBS data, and then developed a reference-free SNP calling strategy for haploid pine genomes. Our GBS procedure proved to be effective in SNP discovery, producing 7000-11 000 and 14 751 SNPs within and among three pine species, respectively, from a PstI library. This investigation provides guidance for the design and analysis of GBS experiments, particularly for organisms for which genomic information is lacking. © 2014 John Wiley & Sons Ltd.

Ancestry-Specific Methylation Patterns in Admixed Offspring from an Experimental Coyote and Gray Wolf Cross.

PubMed

vonHoldt, Bridgett; Heppenheimer, Elizabeth; Petrenko, Vladimir; Croonquist, Paula; Rutledge, Linda Y

2017-06-01

Reduced fitness of admixed individuals is typically attributed to genetic incompatibilities. Although mismatched genomes can lead to fitness changes, in some cases the reduction in hybrid fitness is subtle. The potential role of transcriptional regulation in admixed genomes could provide a mechanistic explanation for these discrepancies, but evidence is lacking for nonmodel organisms. Here, we explored the intersection of genetics and gene regulation in admixed genomes derived from an experimental cross between a western gray wolf and western coyote. We found a significant positive association between methylation and wolf ancestry, and identified outlier genes that have been previously implicated in inbreeding-related, or otherwise deleterious, phenotypes. We describe a pattern of site-specific, rather than genome-wide, methylation driven by inter-specific hybridization. Epigenetic variation is thus suggested to play a nontrivial role in both maintaining and combating mismatched genotypes through putative transcriptional mechanisms. We conclude that the regulation of gene expression is an underappreciated key component of hybrid genome functioning, but could also act as a potential source of novel and beneficial adaptive variation in hybrid offspring. © The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

NATpipe: an integrative pipeline for systematical discovery of natural antisense transcripts (NATs) and phase-distributed nat-siRNAs from de novo assembled transcriptomes

PubMed Central

Yu, Dongliang; Meng, Yijun; Zuo, Ziwei; Xue, Jie; Wang, Huizhong

2016-01-01

Nat-siRNAs (small interfering RNAs originated from natural antisense transcripts) are a class of functional small RNA (sRNA) species discovered in both plants and animals. These siRNAs are highly enriched within the annealed regions of the NAT (natural antisense transcript) pairs. To date, great research efforts have been taken for systematical identification of the NATs in various organisms. However, developing a freely available and easy-to-use program for NAT prediction is strongly demanded by researchers. Here, we proposed an integrative pipeline named NATpipe for systematical discovery of NATs from de novo assembled transcriptomes. By utilizing sRNA sequencing data, the pipeline also allowed users to search for phase-distributed nat-siRNAs within the perfectly annealed regions of the NAT pairs. Additionally, more reliable nat-siRNA loci could be identified based on degradome sequencing data. A case study on the non-model plant Dendrobium officinale was performed to illustrate the utility of NATpipe. Finally, we hope that NATpipe would be a useful tool for NAT prediction, nat-siRNA discovery, and related functional studies. NATpipe is available at www.bioinfolab.cn/NATpipe/NATpipe.zip. PMID:26858106

Differential DNA Methylation Analysis without a Reference Genome.

PubMed

Klughammer, Johanna; Datlinger, Paul; Printz, Dieter; Sheffield, Nathan C; Farlik, Matthias; Hadler, Johanna; Fritsch, Gerhard; Bock, Christoph

2015-12-22

Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS), which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish). Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org). The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Identification of high-efficiency 3'GG gRNA motifs in indexed FASTA files with ngg2.

PubMed

Roberson, Elisha D O

CRISPR/Cas9 is emerging as one of the most-used methods of genome modification in organisms ranging from bacteria to human cells. However, the efficiency of editing varies tremendously site-to-site. A recent report identified a novel motif, called the 3'GG motif, which substantially increases the efficiency of editing at all sites tested in C. elegans . Furthermore, they highlighted that previously published gRNAs with high editing efficiency also had this motif. I designed a python command-line tool, ngg2, to identify 3'GG gRNA sites from indexed FASTA files. As a proof-of-concept, I screened for these motifs in six model genomes: Saccharomyces cerevisiae , Caenorhabditis elegans , Drosophila melanogaster , Danio rerio , Mus musculus , and Homo sapiens. I also scanned the genomes of pig ( Sus scrofa ) and African elephant ( Loxodonta africana ) to demonstrate the utility in non-model organisms. I identified more than 60 million single match 3'GG motifs in these genomes. Greater than 61% of all protein coding genes in the reference genomes had at least one unique 3'GG gRNA site overlapping an exon. In particular, more than 96% of mouse and 93% of human protein coding genes have at least one unique, overlapping 3'GG gRNA. These identified sites can be used as a starting point in gRNA selection, and the ngg2 tool provides an important ability to identify 3'GG editing sites in any species with an available genome sequence.

Membrane Proteomic Insights into the Physiology and Taxonomy of an Oleaginous Green Microalga1

PubMed Central

Vera-Estrella, Rosario

2017-01-01

Ettlia oleoabundans is a nonsequenced oleaginous green microalga. Despite the significant biotechnological interest in producing value-added compounds from the acyl lipids of this microalga, a basic understanding of the physiology and biochemistry of oleaginous microalgae is lacking, especially under nitrogen deprivation conditions known to trigger lipid accumulation. Using an RNA sequencing-based proteomics approach together with manual annotation, we are able to provide, to our knowledge, the first membrane proteome of an oleaginous microalga. This approach allowed the identification of novel proteins in E. oleoabundans, including two photoprotection-related proteins, Photosystem II Subunit S and Maintenance of Photosystem II under High Light1, which were considered exclusive to higher photosynthetic organisms, as well as Retinitis Pigmentosa Type 2-Clathrin Light Chain, a membrane protein with a novel domain architecture. Free-flow zonal electrophoresis of microalgal membranes coupled to liquid chromatography-tandem mass spectrometry proved to be a useful technique for determining the intracellular location of proteins of interest. Carbon-flow compartmentalization in E. oleoabundans was modeled using this information. Molecular phylogenetic analyses of protein markers and 18S ribosomal DNA support the reclassification of E. oleoabundans within the trebouxiophycean microalgae, rather than with the Chlorophyceae class, in which it is currently classified, indicating that it may not be closely related to the model green alga Chlamydomonas reinhardtii. A detailed survey of biological processes taking place in the membranes of nitrogen-deprived E. oleoabundans, including lipid metabolism, provides insights into the basic biology of this nonmodel organism. PMID:27837088

Galaxy tools and workflows for sequence analysis with applications in molecular plant pathology

PubMed Central

Grüning, Björn A.; Paszkiewicz, Konrad; Pritchard, Leighton

2013-01-01

The Galaxy Project offers the popular web browser-based platform Galaxy for running bioinformatics tools and constructing simple workflows. Here, we present a broad collection of additional Galaxy tools for large scale analysis of gene and protein sequences. The motivating research theme is the identification of specific genes of interest in a range of non-model organisms, and our central example is the identification and prediction of “effector” proteins produced by plant pathogens in order to manipulate their host plant. This functional annotation of a pathogen’s predicted capacity for virulence is a key step in translating sequence data into potential applications in plant pathology. This collection includes novel tools, and widely-used third-party tools such as NCBI BLAST+ wrapped for use within Galaxy. Individual bioinformatics software tools are typically available separately as standalone packages, or in online browser-based form. The Galaxy framework enables the user to combine these and other tools to automate organism scale analyses as workflows, without demanding familiarity with command line tools and scripting. Workflows created using Galaxy can be saved and are reusable, so may be distributed within and between research groups, facilitating the construction of a set of standardised, reusable bioinformatic protocols. The Galaxy tools and workflows described in this manuscript are open source and freely available from the Galaxy Tool Shed (http://usegalaxy.org/toolshed or http://toolshed.g2.bx.psu.edu). PMID:24109552

GENE-Counter: A Computational Pipeline for the Analysis of RNA-Seq Data for Gene Expression Differences

PubMed Central

Di, Yanming; Schafer, Daniel W.; Wilhelm, Larry J.; Fox, Samuel E.; Sullivan, Christopher M.; Curzon, Aron D.; Carrington, James C.; Mockler, Todd C.; Chang, Jeff H.

2011-01-01

GENE-counter is a complete Perl-based computational pipeline for analyzing RNA-Sequencing (RNA-Seq) data for differential gene expression. In addition to its use in studying transcriptomes of eukaryotic model organisms, GENE-counter is applicable for prokaryotes and non-model organisms without an available genome reference sequence. For alignments, GENE-counter is configured for CASHX, Bowtie, and BWA, but an end user can use any Sequence Alignment/Map (SAM)-compliant program of preference. To analyze data for differential gene expression, GENE-counter can be run with any one of three statistics packages that are based on variations of the negative binomial distribution. The default method is a new and simple statistical test we developed based on an over-parameterized version of the negative binomial distribution. GENE-counter also includes three different methods for assessing differentially expressed features for enriched gene ontology (GO) terms. Results are transparent and data are systematically stored in a MySQL relational database to facilitate additional analyses as well as quality assessment. We used next generation sequencing to generate a small-scale RNA-Seq dataset derived from the heavily studied defense response of Arabidopsis thaliana and used GENE-counter to process the data. Collectively, the support from analysis of microarrays as well as the observed and substantial overlap in results from each of the three statistics packages demonstrates that GENE-counter is well suited for handling the unique characteristics of small sample sizes and high variability in gene counts. PMID:21998647

Joint assembly and genetic mapping of the Atlantic horseshoe crab genome reveals ancient whole genome duplication

PubMed Central

2014-01-01

Background Horseshoe crabs are marine arthropods with a fossil record extending back approximately 450 million years. They exhibit remarkable morphological stability over their long evolutionary history, retaining a number of ancestral arthropod traits, and are often cited as examples of “living fossils.” As arthropods, they belong to the Ecdysozoa, an ancient super-phylum whose sequenced genomes (including insects and nematodes) have thus far shown more divergence from the ancestral pattern of eumetazoan genome organization than cnidarians, deuterostomes and lophotrochozoans. However, much of ecdysozoan diversity remains unrepresented in comparative genomic analyses. Results Here we apply a new strategy of combined de novo assembly and genetic mapping to examine the chromosome-scale genome organization of the Atlantic horseshoe crab, Limulus polyphemus. We constructed a genetic linkage map of this 2.7 Gbp genome by sequencing the nuclear DNA of 34 wild-collected, full-sibling embryos and their parents at a mean redundancy of 1.1x per sample. The map includes 84,307 sequence markers grouped into 1,876 distinct genetic intervals and 5,775 candidate conserved protein coding genes. Conclusions Comparison with other metazoan genomes shows that the L. polyphemus genome preserves ancestral bilaterian linkage groups, and that a common ancestor of modern horseshoe crabs underwent one or more ancient whole genome duplications 300 million years ago, followed by extensive chromosome fusion. These results provide a counter-example to the often noted correlation between whole genome duplication and evolutionary radiations. The new, low-cost genetic mapping method for obtaining a chromosome-scale view of non-model organism genomes that we demonstrate here does not require laboratory culture, and is potentially applicable to a broad range of other species. PMID:24987520

in silico Whole Genome Sequencer & Analyzer (iWGS): A Computational Pipeline to Guide the Design and Analysis of de novo Genome Sequencing Studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Xiaofan; Peris, David; Kominek, Jacek

The availability of genomes across the tree of life is highly biased toward vertebrates, pathogens, human disease models, and organisms with relatively small and simple genomes. Recent progress in genomics has enabled the de novo decoding of the genome of virtually any organism, greatly expanding its potential for understanding the biology and evolution of the full spectrum of biodiversity. The increasing diversity of sequencing technologies, assays, and de novo assembly algorithms have augmented the complexity of de novo genome sequencing projects in nonmodel organisms. To reduce the costs and challenges in de novo genome sequencing projects and streamline their experimentalmore » design and analysis, we developed iWGS (in silico Whole Genome Sequencer and Analyzer), an automated pipeline for guiding the choice of appropriate sequencing strategy and assembly protocols. iWGS seamlessly integrates the four key steps of a de novo genome sequencing project: data generation (through simulation), data quality control, de novo assembly, and assembly evaluation and validation. The last three steps can also be applied to the analysis of real data. iWGS is designed to enable the user to have great flexibility in testing the range of experimental designs available for genome sequencing projects, and supports all major sequencing technologies and popular assembly tools. Three case studies illustrate how iWGS can guide the design of de novo genome sequencing projects, and evaluate the performance of a wide variety of user-specified sequencing strategies and assembly protocols on genomes of differing architectures. iWGS, along with a detailed documentation, is freely available at https://github.com/zhouxiaofan1983/iWGS.« less

Meneco, a Topology-Based Gap-Filling Tool Applicable to Degraded Genome-Wide Metabolic Networks

PubMed Central

Prigent, Sylvain; Frioux, Clémence; Dittami, Simon M.; Larhlimi, Abdelhalim; Collet, Guillaume; Gutknecht, Fabien; Got, Jeanne; Eveillard, Damien; Bourdon, Jérémie; Plewniak, Frédéric; Tonon, Thierry; Siegel, Anne

2017-01-01

Increasing amounts of sequence data are becoming available for a wide range of non-model organisms. Investigating and modelling the metabolic behaviour of those organisms is highly relevant to understand their biology and ecology. As sequences are often incomplete and poorly annotated, draft networks of their metabolism largely suffer from incompleteness. Appropriate gap-filling methods to identify and add missing reactions are therefore required to address this issue. However, current tools rely on phenotypic or taxonomic information, or are very sensitive to the stoichiometric balance of metabolic reactions, especially concerning the co-factors. This type of information is often not available or at least prone to errors for newly-explored organisms. Here we introduce Meneco, a tool dedicated to the topological gap-filling of genome-scale draft metabolic networks. Meneco reformulates gap-filling as a qualitative combinatorial optimization problem, omitting constraints raised by the stoichiometry of a metabolic network considered in other methods, and solves this problem using Answer Set Programming. Run on several artificial test sets gathering 10,800 degraded Escherichia coli networks Meneco was able to efficiently identify essential reactions missing in networks at high degradation rates, outperforming the stoichiometry-based tools in scalability. To demonstrate the utility of Meneco we applied it to two case studies. Its application to recent metabolic networks reconstructed for the brown algal model Ectocarpus siliculosus and an associated bacterium Candidatus Phaeomarinobacter ectocarpi revealed several candidate metabolic pathways for algal-bacterial interactions. Then Meneco was used to reconstruct, from transcriptomic and metabolomic data, the first metabolic network for the microalga Euglena mutabilis. These two case studies show that Meneco is a versatile tool to complete draft genome-scale metabolic networks produced from heterogeneous data, and to suggest relevant reactions that explain the metabolic capacity of a biological system. PMID:28129330

in silico Whole Genome Sequencer & Analyzer (iWGS): A Computational Pipeline to Guide the Design and Analysis of de novo Genome Sequencing Studies

DOE PAGES

Zhou, Xiaofan; Peris, David; Kominek, Jacek; ...

2016-09-16

The availability of genomes across the tree of life is highly biased toward vertebrates, pathogens, human disease models, and organisms with relatively small and simple genomes. Recent progress in genomics has enabled the de novo decoding of the genome of virtually any organism, greatly expanding its potential for understanding the biology and evolution of the full spectrum of biodiversity. The increasing diversity of sequencing technologies, assays, and de novo assembly algorithms have augmented the complexity of de novo genome sequencing projects in nonmodel organisms. To reduce the costs and challenges in de novo genome sequencing projects and streamline their experimentalmore » design and analysis, we developed iWGS (in silico Whole Genome Sequencer and Analyzer), an automated pipeline for guiding the choice of appropriate sequencing strategy and assembly protocols. iWGS seamlessly integrates the four key steps of a de novo genome sequencing project: data generation (through simulation), data quality control, de novo assembly, and assembly evaluation and validation. The last three steps can also be applied to the analysis of real data. iWGS is designed to enable the user to have great flexibility in testing the range of experimental designs available for genome sequencing projects, and supports all major sequencing technologies and popular assembly tools. Three case studies illustrate how iWGS can guide the design of de novo genome sequencing projects, and evaluate the performance of a wide variety of user-specified sequencing strategies and assembly protocols on genomes of differing architectures. iWGS, along with a detailed documentation, is freely available at https://github.com/zhouxiaofan1983/iWGS.« less

Meneco, a Topology-Based Gap-Filling Tool Applicable to Degraded Genome-Wide Metabolic Networks.

PubMed

Prigent, Sylvain; Frioux, Clémence; Dittami, Simon M; Thiele, Sven; Larhlimi, Abdelhalim; Collet, Guillaume; Gutknecht, Fabien; Got, Jeanne; Eveillard, Damien; Bourdon, Jérémie; Plewniak, Frédéric; Tonon, Thierry; Siegel, Anne

2017-01-01

Increasing amounts of sequence data are becoming available for a wide range of non-model organisms. Investigating and modelling the metabolic behaviour of those organisms is highly relevant to understand their biology and ecology. As sequences are often incomplete and poorly annotated, draft networks of their metabolism largely suffer from incompleteness. Appropriate gap-filling methods to identify and add missing reactions are therefore required to address this issue. However, current tools rely on phenotypic or taxonomic information, or are very sensitive to the stoichiometric balance of metabolic reactions, especially concerning the co-factors. This type of information is often not available or at least prone to errors for newly-explored organisms. Here we introduce Meneco, a tool dedicated to the topological gap-filling of genome-scale draft metabolic networks. Meneco reformulates gap-filling as a qualitative combinatorial optimization problem, omitting constraints raised by the stoichiometry of a metabolic network considered in other methods, and solves this problem using Answer Set Programming. Run on several artificial test sets gathering 10,800 degraded Escherichia coli networks Meneco was able to efficiently identify essential reactions missing in networks at high degradation rates, outperforming the stoichiometry-based tools in scalability. To demonstrate the utility of Meneco we applied it to two case studies. Its application to recent metabolic networks reconstructed for the brown algal model Ectocarpus siliculosus and an associated bacterium Candidatus Phaeomarinobacter ectocarpi revealed several candidate metabolic pathways for algal-bacterial interactions. Then Meneco was used to reconstruct, from transcriptomic and metabolomic data, the first metabolic network for the microalga Euglena mutabilis. These two case studies show that Meneco is a versatile tool to complete draft genome-scale metabolic networks produced from heterogeneous data, and to suggest relevant reactions that explain the metabolic capacity of a biological system.

Red blood cells open promising avenues for longitudinal studies of ageing in laboratory, non-model and wild animals.

PubMed

Stier, Antoine; Reichert, Sophie; Criscuolo, Francois; Bize, Pierre

2015-11-01

Ageing is characterized by a progressive deterioration of multiple physiological and molecular pathways, which impair organismal performance and increase risks of death with advancing age. Hence, ageing studies must identify physiological and molecular pathways that show signs of age-related deterioration, and test their association with the risk of death and longevity. This approach necessitates longitudinal sampling of the same individuals, and therefore requires a minimally invasive sampling technique that provides access to the larger spectrum of physiological and molecular pathways that are putatively associated with ageing. The present paper underlines the interest in using red blood cells (RBCs) as a promising target for longitudinal studies of ageing in vertebrates. RBCs provide valuable information on the following six pathways: cell maintenance and turnover (RBC number, size, and heterogeneity), glucose homeostasis (RBC glycated haemoglobin), oxidative stress parameters, membrane composition and integrity, mitochondrial functioning, and telomere dynamics. The last two pathways are specific to RBCs of non-mammalian species, which possess a nucleus and functional mitochondria. We present the current knowledge about RBCs and age-dependent changes in these pathways in non-model and wild species that are especially suitable to address questions related to ageing using longitudinal studies. We discuss how the different pathways relate with survival and lifespan and give information on their genetic and environmental determinants to appraise their evolutionary potential. Copyright © 2015 Elsevier Inc. All rights reserved.

De novo transcriptome sequencing and comparative analysis of midgut tissues of four non-model insects pertaining to Hemiptera, Coleoptera, Diptera and Lepidoptera.

PubMed

Gazara, Rajesh K; Cardoso, Christiane; Bellieny-Rabelo, Daniel; Ferreira, Clélia; Terra, Walter R; Venancio, Thiago M

2017-09-05

Despite the great morphological diversity of insects, there is a regularity in their digestive functions, which is apparently related to their physiology. In the present work we report the de novo midgut transcriptomes of four non-model insects from four distinct orders: Spodoptera frugiperda (Lepidoptera), Musca domestica (Diptera), Tenebrio molitor (Coleoptera) and Dysdercus peruvianus (Hemiptera). We employed a computational strategy to merge assemblies obtained with two different algorithms, which substantially increased the quality of the final transcriptomes. Unigenes were annotated and analyzed using the eggNOG database, which allowed us to assign some level of functional and evolutionary information to 79.7% to 93.1% of the transcriptomes. We found interesting transcriptional patterns, such as: i) the intense use of lysozymes in digestive functions of M. domestica larvae, which are streamlined and adapted to feed on bacteria; ii) the up-regulation of orthologous UDP-glycosyl transferase and cytochrome P450 genes in the whole midguts different species, supporting the existence of an ancient defense frontline to counter xenobiotics; iii) evidence supporting roles for juvenile hormone binding proteins in the midgut physiology, probably as a way to activate genes that help fight anti-nutritional substances (e.g. protease inhibitors). The results presented here shed light on the digestive and structural properties of the digestive systems of these distantly related species. Furthermore, the produced datasets will also be useful for scientists studying these insects. Copyright © 2017. Published by Elsevier B.V.

Inbred or Outbred? Genetic Diversity in Laboratory Rodent Colonies

PubMed Central

Brekke, Thomas D.; Steele, Katherine A.; Mulley, John F.

2017-01-01

Nonmodel rodents are widely used as subjects for both basic and applied biological research, but the genetic diversity of the study individuals is rarely quantified. University-housed colonies tend to be small and subject to founder effects and genetic drift; so they may be highly inbred or show substantial genetic divergence from other colonies, even those derived from the same source. Disregard for the levels of genetic diversity in an animal colony may result in a failure to replicate results if a different colony is used to repeat an experiment, as different colonies may have fixed alternative variants. Here we use high throughput sequencing to demonstrate genetic divergence in three isolated colonies of Mongolian gerbil (Meriones unguiculatus) even though they were all established recently from the same source. We also show that genetic diversity in allegedly “outbred” colonies of nonmodel rodents (gerbils, hamsters, house mice, deer mice, and rats) varies considerably from nearly no segregating diversity to very high levels of polymorphism. We conclude that genetic divergence in isolated colonies may play an important role in the “replication crisis.” In a more positive light, divergent rodent colonies represent an opportunity to leverage genetically distinct individuals in genetic crossing experiments. In sum, awareness of the genetic diversity of an animal colony is paramount as it allows researchers to properly replicate experiments and also to capitalize on other genetically distinct individuals to explore the genetic basis of a trait. PMID:29242387

Rapid acquisition and model-based analysis of cell-free transcription–translation reactions from nonmodel bacteria

PubMed Central

Wienecke, Sarah; Ishwarbhai, Alka; Tsipa, Argyro; Aw, Rochelle; Kylilis, Nicolas; Bell, David J.; McClymont, David W.; Jensen, Kirsten; Biedendieck, Rebekka

2018-01-01

Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications. PMID:29666238

Monitoring the regulation of gene expression in a growing organ using a fluid mechanics formalism

PubMed Central

2010-01-01

Background Technological advances have enabled the accurate quantification of gene expression, even within single cell types. While transcriptome analyses are routinely performed, most experimental designs only provide snapshots of gene expression. Molecular mechanisms underlying cell fate or positional signalling have been revealed through these discontinuous datasets. However, in developing multicellular structures, temporal and spatial cues, known to directly influence transcriptional networks, get entangled as the cells are displaced and expand. Access to an unbiased view of the spatiotemporal regulation of gene expression occurring during development requires a specific framework that properly quantifies the rate of change of a property in a moving and expanding element, such as a cell or an organ segment. Results We show how the rate of change in gene expression can be quantified by combining kinematics and real-time polymerase chain reaction data in a mechanistic model which considers any organ as a continuum. This framework was applied in order to assess the developmental regulation of the two reference genes Actin11 and Elongation Factor 1-β in the apex of poplar root. The growth field was determined by time-lapse photography and transcript density was obtained at high spatial resolution. The net accumulation rates of the transcripts of the two genes were found to display highly contrasted developmental profiles. Actin11 showed pulses of up and down regulation in the accelerating and decelerating parts of the growth zone while the dynamic of EF1β were much slower. This framework provides key information about gene regulation in a developing organ, such as the location, the duration and the intensity of gene induction/repression. Conclusions We demonstrated that gene expression patterns can be monitored using the continuity equation without using mutants or reporter constructions. Given the rise of imaging technologies, this framework in our view opens a new way to dissect the molecular basis of growth regulation, even in non-model species or complex structures. PMID:20202192

Epigenomics in marine fishes.

PubMed

Metzger, David C H; Schulte, Patricia M

2016-12-01

Epigenetic mechanisms are an underappreciated and often ignored component of an organism's response to environmental change and may underlie many types of phenotypic plasticity. Recent technological advances in methods for detecting epigenetic marks at a whole-genome scale have launched new opportunities for studying epigenomics in ecologically relevant non-model systems. The study of ecological epigenomics holds great promise to better understand the linkages between genotype, phenotype, and the environment and to explore mechanisms of phenotypic plasticity. The many attributes of marine fish species, including their high diversity, variable life histories, high fecundity, impressive plasticity, and economic value provide unique opportunities for studying epigenetic mechanisms in an environmental context. To provide a primer on epigenomic research for fish biologists, we start by describing fundamental aspects of epigenetics, focusing on the most widely studied and most well understood of the epigenetic marks: DNA methylation. We then describe the techniques that have been used to investigate DNA methylation in marine fishes to date and highlight some new techniques that hold great promise for future studies. Epigenomic research in marine fishes is in its early stages, so we first briefly discuss what has been learned about the establishment, maintenance, and function of DNA methylation in fishes from studies in zebrafish and then summarize the studies demonstrating the pervasive effects of the environment on the epigenomes of marine fishes. We conclude by highlighting the potential for ongoing research on the epigenomics of marine fishes to reveal critical aspects of the interaction between organisms and their environments. Copyright © 2016 Elsevier B.V. All rights reserved.

Membrane Proteomic Insights into the Physiology and Taxonomy of an Oleaginous Green Microalga.

PubMed

Garibay-Hernández, Adriana; Barkla, Bronwyn J; Vera-Estrella, Rosario; Martinez, Alfredo; Pantoja, Omar

2017-01-01

Ettlia oleoabundans is a nonsequenced oleaginous green microalga. Despite the significant biotechnological interest in producing value-added compounds from the acyl lipids of this microalga, a basic understanding of the physiology and biochemistry of oleaginous microalgae is lacking, especially under nitrogen deprivation conditions known to trigger lipid accumulation. Using an RNA sequencing-based proteomics approach together with manual annotation, we are able to provide, to our knowledge, the first membrane proteome of an oleaginous microalga. This approach allowed the identification of novel proteins in E. oleoabundans, including two photoprotection-related proteins, Photosystem II Subunit S and Maintenance of Photosystem II under High Light1, which were considered exclusive to higher photosynthetic organisms, as well as Retinitis Pigmentosa Type 2-Clathrin Light Chain, a membrane protein with a novel domain architecture. Free-flow zonal electrophoresis of microalgal membranes coupled to liquid chromatography-tandem mass spectrometry proved to be a useful technique for determining the intracellular location of proteins of interest. Carbon-flow compartmentalization in E. oleoabundans was modeled using this information. Molecular phylogenetic analyses of protein markers and 18S ribosomal DNA support the reclassification of E. oleoabundans within the trebouxiophycean microalgae, rather than with the Chlorophyceae class, in which it is currently classified, indicating that it may not be closely related to the model green alga Chlamydomonas reinhardtii A detailed survey of biological processes taking place in the membranes of nitrogen-deprived E. oleoabundans, including lipid metabolism, provides insights into the basic biology of this nonmodel organism. © 2017 American Society of Plant Biologists. All Rights Reserved.

A flexible bayesian model for testing for transmission ratio distortion.

PubMed

Casellas, Joaquim; Manunza, Arianna; Mercader, Anna; Quintanilla, Raquel; Amills, Marcel

2014-12-01

Current statistical approaches to investigate the nature and magnitude of transmission ratio distortion (TRD) are scarce and restricted to the most common experimental designs such as F2 populations and backcrosses. In this article, we describe a new Bayesian approach to check TRD within a given biallelic genetic marker in a diploid species, providing a highly flexible framework that can accommodate any kind of population structure. This model relies on the genotype of each offspring and thus integrates all available information from either the parents' genotypes or population-specific allele frequencies and yields TRD estimates that can be corroborated by the calculation of a Bayes factor (BF). This approach has been evaluated on simulated data sets with appealing statistical performance. As a proof of concept, we have also tested TRD in a porcine population with five half-sib families and 352 offspring. All boars and piglets were genotyped with the Porcine SNP60 BeadChip, whereas genotypes from the sows were not available. The SNP-by-SNP screening of the pig genome revealed 84 SNPs with decisive evidences of TRD (BF > 100) after accounting for multiple testing. Many of these regions contained genes related to biological processes (e.g., nucleosome assembly and co-organization, DNA conformation and packaging, and DNA complex assembly) that are critically associated with embryonic viability. The implementation of this method, which overcomes many of the limitations of previous approaches, should contribute to fostering research on TRD in both model and nonmodel organisms. Copyright © 2014 by the Genetics Society of America.

The test skeletal matrix of the black sea urchin Arbacia lixula.

PubMed

Kanold, Julia M; Immel, Francoise; Broussard, Cédric; Guichard, Nathalie; Plasseraud, Laurent; Corneillat, Marion; Alcaraz, Gérard; Brümmer, Franz; Marin, Frédéric

2015-03-01

In the field of biomineralization, the past decade has been marked by the increasing use of high throughput techniques, i.e. proteomics, for identifying in one shot the protein content of complex macromolecular mixtures extracted from mineralized tissues. Although crowned with success, this approach has been restricted so far to a limited set of key-organisms, such as the purple sea urchin Strongylocentrotus purpuratus, the pearl oyster or the abalone, leaving in the shadow non-model organisms. As a consequence, it is still unknown to what extent the calcifying repertoire varies, from group to group, at high (phylum, class), median (order, family) or low (genus, species) taxonomic rank. The present paper shows the first biochemical and proteomic characterization of the test matrix of the Mediterranean black sea urchin Arbacia lixula (Arbacioida). Our work suggests that the skeletal repertoire of A. lixula exhibits some similarities but also several differences with that of the few sea urchin species (S. purpuratus, Paracentrotus lividus), for which molecular data are already available. The differences may be attributable to the taxonomic position of the species considered: A. lixula belongs to an order - Arbacioida - that diverged more than one hundred million years ago from the Camarodonta, which includes the two species S. purpuratus and P. lividus. For the echinoid class, we suggest that large-scale proteomic screening should be performed in order to understand which molecular functions related to calcification are conserved and which ones have been co-opted for biomineralization in particular lineages. Copyright © 2014 Elsevier Inc. All rights reserved.

Transgenerational epigenetics: Inheritance of global cytosine methylation and methylation-related epigenetic markers in the shrub Lavandula latifolia.

PubMed

Herrera, Carlos M; Alonso, Conchita; Medrano, Mónica; Pérez, Ricardo; Bazaga, Pilar

2018-04-01

The ecological and evolutionary significance of natural epigenetic variation (i.e., not based on DNA sequence variants) variation will depend critically on whether epigenetic states are transmitted from parents to offspring, but little is known on epigenetic inheritance in nonmodel plants. We present a quantitative analysis of transgenerational transmission of global DNA cytosine methylation (= proportion of all genomic cytosines that are methylated) and individual epigenetic markers (= methylation status of anonymous MSAP markers) in the shrub Lavandula latifolia. Methods based on parent-offspring correlations and parental variance component estimation were applied to epigenetic features of field-growing plants ('maternal parents') and greenhouse-grown progenies. Transmission of genetic markers (AFLP) was also assessed for reference. Maternal parents differed significantly in global DNA cytosine methylation (range = 21.7-36.7%). Greenhouse-grown maternal families differed significantly in global methylation, and their differences were significantly related to maternal origin. Methylation-sensitive amplified polymorphism (MSAP) markers exhibited significant transgenerational transmission, as denoted by significant maternal variance component of marker scores in greenhouse families and significant mother-offspring correlations of marker scores. Although transmission-related measurements for global methylation and MSAP markers were quantitatively lower than those for AFLP markers taken as reference, this study has revealed extensive transgenerational transmission of genome-wide global cytosine methylation and anonymous epigenetic markers in L. latifolia. Similarity of results for global cytosine methylation and epigenetic markers lends robustness to this conclusion, and stresses the value of considering both types of information in epigenetic studies of nonmodel plants. © 2018 Botanical Society of America.

Hemocyte Density Increases with Developmental Stage in an Immune-Challenged Forest Caterpillar

PubMed Central

Stoepler, Teresa M.; Castillo, Julio C.; Lill, John T.; Eleftherianos, Ioannis

2013-01-01

The cellular arm of the insect immune response is mediated by the activity of hemocytes. While hemocytes have been well-characterized morphologically and functionally in model insects, few studies have characterized the hemocytes of non-model insects. Further, the role of ontogeny in mediating immune response is not well understood in non-model invertebrate systems. The goals of the current study were to (1) determine the effects of caterpillar size (and age) on hemocyte density in naïve caterpillars and caterpillars challenged with non-pathogenic bacteria, and (2) characterize the hemocyte activity and diversity of cell types present in two forest caterpillars: Euclea delphinii and Lithacodes fasciola (Limacodidae). We found that although early and late instar (small and large size, respectively) naïve caterpillars had similar constitutive hemocyte densities in both species, late instar Lithacodes caterpillars injected with non-pathogenic E. coli produced more than a twofold greater density of hemocytes than those in early instars. We also found that both caterpillar species contained plasmatocytes, granulocytes and oenocytoids, all of which are found in other lepidopteran species, but lacked spherulocytes. Granulocytes and plasmatocytes were found to be strongly phagocytic in both species, but granulocytes exhibited a higher phagocytic activity than plasmatocytes. Our results strongly suggest that for at least one measure of immunological response, the production of hemocytes in response to infection, response magnitudes can increase over ontogeny. While the underlying raison d’ être for this improvement remains unclear, these findings may be useful in explaining natural patterns of stage-dependent parasitism and pathogen infection. PMID:23940679

Whole-genome sequencing approaches for conservation biology: Advantages, limitations and practical recommendations.

PubMed

Fuentes-Pardo, Angela P; Ruzzante, Daniel E

2017-10-01

Whole-genome resequencing (WGR) is a powerful method for addressing fundamental evolutionary biology questions that have not been fully resolved using traditional methods. WGR includes four approaches: the sequencing of individuals to a high depth of coverage with either unresolved or resolved haplotypes, the sequencing of population genomes to a high depth by mixing equimolar amounts of unlabelled-individual DNA (Pool-seq) and the sequencing of multiple individuals from a population to a low depth (lcWGR). These techniques require the availability of a reference genome. This, along with the still high cost of shotgun sequencing and the large demand for computing resources and storage, has limited their implementation in nonmodel species with scarce genomic resources and in fields such as conservation biology. Our goal here is to describe the various WGR methods, their pros and cons and potential applications in conservation biology. WGR offers an unprecedented marker density and surveys a wide diversity of genetic variations not limited to single nucleotide polymorphisms (e.g., structural variants and mutations in regulatory elements), increasing their power for the detection of signatures of selection and local adaptation as well as for the identification of the genetic basis of phenotypic traits and diseases. Currently, though, no single WGR approach fulfils all requirements of conservation genetics, and each method has its own limitations and sources of potential bias. We discuss proposed ways to minimize such biases. We envision a not distant future where the analysis of whole genomes becomes a routine task in many nonmodel species and fields including conservation biology. © 2017 John Wiley & Sons Ltd.

Genotyping by sequencing resolves shallow population structure to inform conservation of Chinook salmon (Oncorhynchus tshawytscha)

PubMed Central

Larson, Wesley A; Seeb, Lisa W; Everett, Meredith V; Waples, Ryan K; Templin, William D; Seeb, James E

2014-01-01

Recent advances in population genomics have made it possible to detect previously unidentified structure, obtain more accurate estimates of demographic parameters, and explore adaptive divergence, potentially revolutionizing the way genetic data are used to manage wild populations. Here, we identified 10 944 single-nucleotide polymorphisms using restriction-site-associated DNA (RAD) sequencing to explore population structure, demography, and adaptive divergence in five populations of Chinook salmon (Oncorhynchus tshawytscha) from western Alaska. Patterns of population structure were similar to those of past studies, but our ability to assign individuals back to their region of origin was greatly improved (>90% accuracy for all populations). We also calculated effective size with and without removing physically linked loci identified from a linkage map, a novel method for nonmodel organisms. Estimates of effective size were generally above 1000 and were biased downward when physically linked loci were not removed. Outlier tests based on genetic differentiation identified 733 loci and three genomic regions under putative selection. These markers and genomic regions are excellent candidates for future research and can be used to create high-resolution panels for genetic monitoring and population assignment. This work demonstrates the utility of genomic data to inform conservation in highly exploited species with shallow population structure. PMID:24665338

Comparative Transcriptomics Among Four White Pine Species.

PubMed

Baker, Ethan A G; Wegrzyn, Jill L; Sezen, Uzay U; Falk, Taylor; Maloney, Patricia E; Vogler, Detlev R; Delfino-Mix, Annette; Jensen, Camille; Mitton, Jeffry; Wright, Jessica; Knaus, Brian; Rai, Hardeep; Cronn, Richard; Gonzalez-Ibeas, Daniel; Vasquez-Gross, Hans A; Famula, Randi A; Liu, Jun-Jun; Kueppers, Lara M; Neale, David B

2018-05-04

Conifers are the dominant plant species throughout the high latitude boreal forests as well as some lower latitude temperate forests of North America, Europe, and Asia. As such, they play an integral economic and ecological role across much of the world. This study focused on the characterization of needle transcriptomes from four ecologically important and understudied North American white pines within the Pinus subgenus Strobus The populations of many Strobus species are challenged by native and introduced pathogens, native insects, and abiotic factors. RNA from the needles of western white pine ( Pinus monticola ), limber pine ( Pinus flexilis ), whitebark pine ( Pinus albicaulis) , and sugar pine ( Pinus lambertiana ) was sampled, Illumina short read sequenced, and de novo assembled. The assembled transcripts and their subsequent structural and functional annotations were processed through custom pipelines to contend with the challenges of non-model organism transcriptome validation. Orthologous gene family analysis of over 58,000 translated transcripts, implemented through Tribe-MCL, estimated the shared and unique gene space among the four species. This revealed 2025 conserved gene families, of which 408 were aligned to estimate levels of divergence and reveal patterns of selection. Specific candidate genes previously associated with drought tolerance and white pine blister rust resistance in conifers were investigated. Copyright © 2018 Baker et al.

The evaluation of anoxia responsive E2F DNA binding activity in the red eared slider turtle, Trachemys scripta elegans.

PubMed

Biggar, Kyle K; Storey, Kenneth B

2018-01-01

In many cases, the DNA-binding activity of a transcription factor does not change, while its transcriptional activity is greatly influenced by the make-up of bound proteins. In this study, we assessed the protein composition and DNA-binding ability of the E2F transcription factor complex to provide insight into cell cycle control in an anoxia tolerant turtle through the use of a modified ELISA protocol. This modification also permits the use of custom DNA probes that are tailored to a specific DNA binding region, introducing the ability to design capture probes for non-model organisms. Through the use of EMSA and ELISA DNA binding assays, we have successfully determined the in vitro DNA binding activity and complex dynamics of the Rb/E2F cell cycle regulatory mechanisms in an anoxic turtle, Trachemys scripta elegans . Repressive cell cycle proteins (E2F4, Rb, HDAC4 and Suv39H1) were found to significantly increase at E2F DNA-binding sites upon anoxic exposure in anoxic turtle liver. The lack of p130 involvement in the E2F DNA-bound complex indicates that anoxic turtle liver may maintain G 1 arrest for the duration of stress survival.

The evaluation of anoxia responsive E2F DNA binding activity in the red eared slider turtle, Trachemys scripta elegans

PubMed Central

Biggar, Kyle K.

2018-01-01

In many cases, the DNA-binding activity of a transcription factor does not change, while its transcriptional activity is greatly influenced by the make-up of bound proteins. In this study, we assessed the protein composition and DNA-binding ability of the E2F transcription factor complex to provide insight into cell cycle control in an anoxia tolerant turtle through the use of a modified ELISA protocol. This modification also permits the use of custom DNA probes that are tailored to a specific DNA binding region, introducing the ability to design capture probes for non-model organisms. Through the use of EMSA and ELISA DNA binding assays, we have successfully determined the in vitro DNA binding activity and complex dynamics of the Rb/E2F cell cycle regulatory mechanisms in an anoxic turtle, Trachemys scripta elegans. Repressive cell cycle proteins (E2F4, Rb, HDAC4 and Suv39H1) were found to significantly increase at E2F DNA-binding sites upon anoxic exposure in anoxic turtle liver. The lack of p130 involvement in the E2F DNA-bound complex indicates that anoxic turtle liver may maintain G1 arrest for the duration of stress survival. PMID:29770276

Sequence capture by hybridization to explore modern and ancient genomic diversity in model and nonmodel organisms

PubMed Central

Gasc, Cyrielle; Peyretaillade, Eric

2016-01-01

Abstract The recent expansion of next-generation sequencing has significantly improved biological research. Nevertheless, deep exploration of genomes or metagenomic samples remains difficult because of the sequencing depth and the associated costs required. Therefore, different partitioning strategies have been developed to sequence informative subsets of studied genomes. Among these strategies, hybridization capture has proven to be an innovative and efficient tool for targeting and enriching specific biomarkers in complex DNA mixtures. It has been successfully applied in numerous areas of biology, such as exome resequencing for the identification of mutations underlying Mendelian or complex diseases and cancers, and its usefulness has been demonstrated in the agronomic field through the linking of genetic variants to agricultural phenotypic traits of interest. Moreover, hybridization capture has provided access to underexplored, but relevant fractions of genomes through its ability to enrich defined targets and their flanking regions. Finally, on the basis of restricted genomic information, this method has also allowed the expansion of knowledge of nonreference species and ancient genomes and provided a better understanding of metagenomic samples. In this review, we present the major advances and discoveries permitted by hybridization capture and highlight the potency of this approach in all areas of biology. PMID:27105841

Host-Polarized Cell Growth in Animal Symbionts.

PubMed

Pende, Nika; Wang, Jinglan; Weber, Philipp M; Verheul, Jolanda; Kuru, Erkin; Rittmann, Simon K-M R; Leisch, Nikolaus; VanNieuwenhze, Michael S; Brun, Yves V; den Blaauwen, Tanneke; Bulgheresi, Silvia

2018-04-02

To determine the fundamentals of cell growth, we must extend cell biological studies to non-model organisms. Here, we investigated the growth modes of the only two rods known to widen instead of elongating, Candidatus Thiosymbion oneisti and Thiosymbion hypermnestrae. These bacteria are attached by one pole to the surface of their respective nematode hosts. By incubating live Ca. T. oneisti and T. hypermnestrae with a peptidoglycan metabolic probe, we observed that the insertion of new cell wall starts at the poles and proceeds inward, concomitantly with FtsZ-based membrane constriction. Remarkably, in Ca. T. hypermnestrae, the proximal, animal-attached pole grows before the distal, free pole, indicating that the peptidoglycan synthesis machinery is host oriented. Immunostaining of the symbionts with an antibody against the actin homolog MreB revealed that it was arranged medially-that is, parallel to the cell long axis-throughout the symbiont life cycle. Given that depolymerization of MreB abolished newly synthesized peptidoglycan insertion and impaired divisome assembly, we conclude that MreB function is required for symbiont widening and division. In conclusion, our data invoke a reassessment of the localization and function of the bacterial actin homolog. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Sequence capture by hybridization to explore modern and ancient genomic diversity in model and nonmodel organisms.

PubMed

Gasc, Cyrielle; Peyretaillade, Eric; Peyret, Pierre

2016-06-02

The recent expansion of next-generation sequencing has significantly improved biological research. Nevertheless, deep exploration of genomes or metagenomic samples remains difficult because of the sequencing depth and the associated costs required. Therefore, different partitioning strategies have been developed to sequence informative subsets of studied genomes. Among these strategies, hybridization capture has proven to be an innovative and efficient tool for targeting and enriching specific biomarkers in complex DNA mixtures. It has been successfully applied in numerous areas of biology, such as exome resequencing for the identification of mutations underlying Mendelian or complex diseases and cancers, and its usefulness has been demonstrated in the agronomic field through the linking of genetic variants to agricultural phenotypic traits of interest. Moreover, hybridization capture has provided access to underexplored, but relevant fractions of genomes through its ability to enrich defined targets and their flanking regions. Finally, on the basis of restricted genomic information, this method has also allowed the expansion of knowledge of nonreference species and ancient genomes and provided a better understanding of metagenomic samples. In this review, we present the major advances and discoveries permitted by hybridization capture and highlight the potency of this approach in all areas of biology. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Toward an Integrative Understanding of Social Behavior: New Models and New Opportunities

PubMed Central

Blumstein, Daniel T.; Ebensperger, Luis A.; Hayes, Loren D.; Vásquez, Rodrigo A.; Ahern, Todd H.; Burger, Joseph Robert; Dolezal, Adam G.; Dosmann, Andy; González-Mariscal, Gabriela; Harris, Breanna N.; Herrera, Emilio A.; Lacey, Eileen A.; Mateo, Jill; McGraw, Lisa A.; Olazábal, Daniel; Ramenofsky, Marilyn; Rubenstein, Dustin R.; Sakhai, Samuel A.; Saltzman, Wendy; Sainz-Borgo, Cristina; Soto-Gamboa, Mauricio; Stewart, Monica L.; Wey, Tina W.; Wingfield, John C.; Young, Larry J.

2010-01-01

Social interactions among conspecifics are a fundamental and adaptively significant component of the biology of numerous species. Such interactions give rise to group living as well as many of the complex forms of cooperation and conflict that occur within animal groups. Although previous conceptual models have focused on the ecological causes and fitness consequences of variation in social interactions, recent developments in endocrinology, neuroscience, and molecular genetics offer exciting opportunities to develop more integrated research programs that will facilitate new insights into the physiological causes and consequences of social variation. Here, we propose an integrative framework of social behavior that emphasizes relationships between ultimate-level function and proximate-level mechanism, thereby providing a foundation for exploring the full diversity of factors that underlie variation in social interactions, and ultimately sociality. In addition to identifying new model systems for the study of human psychopathologies, this framework provides a mechanistic basis for predicting how social behavior will change in response to environmental variation. We argue that the study of non-model organisms is essential for implementing this integrative model of social behavior because such species can be studied simultaneously in the lab and field, thereby allowing integration of rigorously controlled experimental manipulations with detailed observations of the ecological contexts in which interactions among conspecifics occur. PMID:20661457

Chloroplast genomes of Arabidopsis halleri ssp. gemmifera and Arabidopsis lyrata ssp. petraea: Structures and comparative analysis.

PubMed

Asaf, Sajjad; Khan, Abdul Latif; Khan, Muhammad Aaqil; Waqas, Muhammad; Kang, Sang-Mo; Yun, Byung-Wook; Lee, In-Jung

2017-08-08

We investigated the complete chloroplast (cp) genomes of non-model Arabidopsis halleri ssp. gemmifera and Arabidopsis lyrata ssp. petraea using Illumina paired-end sequencing to understand their genetic organization and structure. Detailed bioinformatics analysis revealed genome sizes of both subspecies ranging between 154.4~154.5 kbp, with a large single-copy region (84,197~84,158 bp), a small single-copy region (17,738~17,813 bp) and pair of inverted repeats (IRa/IRb; 26,264~26,259 bp). Both cp genomes encode 130 genes, including 85 protein-coding genes, eight ribosomal RNA genes and 37 transfer RNA genes. Whole cp genome comparison of A. halleri ssp. gemmifera and A. lyrata ssp. petraea, along with ten other Arabidopsis species, showed an overall high degree of sequence similarity, with divergence among some intergenic spacers. The location and distribution of repeat sequences were determined, and sequence divergences of shared genes were calculated among related species. Comparative phylogenetic analysis of the entire genomic data set and 70 shared genes between both cp genomes confirmed the previous phylogeny and generated phylogenetic trees with the same topologies. The sister species of A. halleri ssp. gemmifera is A. umezawana, whereas the closest relative of A. lyrata spp. petraea is A. arenicola.

Epigenetic differentiation persists after male gametogenesis in natural populations of the perennial herb Helleborus foetidus (Ranunculaceae).

PubMed

Herrera, Carlos M; Medrano, Mónica; Bazaga, Pilar

2013-01-01

Despite the importance of assessing the stability of epigenetic variation in non-model organisms living in real-world scenarios, no studies have been conducted on the transgenerational persistence of epigenetic structure in wild plant populations. This gap in knowledge is hindering progress in the interpretation of natural epigenetic variation. By applying the methylation-sensitive amplified fragment length polymorphism (MSAP) technique to paired plant-pollen (i.e., sporophyte-male gametophyte) DNA samples, and then comparing methylation patterns and epigenetic population differentiation in sporophytes and their descendant gametophytes, we investigated transgenerational constancy of epigenetic structure in three populations of the perennial herb Helleborus foetidus (Ranunculaceae). Single-locus and multilocus analyses revealed extensive epigenetic differentiation between sporophyte populations. Locus-by-locus comparisons of methylation status in individual sporophytes and descendant gametophytes showed that ~75% of epigenetic markers persisted unchanged through gametogenesis. In spite of some epigenetic reorganization taking place during gametogenesis, multilocus epigenetic differentiation between sporophyte populations was preserved in the subsequent gametophyte stage. In addition to illustrating the efficacy of applying the MSAP technique to paired plant-pollen DNA samples to investigate epigenetic gametic inheritance in wild plants, this paper suggests that epigenetic differentiation between adult plant populations of H. foetidus is likely to persist across generations.

Epigenetic Differentiation Persists after Male Gametogenesis in Natural Populations of the Perennial Herb Helleborus foetidus (Ranunculaceae)

PubMed Central

Herrera, Carlos M.; Medrano, Mónica; Bazaga, Pilar

2013-01-01

Despite the importance of assessing the stability of epigenetic variation in non-model organisms living in real-world scenarios, no studies have been conducted on the transgenerational persistence of epigenetic structure in wild plant populations. This gap in knowledge is hindering progress in the interpretation of natural epigenetic variation. By applying the methylation-sensitive amplified fragment length polymorphism (MSAP) technique to paired plant-pollen (i.e., sporophyte-male gametophyte) DNA samples, and then comparing methylation patterns and epigenetic population differentiation in sporophytes and their descendant gametophytes, we investigated transgenerational constancy of epigenetic structure in three populations of the perennial herb Helleborus foetidus (Ranunculaceae). Single-locus and multilocus analyses revealed extensive epigenetic differentiation between sporophyte populations. Locus-by-locus comparisons of methylation status in individual sporophytes and descendant gametophytes showed that ∼75% of epigenetic markers persisted unchanged through gametogenesis. In spite of some epigenetic reorganization taking place during gametogenesis, multilocus epigenetic differentiation between sporophyte populations was preserved in the subsequent gametophyte stage. In addition to illustrating the efficacy of applying the MSAP technique to paired plant-pollen DNA samples to investigate epigenetic gametic inheritance in wild plants, this paper suggests that epigenetic differentiation between adult plant populations of H. foetidus is likely to persist across generations. PMID:23936245

Evolution of eumetazoan nervous systems: insights from cnidarians.

PubMed

Kelava, Iva; Rentzsch, Fabian; Technau, Ulrich

2015-12-19

Cnidarians, the sister group to bilaterians, have a simple diffuse nervous system. This morphological simplicity and their phylogenetic position make them a crucial group in the study of the evolution of the nervous system. The development of their nervous systems is of particular interest, as by uncovering the genetic programme that underlies it, and comparing it with the bilaterian developmental programme, it is possible to make assumptions about the genes and processes involved in the development of ancestral nervous systems. Recent advances in sequencing methods, genetic interference techniques and transgenic technology have enabled us to get a first glimpse into the molecular network underlying the development of a cnidarian nervous system-in particular the nervous system of the anthozoan Nematostella vectensis. It appears that much of the genetic network of the nervous system development is partly conserved between cnidarians and bilaterians, with Wnt and bone morphogenetic protein (BMP) signalling, and Sox genes playing a crucial part in the differentiation of neurons. However, cnidarians possess some specific characteristics, and further studies are necessary to elucidate the full regulatory network. The work on cnidarian neurogenesis further accentuates the need to study non-model organisms in order to gain insights into processes that shaped present-day lineages during the course of evolution. © 2015 The Authors.

The Global Genome Biodiversity Network (GGBN) Data Standard specification.

PubMed

Droege, G; Barker, K; Seberg, O; Coddington, J; Benson, E; Berendsohn, W G; Bunk, B; Butler, C; Cawsey, E M; Deck, J; Döring, M; Flemons, P; Gemeinholzer, B; Güntsch, A; Hollowell, T; Kelbert, P; Kostadinov, I; Kottmann, R; Lawlor, R T; Lyal, C; Mackenzie-Dodds, J; Meyer, C; Mulcahy, D; Nussbeck, S Y; O'Tuama, É; Orrell, T; Petersen, G; Robertson, T; Söhngen, C; Whitacre, J; Wieczorek, J; Yilmaz, P; Zetzsche, H; Zhang, Y; Zhou, X

2016-01-01

Genomic samples of non-model organisms are becoming increasingly important in a broad range of studies from developmental biology, biodiversity analyses, to conservation. Genomic sample definition, description, quality, voucher information and metadata all need to be digitized and disseminated across scientific communities. This information needs to be concise and consistent in today's ever-increasing bioinformatic era, for complementary data aggregators to easily map databases to one another. In order to facilitate exchange of information on genomic samples and their derived data, the Global Genome Biodiversity Network (GGBN) Data Standard is intended to provide a platform based on a documented agreement to promote the efficient sharing and usage of genomic sample material and associated specimen information in a consistent way. The new data standard presented here build upon existing standards commonly used within the community extending them with the capability to exchange data on tissue, environmental and DNA sample as well as sequences. The GGBN Data Standard will reveal and democratize the hidden contents of biodiversity biobanks, for the convenience of everyone in the wider biobanking community. Technical tools exist for data providers to easily map their databases to the standard.Database URL: http://terms.tdwg.org/wiki/GGBN_Data_Standard. © The Author(s) 2016. Published by Oxford University Press.

Transcriptional regulation of brain gene expression in response to a territorial intrusion

PubMed Central

Sanogo, Yibayiri O.; Band, Mark; Blatti, Charles; Sinha, Saurabh; Bell, Alison M.

2012-01-01

Aggressive behaviour associated with territorial defence is widespread and has fitness consequences. However, excess aggression can interfere with other important biological functions such as immunity and energy homeostasis. How the expression of complex behaviours such as aggression is regulated in the brain has long intrigued ethologists, but has only recently become amenable for molecular dissection in non-model organisms. We investigated the transcriptomic response to territorial intrusion in four brain regions in breeding male threespined sticklebacks using expression microarrays and quantitative polymerase chain reaction (qPCR). Each region of the brain had a distinct genomic response to a territorial challenge. We identified a set of genes that were upregulated in the diencephalon and downregulated in the cerebellum and the brain stem. Cis-regulatory network analysis suggested transcription factors that regulated or co-regulated genes that were consistently regulated in all brain regions and others that regulated gene expression in opposing directions across brain regions. Our results support the hypothesis that territorial animals respond to social challenges via transcriptional regulation of genes in different brain regions. Finally, we found a remarkably close association between gene expression and aggressive behaviour at the individual level. This study sheds light on the molecular mechanisms in the brain that underlie the response to social challenges. PMID:23097509

Transcriptome analysis in non-model species: a new method for the analysis of heterologous hybridization on microarrays

PubMed Central

2010-01-01

Background Recent developments in high-throughput methods of analyzing transcriptomic profiles are promising for many areas of biology, including ecophysiology. However, although commercial microarrays are available for most common laboratory models, transcriptome analysis in non-traditional model species still remains a challenge. Indeed, the signal resulting from heterologous hybridization is low and difficult to interpret because of the weak complementarity between probe and target sequences, especially when no microarray dedicated to a genetically close species is available. Results We show here that transcriptome analysis in a species genetically distant from laboratory models is made possible by using MAXRS, a new method of analyzing heterologous hybridization on microarrays. This method takes advantage of the design of several commercial microarrays, with different probes targeting the same transcript. To illustrate and test this method, we analyzed the transcriptome of king penguin pectoralis muscle hybridized to Affymetrix chicken microarrays, two organisms separated by an evolutionary distance of approximately 100 million years. The differential gene expression observed between different physiological situations computed by MAXRS was confirmed by real-time PCR on 10 genes out of 11 tested. Conclusions MAXRS appears to be an appropriate method for gene expression analysis under heterologous hybridization conditions. PMID:20509979

Native xylose-inducible promoter expands the genetic tools for the biomass-degrading, extremely thermophilic bacterium Caldicellulosiruptor bescii.

PubMed

Williams-Rhaesa, Amanda M; Awuku, Nanaakua K; Lipscomb, Gina L; Poole, Farris L; Rubinstein, Gabriel M; Conway, Jonathan M; Kelly, Robert M; Adams, Michael W W

2018-07-01

Regulated control of both homologous and heterologous gene expression is essential for precise genetic manipulation and metabolic engineering of target microorganisms. However, there are often no options available for inducible promoters when working with non-model microorganisms. These include extremely thermophilic, cellulolytic bacteria that are of interest for renewable lignocellulosic conversion to biofuels and chemicals. In fact, improvements to the genetic systems in these organisms often cease once transformation is achieved. This present study expands the tools available for genetically engineering Caldicellulosiruptor bescii, the most thermophilic cellulose-degrader known growing up to 90 °C on unpretreated plant biomass. A native xylose-inducible (P xi ) promoter was utilized to control the expression of the reporter gene (ldh) encoding lactate dehydrogenase. The P xi -ldh construct resulted in a both increased ldh expression (20-fold higher) and lactate dehydrogenase activity (32-fold higher) in the presence of xylose compared to when glucose was used as a substrate. Finally, lactate production during growth of the recombinant C. bescii strain was proportional to the initial xylose concentration, showing that tunable expression of genes is now possible using this xylose-inducible system. This study represents a major step in the use of C. bescii as a potential platform microorganism for biotechnological applications using renewable biomass.

The genetic basis of white tigers.

PubMed

Xu, Xiao; Dong, Gui-Xin; Hu, Xue-Song; Miao, Lin; Zhang, Xue-Li; Zhang, De-Lu; Yang, Han-Dong; Zhang, Tian-You; Zou, Zheng-Ting; Zhang, Ting-Ting; Zhuang, Yan; Bhak, Jong; Cho, Yun Sung; Dai, Wen-Tao; Jiang, Tai-Jiao; Xie, Can; Li, Ruiqiang; Luo, Shu-Jin

2013-06-03

The white tiger, an elusive Bengal tiger (Panthera tigris tigris) variant with white fur and dark stripes, has fascinated humans for centuries ever since its discovery in the jungles of India. Many white tigers in captivity are inbred in order to maintain this autosomal recessive trait and consequently suffer some health problems, leading to the controversial speculation that the white tiger mutation is perhaps a genetic defect. However, the genetic basis of this phenotype remains unknown. Here, we conducted genome-wide association mapping with restriction-site-associated DNA sequencing (RAD-seq) in a pedigree of 16 captive tigers segregating at the putative white locus, followed by whole-genome sequencing (WGS) of the three parents. Validation in 130 unrelated tigers identified the causative mutation to be an amino acid change (A477V) in the transporter protein SLC45A2. Three-dimensional homology modeling suggests that the substitution may partially block the transporter channel cavity and thus affect melanogenesis. We demonstrate the feasibility of combining RAD-seq and WGS to rapidly map exotic variants in nonmodel organisms. Our results identify the basis of the longstanding white tiger mystery as the same gene underlying color variation in human, horse, and chicken and highlight its significance as part of the species' natural polymorphism that is viable in the wild. Copyright © 2013 Elsevier Ltd. All rights reserved.

Advanced technologies for genetically manipulating the silkworm Bombyx mori, a model Lepidopteran insect

PubMed Central

Xu, Hanfu; O'Brochta, David A.

2015-01-01

Genetic technologies based on transposon-mediated transgenesis along with several recently developed genome-editing technologies have become the preferred methods of choice for genetically manipulating many organisms. The silkworm, Bombyx mori, is a Lepidopteran insect of great economic importance because of its use in silk production and because it is a valuable model insect that has greatly enhanced our understanding of the biology of insects, including many agricultural pests. In the past 10 years, great advances have been achieved in the development of genetic technologies in B. mori, including transposon-based technologies that rely on piggyBac-mediated transgenesis and genome-editing technologies that rely on protein- or RNA-guided modification of chromosomes. The successful development and application of these technologies has not only facilitated a better understanding of B. mori and its use as a silk production system, but also provided valuable experiences that have contributed to the development of similar technologies in non-model insects. This review summarizes the technologies currently available for use in B. mori, their application to the study of gene function and their use in genetically modifying B. mori for biotechnology applications. The challenges, solutions and future prospects associated with the development and application of genetic technologies in B. mori are also discussed. PMID:26108630

Making Olympic lizards: the effects of specialised exercise training on performance.

PubMed

Husak, Jerry F; Keith, Allison R; Wittry, Beth N

2015-03-01

Exercise training is well known to affect a suite of physiological and performance traits in mammals, but effects of training in other vertebrate tetrapod groups have been inconsistent. We examined performance and physiological differences among green anole lizards (Anolis carolinensis) that were trained for sprinting or endurance, using an increasingly rigorous training regimen over 8 weeks. Lizards trained for endurance had significantly higher post-training endurance capacity compared with the other treatment groups, but groups did not show post-training differences in sprint speed. Although acclimation to the laboratory environment and training explain some of our results, mechanistic explanations for these results correspond with the observed performance differences. After training, endurance-trained lizards had higher haematocrit and larger fast glycolytic muscle fibres. Despite no detectable change in maximal performance of sprint-trained lizards, we detected that they had significantly larger slow oxidative muscle fibre areas compared with the other treatments. Treatment groups did not differ in the proportion of number of fibre types, nor in the mass of most limb muscles or the heart. Our results offer some caveats for investigators conducting training research on non-model organisms and they reveal that muscle plasticity in response to training may be widespread phylogenetically. © 2015. Published by The Company of Biologists Ltd.

Transcriptome analysis of 20 taxonomically related benzylisoquinoline alkaloid-producing plants.

PubMed

Hagel, Jillian M; Morris, Jeremy S; Lee, Eun-Jeong; Desgagné-Penix, Isabel; Bross, Crystal D; Chang, Limei; Chen, Xue; Farrow, Scott C; Zhang, Ye; Soh, Jung; Sensen, Christoph W; Facchini, Peter J

2015-09-18

Benzylisoquinoline alkaloids (BIAs) represent a diverse class of plant specialized metabolites sharing a common biosynthetic origin beginning with tyrosine. Many BIAs have potent pharmacological activities, and plants accumulating them boast long histories of use in traditional medicine and cultural practices. The decades-long focus on a select number of plant species as model systems has allowed near or full elucidation of major BIA pathways, including those of morphine, sanguinarine and berberine. However, this focus has created a dearth of knowledge surrounding non-model species, which also are known to accumulate a wide-range of BIAs but whose biosynthesis is thus far entirely unexplored. Further, these non-model species represent a rich source of catalyst diversity valuable to plant biochemists and emerging synthetic biology efforts. In order to access the genetic diversity of non-model plants accumulating BIAs, we selected 20 species representing 4 families within the Ranunculales. RNA extracted from each species was processed for analysis by both 1) Roche GS-FLX Titanium and 2) Illumina GA/HiSeq platforms, generating a total of 40 deep-sequencing transcriptome libraries. De novo assembly, annotation and subsequent full-length coding sequence (CDS) predictions indicated greater success for most species using the Illumina-based platform. Assembled data for each transcriptome were deposited into an established web-based BLAST portal ( www.phytometasyn.ca) to allow public access. Homology-based mining of libraries using BIA-biosynthetic enzymes as queries yielded ~850 gene candidates potentially involved in alkaloid biosynthesis. Expression analysis of these candidates was performed using inter-library FPKM normalization methods. These expression data provide a basis for the rational selection of gene candidates, and suggest possible metabolic bottlenecks within BIA metabolism. Phylogenetic analysis was performed for each of 15 different enzyme/protein groupings, highlighting many novel genes with potential involvement in the formation of one or more alkaloid types, including morphinan, aporphine, and phthalideisoquinoline alkaloids. Transcriptome resources were used to design and execute a case study of candidate N-methyltransferases (NMTs) from Glaucium flavum, which revealed predicted and novel enzyme activities. This study establishes an essential resource for the isolation and discovery of 1) functional homologues and 2) entirely novel catalysts within BIA metabolism. Functional analysis of G. flavum NMTs demonstrated the utility of this resource and underscored the importance of empirical determination of proposed enzymatic function. Publically accessible, fully annotated, BLAST-accessible transcriptomes were not previously available for most species included in this report, despite the rich repertoire of bioactive alkaloids found in these plants and their importance to traditional medicine. The results presented herein provide essential sequence information and inform experimental design for the continued elucidation of BIA metabolism.

High-throughput transcriptome sequencing and preliminary functional analysis in four Neotropical tree species.

PubMed

Brousseau, Louise; Tinaut, Alexandra; Duret, Caroline; Lang, Tiange; Garnier-Gere, Pauline; Scotti, Ivan

2014-03-27

The Amazonian rainforest is predicted to suffer from ongoing environmental changes. Despite the need to evaluate the impact of such changes on tree genetic diversity, we almost entirely lack genomic resources. In this study, we analysed the transcriptome of four tropical tree species (Carapa guianensis, Eperua falcata, Symphonia globulifera and Virola michelii) with contrasting ecological features, belonging to four widespread botanical families (respectively Meliaceae, Fabaceae, Clusiaceae and Myristicaceae). We sequenced cDNA libraries from three organs (leaves, stems, and roots) using 454 pyrosequencing. We have developed an R and bioperl-based bioinformatic procedure for de novo assembly, gene functional annotation and marker discovery. Mismatch identification takes into account single-base quality values as well as the likelihood of false variants as a function of contig depth and number of sequenced chromosomes. Between 17103 (for Symphonia globulifera) and 23390 (for Eperua falcata) contigs were assembled. Organs varied in the numbers of unigenes they apparently express, with higher number in roots. Patterns of gene expression were similar across species, with metabolism of aromatic compounds standing out as an overrepresented gene function. Transcripts corresponding to several gene functions were found to be over- or underrepresented in each organ. We identified between 4434 (for Symphonia globulifera) and 9076 (for Virola surinamensis) well-supported mismatches. The resulting overall mismatch density was comprised between 0.89 (S. globulifera) and 1.05 (V. surinamensis) mismatches/100 bp in variation-containing contigs. The relative representation of gene functions in the four transcriptomes suggests that secondary metabolism may be particularly important in tropical trees. The differential representation of transcripts among tissues suggests differential gene expression, which opens the way to functional studies in these non-model, ecologically important species. We found substantial amounts of mismatches in the four species. These newly identified putative variants are a first step towards acquiring much needed genomic resources for tropical tree species.

The aggregate site frequency spectrum for comparative population genomic inference.

PubMed

Xue, Alexander T; Hickerson, Michael J

2015-12-01

Understanding how assemblages of species responded to past climate change is a central goal of comparative phylogeography and comparative population genomics, an endeavour that has increasing potential to integrate with community ecology. New sequencing technology now provides the potential to perform complex demographic inference at unprecedented resolution across assemblages of nonmodel species. To this end, we introduce the aggregate site frequency spectrum (aSFS), an expansion of the site frequency spectrum to use single nucleotide polymorphism (SNP) data sets collected from multiple, co-distributed species for assemblage-level demographic inference. We describe how the aSFS is constructed over an arbitrary number of independent population samples and then demonstrate how the aSFS can differentiate various multispecies demographic histories under a wide range of sampling configurations while allowing effective population sizes and expansion magnitudes to vary independently. We subsequently couple the aSFS with a hierarchical approximate Bayesian computation (hABC) framework to estimate degree of temporal synchronicity in expansion times across taxa, including an empirical demonstration with a data set consisting of five populations of the threespine stickleback (Gasterosteus aculeatus). Corroborating what is generally understood about the recent postglacial origins of these populations, the joint aSFS/hABC analysis strongly suggests that the stickleback data are most consistent with synchronous expansion after the Last Glacial Maximum (posterior probability = 0.99). The aSFS will have general application for multilevel statistical frameworks to test models involving assemblages and/or communities, and as large-scale SNP data from nonmodel species become routine, the aSFS expands the potential for powerful next-generation comparative population genomic inference. © 2015 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

Linkage disequilibrium network analysis (LDna) gives a global view of chromosomal inversions, local adaptation and geographic structure.

PubMed

Kemppainen, Petri; Knight, Christopher G; Sarma, Devojit K; Hlaing, Thaung; Prakash, Anil; Maung Maung, Yan Naung; Somboon, Pradya; Mahanta, Jagadish; Walton, Catherine

2015-09-01

Recent advances in sequencing allow population-genomic data to be generated for virtually any species. However, approaches to analyse such data lag behind the ability to generate it, particularly in nonmodel species. Linkage disequilibrium (LD, the nonrandom association of alleles from different loci) is a highly sensitive indicator of many evolutionary phenomena including chromosomal inversions, local adaptation and geographical structure. Here, we present linkage disequilibrium network analysis (LDna), which accesses information on LD shared between multiple loci genomewide. In LD networks, vertices represent loci, and connections between vertices represent the LD between them. We analysed such networks in two test cases: a new restriction-site-associated DNA sequence (RAD-seq) data set for Anopheles baimaii, a Southeast Asian malaria vector; and a well-characterized single nucleotide polymorphism (SNP) data set from 21 three-spined stickleback individuals. In each case, we readily identified five distinct LD network clusters (single-outlier clusters, SOCs), each comprising many loci connected by high LD. In A. baimaii, further population-genetic analyses supported the inference that each SOC corresponds to a large inversion, consistent with previous cytological studies. For sticklebacks, we inferred that each SOC was associated with a distinct evolutionary phenomenon: two chromosomal inversions, local adaptation, population-demographic history and geographic structure. LDna is thus a useful exploratory tool, able to give a global overview of LD associated with diverse evolutionary phenomena and identify loci potentially involved. LDna does not require a linkage map or reference genome, so it is applicable to any population-genomic data set, making it especially valuable for nonmodel species. © 2015 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

Gene expression divergence and nucleotide differentiation between males of different color morphs and mating strategies in the ruff

PubMed Central

Ekblom, Robert; Farrell, Lindsay L; Lank, David B; Burke, Terry

2012-01-01

By next generation transcriptome sequencing, it is possible to obtain data on both nucleotide sequence variation and gene expression. We have used this approach (RNA-Seq) to investigate the genetic basis for differences in plumage coloration and mating strategies in a non-model bird species, the ruff (Philomachus pugnax). Ruff males show enormous variation in the coloration of ornamental feathers, used for individual recognition. This polymorphism is linked to reproductive strategies, with dark males (Independents) defending territories on leks against other Independents, whereas white morphs (Satellites) co-occupy Independent's courts without agonistic interactions. Previous work found a strong genetic component for mating strategy, but the genes involved were not identified. We present feather transcriptome data of more than 6,000 de-novo sequenced ruff genes (although with limited coverage for many of them). None of the identified genes showed significant expression divergence between males, but many genetic markers showed nucleotide differentiation between different color morphs and mating strategies. These include several feather keratin genes, splicing factors, and the Xg blood-group gene. Many of the genes with significant genetic structure between mating strategies have not yet been annotated and their functions remain to be elucidated. We also conducted in-depth investigations of 28 pre-identified coloration candidate genes. Two of these (EDNRB and TYR) were specifically expressed in black- and rust-colored males, respectively. We have demonstrated the utility of next generation transcriptome sequencing for identifying and genotyping large number of genetic markers in a non-model species without previous genomic resources, and highlight the potential of this approach for addressing the genetic basis of ecologically important variation. PMID:23145334

Colonization and diversification of aquatic insects on three Macaronesian archipelagos using 59 nuclear loci derived from a draft genome.

PubMed

Rutschmann, Sereina; Detering, Harald; Simon, Sabrina; Funk, David H; Gattolliat, Jean-Luc; Hughes, Samantha J; Raposeiro, Pedro M; DeSalle, Rob; Sartori, Michel; Monaghan, Michael T

2017-02-01

The study of processes driving diversification requires a fully sampled and well resolved phylogeny, although a lack of phylogenetic markers remains a limitation for many non-model groups. Multilocus approaches to the study of recent diversification provide a powerful means to study the evolutionary process, but their application remains restricted because multiple unlinked loci with suitable variation for phylogenetic or coalescent analysis are not available for most non-model taxa. Here we identify novel, putative single-copy nuclear DNA (nDNA) phylogenetic markers to study the colonization and diversification of an aquatic insect species complex, Cloeon dipterum L. 1761 (Ephemeroptera: Baetidae), in Macaronesia. Whole-genome sequencing data from one member of the species complex were used to identify 59 nDNA loci (32,213 base pairs), followed by Sanger sequencing of 29 individuals sampled from 13 islands of three Macaronesian archipelagos. Multispecies coalescent analyses established six putative species. Three island species formed a monophyletic clade, with one species occurring on the Azores, Europe and North America. Ancestral state reconstruction indicated at least two colonization events from the mainland (to the Canaries, respectively Azores) and one within the archipelago (between Madeira and the Canaries). Random subsets of the 59 loci showed a positive linear relationship between number of loci and node support. In contrast, node support in the multispecies coalescent tree was negatively correlated with mean number of phylogenetically informative sites per locus, suggesting a complex relationship between tree resolution and marker variability. Our approach highlights the value of combining genomics, coalescent-based phylogeography, species delimitation, and phylogenetic reconstruction to resolve recent diversification events in an archipelago species complex. Copyright © 2016 Elsevier Inc. All rights reserved.

A Single Transcriptome of a Green Toad (Bufo viridis) Yields Candidate Genes for Sex Determination and -Differentiation and Non-Anonymous Population Genetic Markers

PubMed Central

Gerchen, Jörn F.; Reichert, Samuel J.; Röhr, Johannes T.; Dieterich, Christoph; Kloas, Werner

2016-01-01

Large genome size, including immense repetitive and non-coding fractions, still present challenges for capacity, bioinformatics and thus affordability of whole genome sequencing in most amphibians. Here, we test the performance of a single transcriptome to understand whether it can provide a cost-efficient resource for species with large unknown genomes. Using RNA from six different tissues from a single Palearctic green toad (Bufo viridis) specimen and Hiseq2000, we obtained 22,5 Mio reads and publish >100,000 unigene sequences. To evaluate efficacy and quality, we first use this data to identify green toad specific candidate genes, known from other vertebrates for their role in sex determination and differentiation. Of a list of 37 genes, the transcriptome yielded 32 (87%), many of which providing the first such data for this non-model anuran species. However, for many of these genes, only fragments could be retrieved. In order to allow also applications to population genetics, we further used the transcriptome for the targeted development of 21 non-anonymous microsatellites and tested them in genetic families and backcrosses. Eleven markers were specifically developed to be located on the B. viridis sex chromosomes; for eight markers we can indeed demonstrate sex-specific transmission in genetic families. Depending on phylogenetic distance, several markers, which are sex-linked in green toads, show high cross-amplification success across the anuran phylogeny, involving nine systematic anuran families. Our data support the view that single transcriptome sequencing (based on multiple tissues) provides a reliable genomic resource and cost-efficient method for non-model amphibian species with large genome size and, despite limitations, should be considered as long as genome sequencing remains unaffordable for most species. PMID:27232626

Beyond an AFLP genome scan towards the identification of immune genes involved in plague resistance in Rattus rattus from Madagascar.

PubMed

Tollenaere, C; Jacquet, S; Ivanova, S; Loiseau, A; Duplantier, J-M; Streiff, R; Brouat, C

2013-01-01

Genome scans using amplified fragment length polymorphism (AFLP) markers became popular in nonmodel species within the last 10 years, but few studies have tried to characterize the anonymous outliers identified. This study follows on from an AFLP genome scan in the black rat (Rattus rattus), the reservoir of plague (Yersinia pestis infection) in Madagascar. We successfully sequenced 17 of the 22 markers previously shown to be potentially affected by plague-mediated selection and associated with a plague resistance phenotype. Searching these sequences in the genome of the closely related species Rattus norvegicus assigned them to 14 genomic regions, revealing a random distribution of outliers in the genome (no clustering). We compared these results with those of an in silico AFLP study of the R. norvegicus genome, which showed that outlier sequences could not have been inferred by this method in R. rattus (only four of the 15 sequences were predicted). However, in silico analysis allowed the prediction of AFLP markers distribution and the estimation of homoplasy rates, confirming its potential utility for designing AFLP studies in nonmodel species. The 14 genomic regions surrounding AFLP outliers (less than 300 kb from the marker) contained 75 genes encoding proteins of known function, including nine involved in immune function and pathogen defence. We identified the two interleukin 1 genes (Il1a and Il1b) that share homology with an antigen of Y. pestis, as the best candidates for genes subject to plague-mediated natural selection. At least six other genes known to be involved in proinflammatory pathways may also be affected by plague-mediated selection. © 2012 Blackwell Publishing Ltd.

De novo transcriptome sequencing and digital gene expression analysis predict biosynthetic pathway of rhynchophylline and isorhynchophylline from Uncaria rhynchophylla, a non-model plant with potent anti-alzheimer's properties.

PubMed

Guo, Qianqian; Ma, Xiaojun; Wei, Shugen; Qiu, Deyou; Wilson, Iain W; Wu, Peng; Tang, Qi; Liu, Lijun; Dong, Shoukun; Zu, Wei

2014-08-12

The major medicinal alkaloids isolated from Uncaria rhynchophylla (gouteng in chinese) capsules are rhynchophylline (RIN) and isorhynchophylline (IRN). Extracts containing these terpene indole alkaloids (TIAs) can inhibit the formation and destabilize preformed fibrils of amyloid β protein (a pathological marker of Alzheimer's disease), and have been shown to improve the cognitive function of mice with Alzheimer-like symptoms. The biosynthetic pathways of RIN and IRN are largely unknown. In this study, RNA-sequencing of pooled Uncaria capsules RNA samples taken at three developmental stages that accumulate different amount of RIN and IRN was performed. More than 50 million high-quality reads from a cDNA library were generated and de novo assembled. Sequences for all of the known enzymes involved in TIAs synthesis were identified. Additionally, 193 cytochrome P450 (CYP450), 280 methyltransferase and 144 isomerase genes were identified, that are potential candidates for enzymes involved in RIN and IRN synthesis. Digital gene expression profile (DGE) analysis was performed on the three capsule developmental stages, and based on genes possessing expression profiles consistent with RIN and IRN levels; four CYP450s, three methyltransferases and three isomerases were identified as the candidates most likely to be involved in the later steps of RIN and IRN biosynthesis. A combination of de novo transcriptome assembly and DGE analysis was shown to be a powerful method for identifying genes encoding enzymes potentially involved in the biosynthesis of important secondary metabolites in a non-model plant. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the capsule extract from Uncaria, and provides information that may aid in metabolic engineering to increase yields of these important alkaloids.

Skull Development, Ossification Pattern, and Adult Shape in the Emerging Lizard Model Organism Pogona vitticeps: A Comparative Analysis With Other Squamates.

PubMed

Ollonen, Joni; Da Silva, Filipe O; Mahlow, Kristin; Di-Poï, Nicolas

2018-01-01

The rise of the Evo-Devo field and the development of multidisciplinary research tools at various levels of biological organization have led to a growing interest in researching for new non-model organisms. Squamates (lizards and snakes) are particularly important for understanding fundamental questions about the evolution of vertebrates because of their high diversity and evolutionary innovations and adaptations that portrait a striking body plan change that reached its extreme in snakes. Yet, little is known about the intricate connection between phenotype and genotype in squamates, partly due to limited developmental knowledge and incomplete characterization of embryonic development. Surprisingly, squamate models have received limited attention in comparative developmental studies, and only a few species examined so far can be considered as representative and appropriate model organism for mechanistic Evo-Devo studies. Fortunately, the agamid lizard Pogona vitticeps (central bearded dragon) is one of the most popular, domesticated reptile species with both a well-established history in captivity and key advantages for research, thus forming an ideal laboratory model system and justifying his recent use in reptile biology research. We first report here the complete post-oviposition embryonic development for P. vitticeps based on standardized staging systems and external morphological characters previously defined for squamates. Whereas the overall morphological development follows the general trends observed in other squamates, our comparisons indicate major differences in the developmental sequence of several tissues, including early craniofacial characters. Detailed analysis of both embryonic skull development and adult skull shape, using a comparative approach integrating CT-scans and gene expression studies in P. vitticeps as well as comparative embryology and 3D geometric morphometrics in a large dataset of lizards and snakes, highlights the extreme adult skull shape of P. vitticeps and further indicates that heterochrony has played a key role in the early development and ossification of squamate skull bones. Such detailed studies of embryonic character development, craniofacial patterning, and bone formation are essential for the establishment of well-selected squamate species as Evo-Devo model organisms. We expect that P. vitticeps will continue to emerge as a new attractive model organism for understanding developmental and molecular processes underlying tissue formation, morphology, and evolution.

Skull Development, Ossification Pattern, and Adult Shape in the Emerging Lizard Model Organism Pogona vitticeps: A Comparative Analysis With Other Squamates

PubMed Central

Ollonen, Joni; Da Silva, Filipe O.; Mahlow, Kristin; Di-Poï, Nicolas

2018-01-01

The rise of the Evo-Devo field and the development of multidisciplinary research tools at various levels of biological organization have led to a growing interest in researching for new non-model organisms. Squamates (lizards and snakes) are particularly important for understanding fundamental questions about the evolution of vertebrates because of their high diversity and evolutionary innovations and adaptations that portrait a striking body plan change that reached its extreme in snakes. Yet, little is known about the intricate connection between phenotype and genotype in squamates, partly due to limited developmental knowledge and incomplete characterization of embryonic development. Surprisingly, squamate models have received limited attention in comparative developmental studies, and only a few species examined so far can be considered as representative and appropriate model organism for mechanistic Evo-Devo studies. Fortunately, the agamid lizard Pogona vitticeps (central bearded dragon) is one of the most popular, domesticated reptile species with both a well-established history in captivity and key advantages for research, thus forming an ideal laboratory model system and justifying his recent use in reptile biology research. We first report here the complete post-oviposition embryonic development for P. vitticeps based on standardized staging systems and external morphological characters previously defined for squamates. Whereas the overall morphological development follows the general trends observed in other squamates, our comparisons indicate major differences in the developmental sequence of several tissues, including early craniofacial characters. Detailed analysis of both embryonic skull development and adult skull shape, using a comparative approach integrating CT-scans and gene expression studies in P. vitticeps as well as comparative embryology and 3D geometric morphometrics in a large dataset of lizards and snakes, highlights the extreme adult skull shape of P. vitticeps and further indicates that heterochrony has played a key role in the early development and ossification of squamate skull bones. Such detailed studies of embryonic character development, craniofacial patterning, and bone formation are essential for the establishment of well-selected squamate species as Evo-Devo model organisms. We expect that P. vitticeps will continue to emerge as a new attractive model organism for understanding developmental and molecular processes underlying tissue formation, morphology, and evolution. PMID:29643813

Traceability, reproducibility and wiki-exploration for “à-la-carte” reconstructions of genome-scale metabolic models

PubMed Central

Got, Jeanne; Cortés, María Paz; Maass, Alejandro

2018-01-01

Genome-scale metabolic models have become the tool of choice for the global analysis of microorganism metabolism, and their reconstruction has attained high standards of quality and reliability. Improvements in this area have been accompanied by the development of some major platforms and databases, and an explosion of individual bioinformatics methods. Consequently, many recent models result from “à la carte” pipelines, combining the use of platforms, individual tools and biological expertise to enhance the quality of the reconstruction. Although very useful, introducing heterogeneous tools, that hardly interact with each other, causes loss of traceability and reproducibility in the reconstruction process. This represents a real obstacle, especially when considering less studied species whose metabolic reconstruction can greatly benefit from the comparison to good quality models of related organisms. This work proposes an adaptable workspace, AuReMe, for sustainable reconstructions or improvements of genome-scale metabolic models involving personalized pipelines. At each step, relevant information related to the modifications brought to the model by a method is stored. This ensures that the process is reproducible and documented regardless of the combination of tools used. Additionally, the workspace establishes a way to browse metabolic models and their metadata through the automatic generation of ad-hoc local wikis dedicated to monitoring and facilitating the process of reconstruction. AuReMe supports exploration and semantic query based on RDF databases. We illustrate how this workspace allowed handling, in an integrated way, the metabolic reconstructions of non-model organisms such as an extremophile bacterium or eukaryote algae. Among relevant applications, the latter reconstruction led to putative evolutionary insights of a metabolic pathway. PMID:29791443

Comparative study of Hippo pathway genes in cellular conveyor belts of a ctenophore and a cnidarian.

PubMed

Coste, Alicia; Jager, Muriel; Chambon, Jean-Philippe; Manuel, Michaël

2016-01-01

The Hippo pathway regulates growth rate and organ size in fly and mouse, notably through control of cell proliferation. Molecular interactions at the heart of this pathway are known to have originated in the unicellular ancestry of metazoans. They notably involve a cascade of phosphorylations triggered by the kinase Hippo, with subsequent nuclear to cytoplasmic shift of Yorkie localisation, preventing its binding to the transcription factor Scalloped, thereby silencing proliferation genes. There are few comparative expression data of Hippo pathway genes in non-model animal species and notably none in non-bilaterian phyla. All core Hippo pathway genes could be retrieved from the ctenophore Pleurobrachia pileus and the hydrozoan cnidarian Clytia hemisphaerica, with the important exception of Yorkie in ctenophore. Expression study of the Hippo, Salvador and Scalloped genes in tentacle "cellular conveyor belts" of these two organisms revealed striking differences. In P. pileus, their transcripts were detected in areas where undifferentiated progenitors intensely proliferate and where expression of cyclins B and D was also seen. In C. hemisphaerica, these three genes and Yorkie are expressed not only in the proliferating but also in the differentiation zone of the tentacle bulb and in mature tentacle cells. However, using an antibody designed against the C. hemiphaerica Yorkie protein, we show in two distinct cell lineages of the medusa that Yorkie localisation is predominantly nuclear in areas of active cell proliferation and mainly cytoplasmic elsewhere. This is the first evidence of nucleocytoplasmic Yorkie shift in association with the arrest of cell proliferation in a cnidarian, strongly evoking the cell division-promoting role of this protein and its inhibition by the activated Hippo pathway in bilaterian models. Our results furthermore highlight important differences in terms of deployment and regulation of Hippo pathway genes between cnidarians and ctenophores.

Optimized Probe Masking for Comparative Transcriptomics of Closely Related Species

PubMed Central

Poeschl, Yvonne; Delker, Carolin; Trenner, Jana; Ullrich, Kristian Karsten; Quint, Marcel; Grosse, Ivo

2013-01-01

Microarrays are commonly applied to study the transcriptome of specific species. However, many available microarrays are restricted to model organisms, and the design of custom microarrays for other species is often not feasible. Hence, transcriptomics approaches of non-model organisms as well as comparative transcriptomics studies among two or more species often make use of cost-intensive RNAseq studies or, alternatively, by hybridizing transcripts of a query species to a microarray of a closely related species. When analyzing these cross-species microarray expression data, differences in the transcriptome of the query species can cause problems, such as the following: (i) lower hybridization accuracy of probes due to mismatches or deletions, (ii) probes binding multiple transcripts of different genes, and (iii) probes binding transcripts of non-orthologous genes. So far, methods for (i) exist, but these neglect (ii) and (iii). Here, we propose an approach for comparative transcriptomics addressing problems (i) to (iii), which retains only transcript-specific probes binding transcripts of orthologous genes. We apply this approach to an Arabidopsis lyrata expression data set measured on a microarray designed for Arabidopsis thaliana, and compare it to two alternative approaches, a sequence-based approach and a genomic DNA hybridization-based approach. We investigate the number of retained probe sets, and we validate the resulting expression responses by qRT-PCR. We find that the proposed approach combines the benefit of sequence-based stringency and accuracy while allowing the expression analysis of much more genes than the alternative sequence-based approach. As an added benefit, the proposed approach requires probes to detect transcripts of orthologous genes only, which provides a superior base for biological interpretation of the measured expression responses. PMID:24260119

PhytoCRISP-Ex: a web-based and stand-alone application to find specific target sequences for CRISPR/CAS editing.

PubMed

Rastogi, Achal; Murik, Omer; Bowler, Chris; Tirichine, Leila

2016-07-01

With the emerging interest in phytoplankton research, the need to establish genetic tools for the functional characterization of genes is indispensable. The CRISPR/Cas9 system is now well recognized as an efficient and accurate reverse genetic tool for genome editing. Several computational tools have been published allowing researchers to find candidate target sequences for the engineering of the CRISPR vectors, while searching possible off-targets for the predicted candidates. These tools provide built-in genome databases of common model organisms that are used for CRISPR target prediction. Although their predictions are highly sensitive, the applicability to non-model genomes, most notably protists, makes their design inadequate. This motivated us to design a new CRISPR target finding tool, PhytoCRISP-Ex. Our software offers CRIPSR target predictions using an extended list of phytoplankton genomes and also delivers a user-friendly standalone application that can be used for any genome. The software attempts to integrate, for the first time, most available phytoplankton genomes information and provide a web-based platform for Cas9 target prediction within them with high sensitivity. By offering a standalone version, PhytoCRISP-Ex maintains an independence to be used with any organism and widens its applicability in high throughput pipelines. PhytoCRISP-Ex out pars all the existing tools by computing the availability of restriction sites over the most probable Cas9 cleavage sites, which can be ideal for mutant screens. PhytoCRISP-Ex is a simple, fast and accurate web interface with 13 pre-indexed and presently updating phytoplankton genomes. The software was also designed as a UNIX-based standalone application that allows the user to search for target sequences in the genomes of a variety of other species.

Navigating the currents of seascape genomics: how spatial analyses can augment population genomic studies

PubMed Central

Crandall, Eric D.; Liggins, Libby; Bongaerts, Pim; Treml, Eric A.

2016-01-01

Population genomic approaches are making rapid inroads in the study of non-model organisms, including marine taxa. To date, these marine studies have predominantly focused on rudimentary metrics describing the spatial and environmental context of their study region (e.g., geographical distance, average sea surface temperature, average salinity). We contend that a more nuanced and considered approach to quantifying seascape dynamics and patterns can strengthen population genomic investigations and help identify spatial, temporal, and environmental factors associated with differing selective regimes or demographic histories. Nevertheless, approaches for quantifying marine landscapes are complicated. Characteristic features of the marine environment, including pelagic living in flowing water (experienced by most marine taxa at some point in their life cycle), require a well-designed spatial-temporal sampling strategy and analysis. Many genetic summary statistics used to describe populations may be inappropriate for marine species with large population sizes, large species ranges, stochastic recruitment, and asymmetrical gene flow. Finally, statistical approaches for testing associations between seascapes and population genomic patterns are still maturing with no single approach able to capture all relevant considerations. None of these issues are completely unique to marine systems and therefore similar issues and solutions will be shared for many organisms regardless of habitat. Here, we outline goals and spatial approaches for landscape genomics with an emphasis on marine systems and review the growing empirical literature on seascape genomics. We review established tools and approaches and highlight promising new strategies to overcome select issues including a strategy to spatially optimize sampling. Despite the many challenges, we argue that marine systems may be especially well suited for identifying candidate genomic regions under environmentally mediated selection and that seascape genomic approaches are especially useful for identifying robust locus-by-environment associations. PMID:29491947

Integration of Transcriptome, Proteome and Metabolism Data Reveals the Alkaloids Biosynthesis in Macleaya cordata and Macleaya microcarpa

PubMed Central

Liu, Fuqing; Huang, Peng; Zhu, Pengcheng; Chen, Jinjun; Shi, Mingming; Guo, Fang; Cheng, Pi; Zeng, Jing; Liao, Yifang; Gong, Jing; Zhang, Hong-Mei; Wang, Depeng; Guo, An-Yuan; Xiong, Xingyao

2013-01-01

Background The Macleaya spp., including Macleaya cordata and Macleaya microcarpa, are traditional anti-virus, inflammation eliminating, and insecticide herb medicines for their isoquinoline alkaloids. They are also known as the basis of the popular natural animal food addictive in Europe. However, few studies especially at genomics level were conducted on them. Hence, we performed the Macleaya spp. transcriptome and integrated it with iTRAQ proteome analysis in order to identify potential genes involved in alkaloids biosynthesis. Methodology and Principal Findings We elaborately designed the transcriptome, proteome and metabolism profiling for 10 samples of both species to explore their alkaloids biosynthesis. From the transcriptome data, we obtained 69367 and 78255 unigenes for M. cordata and M. microcarpa, in which about two thirds of them were similar to sequences in public databases. By metabolism profiling, reverse patterns for alkaloids sanguinarine, chelerythrine, protopine, and allocryptopine were observed in different organs of two species. We characterized the expressions of enzymes in alkaloid biosynthesis pathways. We also identified more than 1000 proteins from iTRAQ proteome data. Our results strongly suggest that the root maybe the organ for major alkaloids biosynthesis of Macleaya spp. Except for biosynthesis, the alkaloids storage and transport were also important for their accumulation. The ultrastructure of laticifers by SEM helps us to prove the alkaloids maybe accumulated in the mature roots. Conclusions/Significance To our knowledge this is the first study to elucidate the genetic makeup of Macleaya spp. This work provides clues to the identification of the potential modulate genes involved in alkaloids biosynthesis in Macleaya spp., and sheds light on researches for non-model medicinal plants by integrating different high-throughput technologies. PMID:23326424

The evolution of environmental metalloproteomics over the last 15 years through bibliometric techniques.

PubMed

Hauser-Davis, Rachel Ann; Lopes, Renato Matos; Mota, Fábio Batista; Moreira, Josino Costa

2017-06-01

Metalloproteomic studies in environmental scenarios are of significant value in elucidating metal uptake, trafficking, accumulation and metabolism linked to biomolecules in biological systems. The advent of this field occurred in the early 2000s, and it has since become an interesting and growing area of interdisciplinary research, although the number of publications in Environmental Metalloprotemics is still very low compared to other metallomic areas. In this context, the evolution of Environmental Metalloprotemics in the last decades was evaluated herein through the use of bibliometric techniques, identifying variables that may aid researchers in this area to form collaborative networks with established scientists in this regard, such as main authors, published articles, institutions, countries and established collaborations involved in academic research on this subject. Results indicate a growing trend of publications over time, reflecting the interest of the scientific community in Environmental Metalloprotemics, but also demonstrated that the research interactions in this field are still country- and organization-specific. Higher amounts of publications are observed from the late 2000's onwards, related to the increasing technological advances in the area, such as the development of techniques combining atomic spectroscopy and biochemical or proteomic techniques. The retrieved publications also indicate that the recent advances in genomic, proteomic and metallomic areas have allowed for extended applications of Environmental Metalloprotemics in non-model organisms. The results reported herein indicate that Environmental Metalloprotemics seems to now be reaching a more mature stage, in which analytical techniques are now well established and can be routinely applied in environmental scenarios, benefitting researchers and allowing for further insights into this fascinating field. Copyright © 2017 Elsevier Inc. All rights reserved.

Navigating the currents of seascape genomics: how spatial analyses can augment population genomic studies.

PubMed

Riginos, Cynthia; Crandall, Eric D; Liggins, Libby; Bongaerts, Pim; Treml, Eric A

2016-12-01

Population genomic approaches are making rapid inroads in the study of non-model organisms, including marine taxa. To date, these marine studies have predominantly focused on rudimentary metrics describing the spatial and environmental context of their study region (e.g., geographical distance, average sea surface temperature, average salinity). We contend that a more nuanced and considered approach to quantifying seascape dynamics and patterns can strengthen population genomic investigations and help identify spatial, temporal, and environmental factors associated with differing selective regimes or demographic histories. Nevertheless, approaches for quantifying marine landscapes are complicated. Characteristic features of the marine environment, including pelagic living in flowing water (experienced by most marine taxa at some point in their life cycle), require a well-designed spatial-temporal sampling strategy and analysis. Many genetic summary statistics used to describe populations may be inappropriate for marine species with large population sizes, large species ranges, stochastic recruitment, and asymmetrical gene flow. Finally, statistical approaches for testing associations between seascapes and population genomic patterns are still maturing with no single approach able to capture all relevant considerations. None of these issues are completely unique to marine systems and therefore similar issues and solutions will be shared for many organisms regardless of habitat. Here, we outline goals and spatial approaches for landscape genomics with an emphasis on marine systems and review the growing empirical literature on seascape genomics. We review established tools and approaches and highlight promising new strategies to overcome select issues including a strategy to spatially optimize sampling. Despite the many challenges, we argue that marine systems may be especially well suited for identifying candidate genomic regions under environmentally mediated selection and that seascape genomic approaches are especially useful for identifying robust locus-by-environment associations.

Computing and Applying Atomic Regulons to Understand Gene Expression and Regulation

PubMed Central

Faria, José P.; Davis, James J.; Edirisinghe, Janaka N.; Taylor, Ronald C.; Weisenhorn, Pamela; Olson, Robert D.; Stevens, Rick L.; Rocha, Miguel; Rocha, Isabel; Best, Aaron A.; DeJongh, Matthew; Tintle, Nathan L.; Parrello, Bruce; Overbeek, Ross; Henry, Christopher S.

2016-01-01

Understanding gene function and regulation is essential for the interpretation, prediction, and ultimate design of cell responses to changes in the environment. An important step toward meeting the challenge of understanding gene function and regulation is the identification of sets of genes that are always co-expressed. These gene sets, Atomic Regulons (ARs), represent fundamental units of function within a cell and could be used to associate genes of unknown function with cellular processes and to enable rational genetic engineering of cellular systems. Here, we describe an approach for inferring ARs that leverages large-scale expression data sets, gene context, and functional relationships among genes. We computed ARs for Escherichia coli based on 907 gene expression experiments and compared our results with gene clusters produced by two prevalent data-driven methods: Hierarchical clustering and k-means clustering. We compared ARs and purely data-driven gene clusters to the curated set of regulatory interactions for E. coli found in RegulonDB, showing that ARs are more consistent with gold standard regulons than are data-driven gene clusters. We further examined the consistency of ARs and data-driven gene clusters in the context of gene interactions predicted by Context Likelihood of Relatedness (CLR) analysis, finding that the ARs show better agreement with CLR predicted interactions. We determined the impact of increasing amounts of expression data on AR construction and find that while more data improve ARs, it is not necessary to use the full set of gene expression experiments available for E. coli to produce high quality ARs. In order to explore the conservation of co-regulated gene sets across different organisms, we computed ARs for Shewanella oneidensis, Pseudomonas aeruginosa, Thermus thermophilus, and Staphylococcus aureus, each of which represents increasing degrees of phylogenetic distance from E. coli. Comparison of the organism-specific ARs showed that the consistency of AR gene membership correlates with phylogenetic distance, but there is clear variability in the regulatory networks of closely related organisms. As large scale expression data sets become increasingly common for model and non-model organisms, comparative analyses of atomic regulons will provide valuable insights into fundamental regulatory modules used across the bacterial domain. PMID:27933038

Transcriptome Analysis Based on RNA-Seq in Understanding Pathogenic Mechanisms of Diseases and the Immune System of Fish: A Comprehensive Review

PubMed Central

Sudhagar, Arun; El-Matbouli, Mansour

2018-01-01

In recent years, with the advent of next-generation sequencing along with the development of various bioinformatics tools, RNA sequencing (RNA-Seq)-based transcriptome analysis has become much more affordable in the field of biological research. This technique has even opened up avenues to explore the transcriptome of non-model organisms for which a reference genome is not available. This has made fish health researchers march towards this technology to understand pathogenic processes and immune reactions in fish during the event of infection. Recent studies using this technology have altered and updated the previous understanding of many diseases in fish. RNA-Seq has been employed in the understanding of fish pathogens like bacteria, virus, parasites, and oomycetes. Also, it has been helpful in unraveling the immune mechanisms in fish. Additionally, RNA-Seq technology has made its way for future works, such as genetic linkage mapping, quantitative trait analysis, disease-resistant strain or broodstock selection, and the development of effective vaccines and therapies. Until now, there are no reviews that comprehensively summarize the studies which made use of RNA-Seq to explore the mechanisms of infection of pathogens and the defense strategies of fish hosts. This review aims to summarize the contemporary understanding and findings with regard to infectious pathogens and the immune system of fish that have been achieved through RNA-Seq technology. PMID:29342931

Disentangling visual and olfactory signals in mushroom-mimicking Dracula orchids using realistic three-dimensional printed flowers.

PubMed

Policha, Tobias; Davis, Aleah; Barnadas, Melinda; Dentinger, Bryn T M; Raguso, Robert A; Roy, Bitty A

2016-05-01

Flowers use olfactory and visual signals to communicate with pollinators. Disentangling the relative contributions and potential synergies between signals remains a challenge. Understanding the perceptual biases exploited by floral mimicry illuminates the evolution of these signals. Here, we disentangle the olfactory and visual components of Dracula lafleurii, which mimics mushrooms in size, shape, color and scent, and is pollinated by mushroom-associated flies. To decouple signals, we used three-dimensional printing to produce realistic artificial flower molds that were color matched and cast using scent-free surgical silicone, to which we could add scent. We used GC-MS to measure scents in co-occurring mushrooms, and related orchids, and used these scents in field experiments. By combining silicone flower parts with real floral organs, we created chimeras that identified the mushroom-like labellum as a source of volatile attraction. In addition, we showed remarkable overlap in the volatile chemistry between D. lafleurii and co-occurring mushrooms. The characters defining the genus Dracula - a mushroom-like, 'gilled' labellum and a showy, patterned calyx - enhance pollinator attraction by exploiting the visual and chemosensory perceptual biases of drosophilid flies. Our techniques for the manipulation of complex traits in a nonmodel system not conducive to gene silencing or selective breeding are useful for other systems. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Rapid Prediction of Bacterial Heterotrophic Fluxomics Using Machine Learning and Constraint Programming.

PubMed

Wu, Stephen Gang; Wang, Yuxuan; Jiang, Wu; Oyetunde, Tolutola; Yao, Ruilian; Zhang, Xuehong; Shimizu, Kazuyuki; Tang, Yinjie J; Bao, Forrest Sheng

2016-04-01

13C metabolic flux analysis (13C-MFA) has been widely used to measure in vivo enzyme reaction rates (i.e., metabolic flux) in microorganisms. Mining the relationship between environmental and genetic factors and metabolic fluxes hidden in existing fluxomic data will lead to predictive models that can significantly accelerate flux quantification. In this paper, we present a web-based platform MFlux (http://mflux.org) that predicts the bacterial central metabolism via machine learning, leveraging data from approximately 100 13C-MFA papers on heterotrophic bacterial metabolisms. Three machine learning methods, namely Support Vector Machine (SVM), k-Nearest Neighbors (k-NN), and Decision Tree, were employed to study the sophisticated relationship between influential factors and metabolic fluxes. We performed a grid search of the best parameter set for each algorithm and verified their performance through 10-fold cross validations. SVM yields the highest accuracy among all three algorithms. Further, we employed quadratic programming to adjust flux profiles to satisfy stoichiometric constraints. Multiple case studies have shown that MFlux can reasonably predict fluxomes as a function of bacterial species, substrate types, growth rate, oxygen conditions, and cultivation methods. Due to the interest of studying model organism under particular carbon sources, bias of fluxome in the dataset may limit the applicability of machine learning models. This problem can be resolved after more papers on 13C-MFA are published for non-model species.

Genetic architecture of wood properties based on association analysis and co-expression networks in white spruce.

PubMed

Lamara, Mebarek; Raherison, Elie; Lenz, Patrick; Beaulieu, Jean; Bousquet, Jean; MacKay, John

2016-04-01

Association studies are widely utilized to analyze complex traits but their ability to disclose genetic architectures is often limited by statistical constraints, and functional insights are usually minimal in nonmodel organisms like forest trees. We developed an approach to integrate association mapping results with co-expression networks. We tested single nucleotide polymorphisms (SNPs) in 2652 candidate genes for statistical associations with wood density, stiffness, microfibril angle and ring width in a population of 1694 white spruce trees (Picea glauca). Associations mapping identified 229-292 genes per wood trait using a statistical significance level of P < 0.05 to maximize discovery. Over-representation of genes associated for nearly all traits was found in a xylem preferential co-expression group developed in independent experiments. A xylem co-expression network was reconstructed with 180 wood associated genes and several known MYB and NAC regulators were identified as network hubs. The network revealed a link between the gene PgNAC8, wood stiffness and microfibril angle, as well as considerable within-season variation for both genetic control of wood traits and gene expression. Trait associations were distributed throughout the network suggesting complex interactions and pleiotropic effects. Our findings indicate that integration of association mapping and co-expression networks enhances our understanding of complex wood traits. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

COBRA-Seq: Sensitive and Quantitative Methylome Profiling

PubMed Central

Varinli, Hilal; Statham, Aaron L.; Clark, Susan J.; Molloy, Peter L.; Ross, Jason P.

2015-01-01

Combined Bisulfite Restriction Analysis (COBRA) quantifies DNA methylation at a specific locus. It does so via digestion of PCR amplicons produced from bisulfite-treated DNA, using a restriction enzyme that contains a cytosine within its recognition sequence, such as TaqI. Here, we introduce COBRA-seq, a genome wide reduced methylome method that requires minimal DNA input (0.1–1.0 μg) and can either use PCR or linear amplification to amplify the sequencing library. Variants of COBRA-seq can be used to explore CpG-depleted as well as CpG-rich regions in vertebrate DNA. The choice of enzyme influences enrichment for specific genomic features, such as CpG-rich promoters and CpG islands, or enrichment for less CpG dense regions such as enhancers. COBRA-seq coupled with linear amplification has the additional advantage of reduced PCR bias by producing full length fragments at high abundance. Unlike other reduced representative methylome methods, COBRA-seq has great flexibility in the choice of enzyme and can be multiplexed and tuned, to reduce sequencing costs and to interrogate different numbers of sites. Moreover, COBRA-seq is applicable to non-model organisms without the reference genome and compatible with the investigation of non-CpG methylation by using restriction enzymes containing CpA, CpT, and CpC in their recognition site. PMID:26512698

Developmental constraints and convergent evolution in Drosophila sex comb formation.

PubMed

Atallah, Joel; Liu, Nana Hou; Dennis, Peter; Hon, Andy; Larsen, Ellen W

2009-01-01

The most complex and diverse secondary sexual character in Drosophila is the sex comb (SC), an arrangement of modified bristles on the forelegs of a subclade of male fruit flies. We examined SC formation in six representative nonmodel fruit fly species, in an effort to understand how the variation in comb patterning arises. We first compared SC development in two species with relatively small combs, Drosophila takahashii, where the SCs remain approximately transverse, and Drosophila biarmipes, where two rows of SC teeth rotate and move in an anterior direction relative to other bristle landmarks. We then analyzed comb ontogeny in species with prominent extended SCs parallel to the proximodistal axis, including Drosophila ficusphila and species of the montium subgroup. Our study allowed us to identify two general methods of generating longitudinal combs on the tarsus, and we showed that a montium subgroup species (Drosophila nikananu) with a comb convergently similar in size, orientation and position to the model organism Drosophila melanogaster, forms its SC through a different developmental mechanism. We also found that the protein product of the leg patterning gene, dachshund (dac), is strongly reduced in the SC in all species, but not in other bristles. Our results suggest that an apparent constraint on SC position in the adult may be attributable to at least two different lineage-specific developmental processes, although external forces could also play a role.

Is there a link between aging and microbiome diversity in exceptional mammalian longevity?

PubMed Central

Hughes, Graham M.; Leech, John; Puechmaille, Sébastien J.; Lopez, Jose V.

2018-01-01

A changing microbiome has been linked to biological aging in mice and humans, suggesting a possible role of gut flora in pathogenic aging phenotypes. Many bat species have exceptional longevity given their body size and some can live up to ten times longer than expected with little signs of aging. This study explores the anal microbiome of the exceptionally long-lived Myotis myotis bat, investigating bacterial composition in both adult and juvenile bats to determine if the microbiome changes with age in a wild, long-lived non-model organism, using non-lethal sampling. The anal microbiome was sequenced using metabarcoding in more than 50 individuals, finding no significant difference between the composition of juvenile and adult bats, suggesting that age-related microbial shifts previously observed in other mammals may not be present in Myotis myotis. Functional gene categories, inferred from metabarcoding data, expressed in the M. myotis microbiome were categorized identifying pathways involved in metabolism, DNA repair and oxidative phosphorylation. We highlight an abundance of ‘Proteobacteria’ relative to other mammals, with similar patterns compared to other bat microbiomes. Our results suggest that M. myotis may have a relatively stable, unchanging microbiome playing a role in their extended ‘health spans’ with the advancement of age, and suggest a potential link between microbiome and sustained, powered flight. PMID:29333342

Selection on oxidative phosphorylation and ribosomal structure as a multigenerational response to ocean acidification in the common copepod Pseudocalanus acuspes.

PubMed

De Wit, Pierre; Dupont, Sam; Thor, Peter

2016-10-01

Ocean acidification is expected to have dramatic impacts on oceanic ecosystems, yet surprisingly few studies currently examine long-term adaptive and plastic responses of marine invertebrates to p CO 2 stress. Here, we exposed populations of the common copepod Pseudocalanus acuspes to three p CO 2 regimes (400, 900, and 1550 μatm) for two generations, after which we conducted a reciprocal transplant experiment. A de novo transcriptome was assembled, annotated, and gene expression data revealed that genes involved in RNA transcription were strongly down-regulated in populations with long-term exposure to a high p CO 2 environment, even after transplantation back to control levels. In addition, 747 000 SNPs were identified, out of which 1513 showed consistent changes in nucleotide frequency between replicates of control and high p CO 2 populations. Functions involving RNA transcription and ribosomal function, as well as ion transport and oxidative phosphorylation, were highly overrepresented. We thus conclude that p CO 2 stress appears to impose selection in copepods on RNA synthesis and translation, possibly modulated by helicase expression. Using a physiological hypothesis-testing strategy to mine gene expression data, we herein increase the power to detect cellular targets of ocean acidification. This novel approach seems promising for future studies of effects of environmental changes in ecologically important nonmodel organisms.

An integrated chromosome map of microsatellite markers and inversion breakpoints for an Asian malaria mosquito, Anopheles stephensi.

PubMed

Kamali, Maryam; Sharakhova, Maria V; Baricheva, Elina; Karagodin, Dmitrii; Tu, Zhijian; Sharakhov, Igor V

2011-01-01

Anopheles stephensi is one of the major vectors of malaria in the Middle East and Indo-Pakistan subcontinent. Understanding the population genetic structure of malaria mosquitoes is important for developing adequate and successful vector control strategies. Commonly used markers for inferring anopheline taxonomic and population status include microsatellites and chromosomal inversions. Knowledge about chromosomal locations of microsatellite markers with respect to polymorphic inversions could be useful for better understanding a genetic structure of natural populations. However, fragments with microsatellites used in population genetic studies are usually too short for successful labeling and hybridization with chromosomes. We designed new primers for amplification of microsatellite loci identified in the A. stephensi genome sequenced with next-generation technologies. Twelve microsatellites were mapped to polytene chromosomes from ovarian nurse cells of A. stephensi using fluorescent in situ hybridization. All microsatellites hybridized to unique locations on autosomes, and 7 of them localized to the largest arm 2R. Ten microsatellites were mapped inside the previously described polymorphic chromosomal inversions, including 4 loci located inside the widespread inversion 2Rb. We analyzed microsatellite-based population genetic data available for A. stephensi in light of our mapping results. This study demonstrates that the chromosomal position of microsatellites may affect estimates of population genetic parameters and highlights the importance of developing physical maps for nonmodel organisms.

Rapid Prediction of Bacterial Heterotrophic Fluxomics Using Machine Learning and Constraint Programming

PubMed Central

Wu, Stephen Gang; Wang, Yuxuan; Jiang, Wu; Oyetunde, Tolutola; Yao, Ruilian; Zhang, Xuehong; Shimizu, Kazuyuki; Tang, Yinjie J.; Bao, Forrest Sheng

2016-01-01

13C metabolic flux analysis (13C-MFA) has been widely used to measure in vivo enzyme reaction rates (i.e., metabolic flux) in microorganisms. Mining the relationship between environmental and genetic factors and metabolic fluxes hidden in existing fluxomic data will lead to predictive models that can significantly accelerate flux quantification. In this paper, we present a web-based platform MFlux (http://mflux.org) that predicts the bacterial central metabolism via machine learning, leveraging data from approximately 100 13C-MFA papers on heterotrophic bacterial metabolisms. Three machine learning methods, namely Support Vector Machine (SVM), k-Nearest Neighbors (k-NN), and Decision Tree, were employed to study the sophisticated relationship between influential factors and metabolic fluxes. We performed a grid search of the best parameter set for each algorithm and verified their performance through 10-fold cross validations. SVM yields the highest accuracy among all three algorithms. Further, we employed quadratic programming to adjust flux profiles to satisfy stoichiometric constraints. Multiple case studies have shown that MFlux can reasonably predict fluxomes as a function of bacterial species, substrate types, growth rate, oxygen conditions, and cultivation methods. Due to the interest of studying model organism under particular carbon sources, bias of fluxome in the dataset may limit the applicability of machine learning models. This problem can be resolved after more papers on 13C-MFA are published for non-model species. PMID:27092947

Comparative transcriptomic analysis of the evolution and development of flower size in Saltugilia (Polemoniaceae).

PubMed

Landis, Jacob B; Soltis, Douglas E; Soltis, Pamela S

2017-06-23

Flower size varies dramatically across angiosperms, representing innovations over the course of >130 million years of evolution and contributing substantially to relationships with pollinators. However, the genetic underpinning of flower size is not well understood. Saltugilia (Polemoniaceae) provides an excellent non-model system for extending the genetic study of flower size to interspecific differences that coincide with variation in pollinators. Using targeted gene capture methods, we infer phylogenetic relationships among all members of Saltugilia to provide a framework for investigating the genetic control of flower size differences via RNA-Seq de novo assembly. Nuclear concatenation and species tree inference methods provide congruent topologies. The inferred evolutionary trajectory of flower size is from small flowers to larger flowers. We identified 4 to 10,368 transcripts that are differentially expressed during flower development, with many unigenes associated with cell wall modification and components of the auxin and gibberellin pathways. Saltugilia is an excellent model for investigating covarying floral and pollinator evolution. Four candidate genes from model systems (BIG BROTHER, BIG PETAL, GASA, and LONGIFOLIA) show differential expression during development of flowers in Saltugilia, and four other genes (FLOWERING-PROMOTING FACTOR 1, PECTINESTERASE, POLYGALACTURONASE, and SUCROSE SYNTHASE) fit into hypothesized organ size pathways. Together, these gene sets provide a strong foundation for future functional studies to determine their roles in specifying interspecific differences in flower size.

Comparative transcriptomics of Entelegyne spiders (Araneae, Entelegynae), with emphasis on molecular evolution of orphan genes.

PubMed

Carlson, David E; Hedin, Marshal

2017-01-01

Next-generation sequencing technology is rapidly transforming the landscape of evolutionary biology, and has become a cost-effective and efficient means of collecting exome information for non-model organisms. Due to their taxonomic diversity, production of interesting venom and silk proteins, and the relative scarcity of existing genomic resources, spiders in particular are excellent targets for next-generation sequencing (NGS) methods. In this study, the transcriptomes of six entelegyne spider species from three genera (Cicurina travisae, C. vibora, Habronattus signatus, H. ustulatus, Nesticus bishopi, and N. cooperi) were sequenced and de novo assembled. Each assembly was assessed for quality and completeness and functionally annotated using gene ontology information. Approximately 100 transcripts with evidence of homology to venom proteins were discovered. After identifying more than 3,000 putatively orthologous genes across all six taxa, we used comparative analyses to identify 24 instances of positively selected genes. In addition, between ~ 550 and 1,100 unique orphan genes were found in each genus. These unique, uncharacterized genes exhibited elevated rates of amino acid substitution, potentially consistent with lineage-specific adaptive evolution. The data generated for this study represent a valuable resource for future phylogenetic and molecular evolutionary research, and our results provide new insight into the forces driving genome evolution in taxa that span the root of entelegyne spider phylogeny.

With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.

PubMed

Chapman, Joanne R; Waldenström, Jonas

2015-01-01

The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

RADseq provides unprecedented insights into molecular ecology and evolutionary genetics: comment on Breaking RAD by Lowry et al. (2016).

PubMed

McKinney, Garrett J; Larson, Wesley A; Seeb, Lisa W; Seeb, James E

2017-05-01

In their recently corrected manuscript, "Breaking RAD: An evaluation of the utility of restriction site associated DNA sequencing for genome scans of adaptation", Lowry et al. argue that genome scans using RADseq will miss many loci under selection due to a combination of sparse marker density and low levels of linkage disequilibrium in most species. We agree that marker density and levels of LD are important considerations when designing a RADseq study; however, we dispute that RAD-based genome scans are as prone to failure as Lowry et al. suggest. Their arguments ignore the flexible nature of RADseq; the availability of different restriction enzymes and capacity for combining restriction enzymes ensures that a well-designed study should be able to generate enough markers for efficient genome coverage. We further believe that simplifying assumptions about linkage disequilibrium in their simulations are invalid in many species. Finally, it is important to note that the alternative methods proposed by Lowry et al. have limitations equal to or greater than RADseq. The wealth of studies with positive impactful findings that have used RAD genome scans instead supports the argument that properly conducted RAD genome scans are an effective method for gaining insight into ecology and evolution, particularly for non-model organisms and those with large or complex genomes. © 2016 John Wiley & Sons Ltd.

Gene discovery using next-generation pyrosequencing to develop ESTs for Phalaenopsis orchids

PubMed Central

2011-01-01

Background Orchids are one of the most diversified angiosperms, but few genomic resources are available for these non-model plants. In addition to the ecological significance, Phalaenopsis has been considered as an economically important floriculture industry worldwide. We aimed to use massively parallel 454 pyrosequencing for a global characterization of the Phalaenopsis transcriptome. Results To maximize sequence diversity, we pooled RNA from 10 samples of different tissues, various developmental stages, and biotic- or abiotic-stressed plants. We obtained 206,960 expressed sequence tags (ESTs) with an average read length of 228 bp. These reads were assembled into 8,233 contigs and 34,630 singletons. The unigenes were searched against the NCBI non-redundant (NR) protein database. Based on sequence similarity with known proteins, these analyses identified 22,234 different genes (E-value cutoff, e-7). Assembled sequences were annotated with Gene Ontology, Gene Family and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Among these annotations, over 780 unigenes encoding putative transcription factors were identified. Conclusion Pyrosequencing was effective in identifying a large set of unigenes from Phalaenopsis. The informative EST dataset we developed constitutes a much-needed resource for discovery of genes involved in various biological processes in Phalaenopsis and other orchid species. These transcribed sequences will narrow the gap between study of model organisms with many genomic resources and species that are important for ecological and evolutionary studies. PMID:21749684

Q&A: How do gene regulatory networks control environmental responses in plants?

PubMed

Sun, Ying; Dinneny, José R

2018-04-11

A gene regulatory network (GRN) describes the hierarchical relationship between transcription factors, associated proteins, and their target genes. Studying GRNs allows us to understand how a plant's genotype and environment are integrated to regulate downstream physiological responses. Current efforts in plants have focused on defining the GRNs that regulate functions such as development and stress response and have been performed primarily in genetically tractable model plant species such as Arabidopsis thaliana. Future studies will likely focus on how GRNs function in non-model plants and change over evolutionary time to allow for adaptation to extreme environments. This broader understanding will inform efforts to engineer GRNs to create tailored crop traits.

Deep sequencing-based transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus reveals insight into the immune-relevant genes in marine fish

PubMed Central

2010-01-01

Background Systematic research on fish immunogenetics is indispensable in understanding the origin and evolution of immune systems. This has long been a challenging task because of the limited number of deep sequencing technologies and genome backgrounds of non-model fish available. The newly developed Solexa/Illumina RNA-seq and Digital gene expression (DGE) are high-throughput sequencing approaches and are powerful tools for genomic studies at the transcriptome level. This study reports the transcriptome profiling analysis of bacteria-challenged Lateolabrax japonicus using RNA-seq and DGE in an attempt to gain insights into the immunogenetics of marine fish. Results RNA-seq analysis generated 169,950 non-redundant consensus sequences, among which 48,987 functional transcripts with complete or various length encoding regions were identified. More than 52% of these transcripts are possibly involved in approximately 219 known metabolic or signalling pathways, while 2,673 transcripts were associated with immune-relevant genes. In addition, approximately 8% of the transcripts appeared to be fish-specific genes that have never been described before. DGE analysis revealed that the host transcriptome profile of Vibrio harveyi-challenged L. japonicus is considerably altered, as indicated by the significant up- or down-regulation of 1,224 strong infection-responsive transcripts. Results indicated an overall conservation of the components and transcriptome alterations underlying innate and adaptive immunity in fish and other vertebrate models. Analysis suggested the acquisition of numerous fish-specific immune system components during early vertebrate evolution. Conclusion This study provided a global survey of host defence gene activities against bacterial challenge in a non-model marine fish. Results can contribute to the in-depth study of candidate genes in marine fish immunity, and help improve current understanding of host-pathogen interactions and evolutionary history of immunogenetics from fish to mammals. PMID:20707909

Transcriptome and proteome profiling of adventitious root development in hybrid larch (Larix kaempferi × Larix olgensis).

PubMed

Han, Hua; Sun, Xiaomei; Xie, Yunhui; Feng, Jian; Zhang, Shougong

2014-11-26

Hybrids of larch (Larix kaempferi × Larix olgensis) are important afforestation species in northeastern China. They are routinely propagated via rooted stem cuttings. Despite the importance of rooting, little is known about the regulation of adventitious root development in larch hybrids. 454 GS FLX Titanium technology represents a new method for characterizing the transcriptomes of non-model species. This method can be used to identify differentially expressed genes, and then two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analyses can be used to analyze their corresponding proteins. In this study, we analyzed semi-lignified cuttings of two clones of L. kaempferi × L. olgensis with different rooting capacities to study the molecular basis of adventitious root development. We analyzed two clones; clone 25-5, with strong rooting capacity, and clone 23-12, with weak rooting capacity. We constructed four cDNA libraries from 25-5 and 23-12 at two development stages. Sequencing was conducted using the 454 pyrosequencing platform. A total of 957832 raw reads was produced; 95.07% were high-quality reads, and were assembled into 45137 contigs and 61647 singletons. The functions of the unigenes, as indicated by their Gene Ontology annotation, included diverse roles in the molecular functions, biological processes, and cellular component categories. We analyzed 75 protein spots (-fold change ≥ 2, P ≤ 0.05) by 2D-DIGE, and identified the differentially expressed proteins using MALDI-TOF/TOF MS. A joint analysis of transcriptome and proteome showed genes related to two pathways, polyamine synthesis and stress response, might play an important role on adventitious root development. These results provide fundamental and important information for research on the molecular mechanism of adventitious root development. We also demonstrated for the first time the combined use of two important technologies as a powerful approach to advance research on non-model, but otherwise important, larch species.

Tissue Culture as a Source of Replicates in Nonmodel Plants: Variation in Cold Response in Arabidopsis lyrata ssp. petraea.

PubMed

Kenta, Tanaka; Edwards, Jessica E M; Butlin, Roger K; Burke, Terry; Quick, W Paul; Urwin, Peter; Davey, Matthew P

2016-12-07

While genotype-environment interaction is increasingly receiving attention by ecologists and evolutionary biologists, such studies need genetically homogeneous replicates-a challenging hurdle in outcrossing plants. This could be potentially overcome by using tissue culture techniques. However, plants regenerated from tissue culture may show aberrant phenotypes and "somaclonal" variation. Here, we examined somaclonal variation due to tissue culturing using the response to cold treatment of photosynthetic efficiency (chlorophyll fluorescence measurements for F v /F m , F v '/F m ', and Φ PSII , representing maximum efficiency of photosynthesis for dark- and light-adapted leaves, and the actual electron transport operating efficiency, respectively, which are reliable indicators of photoinhibition and damage to the photosynthetic electron transport system). We compared this to variation among half-sibling seedlings from three different families of Arabidopsis lyrata ssp. petraea Somaclonal variation was limited, and we could detect within-family variation in change in chlorophyll fluorescence due to cold shock successfully with the help of tissue-culture derived replicates. Icelandic and Norwegian families exhibited higher chlorophyll fluorescence, suggesting higher performance after cold shock, than a Swedish family. Although the main effect of tissue culture on F v /F m , F v '/F m ', and Φ PSII was small, there were significant interactions between tissue culture and family, suggesting that the effect of tissue culture is genotype-specific. Tissue-cultured plantlets were less affected by cold treatment than seedlings, but to a different extent in each family. These interactive effects, however, were comparable to, or much smaller than the single effect of family. These results suggest that tissue culture is a useful method for obtaining genetically homogenous replicates for studying genotype-environment interaction related to adaptively-relevant phenotypes, such as cold response, in nonmodel outcrossing plants. Copyright © 2016 Kenta et al.

Positional bias in variant calls against draft reference assemblies.

PubMed

Briskine, Roman V; Shimizu, Kentaro K

2017-03-28

Whole genome resequencing projects may implement variant calling using draft reference genomes assembled de novo from short-read libraries. Despite lower quality of such assemblies, they allowed researchers to extend a wide range of population genetic and genome-wide association analyses to non-model species. As the variant calling pipelines are complex and involve many software packages, it is important to understand inherent biases and limitations at each step of the analysis. In this article, we report a positional bias present in variant calling performed against draft reference assemblies constructed from de Bruijn or string overlap graphs. We assessed how frequently variants appeared at each position counted from ends of a contig or scaffold sequence, and discovered unexpectedly high number of variants at the positions related to the length of either k-mers or reads used for the assembly. We detected the bias in both publicly available draft assemblies from Assemblathon 2 competition as well as in the assemblies we generated from our simulated short-read data. Simulations confirmed that the bias causing variants are predominantly false positives induced by reads from spatially distant repeated sequences. The bias is particularly strong in contig assemblies. Scaffolding does not eliminate the bias but tends to mitigate it because of the changes in variants' relative positions and alterations in read alignments. The bias can be effectively reduced by filtering out the variants that reside in repetitive elements. Draft genome sequences generated by several popular assemblers appear to be susceptible to the positional bias potentially affecting many resequencing projects in non-model species. The bias is inherent to the assembly algorithms and arises from their particular handling of repeated sequences. It is recommended to reduce the bias by filtering especially if higher-quality genome assembly cannot be achieved. Our findings can help other researchers to improve the quality of their variant data sets and reduce artefactual findings in downstream analyses.

Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.).

PubMed

Ayyappan, Vasudevan; Kalavacharla, Venu; Thimmapuram, Jyothi; Bhide, Ketaki P; Sripathi, Venkateswara R; Smolinski, Tomasz G; Manoharan, Muthusamy; Thurston, Yaqoob; Todd, Antonette; Kingham, Bruce

2015-01-01

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress.

Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.)

PubMed Central

Thimmapuram, Jyothi; Bhide, Ketaki P.; Sripathi, Venkateswara R.; Smolinski, Tomasz G.; Manoharan, Muthusamy; Thurston, Yaqoob; Todd, Antonette; Kingham, Bruce

2015-01-01

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress. PMID:26167691

Identification and characterisation of Short Interspersed Nuclear Elements in the olive tree (Olea europaea L.) genome.

PubMed

Barghini, Elena; Mascagni, Flavia; Natali, Lucia; Giordani, Tommaso; Cavallini, Andrea

2017-02-01

Short Interspersed Nuclear Elements (SINEs) are nonautonomous retrotransposons in the genome of most eukaryotic species. While SINEs have been intensively investigated in humans and other animal systems, SINE identification has been carried out only in a limited number of plant species. This lack of information is apparent especially in non-model plants whose genome has not been sequenced yet. The aim of this work was to produce a specific bioinformatics pipeline for analysing second generation sequence reads of a non-model species and identifying SINEs. We have identified, for the first time, 227 putative SINEs of the olive tree (Olea europaea), that constitute one of the few sets of such sequences in dicotyledonous species. The identified SINEs ranged from 140 to 362 bp in length and were characterised with regard to the occurrence of the tRNA domain in their sequence. The majority of identified elements resulted in single copy or very lowly repeated, often in association with genic sequences. Analysis of sequence similarity allowed us to identify two major groups of SINEs showing different abundances in the olive tree genome, the former with sequence similarity to SINEs of Scrophulariaceae and Solanaceae and the latter to SINEs of Salicaceae. A comparison of sequence conservation between olive SINEs and LTR retrotransposon families suggested that SINE expansion in the genome occurred especially in very ancient times, before LTR retrotransposon expansion, and presumably before the separation of the rosids (to which Oleaceae belong) from the Asterids. Besides providing data on olive SINEs, our results demonstrate the suitability of the pipeline employed for SINE identification. Applying this pipeline will favour further structural and functional analyses on these relatively unknown elements to be performed also in other plant species, even in the absence of a reference genome, and will allow establishing general evolutionary patterns for this kind of repeats in plants.

Occurrence of Can-SINEs and intron sequence evolution supports robust phylogeny of pinniped carnivores and their terrestrial relatives.

PubMed

Schröder, Christiane; Bleidorn, Christoph; Hartmann, Stefanie; Tiedemann, Ralph

2009-12-15

Investigating the dog genome we found 178965 introns with a moderate length of 200-1000 bp. A screening of these sequences against 23 different repeat libraries to find insertions of short interspersed elements (SINEs) detected 45276 SINEs. Virtually all of these SINEs (98%) belong to the tRNA-derived Can-SINE family. Can-SINEs arose about 55 million years ago before Carnivora split into two basal groups, the Caniformia (dog-like carnivores) and the Feliformia (cat-like carnivores). Genome comparisons of dog and cat recovered 506 putatively informative SINE loci for caniformian phylogeny. In this study we show how to use such genome information of model organisms to research the phylogeny of related non-model species of interest. Investigating a dataset including representatives of all major caniformian lineages, we analysed 24 randomly chosen loci for 22 taxa. All loci were amplifiable and revealed 17 parsimony-informative SINE insertions. The screening for informative SINE insertions yields a large amount of sequence information, in particular of introns, which contain reliable phylogenetic information as well. A phylogenetic analysis of intron- and SINE sequence data provided a statistically robust phylogeny which is congruent with the absence/presence pattern of our SINE markers. This phylogeny strongly supports a sistergroup relationship of Musteloidea and Pinnipedia. Within Pinnipedia, we see strong support from bootstrapping and the presence of a SINE insertion for a sistergroup relationship of the walrus with the Otariidae.

Genomic Characterization of the Evolutionary Potential of the Sea Urchin Strongylocentrotus droebachiensis Facing Ocean Acidification.

PubMed

Runcie, Daniel E; Dorey, Narimane; Garfield, David A; Stumpp, Meike; Dupont, Sam; Wray, Gregory A

2016-12-01

Ocean acidification (OA) is increasing due to anthropogenic CO2 emissions and poses a threat to marine species and communities worldwide. To better project the effects of acidification on organisms' health and persistence, an understanding is needed of the 1) mechanisms underlying developmental and physiological tolerance and 2) potential populations have for rapid evolutionary adaptation. This is especially challenging in nonmodel species where targeted assays of metabolism and stress physiology may not be available or economical for large-scale assessments of genetic constraints. We used mRNA sequencing and a quantitative genetics breeding design to study mechanisms underlying genetic variability and tolerance to decreased seawater pH (-0.4 pH units) in larvae of the sea urchin Strongylocentrotus droebachiensis. We used a gene ontology-based approach to integrate expression profiles into indirect measures of cellular and biochemical traits underlying variation in larval performance (i.e., growth rates). Molecular responses to OA were complex, involving changes to several functions such as growth rates, cell division, metabolism, and immune activities. Surprisingly, the magnitude of pH effects on molecular traits tended to be small relative to variation attributable to segregating functional genetic variation in this species. We discuss how the application of transcriptomics and quantitative genetics approaches across diverse species can enrich our understanding of the biological impacts of climate change. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Gene Expression Variation Resolves Species and Individual Strains among Coral-Associated Dinoflagellates within the Genus Symbiodinium.

PubMed

Parkinson, John E; Baumgarten, Sebastian; Michell, Craig T; Baums, Iliana B; LaJeunesse, Todd C; Voolstra, Christian R

2016-02-11

Reef-building corals depend on symbiotic mutualisms with photosynthetic dinoflagellates in the genus Symbiodinium. This large microalgal group comprises many highly divergent lineages ("Clades A-I") and hundreds of undescribed species. Given their ecological importance, efforts have turned to genomic approaches to characterize the functional ecology of Symbiodinium. To date, investigators have only compared gene expression between representatives from separate clades-the equivalent of contrasting genera or families in other dinoflagellate groups-making it impossible to distinguish between clade-level and species-level functional differences. Here, we examined the transcriptomes of four species within one Symbiodinium clade (Clade B) at ∼20,000 orthologous genes, as well as multiple isoclonal cell lines within species (i.e., cultured strains). These species span two major adaptive radiations within Clade B, each encompassing both host-specialized and ecologically cryptic taxa. Species-specific expression differences were consistently enriched for photosynthesis-related genes, likely reflecting selection pressures driving niche diversification. Transcriptional variation among strains involved fatty acid metabolism and biosynthesis pathways. Such differences among individuals are potentially a major source of physiological variation, contributing to the functional diversity of coral holobionts composed of unique host-symbiont genotype pairings. Our findings expand the genomic resources available for this important symbiont group and emphasize the power of comparative transcriptomics as a method for studying speciation processes and interindividual variation in nonmodel organisms. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Using Next Generation RAD Sequencing to Isolate Multispecies Microsatellites for Pilosocereus (Cactaceae).

PubMed

Bonatelli, Isabel A S; Carstens, Bryan C; Moraes, Evandro M

2015-01-01

Microsatellite markers (also known as SSRs, Simple Sequence Repeats) are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq) on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as di- and trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in non-model organisms.

Using Next Generation RAD Sequencing to Isolate Multispecies Microsatellites for Pilosocereus (Cactaceae)

PubMed Central

Bonatelli, Isabel A. S.; Carstens, Bryan C.; Moraes, Evandro M.

2015-01-01

Microsatellite markers (also known as SSRs, Simple Sequence Repeats) are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq) on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as di- and trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in non-model organisms. PMID:26561396

Isolation and characterization of major histocompatibility class IIβ genes in an endangered North American cyprinid fish, the Rio Grande silvery minnow (Hybognathus amarus).

PubMed

Osborne, Megan J; Turner, Thomas F

2011-06-01

The major histocompatibility complex (MHC) is a critical component of the adaptive immune response in vertebrates. Due to the role that MHC plays in immunity, absence of variation within these genes may cause species to be vulnerable to emerging diseases. The freshwater fish family Cyprinidae comprises the most diverse and species-rich group of freshwater fish in the world, but some are imperiled. Despite considerable species richness and the long evolutionary history of the family, there are very few reports of MHC sequences (apart from a few model species), and no sequences are reported from endemic North American cyprinids (subfamily Leuciscinae). Here we isolate and characterize the MH Class II beta genes from complementary DNA and genomic DNA of the non-model, endangered Rio Grande silvery minnow (Hybognathus amarus), a North American cyprinid. Phylogenetic reconstruction revealed two groups of divergent MH alleles that are paralogous to previously described loci found in deeply divergent cyprinid taxa including common carp, zebrafish, African large barb and bream. Both groups of alleles were under the influence of diversifying selection yet not all individuals had alleles belonging to both allelic groups. We concluded that the general organization and pattern of variation of MH class II genes in Rio Grande silvery minnow is similar to that identified in other cyprinid fishes studied to date, despite distant evolutionary relationships and evidence of a severe genetic bottleneck. Copyright © 2011 Elsevier Ltd. All rights reserved.

Recurrent evolution of melanism in South American felids.

PubMed

Schneider, Alexsandra; Henegar, Corneliu; Day, Kenneth; Absher, Devin; Napolitano, Constanza; Silveira, Leandro; David, Victor A; O'Brien, Stephen J; Menotti-Raymond, Marilyn; Barsh, Gregory S; Eizirik, Eduardo

2015-02-01

Morphological variation in natural populations is a genomic test bed for studying the interface between molecular evolution and population genetics, but some of the most interesting questions involve non-model organisms that lack well annotated reference genomes. Many felid species exhibit polymorphism for melanism but the relative roles played by genetic drift, natural selection, and interspecies hybridization remain uncertain. We identify mutations of Agouti signaling protein (ASIP) or the Melanocortin 1 receptor (MC1R) as independent causes of melanism in three closely related South American species: the pampas cat (Leopardus colocolo), the kodkod (Leopardus guigna), and Geoffroy's cat (Leopardus geoffroyi). To assess population level variation in the regions surrounding the causative mutations we apply genomic resources from the domestic cat to carry out clone-based capture and targeted resequencing of 299 kb and 251 kb segments that contain ASIP and MC1R, respectively, from 54 individuals (13-21 per species), achieving enrichment of ~500-2500-fold and ~150x coverage. Our analysis points to unique evolutionary histories for each of the three species, with a strong selective sweep in the pampas cat, a distinctive but short melanism-specific haplotype in the Geoffroy's cat, and reduced nucleotide diversity for both ancestral and melanism-bearing chromosomes in the kodkod. These results reveal an important role for natural selection in a trait of longstanding interest to ecologists, geneticists, and the lay community, and provide a platform for comparative studies of morphological variation in other natural populations.

Recurrent Evolution of Melanism in South American Felids

PubMed Central

Schneider, Alexsandra; Henegar, Corneliu; Day, Kenneth; Absher, Devin; Napolitano, Constanza; Silveira, Leandro; David, Victor A.; O’Brien, Stephen J.; Menotti-Raymond, Marilyn; Barsh, Gregory S.; Eizirik, Eduardo

2015-01-01

Morphological variation in natural populations is a genomic test bed for studying the interface between molecular evolution and population genetics, but some of the most interesting questions involve non-model organisms that lack well annotated reference genomes. Many felid species exhibit polymorphism for melanism but the relative roles played by genetic drift, natural selection, and interspecies hybridization remain uncertain. We identify mutations of Agouti signaling protein (ASIP) or the Melanocortin 1 receptor (MC1R) as independent causes of melanism in three closely related South American species: the pampas cat (Leopardus colocolo), the kodkod (Leopardus guigna), and Geoffroy’s cat (Leopardus geoffroyi). To assess population level variation in the regions surrounding the causative mutations we apply genomic resources from the domestic cat to carry out clone-based capture and targeted resequencing of 299 kb and 251 kb segments that contain ASIP and MC1R, respectively, from 54 individuals (13–21 per species), achieving enrichment of ~500–2500-fold and ~150x coverage. Our analysis points to unique evolutionary histories for each of the three species, with a strong selective sweep in the pampas cat, a distinctive but short melanism-specific haplotype in the Geoffroy’s cat, and reduced nucleotide diversity for both ancestral and melanism-bearing chromosomes in the kodkod. These results reveal an important role for natural selection in a trait of longstanding interest to ecologists, geneticists, and the lay community, and provide a platform for comparative studies of morphological variation in other natural populations. PMID:25695801

Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei

PubMed Central

Lopez-Zavala, Alonso A.; Carrasco-Miranda, Jesus S.; Ramirez-Aguirre, Claudia D.; López-Hidalgo, Marisol; Benitez-Cardoza, Claudia G.; Ochoa-Leyva, Adrian; Cardona-Felix, Cesar S.; Diaz-Quezada, Corina; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R.; Brieba, Luis G.

2016-01-01

Triosephosphate isomerase (TIM; EC 5.3.1.1) is a key enzyme involved in glycolysis and gluconeogenesis. Glycolysis is one of the most regulated metabolic pathways, however little is known about the structural mechanisms for its regulation in non-model organisms, like crustaceans. To understand the structure and function of this enzyme in invertebrates, we obtained the crystal structure of triosephosphate isomerase from the marine Pacific whiteleg shrimp (Litopenaeus vannamei, LvTIM) in complex with its inhibitor 2-phosphogyceric acid (2-PG) at 1.7 Å resolution. LvTIM assembles as a homodimer with residues 166-176 covering the active site and residue Glu166 interacting with the inhibitor. We found that LvTIM is the least stable TIM characterized to date, with the lowest range of melting temperatures, and with the lowest activation enthalpy associated with the thermal unfolding process reported. In TIMs dimer stabilization is maintained by an interaction of loop 3 by a set of hydrophobic contacts between subunits. Within these contacts, the side chain of a hydrophobic residue of one subunit fits into a cavity created by a set of hydrophobic residues in the neighboring subunit, via a "ball and socket" interaction. LvTIM presents a Cys47 at the "ball" inter-subunit contact indicating that the character of this residue is responsible for the decrease in dimer stability. Mutational studies show that this residue plays a role in dimer stability but is not a solely determinant for dimer formation. PMID:27614148

Historical connectivity, contemporary isolation and local adaptation in a widespread but discontinuously distributed species endemic to Taiwan, Rhododendron oldhamii (Ericaceae)

PubMed Central

Hsieh, Y-C; Chung, J-D; Wang, C-N; Chang, C-T; Chen, C-Y; Hwang, S-Y

2013-01-01

Elucidation of the evolutionary processes that constrain or facilitate adaptive divergence is a central goal in evolutionary biology, especially in non-model organisms. We tested whether changes in dynamics of gene flow (historical vs contemporary) caused population isolation and examined local adaptation in response to environmental selective forces in fragmented Rhododendron oldhamii populations. Variation in 26 expressed sequence tag-simple sequence repeat loci from 18 populations in Taiwan was investigated by examining patterns of genetic diversity, inbreeding, geographic structure, recent bottlenecks, and historical and contemporary gene flow. Selection associated with environmental variables was also examined. Bayesian clustering analysis revealed four regional population groups of north, central, south and southeast with significant genetic differentiation. Historical bottlenecks beginning 9168–13,092 years ago and ending 1584–3504 years ago were revealed by estimates using approximate Bayesian computation for all four regional samples analyzed. Recent migration within and across geographic regions was limited. However, major dispersal sources were found within geographic regions. Altitudinal clines of allelic frequencies of environmentally associated positively selected outliers were found, indicating adaptive divergence. Our results point to a transition from historical population connectivity toward contemporary population isolation and divergence on a regional scale. Spatial and temporal dispersal differences may have resulted in regional population divergence and local adaptation associated with environmental variables, which may have played roles as selective forces at a regional scale. PMID:23591517

Use of genotyping by sequencing data to develop a high-throughput and multifunctional SNP panel for conservation applications in Pacific lamprey.

PubMed

Hess, Jon E; Campbell, Nathan R; Docker, Margaret F; Baker, Cyndi; Jackson, Aaron; Lampman, Ralph; McIlraith, Brian; Moser, Mary L; Statler, David P; Young, William P; Wildbill, Andrew J; Narum, Shawn R

2015-01-01

Next-generation sequencing data can be mined for highly informative single nucleotide polymorphisms (SNPs) to develop high-throughput genomic assays for nonmodel organisms. However, choosing a set of SNPs to address a variety of objectives can be difficult because SNPs are often not equally informative. We developed an optimal combination of 96 high-throughput SNP assays from a total of 4439 SNPs identified in a previous study of Pacific lamprey (Entosphenus tridentatus) and used them to address four disparate objectives: parentage analysis, species identification and characterization of neutral and adaptive variation. Nine of these SNPs are FST outliers, and five of these outliers are localized within genes and significantly associated with geography, run-timing and dwarf life history. Two of the 96 SNPs were diagnostic for two other lamprey species that were morphologically indistinguishable at early larval stages and were sympatric in the Pacific Northwest. The majority (85) of SNPs in the panel were highly informative for parentage analysis, that is, putatively neutral with high minor allele frequency across the species' range. Results from three case studies are presented to demonstrate the broad utility of this panel of SNP markers in this species. As Pacific lamprey populations are undergoing rapid decline, these SNPs provide an important resource to address critical uncertainties associated with the conservation and recovery of this imperiled species. © 2014 John Wiley & Sons Ltd.

Functional and evolutionary implications from the molecular characterization of five spermatophore CHH/MIH/GIH genes in the shrimp Fenneropenaeus merguiensis.

PubMed

Shi, LiLi; Li, Bin; Zhou, Ting Ting; Wang, Wei; Chan, Siuming F

2018-01-01

The recent use of RNA-Seq to study the transcriptomes of different species has helped identify a large number of new genes from different non-model organisms. In this study, five distinctive transcripts encoding for neuropeptide members of the CHH/MIH/GIH family have been identified from the spermatophore transcriptome of the shrimp Fenneropenaeus merguiensis. The size of these transcripts ranged from 531 bp to 1771 bp. Four transcripts encoded different CHH-family subtype I members, and one transcript encoded a subtype II member. RT-PCR and RACE approaches have confirmed the expression of these genes in males. The low degree of amino acid sequence identity among these neuropeptides suggests that they may have different specific function(s). Results from a phylogenetic tree analysis indicated that these neuropeptides were likely derived from a common ancestor gene resulting from mutation and gene duplication. These CHH-family members could be grouped into distinct clusters, indicating a strong structural/functional relationship among these neuropeptides. Eyestalk removal caused a significant increase in the expression of transcript 32710 but decreases in expression for transcript 28020. These findings suggest the possible regulation of these genes by eyestalk factor(s). In summary, the results of this study would justify a re-evaluation of the more generalized and pleiotropic functions of these neuropeptides. This study also represents the first report on the cloning/identification of five CHH family neuropeptides in a non-neuronal tissue from a single crustacean species.

Transcriptional Profiling of Metabolic Transitions during Development and Diapause Preparation in the Copepod Calanus finmarchicus.

PubMed

Tarrant, Ann M; Baumgartner, Mark F; Lysiak, Nadine S J; Altin, Dag; Størseth, Trond R; Hansen, Bjørn Henrik

2016-12-01

Calanus finmarchicus, like many other copepods in the family Calanidae, can enter into a facultative diapause during the last juvenile phase (fifth copepodid, C5) to enable survival during unfavorable periods. Diapause is essential to the persistence of Calanus populations and profoundly impacts energy flow within oceanic ecosystems, yet regulation of diapause is not understood in these animals. Transcriptional profiling has begun to provide insight into metabolic changes occurring as C. finmarchicus prepares for and enters into diapause or skips diapause to prepare for the terminal molt. In particular, components of the glycolysis, pentose phosphate and lipid synthesis pathways are upregulated early in the C5 stage when lipid stores are low. Currently, our ability to identify metabolic patterns is limited by the incomplete functional annotation of the C. finmarchicus transcriptome. Such limitations are widespread among studies of non-model organisms and addressing them should be a priority for future research. In addition, integrating the results across multiple emerging complementary transcriptomic studies will provide a more complete picture of copepod physiology than isolated studies. Ultimately, identifying molecular markers of copepod physiology could enable robust identification of animals preparing to enter into diapause and ultimately lead to a greatly improved understanding of diapause regulation. © The Author 2016. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Abiotic and Biotic Stressors Causing Equivalent Mortality Induce Highly Variable Transcriptional Responses in the Soybean Aphid

PubMed Central

Enders, Laramy S.; Bickel, Ryan D.; Brisson, Jennifer A.; Heng-Moss, Tiffany M.; Siegfried, Blair D.; Zera, Anthony J.; Miller, Nicholas J.

2014-01-01

Environmental stress affects basic organismal functioning and can cause physiological, developmental, and reproductive impairment. However, in many nonmodel organisms, the core molecular stress response remains poorly characterized and the extent to which stress-induced transcriptional changes differ across qualitatively different stress types is largely unexplored. The current study examines the molecular stress response of the soybean aphid (Aphis glycines) using RNA sequencing and compares transcriptional responses to multiple stressors (heat, starvation, and plant defenses) at a standardized stress level (27% adult mortality). Stress-induced transcriptional changes showed remarkable variation, with starvation, heat, and plant defensive stress altering the expression of 3985, 510, and 12 genes, respectively. Molecular responses showed little overlap across all three stressors. However, a common transcriptional stress response was identified under heat and starvation, involved with up-regulation of glycogen biosynthesis and molecular chaperones and down-regulation of bacterial endosymbiont cellular and insect cuticular components. Stressor-specific responses indicated heat affected expression of heat shock proteins and cuticular components, whereas starvation altered a diverse set of genes involved in primary metabolism, oxidative reductive processes, nucleosome and histone assembly, and the regulation of DNA repair and replication. Exposure to host plant defenses elicited the weakest response, of which half of the genes were of unknown function. This study highlights the need for standardizing stress levels when comparing across stress types and provides a basis for understanding the role of general vs. stressor specific molecular responses in aphids. PMID:25538100

Genomics in a changing arctic: critical questions await the molecular ecologist.

PubMed

Wullschleger, Stan D; Breen, Amy L; Iversen, Colleen M; Olson, Matthew S; Näsholm, Torgny; Ganeteg, Ulrika; Wallenstein, Matthew D; Weston, David J

2015-05-01

Molecular ecology is poised to tackle a host of interesting questions in the coming years. The Arctic provides a unique and rapidly changing environment with a suite of emerging research needs that can be addressed through genetics and genomics. Here we highlight recent research on boreal and tundra ecosystems and put forth a series of questions related to plant and microbial responses to climate change that can benefit from technologies and analytical approaches contained within the molecular ecologist's toolbox. These questions include understanding (i) the mechanisms of plant acquisition and uptake of N in cold soils, (ii) how these processes are mediated by root traits, (iii) the role played by the plant microbiome in cycling C and nutrients within high-latitude ecosystems and (iv) plant adaptation to extreme Arctic climates. We highlight how contributions can be made in these areas through studies that target model and nonmodel organisms and emphasize that the sequencing of the Populus and Salix genomes provides a valuable resource for scientific discoveries related to the plant microbiome and plant adaptation in the Arctic. Moreover, there exists an exciting role to play in model development, including incorporating genetic and evolutionary knowledge into ecosystem and Earth System Models. In this regard, the molecular ecologist provides a valuable perspective on plant genetics as a driver for community biodiversity, and how ecological and evolutionary forces govern community dynamics in a rapidly changing climate. © 2015 John Wiley & Sons Ltd.

The floral transcriptomes of four bamboo species (Bambusoideae; Poaceae): support for common ancestry among woody bamboos.

PubMed

Wysocki, William P; Ruiz-Sanchez, Eduardo; Yin, Yanbin; Duvall, Melvin R

2016-05-20

Next-generation sequencing now allows for total RNA extracts to be sequenced in non-model organisms such as bamboos, an economically and ecologically important group of grasses. Bamboos are divided into three lineages, two of which are woody perennials with bisexual flowers, which undergo gregarious monocarpy. The third lineage, which are herbaceous perennials, possesses unisexual flowers that undergo annual flowering events. Transcriptomes were assembled using both reference-based and de novo methods. These two methods were tested by characterizing transcriptome content using sequence alignment to previously characterized reference proteomes and by identifying Pfam domains. Because of the striking differences in floral morphology and phenology between the herbaceous and woody bamboo lineages, MADS-box genes, transcription factors that control floral development and timing, were characterized and analyzed in this study. Transcripts were identified using phylogenetic methods and categorized as A, B, C, D or E-class genes, which control floral development, or SOC or SVP-like genes, which control the timing of flowering events. Putative nuclear orthologues were also identified in bamboos to use as phylogenetic markers. Instances of gene copies exhibiting topological patterns that correspond to shared phenotypes were observed in several gene families including floral development and timing genes. Alignments and phylogenetic trees were generated for 3,878 genes and for all genes in a concatenated analysis. Both the concatenated analysis and those of 2,412 separate gene trees supported monophyly among the woody bamboos, which is incongruent with previous phylogenetic studies using plastid markers.

Recombination rate plasticity: revealing mechanisms by design

PubMed Central

Sefick, Stephen; Rushton, Chase

2017-01-01

For over a century, scientists have known that meiotic recombination rates can vary considerably among individuals, and that environmental conditions can modify recombination rates relative to the background. A variety of external and intrinsic factors such as temperature, age, sex and starvation can elicit ‘plastic’ responses in recombination rate. The influence of recombination rate plasticity on genetic diversity of the next generation has interesting and important implications for how populations evolve. Further, many questions remain regarding the mechanisms and molecular processes that contribute to recombination rate plasticity. Here, we review 100 years of experimental work on recombination rate plasticity conducted in Drosophila melanogaster. We categorize this work into four major classes of experimental designs, which we describe via classic studies in D. melanogaster. Based on these studies, we highlight molecular mechanisms that are supported by experimental results and relate these findings to studies in other systems. We synthesize lessons learned from this model system into experimental guidelines for using recent advances in genotyping technologies, to study recombination rate plasticity in non-model organisms. Specifically, we recommend (1) using fine-scale genome-wide markers, (2) collecting time-course data, (3) including crossover distribution measurements, and (4) using mixed effects models to analyse results. To illustrate this approach, we present an application adhering to these guidelines from empirical work we conducted in Drosophila pseudoobscura. This article is part of the themed issue ‘Evolutionary causes and consequences of recombination rate variation in sexual organisms’. PMID:29109222

Molecular biomarkers for chronological age in animal ecology.

PubMed

Jarman, Simon N; Polanowski, Andrea M; Faux, Cassandra E; Robbins, Jooke; De Paoli-Iseppi, Ricardo; Bravington, Mark; Deagle, Bruce E

2015-10-01

The chronological age of an individual animal predicts many of its biological characteristics, and these in turn influence population-level ecological processes. Animal age information can therefore be valuable in ecological research, but many species have no external features that allow age to be reliably determined. Molecular age biomarkers provide a potential solution to this problem. Research in this area of molecular ecology has so far focused on a limited range of age biomarkers. The most commonly tested molecular age biomarker is change in average telomere length, which predicts age well in a small number of species and tissues, but performs poorly in many other situations. Epigenetic regulation of gene expression has recently been shown to cause age-related modifications to DNA and to cause changes in abundance of several RNA types throughout animal lifespans. Age biomarkers based on these epigenetic changes, and other new DNA-based assays, have already been applied to model organisms, humans and a limited number of wild animals. There is clear potential to apply these marker types more widely in ecological studies. For many species, these new approaches will produce age estimates where this was previously impractical. They will also enable age information to be gathered in cross-sectional studies and expand the range of demographic characteristics that can be quantified with molecular methods. We describe the range of molecular age biomarkers that have been investigated to date and suggest approaches for developing the newer marker types as age assays in nonmodel animal species. © 2015 John Wiley & Sons Ltd.

Genome-wide survey of single-nucleotide polymorphisms reveals fine-scale population structure and signs of selection in the threatened Caribbean elkhorn coral, Acropora palmata

PubMed Central

2017-01-01

The advent of next-generation sequencing tools has made it possible to conduct fine-scale surveys of population differentiation and genome-wide scans for signatures of selection in non-model organisms. Such surveys are of particular importance in sharply declining coral species, since knowledge of population boundaries and signs of local adaptation can inform restoration and conservation efforts. Here, we use genome-wide surveys of single-nucleotide polymorphisms in the threatened Caribbean elkhorn coral, Acropora palmata, to reveal fine-scale population structure and infer the major barrier to gene flow that separates the eastern and western Caribbean populations between the Bahamas and Puerto Rico. The exact location of this break had been subject to discussion because two previous studies based on microsatellite data had come to differing conclusions. We investigate this contradiction by analyzing an extended set of 11 microsatellite markers including the five previously employed and discovered that one of the original microsatellite loci is apparently under selection. Exclusion of this locus reconciles the results from the SNP and the microsatellite datasets. Scans for outlier loci in the SNP data detected 13 candidate loci under positive selection, however there was no correlation between available environmental parameters and genetic distance. Together, these results suggest that reef restoration efforts should use local sources and utilize existing functional variation among geographic regions in ex situ crossing experiments to improve stress resistance of this species. PMID:29181279

xylA and xylB overexpression as a successful strategy for improving xylose utilization and poly-3-hydroxybutyrate production in Burkholderia sacchari.

PubMed

Guamán, Linda P; Oliveira-Filho, Edmar R; Barba-Ostria, Carlos; Gomez, José G C; Taciro, Marilda K; da Silva, Luiziana Ferreira

2018-03-01

Despite the versatility and many advantages of polyhydroxyalkanoates as petroleum-based plastic substitutes, their higher production cost compared to petroleum-based polymers has historically limited their large-scale production. One appealing approach to reducing production costs is to employ less expensive, renewable feedstocks. Xylose, for example is an abundant and inexpensive carbon source derived from hemicellulosic residues abundant in agro-industrial waste (sugarcane bagasse hemicellulosic hydrolysates). In this work, the production of poly-3-hydroxybutyrate P(3HB) from xylose was studied to develop technologies for conversion of agro-industrial waste into high-value chemicals and biopolymers. Specifically, this work elucidates the organization of the xylose assimilation operon of Burkholderia sacchari, a non-model bacterium with high capacity for P(3HB) accumulation. Overexpression of endogenous xylose isomerase and xylulokinase genes was successfully assessed, improving both specific growth rate and P(3HB) production. Compared to control strain (harboring pBBR1MCS-2), xylose utilization in the engineered strain was substantially improved with 25% increase in specific growth rate, 34% increase in P(3HB) production, and the highest P(3HB) yield from xylose reported to date for B. sacchari (Y P3HB/Xil  = 0.35 g/g). This study highlights that xylA and xylB overexpression is an effective strategy to improve xylose utilization and P(3HB) production in B. sacchari.

Engineering microbial electrocatalysis for chemical and fuel production.

PubMed

Rosenbaum, Miriam A; Henrich, Alexander W

2014-10-01

In many biotechnological areas, metabolic engineering and synthetic biology have become core technologies for biocatalyst development. Microbial electrocatalysis for biochemical and fuel production is still in its infancy and reactions rates and the product spectrum are currently very low. Therefore, molecular engineering strategies will be crucial for the advancement and realization of many new bioproduction routes using electroactive microorganisms. The complex and unresolved biochemistry and physiology of extracellular electron transfer and the lack of molecular tools for these new non-model hosts for genetic engineering constitute the major challenges for this effort. This review is providing an insight into the current status, challenges and promising approaches of pathway engineering for microbial electrocatalysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Function and evolution of sex determination mechanisms, genes and pathways in insects

PubMed Central

Gempe, Tanja; Beye, Martin

2011-01-01

Animals have evolved a bewildering diversity of mechanisms to determine the two sexes. Studies of sex determination genes – their history and function – in non-model insects and Drosophila have allowed us to begin to understand the generation of sex determination diversity. One common theme from these studies is that evolved mechanisms produce activities in either males or females to control a shared gene switch that regulates sexual development. Only a few small-scale changes in existing and duplicated genes are sufficient to generate large differences in sex determination systems. This review summarises recent findings in insects, surveys evidence of how and why sex determination mechanisms can change rapidly and suggests fruitful areas of future research. PMID:21110346

Emerging from the bottleneck: Benefits of the comparative approach to modern neuroscience

PubMed Central

Brenowitz, Eliot A.; Zakon, Harold H.

2015-01-01

Neuroscience historically exploited a wide diversity of animal taxa. Recently, however, research focused increasingly on a few model species. This trend accelerated with the genetic revolution, as genomic sequences and genetic tools became available for a few species, which formed a bottleneck. This coalescence on a small set of model species comes with several costs often not considered, especially in the current drive to use mice explicitly as models for human diseases. Comparative studies of strategically chosen non-model species can complement model species research and yield more rigorous studies. As genetic sequences and tools become available for many more species, we are poised to emerge from the bottleneck and once again exploit the rich biological diversity offered by comparative studies. PMID:25800324

Do plants have a segregated germline?

PubMed Central

2018-01-01

For the last 100 years, it has been uncontroversial to state that the plant germline is set aside late in development, but there is surprisingly little evidence to support this view. In contrast, much evolutionary theory and several recent empirical studies seem to suggest the opposite—that the germlines of some and perhaps most plants may be set aside early in development. But is this really the case? How much does it matter? How can we reconcile the new evidence with existing knowledge of plant development? And is there a way to reliably establish the timing of germline segregation in both model and nonmodel plants? Answering these questions is vital to understanding one of the most fundamental aspects of plant development and evolution. PMID:29768400

Nematocytes: Discovery and characterization of a novel anculeate hemocyte in Drosophila falleni and Drosophila phalerata.

PubMed

Bozler, Julianna; Kacsoh, Balint Z; Bosco, Giovanni

2017-01-01

Immune challenges, such as parasitism, can be so pervasive and deleterious that they constitute an existential threat to a species' survival. In response to these ecological pressures, organisms have developed a wide array of novel behavioral, cellular, and molecular adaptations. Research into these immune defenses in model systems has resulted in a revolutionary understanding of evolution and functional biology. As the field has expanded beyond the limited number of model organisms our appreciation of evolutionary innovation and unique biology has widened as well. With this in mind, we have surveyed the hemolymph of several non-model species of Drosophila. Here we identify and describe a novel hemocyte, type-II nematocytes, found in larval stages of numerous Drosophila species. Examined in detail in Drosophila falleni and Drosophila phalerata, we find that these remarkable cells are distinct from previously described hemocytes due to their anucleate state (lacking a nucleus) and unusual morphology. Type-II nematocytes are long, narrow cells with spindle-like projections extending from a cell body with high densities of mitochondria and microtubules, and exhibit the ability to synthesize proteins. These properties are unexpected for enucleated cells, and together with our additional characterization, we demonstrate that these type-II nematocytes represent a biological novelty. Surprisingly, despite the absence of a nucleus, we observe through live cell imaging that these cells remain motile with a highly dynamic cellular shape. Furthermore, these cells demonstrate the ability to form multicellular structures, which we suggest may be a component of the innate immune response to macro-parasites. In addition, live cell imaging points to a large nucleated hemocyte, type-I nematocyte, as the progenitor cell, leading to enucleation through a budding or asymmetrical division process rather than nuclear ejection: This study is the first to report such a process of enucleation. Here we describe these cells in detail for the first time and examine their evolutionary history in Drosophila.

TCW: Transcriptome Computational Workbench

PubMed Central

Soderlund, Carol; Nelson, William; Willer, Mark; Gang, David R.

2013-01-01

Background The analysis of transcriptome data involves many steps and various programs, along with organization of large amounts of data and results. Without a methodical approach for storage, analysis and query, the resulting ad hoc analysis can lead to human error, loss of data and results, inefficient use of time, and lack of verifiability, repeatability, and extensibility. Methodology The Transcriptome Computational Workbench (TCW) provides Java graphical interfaces for methodical analysis for both single and comparative transcriptome data without the use of a reference genome (e.g. for non-model organisms). The singleTCW interface steps the user through importing transcript sequences (e.g. Illumina) or assembling long sequences (e.g. Sanger, 454, transcripts), annotating the sequences, and performing differential expression analysis using published statistical programs in R. The data, metadata, and results are stored in a MySQL database. The multiTCW interface builds a comparison database by importing sequence and annotation from one or more single TCW databases, executes the ESTscan program to translate the sequences into proteins, and then incorporates one or more clusterings, where the clustering options are to execute the orthoMCL program, compute transitive closure, or import clusters. Both singleTCW and multiTCW allow extensive query and display of the results, where singleTCW displays the alignment of annotation hits to transcript sequences, and multiTCW displays multiple transcript alignments with MUSCLE or pairwise alignments. The query programs can be executed on the desktop for fastest analysis, or from the web for sharing the results. Conclusion It is now affordable to buy a multi-processor machine, and easy to install Java and MySQL. By simply downloading the TCW, the user can interactively analyze, query and view their data. The TCW allows in-depth data mining of the results, which can lead to a better understanding of the transcriptome. TCW is freely available from www.agcol.arizona.edu/software/tcw. PMID:23874959

TCW: transcriptome computational workbench.

PubMed

Soderlund, Carol; Nelson, William; Willer, Mark; Gang, David R

2013-01-01

The analysis of transcriptome data involves many steps and various programs, along with organization of large amounts of data and results. Without a methodical approach for storage, analysis and query, the resulting ad hoc analysis can lead to human error, loss of data and results, inefficient use of time, and lack of verifiability, repeatability, and extensibility. The Transcriptome Computational Workbench (TCW) provides Java graphical interfaces for methodical analysis for both single and comparative transcriptome data without the use of a reference genome (e.g. for non-model organisms). The singleTCW interface steps the user through importing transcript sequences (e.g. Illumina) or assembling long sequences (e.g. Sanger, 454, transcripts), annotating the sequences, and performing differential expression analysis using published statistical programs in R. The data, metadata, and results are stored in a MySQL database. The multiTCW interface builds a comparison database by importing sequence and annotation from one or more single TCW databases, executes the ESTscan program to translate the sequences into proteins, and then incorporates one or more clusterings, where the clustering options are to execute the orthoMCL program, compute transitive closure, or import clusters. Both singleTCW and multiTCW allow extensive query and display of the results, where singleTCW displays the alignment of annotation hits to transcript sequences, and multiTCW displays multiple transcript alignments with MUSCLE or pairwise alignments. The query programs can be executed on the desktop for fastest analysis, or from the web for sharing the results. It is now affordable to buy a multi-processor machine, and easy to install Java and MySQL. By simply downloading the TCW, the user can interactively analyze, query and view their data. The TCW allows in-depth data mining of the results, which can lead to a better understanding of the transcriptome. TCW is freely available from www.agcol.arizona.edu/software/tcw.

A deep transcriptomic resource for the copepod crustacean Labidocera madurae: A potential indicator species for assessing near shore ecosystem health

PubMed Central

Christie, Andrew E.; Sommer, Stephanie A.; Cieslak, Matthew C.; Hartline, Daniel K.; Lenz, Petra H.

2017-01-01

Coral reef ecosystems of many sub-tropical and tropical marine coastal environments have suffered significant degradation from anthropogenic sources. Research to inform management strategies that mitigate stressors and promote a healthy ecosystem has focused on the ecology and physiology of coral reefs and associated organisms. Few studies focus on the surrounding pelagic communities, which are equally important to ecosystem function. Zooplankton, often dominated by small crustaceans such as copepods, is an important food source for invertebrates and fishes, especially larval fishes. The reef-associated zooplankton includes a sub-neustonic copepod family that could serve as an indicator species for the community. Here, we describe the generation of a de novo transcriptome for one such copepod, Labidocera madurae, a pontellid from an intensively-studied coral reef ecosystem, Kāne‘ohe Bay, Oahu, Hawai‘i. The transcriptome was assembled using high-throughput sequence data obtained from whole organisms. It comprised 211,002 unique transcripts, including 72,391 with coding regions. It was assessed for quality and completeness using multiple workflows. Bench-marking-universal-single-copy-orthologs (BUSCO) analysis identified transcripts for 88% of expected eukaryotic core proteins. Targeted gene-discovery analyses included searches for transcripts coding full-length “giant” proteins (>4,000 amino acids), proteins and splice variants of voltage-gated sodium channels, and proteins involved in the circadian signaling pathway. Four different reference transcriptomes were generated and compared for the detection of differential gene expression between copepodites and adult females; 6,229 genes were consistently identified as differentially expressed between the two regardless of reference. Automated bioinformatics analyses and targeted manual gene curation suggest that the de novo assembled L. madurae transcriptome is of high quality and completeness. This transcriptome provides a new resource for assessing the global physiological status of a planktonic species inhabiting a coral reef ecosystem that is subjected to multiple anthropogenic stressors. The workflows provide a template for generating and assessing transcriptomes in other non-model species. PMID:29065152

A deep transcriptomic resource for the copepod crustacean Labidocera madurae: A potential indicator species for assessing near shore ecosystem health.

PubMed

Roncalli, Vittoria; Christie, Andrew E; Sommer, Stephanie A; Cieslak, Matthew C; Hartline, Daniel K; Lenz, Petra H

2017-01-01

Coral reef ecosystems of many sub-tropical and tropical marine coastal environments have suffered significant degradation from anthropogenic sources. Research to inform management strategies that mitigate stressors and promote a healthy ecosystem has focused on the ecology and physiology of coral reefs and associated organisms. Few studies focus on the surrounding pelagic communities, which are equally important to ecosystem function. Zooplankton, often dominated by small crustaceans such as copepods, is an important food source for invertebrates and fishes, especially larval fishes. The reef-associated zooplankton includes a sub-neustonic copepod family that could serve as an indicator species for the community. Here, we describe the generation of a de novo transcriptome for one such copepod, Labidocera madurae, a pontellid from an intensively-studied coral reef ecosystem, Kāne'ohe Bay, Oahu, Hawai'i. The transcriptome was assembled using high-throughput sequence data obtained from whole organisms. It comprised 211,002 unique transcripts, including 72,391 with coding regions. It was assessed for quality and completeness using multiple workflows. Bench-marking-universal-single-copy-orthologs (BUSCO) analysis identified transcripts for 88% of expected eukaryotic core proteins. Targeted gene-discovery analyses included searches for transcripts coding full-length "giant" proteins (>4,000 amino acids), proteins and splice variants of voltage-gated sodium channels, and proteins involved in the circadian signaling pathway. Four different reference transcriptomes were generated and compared for the detection of differential gene expression between copepodites and adult females; 6,229 genes were consistently identified as differentially expressed between the two regardless of reference. Automated bioinformatics analyses and targeted manual gene curation suggest that the de novo assembled L. madurae transcriptome is of high quality and completeness. This transcriptome provides a new resource for assessing the global physiological status of a planktonic species inhabiting a coral reef ecosystem that is subjected to multiple anthropogenic stressors. The workflows provide a template for generating and assessing transcriptomes in other non-model species.

Characterization and comparative analysis of the genome of Puccinia sorghi Schwein, the causal agent of maize common rust.

PubMed

Rochi, Lucia; Diéguez, María José; Burguener, Germán; Darino, Martín Alejandro; Pergolesi, María Fernanda; Ingala, Lorena Romina; Cuyeu, Alba Romina; Turjanski, Adrián; Kreff, Enrique Domingo; Sacco, Francisco

2018-03-01

Rust fungi are one of the most devastating pathogens of crop plants. The biotrophic fungus Puccinia sorghi Schwein (Ps) is responsible for maize common rust, an endemic disease of maize (Zea mays L.) in Argentina that causes significant yield losses in corn production. In spite of this, the Ps genomic sequence was not available. We used Illumina sequencing to rapidly produce the 99.6Mbdraft genome sequence of Ps race RO10H11247, derived from a single-uredinial isolate from infected maize leaves collected in the Argentine Corn Belt Region during 2010. High quality reads were obtained from 200bppaired-end and 5000bpmate-paired libraries and assembled in 15,722 scaffolds. A pipeline which combined an ab initio program with homology-based models and homology to in planta enriched ESTs from four cereal pathogenic fungus (the three sequenced wheat rusts and Ustilago maydis) was used to identify 21,087 putative coding sequences, of which 1599 might be part of the Ps RO10H11247 secretome. Among the 458 highly conserved protein families from the euKaryotic Orthologous Groups (KOG) that occur in a wide range of eukaryotic organisms, 97.5% have at least one member with high homology in the Ps assembly (TBlastN, E-value⩽e-10) covering more than 50% of the length of the KOG protein. Comparative studies with the three sequenced wheat rust fungus, and microsynteny analysis involving Puccinia striiformis f. sp. tritici (Pst, wheat stripe rust fungus), support the quality achieved. The results presented here show the effectiveness of the Illumina strategy for sequencing dikaryotic genomes of non-model organisms and provides reliable DNA sequence information for genomic studies, including pathogenic mechanisms of this maize fungus and molecular marker design. Copyright © 2016 Elsevier Inc. All rights reserved.

Improved prediction of peptide detectability for targeted proteomics using a rank-based algorithm and organism-specific data.

PubMed

Qeli, Ermir; Omasits, Ulrich; Goetze, Sandra; Stekhoven, Daniel J; Frey, Juerg E; Basler, Konrad; Wollscheid, Bernd; Brunner, Erich; Ahrens, Christian H

2014-08-28

The in silico prediction of the best-observable "proteotypic" peptides in mass spectrometry-based workflows is a challenging problem. Being able to accurately predict such peptides would enable the informed selection of proteotypic peptides for targeted quantification of previously observed and non-observed proteins for any organism, with a significant impact for clinical proteomics and systems biology studies. Current prediction algorithms rely on physicochemical parameters in combination with positive and negative training sets to identify those peptide properties that most profoundly affect their general detectability. Here we present PeptideRank, an approach that uses learning to rank algorithm for peptide detectability prediction from shotgun proteomics data, and that eliminates the need to select a negative dataset for the training step. A large number of different peptide properties are used to train ranking models in order to predict a ranking of the best-observable peptides within a protein. Empirical evaluation with rank accuracy metrics showed that PeptideRank complements existing prediction algorithms. Our results indicate that the best performance is achieved when it is trained on organism-specific shotgun proteomics data, and that PeptideRank is most accurate for short to medium-sized and abundant proteins, without any loss in prediction accuracy for the important class of membrane proteins. Targeted proteomics approaches have been gaining a lot of momentum and hold immense potential for systems biology studies and clinical proteomics. However, since only very few complete proteomes have been reported to date, for a considerable fraction of a proteome there is no experimental proteomics evidence that would allow to guide the selection of the best-suited proteotypic peptides (PTPs), i.e. peptides that are specific to a given proteoform and that are repeatedly observed in a mass spectrometer. We describe a novel, rank-based approach for the prediction of the best-suited PTPs for targeted proteomics applications. By building on methods developed in the field of information retrieval (e.g. web search engines like Google's PageRank), we circumvent the delicate step of selecting positive and negative training sets and at the same time also more closely reflect the experimentalist´s need for selecting e.g. the 5 most promising peptides for targeting a protein of interest. This approach allows to predict PTPs for not yet observed proteins or for organisms without prior experimental proteomics data such as many non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression and RNA interference of salivary polygalacturonase genes in the tarnished plant bug, Lygus lineolaris.

PubMed

Walker, William B; Allen, Margaret L

2010-01-01

Three genes encoding polygalacturonase (PG) have been identified in Lygus lineolaris (Palisot de Beauvois) (Miridae: Hemiptera). Earlier studies showed that the three PG gene transcripts are exclusively expressed in the feeding stages of L. lineolaris. In this report, it is shown that all three transcripts are specifically expressed in salivary glands indicating that PGs are salivary enzymes. Transcriptional profiles of the three PGs were evaluated with respect to diet, comparing live cotton plant material to artificial diet. PG2 transcript levels were consistently lower in cotton-fed insects than those reared on artificial diet. RNA interference was used to knock down expression of PG1 mRNA in adult salivary glands providing the first demonstration of the use of this method in the non-model insect, L. lineolaris.

An essential cell cycle regulation gene causes hybrid inviability in Drosophila

PubMed Central

Phadnis, Nitin; Baker, EmilyClare P.; Cooper, Jacob C.; Frizzell, Kimberly A.; Hsieh, Emily; de la Cruz, Aida Flor A.; Shendure, Jay; Kitzman, Jacob O.; Malik, Harmit S.

2015-01-01

Speciation, the process by which new biological species arise, involves the evolution of reproductive barriers such as hybrid sterility or inviability between populations. However, identifying hybrid incompatibility genes remains a key obstacle in understanding the molecular basis of reproductive isolation. We devised a genomic screen, which identified a cell cycle regulation gene as the cause of male inviability in hybrids between Drosophila melanogaster and D. simulans. Ablation of the D. simulans allele of this gene is sufficient to rescue the adult viability of hybrid males. This dominantly acting cell cycle regulator causes mitotic arrest and, thereby, inviability of male hybrid larvae. Our genomic method provides a facile means to accelerate the identification of hybrid incompatibility genes in other model and non-model systems. PMID:26680200

Symbiont-mediated RNA interference in insects

PubMed Central

Whitten, Miranda M. A.; Facey, Paul D.; Del Sol, Ricardo; Fernández-Martínez, Lorena T.; Evans, Meirwyn C.; Mitchell, Jacob J.; Bodger, Owen G.

2016-01-01

RNA interference (RNAi) methods for insects are often limited by problems with double-stranded (ds) RNA delivery, which restricts reverse genetics studies and the development of RNAi-based biocides. We therefore delegated to insect symbiotic bacteria the task of: (i) constitutive dsRNA synthesis and (ii) trauma-free delivery. RNaseIII-deficient, dsRNA-expressing bacterial strains were created from the symbionts of two very diverse pest species: a long-lived blood-sucking bug, Rhodnius prolixus, and a short-lived globally invasive polyphagous agricultural pest, western flower thrips (Frankliniella occidentalis). When ingested, the manipulated bacteria colonized the insects, successfully competed with the wild-type microflora, and sustainably mediated systemic knockdown phenotypes that were horizontally transmissible. This represents a significant advance in the ability to deliver RNAi, potentially to a large range of non-model insects. PMID:26911963

Phylogenetic Conflict in Bears Identified by Automated Discovery of Transposable Element Insertions in Low-Coverage Genomes

PubMed Central

Gallus, Susanne; Janke, Axel

2017-01-01

Abstract Phylogenetic reconstruction from transposable elements (TEs) offers an additional perspective to study evolutionary processes. However, detecting phylogenetically informative TE insertions requires tedious experimental work, limiting the power of phylogenetic inference. Here, we analyzed the genomes of seven bear species using high-throughput sequencing data to detect thousands of TE insertions. The newly developed pipeline for TE detection called TeddyPi (TE detection and discovery for Phylogenetic Inference) identified 150,513 high-quality TE insertions in the genomes of ursine and tremarctine bears. By integrating different TE insertion callers and using a stringent filtering approach, the TeddyPi pipeline produced highly reliable TE insertion calls, which were confirmed by extensive in vitro validation experiments. Analysis of single nucleotide substitutions in the flanking regions of the TEs shows that these substitutions correlate with the phylogenetic signal from the TE insertions. Our phylogenomic analyses show that TEs are a major driver of genomic variation in bears and enabled phylogenetic reconstruction of a well-resolved species tree, despite strong signals for incomplete lineage sorting and introgression. The analyses show that the Asiatic black, sun, and sloth bear form a monophyletic clade, in which phylogenetic incongruence originates from incomplete lineage sorting. TeddyPi is open source and can be adapted to various TE and structural variation callers. The pipeline makes it possible to confidently extract thousands of TE insertions even from low-coverage genomes (∼10×) of nonmodel organisms. This opens new possibilities for biologists to study phylogenies and evolutionary processes as well as rates and patterns of (retro-)transposition and structural variation. PMID:28985298

Overview Article: Identifying transcriptional cis-regulatory modules in animal genomes

PubMed Central

Suryamohan, Kushal; Halfon, Marc S.

2014-01-01

Gene expression is regulated through the activity of transcription factors and chromatin modifying proteins acting on specific DNA sequences, referred to as cis-regulatory elements. These include promoters, located at the transcription initiation sites of genes, and a variety of distal cis-regulatory modules (CRMs), the most common of which are transcriptional enhancers. Because regulated gene expression is fundamental to cell differentiation and acquisition of new cell fates, identifying, characterizing, and understanding the mechanisms of action of CRMs is critical for understanding development. CRM discovery has historically been challenging, as CRMs can be located far from the genes they regulate, have few readily-identifiable sequence characteristics, and for many years were not amenable to high-throughput discovery methods. However, the recent availability of complete genome sequences and the development of next-generation sequencing methods has led to an explosion of both computational and empirical methods for CRM discovery in model and non-model organisms alike. Experimentally, CRMs can be identified through chromatin immunoprecipitation directed against transcription factors or histone post-translational modifications, identification of nucleosome-depleted “open” chromatin regions, or sequencing-based high-throughput functional screening. Computational methods include comparative genomics, clustering of known or predicted transcription factor binding sites, and supervised machine-learning approaches trained on known CRMs. All of these methods have proven effective for CRM discovery, but each has its own considerations and limitations, and each is subject to a greater or lesser number of false-positive identifications. Experimental confirmation of predictions is essential, although shortcomings in current methods suggest that additional means of validation need to be developed. PMID:25704908

Testing Convergent Evolution in Auditory Processing Genes between Echolocating Mammals and the Aye-Aye, a Percussive-Foraging Primate

PubMed Central

Jerjos, Michael; Hohman, Baily; Lauterbur, M. Elise; Kistler, Logan

2017-01-01

Abstract Several taxonomically distinct mammalian groups—certain microbats and cetaceans (e.g., dolphins)—share both morphological adaptations related to echolocation behavior and strong signatures of convergent evolution at the amino acid level across seven genes related to auditory processing. Aye-ayes (Daubentonia madagascariensis) are nocturnal lemurs with a specialized auditory processing system. Aye-ayes tap rapidly along the surfaces of trees, listening to reverberations to identify the mines of wood-boring insect larvae; this behavior has been hypothesized to functionally mimic echolocation. Here we investigated whether there are signals of convergence in auditory processing genes between aye-ayes and known mammalian echolocators. We developed a computational pipeline (Basic Exon Assembly Tool) that produces consensus sequences for regions of interest from shotgun genomic sequencing data for nonmodel organisms without requiring de novo genome assembly. We reconstructed complete coding region sequences for the seven convergent echolocating bat–dolphin genes for aye-ayes and another lemur. We compared sequences from these two lemurs in a phylogenetic framework with those of bat and dolphin echolocators and appropriate nonecholocating outgroups. Our analysis reaffirms the existence of amino acid convergence at these loci among echolocating bats and dolphins; some methods also detected signals of convergence between echolocating bats and both mice and elephants. However, we observed no significant signal of amino acid convergence between aye-ayes and echolocating bats and dolphins, suggesting that aye-aye tap-foraging auditory adaptations represent distinct evolutionary innovations. These results are also consistent with a developing consensus that convergent behavioral ecology does not reliably predict convergent molecular evolution. PMID:28810710

The memory remains: application of historical DNA for scaling biodiversity loss.

PubMed

Nielsen, Einar E; Bekkevold, Dorte

2012-04-01

Few species worldwide have attracted as much attention in relation to conservation and sustainable management as Pacific salmon. Most populations have suffered significant reductions, many have disappeared, and even entire evolutionary significant units (ESUs) are believed to have been lost. Until now, no 'smoking gun' in terms of direct genetic evidence of the loss of a salmon ESU has been produced. In this issue of Molecular Ecology, Iwamoto et al. (2012) use microsatellite analysis of historical scale samples of Columbia River sockeye salmon (Oncorhynchus nerka) from 1924 (Fig. 1) to ask the pertinent question: Do the historical samples contain salmon from extirpated populations or ESUs? They identified four genetic groups in the historical samples of which two were almost genetically identical to contemporary ESUs in the river, one showed genetic relationship with a third ESU, but one group was not related to any of the contemporary populations. In association with ecological data, the genetic results suggest that an early migrating Columbia River headwater sockeye salmon ESU has been extirpated. The study has significant importance for conservation and reestablishment of sockeye populations in the Columbia River, but also underpins the general significance of shifting baselines in conservation biology, and how to assess loss of genetic biodiversity. The results clearly illustrate the huge and versatile potential of using historical DNA in population and conservation genetics. Because of the extraordinarily plentiful historical samples and rapid advances in fish genomics, fishes are likely to spearhead future studies of temporal ecological and population genomics in non-model organisms. [Figure: see text]. © 2012 Blackwell Publishing Ltd.

Genomics in a changing arctic: critical questions await the molecular ecologist

DOE PAGES

Wullschleger, Stan D.; Breen, Amy L.; Iversen, Colleen M.; ...

2015-04-20

Molecular ecology is poised to tackle a host of interesting questions in the coming years. Of particular importance to the molecular ecologist are new technologies and analytical approaches that provide opportunities to address questions previously unapproachable.The Arctic provides a unique and rapidly changing environment with a suite of emerging research needs that can be addressed through genetics and genomics. Here we highlight recent research on boreal and tundra ecosystems and put forth a series of questions related to plant and microbial responses to climate change that can benefit from technologies and analytical approaches contained within the molecular ecologist's toolbox. Thesemore » questions include understanding (i) the mechanisms of plant acquisition and uptake of N in cold soils, (ii) how these processes are mediated by root traits, (iii) the role played by the plant microbiome in cycling C and nutrients within high-latitude ecosystems and (iv) plant adaptation to extreme Arctic climates. We highlight how contributions can be made in these areas through studies that target model and nonmodel organisms and emphasize that the sequencing of the Populus and Salix genomes provides a valuable resource for scientific discoveries related to the plant microbiome and plant adaptation in the Arctic. Moreover, there exists an exciting role to play in model development, including incorporating genetic and evolutionary knowledge into ecosystem and Earth System Models. In this regard, the molecular ecologist provides a valuable perspective on plant genetics as a driver for community biodiversity, and how ecological and evolutionary forces govern community dynamics in a rapidly changing climate.« less

Comprehensive Transcriptome Analysis of Phytohormone Biosynthesis and Signaling Genes in the Flowers of Chinese Chinquapin (Castanea henryi).

PubMed

Fan, Xiaoming; Yuan, Deyi; Tian, Xiaoming; Zhu, Zhoujun; Liu, Meilan; Cao, Heping

2017-11-29

Chinese chinquapin (Castanea henryi) nut provides a rich source of starch and nutrients as food and feed, but its yield is restricted by a low ratio of female to male flowers. Little is known about the developmental programs underlying sex differentiation of the flowers. To investigate the involvement of phytohormones during sex differentiation, we described the morphology of male and female floral organs and the cytology of flower sex differentiation, analyzed endogenous levels of indole-3-acetic acid (IAA), gibberellins (GAs), cytokinins (CKs), and abscisic acid (ABA) in the flowers, investigated the effects of exogenous hormones on flower development, and evaluated the expression profiles of genes related to biosyntheses and signaling pathways of these four hormones using RNA-Seq combined with qPCR. Morphological results showed that the flowers consisted of unisexual and bisexual catkins, and could be divided into four developmental stages. HPLC results showed that CK accumulated much more in the female flowers than that in the male flowers, GA and ABA showed the opposite results, while IAA did not show a tendency. The effects of exogenous hormones on sex differentiation were consistent with those of endogenous hormones. RNA-Seq combined with qPCR analyses suggest that several genes may play key roles in hormone biosynthesis and sex differentiation. This study presents the first comprehensive report of phytohormone biosynthesis and signaling during sex differentiation of C. henryi, which should provide a foundation for further mechanistic studies of sex differentiation in Castanea Miller species and other nonmodel plants.

De Novo Assembly, Functional Annotation and Comparative Analysis of Withania somnifera Leaf and Root Transcriptomes to Identify Putative Genes Involved in the Withanolides Biosynthesis

PubMed Central

Gupta, Parul; Goel, Ridhi; Pathak, Sumya; Srivastava, Apeksha; Singh, Surya Pratap; Sangwan, Rajender Singh; Asif, Mehar Hasan; Trivedi, Prabodh Kumar

2013-01-01

Withania somnifera is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicine systems due to bioactive molecules known as withanolides. As genomic information regarding this plant is very limited, little information is available about biosynthesis of withanolides. To facilitate the basic understanding about the withanolide biosynthesis pathways, we performed transcriptome sequencing for Withania leaf (101L) and root (101R) which specifically synthesize withaferin A and withanolide A, respectively. Pyrosequencing yielded 8,34,068 and 7,21,755 reads which got assembled into 89,548 and 1,14,814 unique sequences from 101L and 101R, respectively. A total of 47,885 (101L) and 54,123 (101R) could be annotated using TAIR10, NR, tomato and potato databases. Gene Ontology and KEGG analyses provided a detailed view of all the enzymes involved in withanolide backbone synthesis. Our analysis identified members of cytochrome P450, glycosyltransferase and methyltransferase gene families with unique presence or differential expression in leaf and root and might be involved in synthesis of tissue-specific withanolides. We also detected simple sequence repeats (SSRs) in transcriptome data for use in future genetic studies. Comprehensive sequence resource developed for Withania, in this study, will help to elucidate biosynthetic pathway for tissue-specific synthesis of secondary plant products in non-model plant organisms as well as will be helpful in developing strategies for enhanced biosynthesis of withanolides through biotechnological approaches. PMID:23667511

Comparative transcriptomics reveals genes involved in metabolic and immune pathways in the digestive gland of scallop Chlamys farreri following cadmium exposure

NASA Astrophysics Data System (ADS)

Zhang, Hui; Zhai, Yuxiu; Yao, Lin; Jiang, Yanhua; Li, Fengling

2017-05-01

Chlamys farreri is an economically important mollusk that can accumulate excessive amounts of cadmium (Cd). Studying the molecular mechanism of Cd accumulation in bivalves is difficult because of the lack of genome background. Transcriptomic analysis based on high-throughput RNA sequencing has been shown to be an efficient and powerful method for the discovery of relevant genes in non-model and genome reference-free organisms. Here, we constructed two cDNA libraries (control and Cd exposure groups) from the digestive gland of C. farreri and compared the transcriptomic data between them. A total of 227 673 transcripts were assembled into 105 071 unigenes, most of which shared high similarity with sequences in the NCBI non-redundant protein database. For functional classification, 24 493 unigenes were assigned to Gene Ontology terms. Additionally, EuKaryotic Ortholog Groups and Kyoto Encyclopedia of Genes and Genomes analyses assigned 12 028 unigenes to 26 categories and 7 849 unigenes to five pathways, respectively. Comparative transcriptomics analysis identified 3 800 unigenes that were differentially expressed in the Cd-treated group compared with the control group. Among them, genes associated with heavy metal accumulation were screened, including metallothionein, divalent metal transporter, and metal tolerance protein. The functional genes and predicted pathways identified in our study will contribute to a better understanding of the metabolic and immune system in the digestive gland of C. farreri. In addition, the transcriptomic data will provide a comprehensive resource that may contribute to the understanding of molecular mechanisms that respond to marine pollutants in bivalves.

Inferring the mode of origin of polyploid species from next-generation sequence data.

PubMed

Roux, Camille; Pannell, John R

2015-03-01

Many eukaryote organisms are polyploid. However, despite their importance, evolutionary inference of polyploid origins and modes of inheritance has been limited by a need for analyses of allele segregation at multiple loci using crosses. The increasing availability of sequence data for nonmodel species now allows the application of established approaches for the analysis of genomic data in polyploids. Here, we ask whether approximate Bayesian computation (ABC), applied to realistic traditional and next-generation sequence data, allows correct inference of the evolutionary and demographic history of polyploids. Using simulations, we evaluate the robustness of evolutionary inference by ABC for tetraploid species as a function of the number of individuals and loci sampled, and the presence or absence of an outgroup. We find that ABC adequately retrieves the recent evolutionary history of polyploid species on the basis of both old and new sequencing technologies. The application of ABC to sequence data from diploid and polyploid species of the plant genus Capsella confirms its utility. Our analysis strongly supports an allopolyploid origin of C. bursa-pastoris about 80 000 years ago. This conclusion runs contrary to previous findings based on the same data set but using an alternative approach and is in agreement with recent findings based on whole-genome sequencing. Our results indicate that ABC is a promising and powerful method for revealing the evolution of polyploid species, without the need to attribute alleles to a homeologous chromosome pair. The approach can readily be extended to more complex scenarios involving higher ploidy levels. © 2015 John Wiley & Sons Ltd.

An efficient method to find potentially universal population genetic markers, applied to metazoans

PubMed Central

2010-01-01

Background Despite the impressive growth of sequence databases, the limited availability of nuclear markers that are sufficiently polymorphic for population genetics and phylogeography and applicable across various phyla restricts many potential studies, particularly in non-model organisms. Numerous introns have invariant positions among kingdoms, providing a potential source for such markers. Unfortunately, most of the few known EPIC (Exon Primed Intron Crossing) loci are restricted to vertebrates or belong to multigenic families. Results In order to develop markers with broad applicability, we designed a bioinformatic approach aimed at avoiding multigenic families while identifying intron positions conserved across metazoan phyla. We developed a program facilitating the identification of EPIC loci which allowed slight variation in intron position. From the Homolens databases we selected 29 gene families which contained 52 promising introns for which we designed 93 primer pairs. PCR tests were performed on several ascidians, echinoderms, bivalves and cnidarians. On average, 24 different introns per genus were amplified in bilaterians. Remarkably, five of the introns successfully amplified in all of the metazoan genera tested (a dozen genera, including cnidarians). The influence of several factors on amplification success was investigated. Success rate was not related to the phylogenetic relatedness of a taxon to the groups that most influenced primer design, showing that these EPIC markers are extremely conserved in animals. Conclusions Our new method now makes it possible to (i) rapidly isolate a set of EPIC markers for any phylum, even outside the animal kingdom, and thus, (ii) compare genetic diversity at potentially homologous polymorphic loci between divergent taxa. PMID:20836842

Transcriptome sequencing and microarray development for the woodrat (Neotoma spp.): custom genetic tools for exploring herbivore ecology.

PubMed

Malenke, J R; Milash, B; Miller, A W; Dearing, M D

2013-07-01

Massively parallel sequencing has enabled the creation of novel, in-depth genetic tools for nonmodel, ecologically important organisms. We present the de novo transcriptome sequencing, analysis and microarray development for a vertebrate herbivore, the woodrat (Neotoma spp.). This genus is of ecological and evolutionary interest, especially with respect to ingestion and hepatic metabolism of potentially toxic plant secondary compounds. We generated a liver transcriptome of the desert woodrat (Neotoma lepida) using the Roche 454 platform. The assembled contigs were well annotated using rodent references (99.7% annotation), and biotransformation function was reflected in the gene ontology. The transcriptome was used to develop a custom microarray (eArray, Agilent). We tested the microarray with three experiments: one across species with similar habitat (thus, dietary) niches, one across species with different habitat niches and one across populations within a species. The resulting one-colour arrays had high technical and biological quality. Probes designed from the woodrat transcriptome performed significantly better than functionally similar probes from the Norway rat (Rattus norvegicus). There were a multitude of expression differences across the woodrat treatments, many of which related to biotransformation processes and activities. The pattern and function of the differences indicate shared ecological pressures, and not merely phylogenetic distance, play an important role in shaping gene expression profiles of woodrat species and populations. The quality and functionality of the woodrat transcriptome and custom microarray suggest these tools will be valuable for expanding the scope of herbivore biology, as well as the exploration of conceptual topics in ecology. © 2013 John Wiley & Sons Ltd.

Sequencing-based gene network analysis provides a core set of gene resource for understanding thermal adaptation in Zhikong scallop Chlamys farreri.

PubMed

Fu, X; Sun, Y; Wang, J; Xing, Q; Zou, J; Li, R; Wang, Z; Wang, S; Hu, X; Zhang, L; Bao, Z

2014-01-01

Marine organisms are commonly exposed to variable environmental conditions, and many of them are under threat from increased sea temperatures caused by global climate change. Generating transcriptomic resources under different stress conditions are crucial for understanding molecular mechanisms underlying thermal adaptation. In this study, we conducted transcriptome-wide gene expression profiling of the scallop Chlamys farreri challenged by acute and chronic heat stress. Of the 13 953 unique tags, more than 850 were significantly differentially expressed at each time point after acute heat stress, which was more than the number of tags differentially expressed (320-350) under chronic heat stress. To obtain a systemic view of gene expression alterations during thermal stress, a weighted gene coexpression network was constructed. Six modules were identified as acute heat stress-responsive modules. Among them, four modules involved in apoptosis regulation, mRNA binding, mitochondrial envelope formation and oxidation reduction were downregulated. The remaining two modules were upregulated. One was enriched with chaperone and the other with microsatellite sequences, whose coexpression may originate from a transcription factor binding site. These results indicated that C. farreri triggered several cellular processes to acclimate to elevated temperature. No modules responded to chronic heat stress, suggesting that the scallops might have acclimated to elevated temperature within 3 days. This study represents the first sequencing-based gene network analysis in a nonmodel aquatic species and provides valuable gene resources for the study of thermal adaptation, which should assist in the development of heat-tolerant scallop lines for aquaculture. © 2013 John Wiley & Sons Ltd.

Strategy to Identify and Test Putative Light-Sensitive Non-Opsin G-Protein-Coupled Receptors: A Case Study.

PubMed

Faggionato, Davide; Serb, Jeanne M

2017-08-01

The rise of high-throughput RNA sequencing (RNA-seq) and de novo transcriptome assembly has had a transformative impact on how we identify and study genes in the phototransduction cascade of non-model organisms. But the advantage provided by the nearly automated annotation of RNA-seq transcriptomes may at the same time hinder the possibility for gene discovery and the discovery of new gene functions. For example, standard functional annotation based on domain homology to known protein families can only confirm group membership, not identify the emergence of new biochemical function. In this study, we show the importance of developing a strategy that circumvents the limitations of semiautomated annotation and apply this workflow to photosensitivity as a means to discover non-opsin photoreceptors. We hypothesize that non-opsin G-protein-coupled receptor (GPCR) proteins may have chromophore-binding lysines in locations that differ from opsin. Here, we provide the first case study describing non-opsin light-sensitive GPCRs based on tissue-specific RNA-seq data of the common bay scallop Argopecten irradians (Lamarck, 1819). Using a combination of sequence analysis and three-dimensional protein modeling, we identified two candidate proteins. We tested their photochemical properties and provide evidence showing that these two proteins incorporate 11-cis and/or all-trans retinal and react to light photochemically. Based on this case study, we demonstrate that there is potential for the discovery of new light-sensitive GPCRs, and we have developed a workflow that starts from RNA-seq assemblies to the discovery of new non-opsin, GPCR-based photopigments.

Genomics in a changing arctic: critical questions await the molecular ecologist

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wullschleger, Stan D.; Breen, Amy L.; Iversen, Colleen M.

Molecular ecology is poised to tackle a host of interesting questions in the coming years. Of particular importance to the molecular ecologist are new technologies and analytical approaches that provide opportunities to address questions previously unapproachable.The Arctic provides a unique and rapidly changing environment with a suite of emerging research needs that can be addressed through genetics and genomics. Here we highlight recent research on boreal and tundra ecosystems and put forth a series of questions related to plant and microbial responses to climate change that can benefit from technologies and analytical approaches contained within the molecular ecologist's toolbox. Thesemore » questions include understanding (i) the mechanisms of plant acquisition and uptake of N in cold soils, (ii) how these processes are mediated by root traits, (iii) the role played by the plant microbiome in cycling C and nutrients within high-latitude ecosystems and (iv) plant adaptation to extreme Arctic climates. We highlight how contributions can be made in these areas through studies that target model and nonmodel organisms and emphasize that the sequencing of the Populus and Salix genomes provides a valuable resource for scientific discoveries related to the plant microbiome and plant adaptation in the Arctic. Moreover, there exists an exciting role to play in model development, including incorporating genetic and evolutionary knowledge into ecosystem and Earth System Models. In this regard, the molecular ecologist provides a valuable perspective on plant genetics as a driver for community biodiversity, and how ecological and evolutionary forces govern community dynamics in a rapidly changing climate.« less

Epigenetic estimation of age in humpback whales

PubMed Central

Polanowski, Andrea M; Robbins, Jooke; Chandler, David; Jarman, Simon N

2014-01-01

Age is a fundamental aspect of animal ecology, but is difficult to determine in many species. Humpback whales exemplify this as they have a lifespan comparable to humans, mature sexually as early as 4 years and have no reliable visual age indicators after their first year. Current methods for estimating humpback age cannot be applied to all individuals and populations. Assays for human age have recently been developed based on age-induced changes in DNA methylation of specific genes. We used information on age-associated DNA methylation in human and mouse genes to identify homologous gene regions in humpbacks. Humpback skin samples were obtained from individuals with a known year of birth and employed to calibrate relationships between cytosine methylation and age. Seven of 37 cytosines assayed for methylation level in humpback skin had significant age-related profiles. The three most age-informative cytosine markers were selected for a humpback epigenetic age assay. The assay has an R2 of 0.787 (P = 3.04e−16) and predicts age from skin samples with a standard deviation of 2.991 years. The epigenetic method correctly determined which of parent–offspring pairs is the parent in more than 93% of cases. To demonstrate the potential of this technique, we constructed the first modern age profile of humpback whales off eastern Australia and compared the results to population structure 5 decades earlier. This is the first epigenetic age estimation method for a wild animal species and the approach we took for developing it can be applied to many other nonmodel organisms. PMID:24606053

The transcriptional landscape of seasonal coat colour moult in the snowshoe hare.

PubMed

Ferreira, Mafalda S; Alves, Paulo C; Callahan, Colin M; Marques, João P; Mills, L Scott; Good, Jeffrey M; Melo-Ferreira, José

2017-08-01

Seasonal coat colour change is an important adaptation to seasonally changing environments but the evolution of this and other circannual traits remains poorly understood. In this study, we use gene expression to understand seasonal coat colour moulting in wild snowshoe hares (Lepus americanus). We used hair colour to follow the progression of the moult, simultaneously sampling skin from three moulting stages in hares collected during the peak of the spring moult from white winter to brown summer pelage. Using RNA sequencing, we tested whether patterns of expression were consistent with predictions based on the established phases of the hair growth cycle. We found functionally consistent clustering across skin types, with 766 genes differentially expressed between moult stages. "White" pelage showed more differentially expressed genes that were upregulated relative to other skin types, involved in the transition between late telogen (quiescent stage) and the onset of anagen (proliferative stage). Skin samples from transitional "intermediate" and "brown" pelage were transcriptionally similar and resembled the regressive transition to catagen (regressive stage). We also detected differential expression of several key circadian clock and pigmentation genes, providing important means to dissect the bases of alternate seasonal colour morphs. Our results reveal that pelage colour is a useful biomarker for seasonal change but that there is a consistent lag between the main gene expression waves and change in visible coat colour. These experiments establish that developmental sampling from natural populations of nonmodel organisms can provide a crucial resource to dissect the genetic basis and evolution of complex seasonally changing traits. © 2017 John Wiley & Sons Ltd.

Comparative ultrastructure of fruit plastids in three genetically diverse genotypes of apple (Malus × domestica Borkh.) during development

PubMed Central

Schaeffer, Scott M.; Christian, Ryan; Castro-Velasquez, Nohely; Hyden, Brennan; Lynch-Holm, Valerie

2017-01-01

Plastids are the defining organelle for a plant cell and are critical for myriad metabolic functions. The role of leaf plastid, chloroplast, is extensively documented; however, fruit plastids—chromoplasts—are poorly understood, especially in the context of the diverse metabolic processes operating in these diverse plant organs. Recently, in a comparative study of the predicted plastid-targeted proteomes across seven plant species, we reported that each plant species is predicted to harbor a unique set of plastid-targeted proteins. However, the temporal and developmental context of these processes remains unknown. In this study, an ultrastructural analysis approach was used to characterize fruit plastids in the epidermal and collenchymal cell layers at 11 developmental timepoints in three genotypes of apple (Malus × domestica Borkh.): chlorophyll-predominant ‘Granny Smith’, carotenoid-predominant ‘Golden Delicious’, and anthocyanin-predominant ‘Top Red Delicious’. Plastids transitioned from a proplastid-like plastid to a chromoplast-like plastid in epidermis cells, while in the collenchyma cells, they transitioned from a chloroplast-like plastid to a chloro-chromo-amyloplast plastid. Plastids in the collenchyma cells of the three genotypes demonstrated a diverse array of structures and features. This study enabled the identification of discrete developmental stages during which specific functions are most likely being performed by the plastids as indicated by accumulation of plastoglobuli, starch granules, and other sub-organeller structures. Information regarding the metabolically active developmental stages is expected to facilitate biologically relevant omics studies to unravel the complex biochemistry of plastids in perennial non-model systems. PMID:28698906

Genotyping-by-sequencing for estimating relatedness in nonmodel organisms: Avoiding the trap of precise bias.

PubMed

Attard, Catherine R M; Beheregaray, Luciano B; Möller, Luciana M

2018-05-01

There has been remarkably little attention to using the high resolution provided by genotyping-by-sequencing (i.e., RADseq and similar methods) for assessing relatedness in wildlife populations. A major hurdle is the genotyping error, especially allelic dropout, often found in this type of data that could lead to downward-biased, yet precise, estimates of relatedness. Here, we assess the applicability of genotyping-by-sequencing for relatedness inferences given its relatively high genotyping error rate. Individuals of known relatedness were simulated under genotyping error, allelic dropout and missing data scenarios based on an empirical ddRAD data set, and their true relatedness was compared to that estimated by seven relatedness estimators. We found that an estimator chosen through such analyses can circumvent the influence of genotyping error, with the estimator of Ritland (Genetics Research, 67, 175) shown to be unaffected by allelic dropout and to be the most accurate when there is genotyping error. We also found that the choice of estimator should not rely solely on the strength of correlation between estimated and true relatedness as a strong correlation does not necessarily mean estimates are close to true relatedness. We also demonstrated how even a large SNP data set with genotyping error (allelic dropout or otherwise) or missing data still performs better than a perfectly genotyped microsatellite data set of tens of markers. The simulation-based approach used here can be easily implemented by others on their own genotyping-by-sequencing data sets to confirm the most appropriate and powerful estimator for their data. © 2017 John Wiley & Sons Ltd.

In Vitro vs In Silico Detected SNPs for the Development of a Genotyping Array: What Can We Learn from a Non-Model Species?

PubMed Central

Lepoittevin, Camille; Frigerio, Jean-Marc; Garnier-Géré, Pauline; Salin, Franck; Cervera, María-Teresa; Vornam, Barbara; Harvengt, Luc; Plomion, Christophe

2010-01-01

Background There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size (∼23.8 Gb/C). Methodology/Principal Findings A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates). Conclusions/Significance This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome. PMID:20543950

Heterologous oligonucleotide microarrays for transcriptomics in a non-model species; a proof-of-concept study of drought stress in Musa

PubMed Central

Davey, Mark W; Graham, Neil S; Vanholme, Bartel; Swennen, Rony; May, Sean T; Keulemans, Johan

2009-01-01

Background 'Systems-wide' approaches such as microarray RNA-profiling are ideally suited to the study of the complex overlapping responses of plants to biotic and abiotic stresses. However, commercial microarrays are only available for a limited number of plant species and development costs are so substantial as to be prohibitive for most research groups. Here we evaluate the use of cross-hybridisation to Affymetrix oligonucleotide GeneChip® microarrays to profile the response of the banana (Musa spp.) leaf transcriptome to drought stress using a genomic DNA (gDNA)-based probe-selection strategy to improve the efficiency of detection of differentially expressed Musa transcripts. Results Following cross-hybridisation of Musa gDNA to the Rice GeneChip® Genome Array, ~33,700 gene-specific probe-sets had a sufficiently high degree of homology to be retained for transcriptomic analyses. In a proof-of-concept approach, pooled RNA representing a single biological replicate of control and drought stressed leaves of the Musa cultivar 'Cachaco' were hybridised to the Affymetrix Rice Genome Array. A total of 2,910 Musa gene homologues with a >2-fold difference in expression levels were subsequently identified. These drought-responsive transcripts included many functional classes associated with plant biotic and abiotic stress responses, as well as a range of regulatory genes known to be involved in coordinating abiotic stress responses. This latter group included members of the ERF, DREB, MYB, bZIP and bHLH transcription factor families. Fifty-two of these drought-sensitive Musa transcripts were homologous to genes underlying QTLs for drought and cold tolerance in rice, including in 2 instances QTLs associated with a single underlying gene. The list of drought-responsive transcripts also included genes identified in publicly-available comparative transcriptomics experiments. Conclusion Our results demonstrate that despite the general paucity of nucleotide sequence data in Musa and only distant phylogenetic relations to rice, gDNA probe-based cross-hybridisation to the Rice GeneChip® is a highly promising strategy to study complex biological responses and illustrates the potential of such strategies for gene discovery in non-model species. PMID:19758430

An XML transfer schema for exchange of genomic and genetic mapping data: implementation as a web service in a Taverna workflow.

PubMed

Paterson, Trevor; Law, Andy

2009-08-14

Genomic analysis, particularly for less well-characterized organisms, is greatly assisted by performing comparative analyses between different types of genome maps and across species boundaries. Various providers publish a plethora of on-line resources collating genome mapping data from a multitude of species. Datasources range in scale and scope from small bespoke resources for particular organisms, through larger web-resources containing data from multiple species, to large-scale bioinformatics resources providing access to data derived from genome projects for model and non-model organisms. The heterogeneity of information held in these resources reflects both the technologies used to generate the data and the target users of each resource. Currently there is no common information exchange standard or protocol to enable access and integration of these disparate resources. Consequently data integration and comparison must be performed in an ad hoc manner. We have developed a simple generic XML schema (GenomicMappingData.xsd - GMD) to allow export and exchange of mapping data in a common lightweight XML document format. This schema represents the various types of data objects commonly described across mapping datasources and provides a mechanism for recording relationships between data objects. The schema is sufficiently generic to allow representation of any map type (for example genetic linkage maps, radiation hybrid maps, sequence maps and physical maps). It also provides mechanisms for recording data provenance and for cross referencing external datasources (including for example ENSEMBL, PubMed and Genbank.). The schema is extensible via the inclusion of additional datatypes, which can be achieved by importing further schemas, e.g. a schema defining relationship types. We have built demonstration web services that export data from our ArkDB database according to the GMD schema, facilitating the integration of data retrieval into Taverna workflows. The data exchange standard we present here provides a useful generic format for transfer and integration of genomic and genetic mapping data. The extensibility of our schema allows for inclusion of additional data and provides a mechanism for typing mapping objects via third party standards. Web services retrieving GMD-compliant mapping data demonstrate that use of this exchange standard provides a practical mechanism for achieving data integration, by facilitating syntactically and semantically-controlled access to the data.

An XML transfer schema for exchange of genomic and genetic mapping data: implementation as a web service in a Taverna workflow

PubMed Central

Paterson, Trevor; Law, Andy

2009-01-01

Background Genomic analysis, particularly for less well-characterized organisms, is greatly assisted by performing comparative analyses between different types of genome maps and across species boundaries. Various providers publish a plethora of on-line resources collating genome mapping data from a multitude of species. Datasources range in scale and scope from small bespoke resources for particular organisms, through larger web-resources containing data from multiple species, to large-scale bioinformatics resources providing access to data derived from genome projects for model and non-model organisms. The heterogeneity of information held in these resources reflects both the technologies used to generate the data and the target users of each resource. Currently there is no common information exchange standard or protocol to enable access and integration of these disparate resources. Consequently data integration and comparison must be performed in an ad hoc manner. Results We have developed a simple generic XML schema (GenomicMappingData.xsd – GMD) to allow export and exchange of mapping data in a common lightweight XML document format. This schema represents the various types of data objects commonly described across mapping datasources and provides a mechanism for recording relationships between data objects. The schema is sufficiently generic to allow representation of any map type (for example genetic linkage maps, radiation hybrid maps, sequence maps and physical maps). It also provides mechanisms for recording data provenance and for cross referencing external datasources (including for example ENSEMBL, PubMed and Genbank.). The schema is extensible via the inclusion of additional datatypes, which can be achieved by importing further schemas, e.g. a schema defining relationship types. We have built demonstration web services that export data from our ArkDB database according to the GMD schema, facilitating the integration of data retrieval into Taverna workflows. Conclusion The data exchange standard we present here provides a useful generic format for transfer and integration of genomic and genetic mapping data. The extensibility of our schema allows for inclusion of additional data and provides a mechanism for typing mapping objects via third party standards. Web services retrieving GMD-compliant mapping data demonstrate that use of this exchange standard provides a practical mechanism for achieving data integration, by facilitating syntactically and semantically-controlled access to the data. PMID:19682365

Emerging from the bottleneck: benefits of the comparative approach to modern neuroscience.

PubMed

Brenowitz, Eliot A; Zakon, Harold H

2015-05-01

Neuroscience has historically exploited a wide diversity of animal taxa. Recently, however, research has focused increasingly on a few model species. This trend has accelerated with the genetic revolution, as genomic sequences and genetic tools became available for a few species, which formed a bottleneck. This coalescence on a small set of model species comes with several costs that are often not considered, especially in the current drive to use mice explicitly as models for human diseases. Comparative studies of strategically chosen non-model species can complement model species research and yield more rigorous studies. As genetic sequences and tools become available for many more species, we are poised to emerge from the bottleneck and once again exploit the rich biological diversity offered by comparative studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fundamental CRISPR-Cas9 tools and current applications in microbial systems.

PubMed

Tian, Pingfang; Wang, Jia; Shen, Xiaolin; Rey, Justin Forrest; Yuan, Qipeng; Yan, Yajun

2017-09-01

Derived from the bacterial adaptive immune system, CRISPR technology has revolutionized conventional genetic engineering methods and unprecedentedly facilitated strain engineering. In this review, we outline the fundamental CRISPR tools that have been employed for strain optimization. These tools include CRISPR editing, CRISPR interference, CRISPR activation and protein imaging. To further characterize the CRISPR technology, we present current applications of these tools in microbial systems, including model- and non-model industrial microorganisms. Specially, we point out the major challenges of the CRISPR tools when utilized for multiplex genome editing and sophisticated expression regulation. To address these challenges, we came up with strategies that place emphasis on the amelioration of DNA repair efficiency through CRISPR-Cas9-assisted recombineering. Lastly, multiple promising research directions were proposed, mainly focusing on CRISPR-based construction of microbial ecosystems toward high production of desired chemicals.

Factors That Modulate Neurogenesis: A Top-Down Approach.

PubMed

LaDage, Lara D

2016-08-24

Although hippocampal neurogenesis in the adult brain has been conserved across the vertebrate lineage, laboratory studies have primarily examined this phenomenon in rodent models. This approach has been successful in elucidating important factors and mechanisms that can modulate rates of hippocampal neurogenesis, including hormones, environmental complexity, learning and memory, motor stimulation, and stress. However, recent studies have found that neurobiological research on neurogenesis in rodents may not easily translate to, or explain, neurogenesis patterns in nonrodent systems, particularly in species examined in the field. This review examines some of the evolutionary and ecological variables that may also modulate neurogenesis patterns. This 'top-down' and more naturalistic approach, which incorporates ecology and natural history, particularly of nonmodel species, may allow for a more comprehensive understanding of the functional significance of neurogenesis. © 2016 S. Karger AG, Basel.

Primary Characterization of Small RNAs in Symbiotic Nitrogen-Fixing Bacteria.

PubMed

Robledo, Marta; García-Tomsig, Natalia I; Jiménez-Zurdo, José I

2018-01-01

High-throughput transcriptome profiling (RNAseq) has uncovered large and heterogeneous populations of small noncoding RNA species (sRNAs) with potential regulatory roles in bacteria. A large fraction of sRNAs are differentially regulated and rely on protein-assisted antisense interactions to trans-encoded target mRNAs to fine-tune posttranscriptional reprogramming of gene expression in response to external cues. However, annotation and function of sRNAs are still largely overlooked in nonmodel bacteria with complex lifestyles. Here, we describe experimental protocols successfully applied for the accurate annotation, expression profiling and target mRNA identification of trans-acting sRNAs in the nitrogen-fixing α-rhizobium Sinorhizobium meliloti. The protocols presented here can be similarly applied for the characterization of trans-sRNAs in genetically tractable α-proteobacteria of agronomical or clinical relevance interacting with eukaryotic hosts.

An essential cell cycle regulation gene causes hybrid inviability in Drosophila.

PubMed

Phadnis, Nitin; Baker, EmilyClare P; Cooper, Jacob C; Frizzell, Kimberly A; Hsieh, Emily; de la Cruz, Aida Flor A; Shendure, Jay; Kitzman, Jacob O; Malik, Harmit S

2015-12-18

Speciation, the process by which new biological species arise, involves the evolution of reproductive barriers, such as hybrid sterility or inviability between populations. However, identifying hybrid incompatibility genes remains a key obstacle in understanding the molecular basis of reproductive isolation. We devised a genomic screen, which identified a cell cycle-regulation gene as the cause of male inviability in hybrids resulting from a cross between Drosophila melanogaster and D. simulans. Ablation of the D. simulans allele of this gene is sufficient to rescue the adult viability of hybrid males. This dominantly acting cell cycle regulator causes mitotic arrest and, thereby, inviability of male hybrid larvae. Our genomic method provides a facile means to accelerate the identification of hybrid incompatibility genes in other model and nonmodel systems. Copyright © 2015, American Association for the Advancement of Science.

MHC class I loci of the Bar-Headed goose (Anser indicus)

PubMed Central

2010-01-01

MHC class I proteins mediate functions in anti-pathogen defense. MHC diversity has already been investigated by many studies in model avian species, but here we chose the bar-headed goose, a worldwide migrant bird, as a non-model avian species. Sequences from exons encoding the peptide-binding region (PBR) of MHC class I molecules were isolated from liver genomic DNA, to investigate variation in these genes. These are the first MHC class I partial sequences of the bar-headed goose to be reported. A preliminary analysis suggests the presence of at least four MHC class I genes, which share great similarity with those of the goose and duck. A phylogenetic analysis of bar-headed goose, goose and duck MHC class I sequences using the NJ method supports the idea that they all cluster within the anseriforms clade. PMID:21637434

Embryogenesis and laboratory maintenance of the foam-nesting túngara frogs, genus Engystomops (= Physalaemus)

PubMed Central

Romero-Carvajal, Andrés; Sáenz-Ponce, Natalia; Venegas-Ferrín, Michael; Almeida-Reinoso, Diego; Lee, Chanjae; Bond, Jennifer; Ryan, Michael J.; Wallingford, John B.; del Pino, Eugenia M.

2010-01-01

The vast majority of embryological research on amphibians focuses on just a single genus of frogs, Xenopus. To attain a more comprehensive understanding of amphibian development, experimentation on non-model frogs will be essential. Here, we report on the early development, rearing, and embryological analysis of túngara frogs (genus Engystomops, also called Physaleamus). The frogs Engystomops pustulosus, Engystomops coloradorum and Engystomops randi construct floating foam-nests with small eggs. We define a table of 23 stages for the developmental period in the foam-nest. Embryos were immunostained against Lim1, neural, and somite-specific proteins and the expression pattern of RetinoBlastoma Binding Protein 6 (RBBP6) was analyzed by in situ hybridization. Due to their brief life-cycle, frogs belonging to the genus Engystomops are attractive for comparative and genetic studies of development. PMID:19384855

Expression and RNA Interference of Salivary Polygalacturonase Genes in the Tarnished Plant Bug, Lygus lineolaris

PubMed Central

Walker, William B.; Allen, Margaret L.

2010-01-01

Three genes encoding polygalacturonase (PG) have been identified in Lygus lineolaris (Palisot de Beauvois) (Miridae: Hemiptera). Earlier studies showed that the three PG gene transcripts are exclusively expressed in the feeding stages of L. lineolaris. In this report, it is shown that all three transcripts are specifically expressed in salivary glands indicating that PGs are salivary enzymes. Transcriptional profiles of the three PGs were evaluated with respect to diet, comparing live cotton plant material to artificial diet. PG2 transcript levels were consistently lower in cotton-fed insects than those reared on artificial diet. RNA interference was used to knock down expression of PG1 mRNA in adult salivary glands providing the first demonstration of the use of this method in the non-model insect, L. lineolaris. PMID:21062205

A Scheme for the Integrated Assessment of Mitigation Options

NASA Astrophysics Data System (ADS)

Held, H.; Edenhofer, O.

2003-04-01

After some consensus has been achieved that the global mean temperature will have increased by 1.4 to 5.8^oC at the end of this century in case of continued ``business as usual'' greenhouse gas emissions, society has to decide if or which mitigation measures should be taken. A new integrated assessment project on this very issue will be started at PIK in spring 2003. The assessment will cover economic aspects as well as potential side effects of various measures. In the economic module, the effects of investment decisions on technological innovation will be explicitly taken into account. Special emphasize will be put on the issue of uncertainty. Hereby we distinguish the uncertainty related to the Integrated Assessment modules, including the economic module, from the fact that no over-complex system can be fully captured by a model. Therefore, a scheme for the assessment of the ``residual'', the non-modelled part of the system, needs to be worked out. The scheme must be truly interdisciplinary, i.e. must be applicable to at least the natural science and the economic aspects. A scheme based on meta-principles like minimum persistence, ubiquity, or irreversibility of potential measures appears to be a promising candidate. An implementation of ubiquity as at present successfully operated in environmental chemistry may serve as a guideline [1]. Here, the best-known mechanism within a complex impact chain of potentially harmful chemicals, their transport, is captured by a reaction-diffusion mechanism [2]. begin{thebibliography}{0} bibitem{s} M. Scheringer, Persistence and spatial range as endpoints of an exposure-based assessment of organic chemicals. Environ. Sci. Technol. 30: 1652-1659 (1996). bibitem{h} H. Held, Robustness of spatial ranges of environmental chemicals with respect to model dimension, accepted for publication in Stoch. Environ. Res. Risk Assessment.

Deletion of the Clostridium thermocellum recA gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences.

PubMed

Groom, Joseph; Chung, Daehwan; Kim, Sun-Ki; Guss, Adam; Westpheling, Janet

2018-05-28

A limitation to the engineering of cellulolytic thermophiles is the availability of functional, thermostable (≥ 60 °C) replicating plasmid vectors for rapid expression and testing of genes that provide improved or novel fuel molecule production pathways. A series of plasmid vectors for genetic manipulation of the cellulolytic thermophile Caldicellulosiruptor bescii has recently been extended to Clostridium thermocellum, another cellulolytic thermophile that very efficiently solubilizes plant biomass and produces ethanol. While the C. bescii pBAS2 replicon on these plasmids is thermostable, the use of homologous promoters, signal sequences and genes led to undesired integration into the bacterial chromosome, a result also observed with less thermostable replicating vectors. In an attempt to overcome undesired plasmid integration in C. thermocellum, a deletion of recA was constructed. As expected, C. thermocellum ∆recA showed impaired growth in chemically defined medium and an increased susceptibility to UV damage. Interestingly, we also found that recA is required for replication of the C. bescii thermophilic plasmid pBAS2 in C. thermocellum, but it is not required for replication of plasmid pNW33N. In addition, the C. thermocellum recA mutant retained the ability to integrate homologous DNA into the C. thermocellum chromosome. These data indicate that recA can be required for replication of certain plasmids, and that a recA-independent mechanism exists for the integration of homologous DNA into the C. thermocellum chromosome. Understanding thermophilic plasmid replication is not only important for engineering of these cellulolytic thermophiles, but also for developing genetic systems in similar new potentially useful non-model organisms.

Transcriptome and proteomic analysis of mango (Mangifera indica Linn) fruits.

PubMed

Wu, Hong-xia; Jia, Hui-min; Ma, Xiao-wei; Wang, Song-biao; Yao, Quan-sheng; Xu, Wen-tian; Zhou, Yi-gang; Gao, Zhong-shan; Zhan, Ru-lin

2014-06-13

Here we used Illumina RNA-seq technology for transcriptome sequencing of a mixed fruit sample from 'Zill' mango (Mangifera indica Linn) fruit pericarp and pulp during the development and ripening stages. RNA-seq generated 68,419,722 sequence reads that were assembled into 54,207 transcripts with a mean length of 858bp, including 26,413 clusters and 27,794 singletons. A total of 42,515(78.43%) transcripts were annotated using public protein databases, with a cut-off E-value above 10(-5), of which 35,198 and 14,619 transcripts were assigned to gene ontology terms and clusters of orthologous groups respectively. Functional annotation against the Kyoto Encyclopedia of Genes and Genomes database identified 23,741(43.79%) transcripts which were mapped to 128 pathways. These pathways revealed many previously unknown transcripts. We also applied mass spectrometry-based transcriptome data to characterize the proteome of ripe fruit. LC-MS/MS analysis of the mango fruit proteome was using tandem mass spectrometry (MS/MS) in an LTQ Orbitrap Velos (Thermo) coupled online to the HPLC. This approach enabled the identification of 7536 peptides that matched 2754 proteins. Our study provides a comprehensive sequence for a systemic view of transcriptome during mango fruit development and the most comprehensive fruit proteome to date, which are useful for further genomics research and proteomic studies. Our study provides a comprehensive sequence for a systemic view of both the transcriptome and proteome of mango fruit, and a valuable reference for further research on gene expression and protein identification. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Brown and polar bear Y chromosomes reveal extensive male-biased gene flow within brother lineages.

PubMed

Bidon, Tobias; Janke, Axel; Fain, Steven R; Eiken, Hans Geir; Hagen, Snorre B; Saarma, Urmas; Hallström, Björn M; Lecomte, Nicolas; Hailer, Frank

2014-06-01

Brown and polar bears have become prominent examples in phylogeography, but previous phylogeographic studies relied largely on maternally inherited mitochondrial DNA (mtDNA) or were geographically restricted. The male-specific Y chromosome, a natural counterpart to mtDNA, has remained underexplored. Although this paternally inherited chromosome is indispensable for comprehensive analyses of phylogeographic patterns, technical difficulties and low variability have hampered its application in most mammals. We developed 13 novel Y-chromosomal sequence and microsatellite markers from the polar bear genome and screened these in a broad geographic sample of 130 brown and polar bears. We also analyzed a 390-kb-long Y-chromosomal scaffold using sequencing data from published male ursine genomes. Y chromosome evidence support the emerging understanding that brown and polar bears started to diverge no later than the Middle Pleistocene. Contrary to mtDNA patterns, we found 1) brown and polar bears to be reciprocally monophyletic sister (or rather brother) lineages, without signals of introgression, 2) male-biased gene flow across continents and on phylogeographic time scales, and 3) male dispersal that links the Alaskan ABC islands population to mainland brown bears. Due to female philopatry, mtDNA provides a highly structured estimate of population differentiation, while male-biased gene flow is a homogenizing force for nuclear genetic variation. Our findings highlight the importance of analyzing both maternally and paternally inherited loci for a comprehensive view of phylogeographic history, and that mtDNA-based phylogeographic studies of many mammals should be reevaluated. Recent advances in sequencing technology render the analysis of Y-chromosomal variation feasible, even in nonmodel organisms. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Pervasive, Genome-Wide Transcription in the Organelle Genomes of Diverse Plastid-Bearing Protists.

PubMed

Sanitá Lima, Matheus; Smith, David Roy

2017-11-06

Organelle genomes are among the most sequenced kinds of chromosome. This is largely because they are small and widely used in molecular studies, but also because next-generation sequencing technologies made sequencing easier, faster, and cheaper. However, studies of organelle RNA have not kept pace with those of DNA, despite huge amounts of freely available eukaryotic RNA-sequencing (RNA-seq) data. Little is known about organelle transcription in nonmodel species, and most of the available eukaryotic RNA-seq data have not been mined for organelle transcripts. Here, we use publicly available RNA-seq experiments to investigate organelle transcription in 30 diverse plastid-bearing protists with varying organelle genomic architectures. Mapping RNA-seq data to organelle genomes revealed pervasive, genome-wide transcription, regardless of the taxonomic grouping, gene organization, or noncoding content. For every species analyzed, transcripts covered ≥85% of the mitochondrial and/or plastid genomes (all of which were ≤105 kb), indicating that most of the organelle DNA-coding and noncoding-is transcriptionally active. These results follow earlier studies of model species showing that organellar transcription is coupled and ubiquitous across the genome, requiring significant downstream processing of polycistronic transcripts. Our findings suggest that noncoding organelle DNA can be transcriptionally active, raising questions about the underlying function of these transcripts and underscoring the utility of publicly available RNA-seq data for recovering complete genome sequences. If pervasive transcription is also found in bigger organelle genomes (>105 kb) and across a broader range of eukaryotes, this could indicate that noncoding organelle RNAs are regulating fundamental processes within eukaryotic cells. Copyright © 2017 Sanitá Lima and Smith.

Dealing with AFLP genotyping errors to reveal genetic structure in Plukenetia volubilis (Euphorbiaceae) in the Peruvian Amazon

PubMed Central

Vašek, Jakub; Viehmannová, Iva; Ocelák, Martin; Cachique Huansi, Danter; Vejl, Pavel

2017-01-01

An analysis of the population structure and genetic diversity for any organism often depends on one or more molecular marker techniques. Nonetheless, these techniques are not absolutely reliable because of various sources of errors arising during the genotyping process. Thus, a complex analysis of genotyping error was carried out with the AFLP method in 169 samples of the oil seed plant Plukenetia volubilis L. from small isolated subpopulations in the Peruvian Amazon. Samples were collected in nine localities from the region of San Martin. Analysis was done in eight datasets with a genotyping error from 0 to 5%. Using eleven primer combinations, 102 to 275 markers were obtained according to the dataset. It was found that it is only possible to obtain the most reliable and robust results through a multiple-level filtering process. Genotyping error and software set up influence both the estimation of population structure and genetic diversity, where in our case population number (K) varied between 2–9 depending on the dataset and statistical method used. Surprisingly, discrepancies in K number were caused more by statistical approaches than by genotyping errors themselves. However, for estimation of genetic diversity, the degree of genotyping error was critical because descriptive parameters (He, FST, PLP 5%) varied substantially (by at least 25%). Due to low gene flow, P. volubilis mostly consists of small isolated subpopulations (ΦPT = 0.252–0.323) with some degree of admixture given by socio-economic connectivity among the sites; a direct link between the genetic and geographic distances was not confirmed. The study illustrates the successful application of AFLP to infer genetic structure in non-model plants. PMID:28910307

RNA-Seq Analysis of the Response of the Halophyte, Mesembryanthemum crystallinum (Ice Plant) to High Salinity

PubMed Central

Tsukagoshi, Hironaka; Suzuki, Takamasa; Nishikawa, Kouki; Agarie, Sakae; Ishiguro, Sumie; Higashiyama, Tetsuya

2015-01-01

Understanding the molecular mechanisms that convey salt tolerance in plants is a crucial issue for increasing crop yield. The ice plant (Mesembryanthemum crystallinum) is a halophyte that is capable of growing under high salt conditions. For example, the roots of ice plant seedlings continue to grow in 140 mM NaCl, a salt concentration that completely inhibits Arabidopsis thaliana root growth. Identifying the molecular mechanisms responsible for this high level of salt tolerance in a halophyte has the potential of revealing tolerance mechanisms that have been evolutionarily successful. In the present study, deep sequencing (RNAseq) was used to examine gene expression in ice plant roots treated with various concentrations of NaCl. Sequencing resulted in the identification of 53,516 contigs, 10,818 of which were orthologs of Arabidopsis genes. In addition to the expression analysis, a web-based ice plant database was constructed that allows broad public access to the data. The results obtained from an analysis of the RNAseq data were confirmed by RT-qPCR. Novel patterns of gene expression in response to high salinity within 24 hours were identified in the ice plant when the RNAseq data from the ice plant was compared to gene expression data obtained from Arabidopsis plants exposed to high salt. Although ABA responsive genes and a sodium transporter protein (HKT1), are up-regulated and down-regulated respectively in both Arabidopsis and the ice plant; peroxidase genes exhibit opposite responses. The results of this study provide an important first step towards analyzing environmental tolerance mechanisms in a non-model organism and provide a useful dataset for predicting novel gene functions. PMID:25706745

Immunofluorescent staining reveals hypermethylation of microchromosomes in the central bearded dragon, Pogona vitticeps.

PubMed

Domaschenz, Renae; Livernois, Alexandra M; Rao, Sudha; Ezaz, Tariq; Deakin, Janine E

2015-01-01

Studies of model organisms have demonstrated that DNA cytosine methylation and histone modifications are key regulators of gene expression in biological processes. Comparatively little is known about the presence and distribution of epigenetic marks in non-model amniotes such as non-avian reptiles whose genomes are typically packaged into chromosomes of distinct size classes. Studies of chicken karyotypes have associated the gene-richness and high GC content of microchromosomes with a distinct epigenetic landscape. To determine whether this is likely to be a common feature of amniote microchromosomes, we have analysed the distribution of epigenetic marks using immunofluorescence on metaphase chromosomes of the central bearded dragon (Pogona vitticeps). This study is the first to study the distribution of epigenetic marks on non-avian reptile chromosomes. We observed an enrichment of DNA cytosine methylation, active modifications H3K4me2 and H3K4me3, as well as the repressive mark H3K27me3 in telomeric regions on macro and microchromosomes. Microchromosomes were hypermethylated compared to macrochromosomes, as they are in chicken. However, differences between macro- and microchromosomes for histone modifications associated with actively transcribed or repressed DNA were either less distinct or not detectable. Hypermethylation of microchromosomes compared to macrochromosomes is a shared feature between P. vitticeps and avian species. The lack of the clear distinction between macro- and microchromosome staining patterns for active and repressive histone modifications makes it difficult to determine at this stage whether microchrosome hypermethylation is correlated with greater gene density as it is in aves, or associated with the greater GC content of P. vitticeps microchromosomes compared to macrochromosomes.

Deletion of the Clostridium thermocellum recA Gene Reveals that it is Required for Thermophilic Plasmid Replication but not Plasmid Integration at Homologous DNA Sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chung, Daehwan; Groom, Joseph; Kim, Sun-Ki

A limitation to the engineering of cellulolytic thermophiles is the availability of functional, thermostable (>/= 60 degrees C) replicating plasmid vectors for rapid expression and testing of genes that provide improved or novel fuel molecule production pathways. A series of plasmid vectors for genetic manipulation of the cellulolytic thermophile Caldicellulosiruptor bescii has recently been extended to Clostridium thermocellum, another cellulolytic thermophile that very efficiently solubilizes plant biomass and produces ethanol. While the C. bescii pBAS2 replicon on these plasmids is thermostable, the use of homologous promoters, signal sequences and genes led to undesired integration into the bacterial chromosome, a resultmore » also observed with less thermostable replicating vectors. In an attempt to overcome undesired plasmid integration in C. thermocellum, a deletion of recA was constructed. As expected, C. thermocellum ..delta..recA showed impaired growth in chemically defined medium and an increased susceptibility to UV damage. Interestingly, we also found that recA is required for replication of the C. bescii thermophilic plasmid pBAS2 in C. thermocellum, but it is not required for replication of plasmid pNW33N. In addition, the C. thermocellum recA mutant retained the ability to integrate homologous DNA into the C. thermocellum chromosome. These data indicate that recA can be required for replication of certain plasmids, and that a recA-independent mechanism exists for the integration of homologous DNA into the C. thermocellum chromosome. Understanding thermophilic plasmid replication is not only important for engineering of these cellulolytic thermophiles, but also for developing genetic systems in similar new potentially useful non-model organisms.« less

DETECTING SELECTION IN NATURAL POPULATIONS: MAKING SENSE OF GENOME SCANS AND TOWARDS ALTERNATIVE SOLUTIONS

PubMed Central

Haasl, Ryan J.; Payseur, Bret A.

2016-01-01

Genomewide scans for natural selection (GWSS) have become increasingly common over the last 15 years due to increased availability of genome-scale genetic data. Here, we report a representative survey of GWSS from 1999 to present and find that (i) between 1999 and 2009, 35 of 49 (71%) GWSS focused on human, while from 2010 to present, only 38 of 83 (46%) of GWSS focused on human, indicating increased focus on nonmodel organisms; (ii) the large majority of GWSS incorporate interpopulation or interspecific comparisons using, for example FST, cross-population extended haplotype homozygosity or the ratio of nonsynonymous to synonymous substitutions; (iii) most GWSS focus on detection of directional selection rather than other modes such as balancing selection; and (iv) in human GWSS, there is a clear shift after 2004 from microsatellite markers to dense SNP data. A survey of GWSS meant to identify loci positively selected in response to severe hypoxic conditions support an approach to GWSS in which a list of a priori candidate genes based on potential selective pressures are used to filter the list of significant hits a posteriori. We also discuss four frequently ignored determinants of genomic heterogeneity that complicate GWSS: mutation, recombination, selection and the genetic architecture of adaptive traits. We recommend that GWSS methodology should better incorporate aspects of genomewide heterogeneity using empirical estimates of relevant parameters and/or realistic, whole-chromosome simulations to improve interpretation of GWSS results. Finally, we argue that knowledge of potential selective agents improves interpretation of GWSS results and that new methods focused on correlations between environmental variables and genetic variation can help automate this approach. PMID:26224644

Fifteen years of genomewide scans for selection: trends, lessons and unaddressed genetic sources of complication.

PubMed

Haasl, Ryan J; Payseur, Bret A

2016-01-01

Genomewide scans for natural selection (GWSS) have become increasingly common over the last 15 years due to increased availability of genome-scale genetic data. Here, we report a representative survey of GWSS from 1999 to present and find that (i) between 1999 and 2009, 35 of 49 (71%) GWSS focused on human, while from 2010 to present, only 38 of 83 (46%) of GWSS focused on human, indicating increased focus on nonmodel organisms; (ii) the large majority of GWSS incorporate interpopulation or interspecific comparisons using, for example F(ST), cross-population extended haplotype homozygosity or the ratio of nonsynonymous to synonymous substitutions; (iii) most GWSS focus on detection of directional selection rather than other modes such as balancing selection; and (iv) in human GWSS, there is a clear shift after 2004 from microsatellite markers to dense SNP data. A survey of GWSS meant to identify loci positively selected in response to severe hypoxic conditions support an approach to GWSS in which a list of a priori candidate genes based on potential selective pressures are used to filter the list of significant hits a posteriori. We also discuss four frequently ignored determinants of genomic heterogeneity that complicate GWSS: mutation, recombination, selection and the genetic architecture of adaptive traits. We recommend that GWSS methodology should better incorporate aspects of genomewide heterogeneity using empirical estimates of relevant parameters and/or realistic, whole-chromosome simulations to improve interpretation of GWSS results. Finally, we argue that knowledge of potential selective agents improves interpretation of GWSS results and that new methods focused on correlations between environmental variables and genetic variation can help automate this approach. © 2015 John Wiley & Sons Ltd.

Digital gene expression analysis of the zebra finch genome

PubMed Central

2010-01-01

Background In order to understand patterns of adaptation and molecular evolution it is important to quantify both variation in gene expression and nucleotide sequence divergence. Gene expression profiling in non-model organisms has recently been facilitated by the advent of massively parallel sequencing technology. Here we investigate tissue specific gene expression patterns in the zebra finch (Taeniopygia guttata) with special emphasis on the genes of the major histocompatibility complex (MHC). Results Almost 2 million 454-sequencing reads from cDNA of six different tissues were assembled and analysed. A total of 11,793 zebra finch transcripts were represented in this EST data, indicating a transcriptome coverage of about 65%. There was a positive correlation between the tissue specificity of gene expression and non-synonymous to synonymous nucleotide substitution ratio of genes, suggesting that genes with a specialised function are evolving at a higher rate (or with less constraint) than genes with a more general function. In line with this, there was also a negative correlation between overall expression levels and expression specificity of contigs. We found evidence for expression of 10 different genes related to the MHC. MHC genes showed relatively tissue specific expression levels and were in general primarily expressed in spleen. Several MHC genes, including MHC class I also showed expression in brain. Furthermore, for all genes with highest levels of expression in spleen there was an overrepresentation of several gene ontology terms related to immune function. Conclusions Our study highlights the usefulness of next-generation sequence data for quantifying gene expression in the genome as a whole as well as in specific candidate genes. Overall, the data show predicted patterns of gene expression profiles and molecular evolution in the zebra finch genome. Expression of MHC genes in particular, corresponds well with expression patterns in other vertebrates. PMID:20359325

Clonality, genetic diversity and support for the diversifying selection hypothesis in natural populations of a flower-living yeast.

PubMed

Herrera, C M; Pozo, M I; Bazaga, P

2011-11-01

Vast amounts of effort have been devoted to investigate patterns of genetic diversity and structuring in plants and animals, but similar information is scarce for organisms of other kingdoms. The study of the genetic structure of natural populations of wild yeasts can provide insights into the ecological and genetic correlates of clonality, and into the generality of recent hypotheses postulating that microbial populations lack the potential for genetic divergence and allopatric speciation. Ninety-one isolates of the flower-living yeast Metschnikowia gruessii from southeastern Spain were DNA fingerprinted using amplified fragment length polymorphism (AFLP) markers. Genetic diversity and structuring was investigated with band-based methods and model- and nonmodel-based clustering. Linkage disequilibrium tests were used to assess reproduction mode. Microsite-dependent, diversifying selection was tested by comparing genetic characteristics of isolates from bumble bee vectors and different floral microsites. AFLP polymorphism (91%) and genotypic diversity were very high. Genetic diversity was spatially structured, as shown by amova (Φ(st)  = 0.155) and clustering. The null hypothesis of random mating was rejected, clonality seeming the prevailing reproductive mode in the populations studied. Genetic diversity of isolates declined from bumble bee mouthparts to floral microsites, and frequency of five AFLP markers varied significantly across floral microsites, thus supporting the hypothesis of diversifying selection on clonal lineages. Wild populations of clonal fungal microbes can exhibit levels of genetic diversity and spatial structuring that are not singularly different from those shown by sexually reproducing plants or animals. Microsite-dependent, divergent selection can maintain high local and regional genetic diversity in microbial populations despite extensive clonality. © 2011 Blackwell Publishing Ltd.

Characterisation of the transcriptome of a wild great tit Parus major population by next generation sequencing

PubMed Central

2011-01-01

Background The recent development of next generation sequencing technologies has made it possible to generate very large amounts of sequence data in species with little or no genome information. Combined with the large phenotypic databases available for wild and non-model species, these data will provide an unprecedented opportunity to "genomicise" ecological model organisms and establish the genetic basis of quantitative traits in natural populations. Results This paper describes the sequencing, de novo assembly and analysis from the transcriptome of eight tissues of ten wild great tits. Approximately 4.6 million sequences and 1.4 billion bases of DNA were generated and assembled into 95,979 contigs, one third of which aligned with known Taeniopygia guttata (zebra finch) and Gallus gallus (chicken) transcripts. The majority (78%) of the remaining contigs aligned within or very close to regions of the zebra finch genome containing known genes, suggesting that they represented precursor mRNA rather than untranscribed genomic DNA. More than 35,000 single nucleotide polymorphisms and 10,000 microsatellite repeats were identified. Eleven percent of contigs were expressed in every tissue, while twenty one percent of contigs were expressed in only one tissue. The function of those contigs with strong evidence for tissue specific expression and contigs expressed in every tissue was inferred from the gene ontology (GO) terms associated with these contigs; heart and pancreas had the highest number of highly tissue specific GO terms (21.4% and 28.5% respectively). Conclusions In summary, the transcriptomic data generated in this study will contribute towards efforts to assemble and annotate the great tit genome, as well as providing the markers required to perform gene mapping studies in wild populations. PMID:21635727

Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L.)

PubMed Central

Moazzam Jazi, Maryam; Ghadirzadeh Khorzoghi, Effat; Botanga, Christopher; Seyedi, Seyed Mahdi

2016-01-01

The tree species, Pistacia vera (P. vera) is an important commercial product that is salt-tolerant and long-lived, with a possible lifespan of over one thousand years. Gene expression analysis is an efficient method to explore the possible regulatory mechanisms underlying these characteristics. Therefore, having the most suitable set of reference genes is required for transcript level normalization under different conditions in P. vera. In the present study, we selected eight widely used reference genes, ACT, EF1α, α-TUB, β-TUB, GAPDH, CYP2, UBQ10, and 18S rRNA. Using qRT-PCR their expression was assessed in 54 different samples of three cultivars of P. vera. The samples were collected from different organs under various abiotic treatments (cold, drought, and salt) across three time points. Several statistical programs (geNorm, NormFinder, and BestKeeper) were applied to estimate the expression stability of candidate reference genes. Results obtained from the statistical analysis were then exposed to Rank aggregation package to generate a consensus gene rank. Based on our results, EF1α was found to be the superior reference gene in all samples under all abiotic treatments. In addition to EF1α, ACT and β-TUB were the second best reference genes for gene expression analysis in leaf and root. We recommended β-TUB as the second most stable gene for samples under the cold and drought treatments, while ACT holds the same position in samples analyzed under salt treatment. This report will benefit future research on the expression profiling of P. vera and other members of the Anacardiaceae family. PMID:27308855

Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L.).

PubMed

Moazzam Jazi, Maryam; Ghadirzadeh Khorzoghi, Effat; Botanga, Christopher; Seyedi, Seyed Mahdi

2016-01-01

The tree species, Pistacia vera (P. vera) is an important commercial product that is salt-tolerant and long-lived, with a possible lifespan of over one thousand years. Gene expression analysis is an efficient method to explore the possible regulatory mechanisms underlying these characteristics. Therefore, having the most suitable set of reference genes is required for transcript level normalization under different conditions in P. vera. In the present study, we selected eight widely used reference genes, ACT, EF1α, α-TUB, β-TUB, GAPDH, CYP2, UBQ10, and 18S rRNA. Using qRT-PCR their expression was assessed in 54 different samples of three cultivars of P. vera. The samples were collected from different organs under various abiotic treatments (cold, drought, and salt) across three time points. Several statistical programs (geNorm, NormFinder, and BestKeeper) were applied to estimate the expression stability of candidate reference genes. Results obtained from the statistical analysis were then exposed to Rank aggregation package to generate a consensus gene rank. Based on our results, EF1α was found to be the superior reference gene in all samples under all abiotic treatments. In addition to EF1α, ACT and β-TUB were the second best reference genes for gene expression analysis in leaf and root. We recommended β-TUB as the second most stable gene for samples under the cold and drought treatments, while ACT holds the same position in samples analyzed under salt treatment. This report will benefit future research on the expression profiling of P. vera and other members of the Anacardiaceae family.

Testing Convergent Evolution in Auditory Processing Genes between Echolocating Mammals and the Aye-Aye, a Percussive-Foraging Primate.

PubMed

Bankoff, Richard J; Jerjos, Michael; Hohman, Baily; Lauterbur, M Elise; Kistler, Logan; Perry, George H

2017-07-01

Several taxonomically distinct mammalian groups-certain microbats and cetaceans (e.g., dolphins)-share both morphological adaptations related to echolocation behavior and strong signatures of convergent evolution at the amino acid level across seven genes related to auditory processing. Aye-ayes (Daubentonia madagascariensis) are nocturnal lemurs with a specialized auditory processing system. Aye-ayes tap rapidly along the surfaces of trees, listening to reverberations to identify the mines of wood-boring insect larvae; this behavior has been hypothesized to functionally mimic echolocation. Here we investigated whether there are signals of convergence in auditory processing genes between aye-ayes and known mammalian echolocators. We developed a computational pipeline (Basic Exon Assembly Tool) that produces consensus sequences for regions of interest from shotgun genomic sequencing data for nonmodel organisms without requiring de novo genome assembly. We reconstructed complete coding region sequences for the seven convergent echolocating bat-dolphin genes for aye-ayes and another lemur. We compared sequences from these two lemurs in a phylogenetic framework with those of bat and dolphin echolocators and appropriate nonecholocating outgroups. Our analysis reaffirms the existence of amino acid convergence at these loci among echolocating bats and dolphins; some methods also detected signals of convergence between echolocating bats and both mice and elephants. However, we observed no significant signal of amino acid convergence between aye-ayes and echolocating bats and dolphins, suggesting that aye-aye tap-foraging auditory adaptations represent distinct evolutionary innovations. These results are also consistent with a developing consensus that convergent behavioral ecology does not reliably predict convergent molecular evolution. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

An integrated proteomic and transcriptomic analysis of perivitelline fluid proteins in a freshwater gastropod laying aerial eggs.

PubMed

Mu, Huawei; Sun, Jin; Heras, Horacio; Chu, Ka Hou; Qiu, Jian-Wen

2017-02-23

Proteins of the egg perivitelline fluid (PVF) that surrounds the embryo are critical for embryonic development in many animals, but little is known about their identities. Using an integrated proteomic and transcriptomic approach, we identified 64 proteins from the PVF of Pomacea maculata, a freshwater snail adopting aerial oviposition. Proteins were classified into eight functional groups: major multifunctional perivitellin subunits, immune response, energy metabolism, protein degradation, oxidation-reduction, signaling and binding, transcription and translation, and others. Comparison of gene expression levels between tissues showed that 22 PVF genes were exclusively expressed in albumen gland, the female organ that secretes PVF. Base substitution analysis of PVF and housekeeping genes between P. maculata and its closely related species Pomacea canaliculata showed that the reproductive proteins had a higher mean evolutionary rate. Predicted 3D structures of selected PVF proteins showed that some nonsynonymous substitutions are located at or near the binding regions that may affect protein function. The proteome and sequence divergence analysis revealed a substantial amount of maternal investment in embryonic nutrition and defense, and higher adaptive selective pressure on PVF protein-coding genes when compared with housekeeping genes, providing insight into the adaptations associated with the unusual reproductive strategy in these mollusks. There has been great interest in studying reproduction-related proteins as such studies may not only answer fundamental questions about speciation and evolution, but also solve practical problems of animal infertility and pest outbreak. Our study has demonstrated the effectiveness of an integrated proteomic and transcriptomic approach in understanding the heavy maternal investment of proteins in the eggs of a non-model snail, and how the reproductive proteins may have evolved during the transition from laying underwater eggs to aerial eggs. Copyright © 2017 Elsevier B.V. All rights reserved.

AmpuBase: a transcriptome database for eight species of apple snails (Gastropoda: Ampullariidae).

PubMed

Ip, Jack C H; Mu, Huawei; Chen, Qian; Sun, Jin; Ituarte, Santiago; Heras, Horacio; Van Bocxlaer, Bert; Ganmanee, Monthon; Huang, Xin; Qiu, Jian-Wen

2018-03-05

Gastropoda, with approximately 80,000 living species, is the largest class of Mollusca. Among gastropods, apple snails (family Ampullariidae) are globally distributed in tropical and subtropical freshwater ecosystems and many species are ecologically and economically important. Ampullariids exhibit various morphological and physiological adaptations to their respective habitats, which make them ideal candidates for studying adaptation, population divergence, speciation, and larger-scale patterns of diversity, including the biogeography of native and invasive populations. The limited availability of genomic data, however, hinders in-depth ecological and evolutionary studies of these non-model organisms. Using Illumina Hiseq platforms, we sequenced 1220 million reads for seven species of apple snails. Together with the previously published RNA-Seq data of two apple snails, we conducted de novo transcriptome assembly of eight species that belong to five genera of Ampullariidae, two of which represent Old World lineages and the other three New World lineages. There were 20,730 to 35,828 unigenes with predicted open reading frames for the eight species, with N50 (shortest sequence length at 50% of the unigenes) ranging from 1320 to 1803 bp. 69.7% to 80.2% of these unigenes were functionally annotated by searching against NCBI's non-redundant, Gene Ontology database and the Kyoto Encyclopaedia of Genes and Genomes. With these data we developed AmpuBase, a relational database that features online BLAST functionality for DNA/protein sequences, keyword searching for unigenes/functional terms, and download functions for sequences and whole transcriptomes. In summary, we have generated comprehensive transcriptome data for multiple ampullariid genera and species, and created a publicly accessible database with a user-friendly interface to facilitate future basic and applied studies on ampullariids, and comparative molecular studies with other invertebrates.

Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions.

PubMed

Lo, Te-Wen; Pickle, Catherine S; Lin, Steven; Ralston, Edward J; Gurling, Mark; Schartner, Caitlin M; Bian, Qian; Doudna, Jennifer A; Meyer, Barbara J

2013-10-01

Exploitation of custom-designed nucleases to induce DNA double-strand breaks (DSBs) at genomic locations of choice has transformed our ability to edit genomes, regardless of their complexity. DSBs can trigger either error-prone repair pathways that induce random mutations at the break sites or precise homology-directed repair pathways that generate specific insertions or deletions guided by exogenously supplied DNA. Prior editing strategies using site-specific nucleases to modify the Caenorhabditis elegans genome achieved only the heritable disruption of endogenous loci through random mutagenesis by error-prone repair. Here we report highly effective strategies using TALE nucleases and RNA-guided CRISPR/Cas9 nucleases to induce error-prone repair and homology-directed repair to create heritable, precise insertion, deletion, or substitution of specific DNA sequences at targeted endogenous loci. Our robust strategies are effective across nematode species diverged by 300 million years, including necromenic nematodes (Pristionchus pacificus), male/female species (Caenorhabditis species 9), and hermaphroditic species (C. elegans). Thus, genome-editing tools now exist to transform nonmodel nematode species into genetically tractable model organisms. We demonstrate the utility of our broadly applicable genome-editing strategies by creating reagents generally useful to the nematode community and reagents specifically designed to explore the mechanism and evolution of X chromosome dosage compensation. By developing an efficient pipeline involving germline injection of nuclease mRNAs and single-stranded DNA templates, we engineered precise, heritable nucleotide changes both close to and far from DSBs to gain or lose genetic function, to tag proteins made from endogenous genes, and to excise entire loci through targeted FLP-FRT recombination.

High intralocus variability and interlocus recombination promote immunological diversity in a minimal major histocompatibility system.

PubMed

Wilson, Anthony B; Whittington, Camilla M; Bahr, Angela

2014-12-20

The genes of the major histocompatibility complex (MHC/MH) have attracted considerable scientific interest due to their exceptional levels of variability and important function as part of the adaptive immune system. Despite a large number of studies on MH class II diversity of both model and non-model organisms, most research has focused on patterns of genetic variability at individual loci, failing to capture the functional diversity of the biologically active dimeric molecule. Here, we take a systematic approach to the study of MH variation, analyzing patterns of genetic variation at MH class IIα and IIβ loci of the seahorse, which together form the immunologically active peptide binding cleft of the MH class II molecule. The seahorse carries a minimal class II system, consisting of single copies of both MH class IIα and IIβ, which are physically linked and inherited in a Mendelian fashion. Both genes are ubiquitously expressed and detectible in the brood pouch of male seahorses throughout pregnancy. Genetic variability of the two genes is high, dominated by non-synonymous variation concentrated in their peptide-binding regions. Coding variation outside these regions is negligible, a pattern thought to be driven by intra- and interlocus recombination. Despite the tight physical linkage of MH IIα and IIβ loci, recombination has produced novel composite alleles, increasing functional diversity at sites responsible for antigen recognition. Antigen recognition by the adaptive immune system of the seahorse is enhanced by high variability at both MH class IIα and IIβ loci. Strong positive selection on sites involved in pathogen recognition, coupled with high levels of intra- and interlocus recombination, produce a patchwork pattern of genetic variation driven by genetic hitchhiking. Studies focusing on variation at individual MH loci may unintentionally overlook an important component of ecologically relevant variation.

Genome-wide analyses of the bHLH superfamily in crustaceans: reappraisal of higher-order groupings and evidence for lineage-specific duplications

PubMed Central

2018-01-01

The basic helix-loop-helix (bHLH) proteins represent a key group of transcription factors implicated in numerous eukaryotic developmental and signal transduction processes. Characterization of bHLHs from model species such as humans, fruit flies, nematodes and plants have yielded important information on their functions and evolutionary origin. However, relatively little is known about bHLHs in non-model organisms despite the availability of a vast number of high-throughput sequencing datasets, enabling previously intractable genome-wide and cross-species analyses to be now performed. We extensively searched for bHLHs in 126 crustacean species represented across major Crustacea taxa and identified 3777 putative bHLH orthologues. We have also included seven whole-genome datasets representative of major arthropod lineages to obtain a more accurate prediction of the full bHLH gene complement. With focus on important food crop species from Decapoda, we further defined higher-order groupings and have successfully recapitulated previous observations in other animals. Importantly, we also observed evidence for lineage-specific bHLH expansions in two basal crustaceans (branchiopod and copepod), suggesting a mode of evolution through gene duplication as an adaptation to changing environments. In-depth analysis on bHLH-PAS members confirms the phenomenon coined as ‘modular evolution’ (independently evolved domains) typically seen in multidomain proteins. With the amphipod Parhyale hawaiensis as the exception, our analyses have focused on crustacean transcriptome datasets. Hence, there is a clear requirement for future analyses on whole-genome sequences to overcome potential limitations associated with transcriptome mining. Nonetheless, the present work will serve as a key resource for future mechanistic and biochemical studies on bHLHs in economically important crustacean food crop species. PMID:29657824

De Novo Transcriptomes of a Mixotrophic and a Heterotrophic Ciliate from Marine Plankton

PubMed Central

Santoferrara, Luciana F.; Guida, Stephanie; Zhang, Huan; McManus, George B.

2014-01-01

Studying non-model organisms is crucial in the context of the current development of genomics and transcriptomics for both physiological experimentation and environmental characterization. We investigated the transcriptomes of two marine planktonic ciliates, the mixotrophic oligotrich Strombidium rassoulzadegani and the heterotrophic choreotrich Strombidinopsis sp., and their respective algal food using Illumina RNAseq. Our aim was to characterize the transcriptomes of these contrasting ciliates and to identify genes potentially involved in mixotrophy. We detected approximately 10,000 and 7,600 amino acid sequences for S. rassoulzadegani and Strombidinopsis sp., respectively. About half of these transcripts had significant BLASTP hits (E-value <10−6) against previously-characterized sequences, mostly from the model ciliate Oxytricha trifallax. Transcriptomes from both the mixotroph and the heterotroph species provided similar annotations for GO terms and KEGG pathways. Most of the identified genes were related to housekeeping activity and pathways such as the metabolism of carbohydrates, lipids, amino acids, nucleotides, and vitamins. Although S. rassoulzadegani can keep and use chloroplasts from its prey, we did not find genes clearly linked to chloroplast maintenance and functioning in the transcriptome of this ciliate. While chloroplasts are known sources of reactive oxygen species (ROS), we found the same complement of antioxidant pathways in both ciliates, except for one enzyme possibly linked to ascorbic acid recycling found exclusively in the mixotroph. Contrary to our expectations, we did not find qualitative differences in genes potentially related to mixotrophy. However, these transcriptomes will help to establish a basis for the evaluation of differential gene expression in oligotrichs and choreotrichs and experimental investigation of the costs and benefits of mixotrophy. PMID:24983246

Cuticular bacteria appear detrimental to social spiders in mixed but not monoculture exposure

PubMed Central

Keiser, Carl N.; Shearer, Taylor A.; DeMarco, Alexander E.; Brittingham, Hayley A.; Knutson, Karen A.; Kuo, Candice; Zhao, Katherine; Pruitt, Jonathan N.

2016-01-01

Abstract Much of an animal’s health status, life history, and behavior are dictated by interactions with its endogenous and exogenous bacterial communities. Unfortunately, interactions between hosts and members of their resident bacterial community are often ignored in animal behavior and behavioral ecology. Here, we aim to identify the nature of host–microbe interactions in a nonmodel organism, the African social spider Stegodyphus dumicola. We collected and identified bacteria from the cuticles of spiders in situ and then exposed spiders to bacterial monocultures cultures via topical application or injection. We also topically inoculated spiders with a concomitant “cocktail” of bacteria and measured the behavior of spiders daily for 24 days after inoculation. Lastly, we collected and identified bacteria from the cuticles of prey items in the capture webs of spiders, and then fed spiders domestic crickets which had been injected with these bacteria. We also injected 1 species of prey-borne bacteria into the hemolymph of spiders. Only Bacillus thuringiensis caused increased mortality when injected into the hemolymph of spiders, whereas no bacterial monocultures caused increased mortality when applied topically, relative to control solutions. However, a bacterial cocktail of cuticular bacteria caused weight loss and mortality when applied topically, yet did not detectibly alter spider behavior. Consuming prey injected with prey-borne bacteria was associated with an elongated lifespan in spiders. Thus, indirect evidence from multiple experiments suggests that the effects of these bacteria on spider survivorship appear contingent on their mode of colonization and whether they are applied in monoculture or within a mixed cocktail. We urge that follow-up studies should test these host–microbe interactions across different social contexts to determine the role that microbes play in colony performance. PMID:29491926

Epigenetic estimation of age in humpback whales.

PubMed

Polanowski, Andrea M; Robbins, Jooke; Chandler, David; Jarman, Simon N

2014-09-01

Age is a fundamental aspect of animal ecology, but is difficult to determine in many species. Humpback whales exemplify this as they have a lifespan comparable to humans, mature sexually as early as 4 years and have no reliable visual age indicators after their first year. Current methods for estimating humpback age cannot be applied to all individuals and populations. Assays for human age have recently been developed based on age-induced changes in DNA methylation of specific genes. We used information on age-associated DNA methylation in human and mouse genes to identify homologous gene regions in humpbacks. Humpback skin samples were obtained from individuals with a known year of birth and employed to calibrate relationships between cytosine methylation and age. Seven of 37 cytosines assayed for methylation level in humpback skin had significant age-related profiles. The three most age-informative cytosine markers were selected for a humpback epigenetic age assay. The assay has an R(2) of 0.787 (P = 3.04e-16) and predicts age from skin samples with a standard deviation of 2.991 years. The epigenetic method correctly determined which of parent-offspring pairs is the parent in more than 93% of cases. To demonstrate the potential of this technique, we constructed the first modern age profile of humpback whales off eastern Australia and compared the results to population structure 5 decades earlier. This is the first epigenetic age estimation method for a wild animal species and the approach we took for developing it can be applied to many other nonmodel organisms. © 2014 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

Identification and validation of single nucleotide polymorphisms in growth- and maturation-related candidate genes in sole (Solea solea L.).

PubMed

Diopere, Eveline; Hellemans, Bart; Volckaert, Filip A M; Maes, Gregory E

2013-03-01

Genomic methodologies applied in evolutionary and fisheries research have been of great benefit to understand the marine ecosystem and the management of natural resources. Although single nucleotide polymorphisms (SNPs) are attractive for the study of local adaptation, spatial stock management and traceability, and investigating the effects of fisheries-induced selection, they have rarely been exploited in non-model organisms. This is partly due to difficulties in finding and validating SNPs in species with limited or no genomic resources. Complementary to random genome-scan approaches, a targeted candidate gene approach has the potential to unveil pre-selected functional diversity and provides more in depth information on the action of selection at specific genes. For example genes can be under selective pressure due to climate change and sustained periods of heavy fishing pressure. In this study, we applied a candidate gene approach in sole (Solea solea L.), an important member of the demersal ecosystem. As consumption flatfish it is heavy exploited and has experienced associated life-history changes over the last 60years. To discover novel genetic polymorphisms in or around genes linked to important life history traits in sole, we screened a total of 76 candidate genes related to growth and maturation using a targeted resequencing approach. We identified in total 86 putative SNPs in 22 genes and validated 29 SNPs using a multiplex single-base extension genotyping assay. We found 22 informative SNPs, of which two represent non-synonymous mutations, potentially of functional relevance. These novel markers should be rapidly and broadly applicable in analyses of natural sole populations, as a measure of the evolutionary signature of overfishing and for initiatives on marker assisted selection. Copyright © 2012 Elsevier B.V. All rights reserved.

Stronger transferability but lower variability in transcriptomic- than in anonymous microsatellites: evidence from Hylid frogs.

PubMed

Dufresnes, Christophe; Brelsford, Alan; Béziers, Paul; Perrin, Nicolas

2014-07-01

A simple way to quickly optimize microsatellites in nonmodel organisms is to reuse loci available in closely related taxa; however, this approach can be limited by the stochastic and low cross-amplification success experienced in some groups (e.g. amphibians). An efficient alternative is to develop loci from transcriptome sequences. Transcriptomic microsatellites have been found to vary in their levels of cross-species amplification and variability, but this has to date never been tested in amphibians. Here, we compare the patterns of cross-amplification and levels of polymorphism of 18 published anonymous microsatellites isolated from genomic DNA vs. 17 loci derived from a transcriptome, across nine species of tree frogs (Hyla arborea and Hyla cinerea group). We established a clear negative relationship between divergence time and amplification success, which was much steeper for anonymous than transcriptomic markers, with half-lives (time at which 50% of the markers still amplify) of 1.1 and 37 My, respectively. Transcriptomic markers are significantly less polymorphic than anonymous loci, but remain variable across diverged taxa. We conclude that the exploitation of amphibian transcriptomes for developing microsatellites seems an optimal approach for multispecies surveys (e.g. analyses of hybrid zones, comparative linkage mapping), whereas anonymous microsatellites may be more informative for fine-scale analyses of intraspecific variation. Moreover, our results confirm the pattern that microsatellite cross-amplification is greatly variable among amphibians and should be assessed independently within target lineages. Finally, we provide a bank of microsatellites for Palaearctic tree frogs (so far only available for H. arborea), which will be useful for conservation and evolutionary studies in this radiation. © 2013 John Wiley & Sons Ltd.

Phylogenetic Conflict in Bears Identified by Automated Discovery of Transposable Element Insertions in Low-Coverage Genomes.

PubMed

Lammers, Fritjof; Gallus, Susanne; Janke, Axel; Nilsson, Maria A

2017-10-01

Phylogenetic reconstruction from transposable elements (TEs) offers an additional perspective to study evolutionary processes. However, detecting phylogenetically informative TE insertions requires tedious experimental work, limiting the power of phylogenetic inference. Here, we analyzed the genomes of seven bear species using high-throughput sequencing data to detect thousands of TE insertions. The newly developed pipeline for TE detection called TeddyPi (TE detection and discovery for Phylogenetic Inference) identified 150,513 high-quality TE insertions in the genomes of ursine and tremarctine bears. By integrating different TE insertion callers and using a stringent filtering approach, the TeddyPi pipeline produced highly reliable TE insertion calls, which were confirmed by extensive in vitro validation experiments. Analysis of single nucleotide substitutions in the flanking regions of the TEs shows that these substitutions correlate with the phylogenetic signal from the TE insertions. Our phylogenomic analyses show that TEs are a major driver of genomic variation in bears and enabled phylogenetic reconstruction of a well-resolved species tree, despite strong signals for incomplete lineage sorting and introgression. The analyses show that the Asiatic black, sun, and sloth bear form a monophyletic clade, in which phylogenetic incongruence originates from incomplete lineage sorting. TeddyPi is open source and can be adapted to various TE and structural variation callers. The pipeline makes it possible to confidently extract thousands of TE insertions even from low-coverage genomes (∼10×) of nonmodel organisms. This opens new possibilities for biologists to study phylogenies and evolutionary processes as well as rates and patterns of (retro-)transposition and structural variation. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Reproductive success and contaminant associations in tree swallows (Tachycineta bicolor) used to assess a Beneficial Use Impairment in U.S. and Binational Great Lakes’ Areas of Concern

USGS Publications Warehouse

Custer, Christine M.; Custer, Thomas W.; Etterson, Matthew A.; Dummer, Paul; Goldberg, Diana R.; Franson, J. Christian

2018-01-01

During 2010-2014, tree swallow (Tachycineta bicolor) reproductive success was monitored at 68 sites across all 5 Great Lakes, including 58 sites located within Great Lakes Areas of Concern (AOCs) and 10 non-AOCs. Sample eggs were collected from tree swallow clutches and analyzed for contaminants including polychlorinated biphenyls (PCBs), dioxins and furans, polybrominated diphenyl ethers, and 34 other organic compounds. Contaminant data were available for 360 of the clutches monitored. Markov chain multistate modeling was used to assess the importance of 5 ecological variables and 11 of the dominant contaminants in explaining the pattern of egg and nestling failure rates. Four of 5 ecological variables (Female Age, Date within season, Year, and Site) were important explanatory variables. Of the 11 contaminants, only total dioxin and furan toxic equivalents (TEQs) explained a significant amount of the egg failure probabilities. Neither total PCBs nor PCB TEQs explained the variation in egg failure rates. In a separate analysis, polycyclic aromatic hydrocarbon exposure in nestling diet, used as a proxy for female diet during egg laying, was significantly correlated with the daily probability of egg failure. The 8 sites within AOCs which had poorer reproduction when compared to 10 non-AOC sites, the measure of impaired reproduction as defined by the Great Lakes Restoration Initiative, were associated with exposure to dioxins and furan TEQs, PAHs, or depredation. Only 2 sites had poorer reproduction than the poorest performing non-AOC. Using a classic (non-modeling) approach to estimating reproductive success, 82% of nests hatched at least 1 egg, and 75% of eggs laid, excluding those collected for contaminant analyses, hatched.

Chromosomal Speciation in the Genomics Era: Disentangling Phylogenetic Evolution of Rock-wallabies.

PubMed

Potter, Sally; Bragg, Jason G; Blom, Mozes P K; Deakin, Janine E; Kirkpatrick, Mark; Eldridge, Mark D B; Moritz, Craig

2017-01-01

The association of chromosome rearrangements (CRs) with speciation is well established, and there is a long history of theory and evidence relating to "chromosomal speciation." Genomic sequencing has the potential to provide new insights into how reorganization of genome structure promotes divergence, and in model systems has demonstrated reduced gene flow in rearranged segments. However, there are limits to what we can understand from a small number of model systems, which each only tell us about one episode of chromosomal speciation. Progressing from patterns of association between chromosome (and genic) change, to understanding processes of speciation requires both comparative studies across diverse systems and integration of genome-scale sequence comparisons with other lines of evidence. Here, we showcase a promising example of chromosomal speciation in a non-model organism, the endemic Australian marsupial genus Petrogale . We present initial phylogenetic results from exon-capture that resolve a history of divergence associated with extensive and repeated CRs. Yet it remains challenging to disentangle gene tree heterogeneity caused by recent divergence and gene flow in this and other such recent radiations. We outline a way forward for better integration of comparative genomic sequence data with evidence from molecular cytogenetics, and analyses of shifts in the recombination landscape and potential disruption of meiotic segregation and epigenetic programming. In all likelihood, CRs impact multiple cellular processes and these effects need to be considered together, along with effects of genic divergence. Understanding the effects of CRs together with genic divergence will require development of more integrative theory and inference methods. Together, new data and analysis tools will combine to shed light on long standing questions of how chromosome and genic divergence promote speciation.

Comparative and Evolutionary Analysis of Grass Pollen Allergens Using Brachypodium distachyon as a Model System.

PubMed

Sharma, Akanksha; Sharma, Niharika; Bhalla, Prem; Singh, Mohan

2017-01-01

Comparative genomics have facilitated the mining of biological information from a genome sequence, through the detection of similarities and differences with genomes of closely or more distantly related species. By using such comparative approaches, knowledge can be transferred from the model to non-model organisms and insights can be gained in the structural and evolutionary patterns of specific genes. In the absence of sequenced genomes for allergenic grasses, this study was aimed at understanding the structure, organisation and expression profiles of grass pollen allergens using the genomic data from Brachypodium distachyon as it is phylogenetically related to the allergenic grasses. Combining genomic data with the anther RNA-Seq dataset revealed 24 pollen allergen genes belonging to eight allergen groups mapping on the five chromosomes in B. distachyon. High levels of anther-specific expression profiles were observed for the 24 identified putative allergen-encoding genes in Brachypodium. The genomic evidence suggests that gene encoding the group 5 allergen, the most potent trigger of hay fever and allergic asthma originated as a pollen specific orphan gene in a common grass ancestor of Brachypodium and Triticiae clades. Gene structure analysis showed that the putative allergen-encoding genes in Brachypodium either lack or contain reduced number of introns. Promoter analysis of the identified Brachypodium genes revealed the presence of specific cis-regulatory sequences likely responsible for high anther/pollen-specific expression. With the identification of putative allergen-encoding genes in Brachypodium, this study has also described some important plant gene families (e.g. expansin superfamily, EF-Hand family, profilins etc) for the first time in the model plant Brachypodium. Altogether, the present study provides new insights into structural characterization and evolution of pollen allergens and will further serve as a base for their functional characterization in related grass species.

Comparative ultrastructure of fruit plastids in three genetically diverse genotypes of apple (Malus × domestica Borkh.) during development.

PubMed

Schaeffer, Scott M; Christian, Ryan; Castro-Velasquez, Nohely; Hyden, Brennan; Lynch-Holm, Valerie; Dhingra, Amit

2017-10-01

Comparative ultrastructural developmental time-course analysis has identified discrete stages at which the fruit plastids undergo structural and consequently functional transitions to facilitate subsequent development-guided understanding of the complex plastid biology. Plastids are the defining organelle for a plant cell and are critical for myriad metabolic functions. The role of leaf plastid, chloroplast, is extensively documented; however, fruit plastids-chromoplasts-are poorly understood, especially in the context of the diverse metabolic processes operating in these diverse plant organs. Recently, in a comparative study of the predicted plastid-targeted proteomes across seven plant species, we reported that each plant species is predicted to harbor a unique set of plastid-targeted proteins. However, the temporal and developmental context of these processes remains unknown. In this study, an ultrastructural analysis approach was used to characterize fruit plastids in the epidermal and collenchymal cell layers at 11 developmental timepoints in three genotypes of apple (Malus × domestica Borkh.): chlorophyll-predominant 'Granny Smith', carotenoid-predominant 'Golden Delicious', and anthocyanin-predominant 'Top Red Delicious'. Plastids transitioned from a proplastid-like plastid to a chromoplast-like plastid in epidermis cells, while in the collenchyma cells, they transitioned from a chloroplast-like plastid to a chloro-chromo-amyloplast plastid. Plastids in the collenchyma cells of the three genotypes demonstrated a diverse array of structures and features. This study enabled the identification of discrete developmental stages during which specific functions are most likely being performed by the plastids as indicated by accumulation of plastoglobuli, starch granules, and other sub-organeller structures. Information regarding the metabolically active developmental stages is expected to facilitate biologically relevant omics studies to unravel the complex biochemistry of plastids in perennial non-model systems.

Sequencing and de novo analysis of the hemocytes transcriptome in Litopenaeus vannamei response to white spot syndrome virus infection.

PubMed

Xue, Shuxia; Liu, Yichen; Zhang, Yichen; Sun, Yan; Geng, Xuyun; Sun, Jinsheng

2013-01-01

White spot syndrome virus (WSSV) is a causative pathogen found in most shrimp farming areas of the world and causes large economic losses to the shrimp aquaculture. The mechanism underlying the molecular pathogenesis of the highly virulent WSSV remains unknown. To better understand the virus-host interactions at the molecular level, the transcriptome profiles in hemocytes of unchallenged and WSSV-challenged shrimp (Litopenaeus vannamei) were compared using a short-read deep sequencing method (Illumina). RNA-seq analysis generated more than 25.81 million clean pair end (PE) reads, which were assembled into 52,073 unigenes (mean size = 520 bp). Based on sequence similarity searches, 23,568 (45.3%) genes were identified, among which 6,562 and 7,822 unigenes were assigned to gene ontology (GO) categories and clusters of orthologous groups (COG), respectively. Searches in the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) mapped 14,941 (63.4%) unigenes to 240 KEGG pathways. Among all the annotated unigenes, 1,179 were associated with immune-related genes. Digital gene expression (DGE) analysis revealed that the host transcriptome profile was slightly changed in the early infection (5 hours post injection) of the virus, while large transcriptional differences were identified in the late infection (48 hpi) of WSSV. The differentially expressed genes mainly involved in pattern recognition genes and some immune response factors. The results indicated that antiviral immune mechanisms were probably involved in the recognition of pathogen-associated molecular patterns. This study provided a global survey of host gene activities against virus infection in a non-model organism, pacific white shrimp. Results can contribute to the in-depth study of candidate genes in white shrimp, and help to improve the current understanding of host-pathogen interactions.

Reproductive success and contaminant associations in tree swallows (Tachycineta bicolor) used to assess a Beneficial Use Impairment in U.S. and Binational Great Lakes' Areas of Concern.

PubMed

Custer, Christine M; Custer, Thomas W; Etterson, Matthew A; Dummer, Paul M; Goldberg, Diana; Franson, J Christian

2018-05-01

During 2010-2014, tree swallow (Tachycineta bicolor) reproductive success was monitored at 68 sites across all 5 Great Lakes, including 58 sites located within Great Lakes Areas of Concern (AOCs) and 10 non-AOCs. Sample eggs were collected from tree swallow clutches and analyzed for contaminants including polychlorinated biphenyls (PCBs), dioxins and furans, polybrominated diphenyl ethers, and 34 other organic compounds. Contaminant data were available for 360 of the clutches monitored. Markov chain multistate modeling was used to assess the importance of 5 ecological variables and 11 of the dominant contaminants in explaining the pattern of egg and nestling failure rates. Four of 5 ecological variables (Female Age, Date within season, Year, and Site) were important explanatory variables. Of the 11 contaminants, only total dioxin and furan toxic equivalents (TEQs) explained a significant amount of the egg failure probabilities. Neither total PCBs nor PCB TEQs explained the variation in egg failure rates. In a separate analysis, polycyclic aromatic hydrocarbon exposure in nestling diet, used as a proxy for female diet during egg laying, was significantly correlated with the daily probability of egg failure. The 8 sites within AOCs which had poorer reproduction when compared to 10 non-AOC sites, the measure of impaired reproduction as defined by the Great Lakes Restoration Initiative, were associated with exposure to dioxins and furan TEQs, PAHs, or depredation. Only 2 sites had poorer reproduction than the poorest performing non-AOC. Using a classic (non-modeling) approach to estimating reproductive success, 82% of nests hatched at least 1 egg, and 75% of eggs laid, excluding those collected for contaminant analyses, hatched.

Apomixis is not prevalent in subnival to nival plants of the European Alps

PubMed Central

Hörandl, Elvira; Dobeš, Christoph; Suda, Jan; Vít, Petr; Urfus, Tomáš; Temsch, Eva M.; Cosendai, Anne-Caroline; Wagner, Johanna; Ladinig, Ursula

2011-01-01

Background and Aims High alpine environments are characterized by short growing seasons, stochastic climatic conditions and fluctuating pollinator visits. These conditions are rather unfavourable for sexual reproduction of flowering plants. Apomixis, asexual reproduction via seed, provides reproductive assurance without the need of pollinators and potentially accelerates seed development. Therefore, apomixis is expected to provide selective advantages in high-alpine biota. Indeed, apomictic species occur frequently in the subalpine to alpine grassland zone of the European Alps, but the mode of reproduction of the subnival to nival flora was largely unknown. Methods The mode of reproduction in 14 species belonging to seven families was investigated via flow cytometric seed screen. The sampling comprised 12 species typical for nival to subnival plant communities of the European Alps without any previous information on apomixis (Achillea atrata, Androsace alpina, Arabis caerulea, Erigeron uniflorus, Gnaphalium hoppeanum, Leucanthemopsis alpina, Oxyria digyna, Potentilla frigida, Ranunculus alpestris, R. glacialis, R. pygmaeus and Saxifraga bryoides), and two high-alpine species with apomixis reported from other geographical areas (Leontopodium alpinum and Potentilla crantzii). Key Results Flow cytometric data were clearly interpretable for all 46 population samples, confirming the utility of the method for broad screenings on non-model organisms. Formation of endosperm in all species of Asteraceae was documented. Ratios of endosperm : embryo showed pseudogamous apomixis for Potentilla crantzii (ratio approx. 3), but sexual reproduction for all other species (ratios approx. 1·5). Conclusions The occurrence of apomixis is not correlated to high altitudes, and cannot be readily explained by selective forces due to environmental conditions. The investigated species have probably other adaptations to high altitudes to maintain reproductive assurance via sexuality. We hypothesize that shifts to apomixis are rather connected to frequencies of polyploidization than to ecological conditions. PMID:21724654

Optimizing de novo transcriptome assembly and extending genomic resources for striped catfish (Pangasianodon hypophthalmus).

PubMed

Thanh, Nguyen Minh; Jung, Hyungtaek; Lyons, Russell E; Njaci, Isaac; Yoon, Byoung-Ha; Chand, Vincent; Tuan, Nguyen Viet; Thu, Vo Thi Minh; Mather, Peter

2015-10-01

Striped catfish (Pangasianodon hypophthalmus) is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The culture industry is facing a significant challenge however from saltwater intrusion into many low topographical coastal provinces across the Mekong Delta as a result of predicted climate change impacts. Developing genomic resources for this species can facilitate the production of improved culture lines that can withstand raised salinity conditions, and so we have applied high-throughput Ion Torrent sequencing of transcriptome libraries from six target osmoregulatory organs from striped catfish as a genomic resource for use in future selection strategies. We obtained 12,177,770 reads after trimming and processing with an average length of 97bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 66,451 contigs with an average length of 478bp and N50 length of 506bp. A total of 37,969 contigs (57%) possessed significant similarity with proteins in the non-redundant database. Comparative analyses revealed that a significant number of contigs matched sequences reported in other teleost fishes, ranging in similarity from 45.2% with Atlantic cod to 52% with zebrafish. In addition, 28,879 simple sequence repeats (SSRs) and 55,721 single nucleotide polymorphisms (SNPs) were detected in the striped catfish transcriptome. The sequence collection generated in the current study represents the most comprehensive genomic resource for P. hypophthalmus available to date. Our results illustrate the utility of next-generation sequencing as an efficient tool for constructing a large genomic database for marker development in non-model species. Copyright © 2015 Elsevier B.V. All rights reserved.

RNA-Seq reveals complex genetic response to Deepwater Horizon oil release in Fundulus grandis.

PubMed

Garcia, Tzintzuni I; Shen, Yingjia; Crawford, Douglas; Oleksiak, Marjorie F; Whitehead, Andrew; Walter, Ronald B

2012-09-12

The release of oil resulting from the blowout of the Deepwater Horizon (DH) drilling platform was one of the largest in history discharging more than 189 million gallons of oil and subject to widespread application of oil dispersants. This event impacted a wide range of ecological habitats with a complex mix of pollutants whose biological impact is still not yet fully understood. To better understand the effects on a vertebrate genome, we studied gene expression in the salt marsh minnow Fundulus grandis, which is local to the northern coast of the Gulf of Mexico and is a sister species of the ecotoxicological model Fundulus heteroclitus. To assess genomic changes, we quantified mRNA expression using high throughput sequencing technologies (RNA-Seq) in F. grandis populations in the marshes and estuaries impacted by DH oil release. This application of RNA-Seq to a non-model, wild, and ecologically significant organism is an important evaluation of the technology to quickly assess similar events in the future. Our de novo assembly of RNA-Seq data produced a large set of sequences which included many duplicates and fragments. In many cases several of these could be associated with a common reference sequence using blast to query a reference database. This reduced the set of significant genes to 1,070 down-regulated and 1,251 up-regulated genes. These genes indicate a broad and complex genomic response to DH oil exposure including the expected AHR-mediated response and CYP genes. In addition a response to hypoxic conditions and an immune response are also indicated. Several genes in the choriogenin family were down-regulated in the exposed group; a response that is consistent with AH exposure. These analyses are in agreement with oligonucleotide-based microarray analyses, and describe only a subset of significant genes with aberrant regulation in the exposed set. RNA-Seq may be successfully applied to feral and extremely polymorphic organisms that do not have an underlying genome sequence assembly to address timely environmental problems. Additionally, the observed changes in a large set of transcript expression levels are indicative of a complex response to the varied petroleum components to which the fish were exposed.

Gene-knockdown in the honey bee mite Varroa destructor by a non-invasive approach: studies on a glutathione S-transferase

PubMed Central

2010-01-01

Background The parasitic mite Varroa destructor is considered the major pest of the European honey bee (Apis mellifera) and responsible for declines in honey bee populations worldwide. Exploiting the full potential of gene sequences becoming available for V. destructor requires adaptation of modern molecular biology approaches to this non-model organism. Using a mu-class glutathione S-transferase (VdGST-mu1) as a candidate gene we investigated the feasibility of gene knockdown in V. destructor by double-stranded RNA-interference (dsRNAi). Results Intra-haemocoelic injection of dsRNA-VdGST-mu1 resulted in 97% reduction in VdGST-mu1 transcript levels 48 h post-injection compared to mites injected with a bolus of irrelevant dsRNA (LacZ). This gene suppression was maintained to, at least, 72 h. Total GST catalytic activity was reduced by 54% in VdGST-mu1 gene knockdown mites demonstrating the knockdown was effective at the translation step as well as the transcription steps. Although near total gene knockdown was achieved by intra-haemocoelic injection, only half of such treated mites survived this traumatic method of dsRNA administration and less invasive methods were assessed. V. destructor immersed overnight in 0.9% NaCl solution containing dsRNA exhibited excellent reduction in VdGST-mu1 transcript levels (87% compared to mites immersed in dsRNA-LacZ). Importantly, mites undergoing the immersion approach had greatly improved survival (75-80%) over 72 h, approaching that of mites not undergoing any treatment. Conclusions Our findings on V. destructor are the first report of gene knockdown in any mite species and demonstrate that the small size of such organisms is not a major impediment to applying gene knockdown approaches to the study of such parasitic pests. The immersion in dsRNA solution method provides an easy, inexpensive, relatively high throughput method of gene silencing suitable for studies in V. destructor, other small mites and immature stages of ticks. PMID:20712880

Identification of functional enolase genes of the silkworm Bombyx mori from public databases with a combination of dry and wet bench processes.

PubMed

Kikuchi, Akira; Nakazato, Takeru; Ito, Katsuhiko; Nojima, Yosui; Yokoyama, Takeshi; Iwabuchi, Kikuo; Bono, Hidemasa; Toyoda, Atsushi; Fujiyama, Asao; Sato, Ryoichi; Tabunoki, Hiroko

2017-01-13

Various insect species have been added to genomic databases over the years. Thus, researchers can easily obtain online genomic information on invertebrates and insects. However, many incorrectly annotated genes are included in these databases, which can prevent the correct interpretation of subsequent functional analyses. To address this problem, we used a combination of dry and wet bench processes to select functional genes from public databases. Enolase is an important glycolytic enzyme in all organisms. We used a combination of dry and wet bench processes to identify functional enolases in the silkworm Bombyx mori (BmEno). First, we detected five annotated enolases from public databases using a Hidden Markov Model (HMM) search, and then through cDNA cloning, Northern blotting, and RNA-seq analysis, we revealed three functional enolases in B. mori: BmEno1, BmEno2, and BmEnoC. BmEno1 contained a conserved key amino acid residue for metal binding and substrate binding in other species. However, BmEno2 and BmEnoC showed a change in this key amino acid. Phylogenetic analysis showed that BmEno2 and BmEnoC were distinct from BmEno1 and other enolases, and were distributed only in lepidopteran clusters. BmEno1 was expressed in all of the tissues used in our study. In contrast, BmEno2 was mainly expressed in the testis with some expression in the ovary and suboesophageal ganglion. BmEnoC was weakly expressed in the testis. Quantitative RT-PCR showed that the mRNA expression of BmEno2 and BmEnoC correlated with testis development; thus, BmEno2 and BmEnoC may be related to lepidopteran-specific spermiogenesis. We identified and characterized three functional enolases from public databases with a combination of dry and wet bench processes in the silkworm B. mori. In addition, we determined that BmEno2 and BmEnoC had species-specific functions. Our strategy could be helpful for the detection of minor genes and functional genes in non-model organisms from public databases.

Rapid construction of genome map for large yellow croaker (Larimichthys crocea) by the whole-genome mapping in BioNano Genomics Irys system.

PubMed

Xiao, Shijun; Li, Jiongtang; Ma, Fengshou; Fang, Lujing; Xu, Shuangbin; Chen, Wei; Wang, Zhi Yong

2015-09-03

Large yellow croaker (Larimichthys crocea) is an important commercial fish in China and East-Asia. The annual product of the species from the aqua-farming industry is about 90 thousand tons. In spite of its economic importance, genetic studies of economic traits and genomic selections of the species are hindered by the lack of genomic resources. Specifically, a whole-genome physical map of large yellow croaker is still missing. The traditional BAC-based fingerprint method is extremely time- and labour-consuming. Here we report the first genome map construction using the high-throughput whole-genome mapping technique by nanochannel arrays in BioNano Genomics Irys system. For an optimal marker density of ~10 per 100 kb, the nicking endonuclease Nt.BspQ1 was chosen for the genome map generation. 645,305 DNA molecules with a total length of ~112 Gb were labelled and detected, covering more than 160X of the large yellow croaker genome. Employing IrysView package and signature patterns in raw DNA molecules, a whole-genome map of large yellow croaker was assembled into 686 maps with a total length of 727 Mb, which was consistent with the estimated genome size. The N50 length of the whole-genome map, including 126 maps, was up to 1.7 Mb. The excellent hybrid alignment with large yellow croaker draft genome validated the consensus genome map assembly and highlighted a promising application of whole-genome mapping on draft genome sequence super-scaffolding. The genome map data of large yellow croaker are accessible on lycgenomics.jmu.edu.cn/pm. Using the state-of-the-art whole-genome mapping technique in Irys system, the first whole-genome map for large yellow croaker has been constructed and thus highly facilitates the ongoing genomic and evolutionary studies for the species. To our knowledge, this is the first public report on genome map construction by the whole-genome mapping for aquatic-organisms. Our study demonstrates a promising application of the whole-genome mapping on genome maps construction for other non-model organisms in a fast and reliable manner.

SNP discovery and genotyping using Genotyping-by-Sequencing in Pekin ducks.

PubMed

Zhu, Feng; Cui, Qian-Qian; Hou, Zhuo-Cheng

2016-11-15

Genomic selection and genome-wide association studies need thousands to millions of SNPs. However, many non-model species do not have reference chips for detecting variation. Our goal was to develop and validate an inexpensive but effective method for detecting SNP variation. Genotyping by sequencing (GBS) can be a highly efficient strategy for genome-wide SNP detection, as an alternative to microarray chips. Here, we developed a GBS protocol for ducks and tested it to genotype 49 Pekin ducks. A total of 169,209 SNPs were identified from all animals, with a mean of 55,920 SNPs per individual. The average SNP density reached 1156 SNPs/MB. In this study, the first application of GBS to ducks, we demonstrate the power and simplicity of this method. GBS can be used for genetic studies in to provide an effective method for genome-wide SNP discovery.

Protists and the Wild, Wild West of Gene Expression: New Frontiers, Lawlessness, and Misfits.

PubMed

Smith, David Roy; Keeling, Patrick J

2016-09-08

The DNA double helix has been called one of life's most elegant structures, largely because of its universality, simplicity, and symmetry. The expression of information encoded within DNA, however, can be far from simple or symmetric and is sometimes surprisingly variable, convoluted, and wantonly inefficient. Although exceptions to the rules exist in certain model systems, the true extent to which life has stretched the limits of gene expression is made clear by nonmodel systems, particularly protists (microbial eukaryotes). The nuclear and organelle genomes of protists are subject to the most tangled forms of gene expression yet identified. The complicated and extravagant picture of the underlying genetics of eukaryotic microbial life changes how we think about the flow of genetic information and the evolutionary processes shaping it. Here, we discuss the origins, diversity, and growing interest in noncanonical protist gene expression and its relationship to genomic architecture.

Evolutionary genetics of plant adaptation.

PubMed

Anderson, Jill T; Willis, John H; Mitchell-Olds, Thomas

2011-07-01

Plants provide unique opportunities to study the mechanistic basis and evolutionary processes of adaptation to diverse environmental conditions. Complementary laboratory and field experiments are important for testing hypotheses reflecting long-term ecological and evolutionary history. For example, these approaches can infer whether local adaptation results from genetic tradeoffs (antagonistic pleiotropy), where native alleles are best adapted to local conditions, or if local adaptation is caused by conditional neutrality at many loci, where alleles show fitness differences in one environment, but not in a contrasting environment. Ecological genetics in natural populations of perennial or outcrossing plants can also differ substantially from model systems. In this review of the evolutionary genetics of plant adaptation, we emphasize the importance of field studies for understanding the evolutionary dynamics of model and nonmodel systems, highlight a key life history trait (flowering time) and discuss emerging conservation issues. Copyright © 2011 Elsevier Ltd. All rights reserved.

Recent Advances in Algal Genetic Tool Development

DOE Office of Scientific and Technical Information (OSTI.GOV)

R. Dahlin, Lukas; T. Guarnieri, Michael

The goal of achieving cost-effective biofuels and bioproducts derived from algal biomass will require improvements along the entire value chain, including identification of robust, high-productivity strains and development of advanced genetic tools. Though there have been modest advances in development of genetic systems for the model alga Chlamydomonas reinhardtii, progress in development of algal genetic tools, especially as applied to non-model algae, has generally lagged behind that of more commonly utilized laboratory and industrial microbes. This is in part due to the complex organellar structure of algae, including robust cell walls and intricate compartmentalization of target loci, as well asmore » prevalent gene silencing mechanisms, which hinder facile utilization of conventional genetic engineering tools and methodologies. However, recent progress in global tool development has opened the door for implementation of strain-engineering strategies in industrially-relevant algal strains. Here, we review recent advances in algal genetic tool development and applications in eukaryotic microalgae.« less

Translational plant proteomics: a perspective.

PubMed

Agrawal, Ganesh Kumar; Pedreschi, Romina; Barkla, Bronwyn J; Bindschedler, Laurence Veronique; Cramer, Rainer; Sarkar, Abhijit; Renaut, Jenny; Job, Dominique; Rakwal, Randeep

2012-08-03

Translational proteomics is an emerging sub-discipline of the proteomics field in the biological sciences. Translational plant proteomics aims to integrate knowledge from basic sciences to translate it into field applications to solve issues related but not limited to the recreational and economic values of plants, food security and safety, and energy sustainability. In this review, we highlight the substantial progress reached in plant proteomics during the past decade which has paved the way for translational plant proteomics. Increasing proteomics knowledge in plants is not limited to model and non-model plants, proteogenomics, crop improvement, and food analysis, safety, and nutrition but to many more potential applications. Given the wealth of information generated and to some extent applied, there is the need for more efficient and broader channels to freely disseminate the information to the scientific community. This article is part of a Special Issue entitled: Translational Proteomics. Copyright © 2012 Elsevier B.V. All rights reserved.

Ex vitro composite plants: an inexpensive, rapid method for root biology.

PubMed

Collier, Ray; Fuchs, Beth; Walter, Nathalie; Kevin Lutke, William; Taylor, Christopher G

2005-08-01

Plant transformation technology is frequently the rate-limiting step in gene function analysis in non-model plants. An important tool for root biologists is the Agrobacterium rhizogenes-derived composite plant, which has made possible genetic analyses in a wide variety of transformation recalcitrant dicotyledonous plants. The novel, rapid and inexpensive ex vitro method for producing composite plants described in this report represents a significant advance over existing composite plant induction protocols, which rely on expensive and time-consuming in vitro conditions. The utility of the new system is validated by expression and RNAi silencing of GFP in transgenic roots of composite plants, and is bolstered further by experimental disruption, via RNAi silencing, of endogenous plant resistance to the plant parasitic nematode Meloidogyne incognita in transgenic roots of Lycopersicon esculentum cv. Motelle composite plants. Critical parameters of the method are described and discussed herein.

Recent Advances in Algal Genetic Tool Development

DOE PAGES

R. Dahlin, Lukas; T. Guarnieri, Michael

2016-06-24

The goal of achieving cost-effective biofuels and bioproducts derived from algal biomass will require improvements along the entire value chain, including identification of robust, high-productivity strains and development of advanced genetic tools. Though there have been modest advances in development of genetic systems for the model alga Chlamydomonas reinhardtii, progress in development of algal genetic tools, especially as applied to non-model algae, has generally lagged behind that of more commonly utilized laboratory and industrial microbes. This is in part due to the complex organellar structure of algae, including robust cell walls and intricate compartmentalization of target loci, as well asmore » prevalent gene silencing mechanisms, which hinder facile utilization of conventional genetic engineering tools and methodologies. However, recent progress in global tool development has opened the door for implementation of strain-engineering strategies in industrially-relevant algal strains. Here, we review recent advances in algal genetic tool development and applications in eukaryotic microalgae.« less

Developmental neurogenetics of sexual dimorphism in Aedes aegypti

PubMed Central

Duman-Scheel, Molly; Syed, Zainulabeuddin

2015-01-01

Sexual dimorphism, a poorly understood but crucial aspect of vector mosquito biology, encompasses sex-specific physical, physiological, and behavioral traits related to mosquito reproduction. The study of mosquito sexual dimorphism has largely focused on analysis of the differences between adult female and male mosquitoes, particularly with respect to sex-specific behaviors related to disease transmission. However, sexually dimorphic behaviors are the products of differential gene expression that initiates during development and therefore must also be studied during development. Recent technical advancements are facilitating functional genetic studies in the dengue vector Aedes aegypti, an emerging model for mosquito development. These methodologies, many of which could be extended to other non-model insect species, are facilitating analysis of the development of sexual dimorphism in neural tissues, particularly the olfactory system. These studies are providing insight into the neurodevelopmental genetic basis for sexual dimorphism in vector mosquitoes. PMID:26949699

Poloidal flux profile reconstruction from pointwise measurements via extended Kalman filtering in the DIII-D Tokamak

DOE PAGES

Wang, Hexiang; Barton, Justin E.; Schuster, Eugenio

2015-09-01

The accuracy of the internal states of a tokamak, which usually cannot be measured directly, is of crucial importance for feedback control of the plasma dynamics. A first-principles-driven plasma response model could provide an estimation of the internal states given the boundary conditions on the magnetic axis and at plasma boundary. However, the estimation would highly depend on initial conditions, which may not always be known, disturbances, and non-modeled dynamics. Here in this work, a closed-loop state observer for the poloidal magnetic flux is proposed based on a very limited set of real-time measurements by following an Extended Kalman Filteringmore » (EKF) approach. Comparisons between estimated and measured magnetic flux profiles are carried out for several discharges in the DIII-D tokamak. The experimental results illustrate the capability of the proposed observer in dealing with incorrect initial conditions and measurement noise.« less

Proteins of Unknown Biochemical Function: A Persistent Problem and a Roadmap to Help Overcome It.

PubMed

Niehaus, Thomas D; Thamm, Antje M K; de Crécy-Lagard, Valérie; Hanson, Andrew D

2015-11-01

The number of sequenced genomes is rapidly increasing, but functional annotation of the genes in these genomes lags far behind. Even in Arabidopsis (Arabidopsis thaliana), only approximately 40% of enzyme- and transporter-encoding genes have credible functional annotations, and this number is even lower in nonmodel plants. Functional characterization of unknown genes is a challenge, but various databases (e.g. for protein localization and coexpression) can be mined to provide clues. If homologous microbial genes exist-and about one-half the genes encoding unknown enzymes and transporters in Arabidopsis have microbial homologs-cross-kingdom comparative genomics can powerfully complement plant-based data. Multiple lines of evidence can strengthen predictions and warrant experimental characterization. In some cases, relatively quick tests in genetically tractable microbes can determine whether a prediction merits biochemical validation, which is costly and demands specialized skills. © 2015 American Society of Plant Biologists. All Rights Reserved.

Network Analysis of Students' Use of Representations in Problem Solving

NASA Astrophysics Data System (ADS)

McPadden, Daryl; Brewe, Eric

2016-03-01

We present the preliminary results of a study on student use of representations in problem solving within the Modeling Instruction - Electricity and Magnetism (MI-E&M) course. Representational competence is a critical skill needed for students to develop a sophisticated understanding of college science topics and to succeed in their science courses. In this study, 70 students from the MI-E&M, calculus-based course were given a survey of 25 physics problem statements both pre- and post- instruction, covering both Newtonian Mechanics and Electricity and Magnetism (E&M). For each problem statement, students were asked which representations they would use in that given situation. We analyze the survey results through network analysis, identifying which representations are linked together in which contexts. We also compare the representation networks for those students who had already taken the first-semester Modeling Instruction Mechanics course and those students who had taken a non-Modeling Mechanics course.

Why Assembling Plant Genome Sequences Is So Challenging

PubMed Central

Claros, Manuel Gonzalo; Bautista, Rocío; Guerrero-Fernández, Darío; Benzerki, Hicham; Seoane, Pedro; Fernández-Pozo, Noé

2012-01-01

In spite of the biological and economic importance of plants, relatively few plant species have been sequenced. Only the genome sequence of plants with relatively small genomes, most of them angiosperms, in particular eudicots, has been determined. The arrival of next-generation sequencing technologies has allowed the rapid and efficient development of new genomic resources for non-model or orphan plant species. But the sequencing pace of plants is far from that of animals and microorganisms. This review focuses on the typical challenges of plant genomes that can explain why plant genomics is less developed than animal genomics. Explanations about the impact of some confounding factors emerging from the nature of plant genomes are given. As a result of these challenges and confounding factors, the correct assembly and annotation of plant genomes is hindered, genome drafts are produced, and advances in plant genomics are delayed. PMID:24832233

Rose Scent

PubMed Central

Guterman, Inna; Shalit, Moshe; Menda, Naama; Piestun, Dan; Dafny-Yelin, Mery; Shalev, Gil; Bar, Einat; Davydov, Olga; Ovadis, Mariana; Emanuel, Michal; Wang, Jihong; Adam, Zach; Pichersky, Eran; Lewinsohn, Efraim; Zamir, Dani; Vainstein, Alexander; Weiss, David

2002-01-01

For centuries, rose has been the most important crop in the floriculture industry; its economic importance also lies in the use of its petals as a source of natural fragrances. Here, we used genomics approaches to identify novel scent-related genes, using rose flowers from tetraploid scented and nonscented cultivars. An annotated petal EST database of ∼2100 unique genes from both cultivars was created, and DNA chips were prepared and used for expression analyses of selected clones. Detailed chemical analysis of volatile composition in the two cultivars, together with the identification of secondary metabolism–related genes whose expression coincides with scent production, led to the discovery of several novel flower scent–related candidate genes. The function of some of these genes, including a germacrene D synthase, was biochemically determined using an Escherichia coli expression system. This work demonstrates the advantages of using the high-throughput approaches of genomics to detail traits of interest expressed in a cultivar-specific manner in nonmodel plants. PMID:12368489

Development of Chemical and Metabolite Sensors for Rhodococcus opacus PD630.

PubMed

DeLorenzo, Drew M; Henson, William R; Moon, Tae Seok

2017-10-20

Rhodococcus opacus PD630 is a nonmodel, Gram-positive bacterium that possesses desirable traits for biomass conversion, including consumption capabilities for lignocellulose-based sugars and toxic lignin-derived aromatic compounds, significant triacylglycerol accumulation, relatively rapid growth rate, and genetic tractability. However, few genetic elements have been directly characterized in R. opacus, limiting its application for lignocellulose bioconversion. Here, we report the characterization and development of genetic tools for tunable gene expression in R. opacus, including: (1) six fluorescent reporters for quantifying promoter output, (2) three chemically inducible promoters for variable gene expression, and (3) two classes of metabolite sensors derived from native R. opacus promoters that detect nitrogen levels or aromatic compounds. Using these tools, we also provide insights into native aromatic consumption pathways in R. opacus. Overall, this work expands the ability to control and characterize gene expression in R. opacus for future lignocellulose-based fuel and chemical production.

Analysis of Circadian Leaf Movements.

PubMed

Müller, Niels A; Jiménez-Gómez, José M

2016-01-01

The circadian clock is a molecular timekeeper that controls a wide variety of biological processes. In plants, clock outputs range from the molecular level, with rhythmic gene expression and metabolite content, to physiological processes such as stomatal conductance or leaf movements. Any of these outputs can be used as markers to monitor the state of the circadian clock. In the model plant Arabidopsis thaliana, much of the current knowledge about the clock has been gained from time course experiments profiling expression of endogenous genes or reporter constructs regulated by the circadian clock. Since these methods require labor-intensive sample preparation or transformation, monitoring leaf movements is an interesting alternative, especially in non-model species and for natural variation studies. Technological improvements both in digital photography and image analysis allow cheap and easy monitoring of circadian leaf movements. In this chapter we present a protocol that uses an autonomous point and shoot camera and free software to monitor circadian leaf movements in tomato.

Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems

PubMed Central

2011-01-01

Background Alfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling. Results Cell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. In addition, 341,984 ESTs were generated from ES and PES internodes of genotype 773 using the GS FLX Titanium platform. The first alfalfa (Medicago sativa) gene index (MSGI 1.0) was assembled using the Sanger ESTs available from GenBank, the GS FLX Titanium EST sequences, and the de novo assembled Illumina sequences. MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1,294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Out of 55 SNPs randomly selected for experimental validation, 47 (85%) were polymorphic between the two genotypes. We also identified numerous allelic variations within each genotype. Digital gene expression analysis identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes. Conclusions Our results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a forage crop and cellulosic feedstock. PMID:21504589

Comprehensive Analysis of the Triterpenoid Saponins Biosynthetic Pathway in Anemone flaccida by Transcriptome and Proteome Profiling

PubMed Central

Zhan, Chuansong; Li, Xiaohua; Zhao, Zeying; Yang, Tewu; Wang, Xuekui; Luo, Biaobiao; Zhang, Qiyun; Hu, Yanru; Hu, Xuebo

2016-01-01

Background: Anemone flaccida Fr. Shmidt (Ranunculaceae), commonly known as ‘Di Wu’ in China, is a perennial herb with limited distribution. The rhizome of A. flaccida has long been used to treat arthritis as a tradition in China. Studies disclosed that the plant contains a rich source of triterpenoid saponins. However, little is known about triterpenoid saponins biosynthesis in A. flaccida. Results: In this study, we conducted the tandem transcriptome and proteome profiling of a non-model medicinal plant, A. flaccida. Using Illumina HiSeq 2000 sequencing and iTRAQ technique, a total of 46,962 high-quality unigenes were obtained with an average sequence length of 1,310 bp, along with 1473 unique proteins from A. flaccida. Among the A. flaccida transcripts, 36,617 (77.97%) showed significant similarity (E-value < 1e-5) to the known proteins in the public database. Of the total 46,962 unigenes, 36,617 open reading frame (ORFs) were predicted. By the fragments per kilobases per million reads (FPKM) statistics, 14,004 isoforms/unigenes were found to be upregulated, and 14,090 isoforms/unigenes were down-regulated in the rhizomes as compared to those in the leaves. Based on the bioinformatics analysis, all possible enzymes involved in the triterpenoid saponins biosynthetic pathway of A. flaccida were identified, including cytosolic mevalonate pathway (MVA) and the plastidial methylerythritol pathway (MEP). Additionally, a total of 126 putative cytochrome P450 (CYP450) and 32 putative UDP glycosyltransferases were selected as the candidates of triterpenoid saponins modifiers. Among them, four of them were annotated as the gene of CYP716A subfamily, the key enzyme in the oleanane-type triterpenoid saponins biosynthetic pathway. Furthermore, based on RNA-Seq and proteome analysis, as well as quantitative RT-PCR verification, the expression level of gene and protein committed to triterpenoids biosynthesis in the leaf versus the rhizome was compared. Conclusion: A combination of the de novo transcriptome and proteome profiling based on the Illumina HiSeq 2000 sequencing platform and iTRAQ technique was shown to be a powerful method for the discovery of candidate genes, which encoded enzymes that were responsible for the biosynthesis of novel secondary metabolites in a non-model plant. The transcriptome data of our study provides a very important resource for the understanding of the triterpenoid saponins biosynthesis of A. flaccida. PMID:27504115

Global patterns and controls of soil organic carbon dynamics as simulated by multiple terrestrial biosphere models: Current status and future directions.

PubMed

Tian, Hanqin; Lu, Chaoqun; Yang, Jia; Banger, Kamaljit; Huntzinger, Deborah N; Schwalm, Christopher R; Michalak, Anna M; Cook, Robert; Ciais, Philippe; Hayes, Daniel; Huang, Maoyi; Ito, Akihiko; Jain, Atul K; Lei, Huimin; Mao, Jiafu; Pan, Shufen; Post, Wilfred M; Peng, Shushi; Poulter, Benjamin; Ren, Wei; Ricciuto, Daniel; Schaefer, Kevin; Shi, Xiaoying; Tao, Bo; Wang, Weile; Wei, Yaxing; Yang, Qichun; Zhang, Bowen; Zeng, Ning

2015-06-01

Soil is the largest organic carbon (C) pool of terrestrial ecosystems, and C loss from soil accounts for a large proportion of land-atmosphere C exchange. Therefore, a small change in soil organic C (SOC) can affect atmospheric carbon dioxide (CO 2 ) concentration and climate change. In the past decades, a wide variety of studies have been conducted to quantify global SOC stocks and soil C exchange with the atmosphere through site measurements, inventories, and empirical/process-based modeling. However, these estimates are highly uncertain, and identifying major driving forces controlling soil C dynamics remains a key research challenge. This study has compiled century-long (1901-2010) estimates of SOC storage and heterotrophic respiration (Rh) from 10 terrestrial biosphere models (TBMs) in the Multi-scale Synthesis and Terrestrial Model Intercomparison Project and two observation-based data sets. The 10 TBM ensemble shows that global SOC estimate ranges from 425 to 2111 Pg C (1 Pg = 10 15  g) with a median value of 1158 Pg C in 2010. The models estimate a broad range of Rh from 35 to 69 Pg C yr -1 with a median value of 51 Pg C yr -1 during 2001-2010. The largest uncertainty in SOC stocks exists in the 40-65°N latitude whereas the largest cross-model divergence in Rh are in the tropics. The modeled SOC change during 1901-2010 ranges from -70 Pg C to 86 Pg C, but in some models the SOC change has a different sign from the change of total C stock, implying very different contribution of vegetation and soil pools in determining the terrestrial C budget among models. The model ensemble-estimated mean residence time of SOC shows a reduction of 3.4 years over the past century, which accelerate C cycling through the land biosphere. All the models agreed that climate and land use changes decreased SOC stocks, while elevated atmospheric CO 2 and nitrogen deposition over intact ecosystems increased SOC stocks-even though the responses varied significantly among models. Model representations of temperature and moisture sensitivity, nutrient limitation, and land use partially explain the divergent estimates of global SOC stocks and soil C fluxes in this study. In addition, a major source of systematic error in model estimations relates to nonmodeled SOC storage in wetlands and peatlands, as well as to old C storage in deep soil layers.

Global patterns and controls of soil organic carbon dynamics as simulated by multiple terrestrial biosphere models: Current status and future directions

DOE PAGES

Tian, Hanqin; Lu, Chaoqun; Yang, Jia; ...

2015-06-05

Soil is the largest organic carbon (C) pool of terrestrial ecosystems, and C loss from soil accounts for a large proportion of land-atmosphere C exchange. Therefore, a small change in soil organic C (SOC) can affect atmospheric carbon dioxide (CO₂) concentration and climate change. In the past decades, a wide variety of studies have been conducted to quantify global SOC stocks and soil C exchange with the atmosphere through site measurements, inventories, and empirical/process-based modeling. However, these estimates are highly uncertain, and identifying major driving forces controlling soil C dynamics remains a key research challenge. This study has compiled century-longmore » (1901–2010) estimates of SOC storage and heterotrophic respiration (Rh) from 10 terrestrial biosphere models (TBMs) in the Multi-scale Synthesis and Terrestrial Model Intercomparison Project and two observation-based data sets. The 10 TBM ensemble shows that global SOC estimate ranges from 425 to 2111 Pg C (1 Pg = 10¹⁵ g) with a median value of 1158 Pg C in 2010. The models estimate a broad range of Rh from 35 to 69 Pg C yr⁻¹ with a median value of 51 Pg C yr⁻¹ during 2001–2010. The largest uncertainty in SOC stocks exists in the 40–65°N latitude whereas the largest cross-model divergence in Rh are in the tropics. The modeled SOC change during 1901–2010 ranges from –70 Pg C to 86 Pg C, but in some models the SOC change has a different sign from the change of total C stock, implying very different contribution of vegetation and soil pools in determining the terrestrial C budget among models. The model ensemble-estimated mean residence time of SOC shows a reduction of 3.4 years over the past century, which accelerate C cycling through the land biosphere. All the models agreed that climate and land use changes decreased SOC stocks, while elevated atmospheric CO₂ and nitrogen deposition over intact ecosystems increased SOC stocks—even though the responses varied significantly among models. Model representations of temperature and moisture sensitivity, nutrient limitation, and land use partially explain the divergent estimates of global SOC stocks and soil C fluxes in this study. In addition, a major source of systematic error in model estimations relates to nonmodeled SOC storage in wetlands and peatlands, as well as to old C storage in deep soil layers.« less

Sample sequencing of vascular plants demonstrates widespread conservation and divergence of microRNAs.

PubMed

Chávez Montes, Ricardo A; de Fátima Rosas-Cárdenas, Flor; De Paoli, Emanuele; Accerbi, Monica; Rymarquis, Linda A; Mahalingam, Gayathri; Marsch-Martínez, Nayelli; Meyers, Blake C; Green, Pamela J; de Folter, Stefan

2014-04-23

Small RNAs are pivotal regulators of gene expression that guide transcriptional and post-transcriptional silencing mechanisms in eukaryotes, including plants. Here we report a comprehensive atlas of sRNA and miRNA from 3 species of algae and 31 representative species across vascular plants, including non-model plants. We sequence and quantify sRNAs from 99 different tissues or treatments across species, resulting in a data set of over 132 million distinct sequences. Using miRBase mature sequences as a reference, we identify the miRNA sequences present in these libraries. We apply diverse profiling methods to examine critical sRNA and miRNA features, such as size distribution, tissue-specific regulation and sequence conservation between species, as well as to predict putative new miRNA sequences. We also develop database resources, computational analysis tools and a dedicated website, http://smallrna.udel.edu/. This study provides new insights on plant sRNAs and miRNAs, and a foundation for future studies.

Plant Comparative and Functional Genomics

DOE PAGES

Yang, Xiaohan; Leebens-Mack, Jim; Chen, Feng; ...

2015-01-01

Plants form the foundation for our global ecosystem and are essential for environmental and human health. An increasing number of available plant genomes and tractable experimental systems, comparative and functional plant genomics research is greatly expanding our knowledge of the molecular basis of economically and nutritionally important traits in crop plants. Inferences drawn from comparative genomics are motivating experimental investigations of gene function and gene interactions. In this special issue aims to highlight recent advances made in comparative and functional genomics research in plants. Nine original research articles in this special issue cover five important topics: (1) transcription factor genemore » families relevant to abiotic stress tolerance; (2) plant secondary metabolism; (3) transcriptomebased markers for quantitative trait locus; (4) epigenetic modifications in plant-microbe interactions; and (5) computational prediction of protein-protein interactions. Finally, we studied the plant species in these articles which include model species as well as nonmodel plant species of economic importance (e.g., food crops and medicinal plants).« less

Plant Comparative and Functional Genomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Xiaohan; Leebens-Mack, Jim; Chen, Feng

Plants form the foundation for our global ecosystem and are essential for environmental and human health. An increasing number of available plant genomes and tractable experimental systems, comparative and functional plant genomics research is greatly expanding our knowledge of the molecular basis of economically and nutritionally important traits in crop plants. Inferences drawn from comparative genomics are motivating experimental investigations of gene function and gene interactions. In this special issue aims to highlight recent advances made in comparative and functional genomics research in plants. Nine original research articles in this special issue cover five important topics: (1) transcription factor genemore » families relevant to abiotic stress tolerance; (2) plant secondary metabolism; (3) transcriptomebased markers for quantitative trait locus; (4) epigenetic modifications in plant-microbe interactions; and (5) computational prediction of protein-protein interactions. Finally, we studied the plant species in these articles which include model species as well as nonmodel plant species of economic importance (e.g., food crops and medicinal plants).« less

Gene expression profiling--Opening the black box of plant ecosystem responses to global change

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leakey, A.D.B.; Ainsworth, E.A.; Bernard, S.M.

The use of genomic techniques to address ecological questions is emerging as the field of genomic ecology. Experimentation under environmentally realistic conditions to investigate the molecular response of plants to meaningful changes in growth conditions and ecological interactions is the defining feature of genomic ecology. Since the impact of global change factors on plant performance are mediated by direct effects at the molecular, biochemical and physiological scales, gene expression analysis promises important advances in understanding factors that have previously been consigned to the 'black box' of unknown mechanism. Various tools and approaches are available for assessing gene expression in modelmore » and non-model species as part of global change biology studies. Each approach has its own unique advantages and constraints. A first generation of genomic ecology studies in managed ecosystems and mesocosms have provided a testbed for the approach and have begun to reveal how the experimental design and data analysis of gene expression studies can be tailored for use in an ecological context.« less

Development of Chemical and Metabolite Sensors for Rhodococcus opacus PD630

DOE PAGES

DeLorenzo, Drew M.; Henson, William R.; Moon, Tae Seok

2017-07-26

Rhodococcus opacus PD630 is a non-model, gram positive bacterium that possesses desirable traits for biomass conversion, including consumption capabilities for lignocellulose-based sugars and toxic lignin-derived aromatic compounds, significant triacylglycerol accumulation, relatively rapid growth rate, and genetic tractability. However, few genetic elements have been directly characterized in R. opacus, limiting its application for lignocellulose bioconversion. Here, we report the characterization and development of genetic tools for tunable gene expression in R. opacus, including: 1) six fluorescent reporters for quantifying promoter output, 2) three chemically inducible promoters for variable gene expression, and 3) two classes of metabolite sensors derived from native R.more » opacus promoters that detect nitrogen levels or aromatic compounds. Using these tools, we also provide insights into native aromatic consumption pathways in R. opacus. Overall, this work expands the ability to control and characterize gene expression in R. opacus for future lignocellulose-based fuel and chemical production.« less

Self-similarity in nature

NASA Astrophysics Data System (ADS)

Timashev, S. F.

2000-02-01

A general phenomenological approach to the analysis of experimental temporal, spatial and energetic series for extracting truly physical non-model parameters ("passport data") is presented, which may be used to characterize and distinguish the evolution as well as the spatial and energetic structure of any open nonlinear dissipative system. This methodology is based on a postulate concerning the crucial information contained in the sequences of non-regularities of the measured dynamic variable (temporal, spatial, energetic). In accordance with this approach, multi-parametric formulas for dynamic variable power spectra as well as for structural functions of different orders are identical for every spatial-temporal-energetic level of the system under consideration. In effect, this entails the introduction of a new kind of self-similarity in Nature. An algorithm has been developed for obtaining as many "passport data" as are necessary for the characterization of a dynamic system. Applications of this approach in the analysis of various experimental series (temporal, spatial, energetic) demonstrate its potential for defining adequate phenomenological parameters of different dynamic processes and structures.

[On a contribution of Boris Balinsky to the comparative and ecological embryology of amphibians].

PubMed

Desnitskiĭ, A G

2014-01-01

The outstanding embryologist Boris Ivanovich Balinsky (1905-1997) worked in the Soviet Union up to 1941 and in South Africa since 1949. His experimental studies fulfilled during the Soviet period of his scientific career mainly on the embryos of the caudate amphibians are widely known. After moving to Africa (Johannesburg), he continued the research of amphibian development, with using those possibilities, which were offered by the diverse fauna of local Anura. Other embryologists started complex studies of tropical frog ontogenies (mainly from South and Central America) 30-40 years later than Balinsky. Unfortunately, his pioneering works on numerous African species are poorly known (with the exceptions of the description of the development of endodermal derivatives in Xenopus laevis and the analysis of limb induction in the toad genus Amietophrynus). In this paper, the works of Balinsky are analyzed (with the emphasis on comparative and ecological aspects) and his priority in using of "nonmodel" tropical and subtropical anurans in embryological studies has been shown.

Data of first de-novo transcriptome assembly of a non-model species, hawksbill sea turtle, Eretmochelys imbricate, nesting of the Colombian Caribean.

PubMed

Hernández-Fernández, Javier

2017-12-01

The hawksbill sea turtle, Eretmochelys imbricata, is an endangered species of the Caribbean Colombian coast due to anthropic and natural factors that have decreased their population levels. Little is known about the genes that are involved in their immune system, sex determination, aging and others important functions. The data generated represents RNA sequencing and the first de-novo assembly of transcripts expressed in the blood of the hawksbill sea turtle. The raw FASTQ files were deposited in the NCBI SRA database with accession number SRX2653641. A total of 5.7 Gb raw sequence data were obtained, corresponding to 47,555,108 raw reads. Trinity was used to perform a first de-novo assembly, and we were able to identify 47,586 transcripts of the female hawksbill turtle transcriptome with an N50 of 1100 bp. The obtained transcriptome data will be useful for further studies of the physiology, biochemistry and evolution in this species.

Cholesterol oxidase: ultrahigh-resolution crystal structure and multipolar atom model-based analysis.

PubMed

Zarychta, Bartosz; Lyubimov, Artem; Ahmed, Maqsood; Munshi, Parthapratim; Guillot, Benoît; Vrielink, Alice; Jelsch, Christian

2015-04-01

Examination of protein structure at the subatomic level is required to improve the understanding of enzymatic function. For this purpose, X-ray diffraction data have been collected at 100 K from cholesterol oxidase crystals using synchrotron radiation to an optical resolution of 0.94 Å. After refinement using the spherical atom model, nonmodelled bonding peaks were detected in the Fourier residual electron density on some of the individual bonds. Well defined bond density was observed in the peptide plane after averaging maps on the residues with the lowest thermal motion. The multipolar electron density of the protein-cofactor complex was modelled by transfer of the ELMAM2 charge-density database, and the topology of the intermolecular interactions between the protein and the flavin adenine dinucleotide (FAD) cofactor was subsequently investigated. Taking advantage of the high resolution of the structure, the stereochemistry of main-chain bond lengths and of C=O···H-N hydrogen bonds was analyzed with respect to the different secondary-structure elements.

In the Palm of Your Hand - Normalizing the Use of Mobile Technology for Nurse Practitioner Education and Clinical Practice.

PubMed

Lamarche, Kimberley; Park, Caroline; Fraser, Shawn; Rich, Mariann; MacKenzie, Susan

2016-01-01

The use of mobile devices by nurse practitioners (NPs) to meet an evolving technological landscape is expanding rapidly. A longitudinal study of the ways NP students "normalize" the use of mobile devices in clinical education was completed. This study used researcher-designed survey tools, including sociodemographic questions, and the numerical picture was augmented and interpreted in light of the textual data in the form of selected interviews. Data indicate that mobile technology is normalized in the social realm but still developing in the clinical realm. Progress is hindered by non-modelling by faculty, inconsistent healthcare policy and lack of understanding of the affordances available through this technology. Overall, mobile technology is utilized and normalized in practice; this in turn has influenced their ability to prepare students for practice. Data presented can assist educators and clinicians alike in developing a more fulsome understanding on how to appropriately incorporate mobile technology into education and practice.

Gene Duplication, Population Genomics, and Species-Level Differentiation within a Tropical Mountain Shrub

PubMed Central

Mastretta-Yanes, Alicia; Zamudio, Sergio; Jorgensen, Tove H.; Arrigo, Nils; Alvarez, Nadir; Piñero, Daniel; Emerson, Brent C.

2014-01-01

Gene duplication leads to paralogy, which complicates the de novo assembly of genotyping-by-sequencing (GBS) data. The issue of paralogous genes is exacerbated in plants, because they are particularly prone to gene duplication events. Paralogs are normally filtered from GBS data before undertaking population genomics or phylogenetic analyses. However, gene duplication plays an important role in the functional diversification of genes and it can also lead to the formation of postzygotic barriers. Using populations and closely related species of a tropical mountain shrub, we examine 1) the genomic differentiation produced by putative orthologs, and 2) the distribution of recent gene duplication among lineages and geography. We find high differentiation among populations from isolated mountain peaks and species-level differentiation within what is morphologically described as a single species. The inferred distribution of paralogs among populations is congruent with taxonomy and shows that GBS could be used to examine recent gene duplication as a source of genomic differentiation of nonmodel species. PMID:25223767

Experimental Study on Damage Detection in Timber Specimens Based on an Electromechanical Impedance Technique and RMSD-Based Mahalanobis Distance

PubMed Central

Wang, Dansheng; Wang, Qinghua; Wang, Hao; Zhu, Hongping

2016-01-01

In the electromechanical impedance (EMI) method, the PZT patch performs the functions of both sensor and exciter. Due to the high frequency actuation and non-model based characteristics, the EMI method can be utilized to detect incipient structural damage. In recent years EMI techniques have been widely applied to monitor the health status of concrete and steel materials, however, studies on application to timber are limited. This paper will explore the feasibility of using the EMI technique for damage detection in timber specimens. In addition, the conventional damage index, namely root mean square deviation (RMSD) is employed to evaluate the level of damage. On that basis, a new damage index, Mahalanobis distance based on RMSD, is proposed to evaluate the damage severity of timber specimens. Experimental studies are implemented to detect notch and hole damage in the timber specimens. Experimental results verify the availability and robustness of the proposed damage index and its superiority over the RMSD indexes. PMID:27782088

Experimental Study on Damage Detection in Timber Specimens Based on an Electromechanical Impedance Technique and RMSD-Based Mahalanobis Distance.

PubMed

Wang, Dansheng; Wang, Qinghua; Wang, Hao; Zhu, Hongping

2016-10-22

In the electromechanical impedance (EMI) method, the PZT patch performs the functions of both sensor and exciter. Due to the high frequency actuation and non-model based characteristics, the EMI method can be utilized to detect incipient structural damage. In recent years EMI techniques have been widely applied to monitor the health status of concrete and steel materials, however, studies on application to timber are limited. This paper will explore the feasibility of using the EMI technique for damage detection in timber specimens. In addition, the conventional damage index, namely root mean square deviation (RMSD) is employed to evaluate the level of damage. On that basis, a new damage index, Mahalanobis distance based on RMSD, is proposed to evaluate the damage severity of timber specimens. Experimental studies are implemented to detect notch and hole damage in the timber specimens. Experimental results verify the availability and robustness of the proposed damage index and its superiority over the RMSD indexes.

Expression of zinc and cadmium responsive genes in leaves of willow (Salix caprea L.) genotypes with different accumulation characteristics

PubMed Central

Konlechner, Cornelia; Türktaş, Mine; Langer, Ingrid; Vaculík, Marek; Wenzel, Walter W.; Puschenreiter, Markus; Hauser, Marie-Theres

2013-01-01

Salix caprea is well suited for phytoextraction strategies. In a previous survey we showed that genetically distinct S. caprea plants isolated from metal-polluted and unpolluted sites differed in their zinc (Zn) and cadmium (Cd) tolerance and accumulation abilities. To determine the molecular basis of this difference we examined putative homologues of genes involved in heavy metal responses and identified over 200 new candidates with a suppression subtractive hybridization (SSH) screen. Quantitative expression analyses of 20 genes in leaves revealed that some metallothioneins and cell wall modifying genes were induced irrespective of the genotype's origin and metal uptake capacity while a cysteine biosynthesis gene was expressed constitutively higher in the metallicolous genotype. The third and largest group of genes was only induced in the metallicolous genotype. These data demonstrate that naturally adapted woody non-model species can help to discover potential novel molecular mechanisms for metal accumulation and tolerance. PMID:23562959

Toward a Model-Based Predictive Controller Design in Brain–Computer Interfaces

PubMed Central

Kamrunnahar, M.; Dias, N. S.; Schiff, S. J.

2013-01-01

A first step in designing a robust and optimal model-based predictive controller (MPC) for brain–computer interface (BCI) applications is presented in this article. An MPC has the potential to achieve improved BCI performance compared to the performance achieved by current ad hoc, nonmodel-based filter applications. The parameters in designing the controller were extracted as model-based features from motor imagery task-related human scalp electroencephalography. Although the parameters can be generated from any model-linear or non-linear, we here adopted a simple autoregressive model that has well-established applications in BCI task discriminations. It was shown that the parameters generated for the controller design can as well be used for motor imagery task discriminations with performance (with 8–23% task discrimination errors) comparable to the discrimination performance of the commonly used features such as frequency specific band powers and the AR model parameters directly used. An optimal MPC has significant implications for high performance BCI applications. PMID:21267657

Untargeted Metabolic Quantitative Trait Loci Analyses Reveal a Relationship between Primary Metabolism and Potato Tuber Quality1[W][OA

PubMed Central

Carreno-Quintero, Natalia; Acharjee, Animesh; Maliepaard, Chris; Bachem, Christian W.B.; Mumm, Roland; Bouwmeester, Harro; Visser, Richard G.F.; Keurentjes, Joost J.B.

2012-01-01

Recent advances in -omics technologies such as transcriptomics, metabolomics, and proteomics along with genotypic profiling have permitted dissection of the genetics of complex traits represented by molecular phenotypes in nonmodel species. To identify the genetic factors underlying variation in primary metabolism in potato (Solanum tuberosum), we have profiled primary metabolite content in a diploid potato mapping population, derived from crosses between S. tuberosum and wild relatives, using gas chromatography-time of flight-mass spectrometry. In total, 139 polar metabolites were detected, of which we identified metabolite quantitative trait loci for approximately 72% of the detected compounds. In order to obtain an insight into the relationships between metabolic traits and classical phenotypic traits, we also analyzed statistical associations between them. The combined analysis of genetic information through quantitative trait locus coincidence and the application of statistical learning methods provide information on putative indicators associated with the alterations in metabolic networks that affect complex phenotypic traits. PMID:22223596

Toward a model-based predictive controller design in brain-computer interfaces.

PubMed

Kamrunnahar, M; Dias, N S; Schiff, S J

2011-05-01

A first step in designing a robust and optimal model-based predictive controller (MPC) for brain-computer interface (BCI) applications is presented in this article. An MPC has the potential to achieve improved BCI performance compared to the performance achieved by current ad hoc, nonmodel-based filter applications. The parameters in designing the controller were extracted as model-based features from motor imagery task-related human scalp electroencephalography. Although the parameters can be generated from any model-linear or non-linear, we here adopted a simple autoregressive model that has well-established applications in BCI task discriminations. It was shown that the parameters generated for the controller design can as well be used for motor imagery task discriminations with performance (with 8-23% task discrimination errors) comparable to the discrimination performance of the commonly used features such as frequency specific band powers and the AR model parameters directly used. An optimal MPC has significant implications for high performance BCI applications.

MicroRNA-based biotechnology for plant improvement.

PubMed

Zhang, Baohong; Wang, Qinglian

2015-01-01

MicroRNAs (miRNAs) are an extensive class of newly discovered endogenous small RNAs, which negatively regulate gene expression at the post-transcription levels. As the application of next-generation deep sequencing and advanced bioinformatics, the miRNA-related study has been expended to non-model plant species and the number of identified miRNAs has dramatically increased in the past years. miRNAs play a critical role in almost all biological and metabolic processes, and provide a unique strategy for plant improvement. Here, we first briefly review the discovery, history, and biogenesis of miRNAs, then focus more on the application of miRNAs on plant breeding and the future directions. Increased plant biomass through controlling plant development and phase change has been one achievement for miRNA-based biotechnology; plant tolerance to abiotic and biotic stress was also significantly enhanced by regulating the expression of an individual miRNA. Both endogenous and artificial miRNAs may serve as important tools for plant improvement. © 2014 Wiley Periodicals, Inc.

Parallelism and Epistasis in Skeletal Evolution Identified through Use of Phylogenomic Mapping Strategies

PubMed Central

Daane, Jacob M.; Rohner, Nicolas; Konstantinidis, Peter; Djuranovic, Sergej; Harris, Matthew P.

2016-01-01

The identification of genetic mechanisms underlying evolutionary change is critical to our understanding of natural diversity, but is presently limited by the lack of genetic and genomic resources for most species. Here, we present a new comparative genomic approach that can be applied to a broad taxonomic sampling of nonmodel species to investigate the genetic basis of evolutionary change. Using our analysis pipeline, we show that duplication and divergence of fgfr1a is correlated with the reduction of scales within fishes of the genus Phoxinellus. As a parallel genetic mechanism is observed in scale-reduction within independent lineages of cypriniforms, our finding exposes significant developmental constraint guiding morphological evolution. In addition, we identified fixed variation in fgf20a within Phoxinellus and demonstrated that combinatorial loss-of-function of fgfr1a and fgf20a within zebrafish phenocopies the evolved scalation pattern. Together, these findings reveal epistatic interactions between fgfr1a and fgf20a as a developmental mechanism regulating skeletal variation among fishes. PMID:26452532

The Langley Research Center CSI phase-0 evolutionary model testbed-design and experimental results

NASA Technical Reports Server (NTRS)

Belvin, W. K.; Horta, Lucas G.; Elliott, K. B.

1991-01-01

A testbed for the development of Controls Structures Interaction (CSI) technology is described. The design philosophy, capabilities, and early experimental results are presented to introduce some of the ongoing CSI research at NASA-Langley. The testbed, referred to as the Phase 0 version of the CSI Evolutionary model (CEM), is the first stage of model complexity designed to show the benefits of CSI technology and to identify weaknesses in current capabilities. Early closed loop test results have shown non-model based controllers can provide an order of magnitude increase in damping in the first few flexible vibration modes. Model based controllers for higher performance will need to be robust to model uncertainty as verified by System ID tests. Data are presented that show finite element model predictions of frequency differ from those obtained from tests. Plans are also presented for evolution of the CEM to study integrated controller and structure design as well as multiple payload dynamics.

Damage detection of engine bladed-disks using multivariate statistical analysis

NASA Astrophysics Data System (ADS)

Fang, X.; Tang, J.

2006-03-01

The timely detection of damage in aero-engine bladed-disks is an extremely important and challenging research topic. Bladed-disks have high modal density and, particularly, their vibration responses are subject to significant uncertainties due to manufacturing tolerance (blade-to-blade difference or mistuning), operating condition change and sensor noise. In this study, we present a new methodology for the on-line damage detection of engine bladed-disks using their vibratory responses during spin-up or spin-down operations which can be measured by blade-tip-timing sensing technique. We apply a principle component analysis (PCA)-based approach for data compression, feature extraction, and denoising. The non-model based damage detection is achieved by analyzing the change between response features of the healthy structure and of the damaged one. We facilitate such comparison by incorporating the Hotelling's statistic T2 analysis, which yields damage declaration with a given confidence level. The effectiveness of the method is demonstrated by case studies.

Transcriptome of the inflorescence meristems of the biofuel plant Jatropha curcas treated with cytokinin.

PubMed

Pan, Bang-Zhen; Chen, Mao-Sheng; Ni, Jun; Xu, Zeng-Fu

2014-11-17

Jatropha curcas, whose seed content is approximately 30-40% oil, is an ideal feedstock for producing biodiesel and bio-jet fuels. However, Jatropha plants have a low number of female flowers, which results in low seed yield that cannot meet the needs of the biofuel industry. Thus, increasing the number of female flowers is critical for the improvement of Jatropha seed yield. Our previous findings showed that cytokinin treatment can increase the flower number and female to male ratio and also induce bisexual flowers in Jatropha. The mechanisms underlying the influence of cytokinin on Jatropha flower development and sex determination, however, have not been clarified. This study examined the transcriptional levels of genes involved in the response to cytokinin in Jatropha inflorescence meristems at different time points after cytokinin treatment by 454 sequencing, which gave rise to a total of 294.6 Mb of transcript sequences. Up-regulated and down-regulated annotated and novel genes were identified, and the expression levels of the genes of interest were confirmed by qRT-PCR. The identified transcripts include those encoding genes involved in the biosynthesis, metabolism, and signaling of cytokinin and other plant hormones, flower development and cell division, which may be related to phenotypic changes of Jatropha in response to cytokinin treatment. Our analysis indicated that Jatropha orthologs of the floral organ identity genes known as ABCE model genes, JcAP1,2, JcPI, JcAG, and JcSEP1,2,3, were all significantly repressed, with an exception of one B-function gene JcAP3 that was shown to be up-regulated by BA treatment, indicating different mechanisms to be involved in the floral organ development of unisexual flowers of Jatropha and bisexual flowers of Arabidopsis. Several cell division-related genes, including JcCycA3;2, JcCycD3;1, JcCycD3;2 and JcTSO1, were up-regulated, which may contribute to the increased flower number after cytokinin treatment. This study presents the first report of global expression patterns of cytokinin-regulated transcripts in Jatropha inflorescence meristems. This report laid the foundation for further mechanistic studies on Jatropha and other non-model plants responding to cytokinin. Moreover, the identification of functional candidate genes will be useful for generating superior varieties of high-yielding transgenic Jatropha.

Gastrulation and pre-gastrulation morphogenesis, inductions, and gene expression: similarities and dissimilarities between urodelean and anuran embryos.

PubMed

Kaneda, Teruo; Motoki, Jun-ya Doi

2012-09-01

Studies of meso-endoderm and neural induction and subsequent body plan formation have been analyzed using mainly amphibians as the experimental model. Xenopus is currently the predominant model, because it best enables molecular analysis of these induction processes. However, much of the embryological information on these inductions (e.g., those of the Spemann-Mangold organizer), and on the morphogenetic movements of inductively interacting tissues, derives from research on non-model amphibians, especially urodeles. Although the final body pattern is strongly conserved in vertebrates, and although many of the same developmental genes are expressed, it has become evident that there are individually diverse modes of morphogenesis and timing of developmental events. Whether or not this diversity represents essential differences in the early induction processes remains unclear. The aim of this review is to compare the gastrulation process, induction processes, and gene expressions between a urodele, mainly Cynops pyrrhogaster, and an anura, Xenopus laevis, thereby to clarify conserved and diversified aspects. Cynops gastrulation differs significantly from that of Xenopus in that specification of the regions of the Xenopus dorsal marginal zone (DMZ) are specified before the onset of gastrulation, as marked by blastopore formation, whereas the equivalent state of specification does not occur in Cynops until the middle of gastrulation. Detailed comparison of the germ layer structure and morphogenetic movements during the pre-gastrula and gastrula stages shows that the entire gastrulation process should be divided into two phases of notochord induction and neural induction. Cynops undergoes these processes sequentially after the onset of gastrulation, whereas Xenopus undergoes notochord induction during a series of pre-gastrulation movements, and its traditionally defined period of gastrulation only includes the neural induction phase. Comparing the structure, fate, function and state of commitment of each domain of the DMZ of Xenopus and Cynops has revealed that the true form of the Spemann-Mangold organizer is suprablastoporal gsc-expressing endoderm that has notochord-inducing activity. Gsc-expressing deep endoderm and/or superficial endoderm in Xenopus is involved in inducing notochord during pre-gastrulation morphogenesis, rather than both gsc- and bra-expressing tissues being induced at the same time. Copyright © 2012 Elsevier Inc. All rights reserved.

De novo sequencing, assembly, and analysis of the root transcriptome of Persea americana (Mill.) in response to Phytophthora cinnamomi and flooding.

PubMed

Reeksting, Bianca J; Coetzer, Nanette; Mahomed, Waheed; Engelbrecht, Juanita; van den Berg, Noëlani

2014-01-01

Avocado is a diploid angiosperm containing 24 chromosomes with a genome estimated to be around 920 Mb. It is an important fruit crop worldwide but is susceptible to a root rot caused by the ubiquitous oomycete Phytophthora cinnamomi. Phytophthora root rot (PRR) causes damage to the feeder roots of trees, causing necrosis. This leads to branch-dieback and eventual tree death, resulting in severe losses in production. Control strategies are limited and at present an integrated approach involving the use of phosphite, tolerant rootstocks, and proper nursery management has shown the best results. Disease progression of PRR is accelerated under high soil moisture or flooding conditions. In addition, avocado is highly susceptible to flooding, with even short periods of flooding causing significant losses. Despite the commercial importance of avocado, limited genomic resources are available. Next generation sequencing has provided the means to generate sequence data at a relatively low cost, making this an attractive option for non-model organisms such as avocado. The aims of this study were to generate sequence data for the avocado root transcriptome and identify stress-related genes. Tissue was isolated from avocado infected with P. cinnamomi, avocado exposed to flooding and avocado exposed to a combination of these two stresses. Three separate sequencing runs were performed on the Roche 454 platform and produced approximately 124 Mb of data. This was assembled into 7685 contigs, with 106 448 sequences remaining as singletons. Genes involved in defence pathways such as the salicylic acid and jasmonic acid pathways as well as genes associated with the response to low oxygen caused by flooding, were identified. This is the most comprehensive study of transcripts derived from root tissue of avocado to date and will provide a useful resource for future studies.

De Novo Sequencing, Assembly, and Analysis of the Root Transcriptome of Persea americana (Mill.) in Response to Phytophthora cinnamomi and Flooding

PubMed Central

Reeksting, Bianca J.; Coetzer, Nanette; Mahomed, Waheed; Engelbrecht, Juanita; van den Berg, Noëlani

2014-01-01

Avocado is a diploid angiosperm containing 24 chromosomes with a genome estimated to be around 920 Mb. It is an important fruit crop worldwide but is susceptible to a root rot caused by the ubiquitous oomycete Phytophthora cinnamomi. Phytophthora root rot (PRR) causes damage to the feeder roots of trees, causing necrosis. This leads to branch-dieback and eventual tree death, resulting in severe losses in production. Control strategies are limited and at present an integrated approach involving the use of phosphite, tolerant rootstocks, and proper nursery management has shown the best results. Disease progression of PRR is accelerated under high soil moisture or flooding conditions. In addition, avocado is highly susceptible to flooding, with even short periods of flooding causing significant losses. Despite the commercial importance of avocado, limited genomic resources are available. Next generation sequencing has provided the means to generate sequence data at a relatively low cost, making this an attractive option for non-model organisms such as avocado. The aims of this study were to generate sequence data for the avocado root transcriptome and identify stress-related genes. Tissue was isolated from avocado infected with P. cinnamomi, avocado exposed to flooding and avocado exposed to a combination of these two stresses. Three separate sequencing runs were performed on the Roche 454 platform and produced approximately 124 Mb of data. This was assembled into 7685 contigs, with 106 448 sequences remaining as singletons. Genes involved in defence pathways such as the salicylic acid and jasmonic acid pathways as well as genes associated with the response to low oxygen caused by flooding, were identified. This is the most comprehensive study of transcripts derived from root tissue of avocado to date and will provide a useful resource for future studies. PMID:24563685

Comparing de novo assemblers for 454 transcriptome data

PubMed Central

2010-01-01

Background Roche 454 pyrosequencing has become a method of choice for generating transcriptome data from non-model organisms. Once the tens to hundreds of thousands of short (250-450 base) reads have been produced, it is important to correctly assemble these to estimate the sequence of all the transcripts. Most transcriptome assembly projects use only one program for assembling 454 pyrosequencing reads, but there is no evidence that the programs used to date are optimal. We have carried out a systematic comparison of five assemblers (CAP3, MIRA, Newbler, SeqMan and CLC) to establish best practices for transcriptome assemblies, using a new dataset from the parasitic nematode Litomosoides sigmodontis. Results Although no single assembler performed best on all our criteria, Newbler 2.5 gave longer contigs, better alignments to some reference sequences, and was fast and easy to use. SeqMan assemblies performed best on the criterion of recapitulating known transcripts, and had more novel sequence than the other assemblers, but generated an excess of small, redundant contigs. The remaining assemblers all performed almost as well, with the exception of Newbler 2.3 (the version currently used by most assembly projects), which generated assemblies that had significantly lower total length. As different assemblers use different underlying algorithms to generate contigs, we also explored merging of assemblies and found that the merged datasets not only aligned better to reference sequences than individual assemblies, but were also more consistent in the number and size of contigs. Conclusions Transcriptome assemblies are smaller than genome assemblies and thus should be more computationally tractable, but are often harder because individual contigs can have highly variable read coverage. Comparing single assemblers, Newbler 2.5 performed best on our trial data set, but other assemblers were closely comparable. Combining differently optimal assemblies from different programs however gave a more credible final product, and this strategy is recommended. PMID:20950480

Transcriptome analysis of stem development in the tumourous stem mustard Brassica juncea var. tumida Tsen et Lee by RNA sequencing.

PubMed

Sun, Quan; Zhou, Guanfan; Cai, Yingfan; Fan, Yonghong; Zhu, Xiaoyan; Liu, Yihua; He, Xiaohong; Shen, Jinjuan; Jiang, Huaizhong; Hu, Daiwen; Pan, Zheng; Xiang, Liuxin; He, Guanghua; Dong, Daiwen; Yang, Jianping

2012-04-21

Tumourous stem mustard (Brassica juncea var. tumida Tsen et Lee) is an economically and nutritionally important vegetable crop of the Cruciferae family that also provides the raw material for Fuling mustard. The genetics breeding, physiology, biochemistry and classification of mustards have been extensively studied, but little information is available on tumourous stem mustard at the molecular level. To gain greater insight into the molecular mechanisms underlying stem swelling in this vegetable and to provide additional information for molecular research and breeding, we sequenced the transcriptome of tumourous stem mustard at various stem developmental stages and compared it with that of a mutant variety lacking swollen stems. Using Illumina short-read technology with a tag-based digital gene expression (DGE) system, we performed de novo transcriptome assembly and gene expression analysis. In our analysis, we assembled genetic information for tumourous stem mustard at various stem developmental stages. In addition, we constructed five DGE libraries, which covered the strains Yong'an and Dayejie at various development stages. Illumina sequencing identified 146,265 unigenes, including 11,245 clusters and 135,020 singletons. The unigenes were subjected to a BLAST search and annotated using the GO and KO databases. We also compared the gene expression profiles of three swollen stem samples with those of two non-swollen stem samples. A total of 1,042 genes with significantly different expression levels occurring simultaneously in the six comparison groups were screened out. Finally, the altered expression levels of a number of randomly selected genes were confirmed by quantitative real-time PCR. Our data provide comprehensive gene expression information at the transcriptional level and the first insight into the understanding of the molecular mechanisms and regulatory pathways of stem swelling and development in this plant, and will help define new mechanisms of stem development in non-model plant organisms.

Network and biosignature analysis for the integration of transcriptomic and metabolomic data to characterize leaf senescence process in sunflower.

PubMed

Moschen, Sebastián; Higgins, Janet; Di Rienzo, Julio A; Heinz, Ruth A; Paniego, Norma; Fernandez, Paula

2016-06-06

In recent years, high throughput technologies have led to an increase of datasets from omics disciplines allowing the understanding of the complex regulatory networks associated with biological processes. Leaf senescence is a complex mechanism controlled by multiple genetic and environmental variables, which has a strong impact on crop yield. Transcription factors (TFs) are key proteins in the regulation of gene expression, regulating different signaling pathways; their function is crucial for triggering and/or regulating different aspects of the leaf senescence process. The study of TF interactions and their integration with metabolic profiles under different developmental conditions, especially for a non-model organism such as sunflower, will open new insights into the details of gene regulation of leaf senescence. Weighted Gene Correlation Network Analysis (WGCNA) and BioSignature Discoverer (BioSD, Gnosis Data Analysis, Heraklion, Greece) were used to integrate transcriptomic and metabolomic data. WGCNA allowed the detection of 10 metabolites and 13 TFs whereas BioSD allowed the detection of 1 metabolite and 6 TFs as potential biomarkers. The comparative analysis demonstrated that three transcription factors were detected through both methodologies, highlighting them as potentially robust biomarkers associated with leaf senescence in sunflower. The complementary use of network and BioSignature Discoverer analysis of transcriptomic and metabolomic data provided a useful tool for identifying candidate genes and metabolites which may have a role during the triggering and development of the leaf senescence process. The WGCNA tool allowed us to design and test a hypothetical network in order to infer relationships across selected transcription factor and metabolite candidate biomarkers involved in leaf senescence, whereas BioSignature Discoverer selected transcripts and metabolites which discriminate between different ages of sunflower plants. The methodology presented here would help to elucidate and predict novel networks and potential biomarkers of leaf senescence in sunflower.

Transcriptome- Assisted Label-Free Quantitative Proteomics Analysis Reveals Novel Insights into Piper nigrum—Phytophthora capsici Phytopathosystem

PubMed Central

Mahadevan, Chidambareswaren; Krishnan, Anu; Saraswathy, Gayathri G.; Surendran, Arun; Jaleel, Abdul; Sakuntala, Manjula

2016-01-01

Black pepper (Piper nigrum L.), a tropical spice crop of global acclaim, is susceptible to Phytophthora capsici, an oomycete pathogen which causes the highly destructive foot rot disease. A systematic understanding of this phytopathosystem has not been possible owing to lack of genome or proteome information. In this study, we explain an integrated transcriptome-assisted label-free quantitative proteomics pipeline to study the basal immune components of black pepper when challenged with P. capsici. We report a global identification of 532 novel leaf proteins from black pepper, of which 518 proteins were functionally annotated using BLAST2GO tool. A label-free quantitation of the protein datasets revealed 194 proteins common to diseased and control protein datasets of which 22 proteins showed significant up-regulation and 134 showed significant down-regulation. Ninety-three proteins were identified exclusively on P. capsici infected leaf tissues and 245 were expressed only in mock (control) infected samples. In-depth analysis of our data gives novel insights into the regulatory pathways of black pepper which are compromised during the infection. Differential down-regulation was observed in a number of critical pathways like carbon fixation in photosynthetic organism, cyano-amino acid metabolism, fructose, and mannose metabolism, glutathione metabolism, and phenylpropanoid biosynthesis. The proteomics results were validated with real-time qRT-PCR analysis. We were also able to identify the complete coding sequences for all the proteins of which few selected genes were cloned and sequence characterized for further confirmation. Our study is the first report of a quantitative proteomics dataset in black pepper which provides convincing evidence on the effectiveness of a transcriptome-based label-free proteomics approach for elucidating the host response to biotic stress in a non-model spice crop like P. nigrum, for which genome information is unavailable. Our dataset will serve as a useful resource for future studies in this plant. Data are available via ProteomeXchange with identifier PXD003887. PMID:27379110

Transcriptome- Assisted Label-Free Quantitative Proteomics Analysis Reveals Novel Insights into Piper nigrum-Phytophthora capsici Phytopathosystem.

PubMed

Mahadevan, Chidambareswaren; Krishnan, Anu; Saraswathy, Gayathri G; Surendran, Arun; Jaleel, Abdul; Sakuntala, Manjula

2016-01-01

Black pepper (Piper nigrum L.), a tropical spice crop of global acclaim, is susceptible to Phytophthora capsici, an oomycete pathogen which causes the highly destructive foot rot disease. A systematic understanding of this phytopathosystem has not been possible owing to lack of genome or proteome information. In this study, we explain an integrated transcriptome-assisted label-free quantitative proteomics pipeline to study the basal immune components of black pepper when challenged with P. capsici. We report a global identification of 532 novel leaf proteins from black pepper, of which 518 proteins were functionally annotated using BLAST2GO tool. A label-free quantitation of the protein datasets revealed 194 proteins common to diseased and control protein datasets of which 22 proteins showed significant up-regulation and 134 showed significant down-regulation. Ninety-three proteins were identified exclusively on P. capsici infected leaf tissues and 245 were expressed only in mock (control) infected samples. In-depth analysis of our data gives novel insights into the regulatory pathways of black pepper which are compromised during the infection. Differential down-regulation was observed in a number of critical pathways like carbon fixation in photosynthetic organism, cyano-amino acid metabolism, fructose, and mannose metabolism, glutathione metabolism, and phenylpropanoid biosynthesis. The proteomics results were validated with real-time qRT-PCR analysis. We were also able to identify the complete coding sequences for all the proteins of which few selected genes were cloned and sequence characterized for further confirmation. Our study is the first report of a quantitative proteomics dataset in black pepper which provides convincing evidence on the effectiveness of a transcriptome-based label-free proteomics approach for elucidating the host response to biotic stress in a non-model spice crop like P. nigrum, for which genome information is unavailable. Our dataset will serve as a useful resource for future studies in this plant. Data are available via ProteomeXchange with identifier PXD003887.

A mechanistic understanding of ageing revealed by studying the young.

PubMed

Crespi, Erica J

2012-03-01

A main focus within biomedical research is to understand how adverse environmental conditions experienced during early development affects lifelong health (Barker 1992). Within this context, extensive research in rodent models and humans has shown that intrauterine growth retardation (IUGR) caused by nutrient restriction during early development is often followed by post-natal 'catch-up' growth when access to food resources improves. However, this accelerated growth rate seems to come at a cost, as metabolic and endocrine processes that are programmed during this time cause later-life onset of diseases such as obesity, insulin resistance and cardiovascular disease (reviewed in Crespi & Denver 2005). In this issue Molecular Ecology, Geiger et al. (2012) asked what are the costs of catch-up growth in nutrient-restricted king penguin chicks (Fig. 1) by measuring lengths of telomeres, the protective DNA sequences at the end of chromosomes, before and after catch-up growth, as the amount and rate of telomere sequence loss over time has been associated with reduced lifespan in both model and nonmodel organisms (see reviews of Costantini et al. 2010; Haussmann & Marchetto 2010). Geiger et al. (2011) found that chicks entering the post-winter growth season at a smaller size exhibited increased growth rates (i.e. catch-up growth) at the cost of increased oxidative stress and reduced telomere lengths compared with the chicks entering the growth period at a larger size. Furthermore, chicks that did not survive had drastically shorter telomere lengths and reduced antioxidant capacities at the beginning of the growth period than all other chicks, thereby directly associating telomere length to mortality. These results suggest that while catch-up growth allows smaller chicks to head off into the world on equal footing with chicks that hatched at a larger size, it likely comes at the cost of a shortened lifespan. Thus, this study provides a mechanism that supports the antagonistic pleiotropy theory of senescence (Promislow 2004). © 2012 Blackwell Publishing Ltd.

Comparative and Evolutionary Analysis of Grass Pollen Allergens Using Brachypodium distachyon as a Model System

PubMed Central

Sharma, Akanksha; Sharma, Niharika; Bhalla, Prem; Singh, Mohan

2017-01-01

Comparative genomics have facilitated the mining of biological information from a genome sequence, through the detection of similarities and differences with genomes of closely or more distantly related species. By using such comparative approaches, knowledge can be transferred from the model to non-model organisms and insights can be gained in the structural and evolutionary patterns of specific genes. In the absence of sequenced genomes for allergenic grasses, this study was aimed at understanding the structure, organisation and expression profiles of grass pollen allergens using the genomic data from Brachypodium distachyon as it is phylogenetically related to the allergenic grasses. Combining genomic data with the anther RNA-Seq dataset revealed 24 pollen allergen genes belonging to eight allergen groups mapping on the five chromosomes in B. distachyon. High levels of anther-specific expression profiles were observed for the 24 identified putative allergen-encoding genes in Brachypodium. The genomic evidence suggests that gene encoding the group 5 allergen, the most potent trigger of hay fever and allergic asthma originated as a pollen specific orphan gene in a common grass ancestor of Brachypodium and Triticiae clades. Gene structure analysis showed that the putative allergen-encoding genes in Brachypodium either lack or contain reduced number of introns. Promoter analysis of the identified Brachypodium genes revealed the presence of specific cis-regulatory sequences likely responsible for high anther/pollen-specific expression. With the identification of putative allergen-encoding genes in Brachypodium, this study has also described some important plant gene families (e.g. expansin superfamily, EF-Hand family, profilins etc) for the first time in the model plant Brachypodium. Altogether, the present study provides new insights into structural characterization and evolution of pollen allergens and will further serve as a base for their functional characterization in related grass species. PMID:28103252

Mapping of single-copy genes by TSA-FISH in the codling moth, Cydia pomonella.

PubMed

Carabajal Paladino, Leonela Z; Nguyen, Petr; Síchová, Jindra; Marec, František

2014-01-01

We work on the development of transgenic sexing strains in the codling moth, Cydia pomonella (Tortricidae), which would enable to produce male-only progeny for the population control of this pest using sterile insect technique (SIT). To facilitate this research, we have developed a number of cytogenetic and molecular tools, including a physical map of the codling moth Z chromosome using BAC-FISH (fluorescence in situ hybridization with bacterial artificial chromosome probes). However, chromosomal localization of unique, single-copy sequences such as a transgene cassette by conventional FISH remains challenging. In this study, we adapted a FISH protocol with tyramide signal amplification (TSA-FISH) for detection of single-copy genes in Lepidoptera. We tested the protocol with probes prepared from partial sequences of Z-linked genes in the codling moth. Using a modified TSA-FISH protocol we successfully mapped a partial sequence of the Acetylcholinesterase 1 (Ace-1) gene to the Z chromosome and confirmed thus its Z-linkage. A subsequent combination of BAC-FISH with BAC probes containing anticipated neighbouring Z-linked genes and TSA-FISH with the Ace-1 probe allowed the integration of Ace-1 in the physical map of the codling moth Z chromosome. We also developed a two-colour TSA-FISH protocol which enabled us simultaneous localization of two Z-linked genes, Ace-1 and Notch, to the expected regions of the Z chromosome. We showed that TSA-FISH represents a reliable technique for physical mapping of genes on chromosomes of moths and butterflies. Our results suggest that this technique can be combined with BAC-FISH and in the future used for physical localization of transgene cassettes on chromosomes of transgenic lines in the codling moth or other lepidopteran species. Furthermore, the developed protocol for two-colour TSA-FISH might become a powerful tool for synteny mapping in non-model organisms.

Functional characterization of Pol III U6 promoters for gene knockdown and knockout in Plutella xylostella.

PubMed

Huang, Yuping; Wang, Yajun; Zeng, Baosheng; Liu, Zhaoxia; Xu, Xuejiao; Meng, Qian; Huang, Yongping; Yang, Guang; Vasseur, Liette; Gurr, Geoff M; You, Minsheng

2017-10-01

RNA polymerase type III (Pol-III) promoters such as U6 are commonly used to express small RNAs, including short hairpin RNAs (shRNAs) and single guide RNAs (sgRNAs). Functional U6 promoters are widely used in CRISPR systems, and their characterization can facilitate genome editing of non-model organisms. In the present study, six U6 small nuclear RNA (snRNA) promoters containing two conserved elements of a proximal sequence element (PSEA) and a TATA box, were identified and characterized in the diamondback moth (Plutella xylostella) genome. Relative efficiency of the U6 promoters to express shRNA induced EGFP knockdown was tested in a P. xylostella cell line, revealing that the PxU6:3 promoter had the strongest expression effect. Further work with the PxU6:3 promoter showed its efficacy in EGFP knockout using CRISPR/Cas9 system in the cells. The expression plasmids with versatile Pxabd-A gene specific sgRNA driven by the PxU6:3 promoter, combined with Cas9 mRNA, could induce mutagenesis at specific genomic loci in vivo. The phenotypes induced by sgRNA expression plasmids were similar to those done in vitro transcription sgRNAs. A plasmid with two tandem arranged PxU6:3:sgRNA expression cassettes targeting Pxabd-A loci was generated, which caused a 28,856 bp fragment deletion, suggesting that the multi-sgRNA expression plasmid can be used for multi-targeting. Our work indicates that U6 snRNA promoters can be used for functional studies of genes with the approach of reverse genetics in P. xylostella. These essential promoters also provide valuable potential for CRISPR-derived gene drive as a tactic for population control in this globally significant pest. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mapping of single-copy genes by TSA-FISH in the codling moth, Cydia pomonella

PubMed Central

2014-01-01

Background We work on the development of transgenic sexing strains in the codling moth, Cydia pomonella (Tortricidae), which would enable to produce male-only progeny for the population control of this pest using sterile insect technique (SIT). To facilitate this research, we have developed a number of cytogenetic and molecular tools, including a physical map of the codling moth Z chromosome using BAC-FISH (fluorescence in situ hybridization with bacterial artificial chromosome probes). However, chromosomal localization of unique, single-copy sequences such as a transgene cassette by conventional FISH remains challenging. In this study, we adapted a FISH protocol with tyramide signal amplification (TSA-FISH) for detection of single-copy genes in Lepidoptera. We tested the protocol with probes prepared from partial sequences of Z-linked genes in the codling moth. Results Using a modified TSA-FISH protocol we successfully mapped a partial sequence of the Acetylcholinesterase 1 (Ace-1) gene to the Z chromosome and confirmed thus its Z-linkage. A subsequent combination of BAC-FISH with BAC probes containing anticipated neighbouring Z-linked genes and TSA-FISH with the Ace-1 probe allowed the integration of Ace-1 in the physical map of the codling moth Z chromosome. We also developed a two-colour TSA-FISH protocol which enabled us simultaneous localization of two Z-linked genes, Ace-1 and Notch, to the expected regions of the Z chromosome. Conclusions We showed that TSA-FISH represents a reliable technique for physical mapping of genes on chromosomes of moths and butterflies. Our results suggest that this technique can be combined with BAC-FISH and in the future used for physical localization of transgene cassettes on chromosomes of transgenic lines in the codling moth or other lepidopteran species. Furthermore, the developed protocol for two-colour TSA-FISH might become a powerful tool for synteny mapping in non-model organisms. PMID:25471491

Differential gene expression in the siphonophore Nanomia bijuga (Cnidaria) assessed with multiple next-generation sequencing workflows.

PubMed

Siebert, Stefan; Robinson, Mark D; Tintori, Sophia C; Goetz, Freya; Helm, Rebecca R; Smith, Stephen A; Shaner, Nathan; Haddock, Steven H D; Dunn, Casey W

2011-01-01

We investigated differential gene expression between functionally specialized feeding polyps and swimming medusae in the siphonophore Nanomia bijuga (Cnidaria) with a hybrid long-read/short-read sequencing strategy. We assembled a set of partial gene reference sequences from long-read data (Roche 454), and generated short-read sequences from replicated tissue samples that were mapped to the references to quantify expression. We collected and compared expression data with three short-read expression workflows that differ in sample preparation, sequencing technology, and mapping tools. These workflows were Illumina mRNA-Seq, which generates sequence reads from random locations along each transcript, and two tag-based approaches, SOLiD SAGE and Helicos DGE, which generate reads from particular tag sites. Differences in expression results across workflows were mostly due to the differential impact of missing data in the partial reference sequences. When all 454-derived gene reference sequences were considered, Illumina mRNA-Seq detected more than twice as many differentially expressed (DE) reference sequences as the tag-based workflows. This discrepancy was largely due to missing tag sites in the partial reference that led to false negatives in the tag-based workflows. When only the subset of reference sequences that unambiguously have tag sites was considered, we found broad congruence across workflows, and they all identified a similar set of DE sequences. Our results are promising in several regards for gene expression studies in non-model organisms. First, we demonstrate that a hybrid long-read/short-read sequencing strategy is an effective way to collect gene expression data when an annotated genome sequence is not available. Second, our replicated sampling indicates that expression profiles are highly consistent across field-collected animals in this case. Third, the impacts of partial reference sequences on the ability to detect DE can be mitigated through workflow choice and deeper reference sequencing.

Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows

PubMed Central

Siebert, Stefan; Robinson, Mark D.; Tintori, Sophia C.; Goetz, Freya; Helm, Rebecca R.; Smith, Stephen A.; Shaner, Nathan; Haddock, Steven H. D.; Dunn, Casey W.

2011-01-01

We investigated differential gene expression between functionally specialized feeding polyps and swimming medusae in the siphonophore Nanomia bijuga (Cnidaria) with a hybrid long-read/short-read sequencing strategy. We assembled a set of partial gene reference sequences from long-read data (Roche 454), and generated short-read sequences from replicated tissue samples that were mapped to the references to quantify expression. We collected and compared expression data with three short-read expression workflows that differ in sample preparation, sequencing technology, and mapping tools. These workflows were Illumina mRNA-Seq, which generates sequence reads from random locations along each transcript, and two tag-based approaches, SOLiD SAGE and Helicos DGE, which generate reads from particular tag sites. Differences in expression results across workflows were mostly due to the differential impact of missing data in the partial reference sequences. When all 454-derived gene reference sequences were considered, Illumina mRNA-Seq detected more than twice as many differentially expressed (DE) reference sequences as the tag-based workflows. This discrepancy was largely due to missing tag sites in the partial reference that led to false negatives in the tag-based workflows. When only the subset of reference sequences that unambiguously have tag sites was considered, we found broad congruence across workflows, and they all identified a similar set of DE sequences. Our results are promising in several regards for gene expression studies in non-model organisms. First, we demonstrate that a hybrid long-read/short-read sequencing strategy is an effective way to collect gene expression data when an annotated genome sequence is not available. Second, our replicated sampling indicates that expression profiles are highly consistent across field-collected animals in this case. Third, the impacts of partial reference sequences on the ability to detect DE can be mitigated through workflow choice and deeper reference sequencing. PMID:21829563

Adaptive Genetic Divergence Despite Significant Isolation-by-Distance in Populations of Taiwan Cow-Tail Fir (Keteleeria davidiana var. formosana)

PubMed Central

Shih, Kai-Ming; Chang, Chung-Te; Chung, Jeng-Der; Chiang, Yu-Chung; Hwang, Shih-Ying

2018-01-01

Double digest restriction site-associated DNA sequencing (ddRADseq) is a tool for delivering genome-wide single nucleotide polymorphism (SNP) markers for non-model organisms useful in resolving fine-scale population structure and detecting signatures of selection. This study performs population genetic analysis, based on ddRADseq data, of a coniferous species, Keteleeria davidiana var. formosana, disjunctly distributed in northern and southern Taiwan, for investigation of population adaptive divergence in response to environmental heterogeneity. A total of 13,914 SNPs were detected and used to assess genetic diversity, FST outlier detection, population genetic structure, and individual assignments of five populations (62 individuals) of K. davidiana var. formosana. Principal component analysis (PCA), individual assignments, and the neighbor-joining tree were successful in differentiating individuals between northern and southern populations of K. davidiana var. formosana, but apparent gene flow between the southern DW30 population and northern populations was also revealed. Fifteen of 23 highly differentiated SNPs identified were found to be strongly associated with environmental variables, suggesting isolation-by-environment (IBE). However, multiple matrix regression with randomization analysis revealed strong IBE as well as significant isolation-by-distance. Environmental impacts on divergence were found between populations of the North and South regions and also between the two southern neighboring populations. BLASTN annotation of the sequences flanking outlier SNPs gave significant hits for three of 23 markers that might have biological relevance to mitochondrial homeostasis involved in the survival of locally adapted lineages. Species delimitation between K. davidiana var. formosana and its ancestor, K. davidiana, was also examined (72 individuals). This study has produced highly informative population genomic data for the understanding of population attributes, such as diversity, connectivity, and adaptive divergence associated with large- and small-scale environmental heterogeneity in K. davidiana var. formosana. PMID:29449860

New Insights on Leucine-Rich Repeats Receptor-Like Kinase Orthologous Relationships in Angiosperms

PubMed Central

Dufayard, Jean-François; Bettembourg, Mathilde; Fischer, Iris; Droc, Gaetan; Guiderdoni, Emmanuel; Périn, Christophe; Chantret, Nathalie; Diévart, Anne

2017-01-01

Leucine-Rich Repeats Receptor-Like Kinase (LRR-RLK) genes represent a large and complex gene family in plants, mainly involved in development and stress responses. These receptors are composed of an LRR-containing extracellular domain (ECD), a transmembrane domain (TM) and an intracellular kinase domain (KD). To provide new perspectives on functional analyses of these genes in model and non-model plant species, we performed a phylogenetic analysis on 8,360 LRR-RLK receptors in 31 angiosperm genomes (8 monocots and 23 dicots). We identified 101 orthologous groups (OGs) of genes being conserved among almost all monocot and dicot species analyzed. We observed that more than 10% of these OGs are absent in the Brassicaceae species studied. We show that the ECD structural features are not always conserved among orthologs, suggesting that functions may have diverged in some OG sets. Moreover, we looked at targets of positive selection footprints in 12 pairs of OGs and noticed that depending on the subgroups, positive selection occurred more frequently either in the ECDs or in the KDs. PMID:28424707

Gene duplication, population genomics, and species-level differentiation within a tropical mountain shrub.

PubMed

Mastretta-Yanes, Alicia; Zamudio, Sergio; Jorgensen, Tove H; Arrigo, Nils; Alvarez, Nadir; Piñero, Daniel; Emerson, Brent C

2014-09-14

Gene duplication leads to paralogy, which complicates the de novo assembly of genotyping-by-sequencing (GBS) data. The issue of paralogous genes is exacerbated in plants, because they are particularly prone to gene duplication events. Paralogs are normally filtered from GBS data before undertaking population genomics or phylogenetic analyses. However, gene duplication plays an important role in the functional diversification of genes and it can also lead to the formation of postzygotic barriers. Using populations and closely related species of a tropical mountain shrub, we examine 1) the genomic differentiation produced by putative orthologs, and 2) the distribution of recent gene duplication among lineages and geography. We find high differentiation among populations from isolated mountain peaks and species-level differentiation within what is morphologically described as a single species. The inferred distribution of paralogs among populations is congruent with taxonomy and shows that GBS could be used to examine recent gene duplication as a source of genomic differentiation of nonmodel species. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica.

PubMed

Shabbir Hussain, Murtaza; Gambill, Lauren; Smith, Spencer; Blenner, Mark A

2016-03-18

Eukaryotic promoters have a complex architecture to control both the strength and timing of gene transcription spanning up to thousands of bases from the initiation site. This complexity makes rational fine-tuning of promoters in fungi difficult to predict; however, this very same complexity enables multiple possible strategies for engineering promoter strength. Here, we studied promoter architecture in the oleaginous yeast, Yarrowia lipolytica. While recent studies have focused on upstream activating sequences, we systematically examined various components common in fungal promoters. Here, we examine several promoter components including upstream activating sequences, proximal promoter sequences, core promoters, and the TATA box in autonomously replicating expression plasmids and integrated into the genome. Our findings show that promoter strength can be fine-tuned through the engineering of the TATA box sequence, core promoter, and upstream activating sequences. Additionally, we identified a previously unreported oleic acid responsive transcription enhancement in the XPR2 upstream activating sequences, which illustrates the complexity of fungal promoters. The promoters engineered here provide new genetic tools for metabolic engineering in Y. lipolytica and provide promoter engineering strategies that may be useful in engineering other non-model fungal systems.

A Non-Modeling Exploration of Residential Solar Photovoltaic (PV) Adoption and Non-Adoption

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moezzi, Mithra; Ingle, Aaron; Lutzenhiser, Loren

Although U.S. deployment of residential rooftop solar photovoltaic (PV) systems has accelerated in recent years, PV is still installed on less than 1 percent of single-family homes. Most research on household PV adoption focuses on scaling initial markets and modeling predicted growth rather than considering more broadly why adoption occurs. Among the studies that have investigated the characteristics of PV adoption, most collected data from adopters, sometimes with additional non-adopter data, and rarely from people who considered but did not adopt PV. Yet the vast majority of Americans are non-adopters, and they are a diverse group - understanding their waysmore » of evaluating PV adoption is important. Similarly, PV is a unique consumer product, which makes it difficult to apply findings from studies of other technologies to PV. In addition, little research addresses the experience of households after they install PV. This report helps fill some of these gaps in the existing literature. The results inform a more detailed understanding of residential PV adoption, while helping ensure that adoption is sufficiently beneficial to adopters and even non-adopters.« less

Expanding the view on the evolution of the nematode dauer signalling pathways: refinement through gene gain and pathway co-option.

PubMed

Gilabert, Aude; Curran, David M; Harvey, Simon C; Wasmuth, James D

2016-06-27

Signalling pathways underlie development, behaviour and pathology. To understand patterns in the evolution of signalling pathways, we undertook a comprehensive investigation of the pathways that control the switch between growth and developmentally quiescent dauer in 24 species of nematodes spanning the phylum. Our analysis of 47 genes across these species indicates that the pathways and their interactions are not conserved throughout the Nematoda. For example, the TGF-β pathway was co-opted into dauer control relatively late in a lineage that led to the model species Caenorhabditis elegans. We show molecular adaptations described in C. elegans that are restricted to its genus or even just to the species. Similarly, our analyses both identify species where particular genes have been lost and situations where apparently incorrect orthologues have been identified. Our analysis also highlights the difficulties of working with genome sequences from non-model species as reliance on the published gene models would have significantly restricted our understanding of how signalling pathways evolve. Our approach therefore offers a robust standard operating procedure for genomic comparisons.

Anaerobic gut fungi: Advances in isolation, culture, and cellulolytic enzyme discovery for biofuel production.

PubMed

Haitjema, Charles H; Solomon, Kevin V; Henske, John K; Theodorou, Michael K; O'Malley, Michelle A

2014-08-01

Anaerobic gut fungi are an early branching family of fungi that are commonly found in the digestive tract of ruminants and monogastric herbivores. It is becoming increasingly clear that they are the primary colonizers of ingested plant biomass, and that they significantly contribute to the decomposition of plant biomass into fermentable sugars. As such, anaerobic fungi harbor a rich reservoir of undiscovered cellulolytic enzymes and enzyme complexes that can potentially transform the conversion of lignocellulose into bioenergy products. Despite their unique evolutionary history and cellulolytic activity, few species have been isolated and studied in great detail. As a result, their life cycle, cellular physiology, genetics, and cellulolytic metabolism remain poorly understood compared to aerobic fungi. To help address this limitation, this review briefly summarizes the current body of knowledge pertaining to anaerobic fungal biology, and describes progress made in the isolation, cultivation, molecular characterization, and long-term preservation of these microbes. We also discuss recent cellulase- and cellulosome-discovery efforts from gut fungi, and how these interesting, non-model microbes could be further adapted for biotechnology applications. © 2014 Wiley Periodicals, Inc.

A selection of reference genes and early-warning mRNA biomarkers for environmental monitoring using Mytilus spp. as sentinel species.

PubMed

Lacroix, C; Coquillé, V; Guyomarch, J; Auffret, M; Moraga, D

2014-09-15

mRNA biomarkers are promising tools for environmental health assessment and reference genes are needed to perform relevant qPCR analyses in tissue samples of sentinel species. In the present study, potential reference genes and mRNA biomarkers were tested in the gills and digestive glands of native and caged mussels (Mytilus spp.) exposed to harbor pollution. Results highlighted the difficulty to find stable reference genes in wild, non-model species and suggested the use of normalization indices instead of single genes as they exhibit a higher stability. Several target genes were found differentially expressed between mussel groups, especially in gills where cyp32, π-gst and CuZn-sod mRNA levels could be biomarker candidates. Multivariate analyses confirmed the ability of mRNA levels to highlight site-effects and suggested the use of several combined markers instead of individual ones. These findings support the use of qPCR technology and mRNA levels as early-warning biomarkers in marine monitoring programs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effects of short read quality and quantity on a de novo vertebrate transcriptome assembly.

PubMed

Garcia, T I; Shen, Y; Catchen, J; Amores, A; Schartl, M; Postlethwait, J; Walter, R B

2012-01-01

For many researchers, next generation sequencing data holds the key to answering a category of questions previously unassailable. One of the important and challenging steps in achieving these goals is accurately assembling the massive quantity of short sequencing reads into full nucleic acid sequences. For research groups working with non-model or wild systems, short read assembly can pose a significant challenge due to the lack of pre-existing EST or genome reference libraries. While many publications describe the overall process of sequencing and assembly, few address the topic of how many and what types of reads are best for assembly. The goal of this project was use real world data to explore the effects of read quantity and short read quality scores on the resulting de novo assemblies. Using several samples of short reads of various sizes and qualities we produced many assemblies in an automated manner. We observe how the properties of read length, read quality, and read quantity affect the resulting assemblies and provide some general recommendations based on our real-world data set. Published by Elsevier Inc.

Unsupervised markerless 3-DOF motion tracking in real time using a single low-budget camera.

PubMed

Quesada, Luis; León, Alejandro J

2012-10-01

Motion tracking is a critical task in many computer vision applications. Existing motion tracking techniques require either a great amount of knowledge on the target object or specific hardware. These requirements discourage the wide spread of commercial applications based on motion tracking. In this paper, we present a novel three degrees of freedom motion tracking system that needs no knowledge on the target object and that only requires a single low-budget camera that can be found installed in most computers and smartphones. Our system estimates, in real time, the three-dimensional position of a nonmodeled unmarked object that may be nonrigid, nonconvex, partially occluded, self-occluded, or motion blurred, given that it is opaque, evenly colored, enough contrasting with the background in each frame, and that it does not rotate. Our system is also able to determine the most relevant object to track in the screen. Our proposal does not impose additional constraints, therefore it allows a market-wide implementation of applications that require the estimation of the three position degrees of freedom of an object.

Light in the darkness: New perspective on lanternfish relationships and classification using genomic and morphological data.

PubMed

Martin, Rene P; Olson, Emily E; Girard, Matthew G; Smith, Wm Leo; Davis, Matthew P

2018-04-01

Massive parallel sequencing allows scientists to gather DNA sequences composed of millions of base pairs that can be combined into large datasets and analyzed to infer organismal relationships at a genome-wide scale in non-model organisms. Although the use of these large datasets is becoming more widespread, little to no work has been done in estimating phylogenetic relationships using UCEs in deep-sea fishes. Among deep-sea animals, the 257 species of lanternfishes (Myctophiformes) are among the most important open-ocean lineages, representing half of all mesopelagic vertebrate biomass. With this relative abundance, they are key members of the midwater food web where they feed on smaller invertebrates and fishes in addition to being a primary prey item for other open-ocean animals. Understanding the evolution and relationships of midwater organisms generally, and this dominant group of fishes in particular, is necessary for understanding and preserving the underexplored deep-sea ecosystem. Despite substantial congruence in the evolutionary relationships among deep-sea lanternfishes at higher classification levels in previous studies, the relationships among tribes, genera, and species within Myctophidae often conflict across phylogenetic studies or lack resolution and support. Herein we provide the first genome-scale phylogenetic analysis of lanternfishes, and we integrate these data from across the nuclear genome with additional protein-coding gene sequences and morphological data to further test evolutionary relationships among lanternfishes. Our phylogenetic hypotheses of relationships among lanternfishes are entirely congruent across a diversity of analyses that vary in methods, taxonomic sampling, and data analyzed. Within the Myctophiformes, the Neoscopelidae is inferred to be monophyletic and sister to a monophyletic Myctophidae. The current classification of lanternfishes is incongruent with our phylogenetic tree, so we recommend revisions that retain much of the traditional tribal structure and recognize five subfamilies instead of the traditional two subfamilies. The revised monophyletic taxonomy of myctophids includes the elevation of three former lampanyctine tribes to subfamilies. A restricted Lampanyctinae was recovered sister to Notolychninae. These two clades together were recovered as the sister group to the Gymnoscopelinae. Combined, these three subfamilies were recovered as the sister group to a clade composed of a monophyletic Diaphinae sister to the traditional Myctophinae. Our results corroborate recent multilocus molecular studies that infer a polyphyletic Myctophum in Myctophinae, and a para- or polyphyletic Lampanyctus and Nannobrachium within Lampanyctinae. We resurrect Dasyscopelus and Ctenoscopelus for the independent clades traditionally classified as species of Myctophum, and we place Nannobrachium into the synonymy of Lampanyctus. Copyright © 2017 Elsevier Inc. All rights reserved.

Genome-wide distribution of genetic diversity and linkage disequilibrium in a mass-selected population of maritime pine

PubMed Central

2014-01-01

Background The accessibility of high-throughput genotyping technologies has contributed greatly to the development of genomic resources in non-model organisms. High-density genotyping arrays have only recently been developed for some economically important species such as conifers. The potential for using genomic technologies in association mapping and breeding depends largely on the genome wide patterns of diversity and linkage disequilibrium in current breeding populations. This study aims to deepen our knowledge regarding these issues in maritime pine, the first species used for reforestation in south western Europe. Results Using a new map merging algorithm, we first established a 1,712 cM composite linkage map (comprising 1,838 SNP markers in 12 linkage groups) by bringing together three already available genetic maps. Using rigorous statistical testing based on kernel density estimation and resampling we identified cold and hot spots of recombination. In parallel, 186 unrelated trees of a mass-selected population were genotyped using a 12k-SNP array. A total of 2,600 informative SNPs allowed to describe historical recombination, genetic diversity and genetic structure of this recently domesticated breeding pool that forms the basis of much of the current and future breeding of this species. We observe very low levels of population genetic structure and find no evidence that artificial selection has caused a reduction in genetic diversity. By combining these two pieces of information, we provided the map position of 1,671 SNPs corresponding to 1,192 different loci. This made it possible to analyze the spatial pattern of genetic diversity (H e ) and long distance linkage disequilibrium (LD) along the chromosomes. We found no particular pattern in the empirical variogram of H e across the 12 linkage groups and, as expected for an outcrossing species with large effective population size, we observed an almost complete lack of long distance LD. Conclusions These results are a stepping stone for the development of strategies for studies in population genomics, association mapping and genomic prediction in this economical and ecologically important forest tree species. PMID:24581176

De novo assembly and characterization of the transcriptome in the desiccation-tolerant moss Syntrichia caninervis

PubMed Central

2014-01-01

Background Syntrichia caninervis is a desiccation-tolerant moss and the dominant bryophyte of the Biological Soil Crusts (BSCs) found in the Mojave and Gurbantunggut deserts. Next generation high throughput sequencing technologies offer an efficient and economic choice for characterizing non-model organism transcriptomes with little or no prior molecular information available. Results In this study, we employed next generation, high-throughput, Illumina RNA-Seq to analyze the poly-(A) + mRNA from hydrated, dehydrating and desiccated S. caninervis gametophores. Approximately 58.0 million paired-end short reads were obtained and 92,240 unigenes were assembled with an average size of 493 bp, N50 value of 662 bp and a total size of 45.48 Mbp. Sequence similarity searches against five public databases (NR, Swiss-Prot, COSMOSS, KEGG and COG) found 54,125 unigenes (58.7%) with significant similarity to an existing sequence (E-value ≤ 1e-5) and could be annotated. Gene Ontology (GO) annotation assigned 24,183 unigenes to the three GO terms: Biological Process, Cellular Component or Molecular Function. GO comparison between P. patens and S. caninervis demonstrated similar sequence enrichment across all three GO categories. 29,370 deduced polypeptide sequences were assigned Pfam domain information and categorized into 4,212 Pfam domains/families. Using the PlantTFDB, 778 unigenes were predicted to be involved in the regulation of transcription and were classified into 49 transcription factor families. Annotated unigenes were mapped to the KEGG pathways and further annotated using MapMan. Comparative genomics revealed that 44% of protein families are shared in common by S. caninervis, P. patens and Arabidopsis thaliana and that 80% are shared by both moss species. Conclusions This study is one of the first comprehensive transcriptome analyses of the moss S. caninervis. Our data extends our knowledge of bryophyte transcriptomes, provides an insight to plants adapted to the arid regions of central Asia, and continues the development of S. caninervis as a model for understanding the molecular aspects of desiccation-tolerance. PMID:25086984

Navigating the Interface Between Landscape Genetics and Landscape Genomics.

PubMed

Storfer, Andrew; Patton, Austin; Fraik, Alexandra K

2018-01-01

As next-generation sequencing data become increasingly available for non-model organisms, a shift has occurred in the focus of studies of the geographic distribution of genetic variation. Whereas landscape genetics studies primarily focus on testing the effects of landscape variables on gene flow and genetic population structure, landscape genomics studies focus on detecting candidate genes under selection that indicate possible local adaptation. Navigating the transition between landscape genomics and landscape genetics can be challenging. The number of molecular markers analyzed has shifted from what used to be a few dozen loci to thousands of loci and even full genomes. Although genome scale data can be separated into sets of neutral loci for analyses of gene flow and population structure and putative loci under selection for inference of local adaptation, there are inherent differences in the questions that are addressed in the two study frameworks. We discuss these differences and their implications for study design, marker choice and downstream analysis methods. Similar to the rapid proliferation of analysis methods in the early development of landscape genetics, new analytical methods for detection of selection in landscape genomics studies are burgeoning. We focus on genome scan methods for detection of selection, and in particular, outlier differentiation methods and genetic-environment association tests because they are the most widely used. Use of genome scan methods requires an understanding of the potential mismatches between the biology of a species and assumptions inherent in analytical methods used, which can lead to high false positive rates of detected loci under selection. Key to choosing appropriate genome scan methods is an understanding of the underlying demographic structure of study populations, and such data can be obtained using neutral loci from the generated genome-wide data or prior knowledge of a species' phylogeographic history. To this end, we summarize recent simulation studies that test the power and accuracy of genome scan methods under a variety of demographic scenarios and sampling designs. We conclude with a discussion of additional considerations for future method development, and a summary of methods that show promise for landscape genomics studies but are not yet widely used.

Navigating the Interface Between Landscape Genetics and Landscape Genomics

PubMed Central

Storfer, Andrew; Patton, Austin; Fraik, Alexandra K.

2018-01-01

As next-generation sequencing data become increasingly available for non-model organisms, a shift has occurred in the focus of studies of the geographic distribution of genetic variation. Whereas landscape genetics studies primarily focus on testing the effects of landscape variables on gene flow and genetic population structure, landscape genomics studies focus on detecting candidate genes under selection that indicate possible local adaptation. Navigating the transition between landscape genomics and landscape genetics can be challenging. The number of molecular markers analyzed has shifted from what used to be a few dozen loci to thousands of loci and even full genomes. Although genome scale data can be separated into sets of neutral loci for analyses of gene flow and population structure and putative loci under selection for inference of local adaptation, there are inherent differences in the questions that are addressed in the two study frameworks. We discuss these differences and their implications for study design, marker choice and downstream analysis methods. Similar to the rapid proliferation of analysis methods in the early development of landscape genetics, new analytical methods for detection of selection in landscape genomics studies are burgeoning. We focus on genome scan methods for detection of selection, and in particular, outlier differentiation methods and genetic-environment association tests because they are the most widely used. Use of genome scan methods requires an understanding of the potential mismatches between the biology of a species and assumptions inherent in analytical methods used, which can lead to high false positive rates of detected loci under selection. Key to choosing appropriate genome scan methods is an understanding of the underlying demographic structure of study populations, and such data can be obtained using neutral loci from the generated genome-wide data or prior knowledge of a species' phylogeographic history. To this end, we summarize recent simulation studies that test the power and accuracy of genome scan methods under a variety of demographic scenarios and sampling designs. We conclude with a discussion of additional considerations for future method development, and a summary of methods that show promise for landscape genomics studies but are not yet widely used. PMID:29593776

Transcriptome analysis reveals novel insights into the response of low-dose benzo(a)pyrene exposure in male tilapia.

PubMed

Colli-Dula, Reyna Cristina; Fang, Xiefan; Moraga-Amador, David; Albornoz-Abud, Nacira; Zamora-Bustillos, Roberto; Conesa, Ana; Zapata-Perez, Omar; Moreno, Diego; Hernandez-Nuñez, Emanuel

2018-06-08

Despite a wide number of toxicological studies that describe benzo[a]pyrene (BaP) effects, the metabolic mechanisms that underlie these effects in fish are largely unknown. Of great concern is the presence of BaP in aquatic systems, especially those in close proximity to human activity leading to consumption of potentially contaminated foods. BaP is a known carcinogen and it has been reported to have adverse effects on the survival, development and reproduction of fish. The purpose of this study was to investigate if a low dose of BaP can alter genes and key metabolic pathways in the liver and testis in male adult tilapia, and whether these could be associated with biological endpoints disruption. We used both high-throughput RNA-Sequencing to assess whole genome gene expression following repeated intraperitoneal injections of 3 mg/kg of BaP (every 6 days for 26 days) and morphometric endpoints as indicators of general health. Condition factor (K) along with hepatosomatic and gonadosomatic indices (morphometric parameters) were significantly lower in BaP-treated fish than in controls. BaP exposure induced important changes in the gene expression pattern in liver and testis as revealed by both Pathway and Gene Ontology (GO) analyses. Alterations that were shared by both tissues included arachidonic acid metabolism, androgen receptor to prostate-specific antigen signaling, and insulin-associated effects on lipogenesis. The most salient liver-specific effects included: biological processes involved in detoxification, IL6-associated insulin resistance, mTOR hyperactivation, mitotic cytokinesis, spindle pole and microtubule binding. BaP effects that were confined to the testis included: immune system functions, inflammatory response, estrogen and androgen metabolic pathways. Taken together, gene expression and morphometric end point data indicate that the reproductive success of adult male tilapia could be compromised as a result of BaP exposure. These results constitute new insights on the mechanism of action of low dose BaP in a non-model organism (tilapia). Copyright © 2018 Elsevier B.V. All rights reserved.

Eastern coral snake Micrurus fulvius venom toxicity in mice is mainly determined by neurotoxic phospholipases A2.

PubMed

Vergara, Irene; Pedraza-Escalona, Martha; Paniagua, Dayanira; Restano-Cassulini, Rita; Zamudio, Fernando; Batista, Cesar V F; Possani, Lourival D; Alagón, Alejandro

2014-06-13

Here we show for the first time that the venom from an elapid (Micrurus fulvius) contains three finger toxin (3FTxs) peptides with low toxicity but high content of lethal phospholipases A2 (PLA2). The intravenous venom LD50 in mice was 0.3μg/g. Fractionation on a C18 column yielded 22 fractions; in terms of abundance, 58.3% of them were components of 13-14kDa and 24.9% were molecules of 6-7kDa. Two fractions with PLA2 activity represented 33.4% of the whole venom and were the most lethal fractions. Fractions with low molecular mass (<7000Da) partially and reversibly blocked the nicotinic acetylcholine receptor (nAChR), with the exception of one that blocked it completely. The fraction that blocked 100% contained two protein species whose dose-response was determined; the IC50s were 13±1 and 9.5±0.3nM. Despite the apparent effect on nAChR none of the low molecular mass fractions were lethal in mice, at concentrations of 1μg/g. From 2D-PAGE and LC-MS/MS, we identified fourteen species of PLA2, four protein species of C-type lectin, three zinc metalloproteinases, one phosphodiesterase and one 3FTx. The N-terminal amino acid sequence of fractions with biological interest was obtained. In contrast with coral snake venoms from South America, M. fulvius has minor amounts of low molecular mass components, but high content of PLA2, which is responsible for the venom lethality of this species. The results reported here contribute to better understanding of envenomation development and to improve antivenom design and production. These findings break from the paradigm that neurotoxicity caused by Micrurus venoms is mainly attributable to 3FTx neurotoxins and encourage future studies on Micrurus evolution and venom specialization. This article is part of a Special Issue entitled Non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of differentially expressed placental transcripts during multiple gestations in the Eurasian beaver (Castor fiber L.).

PubMed

Lipka, A; Paukszto, L; Majewska, M; Jastrzebski, J P; Myszczynski, K; Panasiewicz, G; Szafranska, B

2017-09-01

The Eurasian beaver is one of the largest rodents that, despite its high impact on the environment, is a non-model species that lacks a reference genome. Characterising genes critical for pregnancy outcome can serve as a basis for identifying mechanisms underlying effective reproduction, which is required for the success of endangered species conservation programs. In the present study, high-throughput RNA sequencing (RNA-seq) was used to analyse global changes in the Castor fiber subplacenta transcriptome during multiple pregnancy. De novo reconstruction of the C. fiber subplacenta transcriptome was used to identify genes that were differentially expressed in placentas (n=5) from two females (in advanced twin and triple pregnancy). Analyses of the expression values revealed 124 contigs with significantly different expression; of these, 55 genes were identified using MegaBLAST. Within this group of differentially expressed genes (DEGs), 18 were upregulated and 37 were downregulated in twins. Most DEGs were associated with the following gene ontology terms: cellular process, single organism process, response to stimulus, metabolic process and biological regulation. Some genes were also assigned to the developmental process, the reproductive process or reproduction. Among this group, four genes (namely keratin 19 (Krt19) and wingless-type MMTV integration site family - member 2 (Wnt2), which were downregulated in twins, and Nik-related kinase (Nrk) and gap junction protein β2 (Gjb2), which were upregulated in twins) were assigned to placental development and nine (Krt19, Wnt2 and integrin α 7 (Itga7), downregulated in twins, and Nrk, gap junction protein β6 (Gjb6), GATA binding protein 6 (Gata6), apolipoprotein A-I (ApoA1), apolipoprotein B (ApoB) and haemoglobin subunit α 1 (HbA1), upregulated in twins) were assigned to embryo development. The results of the present study indicate that the number of fetuses affects the expression profile in the C. fiber subplacental transcriptome. Enhancement of transcriptomic resources for C. fiber will improve understanding of the pathways relevant to proper placental development and successful reproduction.

Linking mechanistic and behavioral responses to sublethal esfenvalerate exposure in the endangered delta smelt; Hypomesus transpacificus (Fam. Osmeridae)

PubMed Central

2009-01-01

Background The delta smelt (Hypomesus transpacificus) is a pelagic fish species listed as endangered under both the USA Federal and Californian State Endangered Species Acts and considered an indicator of ecosystem health in its habitat range, which is limited to the Sacramento-San Joaquin estuary in California, USA. Anthropogenic contaminants are one of multiple stressors affecting this system, and among them, current-use insecticides are of major concern. Interrogative tools are required to successfully monitor effects of contaminants on the delta smelt, and to research potential causes of population decline in this species. We have created a microarray to investigate genome-wide effects of potentially causative stressors, and applied this tool to assess effects of the pyrethroid insecticide esfenvalerate on larval delta smelt. Selected genes were further investigated as molecular biomarkers using quantitative PCR analyses. Results Exposure to esfenvalerate affected swimming behavior of larval delta smelt at concentrations as low as 0.0625 μg.L-1, and significant differences in expression were measured in genes involved in neuromuscular activity. Alterations in the expression of genes associated with immune responses, along with apoptosis, redox, osmotic stress, detoxification, and growth and development appear to have been invoked by esfenvalerate exposure. Swimming impairment correlated significantly with expression of aspartoacylase (ASPA), an enzyme involved in brain cell function and associated with numerous human diseases. Selected genes were investigated for their use as molecular biomarkers, and strong links were determined between measured downregulation in ASPA and observed behavioral responses in fish exposed to environmentally relevant pyrethroid concentrations. Conclusions The results of this study show that microarray technology is a useful approach in screening for, and generation of molecular biomarkers in endangered, non-model organisms, identifying specific genes that can be directly linked with sublethal toxicological endpoints; such as changes in expression levels of neuromuscular genes resulting in measurable swimming impairments. The developed microarrays were successfully applied on larval fish exposed to esfenvalerate, a known contaminant of the Sacramento-San Joaquin estuary, and has permitted the identification of specific biomarkers which could provide insight into the factors contributing to delta smelt population decline. PMID:20003521

Transcriptomic SNP discovery for custom genotyping arrays: impacts of sequence data, SNP calling method and genotyping technology on the probability of validation success.

PubMed

Humble, Emily; Thorne, Michael A S; Forcada, Jaume; Hoffman, Joseph I

2016-08-26

Single nucleotide polymorphism (SNP) discovery is an important goal of many studies. However, the number of 'putative' SNPs discovered from a sequence resource may not provide a reliable indication of the number that will successfully validate with a given genotyping technology. For this it may be necessary to account for factors such as the method used for SNP discovery and the type of sequence data from which it originates, suitability of the SNP flanking sequences for probe design, and genomic context. To explore the relative importance of these and other factors, we used Illumina sequencing to augment an existing Roche 454 transcriptome assembly for the Antarctic fur seal (Arctocephalus gazella). We then mapped the raw Illumina reads to the new hybrid transcriptome using BWA and BOWTIE2 before calling SNPs with GATK. The resulting markers were pooled with two existing sets of SNPs called from the original 454 assembly using NEWBLER and SWAP454. Finally, we explored the extent to which SNPs discovered using these four methods overlapped and predicted the corresponding validation outcomes for both Illumina Infinium iSelect HD and Affymetrix Axiom arrays. Collating markers across all discovery methods resulted in a global list of 34,718 SNPs. However, concordance between the methods was surprisingly poor, with only 51.0 % of SNPs being discovered by more than one method and 13.5 % being called from both the 454 and Illumina datasets. Using a predictive modeling approach, we could also show that SNPs called from the Illumina data were on average more likely to successfully validate, as were SNPs called by more than one method. Above and beyond this pattern, predicted validation outcomes were also consistently better for Affymetrix Axiom arrays. Our results suggest that focusing on SNPs called by more than one method could potentially improve validation outcomes. They also highlight possible differences between alternative genotyping technologies that could be explored in future studies of non-model organisms.

De Novo Adult Transcriptomes of Two European Brittle Stars: Spotlight on Opsin-Based Photoreception

PubMed Central

Mallefet, Jérôme; Flammang, Patrick

2016-01-01

Next generation sequencing (NGS) technology allows to obtain a deeper and more complete view of transcriptomes. For non-model or emerging model marine organisms, NGS technologies offer a great opportunity for rapid access to genetic information. In this study, paired-end Illumina HiSeqTM technology has been employed to analyse transcriptomes from the arm tissues of two European brittle star species, Amphiura filiformis and Ophiopsila aranea. About 48 million Illumina reads were generated and 136,387 total unigenes were predicted from A. filiformis arm tissues. For O. aranea arm tissues, about 47 million reads were generated and 123,324 total unigenes were obtained. Twenty-four percent of the total unigenes from A. filiformis show significant matches with sequences present in reference online databases, whereas, for O. aranea, this percentage amounts to 23%. In both species, around 50% of the predicted annotated unigenes were significantly similar to transcripts from the purple sea urchin, the closest species to date that has undergone complete genome sequencing and annotation. GO, COG and KEGG analyses were performed on predicted brittle star unigenes. We focused our analyses on the phototransduction actors involved in light perception. Firstly, two new echinoderm opsins were identified in O. aranea: one rhabdomeric opsin (homologous to vertebrate melanopsin) and one RGR opsin. The RGR-opsin is supposed to be involved in retinal regeneration while the r-opsin is suspected to play a role in visual-like behaviour. Secondly, potential phototransduction actors were identified in both transcriptomes using the fly (rhabdomeric) and mammal (ciliary) classical phototransduction pathways as references. Finally, the sensitivity of O.aranea to monochromatic light was investigated to complement data available for A. filiformis. The presence of microlens-like structures at the surface of dorsal arm plate of O. aranea could potentially explain phototactic behaviour differences between the two species. The results confirm (i) the ability of these brittle stars to perceive light using opsin-based photoreception, (ii) suggest the co-occurrence of both rhabdomeric and ciliary photoreceptors, and (iii) emphasise the complexity of light perception in this echinoderm class. PMID:27119739

Linkage maps of the Atlantic salmon (Salmo salar) genome derived from RAD sequencing

PubMed Central

2014-01-01

Background Genetic linkage maps are useful tools for mapping quantitative trait loci (QTL) influencing variation in traits of interest in a population. Genotyping-by-sequencing approaches such as Restriction-site Associated DNA sequencing (RAD-Seq) now enable the rapid discovery and genotyping of genome-wide SNP markers suitable for the development of dense SNP linkage maps, including in non-model organisms such as Atlantic salmon (Salmo salar). This paper describes the development and characterisation of a high density SNP linkage map based on SbfI RAD-Seq SNP markers from two Atlantic salmon reference families. Results Approximately 6,000 SNPs were assigned to 29 linkage groups, utilising markers from known genomic locations as anchors. Linkage maps were then constructed for the four mapping parents separately. Overall map lengths were comparable between male and female parents, but the distribution of the SNPs showed sex-specific patterns with a greater degree of clustering of sire-segregating SNPs to single chromosome regions. The maps were integrated with the Atlantic salmon draft reference genome contigs, allowing the unique assignment of ~4,000 contigs to a linkage group. 112 genome contigs mapped to two or more linkage groups, highlighting regions of putative homeology within the salmon genome. A comparative genomics analysis with the stickleback reference genome identified putative genes closely linked to approximately half of the ordered SNPs and demonstrated blocks of orthology between the Atlantic salmon and stickleback genomes. A subset of 47 RAD-Seq SNPs were successfully validated using a high-throughput genotyping assay, with a correspondence of 97% between the two assays. Conclusions This Atlantic salmon RAD-Seq linkage map is a resource for salmonid genomics research as genotyping-by-sequencing becomes increasingly common. This is aided by the integration of the SbfI RAD-Seq SNPs with existing reference maps and the draft reference genome, as well as the identification of putative genes proximal to the SNPs. Differences in the distribution of recombination events between the sexes is evident, and regions of homeology have been identified which are reflective of the recent salmonid whole genome duplication. PMID:24571138

Photoperiod- and temperature-mediated control of growth cessation and dormancy in trees: a molecular perspective.

PubMed

Maurya, Jay P; Bhalerao, Rishikesh P

2017-09-01

How plants adapt their developmental patterns to regular seasonal changes is an important question in biology. The annual growth cycle in perennial long-lived trees is yet another example of how plants can adapt to seasonal changes. The two main signals that plants rely on to respond to seasonal changes are photoperiod and temperature, and these signals have critical roles in the temporal regulation of the annual growth cycle of trees. This review presents the latest findings to provide insight into the molecular mechanisms that underlie how photoperiodic and temperature signals regulate seasonal growth in trees. The results point to a high level of conservation in the signalling pathways that mediate photoperiodic control of seasonal growth in trees and flowering in annual plants such as arabidopsis. Furthermore, the data indicate that symplastic communication may mediate certain aspects of seasonal growth. Although considerable insight into the control of phenology in model plants such as poplar and spruce has been obtained, the future challenge is extending these studies to other, non-model trees. © The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Approaching Terahertz Range with 3-color Broadband Coherent Raman Micro Spectroscopy

NASA Astrophysics Data System (ADS)

Ujj, Laszlo; Olson, Trevor; Amos, James

The presentation reports the recent progress made on reliable signal recording and processing using 3-color broadband coherent Raman scattering (3C-BCRS). Signals are generated either from nanoparticle structures on surfaces or from bulk samples in transmission and in epi-detected mode. Spectra are recorded with a narrowband (at 532 nm) and a broadband radiation produced by a newly optimized optical parametric oscillator using the signal or idler beams. Vibrational and librational bands are measured over the 0.15-15 THz spectral range from solution and crystalline samples. Volumetric Brag-filter approach is introduced for recording 3C-BCRS spectra at the first time. The technical limitations and advantages of the narrowband filtering relative to the Notch-filter technic is clarified. The signal is proportional to the spectral autocorrelation of the broadband radiation therefore the present scheme gives a better signal-to-noise ratio relative to the traditional multiplex CRS methods. This makes the automation of non-model dependent signal processing more reliable to extract vibrational information which is very crucial in coherent Raman microscopy. Financial support from the Hal Marcus College of Science and Engineering is greatly appreciated.

The Evolution of Arthropod Body Plans: Integrating Phylogeny, Fossils, and Development-An Introduction to the Symposium.

PubMed

Chipman, Ariel D; Erwin, Douglas H

2017-09-01

The last few years have seen a significant increase in the amount of data we have about the evolution of the arthropod body plan. This has come mainly from three separate sources: a new consensus and improved resolution of arthropod phylogeny, based largely on new phylogenomic analyses; a wealth of new early arthropod fossils from a number of Cambrian localities with excellent preservation, as well as a renewed analysis of some older fossils; and developmental data from a range of model and non-model pan-arthropod species that shed light on the developmental origins and homologies of key arthropod traits. However, there has been relatively little synthesis among these different data sources, and the three communities studying them have little overlap. The symposium "The Evolution of Arthropod Body Plans-Integrating Phylogeny, Fossils and Development" brought together leading researchers in these three disciplines and made a significant contribution to the emerging synthesis of arthropod evolution, which will help advance the field and will be useful for years to come. © The Author 2017. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Body image concerns in professional fashion models: are they really an at-risk group?

PubMed

Swami, Viren; Szmigielska, Emilia

2013-05-15

Although professional models are thought to be a high-risk group for body image concerns, only a handful of studies have empirically investigated this possibility. The present study sought to overcome this dearth of information by comparing professional models and a matched sample on key indices of body image and appeared-related concerns. A group of 52 professional fashion models was compared with a matched sample of 51 non-models from London, England, on indices of weight discrepancy, body appreciation, social physique anxiety, body dissatisfaction, drive for thinness, internalization of sociocultural messages about appearance, and dysfunctional investment in appearance. Results indicated that professional models only evidenced significantly higher drive for thinness and dysfunctional investment in appearance than the control group. Greater duration of engagement as a professional model was associated with more positive body appreciation but also greater drive for thinness. These results indicate that models, who are already underweight, have a strong desire to maintain their low body mass or become thinner. Taken together, the present results suggest that interventions aimed at promoting healthy body image among fashion models may require different strategies than those aimed at the general population. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

De novo assembly and transcriptome characterization of the freshwater prawn Palaemonetes argentinus: Implications for a detoxification response.

PubMed

García, C Fernando; Pedrini, Nicolas; Sánchez-Paz, Arturo; Reyna-Blanco, Carlos S; Lavarias, Sabrina; Muhlia-Almazán, Adriana; Fernández-Giménez, Analía; Laino, Aldana; de-la-Re-Vega, Enrique; Lukaszewicz, German; López-Zavala, Alonso A; Brieba, Luis G; Criscitello, Michael F; Carrasco-Miranda, Jesús S; García-Orozco, Karina D; Ochoa-Leyva, Adrian; Rudiño-Piñera, Enrique; Sanchez-Flores, Alejandro; Sotelo-Mundo, Rogerio R

2018-02-01

Palaemonetes argentinus, an abundant freshwater prawn species in the northern and central region of Argentina, has been used as a bioindicator of environmental pollutants as it displays a very high sensitivity to pollutants exposure. Despite their extraordinary ecological relevance, a lack of genomic information has hindered a more thorough understanding of the molecular mechanisms potentially involved in detoxification processes of this species. Thus, transcriptomic profiling studies represent a promising approach to overcome the limitations imposed by the lack of extensive genomic resources for P. argentinus, and may improve the understanding of its physiological and molecular response triggered by pollutants. This work represents the first comprehensive transcriptome-based characterization of the non-model species P. argentinus to generate functional genomic annotations and provides valuable resources for future genetic studies. Trinity de novo assembly consisted of 24,738 transcripts with high representation of detoxification (phase I and II), anti-oxidation, osmoregulation pathways and DNA replication and bioenergetics. This crustacean transcriptome provides valuable molecular information about detoxification and biochemical processes that could be applied as biomarkers in further ecotoxicology studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Deep sequencing and in silico analysis of small RNA library reveals novel miRNA from leaf Persicaria minor transcriptome.

PubMed

Samad, Abdul Fatah A; Nazaruddin, Nazaruddin; Murad, Abdul Munir Abdul; Jani, Jaeyres; Zainal, Zamri; Ismail, Ismanizan

2018-03-01

In current era, majority of microRNA (miRNA) are being discovered through computational approaches which are more confined towards model plants. Here, for the first time, we have described the identification and characterization of novel miRNA in a non-model plant, Persicaria minor ( P . minor ) using computational approach. Unannotated sequences from deep sequencing were analyzed based on previous well-established parameters. Around 24 putative novel miRNAs were identified from 6,417,780 reads of the unannotated sequence which represented 11 unique putative miRNA sequences. PsRobot target prediction tool was deployed to identify the target transcripts of putative novel miRNAs. Most of the predicted target transcripts (mRNAs) were known to be involved in plant development and stress responses. Gene ontology showed that majority of the putative novel miRNA targets involved in cellular component (69.07%), followed by molecular function (30.08%) and biological process (0.85%). Out of 11 unique putative miRNAs, 7 miRNAs were validated through semi-quantitative PCR. These novel miRNAs discoveries in P . minor may develop and update the current public miRNA database.