Biodiversity and molecular ecology of Russula and Lactarius in Alaska based on soil and sporocarp DNA sequences

Treesearch

Geml J.; D.L. Taylor

2013-01-01

Although critical for the functioning of ecosystems, fungi are poorly known in highlatitude regions. This paper summarizes the results of the first genetic diversity assessments of Russula and Lactarius, two of the most diverse and abundant fungal genera in Alaska. SU rDNA sequences from both curated sporocarp collections and soil PCR clone libraries sampled in...

DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis.

PubMed

MacConnell, Andrew B; McEnaney, Patrick J; Cavett, Valerie J; Paegel, Brian M

2015-09-14

The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the "structure elucidation problem": the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS's utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 10(4) molecules/bead and sequencing allowed for elucidation of each compound's synthetic history. We applied DESPS to the combinatorial synthesis of a 75,645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward, and may prompt the chemistry community to revisit the synthesis of more complex and diverse libraries.

Genetic diversity of Entamoeba: Novel ribosomal lineages from cockroaches

PubMed Central

Kawano, Tetsuro; Imada, Mihoko; Chamavit, Pennapa; Kobayashi, Seiki; Hashimoto, Tetsuo

2017-01-01

Our current taxonomic perspective on Entamoeba is largely based on small-subunit ribosomal RNA genes (SSU rDNA) from Entamoeba species identified in vertebrate hosts with minor exceptions such as E. moshkovskii from sewage water and E. marina from marine sediment. Other Entamoeba species have also been morphologically identified and described from non-vertebrate species such as insects; however, their genetic diversity remains unknown. In order to further disclose the diversity of the genus, we investigated Entamoeba spp. in the intestines of three cockroach species: Periplaneta americana, Blaptica dubia, and Gromphadorhina oblongonota. We obtained 134 Entamoeba SSU rDNA sequences from 186 cockroaches by direct nested PCR using the DNA extracts of intestines from cockroaches, followed by scrutinized BLASTn screening and phylogenetic analyses. All the sequences identified in this study were distinct from those reported from known Entamoeba species, and considered as novel Entamoeba ribosomal lineages. Furthermore, they were positioned at the base of the clade of known Entamoeba species and displayed remarkable degree of genetic diversity comprising nine major groups in the three cockroach species. This is the first report of the diversity of SSU rDNA sequences from Entamoeba in non-vertebrate host species, and should help to understand the genetic diversity of the genus Entamoeba. PMID:28934335

Low diversity in the mitogenome of sperm whales revealed by next-generation sequencing

Treesearch

Alana Alexander; Debbie Steel; Beth Slikas; Kendra Hoekzema; Colm Carraher; Matthew Parks; Richard Cronn; C. Scott Baker

2012-01-01

Large population sizes and global distributions generally associate with high mitochondrial DNA control region (CR) diversity. The sperm whale (Physeter macrocephalus) is an exception, showing low CR diversity relative to other cetaceans; however, diversity levels throughout the remainder of the sperm whale mitogenome are unknown. We sequenced 20...

Broad Surveys of DNA Viral Diversity Obtained through Viral Metagenomics of Mosquitoes

PubMed Central

Ng, Terry Fei Fan; Willner, Dana L.; Lim, Yan Wei; Schmieder, Robert; Chau, Betty; Nilsson, Christina; Anthony, Simon; Ruan, Yijun; Rohwer, Forest; Breitbart, Mya

2011-01-01

Viruses are the most abundant and diverse genetic entities on Earth; however, broad surveys of viral diversity are hindered by the lack of a universal assay for viruses and the inability to sample a sufficient number of individual hosts. This study utilized vector-enabled metagenomics (VEM) to provide a snapshot of the diversity of DNA viruses present in three mosquito samples from San Diego, California. The majority of the sequences were novel, suggesting that the viral community in mosquitoes, as well as the animal and plant hosts they feed on, is highly diverse and largely uncharacterized. Each mosquito sample contained a distinct viral community. The mosquito viromes contained sequences related to a broad range of animal, plant, insect and bacterial viruses. Animal viruses identified included anelloviruses, circoviruses, herpesviruses, poxviruses, and papillomaviruses, which mosquitoes may have obtained from vertebrate hosts during blood feeding. Notably, sequences related to human papillomaviruses were identified in one of the mosquito samples. Sequences similar to plant viruses were identified in all mosquito viromes, which were potentially acquired through feeding on plant nectar. Numerous bacteriophages and insect viruses were also detected, including a novel densovirus likely infecting Culex erythrothorax. Through sampling insect vectors, VEM enables broad survey of viral diversity and has significantly increased our knowledge of the DNA viruses present in mosquitoes. PMID:21674005

Genetic diversity and demographic instability in Riftia pachyptila tubeworms from eastern Pacific hydrothermal vents

PubMed Central

2011-01-01

Background Deep-sea hydrothermal vent animals occupy patchy and ephemeral habitats supported by chemosynthetic primary production. Volcanic and tectonic activities controlling the turnover of these habitats contribute to demographic instability that erodes genetic variation within and among colonies of these animals. We examined DNA sequences from one mitochondrial and three nuclear gene loci to assess genetic diversity in the siboglinid tubeworm, Riftia pachyptila, a widely distributed constituent of vents along the East Pacific Rise and Galápagos Rift. Results Genetic differentiation (FST) among populations increased with geographical distances, as expected under a linear stepping-stone model of dispersal. Low levels of DNA sequence diversity occurred at all four loci, allowing us to exclude the hypothesis that an idiosyncratic selective sweep eliminated mitochondrial diversity alone. Total gene diversity declined with tectonic spreading rates. The southernmost populations, which are subjected to superfast spreading rates and high probabilities of extinction, are relatively homogenous genetically. Conclusions Compared to other vent species, DNA sequence diversity is extremely low in R. pachyptila. Though its dispersal abilities appear to be effective, the low diversity, particularly in southern hemisphere populations, is consistent with frequent local extinction and (re)colonization events. PMID:21489281

Genetic diversity and demographic instability in Riftia pachyptila tubeworms from eastern Pacific hydrothermal vents

USGS Publications Warehouse

Coykendall, D.K.; Johnson, S.B.; Karl, S.A.; Lutz, R.A.; Vrijenhoek, R.C.

2011-01-01

Background: Deep-sea hydrothermal vent animals occupy patchy and ephemeral habitats supported by chemosynthetic primary production. Volcanic and tectonic activities controlling the turnover of these habitats contribute to demographic instability that erodes genetic variation within and among colonies of these animals. We examined DNA sequences from one mitochondrial and three nuclear gene loci to assess genetic diversity in the siboglinid tubeworm, Riftia pachyptila, a widely distributed constituent of vents along the East Pacific Rise and Galpagos Rift. Results: Genetic differentiation (FST) among populations increased with geographical distances, as expected under a linear stepping-stone model of dispersal. Low levels of DNA sequence diversity occurred at all four loci, allowing us to exclude the hypothesis that an idiosyncratic selective sweep eliminated mitochondrial diversity alone. Total gene diversity declined with tectonic spreading rates. The southernmost populations, which are subjected to superfast spreading rates and high probabilities of extinction, are relatively homogenous genetically. Conclusions: Compared to other vent species, DNA sequence diversity is extremely low in R. pachyptila. Though its dispersal abilities appear to be effective, the low diversity, particularly in southern hemisphere populations, is consistent with frequent local extinction and (re)colonization events. ?? 2011 Coykendall et al; licensee BioMed Central Ltd.

Analysis of genetic diversity using SNP markers in oat

USDA-ARS?s Scientific Manuscript database

A large-scale single nucleotide polymorphism (SNP) discovery was carried out in cultivated oat using Roche 454 sequencing methods. DNA sequences were generated from cDNAs originating from a panel of 20 diverse oat cultivars, and from Diversity Array Technology (DArT) genomic complexity reductions fr...

Diverse molecular signatures for ribosomally ‘active’ Perkinsea in marine sediments

PubMed Central

2014-01-01

Background Perkinsea are a parasitic lineage within the eukaryotic superphylum Alveolata. Recent studies making use of environmental small sub-unit ribosomal RNA gene (SSU rDNA) sequencing methodologies have detected a significant diversity and abundance of Perkinsea-like phylotypes in freshwater environments. In contrast only a few Perkinsea environmental sequences have been retrieved from marine samples and only two groups of Perkinsea have been cultured and morphologically described and these are parasites of marine molluscs or marine protists. These two marine groups form separate and distantly related phylogenetic clusters, composed of closely related lineages on SSU rDNA trees. Here, we test the hypothesis that Perkinsea are a hitherto under-sampled group in marine environments. Using 454 diversity ‘tag’ sequencing we investigate the diversity and distribution of these protists in marine sediments and water column samples taken from the Deep Chlorophyll Maximum (DCM) and sub-surface using both DNA and RNA as the source template and sampling four European offshore locations. Results We detected the presence of 265 sequences branching with known Perkinsea, the majority of them recovered from marine sediments. Moreover, 27% of these sequences were sampled from RNA derived cDNA libraries. Phylogenetic analyses classify a large proportion of these sequences into 38 cluster groups (including 30 novel marine cluster groups), which share less than 97% sequence similarity suggesting this diversity encompasses a range of biologically and ecologically distinct organisms. Conclusions These results demonstrate that the Perkinsea lineage is considerably more diverse than previously detected in marine environments. This wide diversity of Perkinsea-like protists is largely retrieved in marine sediment with a significant proportion detected in RNA derived libraries suggesting this diversity represents ribosomally ‘active’ and intact cells. Given the phylogenetic range of hosts infected by known Perkinsea parasites, these data suggest that Perkinsea either play a significant but hitherto unrecognized role as parasites in marine sediments and/or members of this group are present in the marine sediment possibly as part of the ‘seed bank’ microbial community. PMID:24779375

The Epigenomic Landscape of Prokaryotes

DOE PAGES

Blow, Matthew J.; Clark, Tyson A.; Daum, Chris G.; ...

2016-02-12

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities ofmore » 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active ‘orphan’ MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.« less

The Epigenomic Landscape of Prokaryotes

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

Blow, Matthew J.; Clark, Tyson A.; Daum, Chris G.

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities ofmore » 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active ‘orphan’ MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.« less

Scaling up discovery of hidden diversity in fungi: impacts of barcoding approaches.

PubMed

Yahr, Rebecca; Schoch, Conrad L; Dentinger, Bryn T M

2016-09-05

The fungal kingdom is a hyperdiverse group of multicellular eukaryotes with profound impacts on human society and ecosystem function. The challenge of documenting and describing fungal diversity is exacerbated by their typically cryptic nature, their ability to produce seemingly unrelated morphologies from a single individual and their similarity in appearance to distantly related taxa. This multiplicity of hurdles resulted in the early adoption of DNA-based comparisons to study fungal diversity, including linking curated DNA sequence data to expertly identified voucher specimens. DNA-barcoding approaches in fungi were first applied in specimen-based studies for identification and discovery of taxonomic diversity, but are now widely deployed for community characterization based on sequencing of environmental samples. Collectively, fungal barcoding approaches have yielded important advances across biological scales and research applications, from taxonomic, ecological, industrial and health perspectives. A major outstanding issue is the growing problem of 'sequences without names' that are somewhat uncoupled from the traditional framework of fungal classification based on morphology and preserved specimens. This review summarizes some of the most significant impacts of fungal barcoding, its limitations, and progress towards the challenge of effective utilization of the exponentially growing volume of data gathered from high-throughput sequencing technologies.This article is part of the themed issue 'From DNA barcodes to biomes'. © 2016 The Authors.

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.

PubMed

Borgogno, María V; Monti, Mariela R; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E; Pezza, Roberto J

2016-03-04

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange*

PubMed Central

Borgogno, María V.; Monti, Mariela R.; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E.; Pezza, Roberto J.

2016-01-01

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. PMID:26709229

Assessing the utility of eDNA as a tool to survey reef-fish communities in the Red Sea

NASA Astrophysics Data System (ADS)

DiBattista, Joseph D.; Coker, Darren J.; Sinclair-Taylor, Tane H.; Stat, Michael; Berumen, Michael L.; Bunce, Michael

2017-12-01

Relatively small volumes of water may contain sufficient environmental DNA (eDNA) to detect target aquatic organisms via genetic sequencing. We therefore assessed the utility of eDNA to document the diversity of coral reef fishes in the central Red Sea. DNA from seawater samples was extracted, amplified using fish-specific 16S mitochondrial DNA primers, and sequenced using a metabarcoding workflow. DNA sequences were assigned to taxa using available genetic repositories or custom genetic databases generated from reference fishes. Our approach revealed a diversity of conspicuous, cryptobenthic, and commercially relevant reef fish at the genus level, with select genera in the family Labridae over-represented. Our approach, however, failed to capture a significant fraction of the fish fauna known to inhabit the Red Sea, which we attribute to limited spatial sampling, amplification stochasticity, and an apparent lack of sequencing depth. Given an increase in fish species descriptions, completeness of taxonomic checklists, and improvement in species-level assignment with custom genetic databases as shown here, we suggest that the Red Sea region may be ideal for further testing of the eDNA approach.

A communal catalogue reveals Earth's multiscale microbial diversity.

PubMed

Thompson, Luke R; Sanders, Jon G; McDonald, Daniel; Amir, Amnon; Ladau, Joshua; Locey, Kenneth J; Prill, Robert J; Tripathi, Anupriya; Gibbons, Sean M; Ackermann, Gail; Navas-Molina, Jose A; Janssen, Stefan; Kopylova, Evguenia; Vázquez-Baeza, Yoshiki; González, Antonio; Morton, James T; Mirarab, Siavash; Zech Xu, Zhenjiang; Jiang, Lingjing; Haroon, Mohamed F; Kanbar, Jad; Zhu, Qiyun; Jin Song, Se; Kosciolek, Tomasz; Bokulich, Nicholas A; Lefler, Joshua; Brislawn, Colin J; Humphrey, Gregory; Owens, Sarah M; Hampton-Marcell, Jarrad; Berg-Lyons, Donna; McKenzie, Valerie; Fierer, Noah; Fuhrman, Jed A; Clauset, Aaron; Stevens, Rick L; Shade, Ashley; Pollard, Katherine S; Goodwin, Kelly D; Jansson, Janet K; Gilbert, Jack A; Knight, Rob

2017-11-23

Our growing awareness of the microbial world's importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth's microbial diversity.

DNA sequences of Pima (Gossypium barbadense L.) cotton leaf for examining transcriptome diversity and SNP biomarker discovery

USDA-ARS?s Scientific Manuscript database

As an initial step to explore the transcriptome genetic diversity and to discover single nucleotide polymorphic (SNP)-biomarkers for marker assisted breeding within Pima (Gossypium barbadense L.) cotton, leaves from 25 day plants of three diverse genotypes were used to develop cDNA libraries. Using ...

Low mitochondrial DNA diversity of Japanese Polled and Kuchinoshima feral cattle.

PubMed

Mannen, Hideyuki; Yonesaka, Riku; Noda, Aoi; Shimogiri, Takeshi; Oshima, Ichiro; Katahira, Kiyomi; Kanemaki, Misao; Kunieda, Tetsuo; Inayoshi, Yousuke; Mukai, Fumio; Sasazaki, Shinji

2017-05-01

This study aims to estimate the mitochondrial genetic diversity and structure of Japanese Polled and Kuchinoshima feral cattle, which are maintained in small populations. We determined the mitochondrial DMA (mtDNA) displacement loop (D-loop) sequences for both cattle populations and analyzed these in conjunction with previously published data from Northeast Asian cattle populations. Our findings showed that Japanese native cattle have a predominant, Asian-specific mtDNA haplogroup T4 with high frequencies (0.43-0.81). This excluded Kuchinoshima cattle (32 animals), which had only one mtDNA haplotype belonging to the haplogroup T3. Japanese Polled showed relatively lower mtDNA diversity in the average sequence divergence (0.0020) than other Wagyu breeds (0.0036-0.0047). Japanese Polled have been maintained in a limited area of Yamaguchi, and the population size is now less than 200. Therefore, low mtDNA diversity in the Japanese Polled could be explained by the decreasing population size in the last three decades. We found low mtDNA diversity in both Japanese Polled and Kuchinoshima cattle. The genetic information obtained in this study will be useful for maintaining these populations and for understanding the origin of Japanese native cattle. © 2016 Japanese Society of Animal Science.

De novo assembly of mitochondrial genomes provides insights into genetic diversity and molecular evolution in wild boars and domestic pigs.

PubMed

Ni, Pan; Bhuiyan, Ali Akbar; Chen, Jian-Hai; Li, Jingjin; Zhang, Cheng; Zhao, Shuhong; Du, Xiaoyong; Li, Hua; Yu, Hui; Liu, Xiangdong; Li, Kui

2018-06-01

Up to date, the scarcity of publicly available complete mitochondrial sequences for European wild pigs hampers deeper understanding about the genetic changes following domestication. Here, we have assembled 26 de novo mtDNA sequences of European wild boars from next generation sequencing (NGS) data and downloaded 174 complete mtDNA sequences to assess the genetic relationship, nucleotide diversity, and selection. The Bayesian consensus tree reveals the clear divergence between the European and Asian clade and a very small portion (10 out of 200 samples) of maternal introgression. The overall nucleotides diversities of the mtDNA sequences have been reduced following domestication. Interestingly, the selection efficiencies in both European and Asian domestic pigs are reduced, probably caused by changes in both selection constraints and maternal population size following domestication. This study suggests that de novo assembled mitogenomes can be a great boon to uncover the genetic turnover following domestication. Further investigation is warranted to include more samples from the ever-increasing amounts of NGS data to help us to better understand the process of domestication.

Low DNA Sequence Diversity of the Intergenic Spacer 1 Region in the Human Skin Commensal Fungi Malassezia sympodialis and M. dermatis Isolated from Patients with Malassezia-Associated Skin Diseases and Healthy Subjects.

PubMed

Cho, Otomi; Sugita, Takashi

2016-12-01

As DNA sequences of the intergenic spacer (IGS) region in the rRNA gene show remarkable intraspecies diversity compared with the small subunit, large subunit, and internal transcribed spacer region, the IGS region has been used as an epidemiological tool in studies on Malassezia globosa and M. restricta, which are responsible for the exacerbation of atopic dermatitis (AD) and seborrheic dermatitis (SD). However, the IGS regions of M. sympodialis and M. dermatis obtained from the skin of patients with AD and SD, as well as healthy subjects, lacked sequence diversity. Of the 105 M. sympodialis strains and the 40 M. dermatis strains, the sequences of 103 (98.1 %) and 39 (97.5 %), respectively, were identical. Thus, given the lack of intraspecies diversity in the IGS regions of M. sympodialis and M. dermatis, studies of the diversity of these species should be performed using appropriate genes and not the IGS.

A “Rosetta Stone” for metazoan zooplankton: DNA barcode analysis of species diversity of the Sargasso Sea (Northwest Atlantic Ocean)

NASA Astrophysics Data System (ADS)

Bucklin, Ann; Ortman, Brian D.; Jennings, Robert M.; Nigro, Lisa M.; Sweetman, Christopher J.; Copley, Nancy J.; Sutton, Tracey; Wiebe, Peter H.

2010-12-01

Species diversity of the metazoan holozooplankton assemblage of the Sargasso Sea, Northwest Atlantic Ocean, was examined through coordinated morphological taxonomic identification of species and DNA sequencing of a ˜650 base-pair region of mitochondrial cytochrome oxidase I (mtCOI) as a DNA barcode (i.e., short sequence for species recognition and discrimination). Zooplankton collections were made from the surface to 5,000 meters during April, 2006 on the R/V R.H. Brown. Samples were examined by a ship-board team of morphological taxonomists; DNA barcoding was carried out in both ship-board and land-based DNA sequencing laboratories. DNA barcodes were determined for a total of 297 individuals of 175 holozooplankton species in four phyla, including: Cnidaria (Hydromedusae, 4 species; Siphonophora, 47); Arthropoda (Amphipoda, 10; Copepoda, 34; Decapoda, 9; Euphausiacea, 10; Mysidacea, 1; Ostracoda, 27); and Mollusca (Cephalopoda, 8; Heteropoda, 6; Pteropoda, 15); and Chaetognatha (4). Thirty species of fish (Teleostei) were also barcoded. For all seven zooplankton groups for which sufficient data were available, Kimura-2-Parameter genetic distances were significantly lower between individuals of the same species (mean=0.0114; S.D. 0.0117) than between individuals of different species within the same group (mean=0.3166; S.D. 0.0378). This difference, known as the barcode gap, ensures that mtCOI sequences are reliable characters for species identification for the oceanic holozooplankton assemblage. In addition, DNA barcodes allow recognition of new or undescribed species, reveal cryptic species within known taxa, and inform phylogeographic and population genetic studies of geographic variation. The growing database of "gold standard" DNA barcodes serves as a Rosetta Stone for marine zooplankton, providing the key for decoding species diversity by linking species names, morphology, and DNA sequence variation. In light of the pivotal position of zooplankton in ocean food webs, their usefulness as rapid responders to environmental change, and the increasing scarcity of taxonomists, the use of DNA barcodes is an important and useful approach for rapid analysis of species diversity and distribution in the pelagic community.

Genetic diversity, genetic structure and demographic history of Cycas simplicipinna (Cycadaceae) assessed by DNA sequences and SSR markers

PubMed Central

2014-01-01

Background Cycas simplicipinna (T. Smitinand) K. Hill. (Cycadaceae) is an endangered species in China. There were seven populations and 118 individuals that we could collect were genotyped in this study. Here, we assessed the genetic diversity, genetic structure and demographic history of this species. Results Analyses of data of DNA sequences (two maternally inherited intergenic spacers of chloroplast, cpDNA and one biparentally inherited internal transcribed spacer region ITS4-ITS5, nrDNA) and sixteen microsatellite loci (SSR) were conducted in the species. Of the 118 samples, 86 individuals from the seven populations were used for DNA sequencing and 115 individuals from six populations were used for the microsatellite study. We found high genetic diversity at the species level, low genetic diversity within each of the seven populations and high genetic differentiation among the populations. There was a clear genetic structure within populations of C. simplicipinna. A demographic history inferred from DNA sequencing data indicates that C. simplicipinna experienced a recent population contraction without retreating to a common refugium during the last glacial period. The results derived from SSR data also showed that C. simplicipinna underwent past effective population contraction, likely during the Pleistocene. Conclusions Some genetic features of C. simplicipinna such as having high genetic differentiation among the populations, a clear genetic structure and a recent population contraction could provide guidelines for protecting this endangered species from extinction. Furthermore, the genetic features with population dynamics of the species in our study would help provide insights and guidelines for protecting other endangered species effectively. PMID:25016306

Algorithms for optimizing cross-overs in DNA shuffling.

PubMed

He, Lu; Friedman, Alan M; Bailey-Kellogg, Chris

2012-03-21

DNA shuffling generates combinatorial libraries of chimeric genes by stochastically recombining parent genes. The resulting libraries are subjected to large-scale genetic selection or screening to identify those chimeras with favorable properties (e.g., enhanced stability or enzymatic activity). While DNA shuffling has been applied quite successfully, it is limited by its homology-dependent, stochastic nature. Consequently, it is used only with parents of sufficient overall sequence identity, and provides no control over the resulting chimeric library. This paper presents efficient methods to extend the scope of DNA shuffling to handle significantly more diverse parents and to generate more predictable, optimized libraries. Our CODNS (cross-over optimization for DNA shuffling) approach employs polynomial-time dynamic programming algorithms to select codons for the parental amino acids, allowing for zero or a fixed number of conservative substitutions. We first present efficient algorithms to optimize the local sequence identity or the nearest-neighbor approximation of the change in free energy upon annealing, objectives that were previously optimized by computationally-expensive integer programming methods. We then present efficient algorithms for more powerful objectives that seek to localize and enhance the frequency of recombination by producing "runs" of common nucleotides either overall or according to the sequence diversity of the resulting chimeras. We demonstrate the effectiveness of CODNS in choosing codons and allocating substitutions to promote recombination between parents targeted in earlier studies: two GAR transformylases (41% amino acid sequence identity), two very distantly related DNA polymerases, Pol X and β (15%), and beta-lactamases of varying identity (26-47%). Our methods provide the protein engineer with a new approach to DNA shuffling that supports substantially more diverse parents, is more deterministic, and generates more predictable and more diverse chimeric libraries.

Diversity of halophilic archaea from six hypersaline environments in Turkey.

PubMed

Ozcan, Birgul; Ozcengiz, Gulay; Coleri, Arzu; Cokmus, Cumhur

2007-06-01

The diversity of archaeal strains from six hypersaline environments in Turkey was analyzed by comparing their phenotypic characteristics and 16S rDNA sequences. Thirty-three isolates were characterized in terms of their phenotypic properties including morphological and biochemical characteristics, susceptibility to different antibiotics, and total lipid and plasmid contents, and finally compared by 16S rDNA gene sequences. The results showed that all isolates belong to the family Halobacteriaceae. Phylogenetic analyses using approximately 1,388 bp comparisions of 16S rDNA sequences demonstrated that all isolates clustered closely to species belonging to 9 genera, namely Halorubrum (8 isolates), Natrinema (5 isolates), Haloarcula (4 isolates), Natronococcus (4 isolates), Natrialba (4 isolates), Haloferax (3 isolates), Haloterrigena (3 isolates), Halalkalicoccus (1 isolate), and Halomicrobium (1 isolate). The results revealed a high diversity among the isolated halophilic strains and indicated that some of these strains constitute new taxa of extremely halophilic archaea.

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats

PubMed Central

de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

2015-01-01

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. PMID:26481363

Mitochondrial DNA diversity of the Amerindian populations living in the Andean Piedmont of Bolivia: Chimane, Moseten, Aymara and Quechua.

PubMed

Corella, Alfons; Bert, Francesc; Pérez-Pérez, Alejandro; Gené, Manel; Turbón, Daniel

2007-01-01

Chimane, Moseten Aymara and Quechua are Amerindian populations living in the Bolivian Piedmont, a characteristic ecoregion between the eastern slope of the Andean mountains and the Amazonian Llanos de Moxos. In both neighbouring areas, dense and complex societies have developed over the centuries. The Piedmont area is especially interesting from a human peopling perspective since there is no clear evidence regarding the genetic influence and peculiarities of these populations. This land has been used extensively as a territory of economic and cultural exchange between the Andes and Amazonia, however Chimane and Moseten populations have been sufficiently isolated from their neighbour groups to be recognized as distinct populations. Genetic information suggests that evolutionary processes, such as genetic drift, natural selection and genetic admixture have formed the history of the Piedmont populations. The objective of this study is to characterize the genetic diversity of the Piedmont populations, analysing the sequence variability of the HVR-I control region in the mitochondrial DNA (mtDNA). Haplogroup mtDNA data available from the whole of Central and South America were utilized to determine the relationship of the Piedmont populations with other Amerindian populations. Hair pulls were obtained in situ, and DNA from non-related individuals was extracted using a standard Chelex 100 method. A 401 bp DNA fragment of HVR-I region was amplified using standard procedures. Two independent 401 and 328 bp DNA fragments were sequenced separately for each sample. The sequence analyses included mismatch distribution and mean pairwise differences, median network analyses, AMOVA and principal component analyses. The genetic diversity of DNA sequences was measured and compared with other South Amerindian populations. The genetic diversity of 401 nucleotide mtDNA sequences, in the hypervariable Control Region, from positions 16 000-16 400, was characterized in a sample of 46 Amerindians living in the Piedmont area in the Beni Department of Bolivia. The results obtained indicate that the genetic diversity in the area is higher than that observed in other American groups living in much larger areas and despite the reduced size of the studied area the human groups analysed show high levels of inter-group variability. In addition, results show that Amerindian populations living in the Piedmont are genetically more related to those in the Andean than in the Amazonian populations.

Continuous Influx of Genetic Material from Host to Virus Populations

PubMed Central

Gilbert, Clément; Peccoud, Jean; Chateigner, Aurélien; Moumen, Bouziane

2016-01-01

Many genes of large double-stranded DNA viruses have a cellular origin, suggesting that host-to-virus horizontal transfer (HT) of DNA is recurrent. Yet, the frequency of these transfers has never been assessed in viral populations. Here we used ultra-deep DNA sequencing of 21 baculovirus populations extracted from two moth species to show that a large diversity of moth DNA sequences (n = 86) can integrate into viral genomes during the course of a viral infection. The majority of the 86 different moth DNA sequences are transposable elements (TEs, n = 69) belonging to 10 superfamilies of DNA transposons and three superfamilies of retrotransposons. The remaining 17 sequences are moth sequences of unknown nature. In addition to bona fide DNA transposition, we uncover microhomology-mediated recombination as a mechanism explaining integration of moth sequences into viral genomes. Many sequences integrated multiple times at multiple positions along the viral genome. We detected a total of 27,504 insertions of moth sequences in the 21 viral populations and we calculate that on average, 4.8% of viruses harbor at least one moth sequence in these populations. Despite this substantial proportion, no insertion of moth DNA was maintained in any viral population after 10 successive infection cycles. Hence, there is a constant turnover of host DNA inserted into viral genomes each time the virus infects a moth. Finally, we found that at least 21 of the moth TEs integrated into viral genomes underwent repeated horizontal transfers between various insect species, including some lepidopterans susceptible to baculoviruses. Our results identify host DNA influx as a potent source of genetic diversity in viral populations. They also support a role for baculoviruses as vectors of DNA HT between insects, and call for an evaluation of possible gene or TE spread when using viruses as biopesticides or gene delivery vectors. PMID:26829124

Continuous Influx of Genetic Material from Host to Virus Populations.

PubMed

Gilbert, Clément; Peccoud, Jean; Chateigner, Aurélien; Moumen, Bouziane; Cordaux, Richard; Herniou, Elisabeth A

2016-02-01

Many genes of large double-stranded DNA viruses have a cellular origin, suggesting that host-to-virus horizontal transfer (HT) of DNA is recurrent. Yet, the frequency of these transfers has never been assessed in viral populations. Here we used ultra-deep DNA sequencing of 21 baculovirus populations extracted from two moth species to show that a large diversity of moth DNA sequences (n = 86) can integrate into viral genomes during the course of a viral infection. The majority of the 86 different moth DNA sequences are transposable elements (TEs, n = 69) belonging to 10 superfamilies of DNA transposons and three superfamilies of retrotransposons. The remaining 17 sequences are moth sequences of unknown nature. In addition to bona fide DNA transposition, we uncover microhomology-mediated recombination as a mechanism explaining integration of moth sequences into viral genomes. Many sequences integrated multiple times at multiple positions along the viral genome. We detected a total of 27,504 insertions of moth sequences in the 21 viral populations and we calculate that on average, 4.8% of viruses harbor at least one moth sequence in these populations. Despite this substantial proportion, no insertion of moth DNA was maintained in any viral population after 10 successive infection cycles. Hence, there is a constant turnover of host DNA inserted into viral genomes each time the virus infects a moth. Finally, we found that at least 21 of the moth TEs integrated into viral genomes underwent repeated horizontal transfers between various insect species, including some lepidopterans susceptible to baculoviruses. Our results identify host DNA influx as a potent source of genetic diversity in viral populations. They also support a role for baculoviruses as vectors of DNA HT between insects, and call for an evaluation of possible gene or TE spread when using viruses as biopesticides or gene delivery vectors.

454 Pyrosequencing to Describe Microbial Eukaryotic Community Composition, Diversity and Relative Abundance: A Test for Marine Haptophytes

PubMed Central

Egge, Elianne; Bittner, Lucie; Andersen, Tom; Audic, Stéphane; de Vargas, Colomban; Edvardsen, Bente

2013-01-01

Next generation sequencing of ribosomal DNA is increasingly used to assess the diversity and structure of microbial communities. Here we test the ability of 454 pyrosequencing to detect the number of species present, and assess the relative abundance in terms of cell numbers and biomass of protists in the phylum Haptophyta. We used a mock community consisting of equal number of cells of 11 haptophyte species and compared targeting DNA and RNA/cDNA, and two different V4 SSU rDNA haptophyte-biased primer pairs. Further, we tested four different bioinformatic filtering methods to reduce errors in the resulting sequence dataset. With sequencing depth of 11000–20000 reads and targeting cDNA with Haptophyta specific primers Hap454 we detected all 11 species. A rarefaction analysis of expected number of species recovered as a function of sampling depth suggested that minimum 1400 reads were required here to recover all species in the mock community. Relative read abundance did not correlate to relative cell numbers. Although the species represented with the largest biomass was also proportionally most abundant among the reads, there was generally a weak correlation between proportional read abundance and proportional biomass of the different species, both with DNA and cDNA as template. The 454 sequencing generated considerable spurious diversity, and more with cDNA than DNA as template. With initial filtering based only on match with barcode and primer we observed 100-fold more operational taxonomic units (OTUs) at 99% similarity than the number of species present in the mock community. Filtering based on quality scores, or denoising with PyroNoise resulted in ten times more OTU99% than the number of species. Denoising with AmpliconNoise reduced the number of OTU99% to match the number of species present in the mock community. Based on our analyses, we propose a strategy to more accurately depict haptophyte diversity using 454 pyrosequencing. PMID:24069303

PuLSE: Quality control and quantification of peptide sequences explored by phage display libraries.

PubMed

Shave, Steven; Mann, Stefan; Koszela, Joanna; Kerr, Alastair; Auer, Manfred

2018-01-01

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use.

Target Site Recognition by a Diversity-Generating Retroelement

PubMed Central

Guo, Huatao; Tse, Longping V.; Nieh, Angela W.; Czornyj, Elizabeth; Williams, Steven; Oukil, Sabrina; Liu, Vincent B.; Miller, Jeff F.

2011-01-01

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification. PMID:22194701

Metagenomic survey of bacterial diversity in the atmosphere of Mexico City using different sampling methods.

PubMed

Serrano-Silva, N; Calderón-Ezquerro, M C

2018-04-01

The identification of airborne bacteria has traditionally been performed by retrieval in culture media, but the bacterial diversity in the air is underestimated using this method because many bacteria are not readily cultured. Advances in DNA sequencing technology have produced a broad knowledge of genomics and metagenomics, which can greatly improve our ability to identify and study the diversity of airborne bacteria. However, researchers are facing several challenges, particularly the efficient retrieval of low-density microorganisms from the air and the lack of standardized protocols for sample collection and processing. In this study, we tested three methods for sampling bioaerosols - a Durham-type spore trap (Durham), a seven-day recording volumetric spore trap (HST), and a high-throughput 'Jet' spore and particle sampler (Jet) - and recovered metagenomic DNA for 16S rDNA sequencing. Samples were simultaneously collected with the three devices during one week, and the sequencing libraries were analyzed. A simple and efficient method for collecting bioaerosols and extracting good quality DNA for high-throughput sequencing was standardized. The Durham sampler collected preferentially Cyanobacteria, the HST Actinobacteria, Proteobacteria and Firmicutes, and the Jet mainly Proteobacteria and Firmicutes. The HST sampler collected the largest amount of airborne bacterial diversity. More experiments are necessary to select the right sampler, depending on study objectives, which may require monitoring and collecting specific airborne bacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Detection and diversity analysis of rumen methanogens in the co-cultures with anaerobic fungi].

PubMed

Cheng, Yan-fen; Mao, Sheng-yong; Pei, Cai-xia; Liu, Jian-xin; Zhu, Wei-yun

2006-12-01

Rumen methanogen diversity in the co-cultures with anaerobic fungi from goat rumen was analyzed. Mix-cultures of anaerobic fungi and methanogens were obtained from goat rumen using anaerobic fungal medium and the addition of penicillin and streptomycin and then subcultured 62 times by transferring cultures every 3 - 4d. Total DNA from the original rumen fluid and subcultured fungal cultures was used for PCR/DGGE and RFLP analysis. 16S rDNA of clones corresponding to representative OTUs were sequenced. Results showed that the diversity index (Shannon index) of the methanogens generated from DGGE profiles reduced from 1.32 to 0.99 from rumen fluid to fungal culture after 45 subculturing, with the lowest similarity of DGGE profiles at 34.7%. The Shannon index increased from 0.99 to 1.15 from the fungal culture after 45 subculturing to that after 62 subculturing, with the lowest similarity at 89.2% . A total of 5 OTUs were obtained from 69. clones using RFLP analysis and six clones representing the 5 OTUs respectively were sequenced. Of the 5 OTUs, three had their cloned 16S rDNA sequences most closely related to uncultured archaeal symbiont PA202 with the same similarity of 95 %, but had not closely related to any identified culturable methanogen. The rest two OTUs had their cloned 16S rDNA sequences sharing the same closest relative, uncultured rumen methanogen 956, with the same similarity of 97% .Their 16S rDNA sequences of these two OTUs also showed 97% similar to the closest identified culturable methanogen Methanobrevibacter sp. NT7. In conclusion, diverse yet unidentified rumen methanogen species exist in the co-cultures with anaerobic fungi isolated from the goat rumen.

Genome sequence diversity and clues to the evolution of variola (smallpox) virus.

PubMed

Esposito, Joseph J; Sammons, Scott A; Frace, A Michael; Osborne, John D; Olsen-Rasmussen, Melissa; Zhang, Ming; Govil, Dhwani; Damon, Inger K; Kline, Richard; Laker, Miriam; Li, Yu; Smith, Geoffrey L; Meyer, Hermann; Leduc, James W; Wohlhueter, Robert M

2006-08-11

Comparative genomics of 45 epidemiologically varied variola virus isolates from the past 30 years of the smallpox era indicate low sequence diversity, suggesting that there is probably little difference in the isolates' functional gene content. Phylogenetic clustering inferred three clades coincident with their geographical origin and case-fatality rate; the latter implicated putative proteins that mediate viral virulence differences. Analysis of the viral linear DNA genome suggests that its evolution involved direct descent and DNA end-region recombination events. Knowing the sequences will help understand the viral proteome and improve diagnostic test precision, therapeutics, and systems for their assessment.

Diversity and distribution of single-stranded DNA phages in the North Atlantic Ocean

PubMed Central

Tucker, Kimberly P; Parsons, Rachel; Symonds, Erin M; Breitbart, Mya

2011-01-01

Knowledge of marine phages is highly biased toward double-stranded DNA (dsDNA) phages; however, recent metagenomic surveys have also identified single-stranded DNA (ssDNA) phages in the oceans. Here, we describe two complete ssDNA phage genomes that were reconstructed from a viral metagenome from 80 m depth at the Bermuda Atlantic Time-series Study (BATS) site in the northwestern Sargasso Sea and examine their spatial and temporal distributions. Both genomes (SARssφ1 and SARssφ2) exhibited similarity to known phages of the Microviridae family in terms of size, GC content, genome organization and protein sequence. PCR amplification of the replication initiation protein (Rep) gene revealed narrow and distinct depth distributions for the newly described ssDNA phages within the upper 200 m of the water column at the BATS site. Comparison of Rep gene sequences obtained from the BATS site over time revealed changes in the diversity of ssDNA phages over monthly time scales, although some nearly identical sequences were recovered from samples collected 4 years apart. Examination of ssDNA phage diversity along transects through the North Atlantic Ocean revealed a positive correlation between genetic distance and geographic distance between sampling sites. Together, the data suggest fundamental differences between the distribution of these ssDNA phages and the distribution of known marine dsDNA phages, possibly because of differences in host range, host distribution, virion stability, or viral evolution mechanisms and rates. Future work needs to elucidate the host ranges for oceanic ssDNA phages and determine their ecological roles in the marine ecosystem. PMID:21124487

Genetic diversity of Burkholderia (Proteobacteria) species from the Caatinga and Atlantic rainforest biomes in Bahia, Brazil.

PubMed

Santini, A C; Santos, H R M; Gross, E; Corrêa, R X

2013-03-11

The genus Burkholderia (β-Proteobacteria) currently comprises more than 60 species, including parasites, symbionts and free-living organisms. Several new species of Burkholderia have recently been described showing a great diversity of phenotypes. We examined the diversity of Burkholderia spp in environmental samples collected from Caatinga and Atlantic rainforest biomes of Bahia, Brazil. Legume nodules were collected from five locations, and 16S rDNA and recA genes of the isolated microorganisms were analyzed. Thirty-three contigs of 16S rRNA genes and four contigs of the recA gene related to the genus Burkholderia were obtained. The genetic dissimilarity of the strains ranged from 0 to 2.5% based on 16S rDNA analysis, indicating two main branches: one distinct branch of the dendrogram for the B. cepacia complex and another branch that rendered three major groups, partially reflecting host plants and locations. A dendrogram designed with sequences of this research and those designed with sequences of Burkholderia-type strains and the first hit BLAST had similar topologies. A dendrogram similar to that constructed by analysis of 16S rDNA was obtained using sequences of the fragment of the recA gene. The 16S rDNA sequences enabled sufficient identification of relevant similarities and groupings amongst isolates and the sequences that we obtained. Only 6 of the 33 isolates analyzed via 16S rDNA sequencing showed high similarity with the B. cepacia complex. Thus, over 3/4 of the isolates have potential for biotechnological applications.

DNA-DNA hybridization values and their relationship to whole-genome sequence similarities.

PubMed

Goris, Johan; Konstantinidis, Konstantinos T; Klappenbach, Joel A; Coenye, Tom; Vandamme, Peter; Tiedje, James M

2007-01-01

DNA-DNA hybridization (DDH) values have been used by bacterial taxonomists since the 1960s to determine relatedness between strains and are still the most important criterion in the delineation of bacterial species. Since the extent of hybridization between a pair of strains is ultimately governed by their respective genomic sequences, we examined the quantitative relationship between DDH values and genome sequence-derived parameters, such as the average nucleotide identity (ANI) of common genes and the percentage of conserved DNA. A total of 124 DDH values were determined for 28 strains for which genome sequences were available. The strains belong to six important and diverse groups of bacteria for which the intra-group 16S rRNA gene sequence identity was greater than 94 %. The results revealed a close relationship between DDH values and ANI and between DNA-DNA hybridization and the percentage of conserved DNA for each pair of strains. The recommended cut-off point of 70 % DDH for species delineation corresponded to 95 % ANI and 69 % conserved DNA. When the analysis was restricted to the protein-coding portion of the genome, 70 % DDH corresponded to 85 % conserved genes for a pair of strains. These results reveal extensive gene diversity within the current concept of "species". Examination of reciprocal values indicated that the level of experimental error associated with the DDH method is too high to reveal the subtle differences in genome size among the strains sampled. It is concluded that ANI can accurately replace DDH values for strains for which genome sequences are available.

An evolution based biosensor receptor DNA sequence generation algorithm.

PubMed

Kim, Eungyeong; Lee, Malrey; Gatton, Thomas M; Lee, Jaewan; Zang, Yupeng

2010-01-01

A biosensor is composed of a bioreceptor, an associated recognition molecule, and a signal transducer that can selectively detect target substances for analysis. DNA based biosensors utilize receptor molecules that allow hybridization with the target analyte. However, most DNA biosensor research uses oligonucleotides as the target analytes and does not address the potential problems of real samples. The identification of recognition molecules suitable for real target analyte samples is an important step towards further development of DNA biosensors. This study examines the characteristics of DNA used as bioreceptors and proposes a hybrid evolution-based DNA sequence generating algorithm, based on DNA computing, to identify suitable DNA bioreceptor recognition molecules for stable hybridization with real target substances. The Traveling Salesman Problem (TSP) approach is applied in the proposed algorithm to evaluate the safety and fitness of the generated DNA sequences. This approach improves efficiency and stability for enhanced and variable-length DNA sequence generation and allows extension to generation of variable-length DNA sequences with diverse receptor recognition requirements.

Comprehensive Survey of Genetic Diversity in Chloroplast Genomes and 45S nrDNAs within Panax ginseng Species

PubMed Central

Kim, Kyunghee; Lee, Sang-Choon; Lee, Junki; Lee, Hyun Oh; Joh, Ho Jun; Kim, Nam-Hoon; Park, Hyun-Seung; Yang, Tae-Jin

2015-01-01

We report complete sequences of chloroplast (cp) genome and 45S nuclear ribosomal DNA (45S nrDNA) for 11 Panax ginseng cultivars. We have obtained complete sequences of cp and 45S nrDNA, the representative barcoding target sequences for cytoplasm and nuclear genome, respectively, based on low coverage NGS sequence of each cultivar. The cp genomes sizes ranged from 156,241 to 156,425 bp and the major size variation was derived from differences in copy number of tandem repeats in the ycf1 gene and in the intergenic regions of rps16-trnUUG and rpl32-trnUAG. The complete 45S nrDNA unit sequences were 11,091 bp, representing a consensus single transcriptional unit with an intergenic spacer region. Comparative analysis of these sequences as well as those previously reported for three Chinese accessions identified very rare but unique polymorphism in the cp genome within P. ginseng cultivars. There were 12 intra-species polymorphisms (six SNPs and six InDels) among 14 cultivars. We also identified five SNPs from 45S nrDNA of 11 Korean ginseng cultivars. From the 17 unique informative polymorphic sites, we developed six reliable markers for analysis of ginseng diversity and cultivar authentication. PMID:26061692

GENETIC STRUCTURE OF CREEK CHUB (SEMOTILUS ATROMACULATUS) POPULATIONS IN COAL MINING-IMPACTED AREAS OF THE EASTERN UNITED STATES, AS DETERMINED BY MTDNA SEQUENCING AND AFLP ANALYSIS

EPA Science Inventory

Analysis of intraspecific patterns in genetic diversity of stream fishes provides a potentially powerful method for assessing the status and trends in the condition of aquatic ecosystems. We analyzed mitochondrial DNA (mtDNA) sequences (590 bases of cytochrome B) and nuclear DNA...

Mitochondrial DNA and Y-chromosomal diversity in ancient populations of domestic sheep (Ovis aries) in Finland: comparison with contemporary sheep breeds.

PubMed

Niemi, Marianna; Bläuer, Auli; Iso-Touru, Terhi; Nyström, Veronica; Harjula, Janne; Taavitsainen, Jussi-Pekka; Storå, Jan; Lidén, Kerstin; Kantanen, Juha

2013-01-22

Several molecular and population genetic studies have focused on the native sheep breeds of Finland. In this work, we investigated their ancestral sheep populations from Iron Age, Medieval and Post-Medieval periods by sequencing a partial mitochondrial DNA D-loop and the 5'-promoter region of the SRY gene. We compared the maternal (mitochondrial DNA haplotypes) and paternal (SNP oY1) genetic diversity of ancient sheep in Finland with modern domestic sheep populations in Europe and Asia to study temporal changes in genetic variation and affinities between ancient and modern populations. A 523-bp mitochondrial DNA sequence was successfully amplified for 26 of 36 sheep ancient samples i.e. five, seven and 14 samples representative of Iron Age, Medieval and Post-Medieval sheep, respectively. Genetic diversity was analyzed within the cohorts. This ancient dataset was compared with present-day data consisting of 94 animals from 10 contemporary European breeds and with GenBank DNA sequence data to carry out a haplotype sharing analysis. Among the 18 ancient mitochondrial DNA haplotypes identified, 14 were present in the modern breeds. Ancient haplotypes were assigned to the highly divergent ovine haplogroups A and B, haplogroup B being the major lineage within the cohorts. Only two haplotypes were detected in the Iron Age samples, while the genetic diversity of the Medieval and Post-Medieval cohorts was higher. For three of the ancient DNA samples, Y-chromosome SRY gene sequences were amplified indicating that they originated from rams. The SRY gene of these three ancient ram samples contained SNP G-oY1, which is frequent in modern north-European sheep breeds. Our study did not reveal any sign of major population replacement of native sheep in Finland since the Iron Age. Variations in the availability of archaeological remains may explain differences in genetic diversity estimates and patterns within the cohorts rather than demographic events that occurred in the past. Our ancient DNA results fit well with the genetic context of domestic sheep as determined by analyses of modern north-European sheep breeds.

Mitochondrial DNA and Y-chromosomal diversity in ancient populations of domestic sheep (Ovis aries) in Finland: comparison with contemporary sheep breeds

PubMed Central

2013-01-01

Background Several molecular and population genetic studies have focused on the native sheep breeds of Finland. In this work, we investigated their ancestral sheep populations from Iron Age, Medieval and Post-Medieval periods by sequencing a partial mitochondrial DNA D-loop and the 5’-promoter region of the SRY gene. We compared the maternal (mitochondrial DNA haplotypes) and paternal (SNP oY1) genetic diversity of ancient sheep in Finland with modern domestic sheep populations in Europe and Asia to study temporal changes in genetic variation and affinities between ancient and modern populations. Results A 523-bp mitochondrial DNA sequence was successfully amplified for 26 of 36 sheep ancient samples i.e. five, seven and 14 samples representative of Iron Age, Medieval and Post-Medieval sheep, respectively. Genetic diversity was analyzed within the cohorts. This ancient dataset was compared with present-day data consisting of 94 animals from 10 contemporary European breeds and with GenBank DNA sequence data to carry out a haplotype sharing analysis. Among the 18 ancient mitochondrial DNA haplotypes identified, 14 were present in the modern breeds. Ancient haplotypes were assigned to the highly divergent ovine haplogroups A and B, haplogroup B being the major lineage within the cohorts. Only two haplotypes were detected in the Iron Age samples, while the genetic diversity of the Medieval and Post-Medieval cohorts was higher. For three of the ancient DNA samples, Y-chromosome SRY gene sequences were amplified indicating that they originated from rams. The SRY gene of these three ancient ram samples contained SNP G-oY1, which is frequent in modern north-European sheep breeds. Conclusions Our study did not reveal any sign of major population replacement of native sheep in Finland since the Iron Age. Variations in the availability of archaeological remains may explain differences in genetic diversity estimates and patterns within the cohorts rather than demographic events that occurred in the past. Our ancient DNA results fit well with the genetic context of domestic sheep as determined by analyses of modern north-European sheep breeds. PMID:23339395

Real-Time DNA Sequencing in the Antarctic Dry Valleys Using the Oxford Nanopore Sequencer

PubMed Central

Johnson, Sarah S.; Zaikova, Elena; Goerlitz, David S.; Bai, Yu; Tighe, Scott W.

2017-01-01

The ability to sequence DNA outside of the laboratory setting has enabled novel research questions to be addressed in the field in diverse areas, ranging from environmental microbiology to viral epidemics. Here, we demonstrate the application of offline DNA sequencing of environmental samples using a hand-held nanopore sequencer in a remote field location: the McMurdo Dry Valleys, Antarctica. Sequencing was performed using a MK1B MinION sequencer from Oxford Nanopore Technologies (ONT; Oxford, United Kingdom) that was equipped with software to operate without internet connectivity. One-direction (1D) genomic libraries were prepared using portable field techniques on DNA isolated from desiccated microbial mats. By adequately insulating the sequencer and laptop, it was possible to run the sequencing protocol for up to 2½ h under arduous conditions. PMID:28337073

The genetic diversity of merozoite surface antigen 1 (MSA-1) among Babesia bovis detected from cattle populations in Thailand, Brazil and Ghana.

PubMed

Nagano, Daisuke; Sivakumar, Thillaiampalam; De De Macedo, Alane Caine Costa; Inpankaew, Tawin; Alhassan, Andy; Igarashi, Ikuo; Yokoyama, Naoaki

2013-11-01

In the present study, we screened blood DNA samples obtained from cattle bred in Brazil (n=164) and Ghana (n=80) for Babesia bovis using a diagnostic PCR assay and found prevalences of 14.6% and 46.3%, respectively. Subsequently, the genetic diversity of B. bovis in Thailand, Brazil and Ghana was analyzed, based on the DNA sequence of merozoite surface antigen-1 (MSA-1). In Thailand, MSA-1 sequences were relatively conserved and found in a single clade of the phylogram, while Brazilian MSA-1 sequences showed high genetic diversity and were dispersed across three different clades. In contrast, the sequences from Ghanaian samples were detected in two different clades, one of which contained only a single Ghanaian sequence. The identities among the MSA-1 sequences from Thailand, Brazil and Ghana were 99.0-100%, 57.5-99.4% and 60.3-100%, respectively, while the similarities among the deduced MSA-1 amino acid sequences within the respective countries were 98.4-100%, 59.4-99.7% and 58.7-100%, respectively. These observations suggested that the genetic diversity of B. bovis based on MSA-1 sequences was higher in Brazil and Ghana than in Thailand. The current data highlight the importance of conducting extensive studies on the genetic diversity of B. bovis before designing immune control strategies in each surveyed country.

Complete Genome Sequences of 38 Gordonia sp. Bacteriophages

PubMed Central

Montgomery, Matthew T.; Bonilla, J. Alfred; Dejong, Randall; Garlena, Rebecca A.; Guerrero Bustamante, Carlos; Klyczek, Karen K.; Russell, Daniel A.; Wertz, John T.; Jacobs-Sera, Deborah; Hatfull, Graham F.

2017-01-01

ABSTRACT We report here the genome sequences of 38 newly isolated bacteriophages using Gordonia terrae 3612 (ATCC 25594) and Gordonia neofelifaecis NRRL59395 as bacterial hosts. All of the phages are double-stranded DNA (dsDNA) tail phages with siphoviral morphologies, with genome sizes ranging from 17,118 bp to 93,843 bp and spanning considerable nucleotide sequence diversity. PMID:28057748

Armillaria phylogeny based on tef-1α sequences suggests ongoing divergent speciation within the boreal floristic kingdom

Treesearch

Ned B. Klopfenstein; John W. Hanna; Amy L. Ross-Davis; Jane E. Stewart; Yuko Ota; Rosario Medel-Ortiz; Miguel Armando Lopez-Ramirez; Ruben Damian Elias-Roman; Dionicio Alvarado-Rosales; Mee-Sook Kim

2013-01-01

Armillaria plays diverse ecological roles in forests worldwide, which has inspired interest in understanding phylogenetic relationships within and among species of this genus. Previous rDNA sequence-based phylogenetic analyses of Armillaria have shown general relationships among widely divergent taxa, but rDNA sequences were not reliable for separating closely related...

GENETIC DIVERSITY OF TYPHA LATIFOLIA (TYPHACEAE) AND THE IMPACT OF POLLUTANTS EXAMINED WITH TANDEM-REPETITIVE DNA PROBES

EPA Science Inventory

Genetic diversity at variable-number-tandem-repeat (VNTR) loci was examined in the common cattail, Typha latifolia (Typhaceae), using three synthetic DNA probes composed of tandemly repeated "core" sequences (GACA, GATA, and GCAC). The principal objectives of this investigation w...

Highly multiplexed targeted DNA sequencing from single nuclei.

PubMed

Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

2016-02-01

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats.

PubMed

de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

2015-11-16

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Significant variance in genetic diversity among populations of Schistosoma haematobium detected using microsatellite DNA loci from a genome-wide database.

PubMed

Glenn, Travis C; Lance, Stacey L; McKee, Anna M; Webster, Bonnie L; Emery, Aidan M; Zerlotini, Adhemar; Oliveira, Guilherme; Rollinson, David; Faircloth, Brant C

2013-10-17

Urogenital schistosomiasis caused by Schistosoma haematobium is widely distributed across Africa and is increasingly being targeted for control. Genome sequences and population genetic parameters can give insight into the potential for population- or species-level drug resistance. Microsatellite DNA loci are genetic markers in wide use by Schistosoma researchers, but there are few primers available for S. haematobium. We sequenced 1,058,114 random DNA fragments from clonal cercariae collected from a snail infected with a single Schistosoma haematobium miracidium. We assembled and aligned the S. haematobium sequences to the genomes of S. mansoni and S. japonicum, identifying microsatellite DNA loci across all three species and designing primers to amplify the loci in S. haematobium. To validate our primers, we screened 32 randomly selected primer pairs with population samples of S. haematobium. We designed >13,790 primer pairs to amplify unique microsatellite loci in S. haematobium, (available at http://www.cebio.org/projetos/schistosoma-haematobium-genome). The three Schistosoma genomes contained similar overall frequencies of microsatellites, but the frequency and length distributions of specific motifs differed among species. We identified 15 primer pairs that amplified consistently and were easily scored. We genotyped these 15 loci in S. haematobium individuals from six locations: Zanzibar had the highest levels of diversity; Malawi, Mauritius, Nigeria, and Senegal were nearly as diverse; but the sample from South Africa was much less diverse. About half of the primers in the database of Schistosoma haematobium microsatellite DNA loci should yield amplifiable and easily scored polymorphic markers, thus providing thousands of potential markers. Sequence conservation among S. haematobium, S. japonicum, and S. mansoni is relatively high, thus it should now be possible to identify markers that are universal among Schistosoma species (i.e., using DNA sequences conserved among species), as well as other markers that are specific to species or species-groups (i.e., using DNA sequences that differ among species). Full genome-sequencing of additional species and specimens of S. haematobium, S. japonicum, and S. mansoni is desirable to better characterize differences within and among these species, to develop additional genetic markers, and to examine genes as well as conserved non-coding elements associated with drug resistance.

Early history of European domestic cattle as revealed by ancient DNA.

PubMed

Bollongino, R; Edwards, C J; Alt, K W; Burger, J; Bradley, D G

2006-03-22

We present an extensive ancient DNA analysis of mainly Neolithic cattle bones sampled from archaeological sites along the route of Neolithic expansion, from Turkey to North-Central Europe and Britain. We place this first reasonable population sample of Neolithic cattle mitochondrial DNA sequence diversity in context to illustrate the continuity of haplotype variation patterns from the first European domestic cattle to the present. Interestingly, the dominant Central European pattern, a starburst phylogeny around the modal sequence, T3, has a Neolithic origin, and the reduced diversity within this cluster in the ancient samples accords with their shorter history of post-domestic accumulation of mutation.

Hybrid selection for sequencing pathogen genomes from clinical samples

PubMed Central

2011-01-01

We have adapted a solution hybrid selection protocol to enrich pathogen DNA in clinical samples dominated by human genetic material. Using mock mixtures of human and Plasmodium falciparum malaria parasite DNA as well as clinical samples from infected patients, we demonstrate an average of approximately 40-fold enrichment of parasite DNA after hybrid selection. This approach will enable efficient genome sequencing of pathogens from clinical samples, as well as sequencing of endosymbiotic organisms such as Wolbachia that live inside diverse metazoan phyla. PMID:21835008

[Bacterial diversity in sequencing batch biofilm reactor (SBBR) for landfill leachate treatment using PCR-DGGE].

PubMed

Xiao, Yong; Yang, Zhao-hui; Zeng, Guang-ming; Ma, Yan-he; Liu, You-sheng; Wang, Rong-juan; Xu, Zheng-yong

2007-05-01

For studying the bacterial diversity and the mechanism of denitrification in sequencing bath biofilm reactor (SBBR) treating landfill leachate to provide microbial evidence for technique improvements, total microbial DNA was extracted from samples which were collected from natural landfill leachate and biofilm of a SBBR that could efficiently remove NH4+ -N and COD of high concentration. 16S rDNA fragments were amplified from the total DNA successfully using a pair of universal bacterial 16S rDNA primer, GC341F and 907R, and then were used for denaturing gradient gel electrophoresis (DGGE) analysis. The bands in the gel were analyzed by statistical methods and excided from the gel for sequencing, and the sequences were used for homology analysis and then two phylogenetic trees were constructed using DNAStar software. Results indicated that the bacterial diversity of the biofilm in SBBR and the landfill leachate was abundant, and no obvious change of community structure happened during running in the biofilm, in which most bacteria came from the landfill leachate. There may be three different modes of denitrification in the reactor because several different nitrifying bacteria, denitrifying bacteria and anaerobic ammonia oxidation bacteria coexisted in it. The results provided some valuable references for studying microbiological mechanism of denitrification in SBBR.

[Influence of PCR cycle number on microbial diversity analysis through next generation sequencing].

PubMed

An, Yunhe; Gao, Lijuan; Li, Junbo; Tian, Yanjie; Wang, Jinlong; Zheng, Xuejuan; Wu, Huijuan

2016-08-25

Using of high throughput sequencing technology to study the microbial diversity in complex samples has become one of the hottest issues in the field of microbial diversity research. In this study, the soil and sheep rumen chyme samples were used to extract DNA, respectively. Then the 25 ng total DNA was used to amplify the 16S rRNA V3 region with 20, 25, 30 PCR cycles, and the final sequencing library was constructed by mixing equal amounts of purified PCR products. Finally, the operational taxonomic unit (OUT) amount, rarefaction curve, microbial number and species were compared through data analysis. It was found that at the same amount of DNA template, the proportion of the community composition was not the best with more numbers of PCR cycle, although the species number was much more. In all, when the PCR cycle number is 25, the number of species and proportion of the community composition were the most optimal both in soil or chyme samples.

Circular replication-associated protein encoding DNA viruses identified in the faecal matter of various animals in New Zealand.

PubMed

Steel, Olivia; Kraberger, Simona; Sikorski, Alyssa; Young, Laura M; Catchpole, Ryan J; Stevens, Aaron J; Ladley, Jenny J; Coray, Dorien S; Stainton, Daisy; Dayaram, Anisha; Julian, Laurel; van Bysterveldt, Katherine; Varsani, Arvind

2016-09-01

In recent years, innovations in molecular techniques and sequencing technologies have resulted in a rapid expansion in the number of known viral sequences, in particular those with circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA genomes. CRESS DNA viruses are present in the virome of many ecosystems and are known to infect a wide range of organisms. A large number of the recently identified CRESS DNA viruses cannot be classified into any known viral families, indicating that the current view of CRESS DNA viral sequence space is greatly underestimated. Animal faecal matter has proven to be a particularly useful source for sampling CRESS DNA viruses in an ecosystem, as it is cost-effective and non-invasive. In this study a viral metagenomic approach was used to explore the diversity of CRESS DNA viruses present in the faeces of domesticated and wild animals in New Zealand. Thirty-eight complete CRESS DNA viral genomes and two circular molecules (that may be defective molecules or single components of multicomponent genomes) were identified from forty-nine individual animal faecal samples. Based on shared genome organisations and sequence similarities, eighteen of the isolates were classified as gemycircularviruses and twelve isolates were classified as smacoviruses. The remaining eight isolates lack significant sequence similarity with any members of known CRESS DNA virus groups. This research adds significantly to our knowledge of CRESS DNA viral diversity in New Zealand, emphasising the prevalence of CRESS DNA viruses in nature, and reinforcing the suggestion that a large proportion of CRESS DNA viruses are yet to be identified. Copyright © 2016 Elsevier B.V. All rights reserved.

Applications of the rep-PCR DNA fingerprinting technique to study microbial diversity, ecology and evolution.

PubMed

Ishii, Satoshi; Sadowsky, Michael J

2009-04-01

A large number of repetitive DNA sequences are found in multiple sites in the genomes of numerous bacteria, archaea and eukarya. While the functions of many of these repetitive sequence elements are unknown, they have proven to be useful as the basis of several powerful tools for use in molecular diagnostics, medical microbiology, epidemiological analyses and environmental microbiology. The repetitive sequence-based PCR or rep-PCR DNA fingerprint technique uses primers targeting several of these repetitive elements and PCR to generate unique DNA profiles or 'fingerprints' of individual microbial strains. Although this technique has been extensively used to examine diversity among variety of prokaryotic microorganisms, rep-PCR DNA fingerprinting can also be applied to microbial ecology and microbial evolution studies since it has the power to distinguish microbes at the strain or isolate level. Recent advancement in rep-PCR methodology has resulted in increased accuracy, reproducibility and throughput. In this minireview, we summarize recent improvements in rep-PCR DNA fingerprinting methodology, and discuss its applications to address fundamentally important questions in microbial ecology and evolution.

Identifying active foraminifera in the Sea of Japan using metatranscriptomic approach

NASA Astrophysics Data System (ADS)

Lejzerowicz, Franck; Voltsky, Ivan; Pawlowski, Jan

2013-02-01

Metagenetics represents an efficient and rapid tool to describe environmental diversity patterns of microbial eukaryotes based on ribosomal DNA sequences. However, the results of metagenetic studies are often biased by the presence of extracellular DNA molecules that are persistent in the environment, especially in deep-sea sediment. As an alternative, short-lived RNA molecules constitute a good proxy for the detection of active species. Here, we used a metatranscriptomic approach based on RNA-derived (cDNA) sequences to study the diversity of the deep-sea benthic foraminifera and compared it to the metagenetic approach. We analyzed 257 ribosomal DNA and cDNA sequences obtained from seven sediments samples collected in the Sea of Japan at depths ranging from 486 to 3665 m. The DNA and RNA-based approaches gave a similar view of the taxonomic composition of foraminiferal assemblage, but differed in some important points. First, the cDNA dataset was dominated by sequences of rotaliids and robertiniids, suggesting that these calcareous species, some of which have been observed in Rose Bengal stained samples, are the most active component of foraminiferal community. Second, the richness of monothalamous (single-chambered) foraminifera was particularly high in DNA extracts from the deepest samples, confirming that this group of foraminifera is abundant but not necessarily very active in the deep-sea sediments. Finally, the high divergence of undetermined sequences in cDNA dataset indicate the limits of our database and lack of knowledge about some active but possibly rare species. Our study demonstrates the capability of the metatranscriptomic approach to detect active foraminiferal species and prompt its use in future high-throughput sequencing-based environmental surveys.

The Targeted Sequencing of Alpha Satellite DNA in Cercopithecus pogonias Provides New Insight into the Diversity and Dynamics of Centromeric Repeats in Old World monkeys.

PubMed

Cacheux, Lauriane; Ponger, Loïc; Gerbault-Seureau, Michèle; Loll, François; Gey, Delphine; Richard, Florence Anne; Escudé, Christophe

2018-06-01

Alpha satellite is the major repeated DNA element of primate centromeres. Specific evolutionary mechanisms have led to a great diversity of sequence families with peculiar genomic organization and distribution, which have till now been studied mostly in great apes. Using high throughput sequencing of alpha satellite monomers obtained by enzymatic digestion followed by computational and cytogenetic analysis, we compare here the diversity and genomic distribution of alpha satellite DNA in two related Old World monkey species, Cercopithecus pogonias and Cercopithecus solatus, which are known to have diverged about seven million years ago. Two main families of monomers, called C1 and C2, are found in both species. A detailed analysis of our datasets revealed the existence of numerous subfamilies within the centromeric C1 family. Although the most abundant subfamily is conserved between both species, our FISH experiments clearly show that some subfamilies are specific for each species and that their distribution is restricted to a subset of chromosomes, thereby pointing to the existence of recurrent amplification/homogenization events. The pericentromeric C2 family is very abundant on the short arm of all acrocentric chromosomes in both species, pointing to specific mechanisms that lead to this distribution. Results obtained using two different restriction enzymes are fully consistent with a predominant monomeric organization of alpha satellite DNA which coexists with higher order organization patterns in the Cercopithecus pogonias genome. Our study suggests a high dynamics of alpha satellite DNA in Cercopithecini, with recurrent apparition of new sequence variants and interchromosomal sequence transfer.

ENVIRONMENTAL INFLUENCES ON GENETIC DIVERSITY OF CREEK CHUBS IN THE MID-ATLANTIC REGION OF THE USA

EPA Science Inventory

Analysis of genetic diversity within and among populations of stream fishes may provide a powerful method for assessing the status and trends in the condition of aquatic ecosystems. We analyzed mitochondrial DNA sequences (590 bases of cytochrome B) and nuclear DNA loci (109 amp...

mtDNA variation of the critically endangered hawksbill turtle (Eretmochelys imbricata) nesting on Iranian islands of the Persian Gulf.

PubMed

Tabib, M; Zolgharnein, H; Mohammadi, M; Salari-Aliabadi, M A; Qasemi, A; Roshani, S; Rajabi-Maham, H; Frootan, F

2011-01-01

Genetic diversity of sea turtles (hawksbill turtle) was studied using sequencing of mitochondrial DNA (mtDNA, D-loop region). Thirty dead embryos were collected from the Kish and Qeshm Islands in the Persian Gulf. Analysis of sequence variation over 890 bp of the mtDNA control region revealed five haplotypes among 30 individuals. This is the first time that Iranian haplotypes have been recorded. Nucleotide and haplotype diversity was 0.77 and 0.001 for Qeshm Island and 0.64 and 0.002 for Kish Island, respectively. Total haplotype diversity was calculated as 0.69, which demonstrates low genetic diversity in this area. The data also indicated very high rates of migration between the populations of these two islands. A comparison of our data with data from previous studies downloaded from a gene bank showed that turtles of the Persian Gulf migrated from the Pacific and the Sea of Oman into this area. On the other hand, evidence of migration from populations to the West was not found.

Diversity of mitochondrial DNA lineages in South Siberia.

PubMed

Derenko, M V; Grzybowski, T; Malyarchuk, B A; Dambueva, I K; Denisova, G A; Czarny, J; Dorzhu, C M; Kakpakov, V T; Miścicka-Sliwka, D; Woźniak, M; Zakharov, I A

2003-09-01

To investigate the origin and evolution of aboriginal populations of South Siberia, a comprehensive mitochondrial DNA (mtDNA) analysis (HVR1 sequencing combined with RFLP typing) of 480 individuals, representing seven Altaic-speaking populations (Altaians, Khakassians, Buryats, Sojots, Tuvinians, Todjins and Tofalars), was performed. Additionally, HVR2 sequence information was obtained for 110 Altaians, providing, in particular, some novel details of the East Asian mtDNA phylogeny. The total sample revealed 81% East Asian (M*, M7, M8, M9, M10, C, D, G, Z, A, B, F, N9a, Y) and 17% West Eurasian (H, U, J, T, I, N1a, X) matrilineal genetic contribution, but with regional differences within South Siberia. The highest influx of West Eurasian mtDNAs was observed in populations from the East Sayan and Altai regions (from 12.5% to 34.5%), whereas in populations from the Baikal region this contribution was markedly lower (less than 10%). The considerable substructure within South Siberian haplogroups B, F, and G, together with the high degree of haplogroup C and D diversity revealed there, allows us to conclude that South Siberians carry the genetic imprint of early-colonization phase of Eurasia. Statistical analyses revealed that South Siberian populations contain high levels of mtDNA diversity and high heterogeneity of mtDNA sequences among populations (Fst = 5.05%) that might be due to geography but not due to language and anthropological features.

DnaSAM: Software to perform neutrality testing for large datasets with complex null models.

PubMed

Eckert, Andrew J; Liechty, John D; Tearse, Brandon R; Pande, Barnaly; Neale, David B

2010-05-01

Patterns of DNA sequence polymorphisms can be used to understand the processes of demography and adaptation within natural populations. High-throughput generation of DNA sequence data has historically been the bottleneck with respect to data processing and experimental inference. Advances in marker technologies have largely solved this problem. Currently, the limiting step is computational, with most molecular population genetic software allowing a gene-by-gene analysis through a graphical user interface. An easy-to-use analysis program that allows both high-throughput processing of multiple sequence alignments along with the flexibility to simulate data under complex demographic scenarios is currently lacking. We introduce a new program, named DnaSAM, which allows high-throughput estimation of DNA sequence diversity and neutrality statistics from experimental data along with the ability to test those statistics via Monte Carlo coalescent simulations. These simulations are conducted using the ms program, which is able to incorporate several genetic parameters (e.g. recombination) and demographic scenarios (e.g. population bottlenecks). The output is a set of diversity and neutrality statistics with associated probability values under a user-specified null model that are stored in easy to manipulate text file. © 2009 Blackwell Publishing Ltd.

A global meta-analysis of Tuber ITS rDNA sequences: species diversity, host associations and long-distance dispersal

Treesearch

Gregory M. Bonito; Andrii P. Gryganskyi; James M. Trappe; Rytas Vilgalys

2010-01-01

Truffles (Tuber) are ectomycorrhizal fungi characterized by hypogeous fruitbodies. Their biodiversity, host associations and geographical distributions are not well documented. ITS rDNA sequences of Tuber are commonly recovered from molecular surveys of fungal communities, but most remain insufficiently identified making it...

Development of polymorphic genic-SSR markers by cDNA library sequencing in boxwood, Buxus spp. (Buxaceae)

USDA-ARS?s Scientific Manuscript database

Genic microsatellites or simple sequence repeat (genic-SSR) markers were developed in boxwood (Buxus taxa) for genetic diversity analysis, identification of taxa, and to facilitate breeding. cDNA libraries were developed from mRNA extracted from leaves of Buxus sempervirens ‘Vardar Valley’ and seque...

Diversity of phytases in the rumen.

PubMed

Nakashima, Brenda A; McAllister, Tim A; Sharma, Ranjana; Selinger, L Brent

2007-01-01

Examples of a new class of phytase related to protein tyrosine phosphatases (PTP) were recently isolated from several anaerobic bacteria from the rumen of cattle. In this study, the diversity of PTP-like phytase gene sequences in the rumen was surveyed by using the polymerase chain reaction (PCR). Two sets of degenerate primers were used to amplify sequences from rumen fluid total community DNA and genomic DNA from nine bacterial isolates. Four novel PTP-like phytase sequences were retrieved from rumen fluid, whereas all nine of the anaerobic bacterial isolates investigated in this work contained PTP-like phytase sequences. One isolate, Selenomonas lacticifex, contained two distinct PTP-like phytase sequences, suggesting that multiple phytate hydrolyzing enzymes are present in this bacterium. The degenerate primer and PCR conditions described here, as well as novel sequences obtained in this study, will provide a valuable resource for future studies on this new class of phytase. The observed diversity of microbial phytases in the rumen may account for the ability of ruminants to derive a significant proportion of their phosphorus requirements from phytate.

Variation in the number of nucleoli and incomplete homogenization of 18S ribosomal DNA sequences in leaf cells of the cultivated Oriental ginseng (Panax ginseng Meyer).

PubMed

Chelomina, Galina N; Rozhkovan, Konstantin V; Voronova, Anastasia N; Burundukova, Olga L; Muzarok, Tamara I; Zhuravlev, Yuri N

2016-04-01

Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440-640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine.

Variation in the number of nucleoli and incomplete homogenization of 18S ribosomal DNA sequences in leaf cells of the cultivated Oriental ginseng (Panax ginseng Meyer)

PubMed Central

Chelomina, Galina N.; Rozhkovan, Konstantin V.; Voronova, Anastasia N.; Burundukova, Olga L.; Muzarok, Tamara I.; Zhuravlev, Yuri N.

2015-01-01

Background Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. Methods The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. Results In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440–640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. Conclusion This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine. PMID:27158239

Detecting differential DNA methylation from sequencing of bisulfite converted DNA of diverse species.

PubMed

Huh, Iksoo; Wu, Xin; Park, Taesung; Yi, Soojin V

2017-07-21

DNA methylation is one of the most extensively studied epigenetic modifications of genomic DNA. In recent years, sequencing of bisulfite-converted DNA, particularly via next-generation sequencing technologies, has become a widely popular method to study DNA methylation. This method can be readily applied to a variety of species, dramatically expanding the scope of DNA methylation studies beyond the traditionally studied human and mouse systems. In parallel to the increasing wealth of genomic methylation profiles, many statistical tools have been developed to detect differentially methylated loci (DMLs) or differentially methylated regions (DMRs) between biological conditions. We discuss and summarize several key properties of currently available tools to detect DMLs and DMRs from sequencing of bisulfite-converted DNA. However, the majority of the statistical tools developed for DML/DMR analyses have been validated using only mammalian data sets, and less priority has been placed on the analyses of invertebrate or plant DNA methylation data. We demonstrate that genomic methylation profiles of non-mammalian species are often highly distinct from those of mammalian species using examples of honey bees and humans. We then discuss how such differences in data properties may affect statistical analyses. Based on these differences, we provide three specific recommendations to improve the power and accuracy of DML and DMR analyses of invertebrate data when using currently available statistical tools. These considerations should facilitate systematic and robust analyses of DNA methylation from diverse species, thus advancing our understanding of DNA methylation. © The Author 2017. Published by Oxford University Press.

Diversity of Basidiomycetes in Michigan Agricultural Soils▿

PubMed Central

Lynch, Michael D. J.; Thorn, R. Greg

2006-01-01

We analyzed the communities of soil basidiomycetes in agroecosystems that differ in tillage history at the Kellogg Biological Station Long-Term Ecological Research site near Battle Creek, Michigan. The approach combined soil DNA extraction through a bead-beating method modified to increase recovery of fungal DNA, PCR amplification with basidiomycete-specific primers, cloning and restriction fragment length polymorphism screening of mixed PCR products, and sequencing of unique clones. Much greater diversity was detected than was anticipated in this habitat on the basis of culture-based methods or surveys of fruiting bodies. With “species” defined as organisms yielding PCR products with ≥99% identity in the 5′ 650 bases of the nuclear large-subunit ribosomal DNA, 241 “species” were detected among 409 unique basidiomycete sequences recovered. Almost all major clades of basidiomycetes from basidiomycetous yeasts and other heterobasidiomycetes through polypores and euagarics (gilled mushrooms and relatives) were represented, with a majority from the latter clade. Only 24 of 241 “species” had 99% or greater sequence similarity to named reference sequences in GenBank, and several clades with multiple “species” could not be identified at the genus level by phylogenetic comparisons with named sequences. The total estimated “species” richness for this 11.2-ha site was 367 “species” of basidiomycetes. Since >99% of the study area has not been sampled, the accuracy of our diversity estimate is uncertain. Replication in time and space is required to detect additional diversity and the underlying community structure. PMID:16950900

Comparison of mitochondrial DNA control region sequence and microsatellite DNA analyses in estimating population structure and gene flow rates in Atlantic sturgeon Acipenser oxyrinchus

USGS Publications Warehouse

Wirgin, I.; Waldman, J.; Stabile, J.; Lubinski, B.; King, T.

2002-01-01

Atlantic sturgeon Acipenser oxyrinchus is large, long-lived, and anadromous with subspecies distributed along the Atlantic (A. oxyrinchus oxyrinchus) and Gulf of Mexico (A. o. desotoi) coasts of North America. Although it is not certain if extirpation of some population units has occurred, because of anthropogenic influences abundances of all populations are low compared with historical levels. Informed management of A. oxyrinchus demands a detailed knowledge of its population structure, levels of genetic diversity, and likelihood to home to natal rivers. We compared the use of mitochondrial DNA (mtDNA) control region sequence and microsatellite nuclear DNA (nDNA) analyses in identifying the stock structure and homing fidelity of Atlantic and Gulf coast populations of A. oxyrinchus. The approaches were concordant in that they revealed moderate to high levels of genetic diversity and suggested that populations of Atlantic sturgeon are highly structured. At least six genetically distinct management units were detected using the two approaches among the rivers surveyed. Mitochondrial DNA sequences revealed a significant cline in haplotype diversity along the Atlantic coast with monomorphism observed in Canadian populations. High levels of nDNA diversity were also observed among populations along the Atlantic coast, including the two Canadian populations, probably resulting from the more rapid rate of mutational and evolutionary change at microsatellite loci. Estimates of gene flow among populations were similar between both approaches with the exception that because of mtDNA monomorphism in Canadian populations, gene flow estimates between them were unobtainable. Analyses of both genomes provided high resolution and confidence in characterizing the population structure of Atlantic sturgeon. Microsatellite analysis was particularly informative in delineating population structure in rivers that were recently glaciated and may prove diagnostic in rivers that are geographically proximal along the south Atlantic coast of the US.

Prospecting Metagenomic Enzyme Subfamily Genes for DNA Family Shuffling by a Novel PCR-based Approach*

PubMed Central

Wang, Qiuyan; Wu, Huili; Wang, Anming; Du, Pengfei; Pei, Xiaolin; Li, Haifeng; Yin, Xiaopu; Huang, Lifeng; Xiong, Xiaolong

2010-01-01

DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64–99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. Our results indicate that homologous sequences of genes captured by TMGS-PCR can be used as suitable genetic material for DNA family shuffling with broad applications in enzyme engineering. PMID:20962349

Identification of Single-Copy Orthologous Genes between Physalis and Solanum lycopersicum and Analysis of Genetic Diversity in Physalis Using Molecular Markers

PubMed Central

Wei, Jingli; Hu, Xiaorong; Yang, Jingjing; Yang, Wencai

2012-01-01

The genus Physalis includes a number of commercially important edible and ornamental species. Its high nutritional value and potential medicinal properties leads to the increased commercial interest in the products of this genus worldwide. However, lack of molecular markers prevents the detailed study of genetics and phylogeny in Physalis, which limits the progress of breeding. In the present study, we compared the DNA sequences between Physalis and tomato, and attempted to analyze genetic diversity in Physalis using tomato markers. Blasting 23180 DNA sequences derived from Physalis against the International Tomato Annotation Group (ITAG) Release2.3 Predicted CDS (SL2.40) discovered 3356 single-copy orthologous genes between them. A total of 38 accessions from at least six species of Physalis were subjected to genetic diversity analysis using 97 tomato markers and 25 SSR markers derived from P. peruviana. Majority (73.2%) of tomato markers could amplify DNA fragments from at least one accession of Physalis. Diversity in Physalis at molecular level was also detected. The average Nei’s genetic distance between accessions was 0.3806 with a range of 0.2865 to 0.7091. These results indicated Physalis and tomato had similarity at both molecular marker and DNA sequence levels. Therefore, the molecular markers developed in tomato can be used in genetic study in Physalis. PMID:23166835

Identification of single-copy orthologous genes between Physalis and Solanum lycopersicum and analysis of genetic diversity in Physalis using molecular markers.

PubMed

Wei, Jingli; Hu, Xiaorong; Yang, Jingjing; Yang, Wencai

2012-01-01

The genus Physalis includes a number of commercially important edible and ornamental species. Its high nutritional value and potential medicinal properties leads to the increased commercial interest in the products of this genus worldwide. However, lack of molecular markers prevents the detailed study of genetics and phylogeny in Physalis, which limits the progress of breeding. In the present study, we compared the DNA sequences between Physalis and tomato, and attempted to analyze genetic diversity in Physalis using tomato markers. Blasting 23180 DNA sequences derived from Physalis against the International Tomato Annotation Group (ITAG) Release2.3 Predicted CDS (SL2.40) discovered 3356 single-copy orthologous genes between them. A total of 38 accessions from at least six species of Physalis were subjected to genetic diversity analysis using 97 tomato markers and 25 SSR markers derived from P. peruviana. Majority (73.2%) of tomato markers could amplify DNA fragments from at least one accession of Physalis. Diversity in Physalis at molecular level was also detected. The average Nei's genetic distance between accessions was 0.3806 with a range of 0.2865 to 0.7091. These results indicated Physalis and tomato had similarity at both molecular marker and DNA sequence levels. Therefore, the molecular markers developed in tomato can be used in genetic study in Physalis.

Estimating Diversity of Florida Keys Zooplankton Using New Environmental DNA Methods

NASA Astrophysics Data System (ADS)

Djurhuus, A.; Goldsmith, D. B.; Sawaya, N. A.; Breitbart, M.

2016-02-01

Zooplankton are of great importance in marine food webs, where they serve to link the phytoplankton and bacteria with higher trophic levels. Zooplankton are a diverse group containing molluscs, crustaceans, fish larvae and many other taxa. The sheer number of species and often minor morphological distinctions between species makes it challenging and exceptionally time consuming to identify the species composition of marine zooplankton samples. As a part of the Marine Biodiversity Observation Network (MBON) project, we have developed and groundtruthed an alternative, relatively time-efficient method for zooplankton identification using environmental DNA (eDNA). Samples were collected from Molasses reef, Looe Key, and Western Sambo along the Florida Keys from five bi-monthly cruises on board the RV Walton Smith. Samples were collected for environmental DNA (eDNA) by filtering 1 L of water on to a 0.22 µm filter and zooplankton samples were collected using nets with three mesh sizes (64μm, 200μm, and 500μm) to catch different size fractions. Half of zooplankton samples were fixed in 70% ethanol and half in 10% formalin, for DNA extraction and morphological identification, respectively. Individuals representing visually abundant taxa were picked into individual wells for PCR with universal 18S rRNA gene primers and subsequent sequencing to build a reference barcode database for zooplankton species commonly found in the study region. PCR and Illumina MiSeq next generation sequencing was applied to the eDNA extracted from the 0.22 μm filters and sequences were be compared to our local custom database as well as publicly available databases to determine zooplankton community composition. Finally, composition and diversity analyses were performed to compare results obtained with the new eDNA approach to standard morphological classification of zooplankton communities. Results show that the eDNA approach can enable the determination of zooplankton diversity through collection of a single water sample, which, when combined with bacterial and archaeal diversity analyses, will help us understand the coupling between different trophic levels and the drivers of plankton dynamics in the sub-tropical Florida Keys.

Land, language, and loci: mtDNA in Native Americans and the genetic history of Peru.

PubMed

Lewis, Cecil M; Tito, Raúl Y; Lizárraga, Beatriz; Stone, Anne C

2005-07-01

Despite a long history of complex societies and despite extensive present-day linguistic and ethnic diversity, relatively few populations in Peru have been sampled for population genetic investigations. In order to address questions about the relationships between South American populations and about the extent of correlation between genetic distance, language, and geography in the region, mitochondrial DNA (mtDNA) hypervariable region I sequences and mtDNA haplogroup markers were examined in 33 individuals from the state of Ancash, Peru. These sequences were compared to those from 19 American Indian populations using diversity estimates, AMOVA tests, mismatch distributions, a multidimensional scaling plot, and regressions. The results show correlations between genetics, linguistics, and geographical affinities, with stronger correlations between genetics and language. Additionally, the results suggest a pattern of differential gene flow and drift in western vs. eastern South America, supporting previous mtDNA and Y chromosome investigations. (c) 2004 Wiley-Liss, Inc

Calibrating snakehead diversity with DNA barcodes: expanding taxonomic coverage to enable identification of potential and established invasive species.

PubMed

Serrao, Natasha R; Steinke, Dirk; Hanner, Robert H

2014-01-01

Detecting and documenting the occurrence of invasive species outside their native range requires tools to support their identification. This can be challenging for taxa with diverse life stages and/or problematic or unresolved morphological taxonomies. DNA barcoding provides a potent method for identifying invasive species, as it allows for species identification at all life stages, including fragmentary remains. It also provides an efficient interim taxonomic framework for quantifying cryptic genetic diversity by parsing barcode sequences into discontinuous haplogroup clusters (typical of reproductively isolated species) and labelling them with unique alphanumeric identifiers. Snakehead fishes are a diverse group of opportunistic predators endemic to Asia and Africa that may potentially pose significant threats as aquatic invasive species. At least three snakehead species (Channa argus, C. maculata, and C. marulius) are thought to have entered North America through the aquarium and live-food fish markets, and have established populations, yet their origins remain unclear. The objectives of this study were to assemble a library of DNA barcode sequences derived from expert identified reference specimens in order to determine the identity and aid invasion pathway analysis of the non-indigenous species found in North America using DNA barcodes. Sequences were obtained from 121 tissue samples representing 25 species and combined with public records from GenBank for a total of 36 putative species, which then partitioned into 49 discrete haplogroups. Multiple divergent clusters were observed within C. gachua, C. marulius, C. punctata and C. striata suggesting the potential presence of cryptic species diversity within these lineages. Our findings demonstrate that DNA barcoding is a valuable tool for species identification in challenging and under-studied taxonomic groups such as snakeheads, and provides a useful framework for inferring invasion pathway analysis.

Four new bisabolane-type sesquiterpenes from Ligularia lankongensis.

PubMed

Hirota, Hiroshi; Horiguchi, Yurie; Kawaii, Satoru; Kuroda, Chiaki; Hanai, Ryo; Gong, Xun

2012-04-01

The chemical constituents of the roots of two Ligularia lankongensis samples collected in Yunnan and Sichuan Provinces, China, were investigated, together with the DNA sequence of the atpB-rbcL and ITS regions. Four new highly oxygenated bisabolane-type sesquiterpenes (1 - 4) were obtained. Intraspecific diversity in the DNA sequence was found to be limited.

Identification of Bacterial Species in Kuwaiti Waters Through DNA Sequencing

NASA Astrophysics Data System (ADS)

Chen, K.

2017-01-01

With an objective of identifying the bacterial diversity associated with ecosystem of various Kuwaiti Seas, bacteria were cultured and isolated from 3 water samples. Due to the difficulties for cultured and isolated fecal coliforms on the selective agar plates, bacterial isolates from marine agar plates were selected for molecular identification. 16S rRNA genes were successfully amplified from the genome of the selected isolates using Universal Eubacterial 16S rRNA primers. The resulted amplification products were subjected to automated DNA sequencing. Partial 16S rDNA sequences obtained were compared directly with sequences in the NCBI database using BLAST as well as with the sequences available with Ribosomal Database Project (RDP).

Combined Use of 16S Ribosomal DNA and 16S rRNA To Study the Bacterial Community of Polychlorinated Biphenyl-Polluted Soil

PubMed Central

Nogales, Balbina; Moore, Edward R. B.; Llobet-Brossa, Enrique; Rossello-Mora, Ramon; Amann, Rudolf; Timmis, Kenneth N.

2001-01-01

The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries. More than 60% of the total cloned sequence types analyzed were grouped in phylogenetic groups (a clone group with sequence similarity higher than 97% [98% for Burkholderia and Pseudomonas-type clones]) represented in both types of libraries. Some of those phylogenetic groups, mostly represented by a single (or pair) of cloned sequence type(s), were observed in only one of the types of library. An important difference between the libraries was the lack of clones representative of the Actinobacteria in the rDNA library. The PCB-polluted soil exhibited a high bacterial diversity which included representatives of two novel lineages. The apparent abundance of bacteria affiliated to the beta-subclass of the Proteobacteria, and to the genus Burkholderia in particular, was confirmed by fluorescence in situ hybridization analysis. The possible influence on apparent diversity of low template concentrations was assessed by dilution of the RNA template prior to amplification by reverse transcription-PCR. Although differences in the composition of the two rRNA libraries obtained from high and low RNA concentrations were observed, the main components of the bacterial community were represented in both libraries, and therefore their detection was not compromised by the lower concentrations of template used in this study. PMID:11282645

First Molecular Identification and Phylogeny of Moroccan Anopheles sergentii (Diptera: Culicidae) Based on Second Internal Transcribed Spencer (ITS2) and Cytochrome c Oxidase I (COI) Sequences.

PubMed

Benabdelkrim Filali, Oumama; Kabine, Mostafa; El Hamouchi, Adil; Lemrani, Meryem; Debboun, Mustapha; Sarih, M'hammed

2018-06-05

Anopheles sergentii known as the "oasis vector" or the "desert malaria vector" is considered the main vector of malaria in the southern parts of Morocco. Its presence in Morocco is confirmed for the first time through sequencing of mitochondrial DNA (mDNA) cytochrome c oxidase subunit I (COI) barcodes and nuclear ribosomal DNA (rDNA) second internal transcribed spacer (ITS2) sequences and direct comparison with specimens of A. sergentii of other countries. The DNA barcodes (n = 39) obtained from A. sergentii collected in 2015 and 2016 showed more diversity with 10 haplotypes, compared with 3 haplotypes obtained from ITS2 sequences (n = 59). Moreover, the comparison using the ITS2 sequences showed closer evolutionary relationship between the Moroccan and Egyptian strains than the Iranian strain. Nevertheless, genetic differences due to geographical segregation were also observed. This study provides the first report on the sequence of rDNA-ITS2 and mtDNA COI, which could be used to better understand the biodiversity of A. sergentii.

Proteopedia: 3D Visualization and Annotation of Transcription Factor-DNA Readout Modes

ERIC Educational Resources Information Center

Dantas Machado, Ana Carolina; Saleebyan, Skyler B.; Holmes, Bailey T.; Karelina, Maria; Tam, Julia; Kim, Sharon Y.; Kim, Keziah H.; Dror, Iris; Hodis, Eran; Martz, Eric; Compeau, Patricia A.; Rohs, Remo

2012-01-01

3D visualization assists in identifying diverse mechanisms of protein-DNA recognition that can be observed for transcription factors and other DNA binding proteins. We used Proteopedia to illustrate transcription factor-DNA readout modes with a focus on DNA shape, which can be a function of either nucleotide sequence (Hox proteins) or base pairing…

TaxI: a software tool for DNA barcoding using distance methods

PubMed Central

Steinke, Dirk; Vences, Miguel; Salzburger, Walter; Meyer, Axel

2005-01-01

DNA barcoding is a promising approach to the diagnosis of biological diversity in which DNA sequences serve as the primary key for information retrieval. Most existing software for evolutionary analysis of DNA sequences was designed for phylogenetic analyses and, hence, those algorithms do not offer appropriate solutions for the rapid, but precise analyses needed for DNA barcoding, and are also unable to process the often large comparative datasets. We developed a flexible software tool for DNA taxonomy, named TaxI. This program calculates sequence divergences between a query sequence (taxon to be barcoded) and each sequence of a dataset of reference sequences defined by the user. Because the analysis is based on separate pairwise alignments this software is also able to work with sequences characterized by multiple insertions and deletions that are difficult to align in large sequence sets (i.e. thousands of sequences) by multiple alignment algorithms because of computational restrictions. Here, we demonstrate the utility of this approach with two datasets of fish larvae and juveniles from Lake Constance and juvenile land snails under different models of sequence evolution. Sets of ribosomal 16S rRNA sequences, characterized by multiple indels, performed as good as or better than cox1 sequence sets in assigning sequences to species, demonstrating the suitability of rRNA genes for DNA barcoding. PMID:16214755

Plant DNA sequences from feces: potential means for assessing diets of wild primates.

PubMed

Bradley, Brenda J; Stiller, Mathias; Doran-Sheehy, Diane M; Harris, Tara; Chapman, Colin A; Vigilant, Linda; Poinar, Hendrik

2007-06-01

Analyses of plant DNA in feces provides a promising, yet largely unexplored, means of documenting the diets of elusive primates. Here we demonstrate the promise and pitfalls of this approach using DNA extracted from fecal samples of wild western gorillas (Gorilla gorilla) and black and white colobus monkeys (Colobus guereza). From these DNA extracts we amplified, cloned, and sequenced small segments of chloroplast DNA (part of the rbcL gene) and plant nuclear DNA (ITS-2). The obtained sequences were compared to sequences generated from known plant samples and to those in GenBank to identify plant taxa in the feces. With further optimization, this method could provide a basic evaluation of minimum primate dietary diversity even when knowledge of local flora is limited. This approach may find application in studies characterizing the diets of poorly-known, unhabituated primate species or assaying consumer-resource relationships in an ecosystem. (c) 2007 Wiley-Liss, Inc.

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

PubMed

Massana, Ramon; Gobet, Angélique; Audic, Stéphane; Bass, David; Bittner, Lucie; Boutte, Christophe; Chambouvet, Aurélie; Christen, Richard; Claverie, Jean-Michel; Decelle, Johan; Dolan, John R; Dunthorn, Micah; Edvardsen, Bente; Forn, Irene; Forster, Dominik; Guillou, Laure; Jaillon, Olivier; Kooistra, Wiebe H C F; Logares, Ramiro; Mahé, Frédéric; Not, Fabrice; Ogata, Hiroyuki; Pawlowski, Jan; Pernice, Massimo C; Probert, Ian; Romac, Sarah; Richards, Thomas; Santini, Sébastien; Shalchian-Tabrizi, Kamran; Siano, Raffaele; Simon, Nathalie; Stoeck, Thorsten; Vaulot, Daniel; Zingone, Adriana; de Vargas, Colomban

2015-10-01

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Barcode extension for analysis and reconstruction of structures

NASA Astrophysics Data System (ADS)

Myhrvold, Cameron; Baym, Michael; Hanikel, Nikita; Ong, Luvena L.; Gootenberg, Jonathan S.; Yin, Peng

2017-03-01

Collections of DNA sequences can be rationally designed to self-assemble into predictable three-dimensional structures. The geometric and functional diversity of DNA nanostructures created to date has been enhanced by improvements in DNA synthesis and computational design. However, existing methods for structure characterization typically image the final product or laboriously determine the presence of individual, labelled strands using gel electrophoresis. Here we introduce a new method of structure characterization that uses barcode extension and next-generation DNA sequencing to quantitatively measure the incorporation of every strand into a DNA nanostructure. By quantifying the relative abundances of distinct DNA species in product and monomer bands, we can study the influence of geometry and sequence on assembly. We have tested our method using 2D and 3D DNA brick and DNA origami structures. Our method is general and should be extensible to a wide variety of DNA nanostructures.

Barcode extension for analysis and reconstruction of structures.

PubMed

Myhrvold, Cameron; Baym, Michael; Hanikel, Nikita; Ong, Luvena L; Gootenberg, Jonathan S; Yin, Peng

2017-03-13

Collections of DNA sequences can be rationally designed to self-assemble into predictable three-dimensional structures. The geometric and functional diversity of DNA nanostructures created to date has been enhanced by improvements in DNA synthesis and computational design. However, existing methods for structure characterization typically image the final product or laboriously determine the presence of individual, labelled strands using gel electrophoresis. Here we introduce a new method of structure characterization that uses barcode extension and next-generation DNA sequencing to quantitatively measure the incorporation of every strand into a DNA nanostructure. By quantifying the relative abundances of distinct DNA species in product and monomer bands, we can study the influence of geometry and sequence on assembly. We have tested our method using 2D and 3D DNA brick and DNA origami structures. Our method is general and should be extensible to a wide variety of DNA nanostructures.

Barcode extension for analysis and reconstruction of structures

PubMed Central

Myhrvold, Cameron; Baym, Michael; Hanikel, Nikita; Ong, Luvena L; Gootenberg, Jonathan S; Yin, Peng

2017-01-01

Collections of DNA sequences can be rationally designed to self-assemble into predictable three-dimensional structures. The geometric and functional diversity of DNA nanostructures created to date has been enhanced by improvements in DNA synthesis and computational design. However, existing methods for structure characterization typically image the final product or laboriously determine the presence of individual, labelled strands using gel electrophoresis. Here we introduce a new method of structure characterization that uses barcode extension and next-generation DNA sequencing to quantitatively measure the incorporation of every strand into a DNA nanostructure. By quantifying the relative abundances of distinct DNA species in product and monomer bands, we can study the influence of geometry and sequence on assembly. We have tested our method using 2D and 3D DNA brick and DNA origami structures. Our method is general and should be extensible to a wide variety of DNA nanostructures. PMID:28287117

An atlas of DNA methylation in diverse bovine tissues

USDA-ARS?s Scientific Manuscript database

We launched an effort to produce a reference cattle DNA methylation resource to improve animal production. We will employ experimental pipelines built around next generation sequencing technologies to map DNA methylation in cultured cells and primary tissues systems frequently involved in animal pro...

Assessment of species diversity and distribution of an ancient diatom lineage using a DNA metabarcoding approach.

PubMed

Nanjappa, Deepak; Audic, Stephane; Romac, Sarah; Kooistra, Wiebe H C F; Zingone, Adriana

2014-01-01

Continuous efforts to estimate actual diversity and to trace the species distribution and ranges in the natural environments have gone in equal pace with advancements of the technologies in the study of microbial species diversity from microscopic observations to DNA-based barcoding. DNA metabarcoding based on Next Generation Sequencing (NGS) constitutes the latest advancement in these efforts. Here we use NGS data from different sites to investigate the geographic range of six species of the diatom family Leptocylindraceae and to identify possible new taxa within the family. We analysed the V4 and V9 regions of the nuclear-encoded SSU rDNA gene region in the NGS database of the European ERA-Biodiversa project BioMarKs, collected in plankton and sediments at six coastal sites in European coastal waters, as well as environmental sequences from the NCBI database. All species known in the family Leptocylindraceae were detected in both datasets, but the much larger Illumina V9 dataset showed a higher species coverage at the various sites than the 454 V4 dataset. Sequences identical or similar to the references of Leptocylindrus aporus, L. convexus, L. danicus/hargravesii and Tenuicylindrus belgicus were found in the Mediterranean Sea, North Atlantic Ocean and Black Sea as well as at locations outside Europe. Instead, sequences identical or close to that of L. minimus were found in the North Atlantic Ocean and the Black Sea but not in the Mediterranean Sea, while sequences belonging to a yet undescribed taxon were encountered only in Oslo Fjord and Baffin Bay. Identification of Leptocylindraceae species in NGS datasets has expanded our knowledge of the species biogeographic distribution and of the overall diversity of this diatom family. Individual species appear to be widespread, but not all of them are found everywhere. Despite the sequencing depth allowed by NGS and the wide geographic area covered by this study, the diversity of this ancient diatom family appears to be low, at least at the level of the marker used in this study.

Assessment of Species Diversity and Distribution of an Ancient Diatom Lineage Using a DNA Metabarcoding Approach

PubMed Central

Nanjappa, Deepak; Audic, Stephane; Romac, Sarah; Kooistra, Wiebe H. C. F.; Zingone, Adriana

2014-01-01

Background Continuous efforts to estimate actual diversity and to trace the species distribution and ranges in the natural environments have gone in equal pace with advancements of the technologies in the study of microbial species diversity from microscopic observations to DNA-based barcoding. DNA metabarcoding based on Next Generation Sequencing (NGS) constitutes the latest advancement in these efforts. Here we use NGS data from different sites to investigate the geographic range of six species of the diatom family Leptocylindraceae and to identify possible new taxa within the family. Methodology/Principal Findings We analysed the V4 and V9 regions of the nuclear-encoded SSU rDNA gene region in the NGS database of the European ERA-Biodiversa project BioMarKs, collected in plankton and sediments at six coastal sites in European coastal waters, as well as environmental sequences from the NCBI database. All species known in the family Leptocylindraceae were detected in both datasets, but the much larger Illumina V9 dataset showed a higher species coverage at the various sites than the 454 V4 dataset. Sequences identical or similar to the references of Leptocylindrus aporus, L. convexus, L. danicus/hargravesii and Tenuicylindrus belgicus were found in the Mediterranean Sea, North Atlantic Ocean and Black Sea as well as at locations outside Europe. Instead, sequences identical or close to that of L. minimus were found in the North Atlantic Ocean and the Black Sea but not in the Mediterranean Sea, while sequences belonging to a yet undescribed taxon were encountered only in Oslo Fjord and Baffin Bay. Conclusions/Significance Identification of Leptocylindraceae species in NGS datasets has expanded our knowledge of the species biogeographic distribution and of the overall diversity of this diatom family. Individual species appear to be widespread, but not all of them are found everywhere. Despite the sequencing depth allowed by NGS and the wide geographic area covered by this study, the diversity of this ancient diatom family appears to be low, at least at the level of the marker used in this study. PMID:25133638

Improved serial analysis of V1 ribosomal sequence tags (SARST-V1) provides a rapid, comprehensive, sequence-based characterization of bacterial diversity and community composition.

PubMed

Yu, Zhongtang; Yu, Marie; Morrison, Mark

2006-04-01

Serial analysis of ribosomal sequence tags (SARST) is a recently developed technology that can generate large 16S rRNA gene (rrs) sequence data sets from microbiomes, but there are numerous enzymatic and purification steps required to construct the ribosomal sequence tag (RST) clone libraries. We report here an improved SARST method, which still targets the V1 hypervariable region of rrs genes, but reduces the number of enzymes, oligonucleotides, reagents, and technical steps needed to produce the RST clone libraries. The new method, hereafter referred to as SARST-V1, was used to examine the eubacterial diversity present in community DNA recovered from the microbiome resident in the ovine rumen. The 190 sequenced clones contained 1055 RSTs and no less than 236 unique phylotypes (based on > or = 95% sequence identity) that were assigned to eight different eubacterial phyla. Rarefaction and monomolecular curve analyses predicted that the complete RST clone library contains 99% of the 353 unique phylotypes predicted to exist in this microbiome. When compared with ribosomal intergenic spacer analysis (RISA) of the same community DNA sample, as well as a compilation of nine previously published conventional rrs clone libraries prepared from the same type of samples, the RST clone library provided a more comprehensive characterization of the eubacterial diversity present in rumen microbiomes. As such, SARST-V1 should be a useful tool applicable to comprehensive examination of diversity and composition in microbiomes and offers an affordable, sequence-based method for diversity analysis.

ComM is a hexameric helicase that promotes branch migration during natural transformation in diverse Gram-negative species.

PubMed

Nero, Thomas M; Dalia, Triana N; Wang, Joseph Che-Yen; Kysela, David T; Bochman, Matthew L; Dalia, Ankur B

2018-05-02

Acquisition of foreign DNA by natural transformation is an important mechanism of adaptation and evolution in diverse microbial species. Here, we characterize the mechanism of ComM, a broadly conserved AAA+ protein previously implicated in homologous recombination of transforming DNA (tDNA) in naturally competent Gram-negative bacterial species. In vivo, we found that ComM was required for efficient comigration of linked genetic markers in Vibrio cholerae and Acinetobacter baylyi, which is consistent with a role in branch migration. Also, ComM was particularly important for integration of tDNA with increased sequence heterology, suggesting that its activity promotes the acquisition of novel DNA sequences. In vitro, we showed that purified ComM binds ssDNA, oligomerizes into a hexameric ring, and has bidirectional helicase and branch migration activity. Based on these data, we propose a model for tDNA integration during natural transformation. This study provides mechanistic insight into the enigmatic steps involved in tDNA integration and uncovers the function of a protein required for this conserved mechanism of horizontal gene transfer.

Blow flies as urban wildlife sensors.

PubMed

Hoffmann, Constanze; Merkel, Kevin; Sachse, Andreas; Rodríguez, Pablo; Leendertz, Fabian H; Calvignac-Spencer, Sébastien

2018-05-01

Wildlife detection in urban areas is very challenging. Conventional monitoring techniques such as direct observation are faced with the limitation that urban wildlife is extremely elusive. It was recently shown that invertebrate-derived DNA (iDNA) can be used to assess wildlife diversity in tropical rainforests. Flies, which are ubiquitous and very abundant in most cities, may also be used to detect wildlife in urban areas. In urban ecosystems, however, overwhelming quantities of domestic mammal DNA could completely mask the presence of wild mammal DNA. To test whether urban wild mammals can be detected using fly iDNA, we performed DNA metabarcoding of pools of flies captured in Berlin, Germany, using three combinations of blocking primers. Our results show that domestic animal sequences are, as expected, very dominant in urban environments. Nevertheless, wild mammal sequences can often be retrieved, although they usually only represent a minor fraction of the sequence reads. Fly iDNA metabarcoding is therefore a viable approach for quick scans of urban wildlife diversity. Interestingly, our study also shows that blocking primers can interact with each other in ways that affect the outcome of metabarcoding. We conclude that the use of complex combinations of blocking primers, although potentially powerful, should be carefully planned when designing experiments. © 2018 John Wiley & Sons Ltd.

Enlightenment of Yeast Mitochondrial Homoplasmy: Diversified Roles of Gene Conversion

PubMed Central

Ling, Feng; Mikawa, Tsutomu; Shibata, Takehiko

2011-01-01

Mitochondria have their own genomic DNA. Unlike the nuclear genome, each cell contains hundreds to thousands of copies of mitochondrial DNA (mtDNA). The copies of mtDNA tend to have heterogeneous sequences, due to the high frequency of mutagenesis, but are quickly homogenized within a cell (“homoplasmy”) during vegetative cell growth or through a few sexual generations. Heteroplasmy is strongly associated with mitochondrial diseases, diabetes and aging. Recent studies revealed that the yeast cell has the machinery to homogenize mtDNA, using a common DNA processing pathway with gene conversion; i.e., both genetic events are initiated by a double-stranded break, which is processed into 3′ single-stranded tails. One of the tails is base-paired with the complementary sequence of the recipient double-stranded DNA to form a D-loop (homologous pairing), in which repair DNA synthesis is initiated to restore the sequence lost by the breakage. Gene conversion generates sequence diversity, depending on the divergence between the donor and recipient sequences, especially when it occurs among a number of copies of a DNA sequence family with some sequence variations, such as in immunoglobulin diversification in chicken. MtDNA can be regarded as a sequence family, in which the members tend to be diversified by a high frequency of spontaneous mutagenesis. Thus, it would be interesting to determine why and how double-stranded breakage and D-loop formation induce sequence homogenization in mitochondria and sequence diversification in nuclear DNA. We will review the mechanisms and roles of mtDNA homoplasmy, in contrast to nuclear gene conversion, which diversifies gene and genome sequences, to provide clues toward understanding how the common DNA processing pathway results in such divergent outcomes. PMID:24710143

Historically low mitochondrial DNA diversity in koalas (Phascolarctos cinereus)

PubMed Central

2012-01-01

Background The koala (Phascolarctos cinereus) is an arboreal marsupial that was historically widespread across eastern Australia until the end of the 19th century when it suffered a steep population decline. Hunting for the fur trade, habitat conversion, and disease contributed to a precipitous reduction in koala population size during the late 1800s and early 1900s. To examine the effects of these reductions in population size on koala genetic diversity, we sequenced part of the hypervariable region of mitochondrial DNA (mtDNA) in koala museum specimens collected in the 19th and 20th centuries, hypothesizing that the historical samples would exhibit greater genetic diversity. Results The mtDNA haplotypes present in historical museum samples were identical to haplotypes found in modern koala populations, and no novel haplotypes were detected. Rarefaction analyses suggested that the mtDNA genetic diversity present in the museum samples was similar to that of modern koalas. Conclusions Low mtDNA diversity may have been present in koala populations prior to recent population declines. When considering management strategies, low genetic diversity of the mtDNA hypervariable region may not indicate recent inbreeding or founder events but may reflect an older historical pattern for koalas. PMID:23095716

Wolbachia association with the tsetse fly, Glossina fuscipes fuscipes, reveals high levels of genetic diversity and complex evolutionary dynamics

PubMed Central

2013-01-01

Background Wolbachia pipientis, a diverse group of α-proteobacteria, can alter arthropod host reproduction and confer a reproductive advantage to Wolbachia-infected females (cytoplasmic incompatibility (CI)). This advantage can alter host population genetics because Wolbachia-infected females produce more offspring with their own mitochondrial DNA (mtDNA) haplotypes than uninfected females. Thus, these host haplotypes become common or fixed (selective sweep). Although simulations suggest that for a CI-mediated sweep to occur, there must be a transient phase with repeated initial infections of multiple individual hosts by different Wolbachia strains, this has not been observed empirically. Wolbachia has been found in the tsetse fly, Glossina fuscipes fuscipes, but it is not limited to a single host haplotype, suggesting that CI did not impact its population structure. However, host population genetic differentiation could have been generated if multiple Wolbachia strains interacted in some populations. Here, we investigated Wolbachia genetic variation in G. f. fuscipes populations of known host genetic composition in Uganda. We tested for the presence of multiple Wolbachia strains using Multi-Locus Sequence Typing (MLST) and for an association between geographic region and host mtDNA haplotype using Wolbachia DNA sequence from a variable locus, groEL (heat shock protein 60). Results MLST demonstrated that some G. f. fuscipes carry Wolbachia strains from two lineages. GroEL revealed high levels of sequence diversity within and between individuals (Haplotype diversity = 0.945). We found Wolbachia associated with 26 host mtDNA haplotypes, an unprecedented result. We observed a geographical association of one Wolbachia lineage with southern host mtDNA haplotypes, but it was non-significant (p = 0.16). Though most Wolbachia-infected host haplotypes were those found in the contact region between host mtDNA groups, this association was non-significant (p = 0.17). Conclusions High Wolbachia sequence diversity and the association of Wolbachia with multiple host haplotypes suggest that different Wolbachia strains infected G. f. fuscipes multiple times independently. We suggest that these observations reflect a transient phase in Wolbachia evolution that is influenced by the long gestation and low reproductive output of tsetse. Although G. f. fuscipes is superinfected with Wolbachia, our data does not support that bidirectional CI has influenced host genetic diversity in Uganda. PMID:23384159

Raw Sewage Harbors Diverse Viral Populations

PubMed Central

Cantalupo, Paul G.; Calgua, Byron; Zhao, Guoyan; Hundesa, Ayalkibet; Wier, Adam D.; Katz, Josh P.; Grabe, Michael; Hendrix, Roger W.; Girones, Rosina; Wang, David; Pipas, James M.

2011-01-01

ABSTRACT At this time, about 3,000 different viruses are recognized, but metagenomic studies suggest that these viruses are a small fraction of the viruses that exist in nature. We have explored viral diversity by deep sequencing nucleic acids obtained from virion populations enriched from raw sewage. We identified 234 known viruses, including 17 that infect humans. Plant, insect, and algal viruses as well as bacteriophages were also present. These viruses represented 26 taxonomic families and included viruses with single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), positive-sense ssRNA [ssRNA(+)], and dsRNA genomes. Novel viruses that could be placed in specific taxa represented 51 different families, making untreated wastewater the most diverse viral metagenome (genetic material recovered directly from environmental samples) examined thus far. However, the vast majority of sequence reads bore little or no sequence relation to known viruses and thus could not be placed into specific taxa. These results show that the vast majority of the viruses on Earth have not yet been characterized. Untreated wastewater provides a rich matrix for identifying novel viruses and for studying virus diversity. Importance At this time, virology is focused on the study of a relatively small number of viral species. Specific viruses are studied either because they are easily propagated in the laboratory or because they are associated with disease. The lack of knowledge of the size and characteristics of the viral universe and the diversity of viral genomes is a roadblock to understanding important issues, such as the origin of emerging pathogens and the extent of gene exchange among viruses. Untreated wastewater is an ideal system for assessing viral diversity because virion populations from large numbers of individuals are deposited and because raw sewage itself provides a rich environment for the growth of diverse host species and thus their viruses. These studies suggest that the viral universe is far more vast and diverse than previously suspected. PMID:21972239

Screening and Characterization of RAPD Markers in Viscerotropic Leishmania Parasites

PubMed Central

Mkada–Driss, Imen; Talbi, Chiraz; Guerbouj, Souheila; Driss, Mehdi; Elamine, Elwaleed M.; Cupolillo, Elisa; Mukhtar, Moawia M.; Guizani, Ikram

2014-01-01

Visceral leishmaniasis (VL) is mainly due to the Leishmania donovani complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL Leishmania parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 L. donovani, L. archibaldi and L. infantum strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5′ end transversions, and presence of inter– and intra– taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between L. infantum and L. donovani. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents. PMID:25313833

Molecular genetic diversity in populations of the stingless bee Plebeia remota: A case study

PubMed Central

de Oliveira Francisco, Flávio; Santiago, Leandro Rodrigues; Arias, Maria Cristina

2013-01-01

Genetic diversity is a major component of the biological diversity of an ecosystem. The survival of a population may be seriously threatened if its genetic diversity values are low. In this work, we measured the genetic diversity of the stingless bee Plebeia remota based on molecular data obtained by analyzing 15 microsatellite loci and sequencing two mitochondrial genes. Population structure and genetic diversity differed depending on the molecular marker analyzed: microsatellites showed low population structure and moderate to high genetic diversity, while mitochondrial DNA (mtDNA) showed high population structure and low diversity in three populations. Queen philopatry and male dispersal behavior are discussed as the main reasons for these findings. PMID:23569417

Molecular genetic diversity in populations of the stingless bee Plebeia remota: A case study.

PubMed

de Oliveira Francisco, Flávio; Santiago, Leandro Rodrigues; Arias, Maria Cristina

2013-03-01

Genetic diversity is a major component of the biological diversity of an ecosystem. The survival of a population may be seriously threatened if its genetic diversity values are low. In this work, we measured the genetic diversity of the stingless bee Plebeia remota based on molecular data obtained by analyzing 15 microsatellite loci and sequencing two mitochondrial genes. Population structure and genetic diversity differed depending on the molecular marker analyzed: microsatellites showed low population structure and moderate to high genetic diversity, while mitochondrial DNA (mtDNA) showed high population structure and low diversity in three populations. Queen philopatry and male dispersal behavior are discussed as the main reasons for these findings.

Grassland management regimens reduce small-scale heterogeneity and species diversity of beta-proteobacterial ammonia pxidizer populations.

PubMed

Webster, Gordon; Embley, T Martin; Prosser, James I

2002-01-01

The impact of soil management practices on ammonia oxidizer diversity and spatial heterogeneity was determined in improved (addition of N fertilizer), unimproved (no additions), and semi-improved (intermediate management) grassland pastures at the Sourhope Research Station in Scotland. Ammonia oxidizer diversity within each grassland soil was assessed by PCR amplification of microbial community DNA with both ammonia oxidizer-specific, 16S rRNA gene (rDNA) and functional, amoA, gene primers. PCR products were analysed by denaturing gradient gel electrophoresis, phylogenetic analysis of partial 16S rDNA and amoA sequences, and hybridization with ammonia oxidizer-specific oligonucleotide probes. Ammonia oxidizer populations in unimproved soils were more diverse than those in improved soils and were dominated by organisms representing Nitrosospira clusters 1 and 3 and Nitrosomonas cluster 7 (closely related phylogenetically to Nitrosomonas europaea). Improved soils were only dominated by Nitrosospira cluster 3 and Nitrosomonas cluster 7. These differences were also reflected in functional gene (amoA) diversity, with amoA gene sequences of both Nitrosomonas and Nitrosospira species detected. Replicate 0.5-g samples of unimproved soil demonstrated significant spatial heterogeneity in 16S rDNA-defined ammonia oxidizer clusters, which was reflected in heterogeneity in ammonium concentration and pH. Heterogeneity in soil characteristics and ammonia oxidizer diversity were lower in improved soils. The results therefore demonstrate significant effects of soil management on diversity and heterogeneity of ammonia oxidizer populations that are related to similar changes in relevant soil characteristics.

A new family of satellite DNA sequences as a major component of centromeric heterochromatin in owls (Strigiformes).

PubMed

Yamada, Kazuhiko; Nishida-Umehara, Chizuko; Matsuda, Yoichi

2004-03-01

We isolated a new family of satellite DNA sequences from HaeIII- and EcoRI-digested genomic DNA of the Blakiston's fish owl ( Ketupa blakistoni). The repetitive sequences were organized in tandem arrays of the 174 bp element, and localized to the centromeric regions of all macrochromosomes, including the Z and W chromosomes, and microchromosomes. This hybridization pattern was consistent with the distribution of C-band-positive centromeric heterochromatin, and the satellite DNA sequences occupied 10% of the total genome as a major component of centromeric heterochromatin. The sequences were homogenized between macro- and microchromosomes in this species, and therefore intraspecific divergence of the nucleotide sequences was low. The 174 bp element cross-hybridized to the genomic DNA of six other Strigidae species, but not to that of the Tytonidae, suggesting that the satellite DNA sequences are conserved in the same family but fairly divergent between the different families in the Strigiformes. Secondly, the centromeric satellite DNAs were cloned from eight Strigidae species, and the nucleotide sequences of 41 monomer fragments were compared within and between species. Molecular phylogenetic relationships of the nucleotide sequences were highly correlated with both the taxonomy based on morphological traits and the phylogenetic tree constructed by DNA-DNA hybridization. These results suggest that the satellite DNA sequence has evolved by concerted evolution in the Strigidae and that it is a good taxonomic and phylogenetic marker to examine genetic diversity between Strigiformes species.

Genetic variations in two seahorse species (Hippocampus mohnikei and Hippocampus trimaculatus): evidence for middle Pleistocene population expansion.

PubMed

Zhang, Yanhong; Pham, Nancy Kim; Zhang, Huixian; Lin, Junda; Lin, Qiang

2014-01-01

Population genetic of seahorses is confidently influenced by their species-specific ecological requirements and life-history traits. In the present study, partial sequences of mitochondrial cytochrome b (cytb) and control region (CR) were obtained from 50 Hippocampus mohnikei and 92 H. trimaculatus from four zoogeographical zones. A total of 780 base pairs of cytb gene were sequenced to characterize mitochondrial DNA (mtDNA) diversity. The mtDNA marker revealed high haplotype diversity, low nucleotide diversity, and a lack of population structure across both populations of H. mohnikei and H. trimaculatus. A neighbour-joining (NJ) tree of cytb gene sequences showed that H. mohnikei haplotypes formed one cluster. A maximum likelihood (ML) tree of cytb gene sequences showed that H. trimaculatus belonged to one lineage. The star-like pattern median-joining network of cytb and CR markers indicated a previous demographic expansion of H. mohnikei and H. trimaculatus. The cytb and CR data sets exhibited a unimodal mismatch distribution, which may have resulted from population expansion. Mismatch analysis suggested that the expansion was initiated about 276,000 years ago for H. mohnikei and about 230,000 years ago for H. trimaculatus during the middle Pleistocene period. This study indicates a possible signature of genetic variation and population expansion in two seahorses under complex marine environments.

Pay Attention to the Overlooked Cryptic Diversity in Existing Barcoding Data: the Case of Mollusca with Character-Based DNA Barcoding.

PubMed

Zou, Shanmei; Li, Qi

2016-06-01

With the global biodiversity crisis, DNA barcoding aims for fast species identification and cryptic species diversity revelation. For more than 10 years, large amounts of DNA barcode data have been accumulating in publicly available databases, most of which were conducted by distance or tree-building methods that have often been argued, especially for cryptic species revelation. In this context, overlooked cryptic diversity may exist in the available barcoding data. The character-based DNA barcoding, however, has a good chance for detecting the overlooked cryptic diversity. In this study, marine mollusk was as the ideal case for detecting the overlooked potential cryptic species from existing cytochrome c oxidase I (COI) sequences with character-based DNA barcode. A total of 1081 COI sequences of mollusks, belonging to 176 species of 25 families of Gastropoda, Cephalopoda, and Lamellibranchia, were conducted by character analysis. As a whole, the character-based barcoding results were consistent with previous distance and tree-building analysis for species discrimination. More importantly, quite a number of species analyzed were divided into distinct clades with unique diagnostical characters. Based on the concept of cryptic species revelation of character-based barcoding, these species divided into separate taxonomic groups might be potential cryptic species. The detection of the overlooked potential cryptic diversity proves that the character-based barcoding mode possesses more advantages of revealing cryptic biodiversity. With the development of DNA barcoding, making the best use of barcoding data is worthy of our attention for species conservation.

Artificial Intelligence, DNA Mimicry, and Human Health.

PubMed

Stefano, George B; Kream, Richard M

2017-08-14

The molecular evolution of genomic DNA across diverse plant and animal phyla involved dynamic registrations of sequence modifications to maintain existential homeostasis to increasingly complex patterns of environmental stressors. As an essential corollary, driver effects of positive evolutionary pressure are hypothesized to effect concerted modifications of genomic DNA sequences to meet expanded platforms of regulatory controls for successful implementation of advanced physiological requirements. It is also clearly apparent that preservation of updated registries of advantageous modifications of genomic DNA sequences requires coordinate expansion of convergent cellular proofreading/error correction mechanisms that are encoded by reciprocally modified genomic DNA. Computational expansion of operationally defined DNA memory extends to coordinate modification of coding and previously under-emphasized noncoding regions that now appear to represent essential reservoirs of untapped genetic information amenable to evolutionary driven recruitment into the realm of biologically active domains. Additionally, expansion of DNA memory potential via chemical modification and activation of noncoding sequences is targeted to vertical augmentation and integration of an expanded cadre of transcriptional and epigenetic regulatory factors affecting linear coding of protein amino acid sequences within open reading frames.

Towards a comprehensive barcode library for arctic life - Ephemeroptera, Plecoptera, and Trichoptera of Churchill, Manitoba, Canada

PubMed Central

2009-01-01

Background This study reports progress in assembling a DNA barcode reference library for Ephemeroptera, Plecoptera, and Trichoptera ("EPTs") from a Canadian subarctic site, which is the focus of a comprehensive biodiversity inventory using DNA barcoding. These three groups of aquatic insects exhibit a moderate level of species diversity, making them ideal for testing the feasibility of DNA barcoding for routine biotic surveys. We explore the correlation between the morphological species delineations, DNA barcode-based haplotype clusters delimited by a sequence threshold (2%), and a threshold-free approach to biodiversity quantification--phylogenetic diversity. Results A DNA barcode reference library is built for 112 EPT species for the focal region, consisting of 2277 COI sequences. Close correspondence was found between EPT morphospecies and haplotype clusters as designated using a standard threshold value. Similarly, the shapes of taxon accumulation curves based upon haplotype clusters were very similar to those generated using phylogenetic diversity accumulation curves, but were much more computationally efficient. Conclusion The results of this study will facilitate other lines of research on northern EPTs and also bode well for rapidly conducting initial biodiversity assessments in unknown EPT faunas. PMID:20003245

High Bacterial Diversity in Permanently Cold Marine Sediments

PubMed Central

Ravenschlag, Katrin; Sahm, Kerstin; Pernthaler, Jakob; Amann, Rudolf

1999-01-01

A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. Screening 353 clones by dot blot hybridization with group-specific oligonucleotide probes suggested a predominance of sequences related to bacteria of the sulfur cycle (43.4% potential sulfate reducers). Within this fraction, the major cluster (19.0%) was affiliated with Desulfotalea sp. and other closely related psychrophilic sulfate reducers isolated from the same habitat. The cloned sequences showed between 93 and 100% similarity to these bacteria. Two additional groups were frequently encountered: 13% of the clones were related to Desulfuromonas palmitatis, and a second group was affiliated with Myxobacteria spp. and Bdellovibrio spp. Many clones (18.1%) belonged to the γ subclass of the class Proteobacteria and were closest to symbiotic or free-living sulfur oxidizers. Probe target groups were further characterized by amplified rDNA restriction analysis to determine diversity within the groups and within the clone library. Rarefaction analysis suggested that the total diversity assessed by 16S rDNA analysis was very high in these permanently cold sediments and was only partially revealed by screening of 353 clones. PMID:10473405

Reduced Mtdna Diversity in the Ngobe Amerinds of Panama

PubMed Central

Kolman, C. J.; Bermingham, E.; Cooke, R.; Ward, R. H.; Arias, T. D.; Guionneau-Sinclair, F.

1995-01-01

Mitochondrial DNA (mtDNA) haplotype diversity was determined for 46 Ngobe Amerinds sampled widely across their geographic range in western Panama. The Ngobe data were compared with mtDNA control region I sequences from two additional Amerind groups located at the northern and southern extremes of Amerind distribution, the Nuu-Chah-Nulth of the Pacific Northwest and the Chilean Mapuche and from one Na-Dene group, the Haida of the Pacific Northwest. The Ngobe exhibit the lowest mtDNA control region sequence diversity yet reported for an Amerind group. Moreover, they carry only two of the four Amerind founding lineages first described by Wallace and coworkers. We posit that the Ngobe passed through a population bottleneck caused by ethnogenesis from a small founding population and/or European conquest and colonization. Dating of the Ngobe population expansion using the HARPENDING et al. approach to the analysis of pairwise genetic differences indicates a Ngobe expansion at roughly 6800 years before present (range: 1850-14,000 years before present), a date more consistent with a bottleneck at Chibcha ethnogenesis than a conquest-based event. PMID:7635293

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

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

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE PAGES

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; ...

2016-03-09

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

Identification of Active Bacterial Communities in Drinking Water Using 16S rRNA-Based Sequence Analyses

EPA Science Inventory

DNA-based methods have considerably increased our understanding of the bacterial diversity of water distribution systems (WDS). However, as DNA may persist after cell death, the use of DNA-based methods cannot be used to describe metabolically-active microbes. In contrast, intra...

Evaluating the Impact of DNA Extraction Method on the Representation of Human Oral Bacterial and Fungal Communities

PubMed Central

Biswas, Kristi; Taylor, Michael W.; Gear, Kim

2017-01-01

The application of high-throughput, next-generation sequencing technologies has greatly improved our understanding of the human oral microbiome. While deciphering this diverse microbial community using such approaches is more accurate than traditional culture-based methods, experimental bias introduced during critical steps such as DNA extraction may compromise the results obtained. Here, we systematically evaluate four commonly used microbial DNA extraction methods (MoBio PowerSoil® DNA Isolation Kit, QIAamp® DNA Mini Kit, Zymo Bacterial/Fungal DNA Mini PrepTM, phenol:chloroform-based DNA isolation) based on the following criteria: DNA quality and yield, and microbial community structure based on Illumina amplicon sequencing of the V3–V4 region of the 16S rRNA gene of bacteria and the internal transcribed spacer (ITS) 1 region of fungi. Our results indicate that DNA quality and yield varied significantly with DNA extraction method. Representation of bacterial genera in plaque and saliva samples did not significantly differ across DNA extraction methods and DNA extraction method showed no effect on the recovery of fungal genera from plaque. By contrast, fungal diversity from saliva was affected by DNA extraction method, suggesting that not all protocols are suitable to study the salivary mycobiome. PMID:28099455

How many novel eukaryotic 'kingdoms'? Pitfalls and limitations of environmental DNA surveys

PubMed Central

Berney, Cédric; Fahrni, José; Pawlowski, Jan

2004-01-01

Background Over the past few years, the use of molecular techniques to detect cultivation-independent, eukaryotic diversity has proven to be a powerful approach. Based on small-subunit ribosomal RNA (SSU rRNA) gene analyses, these studies have revealed the existence of an unexpected variety of new phylotypes. Some of them represent novel diversity in known eukaryotic groups, mainly stramenopiles and alveolates. Others do not seem to be related to any molecularly described lineage, and have been proposed to represent novel eukaryotic kingdoms. In order to review the evolutionary importance of this novel high-level eukaryotic diversity critically, and to test the potential technical and analytical pitfalls and limitations of eukaryotic environmental DNA surveys (EES), we analysed 484 environmental SSU rRNA gene sequences, including 81 new sequences from sediments of the small river, the Seymaz (Geneva, Switzerland). Results Based on a detailed screening of an exhaustive alignment of eukaryotic SSU rRNA gene sequences and the phylogenetic re-analysis of previously published environmental sequences using Bayesian methods, our results suggest that the number of novel higher-level taxa revealed by previously published EES was overestimated. Three main sources of errors are responsible for this situation: (1) the presence of undetected chimeric sequences; (2) the misplacement of several fast-evolving sequences; and (3) the incomplete sampling of described, but yet unsequenced eukaryotes. Additionally, EES give a biased view of the diversity present in a given biotope because of the difficult amplification of SSU rRNA genes in some taxonomic groups. Conclusions Environmental DNA surveys undoubtedly contribute to reveal many novel eukaryotic lineages, but there is no clear evidence for a spectacular increase of the diversity at the kingdom level. After re-analysis of previously published data, we found only five candidate lineages of possible novel high-level eukaryotic taxa, two of which comprise several phylotypes that were found independently in different studies. To ascertain their taxonomic status, however, the organisms themselves have now to be identified. PMID:15176975

DNA barcoding as an aid for species identification in austral black flies (Insecta: Diptera: Simuliidae).

PubMed

Hernández-Triana, Luis M; Montes De Oca, Fernanda; Prosser, Sean W J; Hebert, Paul D N; Gregory, T Ryan; McMurtrie, Shelley

2017-04-01

In this paper, the utility of a partial sequence of the COI gene, the DNA barcoding region, for the identification of species of black flies in the austral region was assessed. Twenty-eight morphospecies were analyzed: eight of the genus Austrosimulium (four species in the subgenus Austrosimulium s. str., three species in the subgenus Novaustrosimulium, and one species unassigned to subgenus), two of the genus Cnesia, eight of Gigantodax, three of Paracnephia, one of Paraustrosimulium, and six of Simulium (subgenera Morops, Nevermannia, and Pternaspatha). The neighbour-joining tree derived from the DNA barcode sequences grouped most specimens according to species or species groups recognized by morphotaxonomic studies. Intraspecific sequence divergences within morphologically distinct species ranged from 0% to 1.8%, while higher divergences (2%-4.2%) in certain species suggested the presence of cryptic diversity. The existence of well-defined groups within S. simile revealed the likely inclusion of cryptic diversity. DNA barcodes also showed that specimens identified as C. dissimilis, C. nr. pussilla, and C. ornata might be conspecific, suggesting possible synonymy. DNA barcoding combined with a sound morphotaxonomic framework would provide an effective approach for the identification of black flies in the region.

Diversity in Requirement of Genetic and Epigenetic Factors for Centromere Function in Fungi ▿

PubMed Central

Roy, Babhrubahan; Sanyal, Kaustuv

2011-01-01

A centromere is a chromosomal region on which several proteins assemble to form the kinetochore. The centromere-kinetochore complex helps in the attachment of chromosomes to spindle microtubules to mediate segregation of chromosomes to daughter cells during mitosis and meiosis. In several budding yeast species, the centromere forms in a DNA sequence-dependent manner, whereas in most other fungi, factors other than the DNA sequence also determine the centromere location, as centromeres were able to form on nonnative sequences (neocentromeres) when native centromeres were deleted in engineered strains. Thus, in the absence of a common DNA sequence, the cues that have facilitated centromere formation on a specific DNA sequence for millions of years remain a mystery. Kinetochore formation is facilitated by binding of a centromere-specific histone protein member of the centromeric protein A (CENP-A) family that replaces a canonical histone H3 to form a specialized centromeric chromatin structure. However, the process of kinetochore formation on the rapidly evolving and seemingly diverse centromere DNAs in different fungal species is largely unknown. More interestingly, studies in various yeasts suggest that the factors required for de novo centromere formation (establishment) may be different from those required for maintenance (propagation) of an already established centromere. Apart from the DNA sequence and CENP-A, many other factors, such as posttranslational modification (PTM) of histones at centric and pericentric chromatin, RNA interference, and DNA methylation, are also involved in centromere formation, albeit in a species-specific manner. In this review, we discuss how several genetic and epigenetic factors influence the evolution of structure and function of centromeres in fungal species. PMID:21908596

Environmental DNA sequencing primers for eutardigrades and bdelloid rotifers

PubMed Central

2009-01-01

Background The time it takes to isolate individuals from environmental samples and then extract DNA from each individual is one of the problems with generating molecular data from meiofauna such as eutardigrades and bdelloid rotifers. The lack of consistent morphological information and the extreme abundance of these classes makes morphological identification of rare, or even common cryptic taxa a large and unwieldy task. This limits the ability to perform large-scale surveys of the diversity of these organisms. Here we demonstrate a culture-independent molecular survey approach that enables the generation of large amounts of eutardigrade and bdelloid rotifer sequence data directly from soil. Our PCR primers, specific to the 18s small-subunit rRNA gene, were developed for both eutardigrades and bdelloid rotifers. Results The developed primers successfully amplified DNA of their target organism from various soil DNA extracts. This was confirmed by both the BLAST similarity searches and phylogenetic analyses. Tardigrades showed much better phylogenetic resolution than bdelloids. Both groups of organisms exhibited varying levels of endemism. Conclusion The development of clade-specific primers for characterizing eutardigrades and bdelloid rotifers from environmental samples should greatly increase our ability to characterize the composition of these taxa in environmental samples. Environmental sequencing as shown here differs from other molecular survey methods in that there is no need to pre-isolate the organisms of interest from soil in order to amplify their DNA. The DNA sequences obtained from methods that do not require culturing can be identified post-hoc and placed phylogenetically as additional closely related sequences are obtained from morphologically identified conspecifics. Our non-cultured environmental sequence based approach will be able to provide a rapid and large-scale screening of the presence, absence and diversity of Bdelloidea and Eutardigrada in a variety of soils. PMID:20003362

Physical mapping of repetitive DNA suggests 2n reduction in Amazon turtles Podocnemis (Testudines: Podocnemididae)

PubMed Central

Cavalcante, Manoella Gemaque; Bastos, Carlos Eduardo Matos Carvalho; Nagamachi, Cleusa Yoshiko; Pieczarka, Julio Cesar; Vicari, Marcelo Ricardo; Noronha, Renata Coelho Rodrigues

2018-01-01

Cytogenetic studies show that there is great karyotypic diversity in order Testudines (2n = 26–68), and that this may be mainly attributed to the presence/absence of microchromosomes. Members of the Podocnemididae family have the smallest diploid numbers of this order (2n = 26–28), which may be a derived condition of the group. Diverse studies suggest that repetitive-DNA-rich sites generally act as hotspots for double-strand breaks and chromosomal reorganization. In this context, we used fluorescent in situ hybridization (FISH) to map telomeric sequences (TTAGGG)n, 45S rDNA, and the genes encoding histones H1 and H3 in two species of genus Podocnemis. We also observed conservation of the 45S rDNA and H1 histone sequences (probable case of conserved synteny), but multiple conserved and non-conserved clusters of H3 genes, which colocalized with the interstitial telomeric sequences in the Podocnemis genome. Our results suggest that fusions have occurred between macro and microchromosomes or between microchromosomes, leading to the observed reduction in diploid number in the family Podocnemididae. PMID:29813087

Physical mapping of repetitive DNA suggests 2n reduction in Amazon turtles Podocnemis (Testudines: Podocnemididae).

PubMed

Cavalcante, Manoella Gemaque; Bastos, Carlos Eduardo Matos Carvalho; Nagamachi, Cleusa Yoshiko; Pieczarka, Julio Cesar; Vicari, Marcelo Ricardo; Noronha, Renata Coelho Rodrigues

2018-01-01

Cytogenetic studies show that there is great karyotypic diversity in order Testudines (2n = 26-68), and that this may be mainly attributed to the presence/absence of microchromosomes. Members of the Podocnemididae family have the smallest diploid numbers of this order (2n = 26-28), which may be a derived condition of the group. Diverse studies suggest that repetitive-DNA-rich sites generally act as hotspots for double-strand breaks and chromosomal reorganization. In this context, we used fluorescent in situ hybridization (FISH) to map telomeric sequences (TTAGGG)n, 45S rDNA, and the genes encoding histones H1 and H3 in two species of genus Podocnemis. We also observed conservation of the 45S rDNA and H1 histone sequences (probable case of conserved synteny), but multiple conserved and non-conserved clusters of H3 genes, which colocalized with the interstitial telomeric sequences in the Podocnemis genome. Our results suggest that fusions have occurred between macro and microchromosomes or between microchromosomes, leading to the observed reduction in diploid number in the family Podocnemididae.

Molecular Analysis of Date Palm Genetic Diversity Using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSRs).

PubMed

El Sharabasy, Sherif F; Soliman, Khaled A

2017-01-01

The date palm is an ancient domesticated plant with great diversity and has been cultivated in the Middle East and North Africa for at last 5000 years. Date palm cultivars are classified based on the fruit moisture content, as dry, semidry, and soft dates. There are a number of biochemical and molecular techniques available for characterization of the date palm variation. This chapter focuses on the DNA-based markers random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) techniques, in addition to biochemical markers based on isozyme analysis. These techniques coupled with appropriate statistical tools proved useful for determining phylogenetic relationships among date palm cultivars and provide information resources for date palm gene banks.

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae).

PubMed

Begum, Rabeya; Zakrzewski, Falk; Menzel, Gerhard; Weber, Beatrice; Alam, Sheikh Shamimul; Schmidt, Thomas

2013-07-01

The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100-500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S-5·8S-25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.

High-resolution mapping, characterization, and optimization of autonomously replicating sequences in yeast

PubMed Central

Liachko, Ivan; Youngblood, Rachel A.; Keich, Uri; Dunham, Maitreya J.

2013-01-01

DNA replication origins are necessary for the duplication of genomes. In addition, plasmid-based expression systems require DNA replication origins to maintain plasmids efficiently. The yeast autonomously replicating sequence (ARS) assay has been a valuable tool in dissecting replication origin structure and function. However, the dearth of information on origins in diverse yeasts limits the availability of efficient replication origin modules to only a handful of species and restricts our understanding of origin function and evolution. To enable rapid study of origins, we have developed a sequencing-based suite of methods for comprehensively mapping and characterizing ARSs within a yeast genome. Our approach finely maps genomic inserts capable of supporting plasmid replication and uses massively parallel deep mutational scanning to define molecular determinants of ARS function with single-nucleotide resolution. In addition to providing unprecedented detail into origin structure, our data have allowed us to design short, synthetic DNA sequences that retain maximal ARS function. These methods can be readily applied to understand and modulate ARS function in diverse systems. PMID:23241746

HLA DNA Sequence Variation among Human Populations: Molecular Signatures of Demographic and Selective Events

PubMed Central

Buhler, Stéphane; Sanchez-Mazas, Alicia

2011-01-01

Molecular differences between HLA alleles vary up to 57 nucleotides within the peptide binding coding region of human Major Histocompatibility Complex (MHC) genes, but it is still unclear whether this variation results from a stochastic process or from selective constraints related to functional differences among HLA molecules. Although HLA alleles are generally treated as equidistant molecular units in population genetic studies, DNA sequence diversity among populations is also crucial to interpret the observed HLA polymorphism. In this study, we used a large dataset of 2,062 DNA sequences defined for the different HLA alleles to analyze nucleotide diversity of seven HLA genes in 23,500 individuals of about 200 populations spread worldwide. We first analyzed the HLA molecular structure and diversity of these populations in relation to geographic variation and we further investigated possible departures from selective neutrality through Tajima's tests and mismatch distributions. All results were compared to those obtained by classical approaches applied to HLA allele frequencies. Our study shows that the global patterns of HLA nucleotide diversity among populations are significantly correlated to geography, although in some specific cases the molecular information reveals unexpected genetic relationships. At all loci except HLA-DPB1, populations have accumulated a high proportion of very divergent alleles, suggesting an advantage of heterozygotes expressing molecularly distant HLA molecules (asymmetric overdominant selection model). However, both different intensities of selection and unequal levels of gene conversion may explain the heterogeneous mismatch distributions observed among the loci. Also, distinctive patterns of sequence divergence observed at the HLA-DPB1 locus suggest current neutrality but old selective pressures on this gene. We conclude that HLA DNA sequences advantageously complement HLA allele frequencies as a source of data used to explore the genetic history of human populations, and that their analysis allows a more thorough investigation of human MHC molecular evolution. PMID:21408106

DNA-based approaches to identify forest fungi in Pacific Islands: A pilot study

Treesearch

Anna E. Case; Sara M. Ashiglar; Phil G. Cannon; Ernesto P. Militante; Edwin R. Tadiosa; Mutya Quintos-Manalo; Nelson M. Pampolina; John W. Hanna; Fred E. Brooks; Amy L. Ross-Davis; Mee-Sook Kim; Ned B. Klopfenstein

2013-01-01

DNA-based diagnostics have been successfully used to characterize diverse forest fungi (e.g., Hoff et al. 2004, Kim et al. 2006, Glaeser & Lindner 2011). DNA sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) regions of nuclear ribosomal DNA (rDNA) has proved especially useful (Sonnenberg et al. 2007, Seifert 2009, Schoch et al. 2012) for...

Genetic diversity and classification of Tibetan yak populations based on the mtDNA COIII gene.

PubMed

Song, Q Q; Chai, Z X; Xin, J W; Zhao, S J; Ji, Q M; Zhang, C F; Ma, Z J; Zhong, J C

2015-03-13

To determine the level of genetic diversity and phylogenetic relationships among Tibetan yak populations, the mitochondrial DNA cytochrome c oxidase subunit 3 (COIII) genes of 378 yak individuals from 16 populations were analyzed in this study. The results showed that the length of cytochrome c oxidase subunit 3 gene sequences was 781 bp, with nucleotide frequencies of 29.2, 29.4, 26.1, and 15.2% for T, C, A, and G, respectively. A total of 26 haplotypes were identified, with 69 polymorphic sites, including 11 parsimony-informative sites and 58 single-nucleotide polymorphism sites. No deletions/insertions were found in sequence comparison, indicating that nucleotide mutation types were transitions and transversions. Haplotype and nucleotide diversities were 0.562 and 0.00138, respectively, indicating a high level of genetic diversity in Tibetan yak populations. Phylogenetic relationship analysis indicated that Tibetan yak populations are divided into 2 groups.

The Vaginal Eukaryotic DNA Virome and Preterm Birth.

PubMed

Wylie, Kristine M; Wylie, Todd N; Cahill, Alison G; Macones, George A; Tuuli, Methodius G; Stout, Molly J

2018-05-05

Despite decades of attempts to link infectious agents to preterm birth, an exact causative microbe or community of microbes remains elusive. Culture-independent sequencing of vaginal bacterial communities demonstrates community characteristics are associated with preterm birth, although none are specific enough to apply clinically. Viruses are important components of the vaginal microbiome and have dynamic relationships with vaginal bacterial communities. We hypothesized that vaginal eukaryotic DNA viral communities (the "vaginal virome") either alone or in the context of bacterial communities are associated with preterm birth. The objective of this study was to use high-throughput sequencing to examine the vaginal eukaryotic DNA virome in a cohort of pregnant women and examine associations between vaginal community characteristics and preterm birth. This is a nested case-control study within a prospective cohort study of women with singleton pregnancies, not on supplemental progesterone, and without cervical cerclage in situ. Serial mid-vaginal swabs were obtained at routine prenatal visits. DNA was extracted, bacterial communities were characterized by 16S rRNA gene sequencing, and eukaryotic viral communities were characterized by enrichment of viral nucleic acid with the ViroCap targeted sequence capture panel followed by nucleic acid sequencing. Viral communities were analyzed according to presence/absence of viruses, diversity, dynamics over time, and association with bacterial community data obtained from the same specimens. Sixty subjects contributed 128 vaginal swabs longitudinally across pregnancy. Twenty-four patients delivered preterm. Participants were predominantly African-American (65%). Six families of eukaryotic DNA viruses were detected in the vaginal samples. At least 1 virus was detected in 80% of women. No specific virus or group of viruses was associated with preterm delivery. Higher viral richness was significantly associated with preterm delivery in the full group and in the African American subgroup (P=0.0005 and P=0.0003, respectively). Having both high bacterial diversity and high viral diversity in the first trimester was associated with the highest risk for preterm birth. Higher vaginal viral diversity is associated with preterm birth. Changes in vaginal virome diversity appear similar to changes in the vaginal bacterial microbiome over pregnancy, suggesting that underlying physiology of pregnancy may regulate both bacterial and viral communities. Copyright © 2018 Elsevier Inc. All rights reserved.

Diversity and function in microbial mats from the Lucky Strike hydrothermal vent field.

PubMed

Crépeau, Valentin; Cambon Bonavita, Marie-Anne; Lesongeur, Françoise; Randrianalivelo, Henintsoa; Sarradin, Pierre-Marie; Sarrazin, Jozée; Godfroy, Anne

2011-06-01

Diversity and function in microbial mats from the Lucky Strike hydrothermal vent field (Mid-Atlantic Ridge) were investigated using molecular approaches. DNA and RNA were extracted from mat samples overlaying hydrothermal deposits and Bathymodiolus azoricus mussel assemblages. We constructed and analyzed libraries of 16S rRNA gene sequences and sequences of functional genes involved in autotrophic carbon fixation [forms I and II RuBisCO (cbbL/M), ATP-citrate lyase B (aclB)]; methane oxidation [particulate methane monooxygenase (pmoA)] and sulfur oxidation [adenosine-5'-phosphosulfate reductase (aprA) and soxB]. To gain new insights into the relationships between mats and mussels, we also used new domain-specific 16S rRNA gene primers targeting Bathymodiolus sp. symbionts. All identified archaeal sequences were affiliated with a single group: the marine group 1 Thaumarchaeota. In contrast, analyses of bacterial sequences revealed much higher diversity, although two phyla Proteobacteria and Bacteroidetes were largely dominant. The 16S rRNA gene sequence library revealed that species affiliated to Beggiatoa Gammaproteobacteria were the dominant active population. Analyses of DNA and RNA functional gene libraries revealed a diverse and active chemolithoautotrophic population. Most of these sequences were affiliated with Gammaproteobacteria, including hydrothermal fauna symbionts, Thiotrichales and Methylococcales. PCR and reverse transcription-PCR using 16S rRNA gene primers targeted to Bathymodiolus sp. symbionts revealed sequences affiliated with both methanotrophic and thiotrophic endosymbionts. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Patterns of population subdivision, gene flow and genetic variability in the African wild dog (Lycaon pictus).

PubMed

Girman, D J; Vilà, C; Geffen, E; Creel, S; Mills, M G; McNutt, J W; Ginsberg, J; Kat, P W; Mamiya, K H; Wayne, R K

2001-07-01

African wild dogs are large, highly mobile carnivores that are known to disperse over considerable distances and are rare throughout much of their geographical range. Consequently, genetic variation within and differentiation between geographically separated populations is predicted to be minimal. We determined the genetic diversity of mitochondrial DNA (mtDNA) control region sequences and microsatellite loci in seven populations of African wild dogs. Analysis of mtDNA nucleotide diversity suggests that, historically, wild dog populations have been small relative to other large carnivores. However, population declines due to recent habitat loss have not caused a dramatic reduction in genetic diversity. We found one historical and eight recent mtDNA genotypes in 280 individuals that defined two highly divergent clades. In contrast to a previous, more limited, mtDNA analysis, sequences from these clades are not geographically restricted to eastern or southern African populations. Rather, we found a large admixture zone spanning populations from Botswana, Zimbabwe and south-eastern Tanzania. Mitochondrial and microsatellite differentiation between populations was significant and unique mtDNA genotypes and alleles characterized the populations. However, gene flow estimates (Nm) based on microsatellite data were generally greater than one migrant per generation. In contrast, gene flow estimates based on the mtDNA control region were lower than expected given differences in the mode of inheritance of mitochondrial and nuclear markers which suggests a male bias in long-distance dispersal.

Rhizobial characterization in revegetated areas after bauxite mining.

PubMed

Borges, Wardsson Lustrino; Prin, Yves; Ducousso, Marc; Le Roux, Christine; de Faria, Sergio Miana

2016-01-01

Little is known regarding how the increased diversity of nitrogen-fixing bacteria contributes to the productivity and diversity of plants in complex communities. However, some authors have shown that the presence of a diverse group of nodulating bacteria is required for different plant species to coexist. A better understanding of the plant symbiotic organism diversity role in natural ecosystems can be extremely useful to define recovery strategies of environments that were degraded by human activities. This study used ARDRA, BOX-PCR fingerprinting and sequencing of the 16S rDNA gene to assess the diversity of root nodule nitrogen-fixing bacteria in former bauxite mining areas that were replanted in 1981, 1985, 1993, 1998, 2004 and 2006 and in a native forest. Among the 12 isolates for which the 16S rDNA gene was partially sequenced, eight, three and one isolate(s) presented similarity with sequences of the genera Bradyrhizobium, Rhizobium and Mesorhizobium, respectively. The richness, Shannon and evenness indices were the highest in the area that was replanted the earliest (1981) and the lowest in the area that was replanted most recently (2006). Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

i-rDNA: alignment-free algorithm for rapid in silico detection of ribosomal gene fragments from metagenomic sequence data sets.

PubMed

Mohammed, Monzoorul Haque; Ghosh, Tarini Shankar; Chadaram, Sudha; Mande, Sharmila S

2011-11-30

Obtaining accurate estimates of microbial diversity using rDNA profiling is the first step in most metagenomics projects. Consequently, most metagenomic projects spend considerable amounts of time, money and manpower for experimentally cloning, amplifying and sequencing the rDNA content in a metagenomic sample. In the second step, the entire genomic content of the metagenome is extracted, sequenced and analyzed. Since DNA sequences obtained in this second step also contain rDNA fragments, rapid in silico identification of these rDNA fragments would drastically reduce the cost, time and effort of current metagenomic projects by entirely bypassing the experimental steps of primer based rDNA amplification, cloning and sequencing. In this study, we present an algorithm called i-rDNA that can facilitate the rapid detection of 16S rDNA fragments from amongst millions of sequences in metagenomic data sets with high detection sensitivity. Performance evaluation with data sets/database variants simulating typical metagenomic scenarios indicates the significantly high detection sensitivity of i-rDNA. Moreover, i-rDNA can process a million sequences in less than an hour on a simple desktop with modest hardware specifications. In addition to the speed of execution, high sensitivity and low false positive rate, the utility of the algorithmic approach discussed in this paper is immense given that it would help in bypassing the entire experimental step of primer-based rDNA amplification, cloning and sequencing. Application of this algorithmic approach would thus drastically reduce the cost, time and human efforts invested in all metagenomic projects. A web-server for the i-rDNA algorithm is available at http://metagenomics.atc.tcs.com/i-rDNA/

Methods for decoding Cas9 protospacer adjacent motif (PAM) sequences: A brief overview.

PubMed

Karvelis, Tautvydas; Gasiunas, Giedrius; Siksnys, Virginijus

2017-05-15

Recently the Cas9, an RNA guided DNA endonuclease, emerged as a powerful tool for targeted genome manipulations. Cas9 protein can be reprogrammed to cleave, bind or nick any DNA target by simply changing crRNA sequence, however a short nucleotide sequence, termed PAM, is required to initiate crRNA hybridization to the DNA target. PAM sequence is recognized by Cas9 protein and must be determined experimentally for each Cas9 variant. Exploration of Cas9 orthologs could offer a diversity of PAM sequences and novel biochemical properties that may be beneficial for genome editing applications. Here we briefly review and compare Cas9 PAM identification assays that can be adopted for other PAM-dependent CRISPR-Cas systems. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA barcoding Indian freshwater fishes.

PubMed

Lakra, Wazir Singh; Singh, M; Goswami, Mukunda; Gopalakrishnan, A; Lal, K K; Mohindra, V; Sarkar, U K; Punia, P P; Singh, K V; Bhatt, J P; Ayyappan, S

2016-11-01

DNA barcoding is a promising technique for species identification using a short mitochondrial DNA sequence of cytochrome c oxidase I (COI) gene. In the present study, DNA barcodes were generated from 72 species of freshwater fish covering the Orders Cypriniformes, Siluriformes, Perciformes, Synbranchiformes, and Osteoglossiformes representing 50 genera and 19 families. All the samples were collected from diverse sites except the species endemic to a particular location. Species were represented by multiple specimens in the great majority of the barcoded species. A total of 284 COI sequences were generated. After amplification and sequencing of 700 base pair fragment of COI, primers were trimmed which invariably generated a 655 base pair barcode sequence. The average Kimura two-parameter (K2P) distances within-species, genera, families, and orders were 0.40%, 9.60%, 13.10%, and 17.16%, respectively. DNA barcode discriminated congeneric species without any confusion. The study strongly validated the efficiency of COI as an ideal marker for DNA barcoding of Indian freshwater fishes.

Application of rDNA-PCR amplification and DGGE fingerprinting for detection of microbial diversity in a Malaysian crude oil.

PubMed

Liew, Pauline Woanying; Jong, Bor Chyan

2008-05-01

Two culture-independent methods, namely ribosomal DNA libraries and denaturing gradient gel electrophoresis (DGGE), were adopted to examine the microbial community of a Malaysian light crude oil. In this study, both 16S and 18S rDNAs were PCR-amplified from bulk DNA of crude oil samples, cloned, and sequenced. Analyses of restriction fragment length polymorphism (RFLP) and phylogenetics clustered the 16S and 18S rDNA sequences into seven and six groups, respectively. The ribosomal DNA sequences obtained showed sequence similarity between 90 to 100% to those available in the GenBank database. The closest relatives documented for the 16S rDNAs include member species of Thermoincola and Rhodopseudomonas, whereas the closest fungal relatives include Acremonium, Ceriporiopsis, Xeromyces, Lecythophora, and Candida. Others were affiliated to uncultured bacteria and uncultured ascomycete. The 16S rDNA library demonstrated predomination by a single uncultured bacterial type by >80% relative abundance. The predomination was confirmed by DGGE analysis.

Limited performance of DNA barcoding in a diverse community of tropical butterflies

PubMed Central

Elias, Marianne; Hill, Ryan I; Willmott, Keith R; Dasmahapatra, Kanchon K; Brower, Andrew V.Z; Mallet, James; Jiggins, Chris D

2007-01-01

DNA ‘barcoding’ relies on a short fragment of mitochondrial DNA to infer identification of specimens. The method depends on genetic diversity being markedly lower within than between species. Closely related species are most likely to share genetic variation in communities where speciation rates are rapid and effective population sizes are large, such that coalescence times are long. We assessed the applicability of DNA barcoding (here the 5′ half of the cytochrome c oxidase I) to a diverse community of butterflies from the upper Amazon, using a group with a well-established morphological taxonomy to serve as a reference. Only 77% of species could be accurately identified using the barcode data, a figure that dropped to 68% in species represented in the analyses by more than one geographical race and at least one congener. The use of additional mitochondrial sequence data hardly improved species identification, while a fragment of a nuclear gene resolved issues in some of the problematic species. We acknowledge the utility of barcodes when morphological characters are ambiguous or unknown, but we also recommend the addition of nuclear sequence data, and caution that species-level identification rates might be lower in the most diverse habitats of our planet. PMID:17785265

From Environmental Sequences to Morphology: Observation and Characterisation of a Paulinellid Testate Amoeba (Micropyxidiella edaphonis gen. nov. sp. nov. Euglyphida, Paulinellidae) from Soil using Fluorescent in situ Hybridization.

PubMed

Tarnawski, Sonia-Estelle; Lara, Enrique

2015-05-01

High microbial diversity is revealed by environmental DNA surveys. However, nothing is known about the morphology and function of these potentially new organisms. In the course of an environmental soil diversity study, we found for the first time environmental sequences that reveal the presence of Paulinellidae (a mostly marine and marginally freshwater family of euglyphid testate amoebae) in samples of forest litter from different geographic origins. The new sequences form a basal, robust clade in the family. We used fluorescent in situ hybridization (FISH) to detect the organisms from which these sequences derived. We isolated the cells and documented them with light and scanning electron microscopy. Based on these observations, we described these organisms as Micropyxidiella edaphonis gen. nov. sp. nov. The organisms were very small testate amoebae (generally less than 10μm) with an irregular proteinaceous test. This suggests an unknown diversity in testate amoebae, and calls for extending this type of investigations to other protist groups which are known only as environmental DNA sequences. Copyright © 2015 Elsevier GmbH. All rights reserved.

Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection.

PubMed

Weiss, Eric R; Lamers, Susanna L; Henderson, Jennifer L; Melnikov, Alexandre; Somasundaran, Mohan; Garber, Manuel; Selin, Liisa; Nusbaum, Chad; Luzuriaga, Katherine

2018-01-15

Over 90% of the world's population is persistently infected with Epstein-Barr virus. While EBV does not cause disease in most individuals, it is the common cause of acute infectious mononucleosis (AIM) and has been associated with several cancers and autoimmune diseases, highlighting a need for a preventive vaccine. At present, very few primary, circulating EBV genomes have been sequenced directly from infected individuals. While low levels of diversity and low viral evolution rates have been predicted for double-stranded DNA (dsDNA) viruses, recent studies have demonstrated appreciable diversity in common dsDNA pathogens (e.g., cytomegalovirus). Here, we report 40 full-length EBV genome sequences obtained from matched oral wash and B cell fractions from a cohort of 10 AIM patients. Both intra- and interpatient diversity were observed across the length of the entire viral genome. Diversity was most pronounced in viral genes required for establishing latent infection and persistence, with appreciable levels of diversity also detected in structural genes, including envelope glycoproteins. Interestingly, intrapatient diversity declined significantly over time ( P < 0.01), and this was particularly evident on comparison of viral genomes sequenced from B cell fractions in early primary infection and convalescence ( P < 0.001). B cell-associated viral genomes were observed to converge, becoming nearly identical to the B95.8 reference genome over time (Spearman rank-order correlation test; r = -0.5589, P = 0.0264). The reduction in diversity was most marked in the EBV latency genes. In summary, our data suggest independent convergence of diverse viral genome sequences toward a reference-like strain within a relatively short period following primary EBV infection. IMPORTANCE Identification of viral proteins with low variability and high immunogenicity is important for the development of a protective vaccine. Knowledge of genome diversity within circulating viral populations is a key step in this process, as is the expansion of intrahost genomic variation during infection. We report full-length EBV genomes sequenced from the blood and oral wash of 10 individuals early in primary infection and during convalescence. Our data demonstrate considerable diversity within the pool of circulating EBV strains, as well as within individual patients. Overall viral diversity decreased from early to persistent infection, particularly in latently infected B cells, which serve as the viral reservoir. Reduction in B cell-associated viral genome diversity coincided with a convergence toward a reference-like EBV genotype. Greater convergence positively correlated with time after infection, suggesting that the reference-like genome is the result of selection. Copyright © 2018 American Society for Microbiology.

Strain diversity and host specificity in bee gut symbionts revealed by deep sampling of single copy protein-coding sequences

PubMed Central

Powell, J. Elijah; Ratnayeke, Nalin; Moran, Nancy A.

2017-01-01

High throughput rRNA amplicon surveys of bacterial communities provide a rapid snapshot of taxonomic composition. But strains with nearly identical rRNA sequences often differ in gene repertoires and metabolic capabilities. To assess strain-level variation within Snodgrassella alvi, a gut symbiont of corbiculate bees, we performed deep sequencing on amplicons of a single copy coding gene (minD) as well as the 16S rDNA V4 region. We surveyed honey bees (Apis mellifera) sampled globally and 12 bumble bee species (Bombus) sampled from two regions of the USA. The minD analyses reveal that S. alvi contains far more strain diversity than is evident from 16S rDNA analysis. Many taxa inferred on the basis of 16S rDNA are shared between A. mellifera and Bombus species, but taxa inferred on the basis of minD are never shared and often are restricted to particular Bombus species. Clustering based on minD revealed that gut communities often reflect host species and geographic location. Both minD and 16S rDNA analyses indicate that strain diversity is higher in A. mellifera than in Bombus species. The minD locus flanks a 16S gene, enabling development of strain-specific 16S fluorescent probes to illuminate the spatial relationship of strains within the bee gut. PMID:27482856

Characterization of Microbial Population Structures in Recreational Waters and Primary Sources of Fecal Pollution with a Next-Generation Sequencing Approach

EPA Science Inventory

The invention of new approaches to DNA sequencing commonly referred to as next generation sequencing technologies is revolutionizing the study of microbial diversity. In this chapter, we discuss the characterization of microbial population structures in recreational waters and p...

DNA mimic proteins: functions, structures, and bioinformatic analysis.

PubMed

Wang, Hao-Ching; Ho, Chun-Han; Hsu, Kai-Cheng; Yang, Jinn-Moon; Wang, Andrew H-J

2014-05-13

DNA mimic proteins have DNA-like negative surface charge distributions, and they function by occupying the DNA binding sites of DNA binding proteins to prevent these sites from being accessed by DNA. DNA mimic proteins control the activities of a variety of DNA binding proteins and are involved in a wide range of cellular mechanisms such as chromatin assembly, DNA repair, transcription regulation, and gene recombination. However, the sequences and structures of DNA mimic proteins are diverse, making them difficult to predict by bioinformatic search. To date, only a few DNA mimic proteins have been reported. These DNA mimics were not found by searching for functional motifs in their sequences but were revealed only by structural analysis of their charge distribution. This review highlights the biological roles and structures of 16 reported DNA mimic proteins. We also discuss approaches that might be used to discover new DNA mimic proteins.

With a little help from DNA barcoding: investigating the diversity of Gastropoda from the Portuguese coast

PubMed Central

Borges, Luísa M. S.; Hollatz, Claudia; Lobo, Jorge; Cunha, Ana M.; Vilela, Ana P.; Calado, Gonçalo; Coelho, Rita; Costa, Ana C.; Ferreira, Maria S. G.; Costa, Maria H.; Costa, Filipe O.

2016-01-01

The Gastropoda is one of the best studied classes of marine invertebrates. Yet, most species have been delimited based on morphology only. The application of DNA barcodes has shown to be greatly useful to help delimiting species. Therefore, sequences of the cytochrome c oxidase I gene from 108 specimens of 34 morpho-species were used to investigate the molecular diversity within the gastropods from the Portuguese coast. To the above dataset, we added available COI-5P sequences of taxonomically close species, in a total of 58 morpho-species examined. There was a good match between ours and sequences from independent studies, in public repositories. We found 32 concordant (91.4%) out of the 35 Barcode Index Numbers (BINs) generated from our sequences. The application of a ranking system to the barcodes yield over 70% with top taxonomic congruence, while 14.2% of the species barcodes had insufficient data. In the majority of the cases, there was a good concordance between morphological identification and DNA barcodes. Nonetheless, the discordance between morphological and molecular data is a reminder that even the comparatively well-known European marine gastropods can benefit from being probed using the DNA barcode approach. Discordant cases should be reviewed with more integrative studies. PMID:26876495

With a little help from DNA barcoding: investigating the diversity of Gastropoda from the Portuguese coast.

PubMed

Borges, Luísa M S; Hollatz, Claudia; Lobo, Jorge; Cunha, Ana M; Vilela, Ana P; Calado, Gonçalo; Coelho, Rita; Costa, Ana C; Ferreira, Maria S G; Costa, Maria H; Costa, Filipe O

2016-02-15

The Gastropoda is one of the best studied classes of marine invertebrates. Yet, most species have been delimited based on morphology only. The application of DNA barcodes has shown to be greatly useful to help delimiting species. Therefore, sequences of the cytochrome c oxidase I gene from 108 specimens of 34 morpho-species were used to investigate the molecular diversity within the gastropods from the Portuguese coast. To the above dataset, we added available COI-5P sequences of taxonomically close species, in a total of 58 morpho-species examined. There was a good match between ours and sequences from independent studies, in public repositories. We found 32 concordant (91.4%) out of the 35 Barcode Index Numbers (BINs) generated from our sequences. The application of a ranking system to the barcodes yield over 70% with top taxonomic congruence, while 14.2% of the species barcodes had insufficient data. In the majority of the cases, there was a good concordance between morphological identification and DNA barcodes. Nonetheless, the discordance between morphological and molecular data is a reminder that even the comparatively well-known European marine gastropods can benefit from being probed using the DNA barcode approach. Discordant cases should be reviewed with more integrative studies.

Functional and mechanistic diversity of distal transcription enhancers

PubMed Central

Bulger, Michael; Groudine, Mark

2013-01-01

Biological differences among metazoans, and between cell types in a given organism, arise in large part due to differences in gene expression patterns. The sequencing of multiple metazoan genomes, coupled with recent advances in genome-wide analysis of histone modifications and transcription factor binding, has revealed that among regulatory DNA sequences, gene-distal enhancers appear to exhibit the greatest diversity and cell-type specificity. Moreover, such elements are emerging as important targets for mutations that can give rise to disease and to genetic variability that underlies evolutionary change. Studies of long-range interactions between distal genomic sequences in the nucleus indicate that enhancers are often important determinants of nuclear organization, contributing to a general model for enhancer function that involves direct enhancer-promoter contact. In a number of systems, however, mechanisms for enhancer function are emerging that do not fit solely within such a model, suggesting that enhancers as a class of DNA regulatory element may be functionally and mechanistically diverse. PMID:21295696

Diversity of Babesia bovis merozoite surface antigen genes in the Philippines.

PubMed

Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Ybanez, Adrian Patalinghug; Ybanez, Rochelle Haidee Daclan; Perez, Zandro Obligado; Guswanto, Azirwan; Igarashi, Ikuo; Yokoyama, Naoaki

2014-02-01

Babesia bovis is the causative agent of fatal babesiosis in cattle. In the present study, we investigated the genetic diversity of B. bovis among Philippine cattle, based on the genes that encode merozoite surface antigens (MSAs). Forty-one B. bovis-positive blood DNA samples from cattle were used to amplify the msa-1, msa-2b, and msa-2c genes. In phylogenetic analyses, the msa-1, msa-2b, and msa-2c gene sequences generated from Philippine B. bovis-positive DNA samples were found in six, three, and four different clades, respectively. All of the msa-1 and most of the msa-2b sequences were found in clades that were formed only by Philippine msa sequences in the respective phylograms. While all the msa-1 sequences from the Philippines showed similarity to those formed by Australian msa-1 sequences, the msa-2b sequences showed similarity to either Australian or Mexican msa-2b sequences. In contrast, msa-2c sequences from the Philippines were distributed across all the clades of the phylogram, although one clade was formed exclusively by Philippine msa-2c sequences. Similarities among the deduced amino acid sequences of MSA-1, MSA-2b, and MSA-2c from the Philippines were 62.2-100, 73.1-100, and 67.3-100%, respectively. The present findings demonstrate that B. bovis populations are genetically diverse in the Philippines. This information will provide a good foundation for the future design and implementation of improved immunological preventive methodologies against bovine babesiosis in the Philippines. The study has also generated a set of data that will be useful for futher understanding of the global genetic diversity of this important parasite. © 2013.

A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria

NASA Astrophysics Data System (ADS)

Kauffman, Kathryn M.; Hussain, Fatima A.; Yang, Joy; Arevalo, Philip; Brown, Julia M.; Chang, William K.; Vaninsberghe, David; Elsherbini, Joseph; Sharma, Radhey S.; Cutler, Michael B.; Kelly, Libusha; Polz, Martin F.

2018-02-01

The most abundant viruses on Earth are thought to be double-stranded DNA (dsDNA) viruses that infect bacteria. However, tailed bacterial dsDNA viruses (Caudovirales), which dominate sequence and culture collections, are not representative of the environmental diversity of viruses. In fact, non-tailed viruses often dominate ocean samples numerically, raising the fundamental question of the nature of these viruses. Here we characterize a group of marine dsDNA non-tailed viruses with short 10-kb genomes isolated during a study that quantified the diversity of viruses infecting Vibrionaceae bacteria. These viruses, which we propose to name the Autolykiviridae, represent a novel family within the ancient lineage of double jelly roll (DJR) capsid viruses. Ecologically, members of the Autolykiviridae have a broad host range, killing on average 34 hosts in four Vibrio species, in contrast to tailed viruses which kill on average only two hosts in one species. Biochemical and physical characterization of autolykiviruses reveals multiple virion features that cause systematic loss of DJR viruses in sequencing and culture-based studies, and we describe simple procedural adjustments to recover them. We identify DJR viruses in the genomes of diverse major bacterial and archaeal phyla, and in marine water column and sediment metagenomes, and find that their diversity greatly exceeds the diversity that is currently captured by the three recognized families of such viruses. Overall, these data suggest that viruses of the non-tailed dsDNA DJR lineage are important but often overlooked predators of bacteria and archaea that impose fundamentally different predation and gene transfer regimes on microbial systems than on tailed viruses, which form the basis of all environmental models of bacteria-virus interactions.

High levels of Y-chromosome nucleotide diversity in the genus Pan

PubMed Central

Stone, Anne C.; Griffiths, Robert C.; Zegura, Stephen L.; Hammer, Michael F.

2002-01-01

Although some mitochondrial, X chromosome, and autosomal sequence diversity data are available for our closest relatives, Pan troglodytes and Pan paniscus, data from the nonrecombining portion of the Y chromosome (NRY) are more limited. We examined ≈3 kb of NRY DNA from 101 chimpanzees, seven bonobos, and 42 humans to investigate: (i) relative levels of intraspecific diversity; (ii) the degree of paternal lineage sorting among species and subspecies of the genus Pan; and (iii) the date of the chimpanzee/bonobo divergence. We identified 10 informative sequence-tagged sites associated with 23 polymorphisms on the NRY from the genus Pan. Nucleotide diversity was significantly higher on the NRY of chimpanzees and bonobos than on the human NRY. Similar to mtDNA, but unlike X-linked and autosomal loci, lineages defined by mutations on the NRY were not shared among subspecies of P. troglodytes. Comparisons with mtDNA ND2 sequences from some of the same individuals revealed a larger female versus male effective population size for chimpanzees. The NRY-based divergence time between chimpanzees and bonobos was estimated at ≈1.8 million years ago. In contrast to human populations who appear to have had a low effective size and a recent origin with subsequent population growth, some taxa within the genus Pan may be characterized by large populations of relatively constant size, more ancient origins, and high levels of subdivision. PMID:11756656

Individual sequences in large sets of gene sequences may be distinguished efficiently by combinations of shared sub-sequences

PubMed Central

Gibbs, Mark J; Armstrong, John S; Gibbs, Adrian J

2005-01-01

Background Most current DNA diagnostic tests for identifying organisms use specific oligonucleotide probes that are complementary in sequence to, and hence only hybridise with the DNA of one target species. By contrast, in traditional taxonomy, specimens are usually identified by 'dichotomous keys' that use combinations of characters shared by different members of the target set. Using one specific character for each target is the least efficient strategy for identification. Using combinations of shared bisectionally-distributed characters is much more efficient, and this strategy is most efficient when they separate the targets in a progressively binary way. Results We have developed a practical method for finding minimal sets of sub-sequences that identify individual sequences, and could be targeted by combinations of probes, so that the efficient strategy of traditional taxonomic identification could be used in DNA diagnosis. The sizes of minimal sub-sequence sets depended mostly on sequence diversity and sub-sequence length and interactions between these parameters. We found that 201 distinct cytochrome oxidase subunit-1 (CO1) genes from moths (Lepidoptera) were distinguished using only 15 sub-sequences 20 nucleotides long, whereas only 8–10 sub-sequences 6–10 nucleotides long were required to distinguish the CO1 genes of 92 species from the 9 largest orders of insects. Conclusion The presence/absence of sub-sequences in a set of gene sequences can be used like the questions in a traditional dichotomous taxonomic key; hybridisation probes complementary to such sub-sequences should provide a very efficient means for identifying individual species, subtypes or genotypes. Sequence diversity and sub-sequence length are the major factors that determine the numbers of distinguishing sub-sequences in any set of sequences. PMID:15817134

Beyond Bacteria: A Study of the Enteric Microbial Consortium in Extremely Low Birth Weight Infants

PubMed Central

Cotton, Charles Michael; Goldberg, Ronald N.; Wynn, James L.; Jackson, Robert B.; Seed, Patrick C.

2011-01-01

Extremely low birth weight (ELBW) infants have high morbidity and mortality, frequently due to invasive infections from bacteria, fungi, and viruses. The microbial communities present in the gastrointestinal tracts of preterm infants may serve as a reservoir for invasive organisms and remain poorly characterized. We used deep pyrosequencing to examine the gut-associated microbiome of 11 ELBW infants in the first postnatal month, with a first time determination of the eukaryote microbiota such as fungi and nematodes, including bacteria and viruses that have not been previously described. Among the fungi observed, Candida sp. and Clavispora sp. dominated the sequences, but a range of environmental molds were also observed. Surprisingly, seventy-one percent of the infant fecal samples tested contained ribosomal sequences corresponding to the parasitic organism Trichinella. Ribosomal DNA sequences for the roundworm symbiont Xenorhabdus accompanied these sequences in the infant with the greatest proportion of Trichinella sequences. When examining ribosomal DNA sequences in aggregate, Enterobacteriales, Pseudomonas, Staphylococcus, and Enterococcus were the most abundant bacterial taxa in a low diversity bacterial community (mean Shannon-Weaver Index of 1.02±0.69), with relatively little change within individual infants through time. To supplement the ribosomal sequence data, shotgun sequencing was performed on DNA from multiple displacement amplification (MDA) of total fecal genomic DNA from two infants. In addition to the organisms mentioned previously, the metagenome also revealed sequences for gram positive and gram negative bacteriophages, as well as human adenovirus C. Together, these data reveal surprising eukaryotic and viral microbial diversity in ELBW enteric microbiota dominated bytypes of bacteria known to cause invasive disease in these infants. PMID:22174751

Previously unknown and highly divergent ssDNA viruses populate the oceans.

PubMed

Labonté, Jessica M; Suttle, Curtis A

2013-11-01

Single-stranded DNA (ssDNA) viruses are economically important pathogens of plants and animals, and are widespread in oceans; yet, the diversity and evolutionary relationships among marine ssDNA viruses remain largely unknown. Here we present the results from a metagenomic study of composite samples from temperate (Saanich Inlet, 11 samples; Strait of Georgia, 85 samples) and subtropical (46 samples, Gulf of Mexico) seawater. Most sequences (84%) had no evident similarity to sequenced viruses. In total, 608 putative complete genomes of ssDNA viruses were assembled, almost doubling the number of ssDNA viral genomes in databases. These comprised 129 genetically distinct groups, each represented by at least one complete genome that had no recognizable similarity to each other or to other virus sequences. Given that the seven recognized families of ssDNA viruses have considerable sequence homology within them, this suggests that many of these genetic groups may represent new viral families. Moreover, nearly 70% of the sequences were similar to one of these genomes, indicating that most of the sequences could be assigned to a genetically distinct group. Most sequences fell within 11 well-defined gene groups, each sharing a common gene. Some of these encoded putative replication and coat proteins that had similarity to sequences from viruses infecting eukaryotes, suggesting that these were likely from viruses infecting eukaryotic phytoplankton and zooplankton.

Genotyping of Leptospira directly in urine samples of cattle demonstrates a diversity of species and strains in Brazil.

PubMed

Hamond, C; Pestana, C P; Medeiros, M A; Lilenbaum, W

2016-01-01

The aim of this study was to identify Leptospira in urine samples of cattle by direct sequencing of the secY gene. The validity of this approach was assessed using ten Leptospira strains obtained from cattle in Brazil and 77 DNA samples previously extracted from cattle urine, that were positive by PCR for the genus-specific lipL32 gene of Leptospira. Direct sequencing identified 24 (31·1%) interpretable secY sequences and these were identical to those obtained from direct DNA sequencing of the urine samples from which they were recovered. Phylogenetic analyses identified four species: L. interrogans, L. borgpetersenii, L. noguchii, and L. santarosai with the most prevalent genotypes being associated with L. borgpetersenii. While direct sequencing cannot, as yet, replace culturing of leptospires, it is a valid additional tool for epidemiological studies. An unexpected finding from this study was the genetic diversity of Leptospira infecting Brazilian cattle.

New high through put approach to study ancient microbial phylogenetic diversity in permafrost

NASA Astrophysics Data System (ADS)

Spirina, E.; Cole, J.; Chai, B.; Gilichinksy, D.; Tiedje, J.

2003-04-01

The study of microbial diversity in the deep ancient permafrost can help to answer many questions: (1) what kind of mechanisms keeps microbial cells alive, (2) how many of phylogenetic groups exist in situ and never had been cultivated, (3) what is the difference between modern and ancient microorganisms? From this point, distinct environments were examined: Arctic and Antarctic modern soil and permafrost. 16S rDNA genes were amplified from genomic DNA extracted from both original frozen samples and the same samples incubated at 10oC for 8 weeks under both aerobic and anaerobic conditions to determine those capable to grow. High throughput DNA sequencing was performed on the cloned PCR products to obtain partial 16S rDNA gene sequences. The unique script was written to automatically compare over 2,000 partial sequences with those rrn sequences in the Ribosomal Database Project (RDP) release 8.1 using the SEQUENCE MATCH. Sequences were grouped into categories from the RDPs phylogenetic hierarchy based on the closest database matches. Investigation revealed significant microbial diversity; two phylogenetic groups were predominant in all samples: Proteobacteria and Gram Positive Bacteria. Microbial community composition within those groups is different from sample to sample. However, similar genera, such as Arthrobacter, Bacillus, Citrobacter, Caulobacter, Comamonas, Flavobacterium, Nocardioides, Pseudomonas, Rhodocyclus, Rhodococcus, Sphingobacterium, Sphingomonas, Streptococcus, Terrabacter appeared in both polar regions. The greatest microbial diversity was detected in Arctic surface samples. According to RDPs phylogenetic hierarchy those organisms are related to Proteobacteria_SD, Gram Positive Bacteria_SD, Leptospirillum-Nitrospira, Nitrospina_SD, Flexibacter-Cytophaga-Bacteroides, Planctomyces and Relatives. Both the aerobic and anaerobic low temperatures soil incubation yielded some microbes not detected in the original samples. It should be possible, using phylogenetic diversity from the same organisms from modern top layers to the several millions years old, to find out what are the differences among members of the same species as we go back in time. Then, if we compare those mutations rate with geological time, we can speculate on how fast or slow evolution or adaptation takes place and for that particular type of organism. This is a beginning of studies concerning the biological clocks extending back the duration of the permanently frozen state in the terrestrial and extraterrestrial soils, i. e. the age of biota.

Genetic Variations in Two Seahorse Species (Hippocampus mohnikei and Hippocampus trimaculatus): Evidence for Middle Pleistocene Population Expansion

PubMed Central

Zhang, Yanhong; Pham, Nancy Kim; Zhang, Huixian; Lin, Junda; Lin, Qiang

2014-01-01

Population genetic of seahorses is confidently influenced by their species-specific ecological requirements and life-history traits. In the present study, partial sequences of mitochondrial cytochrome b (cytb) and control region (CR) were obtained from 50 Hippocampus mohnikei and 92 H. trimaculatus from four zoogeographical zones. A total of 780 base pairs of cytb gene were sequenced to characterize mitochondrial DNA (mtDNA) diversity. The mtDNA marker revealed high haplotype diversity, low nucleotide diversity, and a lack of population structure across both populations of H. mohnikei and H. trimaculatus. A neighbour-joining (NJ) tree of cytb gene sequences showed that H. mohnikei haplotypes formed one cluster. A maximum likelihood (ML) tree of cytb gene sequences showed that H. trimaculatus belonged to one lineage. The star-like pattern median-joining network of cytb and CR markers indicated a previous demographic expansion of H. mohnikei and H. trimaculatus. The cytb and CR data sets exhibited a unimodal mismatch distribution, which may have resulted from population expansion. Mismatch analysis suggested that the expansion was initiated about 276,000 years ago for H. mohnikei and about 230,000 years ago for H. trimaculatus during the middle Pleistocene period. This study indicates a possible signature of genetic variation and population expansion in two seahorses under complex marine environments. PMID:25144384

Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR

PubMed Central

Miner, Brooks E.; Stöger, Reinhard J.; Burden, Alice F.; Laird, Charles D.; Hansen, R. Scott

2004-01-01

PCR amplification of limited amounts of DNA template carries an increased risk of product redundancy and contamination. We use molecular barcoding to label each genomic DNA template with an individual sequence tag prior to PCR amplification. In addition, we include molecular ‘batch-stamps’ that effectively label each genomic template with a sample ID and analysis date. This highly sensitive method identifies redundant and contaminant sequences and serves as a reliable method for positive identification of desired sequences; we can therefore capture accurately the genomic template diversity in the sample analyzed. Although our application described here involves the use of hairpin-bisulfite PCR for amplification of double-stranded DNA, the method can readily be adapted to single-strand PCR. Useful applications will include analyses of limited template DNA for biomedical, ancient DNA and forensic purposes. PMID:15459281

Limited Genetic Diversity Preceded Extinction of the Tasmanian Tiger

PubMed Central

Menzies, Brandon R.; Renfree, Marilyn B.; Heider, Thomas; Mayer, Frieder; Hildebrandt, Thomas B.; Pask, Andrew J.

2012-01-01

The Tasmanian tiger or thylacine was the largest carnivorous marsupial when Europeans first reached Australia. Sadly, the last known thylacine died in captivity in 1936. A recent analysis of the genome of the closely related and extant Tasmanian devil demonstrated limited genetic diversity between individuals. While a similar lack of diversity has been reported for the thylacine, this analysis was based on just two individuals. Here we report the sequencing of an additional 12 museum-archived specimens collected between 102 and 159 years ago. We examined a portion of the mitochondrial DNA hyper-variable control region and determined that all sequences were on average 99.5% identical at the nucleotide level. As a measure of accuracy we also sequenced mitochondrial DNA from a mother and two offspring. As expected, these samples were found to be 100% identical, validating our methods. We also used 454 sequencing to reconstruct 2.1 kilobases of the mitochondrial genome, which shared 99.91% identity with the two complete thylacine mitochondrial genomes published previously. Our thylacine genomic data also contained three highly divergent putative nuclear mitochondrial sequences, which grouped phylogenetically with the published thylacine mitochondrial homologs but contained 100-fold more polymorphisms than the conserved fragments. Together, our data suggest that the thylacine population in Tasmania had limited genetic diversity prior to its extinction, possibly as a result of their geographic isolation from mainland Australia approximately 10,000 years ago. PMID:22530022

Evidence for a Complex Class of Nonadenylated mRNA in Drosophila

PubMed Central

Zimmerman, J. Lynn; Fouts, David L.; Manning, Jerry E.

1980-01-01

The amount, by mass, of poly(A+) mRNA present in the polyribosomes of third-instar larvae of Drosophila melanogaster, and the relative contribution of the poly(A+) mRNA to the sequence complexity of total polysomal RNA, has been determined. Selective removal of poly(A+) mRNA from total polysomal RNA by use of either oligo-dT-cellulose, or poly(U)-sepharose affinity chromatography, revealed that only 0.15% of the mass of the polysomal RNA was present as poly(A+) mRNA. The present study shows that this RNA hybridized at saturation with 3.3% of the single-copy DNA in the Drosophila genome. After correction for asymmetric transcription and reactability of the DNA, 7.4% of the single-copy DNA in the Drosophila genome is represented in larval poly(A+) mRNA. This corresponds to 6.73 x 106 nucleotides of mRNA coding sequences, or approximately 5,384 diverse RNA sequences of average size 1,250 nucleotides. However, total polysomal RNA hybridizes at saturation to 10.9% of the single-copy DNA sequences. After correcting this value for asymmetric transcription and tracer DNA reactability, 24% of the single-copy DNA in Drosophila is represented in total polysomal RNA. This corresponds to 2.18 x 107 nucleotides of RNA coding sequences or 17,440 diverse RNA molecules of size 1,250 nucleotides. This value is 3.2 times greater than that observed for poly(A+) mRNA, and indicates that ≃69% of the polysomal RNA sequence complexity is contributed by nonadenylated RNA. Furthermore, if the number of different structural genes represented in total polysomal RNA is ≃1.7 x 104, then the number of genes expressed in third-instar larvae exceeds the number of chromomeres in Drosophila by about a factor of three. This numerology indicates that the number of chromomeres observed in polytene chromosomes does not reflect the number of structural gene sequences in the Drosophila genome. PMID:6777246

Sequence-Specific Recognition of DNA by Proteins: Binding Motifs Discovered Using a Novel Statistical/Computational Analysis

PubMed Central

Jakubec, David; Laskowski, Roman A.; Vondrasek, Jiri

2016-01-01

Decades of intensive experimental studies of the recognition of DNA sequences by proteins have provided us with a view of a diverse and complicated world in which few to no features are shared between individual DNA-binding protein families. The originally conceived direct readout of DNA residue sequences by amino acid side chains offers very limited capacity for sequence recognition, while the effects of the dynamic properties of the interacting partners remain difficult to quantify and almost impossible to generalise. In this work we investigated the energetic characteristics of all DNA residue—amino acid side chain combinations in the conformations found at the interaction interface in a very large set of protein—DNA complexes by the means of empirical potential-based calculations. General specificity-defining criteria were derived and utilised to look beyond the binding motifs considered in previous studies. Linking energetic favourability to the observed geometrical preferences, our approach reveals several additional amino acid motifs which can distinguish between individual DNA bases. Our results remained valid in environments with various dielectric properties. PMID:27384774

Next-Generation Sequencing Reveals Significant Bacterial Diversity of Botrytized Wine

PubMed Central

Bokulich, Nicholas A.; Joseph, C. M. Lucy; Allen, Greg; Benson, Andrew K.; Mills, David A.

2012-01-01

While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies. PMID:22563494

Culture-dependent and culture-independent diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve from the South China Sea.

PubMed

Sun, Wei; Dai, Shikun; Jiang, Shumei; Wang, Guanghua; Liu, Guohui; Wu, Houbo; Li, Xiang

2010-06-01

In this report, the diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve collected from a remote island of the South China Sea was investigated employing classical cultivation and characterization, 16S rDNA library construction, 16S rDNA-restriction fragment length polymorphism (rDNA-RFLP) and phylogenetic analysis. A total of 184 strains were isolated using seven different media and 24 isolates were selected according to their morphological characteristics for phylogenetic analysis on the basis of their 16S rRNA gene sequences. Results showed that the 24 isolates were assigned to six genera including Salinispora, Gordonia, Mycobacterium, Nocardia, Rhodococcus and Streptomyces. This is the first report that Salinispora is present in a marine sponge from the South China Sea. Subsequently, 26 rDNA clones were selected from 191 clones in an Actinobacteria-specific 16S rDNA library of the H. perleve sample, using the RFLP technique for sequencing and phylogenetic analysis. In total, 26 phylotypes were clustered in eight known genera of Actinobacteria including Mycobacterium, Amycolatopsis, Arthrobacter, Brevibacterium, Microlunatus, Nocardioides, Pseudonocardia and Streptomyces. This study contributes to our understanding of actinobacterial diversity in the marine sponge H. perleve from the South China Sea.

Deep COI sequencing of standardized benthic samples unveils overlooked diversity of Jordanian coral reefs in the northern Red Sea.

PubMed

Al-Rshaidat, Mamoon M D; Snider, Allison; Rosebraugh, Sydney; Devine, Amanda M; Devine, Thomas D; Plaisance, Laetitia; Knowlton, Nancy; Leray, Matthieu

2016-09-01

High-throughput sequencing (HTS) of DNA barcodes (metabarcoding), particularly when combined with standardized sampling protocols, is one of the most promising approaches for censusing overlooked cryptic invertebrate communities. We present biodiversity estimates based on sequencing of the cytochrome c oxidase subunit 1 (COI) gene for coral reefs of the Gulf of Aqaba, a semi-enclosed system in the northern Red Sea. Samples were obtained from standardized sampling devices (Autonomous Reef Monitoring Structures (ARMS)) deployed for 18 months. DNA barcoding of non-sessile specimens >2 mm revealed 83 OTUs in six phyla, of which only 25% matched a reference sequence in public databases. Metabarcoding of the 2 mm - 500 μm and sessile bulk fractions revealed 1197 OTUs in 15 animal phyla, of which only 4.9% matched reference barcodes. These results highlight the scarcity of COI data for cryptobenthic organisms of the Red Sea. Compared with data obtained using similar methods, our results suggest that Gulf of Aqaba reefs are less diverse than two Pacific coral reefs but much more diverse than an Atlantic oyster reef at a similar latitude. The standardized approaches used here show promise for establishing baseline data on biodiversity, monitoring the impacts of environmental change, and quantifying patterns of diversity at regional and global scales.

AmericaPlex26: A SNaPshot Multiplex System for Genotyping the Main Human Mitochondrial Founder Lineages of the Americas

PubMed Central

Coutinho, Alexandra; Valverde, Guido; Fehren-Schmitz, Lars; Cooper, Alan; Barreto Romero, Maria Inés; Espinoza, Isabel Flores; Llamas, Bastien; Haak, Wolfgang

2014-01-01

Phylogeographic studies have described a reduced genetic diversity in Native American populations, indicative of one or more bottleneck events during the peopling and prehistory of the Americas. Classical sequencing approaches targeting the mitochondrial diversity have reported the presence of five major haplogroups, namely A, B, C, D and X, whereas the advent of complete mitochondrial genome sequencing has recently refined the number of founder lineages within the given diversity to 15 sub-haplogroups. We developed and optimized a SNaPshot assay to study the mitochondrial diversity in pre-Columbian Native American populations by simultaneous typing of 26 single nucleotide polymorphisms (SNPs) characterising Native American sub-haplogroups. Our assay proved to be highly sensitive with respect to starting concentrations of target DNA and could be applied successfully to a range of ancient human skeletal material from South America from various time periods. The AmericaPlex26 is a powerful assay with enhanced phylogenetic resolution that allows time- and cost-efficient mitochondrial DNA sub-typing from valuable ancient specimens. It can be applied in addition or alternative to standard sequencing of the D-loop region in forensics, ancestry testing, and population studies, or where full-resolution mitochondrial genome sequencing is not feasible. PMID:24671218

AmericaPlex26: a SNaPshot multiplex system for genotyping the main human mitochondrial founder lineages of the Americas.

PubMed

Coutinho, Alexandra; Valverde, Guido; Fehren-Schmitz, Lars; Cooper, Alan; Barreto Romero, Maria Inés; Espinoza, Isabel Flores; Llamas, Bastien; Haak, Wolfgang

2014-01-01

Phylogeographic studies have described a reduced genetic diversity in Native American populations, indicative of one or more bottleneck events during the peopling and prehistory of the Americas. Classical sequencing approaches targeting the mitochondrial diversity have reported the presence of five major haplogroups, namely A, B, C, D and X, whereas the advent of complete mitochondrial genome sequencing has recently refined the number of founder lineages within the given diversity to 15 sub-haplogroups. We developed and optimized a SNaPshot assay to study the mitochondrial diversity in pre-Columbian Native American populations by simultaneous typing of 26 single nucleotide polymorphisms (SNPs) characterising Native American sub-haplogroups. Our assay proved to be highly sensitive with respect to starting concentrations of target DNA and could be applied successfully to a range of ancient human skeletal material from South America from various time periods. The AmericaPlex26 is a powerful assay with enhanced phylogenetic resolution that allows time- and cost-efficient mitochondrial DNA sub-typing from valuable ancient specimens. It can be applied in addition or alternative to standard sequencing of the D-loop region in forensics, ancestry testing, and population studies, or where full-resolution mitochondrial genome sequencing is not feasible.

DNA-based species level detection of Glomeromycota: one PCR primer set for all arbuscular mycorrhizal fungi.

PubMed

Krüger, Manuela; Stockinger, Herbert; Krüger, Claudia; Schüssler, Arthur

2009-01-01

* At present, molecular ecological studies of arbuscular mycorrhizal fungi (AMF) are only possible above species level when targeting entire communities. To improve molecular species characterization and to allow species level community analyses in the field, a set of newly designed AMF specific PCR primers was successfully tested. * Nuclear rDNA fragments from diverse phylogenetic AMF lineages were sequenced and analysed to design four primer mixtures, each targeting one binding site in the small subunit (SSU) or large subunit (LSU) rDNA. To allow species resolution, they span a fragment covering the partial SSU, whole internal transcribed spacer (ITS) rDNA region and partial LSU. * The new primers are suitable for specifically amplifying AMF rDNA from material that may be contaminated by other organisms (e.g., samples from pot cultures or the field), characterizing the diversity of AMF species from field samples, and amplifying a SSU-ITS-LSU fragment that allows phylogenetic analyses with species level resolution. * The PCR primers can be used to monitor entire AMF field communities, based on a single rDNA marker region. Their application will improve the base for deep sequencing approaches; moreover, they can be efficiently used as DNA barcoding primers.

Cryptic diversity in European bats.

PubMed Central

Mayer, F.; von Helversen, O.

2001-01-01

Different species of bat can be morphologically very similar. In order to estimate the amount of cryptic diversity among European bats we screened the intra- and interspecific genetic variation in 26 European vespertilionid bat species. We sequenced the DNA of subunit 1 of the mitochondrial protein NADH dehydrogenase (ND1) from several individuals of a species, which were sampled in a variety of geographical regions. A phylogeny based on the mitochondrial (mt) DNA data is in good agreement with the current classification in the family. Highly divergent mitochondrial lineages were found in two taxa, which differed in at least 11% of their ND1 sequence. The two mtDNA lineages in Plecotus austriacus correlated with the two subspecies Plecotus austriacus austriacus and Plecotus austriacus kolombatovici. The two mtDNA lineages in Myotis mystacinus were partitioned among two morphotypes. The evidence for two new bat species within Europe is discussed. Convergent adaptive evolution might have contributed to the morphological similarity among distantly related species if they occupy similar ecological niches. Closely related species may differ in their ecology but not necessarily in their morphology. On the other hand, two morphologically clearly different species (Eptesicus serotinus and Eptesicus nilssonii) were found to be genetically very similar. Neither morphological nor mitochondrial DNA sequence analysis alone can be guaranteed to identify species. PMID:11522202

Evolution of EF-hand calcium-modulated proteins. III. Exon sequences confirm most dendrograms based on protein sequences: calmodulin dendrograms show significant lack of parallelism

NASA Technical Reports Server (NTRS)

Nakayama, S.; Kretsinger, R. H.

1993-01-01

In the first report in this series we presented dendrograms based on 152 individual proteins of the EF-hand family. In the second we used sequences from 228 proteins, containing 835 domains, and showed that eight of the 29 subfamilies are congruent and that the EF-hand domains of the remaining 21 subfamilies have diverse evolutionary histories. In this study we have computed dendrograms within and among the EF-hand subfamilies using the encoding DNA sequences. In most instances the dendrograms based on protein and on DNA sequences are very similar. Significant differences between protein and DNA trees for calmodulin remain unexplained. In our fourth report we evaluate the sequences and the distribution of introns within the EF-hand family and conclude that exon shuffling did not play a significant role in its evolution.

DNA sequence database as a tool to identify decapod crustaceans on the São Paulo coastline.

PubMed

Mantelatto, Fernando L; Terossi, Mariana; Negri, Mariana; Buranelli, Raquel C; Robles, Rafael; Magalhães, Tatiana; Tamburus, Ana Francisca; Rossi, Natália; Miyazaki, Mayara J

2017-09-05

DNA barcoding has emerged as an efficient tool for taxonomy and other biodiversity fields. The vast and speciose group of decapod crustaceans is not an exception in the current scenario and comparing short DNA fragments has enabled researchers to overcome some taxonomic impediments to help broadening knowledge on the diversity of this group of crustaceans. Brazil is considered as an important area in terms of global marine biodiversity and some regions stand out in terms of decapod fauna, such as the São Paulo coastline. Thus, the aim of this study is to obtain sequences of the mitochondrial markers (COI and 16S) for decapod crustaceans distributed at the São Paulo coastline and to test the accuracy of these markers for species identification from this region by comparing our sequences to those already present in the GenBank database. We sampled along almost the 300 km of the São Paulo coastline from estuaries to offshore islands during the development of a multidisciplinary research project that took place for 5 years. All the species were processed to obtain the DNA sequences. The diversity of the decapod fauna on the São Paulo coastline comprises at least 404 species. We were able to collect 256 of those species and sequence of at least one of the target genes from 221. By testing the accuracy of these two DNA markers as a tool for identification, we were able to check our own identifications, including new records in GenBank, spot potential mistakes in GenBank, and detect potential new species.

Sequence-based prediction of protein-binding sites in DNA: comparative study of two SVM models.

PubMed

Park, Byungkyu; Im, Jinyong; Tuvshinjargal, Narankhuu; Lee, Wook; Han, Kyungsook

2014-11-01

As many structures of protein-DNA complexes have been known in the past years, several computational methods have been developed to predict DNA-binding sites in proteins. However, its inverse problem (i.e., predicting protein-binding sites in DNA) has received much less attention. One of the reasons is that the differences between the interaction propensities of nucleotides are much smaller than those between amino acids. Another reason is that DNA exhibits less diverse sequence patterns than protein. Therefore, predicting protein-binding DNA nucleotides is much harder than predicting DNA-binding amino acids. We computed the interaction propensity (IP) of nucleotide triplets with amino acids using an extensive dataset of protein-DNA complexes, and developed two support vector machine (SVM) models that predict protein-binding nucleotides from sequence data alone. One SVM model predicts protein-binding nucleotides using DNA sequence data alone, and the other SVM model predicts protein-binding nucleotides using both DNA and protein sequences. In a 10-fold cross-validation with 1519 DNA sequences, the SVM model that uses DNA sequence data only predicted protein-binding nucleotides with an accuracy of 67.0%, an F-measure of 67.1%, and a Matthews correlation coefficient (MCC) of 0.340. With an independent dataset of 181 DNAs that were not used in training, it achieved an accuracy of 66.2%, an F-measure 66.3% and a MCC of 0.324. Another SVM model that uses both DNA and protein sequences achieved an accuracy of 69.6%, an F-measure of 69.6%, and a MCC of 0.383 in a 10-fold cross-validation with 1519 DNA sequences and 859 protein sequences. With an independent dataset of 181 DNAs and 143 proteins, it showed an accuracy of 67.3%, an F-measure of 66.5% and a MCC of 0.329. Both in cross-validation and independent testing, the second SVM model that used both DNA and protein sequence data showed better performance than the first model that used DNA sequence data. To the best of our knowledge, this is the first attempt to predict protein-binding nucleotides in a given DNA sequence from the sequence data alone. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

ScaffoldSeq: Software for characterization of directed evolution populations.

PubMed

Woldring, Daniel R; Holec, Patrick V; Hackel, Benjamin J

2016-07-01

ScaffoldSeq is software designed for the numerous applications-including directed evolution analysis-in which a user generates a population of DNA sequences encoding for partially diverse proteins with related functions and would like to characterize the single site and pairwise amino acid frequencies across the population. A common scenario for enzyme maturation, antibody screening, and alternative scaffold engineering involves naïve and evolved populations that contain diversified regions, varying in both sequence and length, within a conserved framework. Analyzing the diversified regions of such populations is facilitated by high-throughput sequencing platforms; however, length variability within these regions (e.g., antibody CDRs) encumbers the alignment process. To overcome this challenge, the ScaffoldSeq algorithm takes advantage of conserved framework sequences to quickly identify diverse regions. Beyond this, unintended biases in sequence frequency are generated throughout the experimental workflow required to evolve and isolate clones of interest prior to DNA sequencing. ScaffoldSeq software uniquely handles this issue by providing tools to quantify and remove background sequences, cluster similar protein families, and dampen the impact of dominant clones. The software produces graphical and tabular summaries for each region of interest, allowing users to evaluate diversity in a site-specific manner as well as identify epistatic pairwise interactions. The code and detailed information are freely available at http://research.cems.umn.edu/hackel. Proteins 2016; 84:869-874. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Genetic variations in merozoite surface antigen genes of Babesia bovis detected in Vietnamese cattle and water buffaloes.

PubMed

Yokoyama, Naoaki; Sivakumar, Thillaiampalam; Tuvshintulga, Bumduuren; Hayashida, Kyoko; Igarashi, Ikuo; Inoue, Noboru; Long, Phung Thang; Lan, Dinh Thi Bich

2015-03-01

The genes that encode merozoite surface antigens (MSAs) in Babesia bovis are genetically diverse. In this study, we analyzed the genetic diversity of B. bovis MSA-1, MSA-2b, and MSA-2c genes in Vietnamese cattle and water buffaloes. Blood DNA samples from 258 cattle and 49 water buffaloes reared in the Thua Thien Hue province of Vietnam were screened with a B. bovis-specific diagnostic PCR assay. The B. bovis-positive DNA samples (23 cattle and 16 water buffaloes) were then subjected to PCR assays to amplify the MSA-1, MSA-2b, and MSA-2c genes. Sequencing analyses showed that the Vietnamese MSA-1 and MSA-2b sequences are genetically diverse, whereas MSA-2c is relatively conserved. The nucleotide identity values for these MSA gene sequences were similar in the cattle and water buffaloes. Consistent with the sequencing data, the Vietnamese MSA-1 and MSA-2b sequences were dispersed across several clades in the corresponding phylogenetic trees, whereas the MSA-2c sequences occurred in a single clade. Cattle- and water-buffalo-derived sequences also often clustered together on the phylogenetic trees. The Vietnamese MSA-1, MSA-2b, and MSA-2c sequences were then screened for recombination with automated methods. Of the seven recombination events detected, five and two were associated with the MSA-2b and MSA-2c recombinant sequences, respectively, whereas no MSA-1 recombinants were detected among the sequences analyzed. Recombination between the sequences derived from cattle and water buffaloes was very common, and the resultant recombinant sequences were found in both host animals. These data indicate that the genetic diversity of the MSA sequences does not differ between cattle and water buffaloes in Vietnam. They also suggest that recombination between the B. bovis MSA sequences in both cattle and water buffaloes might contribute to the genetic variation in these genes in Vietnam. Copyright © 2015 Elsevier B.V. All rights reserved.

PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy.

PubMed

Decelle, Johan; Romac, Sarah; Stern, Rowena F; Bendif, El Mahdi; Zingone, Adriana; Audic, Stéphane; Guiry, Michael D; Guillou, Laure; Tessier, Désiré; Le Gall, Florence; Gourvil, Priscillia; Dos Santos, Adriana L; Probert, Ian; Vaulot, Daniel; de Vargas, Colomban; Christen, Richard

2015-11-01

Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing. © 2015 John Wiley & Sons Ltd.

Whose genome is it, anyway?

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

Marshall, E.

1996-09-27

The genome program has issued guidelines to ensure that sequencing is done on DNA from diverse sources who have given informed consent and are anonymous. Most current sources don`t meet those criteria. It may be the first question every nonexpert asks on learning about the Human Genome Project: Whose genome are we studying, anyway? It sounds naive, says one government scientist-so naive, in fact, that {open_quotes}we chuckle as we explain that we aren`t sequencing anyone`s genome in particular; we`re sequencing a representative genome{close_quotes} made up of a mosaic of DNA from a variety of anonymous sources. And Bruce Birren, amore » clone-maker now at the Massachusetts Institute of Technology`s (MIT`s) Whitehead Center for Genome Research says: {open_quotes}We spent many years pooh-poohing the question{close_quotes} of whose genome would be stored in the database. But now that labs have begun working on large stretches of human DNA-aiming to identify all 3 billion base pairs in the genetic code-the question no longer seems to laughable. To the distress of program managers in Bethesda, Maryland, the initial sources of DNA are not as diverse or as anonymous as they had assumed.« less

Using sheep genomes from diverse U.S. breeds to identify missense variants in genes affecting fecundity

USDA-ARS?s Scientific Manuscript database

Background: Access to sheep genome sequences significantly improves the chances of identifying genes that may influence the health, welfare, and productivity of these animals. Methods: A public, searchable DNA sequence resource for U.S. sheep was created with whole genome sequence (WGS) of 96 rams. ...

Towards writing the encyclopaedia of life: an introduction to DNA barcoding

PubMed Central

Savolainen, Vincent; Cowan, Robyn S; Vogler, Alfried P; Roderick, George K; Lane, Richard

2005-01-01

An international consortium of major natural history museums, herbaria and other organizations has launched an ambitious project, the ‘Barcode of Life Initiative’, to promote a process enabling the rapid and inexpensive identification of the estimated 10 million species on Earth. DNA barcoding is a diagnostic technique in which short DNA sequence(s) can be used for species identification. The first international scientific conference on Barcoding of Life was held at the Natural History Museum in London in February 2005, and here we review the scientific challenges discussed during this conference and in previous publications. Although still controversial, the scientific benefits of DNA barcoding include: (i) enabling species identification, including any life stage or fragment, (ii) facilitating species discoveries based on cluster analyses of gene sequences (e.g. cox1=CO1, in animals), (iii) promoting development of handheld DNA sequencing technology that can be applied in the field for biodiversity inventories and (iv) providing insight into the diversity of life. PMID:16214739

First genomic survey of human skin fungal diversity

Cancer.gov

Fungal infections of the skin affect 29 million people in the United States. In the first study of human fungal skin diversity, National Institutes of Health researchers sequenced the DNA of fungi that thrive at different skin sites of healthy adults to d

Mitochondrial Genome Diversity of Native Americans Supports a Single Early Entry of Founder Populations into America

PubMed Central

Silva Jr., Wilson A.; Bonatto, Sandro L.; Holanda, Adriano J.; Ribeiro-dos-Santos, Andrea K.; Paixão, Beatriz M.; Goldman, Gustavo H.; Abe-Sandes, Kiyoko; Rodriguez-Delfin, Luis; Barbosa, Marcela; Paçó-Larson, Maria Luiza; Petzl-Erler, Maria Luiza; Valente, Valeria; Santos, Sidney E. B.; Zago, Marco A.

2002-01-01

There is general agreement that the Native American founder populations migrated from Asia into America through Beringia sometime during the Pleistocene, but the hypotheses concerning the ages and the number of these migrations and the size of the ancestral populations are surrounded by controversy. DNA sequence variations of several regions of the genome of Native Americans, especially in the mitochondrial DNA (mtDNA) control region, have been studied as a tool to help answer these questions. However, the small number of nucleotides studied and the nonclocklike rate of mtDNA control-region evolution impose several limitations to these results. Here we provide the sequence analysis of a continuous region of 8.8 kb of the mtDNA outside the D-loop for 40 individuals, 30 of whom are Native Americans whose mtDNA belongs to the four founder haplogroups. Haplogroups A, B, and C form monophyletic clades, but the five haplogroup D sequences have unstable positions and usually do not group together. The high degree of similarity in the nucleotide diversity and time of differentiation (i.e., ∼21,000 years before present) of these four haplogroups support a common origin for these sequences and suggest that the populations who harbor them may also have a common history. Additional evidence supports the idea that this age of differentiation coincides with the process of colonization of the New World and supports the hypothesis of a single and early entry of the ancestral Asian population into the Americas. PMID:12022039

Mitochondrial DNA control region sequences from Nairobi (Kenya): inferring phylogenetic parameters for the establishment of a forensic database.

PubMed

Brandstätter, Anita; Peterson, Christine T; Irwin, Jodi A; Mpoke, Solomon; Koech, Davy K; Parson, Walther; Parsons, Thomas J

2004-10-01

Large forensic mtDNA databases which adhere to strict guidelines for generation and maintenance, are not available for many populations outside of the United States and western Europe. We have established a high quality mtDNA control region sequence database for urban Nairobi as both a reference database for forensic investigations, and as a tool to examine the genetic variation of Kenyan sequences in the context of known African variation. The Nairobi sequences exhibited high variation and a low random match probability, indicating utility for forensic testing. Haplogroup identification and frequencies were compared with those reported from other published studies on African, or African-origin populations from Mozambique, Sierra Leone, and the United States, and suggest significant differences in the mtDNA compositions of the various populations. The quality of the sequence data in our study was investigated and supported using phylogenetic measures. Our data demonstrate the diversity and distinctiveness of African populations, and underline the importance of establishing additional forensic mtDNA databases of indigenous African populations.

A multiple-alignment based primer design algorithm for genetically highly variable DNA targets

PubMed Central

2013-01-01

Background Primer design for highly variable DNA sequences is difficult, and experimental success requires attention to many interacting constraints. The advent of next-generation sequencing methods allows the investigation of rare variants otherwise hidden deep in large populations, but requires attention to population diversity and primer localization in relatively conserved regions, in addition to recognized constraints typically considered in primer design. Results Design constraints include degenerate sites to maximize population coverage, matching of melting temperatures, optimizing de novo sequence length, finding optimal bio-barcodes to allow efficient downstream analyses, and minimizing risk of dimerization. To facilitate primer design addressing these and other constraints, we created a novel computer program (PrimerDesign) that automates this complex procedure. We show its powers and limitations and give examples of successful designs for the analysis of HIV-1 populations. Conclusions PrimerDesign is useful for researchers who want to design DNA primers and probes for analyzing highly variable DNA populations. It can be used to design primers for PCR, RT-PCR, Sanger sequencing, next-generation sequencing, and other experimental protocols targeting highly variable DNA samples. PMID:23965160

The genetic diversity of Epstein-Barr virus in the setting of transplantation relative to non-transplant settings: A feasibility study.

PubMed

Allen, Upton D; Hu, Pingzhao; Pereira, Sergio L; Robinson, Joan L; Paton, Tara A; Beyene, Joseph; Khodai-Booran, Nasser; Dipchand, Anne; Hébert, Diane; Ng, Vicky; Nalpathamkalam, Thomas; Read, Stanley

2016-02-01

This study examines EBV strains from transplant patients and patients with IM by sequencing major EBV genes. We also used NGS to detect EBV DNA within total genomic DNA, and to evaluate its genetic variation. Sanger sequencing of major EBV genes was used to compare SNVs from samples taken from transplant patients vs. patients with IM. We sequenced EBV DNA from a healthy EBV-seropositive individual on a HiSeq 2000 instrument. Data were mapped to the EBV reference genomes (AG876 and B95-8). The number of EBNA2 SNVs was higher than for EBNA1 and the other genes sequenced within comparable reference coordinates. For EBNA2, there was a median of 15 SNV among transplant samples compared with 10 among IM samples (p = 0.036). EBNA1 showed little variation between samples. For NGS, we identified 640 and 892 variants at an unadjusted p value of 5 × 10(-8) for AG876 and B95-8 genomes, respectively. We used complementary sequence strategies to examine EBV genetic diversity and its application to transplantation. The results provide the framework for further characterization of EBV strains and related outcomes after organ transplantation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer (ITS) region of peritrich ciliates in environmental samples

NASA Astrophysics Data System (ADS)

Su, Lei; Zhang, Qianqian; Gong, Jun

2017-07-01

Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifications of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specific PCR primers were newly designed to amplify a fragment including the internal transcribed spacer (ITS) region of ribosomal rDNA from environmental samples. The primers showed high specificity in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18S rDNA in peritrichs. The newly designed specific primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in different systems.

Microbial eukaryotic diversity and distribution in a river plume and cyclonic eddy-influenced ecosystem in the South China Sea

PubMed Central

Wu, Wenxue; Wang, Lei; Liao, Yu; Huang, Bangqin

2015-01-01

To evaluate microbial eukaryotic diversity and distribution in mesoscale processes, we investigated 18S rDNA diversity in a river plume and cyclonic eddy-influenced ecosystem in the southwestern South China Sea (SCS). Restriction fragment length polymorphism analysis was carried out using multiple primer sets. Relative to a wide range of previous similar studies, we observed a significantly higher proportion of sequences of pigmented taxa. Among the photosynthetic groups, Haptophyta accounted for 27.7% of the sequenced clones, which belonged primarily to Prymnesiophyceae. Unexpectedly, five operational taxonomic units of Cryptophyta were closely related to freshwater species. The Chlorophyta mostly fell within the Prasinophyceae, which was comprised of six clades, including Clade III, which is detected in the SCS for the first time in this study. Among the photosynthetic stramenopiles, Chrysophyceae was the most diverse taxon, which included seven clades. The majority of 18S rDNA sequences affiliated with the Dictyochophyceae, Eustigmatophyceae, and Pelagophyceae were closely related to those of pure cultures. The results of redundancy analysis and the permutation Mantel test based on unweighted UniFrac distances, conducted for spatial analyses of the Haptophyta subclades suggested that the Mekong River plume and cyclonic eddy play important roles in regulating microbial eukaryotic diversity and distribution in the southwestern SCS. PMID:26268071

Dust Rains Deliver Diverse Assemblages of Microorganisms to the Eastern Mediterranean

PubMed Central

Itani, Ghida Nouhad; Smith, Colin Andrew

2016-01-01

Dust rains may be particularly effective at delivering microorganisms, yet their biodiversities have been seldom examined. During 2011 and 2012 in Beirut, Lebanon, 16 of 21 collected rainfalls appeared dusty. Trajectory modelling of air mass origins was consistent with North African sources and at least one Southwest Asian source. As much as ~4 g particulate matter, ~20 μg DNA, and 50 million colony forming units were found deposited per square meter during rainfalls each lasting less than one day. Sequencing of 93 bacteria and 25 fungi cultured from rain samples revealed diverse bacterial phyla, both Gram positive and negative, and Ascomycota fungi. Denaturing Gradient Gel Electrophoresis of amplified 16S rDNA of 13 rains revealed distinct and diverse assemblages of bacteria. Dust rain 16S libraries yielded 131 sequences matching, in decreasing order of abundance, Betaproteobacteria, Alphaproteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Cyanobacteria, Epsilonproteobacteria, Gammaproteobacteria, and Deltaproteobacteria. Clean rain 16S libraries yielded 33 sequences matching only Betaproteobacteria family Oxalobacteraceae. Microbial composition varied between dust rains, and more diverse and different microbes were found in dust rains than clean rains. These results show that dust rains deliver diverse communities of microorganisms that may be complex products of revived desert soil species and fertilized cloud species. PMID:26939571

Dust Rains Deliver Diverse Assemblages of Microorganisms to the Eastern Mediterranean

NASA Astrophysics Data System (ADS)

Itani, Ghida Nouhad; Smith, Colin Andrew

2016-03-01

Dust rains may be particularly effective at delivering microorganisms, yet their biodiversities have been seldom examined. During 2011 and 2012 in Beirut, Lebanon, 16 of 21 collected rainfalls appeared dusty. Trajectory modelling of air mass origins was consistent with North African sources and at least one Southwest Asian source. As much as ~4 g particulate matter, ~20 μg DNA, and 50 million colony forming units were found deposited per square meter during rainfalls each lasting less than one day. Sequencing of 93 bacteria and 25 fungi cultured from rain samples revealed diverse bacterial phyla, both Gram positive and negative, and Ascomycota fungi. Denaturing Gradient Gel Electrophoresis of amplified 16S rDNA of 13 rains revealed distinct and diverse assemblages of bacteria. Dust rain 16S libraries yielded 131 sequences matching, in decreasing order of abundance, Betaproteobacteria, Alphaproteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Cyanobacteria, Epsilonproteobacteria, Gammaproteobacteria, and Deltaproteobacteria. Clean rain 16S libraries yielded 33 sequences matching only Betaproteobacteria family Oxalobacteraceae. Microbial composition varied between dust rains, and more diverse and different microbes were found in dust rains than clean rains. These results show that dust rains deliver diverse communities of microorganisms that may be complex products of revived desert soil species and fertilized cloud species.

DNA record of some traditional small millet landraces in India and Nepal.

PubMed

Ragupathy, Subramanyam; Dhivya, Shanmughanandhan; Patel, Kirit; Sritharan, Abiran; Sambandan, Kathirvelu; Gartaula, Hom; Sathishkumar, Ramalingam; Khadka, Kamal; Nirmala, Balasubramanian C; Kumari, A Nirmala; Newmaster, Steven G

2016-12-01

Despite the extensive use of small millet landraces as an important source of nutrition for people living in semi-arid regions, they are presently marginalized and their diversity and distribution are threatened at a global scale. Local farmers have developed ancient breeding programs entrenched in traditional knowledge (TK) that has sustained rural cultures for thousands of years. The convention on biological diversity seeks fair and equitable sharing of genetic resources arising from local knowledge and requires signatory nations to provide appropriate policy and legal framework to farmers' rights over plant genetic resources and associated TK. DNA barcoding employed in this study is proposed as a model for conservation of genetic diversity and an essential step towards documenting and protecting farmers' rights and TK. Our study focuses on 32 landraces of small millets that are still used by indigenous farmers located in the rain fed areas of rural India and Nepal. Traditional knowledge of traits and utility was gathered using participatory methods and semi-structured interviews with key informants. DNA was extracted and sequenced (rbcL, trnH-psbA and ITS2) from 160 samples. Both multivariate analysis of traits and phylogenetic analyses were used to assess diversity among small millet landraces. Our research revealed considerable variation in traits and DNA sequences among the 32 small millet landraces. We utilized a tiered approach using ITS2 DNA barcode to make 100 % accurate landrace (32 landraces) and species (six species) assignments for all 160 blind samples in our study. We have also recorded precious TK of nutritional value, ecological and agricultural traits used by local farmers for each of these traditional landraces. This research demonstrates the potential of DNA barcoding as a reliable identification tool and for use in evaluating and conserving genetic diversity of small millets. We suggest ways in which DNA barcodes could be used in the Protection of Plant Varieties and Farmers' Rights in India and Nepal.

DNA barcoding of Bemisia tabaci complex (Hemiptera: Aleyrodidae) reveals southerly expansion of the dominant whitefly species on cotton in Pakistan.

PubMed

Ashfaq, Muhammad; Hebert, Paul D N; Mirza, M Sajjad; Khan, Arif M; Mansoor, Shahid; Shah, Ghulam S; Zafar, Yusuf

2014-01-01

Although whiteflies (Bemisia tabaci complex) are an important pest of cotton in Pakistan, its taxonomic diversity is poorly understood. As DNA barcoding is an effective tool for resolving species complexes and analyzing species distributions, we used this approach to analyze genetic diversity in the B. tabaci complex and map the distribution of B. tabaci lineages in cotton growing areas of Pakistan. Sequence diversity in the DNA barcode region (mtCOI-5') was examined in 593 whiteflies from Pakistan to determine the number of whitefly species and their distributions in the cotton-growing areas of Punjab and Sindh provinces. These new records were integrated with another 173 barcode sequences for B. tabaci, most from India, to better understand regional whitefly diversity. The Barcode Index Number (BIN) System assigned the 766 sequences to 15 BINs, including nine from Pakistan. Representative specimens of each Pakistan BIN were analyzed for mtCOI-3' to allow their assignment to one of the putative species in the B. tabaci complex recognized on the basis of sequence variation in this gene region. This analysis revealed the presence of Asia II 1, Middle East-Asia Minor 1, Asia 1, Asia II 5, Asia II 7, and a new lineage "Pakistan". The first two taxa were found in both Punjab and Sindh, but Asia 1 was only detected in Sindh, while Asia II 5, Asia II 7 and "Pakistan" were only present in Punjab. The haplotype networks showed that most haplotypes of Asia II 1, a species implicated in transmission of the cotton leaf curl virus, occurred in both India and Pakistan. DNA barcodes successfully discriminated cryptic species in B. tabaci complex. The dominant haplotypes in the B. tabaci complex were shared by India and Pakistan. Asia II 1 was previously restricted to Punjab, but is now the dominant lineage in southern Sindh; its southward spread may have serious implications for cotton plantations in this region.

Biodiversity hot spot on a hot spot: novel extremophile diversity in Hawaiian fumaroles.

PubMed

Wall, Kate; Cornell, Jennifer; Bizzoco, Richard W; Kelley, Scott T

2015-01-06

Fumaroles (steam vents) are the most common, yet least understood, microbial habitat in terrestrial geothermal settings. Long believed too extreme for life, recent advances in sample collection and DNA extraction methods have found that fumarole deposits and subsurface waters harbor a considerable diversity of viable microbes. In this study, we applied culture-independent molecular methods to explore fumarole deposit microbial assemblages in 15 different fumaroles in four geographic locations on the Big Island of Hawai'i. Just over half of the vents yielded sufficient high-quality DNA for the construction of 16S ribosomal RNA gene sequence clone libraries. The bacterial clone libraries contained sequences belonging to 11 recognized bacterial divisions and seven other division-level phylogenetic groups. Archaeal sequences were less numerous, but similarly diverse. The taxonomic composition among fumarole deposits was highly heterogeneous. Phylogenetic analysis found cloned fumarole sequences were related to microbes identified from a broad array of globally distributed ecotypes, including hot springs, terrestrial soils, and industrial waste sites. Our results suggest that fumarole deposits function as an "extremophile collector" and may be a hot spot of novel extremophile biodiversity. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Biodiversity hot spot on a hot spot: novel extremophile diversity in Hawaiian fumaroles

PubMed Central

Wall, Kate; Cornell, Jennifer; Bizzoco, Richard W; Kelley, Scott T

2015-01-01

Fumaroles (steam vents) are the most common, yet least understood, microbial habitat in terrestrial geothermal settings. Long believed too extreme for life, recent advances in sample collection and DNA extraction methods have found that fumarole deposits and subsurface waters harbor a considerable diversity of viable microbes. In this study, we applied culture-independent molecular methods to explore fumarole deposit microbial assemblages in 15 different fumaroles in four geographic locations on the Big Island of Hawai'i. Just over half of the vents yielded sufficient high-quality DNA for the construction of 16S ribosomal RNA gene sequence clone libraries. The bacterial clone libraries contained sequences belonging to 11 recognized bacterial divisions and seven other division-level phylogenetic groups. Archaeal sequences were less numerous, but similarly diverse. The taxonomic composition among fumarole deposits was highly heterogeneous. Phylogenetic analysis found cloned fumarole sequences were related to microbes identified from a broad array of globally distributed ecotypes, including hot springs, terrestrial soils, and industrial waste sites. Our results suggest that fumarole deposits function as an “extremophile collector” and may be a hot spot of novel extremophile biodiversity. PMID:25565172

Protistan diversity and activity inferred from RNA and DNA at a coastal ocean site in the eastern North Pacific.

PubMed

Hu, Sarah K; Campbell, Victoria; Connell, Paige; Gellene, Alyssa G; Liu, Zhenfeng; Terrado, Ramon; Caron, David A

2016-04-01

Microbial eukaryotes fulfill key ecological positions in marine food webs. Molecular approaches that connect protistan diversity and biogeography to their diverse metabolisms will greatly improve our understanding of marine ecosystem function. The majority of molecular-based studies to date use 18S rRNA gene sequencing to characterize natural microbial assemblages, but this approach does not necessarily discriminate between active and non-active cells. We incorporated RNA sequencing into standard 18S rRNA gene sequence surveys with the purpose of assessing those members of the protistan community contributing to biogeochemical cycling (active organisms), using the ratio of cDNA (reverse transcribed from total RNA) to 18S rRNA gene sequences within major protistan taxonomic groups. Trophically important phytoplankton, such as diatoms and chlorophytes exhibited seasonal trends in relative activity. Additionally, both radiolaria and ciliates displayed previously unreported high relative activities below the euphotic zone. This study sheds new light on the relative metabolic activity of specific protistan groups and how microbial communities respond to changing environmental conditions. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

[The use of 16S rDNA sequencing in species diversity analysis for sputum of patients with ventilator-associated pneumonia].

PubMed

Yang, Xiaojun; Wang, Xiaohong; Liang, Zhijuan; Zhang, Xiaoya; Wang, Yanbo; Wang, Zhenhai

2014-05-01

To study the species and amount of bacteria in sputum of patients with ventilator-associated pneumonia (VAP) by using 16S rDNA sequencing analysis, and to explore the new method for etiologic diagnosis of VAP. Bronchoalveolar lavage sputum samples were collected from 31 patients with VAP. Bacterial DNA of the samples were extracted and identified by polymerase chain reaction (PCR). At the same time, sputum specimens were processed for routine bacterial culture. The high flux sequencing experiment was conducted on PCR positive samples with 16S rDNA macro genome sequencing technology, and sequencing results were analyzed using bioinformatics, then the results between the sequencing and bacteria culture were compared. (1) 550 bp of specific DNA sequences were amplified in sputum specimens from 27 cases of the 31 patients with VAP, and they were used for sequencing analysis. 103 856 sequences were obtained from those sputum specimens using 16S rDNA sequencing, yielding approximately 39 Mb of raw data. Tag sequencing was able to inform genus level in all 27 samples. (2) Alpha-diversity analysis showed that sputum samples of patients with VAP had significantly higher variability and richness in bacterial species (Shannon index values 1.20, Simpson index values 0.48). Rarefaction curve analysis showed that there were more species that were not detected by sequencing from some VAP sputum samples. (3) Analysis of 27 sputum samples with VAP by using 16S rDNA sequences yielded four phyla: namely Acitinobacteria, Bacteroidetes, Firmicutes, Proteobacteria. With genus as a classification, it was found that the dominant species included Streptococcus 88.9% (24/27), Limnohabitans 77.8% (21/27), Acinetobacter 70.4% (19/27), Sphingomonas 63.0% (17/27), Prevotella 63.0% (17/27), Klebsiella 55.6% (15/27), Pseudomonas 55.6% (15/27), Aquabacterium 55.6% (15/27), and Corynebacterium 55.6% (15/27). (4) Pyrophosphate sequencing discovered that Prevotella, Limnohabitans, Aquabacterium, Sphingomonas might not be detected by routine bacteria culture. Among seven species which were identified by both methods, pyrophosphate sequencing yielded higher positive rate than that of ordinary bacteria culture [Streptococcus: 88.9% (24/27) vs. 18.5% (5/27), Klebsiella: 55.6% (15/27) vs. 18.5% (5/27), Acinetobacter: 70.4% (19/27) vs. 37.0% (10/27), Corynebacterium: 55.6% (15/27) vs. 7.4% (2/27), P<0.05 or P<0.01]. Sequencing positive rate was found to increase positive rate for culture of Pseudomonas [55.6% (15/27) vs. 25.9% (7/27), P=0.050]. No significant differences were observed between sequencing and ordinary bacteria culture for detection Staphylococcus [7.4% (2/27) vs. 11.1% (3/27)] and Neisseria bacteria genera [18.5% (5/27) vs. 3.7% (1/27), both P>0.05]. 16S rDNA sequencing analysis confirmed that pathogenic bacteria in sputum of VAP were complicated with multiple drug resistant strains. Compared with routine bacterial culture, pyrophosphate sequencing had higher positive rate in detecting pathogens. 16S rDNA gene sequencing technology may become a new method for etiological diagnosis of VAP.

[Investigation of bacterial diversity in the biological desulfurization reactor for treating high salinity wastewater by the 16S rDNA cloning method].

PubMed

Liu, Wei-Guo; Liang, Cun-Zhen; Yang, Jin-Sheng; Wang, Gui-Ping; Liu, Miao-Miao

2013-02-01

The bacterial diversity in the biological desulfurization reactor operated continuously for 1 year was studied by the 16S rDNA cloning and sequencing method. Forty clones were randomly selected and their partial 16S rDNA genes (ca. 1,400 bp) were sequenced and blasted. The results indicated that there were dominant bacterias in the biological desulfurization reactor, where 33 clones belonged to 3 different published phyla, while 1 clone belonged to unknown phylum. The dominant bacterial community in the system was Proteobacteria, which accounted for 85.3%. The bacterial community succession was as follows: the gamma-Proteobacteria(55.9%), beta-Proteobacteria(17.6%), Actinobacteridae (8.8%), delta-Proteobacteria (5.9%) , alpha-Proteobacteria(5.9%), and Sphingobacteria (2.9%). Halothiobacillus sp. ST15 and Thiobacillus sp. UAM-I were the major desulfurization strains.

IM-TORNADO: a tool for comparison of 16S reads from paired-end libraries.

PubMed

Jeraldo, Patricio; Kalari, Krishna; Chen, Xianfeng; Bhavsar, Jaysheel; Mangalam, Ashutosh; White, Bryan; Nelson, Heidi; Kocher, Jean-Pierre; Chia, Nicholas

2014-01-01

16S rDNA hypervariable tag sequencing has become the de facto method for accessing microbial diversity. Illumina paired-end sequencing, which produces two separate reads for each DNA fragment, has become the platform of choice for this application. However, when the two reads do not overlap, existing computational pipelines analyze data from read separately and underutilize the information contained in the paired-end reads. We created a workflow known as Illinois Mayo Taxon Organization from RNA Dataset Operations (IM-TORNADO) for processing non-overlapping reads while retaining maximal information content. Using synthetic mock datasets, we show that the use of both reads produced answers with greater correlation to those from full length 16S rDNA when looking at taxonomy, phylogeny, and beta-diversity. IM-TORNADO is freely available at http://sourceforge.net/projects/imtornado and produces BIOM format output for cross compatibility with other pipelines such as QIIME, mothur, and phyloseq.

In vivo generation of DNA sequence diversity for cellular barcoding

PubMed Central

Peikon, Ian D.; Gizatullina, Diana I.; Zador, Anthony M.

2014-01-01

Heterogeneity is a ubiquitous feature of biological systems. A complete understanding of such systems requires a method for uniquely identifying and tracking individual components and their interactions with each other. We have developed a novel method of uniquely tagging individual cells in vivo with a genetic ‘barcode’ that can be recovered by DNA sequencing. Our method is a two-component system comprised of a genetic barcode cassette whose fragments are shuffled by Rci, a site-specific DNA invertase. The system is highly scalable, with the potential to generate theoretical diversities in the billions. We demonstrate the feasibility of this technique in Escherichia coli. Currently, this method could be employed to track the dynamics of populations of microbes through various bottlenecks. Advances of this method should prove useful in tracking interactions of cells within a network, and/or heterogeneity within complex biological samples. PMID:25013177

Diversity of Bacterial Communities in Container Habitats of Mosquitoes

PubMed Central

Ponnusamy, Loganathan; Xu, Ning; Stav, Gil; Wesson, Dawn M.; Schal, Coby

2010-01-01

We investigated the bacterial diversity of microbial communities in water-filled, human-made and natural container habitats of the mosquitoes Aedes aegypti and Aedes albopictus in suburban landscapes of New Orleans, Louisiana in 2003. We collected water samples from three classes of containers, including tires (n=12), cemetery urns (n=23), and miscellaneous containers that included two tree holes (n=19). Total genomic DNA was extracted from water samples, and 16S ribosomal DNA fragments (operational taxonomic units, OTUs) were amplified by PCR and separated by denaturing gradient gel electrophoresis (DGGE). The bacterial communities in containers represented diverse DGGE-DNA banding patterns that were not related to the class of container or to the local spatial distribution of containers. Mean richness and evenness of OTUs were highest in water samples from tires. Bacterial phylotypes were identified by comparative sequence analysis of 90 16S rDNA DGGE band amplicons. The majority of sequences were placed in five major taxa: Alpha-, Beta- and Gammaproteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and an unclassified group; Proteobacteria and Bacteroidetes were the predominant heterotrophic bacteria in containers. The bacterial communities in human-made containers consisted mainly of undescribed species, and a phylogenetic analysis based on 16S rRNA sequences suggested that species composition was independent of both container type and the spatial distribution of containers. Comparative PCR-based, cultivation-independent rRNA surveys of microbial communities associated with mosquito habitats can provide significant insight into community organization and dynamics of bacterial species. PMID:18373113

Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer

PubMed Central

Sanz, Yolanda

2017-01-01

Abstract The miniaturized and portable DNA sequencer MinION™ has demonstrated great potential in different analyses such as genome-wide sequencing, pathogen outbreak detection and surveillance, human genome variability, and microbial diversity. In this study, we tested the ability of the MinION™ platform to perform long amplicon sequencing in order to design new approaches to study microbial diversity using a multi-locus approach. After compiling a robust database by parsing and extracting the rrn bacterial region from more than 67000 complete or draft bacterial genomes, we demonstrated that the data obtained during sequencing of the long amplicon in the MinION™ device using R9 and R9.4 chemistries were sufficient to study 2 mock microbial communities in a multiplex manner and to almost completely reconstruct the microbial diversity contained in the HM782D and D6305 mock communities. Although nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, we presented a novel approach consisting of multi-locus and long amplicon sequencing using the MinION™ MkIb DNA sequencer and R9 and R9.4 chemistries that help to overcome the main disadvantage of this portable sequencing platform. Furthermore, the nanopore sequencing library, constructed with the last releases of pore chemistry (R9.4) and sequencing kit (SQK-LSK108), permitted the retrieval of the higher level of 1D read accuracy sufficient to characterize the microbial species present in each mock community analysed. Improvements in nanopore chemistry, such as minimizing base-calling errors and new library protocols able to produce rapid 1D libraries, will provide more reliable information in the near future. Such data will be useful for more comprehensive and faster specific detection of microbial species and strains in complex ecosystems. PMID:28605506

Suitability of partial 16S ribosomal RNA gene sequence analysis for the identification of dangerous bacterial pathogens.

PubMed

Ruppitsch, W; Stöger, A; Indra, A; Grif, K; Schabereiter-Gurtner, C; Hirschl, A; Allerberger, F

2007-03-01

In a bioterrorism event a rapid tool is needed to identify relevant dangerous bacteria. The aim of the study was to assess the usefulness of partial 16S rRNA gene sequence analysis and the suitability of diverse databases for identifying dangerous bacterial pathogens. For rapid identification purposes a 500-bp fragment of the 16S rRNA gene of 28 isolates comprising Bacillus anthracis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, and eight genus-related and unrelated control strains was amplified and sequenced. The obtained sequence data were submitted to three public and two commercial sequence databases for species identification. The most frequent reason for incorrect identification was the lack of the respective 16S rRNA gene sequences in the database. Sequence analysis of a 500-bp 16S rDNA fragment allows the rapid identification of dangerous bacterial species. However, for discrimination of closely related species sequencing of the entire 16S rRNA gene, additional sequencing of the 23S rRNA gene or sequencing of the 16S-23S rRNA intergenic spacer is essential. This work provides comprehensive information on the suitability of partial 16S rDNA analysis and diverse databases for rapid and accurate identification of dangerous bacterial pathogens.

The structures of non-CG-repeat Z-DNAs co-crystallized with the Z-DNA-binding domain, hZ alpha(ADAR1).

PubMed

Ha, Sung Chul; Choi, Jongkeun; Hwang, Hye-Yeon; Rich, Alexander; Kim, Yang-Gyun; Kim, Kyeong Kyu

2009-02-01

The Z-DNA conformation preferentially occurs at alternating purine-pyrimidine repeats, and is specifically recognized by Z alpha domains identified in several Z-DNA-binding proteins. The binding of Z alpha to foreign or chromosomal DNA in various sequence contexts is known to influence various biological functions, including the DNA-mediated innate immune response and transcriptional modulation of gene expression. For these reasons, understanding its binding mode and the conformational diversity of Z alpha bound Z-DNAs is of considerable importance. However, structural studies of Z alpha bound Z-DNA have been mostly limited to standard CG-repeat DNAs. Here, we have solved the crystal structures of three representative non-CG repeat DNAs, d(CACGTG)(2), d(CGTACG)(2) and d(CGGCCG)(2) complexed to hZ alpha(ADAR1) and compared those structures with that of hZ alpha(ADAR1)/d(CGCGCG)(2) and the Z alpha-free Z-DNAs. hZ alpha(ADAR1) bound to each of the three Z-DNAs showed a well conserved binding mode with very limited structural deviation irrespective of the DNA sequence, although varying numbers of residues were in contact with Z-DNA. Z-DNAs display less structural alterations in the Z alpha-bound state than in their free form, thereby suggesting that conformational diversities of Z-DNAs are restrained by the binding pocket of Z alpha. These data suggest that Z-DNAs are recognized by Z alpha through common conformational features regardless of the sequence and structural alterations.

In silico assessment of primers for eDNA studies using PrimerTree and application to characterize the biodiversity surrounding the Cuyahoga River

NASA Astrophysics Data System (ADS)

Cannon, M. V.; Hester, J.; Shalkhauser, A.; Chan, E. R.; Logue, K.; Small, S. T.; Serre, D.

2016-03-01

Analysis of environmental DNA (eDNA) enables the detection of species of interest from water and soil samples, typically using species-specific PCR. Here, we describe a method to characterize the biodiversity of a given environment by amplifying eDNA using primer pairs targeting a wide range of taxa and high-throughput sequencing for species identification. We tested this approach on 91 water samples of 40 mL collected along the Cuyahoga River (Ohio, USA). We amplified eDNA using 12 primer pairs targeting mammals, fish, amphibians, birds, bryophytes, arthropods, copepods, plants and several microorganism taxa and sequenced all PCR products simultaneously by high-throughput sequencing. Overall, we identified DNA sequences from 15 species of fish, 17 species of mammals, 8 species of birds, 15 species of arthropods, one turtle and one salamander. Interestingly, in addition to aquatic and semi-aquatic animals, we identified DNA from terrestrial species that live near the Cuyahoga River. We also identified DNA from one Asian carp species invasive to the Great Lakes but that had not been previously reported in the Cuyahoga River. Our study shows that analysis of eDNA extracted from small water samples using wide-range PCR amplification combined with high-throughput sequencing can provide a broad perspective on biological diversity.

Background sequence characteristics influence the occurrence and severity of disease-causing mtDNA mutations

PubMed Central

Wei, Wei; Hudson, Gavin

2017-01-01

Inherited mitochondrial DNA (mtDNA) mutations have emerged as a common cause of human disease, with mutations occurring multiple times in the world population. The clinical presentation of three pathogenic mtDNA mutations is strongly associated with a background mtDNA haplogroup, but it is not clear whether this is limited to a handful of examples or is a more general phenomenon. To address this, we determined the characteristics of 30,506 mtDNA sequences sampled globally. After performing several quality control steps, we ascribed an established pathogenicity score to the major alleles for each sequence. The mean pathogenicity score for known disease-causing mutations was significantly different between mtDNA macro-haplogroups. Several mutations were observed across all haplogroup backgrounds, whereas others were only observed on specific clades. In some instances this reflected a founder effect, but in others, the mutation recurred but only within the same phylogenetic cluster. Sequence diversity estimates showed that disease-causing mutations were more frequent on young sequences, and genomes with two or more disease-causing mutations were more common than expected by chance. These findings implicate the mtDNA background more generally in recurrent mutation events that have been purified through natural selection in older populations. This provides an explanation for the low frequency of mtDNA disease reported in specific ethnic groups. PMID:29253894

In silico assessment of primers for eDNA studies using PrimerTree and application to characterize the biodiversity surrounding the Cuyahoga River

PubMed Central

Cannon, M. V.; Hester, J.; Shalkhauser, A.; Chan, E. R.; Logue, K.; Small, S. T.; Serre, D.

2016-01-01

Analysis of environmental DNA (eDNA) enables the detection of species of interest from water and soil samples, typically using species-specific PCR. Here, we describe a method to characterize the biodiversity of a given environment by amplifying eDNA using primer pairs targeting a wide range of taxa and high-throughput sequencing for species identification. We tested this approach on 91 water samples of 40 mL collected along the Cuyahoga River (Ohio, USA). We amplified eDNA using 12 primer pairs targeting mammals, fish, amphibians, birds, bryophytes, arthropods, copepods, plants and several microorganism taxa and sequenced all PCR products simultaneously by high-throughput sequencing. Overall, we identified DNA sequences from 15 species of fish, 17 species of mammals, 8 species of birds, 15 species of arthropods, one turtle and one salamander. Interestingly, in addition to aquatic and semi-aquatic animals, we identified DNA from terrestrial species that live near the Cuyahoga River. We also identified DNA from one Asian carp species invasive to the Great Lakes but that had not been previously reported in the Cuyahoga River. Our study shows that analysis of eDNA extracted from small water samples using wide-range PCR amplification combined with high-throughput sequencing can provide a broad perspective on biological diversity. PMID:26965911

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era

PubMed Central

d’Avila-Levy, Claudia Masini; Boucinha, Carolina; Kostygov, Alexei; Santos, Helena Lúcia Carneiro; Morelli, Karina Alessandra; Grybchuk-Ieremenko, Anastasiia; Duval, Linda; Votýpka, Jan; Yurchenko, Vyacheslav; Grellier, Philippe; Lukeš, Julius

2015-01-01

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists. PMID:26602872

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae)

PubMed Central

Begum, Rabeya; Zakrzewski, Falk; Menzel, Gerhard; Weber, Beatrice; Alam, Sheikh Shamimul; Schmidt, Thomas

2013-01-01

Background and Aims The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. Methods A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100–500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. Key Results Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S–5·8S–25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. Conclusions The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species. PMID:23666888

Quantitative analysis and prediction of G-quadruplex forming sequences in double-stranded DNA

PubMed Central

Kim, Minji; Kreig, Alex; Lee, Chun-Ying; Rube, H. Tomas; Calvert, Jacob; Song, Jun S.; Myong, Sua

2016-01-01

Abstract G-quadruplex (GQ) is a four-stranded DNA structure that can be formed in guanine-rich sequences. GQ structures have been proposed to regulate diverse biological processes including transcription, replication, translation and telomere maintenance. Recent studies have demonstrated the existence of GQ DNA in live mammalian cells and a significant number of potential GQ forming sequences in the human genome. We present a systematic and quantitative analysis of GQ folding propensity on a large set of 438 GQ forming sequences in double-stranded DNA by integrating fluorescence measurement, single-molecule imaging and computational modeling. We find that short minimum loop length and the thymine base are two main factors that lead to high GQ folding propensity. Linear and Gaussian process regression models further validate that the GQ folding potential can be predicted with high accuracy based on the loop length distribution and the nucleotide content of the loop sequences. Our study provides important new parameters that can inform the evaluation and classification of putative GQ sequences in the human genome. PMID:27095201

Integrating DNA barcode data and taxonomic practice: determination, discovery, and description.

PubMed

Goldstein, Paul Z; DeSalle, Rob

2011-02-01

DNA barcodes, like traditional sources of taxonomic information, are potentially powerful heuristics in the identification of described species but require mindful analytical interpretation. The role of DNA barcoding in generating hypotheses of new taxa in need of formal taxonomic treatment is discussed, and it is emphasized that the recursive process of character evaluation is both necessary and best served by understanding the empirical mechanics of the discovery process. These undertakings carry enormous ramifications not only for the translation of DNA sequence data into taxonomic information but also for our comprehension of the magnitude of species diversity and its disappearance. This paper examines the potential strengths and pitfalls of integrating DNA sequence data, specifically in the form of DNA barcodes as they are currently generated and analyzed, with taxonomic practice.

The Diversity Present in 5140 Human Mitochondrial Genomes

PubMed Central

Pereira, Luísa; Freitas, Fernando; Fernandes, Verónica; Pereira, Joana B.; Costa, Marta D.; Costa, Stephanie; Máximo, Valdemar; Macaulay, Vincent; Rocha, Ricardo; Samuels, David C.

2009-01-01

We analyzed the current status (as of the end of August 2008) of human mitochondrial genomes deposited in GenBank, amounting to 5140 complete or coding-region sequences, in order to present an overall picture of the diversity present in the mitochondrial DNA of the global human population. To perform this task, we developed mtDNA-GeneSyn, a computer tool that identifies and exhaustedly classifies the diversity present in large genetic data sets. The diversity observed in the 5140 human mitochondrial genomes was compared with all possible transitions and transversions from the standard human mitochondrial reference genome. This comparison showed that tRNA and rRNA secondary structures have a large effect in limiting the diversity of the human mitochondrial sequences, whereas for the protein-coding genes there is a bias toward less variation at the second codon positions. The analysis of the observed amino acid variations showed a tolerance of variations that convert between the amino acids V, I, A, M, and T. This defines a group of amino acids with similar chemical properties that can interconvert by a single transition. PMID:19426953

Protein Science by DNA Sequencing: How Advances in Molecular Biology Are Accelerating Biochemistry.

PubMed

Higgins, Sean A; Savage, David F

2018-01-09

A fundamental goal of protein biochemistry is to determine the sequence-function relationship, but the vastness of sequence space makes comprehensive evaluation of this landscape difficult. However, advances in DNA synthesis and sequencing now allow researchers to assess the functional impact of every single mutation in many proteins, but challenges remain in library construction and the development of general assays applicable to a diverse range of protein functions. This Perspective briefly outlines the technical innovations in DNA manipulation that allow massively parallel protein biochemistry and then summarizes the methods currently available for library construction and the functional assays of protein variants. Areas in need of future innovation are highlighted with a particular focus on assay development and the use of computational analysis with machine learning to effectively traverse the sequence-function landscape. Finally, applications in the fundamentals of protein biochemistry, disease prediction, and protein engineering are presented.

The"minimum information about an environmental sequence" (MIENS) specification

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

Yilmaz, P.; Kottmann, R.; Field, D.

We present the Genomic Standards Consortium's (GSC) 'Minimum Information about an ENvironmental Sequence' (MIENS) standard for describing marker genes. Adoption of MIENS will enhance our ability to analyze natural genetic diversity across the Tree of Life as it is currently being documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.

Characterizing novel endogenous retroviruses from genetic variation inferred from short sequence reads

PubMed Central

Mourier, Tobias; Mollerup, Sarah; Vinner, Lasse; Hansen, Thomas Arn; Kjartansdóttir, Kristín Rós; Guldberg Frøslev, Tobias; Snogdal Boutrup, Torsten; Nielsen, Lars Peter; Willerslev, Eske; Hansen, Anders J.

2015-01-01

From Illumina sequencing of DNA from brain and liver tissue from the lion, Panthera leo, and tumor samples from the pike-perch, Sander lucioperca, we obtained two assembled sequence contigs with similarity to known retroviruses. Phylogenetic analyses suggest that the pike-perch retrovirus belongs to the epsilonretroviruses, and the lion retrovirus to the gammaretroviruses. To determine if these novel retroviral sequences originate from an endogenous retrovirus or from a recently integrated exogenous retrovirus, we assessed the genetic diversity of the parental sequences from which the short Illumina reads are derived. First, we showed by simulations that we can robustly infer the level of genetic diversity from short sequence reads. Second, we find that the measures of nucleotide diversity inferred from our retroviral sequences significantly exceed the level observed from Human Immunodeficiency Virus infections, prompting us to conclude that the novel retroviruses are both of endogenous origin. Through further simulations, we rule out the possibility that the observed elevated levels of nucleotide diversity are the result of co-infection with two closely related exogenous retroviruses. PMID:26493184

Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay

EPA Science Inventory

Using rDNA sequencing analysis, we examined the bacterial diversity and the presence of opportunistic bacterial pathogens (i.e., Campylobacter and Helicobacter) in Red Knot (Calidris canutus, n=40), Ruddy Turnstone (Arenaria interpres, n=35), and Semipalmated Sandpiper (Calidris ...

Unusually high genetic diversity in COI sequences of Chimarra obscura (Trichoptera: Philopotamidae)

EPA Science Inventory

Chimarra obscura (Walker 1852) is a philopotamid caddisfly found throughout much of North America. Using the COI DNA barcode locus, we have found unexpectedly high amounts of genetic diversity and distances within C. obscura. Of the approximately 150 specimens sampled, we have fo...

Molecular diversity and distribution of marine fungi across 130 European environmental samples.

PubMed

Richards, Thomas A; Leonard, Guy; Mahé, Frédéric; Del Campo, Javier; Romac, Sarah; Jones, Meredith D M; Maguire, Finlay; Dunthorn, Micah; De Vargas, Colomban; Massana, Ramon; Chambouvet, Aurélie

2015-11-22

Environmental DNA and culture-based analyses have suggested that fungi are present in low diversity and in low abundance in many marine environments, especially in the upper water column. Here, we use a dual approach involving high-throughput diversity tag sequencing from both DNA and RNA templates and fluorescent cell counts to evaluate the diversity and relative abundance of fungi across marine samples taken from six European near-shore sites. We removed very rare fungal operational taxonomic units (OTUs) selecting only OTUs recovered from multiple samples for a detailed analysis. This approach identified a set of 71 fungal 'OTU clusters' that account for 66% of all the sequences assigned to the Fungi. Phylogenetic analyses demonstrated that this diversity includes a significant number of chytrid-like lineages that had not been previously described, indicating that the marine environment encompasses a number of zoosporic fungi that are new to taxonomic inventories. Using the sequence datasets, we identified cases where fungal OTUs were sampled across multiple geographical sites and between different sampling depths. This was especially clear in one relatively abundant and diverse phylogroup tentatively named Novel Chytrid-Like-Clade 1 (NCLC1). For comparison, a subset of the water column samples was also investigated using fluorescent microscopy to examine the abundance of eukaryotes with chitin cell walls. Comparisons of relative abundance of RNA-derived fungal tag sequences and chitin cell-wall counts demonstrate that fungi constitute a low fraction of the eukaryotic community in these water column samples. Taken together, these results demonstrate the phylogenetic position and environmental distribution of 71 lineages, improving our understanding of the diversity and abundance of fungi in marine environments. © 2015 The Authors.

Molecular diversity and distribution of marine fungi across 130 European environmental samples

PubMed Central

Richards, Thomas A.; Leonard, Guy; Mahé, Frédéric; del Campo, Javier; Romac, Sarah; Jones, Meredith D. M.; Maguire, Finlay; Dunthorn, Micah; De Vargas, Colomban; Massana, Ramon; Chambouvet, Aurélie

2015-01-01

Environmental DNA and culture-based analyses have suggested that fungi are present in low diversity and in low abundance in many marine environments, especially in the upper water column. Here, we use a dual approach involving high-throughput diversity tag sequencing from both DNA and RNA templates and fluorescent cell counts to evaluate the diversity and relative abundance of fungi across marine samples taken from six European near-shore sites. We removed very rare fungal operational taxonomic units (OTUs) selecting only OTUs recovered from multiple samples for a detailed analysis. This approach identified a set of 71 fungal ‘OTU clusters' that account for 66% of all the sequences assigned to the Fungi. Phylogenetic analyses demonstrated that this diversity includes a significant number of chytrid-like lineages that had not been previously described, indicating that the marine environment encompasses a number of zoosporic fungi that are new to taxonomic inventories. Using the sequence datasets, we identified cases where fungal OTUs were sampled across multiple geographical sites and between different sampling depths. This was especially clear in one relatively abundant and diverse phylogroup tentatively named Novel Chytrid-Like-Clade 1 (NCLC1). For comparison, a subset of the water column samples was also investigated using fluorescent microscopy to examine the abundance of eukaryotes with chitin cell walls. Comparisons of relative abundance of RNA-derived fungal tag sequences and chitin cell-wall counts demonstrate that fungi constitute a low fraction of the eukaryotic community in these water column samples. Taken together, these results demonstrate the phylogenetic position and environmental distribution of 71 lineages, improving our understanding of the diversity and abundance of fungi in marine environments. PMID:26582030

Application of Sequence-based Methods in Human MicrobialEcology

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

Weng, Li; Rubin, Edward M.; Bristow, James

2005-08-29

Ecologists studying microbial life in the environment have recognized the enormous complexity of microbial diversity for many years, and the development of a variety of culture-independent methods, many of them coupled with high-throughput DNA sequencing, has allowed this diversity to be explored in ever greater detail. Despite the widespread application of these new techniques to the characterization of uncultivated microbes and microbial communities in the environment, their application to human health and disease has lagged behind. Because DNA based-techniques for defining uncultured microbes allow not only cataloging of microbial diversity, but also insight into microbial functions, investigators are beginning tomore » apply these tools to the microbial communities that abound on and within us, in what has aptly been called the second Human Genome Project. In this review we discuss the sequence-based methods for microbial analysis that are currently available and their application to identify novel human pathogens, improve diagnosis of known infectious diseases, and to advance understanding of our relationship with microbial communities that normally reside in and on the human body.« less

Endophytic bacterial diversity in grapevine (Vitis vinifera L.) leaves described by 16S rRNA gene sequence analysis and length heterogeneity-PCR.

PubMed

Bulgari, Daniela; Casati, Paola; Brusetti, Lorenzo; Quaglino, Fabio; Brasca, Milena; Daffonchio, Daniele; Bianco, Piero Attilio

2009-08-01

Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. In order to identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Sequence analysis of 16S rRNA gene libraries underscored five diverse Operational Taxonomic Units (OTUs), showing best sequence matches with gamma-Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Bacteria isolation through cultivation revealed the presence of six OTUs, showing best sequence matches with Actinobacteria, genus Curtobacterium, and with Firmicutes genera Bacillus and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Analysis of healthy and phytoplasma-infected grapevine plants showed that LH-PCR could be a useful complementary tool for examining the diversity of bacterial endophytes especially for diversity survey on a large number of samples.

HIV sequence diversity during the early phase of infection is associated with HIV DNA reductions during antiretroviral therapy.

PubMed

Wang, Nidan; Li, Yijia; Han, Yang; Xie, Jing; Li, Taisheng

2017-06-01

The association between baseline human immunodeficiency virus (HIV) sequence diversity and HIV DNA decay after the initiation of antiretroviral therapy (ART) remains uncharacterized during the early stages of HIV infection. Samples were obtained from a cohort of 17 patients with early HIV infection (<6 months after infection) who initiated ART, and the C2V5 region of the HIV-1 envelope (env) gene was amplified via single genome amplification (SGA) to determine the peripheral plasma HIV quasispecies. We categorized HIV quasispecies into two groups according to baseline viral sequence genetic distance, which was determined by the Poisson-Fitter tool. Total HIV DNA in peripheral blood mononuclear cells (PBMCs), viral load, and T cell subsets were measured prior to and after the initiation of ART. The median SGA sequence number was 17 (range 6-28). At baseline, we identified 7 patients with homogeneous viral populations (designated the Homogeneous group) and 10 patients with heterogeneous viral populations (designated the Heterogeneous group) based on SGA sequences. Both groups exhibited similar HIV DNA decay rates during the first 6 months of ART (P > 0.99), but the Homogenous group experienced more prominent decay than the Heterogeneous group after 6 months (P = 0.037). The Heterogeneous group had higher CD4 cell counts after ART initiation; however, both groups had comparable recovery in terms of CD4/CD8 ratios and CD8 T cell activation levels. Viral population homogeneity upon the initiation of ART is associated with a decrease in HIV DNA levels during ART. J. Med. Virol. 89:982-988, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Assessment of genome origins and genetic diversity in the genus Eleusine with DNA markers.

PubMed

Salimath, S S; de Oliveira, A C; Godwin, I D; Bennetzen, J L

1995-08-01

Finger millet (Eleusine coracana), an allotetraploid cereal, is widely cultivated in the arid and semiarid regions of the world. Three DNA marker techniques, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), and inter simple sequence repeat amplification (ISSR), were employed to analyze 22 accessions belonging to 5 species of Eleusine. An 8 probe--3 enzyme RFLP combination, 18 RAPD primers, and 6 ISSR primers, respectively, revealed 14, 10, and 26% polymorphism in 17 accessions of E. coracana from Africa and Asia. These results indicated a very low level of DNA sequence variability in the finger millets but did allow each line to be distinguished. The different Eleusine species could be easily identified by DNA marker technology and the 16% intraspecific polymorphism exhibited by the two analyzed accessions of E. floccifolia suggested a much higher level of diversity in this species than in E. coracana. Between species, E. coracana and E. indica shared the most markers, while E. indica and E. tristachya shared a considerable number of markers, indicating that these three species form a close genetic assemblage within the Eleusine. Eleusine floccifolia and E. compressa were found to be the most divergent among the species examined. Comparison of RFLP, RAPD, and ISSR technologies, in terms of the quantity and quality of data output, indicated that ISSRs are particularly promising for the analysis of plant genome diversity.

Genome-wide characterization of centromeric satellites from multiple mammalian genomes.

PubMed

Alkan, Can; Cardone, Maria Francesca; Catacchio, Claudia Rita; Antonacci, Francesca; O'Brien, Stephen J; Ryder, Oliver A; Purgato, Stefania; Zoli, Monica; Della Valle, Giuliano; Eichler, Evan E; Ventura, Mario

2011-01-01

Despite its importance in cell biology and evolution, the centromere has remained the final frontier in genome assembly and annotation due to its complex repeat structure. However, isolation and characterization of the centromeric repeats from newly sequenced species are necessary for a complete understanding of genome evolution and function. In recent years, various genomes have been sequenced, but the characterization of the corresponding centromeric DNA has lagged behind. Here, we present a computational method (RepeatNet) to systematically identify higher-order repeat structures from unassembled whole-genome shotgun sequence and test whether these sequence elements correspond to functional centromeric sequences. We analyzed genome datasets from six species of mammals representing the diversity of the mammalian lineage, namely, horse, dog, elephant, armadillo, opossum, and platypus. We define candidate monomer satellite repeats and demonstrate centromeric localization for five of the six genomes. Our analysis revealed the greatest diversity of centromeric sequences in horse and dog in contrast to elephant and armadillo, which showed high-centromeric sequence homogeneity. We could not isolate centromeric sequences within the platypus genome, suggesting that centromeres in platypus are not enriched in satellite DNA. Our method can be applied to the characterization of thousands of other vertebrate genomes anticipated for sequencing in the near future, providing an important tool for annotation of centromeres.

Metagenomic characterization of airborne viral DNA diversity in the near-surface atmosphere.

PubMed

Whon, Tae Woong; Kim, Min-Soo; Roh, Seong Woon; Shin, Na-Ri; Lee, Hae-Won; Bae, Jin-Woo

2012-08-01

Airborne viruses are expected to be ubiquitous in the atmosphere but they still remain poorly understood. This study investigated the temporal and spatial dynamics of airborne viruses and their genotypic characteristics in air samples collected from three distinct land use types (a residential district [RD], a forest [FR], and an industrial complex [IC]) and from rainwater samples freshly precipitated at the RD site (RD-rain). Viral abundance exhibited a seasonal fluctuation in the range between 1.7 × 10(6) and 4.0 × 10(7) viruses m(-3), which increased from autumn to winter and decreased toward spring, but no significant spatial differences were observed. Temporal variations in viral abundance were inversely correlated with seasonal changes in temperature and absolute humidity. Metagenomic analysis of air viromes amplified by rolling-circle phi29 polymerase-based random hexamer priming indicated the dominance of plant-associated single-stranded DNA (ssDNA) geminivirus-related viruses, followed by animal-infecting circovirus-related sequences, with low numbers of nanoviruses and microphages-related genomes. Particularly, the majority of the geminivirus-related viruses were closely related to ssDNA mycoviruses that infect plant-pathogenic fungi. Phylogenetic analysis based on the replication initiator protein sequence indicated that the airborne ssDNA viruses were distantly related to known ssDNA viruses, suggesting that a high diversity of viruses were newly discovered. This research is the first to report the seasonality of airborne viruses and their genetic diversity, which enhances our understanding of viral ecology in temperate regions.

Metagenomic Characterization of Airborne Viral DNA Diversity in the Near-Surface Atmosphere

PubMed Central

Whon, Tae Woong; Kim, Min-Soo; Roh, Seong Woon; Shin, Na-Ri; Lee, Hae-Won

2012-01-01

Airborne viruses are expected to be ubiquitous in the atmosphere but they still remain poorly understood. This study investigated the temporal and spatial dynamics of airborne viruses and their genotypic characteristics in air samples collected from three distinct land use types (a residential district [RD], a forest [FR], and an industrial complex [IC]) and from rainwater samples freshly precipitated at the RD site (RD-rain). Viral abundance exhibited a seasonal fluctuation in the range between 1.7 × 106 and 4.0 × 107 viruses m−3, which increased from autumn to winter and decreased toward spring, but no significant spatial differences were observed. Temporal variations in viral abundance were inversely correlated with seasonal changes in temperature and absolute humidity. Metagenomic analysis of air viromes amplified by rolling-circle phi29 polymerase-based random hexamer priming indicated the dominance of plant-associated single-stranded DNA (ssDNA) geminivirus-related viruses, followed by animal-infecting circovirus-related sequences, with low numbers of nanoviruses and microphages-related genomes. Particularly, the majority of the geminivirus-related viruses were closely related to ssDNA mycoviruses that infect plant-pathogenic fungi. Phylogenetic analysis based on the replication initiator protein sequence indicated that the airborne ssDNA viruses were distantly related to known ssDNA viruses, suggesting that a high diversity of viruses were newly discovered. This research is the first to report the seasonality of airborne viruses and their genetic diversity, which enhances our understanding of viral ecology in temperate regions. PMID:22623790

Genetic composition and connectivity of the Antillean manatee (Trichechus manatus manatus) in Panama

USGS Publications Warehouse

Díaz-Ferguson, Edgardo; Hunter, Margaret; Guzmán, Héctor M.

2017-01-01

Genetic diversity and haplotype composition of the West Indian manatee (Trichechus manatus) population from the San San Pond Sak wetland in Bocas del Toro, Panama was studied using a segment of mitochondrial DNA (D’loop). No genetic information has been published to date for Panamanian populations. Due to the secretive behavior and small population size of the species in the area, DNA extraction was conducted from opportunistically collected fecal (N=20), carcass tissue (N=4) and bone (N=4) samples. However, after DNA processing only 10 samples provided good quality DNA for sequencing (3 fecal, 4 tissue and 3 bone samples). We found three haplotypes in total; two of these haplotypes are reported for the first time, J02 (N=3) and J03 (N=4), and one J01 was previously published (N=3). Genetic diversity showed similar values to previous studies conducted in other Caribbean regions with moderate values of nucleotide diversity (π= 0.00152) and haplotipic diversity (Hd= 0.57). Connectivity assessment was based on sequence similarity, genetic distance and genetic differentiation between San San population and other manatee populations previously studied. The J01 haplotype found in the Panamanian population is shared with populations in the Caribbean mainland and the Gulf of Mexico showing a reduced differentiation corroborated with Fst value between HSSPS and this region of 0.0094. In contrast, comparisons between our sequences and populations in the Eastern Caribbean (South American populations) and North Western Caribbean showed fewer similarities (Fst =0.049 and 0.058, respectively). These results corroborate previous phylogeographic patterns already established for manatee populations and situate Panamanian populations into the Belize and Mexico cluster. In addition, these findings will be a baseline for future studies and comparisons with manatees in other areas of Panama and Central America. These results should be considered to inform management decisions regarding conservation of genetic diversity, future controlled introductions, connectivity and effective population size of the West Indian manatee along the Central American corridor.

msgbsR: An R package for analysing methylation-sensitive restriction enzyme sequencing data.

PubMed

Mayne, Benjamin T; Leemaqz, Shalem Y; Buckberry, Sam; Rodriguez Lopez, Carlos M; Roberts, Claire T; Bianco-Miotto, Tina; Breen, James

2018-02-01

Genotyping-by-sequencing (GBS) or restriction-site associated DNA marker sequencing (RAD-seq) is a practical and cost-effective method for analysing large genomes from high diversity species. This method of sequencing, coupled with methylation-sensitive enzymes (often referred to as methylation-sensitive restriction enzyme sequencing or MRE-seq), is an effective tool to study DNA methylation in parts of the genome that are inaccessible in other sequencing techniques or are not annotated in microarray technologies. Current software tools do not fulfil all methylation-sensitive restriction sequencing assays for determining differences in DNA methylation between samples. To fill this computational need, we present msgbsR, an R package that contains tools for the analysis of methylation-sensitive restriction enzyme sequencing experiments. msgbsR can be used to identify and quantify read counts at methylated sites directly from alignment files (BAM files) and enables verification of restriction enzyme cut sites with the correct recognition sequence of the individual enzyme. In addition, msgbsR assesses DNA methylation based on read coverage, similar to RNA sequencing experiments, rather than methylation proportion and is a useful tool in analysing differential methylation on large populations. The package is fully documented and available freely online as a Bioconductor package ( https://bioconductor.org/packages/release/bioc/html/msgbsR.html ).

The Mitochondrial Genome of Chara vulgaris: Insights into the Mitochondrial DNA Architecture of the Last Common Ancestor of Green Algae and Land PlantsW⃞

PubMed Central

Turmel, Monique; Otis, Christian; Lemieux, Claude

2003-01-01

Mitochondrial DNA (mtDNA) has undergone radical changes during the evolution of green plants, yet little is known about the dynamics of mtDNA evolution in this phylum. Land plant mtDNAs differ from the few green algal mtDNAs that have been analyzed to date by their expanded size, long spacers, and diversity of introns. We have determined the mtDNA sequence of Chara vulgaris (Charophyceae), a green alga belonging to the charophycean order (Charales) that is thought to be the most closely related alga to land plants. This 67,737-bp mtDNA sequence, displaying 68 conserved genes and 27 introns, was compared with those of three angiosperms, the bryophyte Marchantia polymorpha, the charophycean alga Chaetosphaeridium globosum (Coleochaetales), and the green alga Mesostigma viride. Despite important differences in size and intron composition, Chara mtDNA strikingly resembles Marchantia mtDNA; for instance, all except 9 of 68 conserved genes lie within blocks of colinear sequences. Overall, our genome comparisons and phylogenetic analyses provide unequivocal support for a sister-group relationship between the Charales and the land plants. Only four introns in land plant mtDNAs appear to have been inherited vertically from a charalean algar ancestor. We infer that the common ancestor of green algae and land plants harbored a tightly packed, gene-rich, and relatively intron-poor mitochondrial genome. The group II introns in this ancestral genome appear to have spread to new mtDNA sites during the evolution of bryophytes and charalean green algae, accounting for part of the intron diversity found in Chara and land plant mitochondria. PMID:12897260

A high-throughput Sanger strategy for human mitochondrial genome sequencing

PubMed Central

2013-01-01

Background A population reference database of complete human mitochondrial genome (mtGenome) sequences is needed to enable the use of mitochondrial DNA (mtDNA) coding region data in forensic casework applications. However, the development of entire mtGenome haplotypes to forensic data quality standards is difficult and laborious. A Sanger-based amplification and sequencing strategy that is designed for automated processing, yet routinely produces high quality sequences, is needed to facilitate high-volume production of these mtGenome data sets. Results We developed a robust 8-amplicon Sanger sequencing strategy that regularly produces complete, forensic-quality mtGenome haplotypes in the first pass of data generation. The protocol works equally well on samples representing diverse mtDNA haplogroups and DNA input quantities ranging from 50 pg to 1 ng, and can be applied to specimens of varying DNA quality. The complete workflow was specifically designed for implementation on robotic instrumentation, which increases throughput and reduces both the opportunities for error inherent to manual processing and the cost of generating full mtGenome sequences. Conclusions The described strategy will assist efforts to generate complete mtGenome haplotypes which meet the highest data quality expectations for forensic genetic and other applications. Additionally, high-quality data produced using this protocol can be used to assess mtDNA data developed using newer technologies and chemistries. Further, the amplification strategy can be used to enrich for mtDNA as a first step in sample preparation for targeted next-generation sequencing. PMID:24341507

DNA Barcoding the Geometrid Fauna of Bavaria (Lepidoptera): Successes, Surprises, and Questions

PubMed Central

Hausmann, Axel; Haszprunar, Gerhard; Hebert, Paul D. N.

2011-01-01

Background The State of Bavaria is involved in a research program that will lead to the construction of a DNA barcode library for all animal species within its territorial boundaries. The present study provides a comprehensive DNA barcode library for the Geometridae, one of the most diverse of insect families. Methodology/Principal Findings This study reports DNA barcodes for 400 Bavarian geometrid species, 98 per cent of the known fauna, and approximately one per cent of all Bavarian animal species. Although 98.5% of these species possess diagnostic barcode sequences in Bavaria, records from neighbouring countries suggest that species-level resolution may be compromised in up to 3.5% of cases. All taxa which apparently share barcodes are discussed in detail. One case of modest divergence (1.4%) revealed a species overlooked by the current taxonomic system: Eupithecia goossensiata Mabille, 1869 stat.n. is raised from synonymy with Eupithecia absinthiata (Clerck, 1759) to species rank. Deep intraspecific sequence divergences (>2%) were detected in 20 traditionally recognized species. Conclusions/Significance The study emphasizes the effectiveness of DNA barcoding as a tool for monitoring biodiversity. Open access is provided to a data set that includes records for 1,395 geometrid specimens (331 species) from Bavaria, with 69 additional species from neighbouring regions. Taxa with deep intraspecific sequence divergences are undergoing more detailed analysis to ascertain if they represent cases of cryptic diversity. PMID:21423340

DNA barcoding applied to ex situ tropical amphibian conservation programme reveals cryptic diversity in captive populations.

PubMed

Crawford, Andrew J; Cruz, Catalina; Griffith, Edgardo; Ross, Heidi; Ibáñez, Roberto; Lips, Karen R; Driskell, Amy C; Bermingham, Eldredge; Crump, Paul

2013-11-01

Amphibians constitute a diverse yet still incompletely characterized clade of vertebrates, in which new species are still being discovered and described at a high rate. Amphibians are also increasingly endangered, due in part to disease-driven threats of extinctions. As an emergency response, conservationists have begun ex situ assurance colonies for priority species. The abundance of cryptic amphibian diversity, however, may cause problems for ex situ conservation. In this study we used a DNA barcoding approach to survey mitochondrial DNA (mtDNA) variation in captive populations of 10 species of Neotropical amphibians maintained in an ex situ assurance programme at El Valle Amphibian Conservation Center (EVACC) in the Republic of Panama. We combined these mtDNA sequences with genetic data from presumably conspecific wild populations sampled from across Panama, and applied genetic distance-based and character-based analyses to identify cryptic lineages. We found that three of ten species harboured substantial cryptic genetic diversity within EVACC, and an additional three species harboured cryptic diversity among wild populations, but not in captivity. Ex situ conservation efforts focused on amphibians are therefore vulnerable to an incomplete taxonomy leading to misidentification among cryptic species. DNA barcoding may therefore provide a simple, standardized protocol to identify cryptic diversity readily applicable to any amphibian community. © 2012 John Wiley & Sons Ltd.

Phylogenetic Diversity of Lactic Acid Bacteria Associated with Paddy Rice Silage as Determined by 16S Ribosomal DNA Analysis

PubMed Central

Ennahar, Saïd; Cai, Yimin; Fujita, Yasuhito

2003-01-01

A total of 161 low-G+C-content gram-positive bacteria isolated from whole-crop paddy rice silage were classified and subjected to phenotypic and genetic analyses. Based on morphological and biochemical characters, these presumptive lactic acid bacterium (LAB) isolates were divided into 10 groups that included members of the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Weissella. Analysis of the 16S ribosomal DNA (rDNA) was used to confirm the presence of the predominant groups indicated by phenotypic analysis and to determine the phylogenetic affiliation of representative strains. The virtually complete 16S rRNA gene was PCR amplified and sequenced. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank reference strains (between 98.7 and 99.8%). Phylogenetic trees based on the 16S rDNA sequence displayed high consistency, with nodes supported by high bootstrap values. With the exception of one species, the genetic data was in agreement with the phenotypic identification. The prevalent LAB, predominantly homofermentative (66%), consisted of Lactobacillus plantarum (24%), Lactococcus lactis (22%), Leuconostoc pseudomesenteroides (20%), Pediococcus acidilactici (11%), Lactobacillus brevis (11%), Enterococcus faecalis (7%), Weissella kimchii (3%), and Pediococcus pentosaceus (2%). The present study, the first to fully document rice-associated LAB, showed a very diverse community of LAB with a relatively high number of species involved in the fermentation process of paddy rice silage. The comprehensive 16S rDNA-based approach to describing LAB community structure was valuable in revealing the large diversity of bacteria inhabiting paddy rice silage and enabling the future design of appropriate inoculants aimed at improving its fermentation quality. PMID:12514026

Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis.

PubMed

Ennahar, Saïd; Cai, Yimin; Fujita, Yasuhito

2003-01-01

A total of 161 low-G+C-content gram-positive bacteria isolated from whole-crop paddy rice silage were classified and subjected to phenotypic and genetic analyses. Based on morphological and biochemical characters, these presumptive lactic acid bacterium (LAB) isolates were divided into 10 groups that included members of the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and WEISSELLA: Analysis of the 16S ribosomal DNA (rDNA) was used to confirm the presence of the predominant groups indicated by phenotypic analysis and to determine the phylogenetic affiliation of representative strains. The virtually complete 16S rRNA gene was PCR amplified and sequenced. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank reference strains (between 98.7 and 99.8%). Phylogenetic trees based on the 16S rDNA sequence displayed high consistency, with nodes supported by high bootstrap values. With the exception of one species, the genetic data was in agreement with the phenotypic identification. The prevalent LAB, predominantly homofermentative (66%), consisted of Lactobacillus plantarum (24%), Lactococcus lactis (22%), Leuconostoc pseudomesenteroides (20%), Pediococcus acidilactici (11%), Lactobacillus brevis (11%), Enterococcus faecalis (7%), Weissella kimchii (3%), and Pediococcus pentosaceus (2%). The present study, the first to fully document rice-associated LAB, showed a very diverse community of LAB with a relatively high number of species involved in the fermentation process of paddy rice silage. The comprehensive 16S rDNA-based approach to describing LAB community structure was valuable in revealing the large diversity of bacteria inhabiting paddy rice silage and enabling the future design of appropriate inoculants aimed at improving its fermentation quality.

Diversity in a Cold Hot-Spot: DNA-Barcoding Reveals Patterns of Evolution among Antarctic Demosponges (Class Demospongiae, Phylum Porifera).

PubMed

Vargas, Sergio; Kelly, Michelle; Schnabel, Kareen; Mills, Sadie; Bowden, David; Wörheide, Gert

2015-01-01

The approximately 350 demosponge species that have been described from Antarctica represent a faunistic component distinct from that of neighboring regions. Sponges provide structure to the Antarctic benthos and refuge to other invertebrates, and can be dominant in some communities. Despite the importance of sponges in the Antarctic subtidal environment, sponge DNA barcodes are scarce but can provide insight into the evolutionary relationships of this unique biogeographic province. We sequenced the standard barcoding COI region for a comprehensive selection of sponges collected during expeditions to the Ross Sea region in 2004 and 2008, and produced DNA-barcodes for 53 demosponge species covering about 60% of the species collected. The Antarctic sponge communities are phylogenetically diverse, matching the diversity of well-sampled sponge communities in the Lusitanic and Mediterranean marine provinces in the Temperate Northern Atlantic for which molecular data are readily available. Additionally, DNA-barcoding revealed levels of in situ molecular evolution comparable to those present among Caribbean sponges. DNA-barcoding using the Segregating Sites Algorithm correctly assigned approximately 54% of the barcoded species to the morphologically determined species. A barcode library for Antarctic sponges was assembled and used to advance the systematic and evolutionary research of Antarctic sponges. We provide insights on the evolutionary forces shaping Antarctica's diverse sponge communities, and a barcode library against which future sequence data from other regions or depth strata of Antarctica can be compared. The opportunity for rapid taxonomic identification of sponge collections for ecological research is now at the horizon.

Diversity of DNA and RNA Viruses in Indoor Air As Assessed via Metagenomic Sequencing.

PubMed

Rosario, Karyna; Fierer, Noah; Miller, Shelly; Luongo, Julia; Breitbart, Mya

2018-02-06

Diverse bacterial and fungal communities inhabit human-occupied buildings and circulate in indoor air; however, viral diversity in these man-made environments remains largely unknown. Here we investigated DNA and RNA viruses circulating in the air of 12 university dormitory rooms by analyzing dust accumulated over a one-year period on heating, ventilation, and air conditioning (HVAC) filters. A metagenomic sequencing approach was used to determine the identity and diversity of viral particles extracted from the HVAC filters. We detected a broad diversity of viruses associated with a range of hosts, including animals, arthropods, bacteria, fungi, humans, plants, and protists, suggesting that disparate organisms can contribute to indoor airborne viral communities. Viral community composition and the distribution of human-infecting papillomaviruses and polyomaviruses were distinct in the different dormitory rooms, indicating that airborne viral communities are variable in human-occupied spaces and appear to reflect differential rates of viral shedding from room occupants. This work significantly expands the known airborne viral diversity found indoors, enabling the design of sensitive and quantitative assays to further investigate specific viruses of interest and providing new insight into the likely sources of viruses found in indoor air.

Selection and Screening of DNA Aptamers for Inorganic Nanomaterials.

PubMed

Zhou, Yibo; Huang, Zhicheng; Yang, Ronghua; Liu, Juewen

2018-02-21

Searching for DNA sequences that can strongly and selectively bind to inorganic surfaces is a long-standing topic in bionanotechnology, analytical chemistry and biointerface research. This can be achieved either by aptamer selection starting with a very large library of ≈10 14 random DNA sequences, or by careful screening of a much smaller library (usually from a few to a few hundred) with rationally designed sequences. Unlike typical molecular targets, inorganic surfaces often have quite strong DNA adsorption affinities due to polyvalent binding and even chemical interactions. This leads to a very high background binding making aptamer selection difficult. Screening, on the other hand, can be designed to compare relative binding affinities of different DNA sequences and could be more appropriate for inorganic surfaces. The resulting sequences have been used for DNA-directed assembly, sorting of carbon nanotubes, and DNA-controlled growth of inorganic nanomaterials. It was recently discovered that poly-cytosine (C) DNA can strongly bind to a diverse range of nanomaterials including nanocarbons (graphene oxide and carbon nanotubes), various metal oxides and transition-metal dichalcogenides. In this Concept article, we articulate the need for screening and potential artifacts associated with traditional aptamer selection methods for inorganic surfaces. Representative examples of application are discussed, and a few future research opportunities are proposed towards the end of this article. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microbial community analysis of the hypersaline water of the Dead Sea using high-throughput amplicon sequencing.

PubMed

Jacob, Jacob H; Hussein, Emad I; Shakhatreh, Muhamad Ali K; Cornelison, Christopher T

2017-10-01

Amplicon sequencing using next-generation technology (bTEFAP ® ) has been utilized in describing the diversity of Dead Sea microbiota. The investigated area is a well-known salt lake in the western part of Jordan found in the lowest geographical location in the world (more than 420 m below sea level) and characterized by extreme salinity (approximately, 34%) in addition to other extreme conditions (low pH, unique ionic composition different from sea water). DNA was extracted from Dead Sea water. A total of 314,310 small subunit RNA (SSU rRNA) sequences were parsed, and 288,452 sequences were then clustered. For alpha diversity analysis, sample was rarefied to 3,000 sequences. The Shannon-Wiener index curve plot reached a plateau at approximately 3,000 sequences indicating that sequencing depth was sufficient to capture the full scope of microbial diversity. Archaea was found to be dominating the sequences (52%), whereas Bacteria constitute 45% of the sequences. Altogether, prokaryotic sequences (which constitute 97% of all sequences) were found to predominate. The findings expand on previous studies by using high-throughput amplicon sequencing to describe the microbial community in an environment which in recent years has been shown to hide some interesting diversity. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Reconstructing the history of a fragmented and heavily exploited red deer population using ancient and contemporary DNA.

PubMed

Rosvold, Jørgen; Røed, Knut H; Hufthammer, Anne Karin; Andersen, Reidar; Stenøien, Hans K

2012-09-26

Red deer (Cervus elaphus) have been an important human resource for millennia, experiencing intensive human influence through habitat alterations, hunting and translocation of animals. In this study we investigate a time series of ancient and contemporary DNA from Norwegian red deer spanning about 7,000 years. Our main aim was to investigate how increasing agricultural land use, hunting pressure and possibly human mediated translocation of animals have affected the genetic diversity on a long-term scale. We obtained mtDNA (D-loop) sequences from 73 ancient specimens. These show higher genetic diversity in ancient compared to extant samples, with the highest diversity preceding the onset of agricultural intensification in the Early Iron Age. Using standard diversity indices, Bayesian skyline plot and approximate Bayesian computation, we detected a population reduction which was more prolonged than, but not as severe as, historic documents indicate. There are signs of substantial changes in haplotype frequencies primarily due to loss of haplotypes through genetic drift. There is no indication of human mediated translocations into the Norwegian population. All the Norwegian sequences show a western European origin, from which the Norwegian lineage diverged approximately 15,000 years ago. Our results provide direct insight into the effects of increasing habitat fragmentation and human hunting pressure on genetic diversity and structure of red deer populations. They also shed light on the northward post-glacial colonisation process of red deer in Europe and suggest increased precision in inferring past demographic events when including both ancient and contemporary DNA.

Ultraaccurate genome sequencing and haplotyping of single human cells.

PubMed

Chu, Wai Keung; Edge, Peter; Lee, Ho Suk; Bansal, Vikas; Bafna, Vineet; Huang, Xiaohua; Zhang, Kun

2017-11-21

Accurate detection of variants and long-range haplotypes in genomes of single human cells remains very challenging. Common approaches require extensive in vitro amplification of genomes of individual cells using DNA polymerases and high-throughput short-read DNA sequencing. These approaches have two notable drawbacks. First, polymerase replication errors could generate tens of thousands of false-positive calls per genome. Second, relatively short sequence reads contain little to no haplotype information. Here we report a method, which is dubbed SISSOR (single-stranded sequencing using microfluidic reactors), for accurate single-cell genome sequencing and haplotyping. A microfluidic processor is used to separate the Watson and Crick strands of the double-stranded chromosomal DNA in a single cell and to randomly partition megabase-size DNA strands into multiple nanoliter compartments for amplification and construction of barcoded libraries for sequencing. The separation and partitioning of large single-stranded DNA fragments of the homologous chromosome pairs allows for the independent sequencing of each of the complementary and homologous strands. This enables the assembly of long haplotypes and reduction of sequence errors by using the redundant sequence information and haplotype-based error removal. We demonstrated the ability to sequence single-cell genomes with error rates as low as 10 -8 and average 500-kb-long DNA fragments that can be assembled into haplotype contigs with N50 greater than 7 Mb. The performance could be further improved with more uniform amplification and more accurate sequence alignment. The ability to obtain accurate genome sequences and haplotype information from single cells will enable applications of genome sequencing for diverse clinical needs. Copyright © 2017 the Author(s). Published by PNAS.

High-Throughput Sequencing of 16S rRNA Gene Amplicons: Effects of Extraction Procedure, Primer Length and Annealing Temperature

PubMed Central

Sergeant, Martin J.; Constantinidou, Chrystala; Cogan, Tristan; Penn, Charles W.; Pallen, Mark J.

2012-01-01

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers. PMID:22666455

High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature.

PubMed

Sergeant, Martin J; Constantinidou, Chrystala; Cogan, Tristan; Penn, Charles W; Pallen, Mark J

2012-01-01

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.

The mtDNA haplogroup P of modern Asian cattle: A genetic legacy of Asian aurochs?

PubMed

Noda, Aoi; Yonesaka, Riku; Sasazaki, Shinji; Mannen, Hideyuki

2018-01-01

Aurochs (Bos primigenius) were distributed throughout large parts of Eurasia and Northern Africa during the late Pleistocene and the early Holocene, and all modern cattle are derived from the aurochs. Although the mtDNA haplogroups of most modern cattle belong to haplogroups T and I, several additional haplogroups (P, Q, R, C and E) have been identified in modern cattle and aurochs. Haplogroup P was the most common haplogroup in European aurochs, but so far, it has been identified in only three of >3,000 submitted haplotypes of modern Asian cattle. We sequenced the complete mtDNA D-loop region of 181 Japanese Shorthorn cattle and analyzed these together with representative bovine mtDNA sequences. The haplotype P of Japanese Shorthorn cattle was analyzed along with that of 36 previously published European aurochs and three modern Asian cattle sequences using the hypervariable 410 bp of the D-loop region. We detected the mtDNA haplogroup P in Japanese Shorthorn cattle with an extremely high frequency (83/181). Phylogenetic networks revealed two main clusters, designated as Pa for haplogroup P in European aurochs and Pc in modern Asian cattle. We also report the genetic diversity of haplogroup P compared with the sequences of extinct aurochs. No shared haplotypes are observed between the European aurochs and the modern Asian cattle. This finding suggests the possibility of local and secondary introgression events of haplogroup P in northeast Asian cattle, and will contribute to a better understanding of its origin and genetic diversity.

The mtDNA haplogroup P of modern Asian cattle: A genetic legacy of Asian aurochs?

PubMed Central

Noda, Aoi; Yonesaka, Riku; Sasazaki, Shinji

2018-01-01

Background Aurochs (Bos primigenius) were distributed throughout large parts of Eurasia and Northern Africa during the late Pleistocene and the early Holocene, and all modern cattle are derived from the aurochs. Although the mtDNA haplogroups of most modern cattle belong to haplogroups T and I, several additional haplogroups (P, Q, R, C and E) have been identified in modern cattle and aurochs. Haplogroup P was the most common haplogroup in European aurochs, but so far, it has been identified in only three of >3,000 submitted haplotypes of modern Asian cattle. Methodology We sequenced the complete mtDNA D-loop region of 181 Japanese Shorthorn cattle and analyzed these together with representative bovine mtDNA sequences. The haplotype P of Japanese Shorthorn cattle was analyzed along with that of 36 previously published European aurochs and three modern Asian cattle sequences using the hypervariable 410 bp of the D-loop region. Conclusions We detected the mtDNA haplogroup P in Japanese Shorthorn cattle with an extremely high frequency (83/181). Phylogenetic networks revealed two main clusters, designated as Pa for haplogroup P in European aurochs and Pc in modern Asian cattle. We also report the genetic diversity of haplogroup P compared with the sequences of extinct aurochs. No shared haplotypes are observed between the European aurochs and the modern Asian cattle. This finding suggests the possibility of local and secondary introgression events of haplogroup P in northeast Asian cattle, and will contribute to a better understanding of its origin and genetic diversity. PMID:29304129

Comparative analyses of genetic/epigenetic diversities and structures in a wild barley species (Hordeum brevisubulatum) using MSAP, SSAP and AFLP.

PubMed

Shan, X H; Li, Y D; Liu, X M; Wu, Y; Zhang, M Z; Guo, W L; Liu, B; Yuan, Y P

2012-08-17

We analyzed genetic diversity and population genetic structure of four artificial populations of wild barley (Hordeum brevisubulatum); 96 plants collected from the Songnen Prairie in northeastern China were analyzed using amplified fragment length polymorphism (AFLP), specific-sequence amplified polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) markers. Indices of (epi-)genetic diversity, (epi-)genetic distance, gene flow, genotype frequency, cluster analysis, PCA analysis and AMOVA analysis generated from MSAP, AFLP and SSAP markers had the same trend. We found a high level of correlation in the artificial populations between MSAP, SSAP and AFLP markers by the Mantel test (r > 0.8). This is incongruent with previous findings showing that there is virtually no correlation between DNA methylation polymorphism and classical genetic variation; the high level of genetic polymorphism could be a result of epigenetic regulation. We compared our results with data from natural populations. The population diversity of the artificial populations was lower. However, different from what was found using AFLP and SSAP, based on MSAP results the methylation polymorphism of the artificial populations was not significantly reduced. This leads us to suggest that the DNA methylation pattern change in H. brevisubulatum populations is not only related to DNA sequence variation, but is also regulated by other controlling systems.

Genetic diversity of Guangxi chicken breeds assessed with microsatellites and the mitochondrial DNA D-loop region.

PubMed

Liao, Yuying; Mo, Guodong; Sun, Junli; Wei, Fengying; Liao, Dezhong Joshua

2016-05-01

The domestic chicken (Gallus gallus domesticus) is an excellent model for genetic studies of phenotypic diversity. The Guangxi Region of China possesses several native chicken breeds displaying a broad range of phenotypes well adapted to the extreme hot-and-wet environments in the region. We thus evaluated the genetic diversity and relationships among six native chicken populations of the Guangxi region and also evaluated two commercial breeds (Arbor Acres and Roman chickens). We analyzed the sequences of the D-loop region of the mitochondrial DNA (mtDNA) and 18 microsatellite loci of 280 blood samples from six Guangxi native chicken breeds and from Arbor Acres and Roman chickens, and used the neighbor-joining method to construct the phylogenetic tree of these eight breeds. Our results showed that the genetic diversity of Guangxi native breeds was relatively rich. The phylogenetic tree using the unweighed pair-group method with arithmetic means (UPGAM) on microsatellite marks revealed two main clusters. Arbor Acres chicken and Roman chicken were in one cluster, while the Guangxi breeds were in the other cluster. Moreover, the UPGAM tree of Guangxi native breeds based on microsatellite loci was more consistent with the genesis, breeding history, differentiation and location than the mtDNA D-loop region. STRUCTURE analysis further confirmed the genetic structure of Guangxi native breeds in the Neighbor-Net dendrogram. The nomenclature of mtDNA sequence polymorphisms suggests that the Guangxi native chickens are distributed across four clades, but most of them are clustered in two main clades (B and E), with the other haplotypes within the clades A and C. The Guangxi native breeds revealed abundant genetic diversity not only on microsatellite loci but also on mtDNA D-loop region, and contained multiple maternal lineages, including one from China and another from Europe or the Middle East.

Diverse Applications of Environmental DNA Methods in Parasitology.

PubMed

Bass, David; Stentiford, Grant D; Littlewood, D T J; Hartikainen, Hanna

2015-10-01

Nucleic acid extraction and sequencing of genes from organisms within environmental samples encompasses a variety of techniques collectively referred to as environmental DNA or 'eDNA'. The key advantages of eDNA analysis include the detection of cryptic or otherwise elusive organisms, large-scale sampling with fewer biases than specimen-based methods, and generation of data for molecular systematics. These are particularly relevant for parasitology because parasites can be difficult to locate and are morphologically intractable and genetically divergent. However, parasites have rarely been the focus of eDNA studies. Focusing on eukaryote parasites, we review the increasing diversity of the 'eDNA toolbox'. Combining eDNA methods with complementary tools offers much potential to understand parasite communities, disease risk, and parasite roles in broader ecosystem processes such as food web structuring and community assembly. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Genetic Diversity in Endangered Guizhou Snub-Nosed Monkeys (Rhinopithecus brelichi): Contrasting Results from Microsatellite and Mitochondrial DNA Data

PubMed Central

Kolleck, Jakob; Yang, Mouyu; Zinner, Dietmar; Roos, Christian

2013-01-01

To evaluate the conservation status of a species or population it is necessary to gain insight into its ecological requirements, reproduction, genetic population structure, and overall genetic diversity. In our study we examined the genetic diversity of Rhinopithecus brelichi by analyzing microsatellite data and compared them with already existing data derived from mitochondrial DNA, which revealed that R. brelichi exhibits the lowest mitochondrial diversity of all so far studied Rhinopithecus species. In contrast, the genetic diversity of nuclear DNA is high and comparable to other Rhinopithecus species, i.e. the examined microsatellite loci are similarly highly polymorphic as in other species of the genus. An explanation for these differences in mitochondrial and nuclear genetic diversity could be a male biased dispersal. Females most likely stay within their natal band and males migrate between bands, thus mitochondrial DNA will not be exchanged between bands but nuclear DNA via males. A Bayesian Skyline Plot based on mitochondrial DNA sequences shows a strong decrease of the female effective population size (Nef) starting about 3,500 to 4,000 years ago, which concurs with the increasing human population in the area and respective expansion of agriculture. Given that we found no indication for a loss of nuclear DNA diversity in R. brelichi it seems that this factor does not represent the most prominent conservation threat for the long-term survival of the species. Conservation efforts should therefore focus more on immediate threats such as development of tourism and habitat destruction. PMID:24009761

The wheat cytochrome oxidase subunit II gene has an intron insert and three radical amino acid changes relative to maize

PubMed Central

Bonen, Linda; Boer, Poppo H.; Gray, Michael W.

1984-01-01

We have determined the sequence of the wheat mitochondrial gene for cytochrome oxidase subunit II (COII) and find that its derived protein sequence differs from that of maize at only three amino acid positions. Unexpectedly, all three replacements are non-conservative ones. The wheat COII gene has a highly-conserved intron at the same position as in maize, but the wheat intron is 1.5 times longer because of an insert relative to its maize counterpart. Hybridization analysis of mitochondrial DNA from rye, pea, broad bean and cucumber indicates strong sequence conservation of COII coding sequences among all these higher plants. However, only rye and maize mitochondrial DNA show homology with wheat COII intron sequences and rye alone with intron-insert sequences. We find that a sequence identical to the region of the 5' exon corresponding to the transmembrane domain of the COII protein is present at a second genomic location in wheat mitochondria. These variations in COII gene structure and size, as well as the presence of repeated COII sequences, illustrate at the DNA sequence level, factors which contribute to higher plant mitochondrial DNA diversity and complexity. ImagesFig. 3.Fig. 4.Fig. 5. PMID:16453565

Widespread Transient Hoogsteen Base-Pairs in Canonical Duplex DNA with Variable Energetics

PubMed Central

Alvey, Heidi S.; Gottardo, Federico L.; Nikolova, Evgenia N.; Al-Hashimi, Hashim M.

2015-01-01

Hoogsteen base-pairing involves a 180 degree rotation of the purine base relative to Watson-Crick base-pairing within DNA duplexes, creating alternative DNA conformations that can play roles in recognition, damage induction, and replication. Here, using Nuclear Magnetic Resonance R1ρ relaxation dispersion, we show that transient Hoogsteen base-pairs occur across more diverse sequence and positional contexts than previously anticipated. We observe sequence-specific variations in Hoogsteen base-pair energetic stabilities that are comparable to variations in Watson-Crick base-pair stability, with Hoogsteen base-pairs being more abundant for energetically less favorable Watson-Crick base-pairs. Our results suggest that the variations in Hoogsteen stabilities and rates of formation are dominated by variations in Watson-Crick base pair stability, suggesting a late transition state for the Watson-Crick to Hoogsteen conformational switch. The occurrence of sequence and position-dependent Hoogsteen base-pairs provide a new potential mechanism for achieving sequence-dependent DNA transactions. PMID:25185517

Deep sampling of the Palomero maize transcriptome by a high throughput strategy of pyrosequencing.

PubMed

Vega-Arreguín, Julio C; Ibarra-Laclette, Enrique; Jiménez-Moraila, Beatriz; Martínez, Octavio; Vielle-Calzada, Jean Philippe; Herrera-Estrella, Luis; Herrera-Estrella, Alfredo

2009-07-06

In-depth sequencing analysis has not been able to determine the overall complexity of transcriptional activity of a plant organ or tissue sample. In some cases, deep parallel sequencing of Expressed Sequence Tags (ESTs), although not yet optimized for the sequencing of cDNAs, has represented an efficient procedure for validating gene prediction and estimating overall gene coverage. This approach could be very valuable for complex plant genomes. In addition, little emphasis has been given to efforts aiming at an estimation of the overall transcriptional universe found in a multicellular organism at a specific developmental stage. To explore, in depth, the transcriptional diversity in an ancient maize landrace, we developed a protocol to optimize the sequencing of cDNAs and performed 4 consecutive GS20-454 pyrosequencing runs of a cDNA library obtained from 2 week-old Palomero Toluqueño maize plants. The protocol reported here allowed obtaining over 90% of informative sequences. These GS20-454 runs generated over 1.5 Million reads, representing the largest amount of sequences reported from a single plant cDNA library. A collection of 367,391 quality-filtered reads (30.09 Mb) from a single run was sufficient to identify transcripts corresponding to 34% of public maize ESTs databases; total sequences generated after 4 filtered runs increased this coverage to 50%. Comparisons of all 1.5 Million reads to the Maize Assembled Genomic Islands (MAGIs) provided evidence for the transcriptional activity of 11% of MAGIs. We estimate that 5.67% (86,069 sequences) do not align with public ESTs or annotated genes, potentially representing new maize transcripts. Following the assembly of 74.4% of the reads in 65,493 contigs, real-time PCR of selected genes confirmed a predicted correlation between the abundance of GS20-454 sequences and corresponding levels of gene expression. A protocol was developed that significantly increases the number, length and quality of cDNA reads using massive 454 parallel sequencing. We show that recurrent 454 pyrosequencing of a single cDNA sample is necessary to attain a thorough representation of the transcriptional universe present in maize, that can also be used to estimate transcript abundance of specific genes. This data suggests that the molecular and functional diversity contained in the vast native landraces remains to be explored, and that large-scale transcriptional sequencing of a presumed ancestor of the modern maize varieties represents a valuable approach to characterize the functional diversity of maize for future agricultural and evolutionary studies.

Molecular Phylogenetic Diversity of Dermatologic and Other Human Pathogenic Fusaria from Hospitals in Northern Italy

USDA-ARS?s Scientific Manuscript database

Fifty-eight fusaria isolated from 52 Italian patients between 2004 and 2007 were subject to multilocus DNA sequence typing to characterize the spectrum of species and circulating sequence types (STs) associated with dermatological infections, especially onychomycoses and paronychia, and other fusari...

Analysis of human mitochondrial DNA sequences from fecally polluted environmental waters as a tool to study population diversity

EPA Science Inventory

Mitochondrial signature sequences have frequently been used to study the demographics of many different populations around the world. Traditionally, this requires obtaining samples directly from individuals which is cumbersome, time consuming and limited to the number of individu...

Microbial dynamics in upflow anaerobic sludge blanket (UASB) bioreactor granules in response to short-term changes in substrate feed

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

Kovacik, William P.; Scholten, Johannes C.; Culley, David E.

2010-08-01

The complexity and diversity of the microbial communities in biogranules from an upflow anaerobic sludge blanket (UASB) bioreactor were determined in response to short-term changes in substrate feeds. The reactor was fed simulated brewery wastewater (SBWW) (70% ethanol, 15% acetate, 15% propionate) for 1.5 months (phase 1), acetate / sulfate for 2 months (phase 2), acetate-alone for 3 months (phase 3), and then a return to SBWW for 2 months (phase 4). Performance of the reactor remained relatively stable throughout the experiment as shown by COD removal and gas production. 16S rDNA, methanogen-associated mcrA and sulfate reducer-associated dsrAB genes weremore » PCR amplified, then cloned and sequenced. Sequence analysis of 16S clone libraries showed a relatively simple community composed mainly of the methanogenic Archaea (Methanobacterium and Methanosaeta), members of the Green Non-Sulfur (Chloroflexi) group of Bacteria, followed by fewer numbers of Syntrophobacter, Spirochaeta, Acidobacteria and Cytophaga-related Bacterial sequences. Methanogen-related mcrA clone libraries were dominated throughout by Methanobacter and Methanospirillum related sequences. Although not numerous enough to be detected in our 16S rDNA libraries, sulfate reducers were detected in dsrAB clone libraries, with sequences related to Desulfovibrio and Desulfomonile. Community diversity levels (Shannon-Weiner index) generally decreased for all libraries in response to a change from SBWW to acetate-alone feed. But there was a large transitory increase noted in 16S diversity at the two-month sampling on acetate-alone, entirely related to an increase in Bacterial diversity. Upon return to SBWW conditions in phase 4, all diversity measures returned to near phase 1 levels.« less

Molecular and Cytogenetic Characterization of Wild Musa Species

PubMed Central

Čížková, Jana; Hřibová, Eva; Christelová, Pavla; Van den Houwe, Ines; Häkkinen, Markku; Roux, Nicolas; Swennen, Rony; Doležel, Jaroslav

2015-01-01

The production of bananas is threatened by rapid spreading of various diseases and adverse environmental conditions. The preservation and characterization of banana diversity is essential for the purposes of crop improvement. The world's largest banana germplasm collection maintained at the Bioversity International Transit Centre (ITC) in Belgium is continuously expanded by new accessions of edible cultivars and wild species. Detailed morphological and molecular characterization of the accessions is necessary for efficient management of the collection and utilization of banana diversity. In this work, nuclear DNA content and genomic distribution of 45S and 5S rDNA were examined in 21 diploid accessions recently added to ITC collection, representing both sections of the genus Musa. 2C DNA content in the section Musa ranged from 1.217 to 1.315 pg. Species belonging to section Callimusa had 2C DNA contents ranging from 1.390 to 1.772 pg. While the number of 45S rDNA loci was conserved in the section Musa, it was highly variable in Callimusa species. 5S rRNA gene clusters were found on two to eight chromosomes per diploid cell. The accessions were genotyped using a set of 19 microsatellite markers to establish their relationships with the remaining accessions held at ITC. Genetic diversity done by SSR genotyping platform was extended by phylogenetic analysis of ITS region. ITS sequence data supported the clustering obtained by SSR analysis for most of the accessions. High level of nucleotide diversity and presence of more than two types of ITS sequences in eight wild diploids pointed to their origin by hybridization of different genotypes. This study significantly expands the number of wild Musa species where nuclear genome size and genomic distribution of rDNA loci is known. SSR genotyping identified Musa species that are closely related to the previously characterized accessions and provided data to aid in their classification. Sequence analysis of ITS region provided further information about evolutionary relationships between individual accessions and suggested that some of analyzed accessions were interspecific hybrids and/or backcross progeny. PMID:26252482

Multiple DNA and protein sequence alignment on a workstation and a supercomputer.

PubMed

Tajima, K

1988-11-01

This paper describes a multiple alignment method using a workstation and supercomputer. The method is based on the alignment of a set of aligned sequences with the new sequence, and uses a recursive procedure of such alignment. The alignment is executed in a reasonable computation time on diverse levels from a workstation to a supercomputer, from the viewpoint of alignment results and computational speed by parallel processing. The application of the algorithm is illustrated by several examples of multiple alignment of 12 amino acid and DNA sequences of HIV (human immunodeficiency virus) env genes. Colour graphic programs on a workstation and parallel processing on a supercomputer are discussed.

Deep-sequencing to resolve complex diversity of apicomplexan parasites in platypuses and echidnas: Proof of principle for wildlife disease investigation.

PubMed

Šlapeta, Jan; Saverimuttu, Stefan; Vogelnest, Larry; Sangster, Cheryl; Hulst, Frances; Rose, Karrie; Thompson, Paul; Whittington, Richard

2017-11-01

The short-beaked echidna (Tachyglossus aculeatus) and the platypus (Ornithorhynchus anatinus) are iconic egg-laying monotremes (Mammalia: Monotremata) from Australasia. The aim of this study was to demonstrate the utility of diversity profiles in disease investigations of monotremes. Using small subunit (18S) rDNA amplicon deep-sequencing we demonstrated the presence of apicomplexan parasites and confirmed by direct and cloned amplicon gene sequencing Theileria ornithorhynchi, Theileria tachyglossi, Eimeria echidnae and Cryptosporidium fayeri. Using a combination of samples from healthy and diseased animals, we show a close evolutionary relationship between species of coccidia (Eimeria) and piroplasms (Theileria) from the echidna and platypus. The presence of E. echidnae was demonstrated in faeces and tissues affected by disseminated coccidiosis. Moreover, the presence of E. echidnae DNA in the blood of echidnas was associated with atoxoplasma-like stages in white blood cells, suggesting Hepatozoon tachyglossi blood stages are disseminated E. echidnae stages. These next-generation DNA sequencing technologies are suited to material and organisms that have not been previously characterised and for which the material is scarce. The deep sequencing approach supports traditional diagnostic methods, including microscopy, clinical pathology and histopathology, to better define the status quo. This approach is particularly suitable for wildlife disease investigation. Copyright © 2017 Elsevier B.V. All rights reserved.

Colombian Creole horse breeds: Same origin but different diversity

PubMed Central

Jimenez, Ligia Mercedes; Mendez, Susy; Dunner, Susana; Cañón, Javier; Cortés, Óscar

2012-01-01

In order to understand the genetic ancestry and mitochondrial DNA (mtDNA) diversity of current Colombian horse breeds we sequenced a 364-bp fragment of the mitocondrial DNA D-loop in 116 animals belonging to five Spanish horse breeds and the Colombian Paso Fino and Colombian Creole cattle horse breeds. Among Colombian horse breeds, haplogroup D had the highest frequency (53%), followed by haplogroups A (19%), C (8%) and F (6%). The higher frequency of haplogroup D in Colombian horse breeds supports the theory of an ancestral Iberian origin for these breeds. These results also indicate that different selective pressures among the Colombian breeds could explain the relatively higher genetic diversity found in the Colombian Creole cattle horse when compared with the Colombian Paso Fino. PMID:23271940

Comparison of a High-Resolution Melting Assay to Next-Generation Sequencing for Analysis of HIV Diversity

PubMed Central

Cousins, Matthew M.; Ou, San-San; Wawer, Maria J.; Munshaw, Supriya; Swan, David; Magaret, Craig A.; Mullis, Caroline E.; Serwadda, David; Porcella, Stephen F.; Gray, Ronald H.; Quinn, Thomas C.; Donnell, Deborah; Eshleman, Susan H.

2012-01-01

Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution. PMID:22785188

Environmental DNA metabarcoding: Transforming how we survey animal and plant communities.

PubMed

Deiner, Kristy; Bik, Holly M; Mächler, Elvira; Seymour, Mathew; Lacoursière-Roussel, Anaïs; Altermatt, Florian; Creer, Simon; Bista, Iliana; Lodge, David M; de Vere, Natasha; Pfrender, Michael E; Bernatchez, Louis

2017-11-01

The genomic revolution has fundamentally changed how we survey biodiversity on earth. High-throughput sequencing ("HTS") platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed "environmental DNA" or "eDNA"). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called "eDNA metabarcoding" and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education. © 2017 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

Validation of the Hirst-Type Spore Trap for Simultaneous Monitoring of Prokaryotic and Eukaryotic Biodiversities in Urban Air Samples by Next-Generation Sequencing.

PubMed

Núñez, Andrés; Amo de Paz, Guillermo; Ferencova, Zuzana; Rastrojo, Alberto; Guantes, Raúl; García, Ana M; Alcamí, Antonio; Gutiérrez-Bustillo, A Montserrat; Moreno, Diego A

2017-07-01

Pollen, fungi, and bacteria are the main microscopic biological entities present in outdoor air, causing allergy symptoms and disease transmission and having a significant role in atmosphere dynamics. Despite their relevance, a method for monitoring simultaneously these biological particles in metropolitan environments has not yet been developed. Here, we assessed the use of the Hirst-type spore trap to characterize the global airborne biota by high-throughput DNA sequencing, selecting regions of the 16S rRNA gene and internal transcribed spacer for the taxonomic assignment. We showed that aerobiological communities are well represented by this approach. The operational taxonomic units (OTUs) of two traps working synchronically compiled >87% of the total relative abundance for bacterial diversity collected in each sampler, >89% for fungi, and >97% for pollen. We found a good correspondence between traditional characterization by microscopy and genetic identification, obtaining more-accurate taxonomic assignments and detecting a greater diversity using the latter. We also demonstrated that DNA sequencing accurately detects differences in biodiversity between samples. We concluded that high-throughput DNA sequencing applied to aerobiological samples obtained with Hirst spore traps provides reliable results and can be easily implemented for monitoring prokaryotic and eukaryotic entities present in the air of urban areas. IMPORTANCE Detection, monitoring, and characterization of the wide diversity of biological entities present in the air are difficult tasks that require time and expertise in different disciplines. We have evaluated the use of the Hirst spore trap (an instrument broadly employed in aerobiological studies) to detect and identify these organisms by DNA-based analyses. Our results showed a consistent collection of DNA and a good concordance with traditional methods for identification, suggesting that these devices can be used as a tool for continuous monitoring of the airborne biodiversity, improving taxonomic resolution and characterization together. They are also suitable for acquiring novel DNA amplicon-based information in order to gain a better understanding of the biological particles present in a scarcely known environment such as the air. Copyright © 2017 American Society for Microbiology.

Validation of the Hirst-Type Spore Trap for Simultaneous Monitoring of Prokaryotic and Eukaryotic Biodiversities in Urban Air Samples by Next-Generation Sequencing

PubMed Central

Núñez, Andrés; Amo de Paz, Guillermo; Ferencova, Zuzana; Rastrojo, Alberto; Guantes, Raúl; García, Ana M.; Alcamí, Antonio; Gutiérrez-Bustillo, A. Montserrat

2017-01-01

ABSTRACT Pollen, fungi, and bacteria are the main microscopic biological entities present in outdoor air, causing allergy symptoms and disease transmission and having a significant role in atmosphere dynamics. Despite their relevance, a method for monitoring simultaneously these biological particles in metropolitan environments has not yet been developed. Here, we assessed the use of the Hirst-type spore trap to characterize the global airborne biota by high-throughput DNA sequencing, selecting regions of the 16S rRNA gene and internal transcribed spacer for the taxonomic assignment. We showed that aerobiological communities are well represented by this approach. The operational taxonomic units (OTUs) of two traps working synchronically compiled >87% of the total relative abundance for bacterial diversity collected in each sampler, >89% for fungi, and >97% for pollen. We found a good correspondence between traditional characterization by microscopy and genetic identification, obtaining more-accurate taxonomic assignments and detecting a greater diversity using the latter. We also demonstrated that DNA sequencing accurately detects differences in biodiversity between samples. We concluded that high-throughput DNA sequencing applied to aerobiological samples obtained with Hirst spore traps provides reliable results and can be easily implemented for monitoring prokaryotic and eukaryotic entities present in the air of urban areas. IMPORTANCE Detection, monitoring, and characterization of the wide diversity of biological entities present in the air are difficult tasks that require time and expertise in different disciplines. We have evaluated the use of the Hirst spore trap (an instrument broadly employed in aerobiological studies) to detect and identify these organisms by DNA-based analyses. Our results showed a consistent collection of DNA and a good concordance with traditional methods for identification, suggesting that these devices can be used as a tool for continuous monitoring of the airborne biodiversity, improving taxonomic resolution and characterization together. They are also suitable for acquiring novel DNA amplicon-based information in order to gain a better understanding of the biological particles present in a scarcely known environment such as the air. PMID:28455334

Efficient isolation method for high-quality genomic DNA from cicada exuviae.

PubMed

Nguyen, Hoa Quynh; Kim, Ye Inn; Borzée, Amaël; Jang, Yikweon

2017-10-01

In recent years, animal ethics issues have led researchers to explore nondestructive methods to access materials for genetic studies. Cicada exuviae are among those materials because they are cast skins that individuals left after molt and are easily collected. In this study, we aim to identify the most efficient extraction method to obtain high quantity and quality of DNA from cicada exuviae. We compared relative DNA yield and purity of six extraction protocols, including both manual protocols and available commercial kits, extracting from four different exoskeleton parts. Furthermore, amplification and sequencing of genomic DNA were evaluated in terms of availability of sequencing sequence at the expected genomic size. Both the choice of protocol and exuvia part significantly affected DNA yield and purity. Only samples that were extracted using the PowerSoil DNA Isolation kit generated gel bands of expected size as well as successful sequencing results. The failed attempts to extract DNA using other protocols could be partially explained by a low DNA yield from cicada exuviae and partly by contamination with humic acids that exist in the soil where cicada nymphs reside before emergence, as shown by spectroscopic measurements. Genomic DNA extracted from cicada exuviae could provide valuable information for species identification, allowing the investigation of genetic diversity across consecutive broods, or spatiotemporal variation among various populations. Consequently, we hope to provide a simple method to acquire pure genomic DNA applicable for multiple research purposes.

Dinoflagellates associated with freshwater sponges from the ancient lake baikal.

PubMed

Annenkova, Natalia V; Lavrov, Dennis V; Belikov, Sergey I

2011-04-01

Dinoflagellates are a diverse group of protists that are common in both marine and freshwater environments. While the biology of marine dinoflagellates has been the focus of several recent studies, their freshwater relatives remain little-investigated. In the present study we explore the diversity of dinoflagellates in Lake Baikal by identifying and analyzing dinoflagellate sequences for 18S rDNA and ITS-2 from total DNA extracted from three species of endemic Baikalian sponges (Baikalospongia intermedia,Baikalospongia rectaand Lubomirskia incrustans). Phylogenetic analyses of these sequences revealed extensive dinoflagellate diversity in Lake Baikal. We found two groups of sequences clustering within the order Suessiales, known for its symbiotic relationships with various invertebrates. Thus they may be regarded as potential symbionts of Baikalian sponges. In addition,Gyrodinium helveticum, representatives from the genus Gymnodinium, dinoflagellates close to the family Pfiesteriaceae, and a few dinoflagellates without definite affiliation were detected. No pronounced difference in the distribution of dinoflagellates among the studied sponges was found, except for the absence of the Piscinoodinium-like dinoflagellates inL. incrustans. To the best of our knowledge, this is the first study of the diversity of dinoflagellates in freshwater sponges, the first systematic investigation of dinoflagellate molecular diversity in Lake Baikal and the first finding of members of the order Suessiales as symbionts of freshwater invertebrates. Copyright © 2010 Elsevier GmbH. All rights reserved.

Microbial eukaryotic diversity and distribution in a river plume and cyclonic eddy-influenced ecosystem in the South China Sea.

PubMed

Wu, Wenxue; Wang, Lei; Liao, Yu; Huang, Bangqin

2015-10-01

To evaluate microbial eukaryotic diversity and distribution in mesoscale processes, we investigated 18S rDNA diversity in a river plume and cyclonic eddy-influenced ecosystem in the southwestern South China Sea (SCS). Restriction fragment length polymorphism analysis was carried out using multiple primer sets. Relative to a wide range of previous similar studies, we observed a significantly higher proportion of sequences of pigmented taxa. Among the photosynthetic groups, Haptophyta accounted for 27.7% of the sequenced clones, which belonged primarily to Prymnesiophyceae. Unexpectedly, five operational taxonomic units of Cryptophyta were closely related to freshwater species. The Chlorophyta mostly fell within the Prasinophyceae, which was comprised of six clades, including Clade III, which is detected in the SCS for the first time in this study. Among the photosynthetic stramenopiles, Chrysophyceae was the most diverse taxon, which included seven clades. The majority of 18S rDNA sequences affiliated with the Dictyochophyceae, Eustigmatophyceae, and Pelagophyceae were closely related to those of pure cultures. The results of redundancy analysis and the permutation Mantel test based on unweighted UniFrac distances, conducted for spatial analyses of the Haptophyta subclades suggested that the Mekong River plume and cyclonic eddy play important roles in regulating microbial eukaryotic diversity and distribution in the southwestern SCS. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Genetic diversity and genetic structure of farmed and wild Chinese mitten crab (Eriocheir sinensis) populations from three major basins by mitochondrial DNA COI and Cyt b gene sequences.

PubMed

Zhang, Cheng; Li, Qingqing; Wu, Xugan; Liu, Qing; Cheng, Yongxu

2017-11-20

The Chinese mitten crab, Eriocheir sinensis, is one of the important native crab species in East Asian region, which has been widely cultured throughout China, particularly in river basins of Yangtze, Huanghe and Liaohe. This study was designed to evaluate the genetic diversity and genetic structure of cultured and wild E. sinensis populations from the three river basins based on mitochondrial DNA (mtDNA) cytochrome oxidase subunit I (COI) and cytochrome b (Cyt b). The results showed that there were 62 variable sites and 30 parsimony informative sites in the 647 bp of sequenced mtDNA COI from 335 samples. Similarly, a 637 bp segment of Cyt b provided 59 variable sites and 26 parsimony informative sites. AMOVA showed that the levels of genetic differentiation were low among six populations. Although the haplotype diversity and nucleotide diversity of Huanghe wild population had slightly higher than the other populations, there were no significant differences. There was no significant differentiation between the genetic and geographic distance of the six populations, and haplotype network diagram indicated that there may exist genetic hybrids of E. sinensis from different river basins. The results of clustering and neutrality tests revealed that the distance of geographical locations were not completely related to their genetic distance values for the six populations. In conclusion, these results have great significance for the evaluation and exploitation of germplasm resources of E. sinensis.

ngs.plot: Quick mining and visualization of next-generation sequencing data by integrating genomic databases.

PubMed

Shen, Li; Shao, Ningyi; Liu, Xiaochuan; Nestler, Eric

2014-04-15

Understanding the relationship between the millions of functional DNA elements and their protein regulators, and how they work in conjunction to manifest diverse phenotypes, is key to advancing our understanding of the mammalian genome. Next-generation sequencing technology is now used widely to probe these protein-DNA interactions and to profile gene expression at a genome-wide scale. As the cost of DNA sequencing continues to fall, the interpretation of the ever increasing amount of data generated represents a considerable challenge. We have developed ngs.plot - a standalone program to visualize enrichment patterns of DNA-interacting proteins at functionally important regions based on next-generation sequencing data. We demonstrate that ngs.plot is not only efficient but also scalable. We use a few examples to demonstrate that ngs.plot is easy to use and yet very powerful to generate figures that are publication ready. We conclude that ngs.plot is a useful tool to help fill the gap between massive datasets and genomic information in this era of big sequencing data.

ngs.plot: Quick mining and visualization of next-generation sequencing data by integrating genomic databases

PubMed Central

2014-01-01

Background Understanding the relationship between the millions of functional DNA elements and their protein regulators, and how they work in conjunction to manifest diverse phenotypes, is key to advancing our understanding of the mammalian genome. Next-generation sequencing technology is now used widely to probe these protein-DNA interactions and to profile gene expression at a genome-wide scale. As the cost of DNA sequencing continues to fall, the interpretation of the ever increasing amount of data generated represents a considerable challenge. Results We have developed ngs.plot – a standalone program to visualize enrichment patterns of DNA-interacting proteins at functionally important regions based on next-generation sequencing data. We demonstrate that ngs.plot is not only efficient but also scalable. We use a few examples to demonstrate that ngs.plot is easy to use and yet very powerful to generate figures that are publication ready. Conclusions We conclude that ngs.plot is a useful tool to help fill the gap between massive datasets and genomic information in this era of big sequencing data. PMID:24735413

High-resolution mitochondrial DNA analysis sheds light on human diversity, cultural interactions, and population mobility in Northwestern Amazonia.

PubMed

Arias, Leonardo; Barbieri, Chiara; Barreto, Guillermo; Stoneking, Mark; Pakendorf, Brigitte

2018-02-01

Northwestern Amazonia (NWA) is a center of high linguistic and cultural diversity. Several language families and linguistic isolates occur in this region, as well as different subsistence patterns, with some groups being foragers and others agriculturalists. In addition, speakers of Eastern Tukanoan languages are known for practicing linguistic exogamy, a marriage system in which partners are taken from different language groups. In this study, we use high-resolution mitochondrial DNA sequencing to investigate the impact of this linguistic and cultural diversity on the genetic relationships and population structure of NWA groups. We collected saliva samples from individuals representing 40 different NWA ethnolinguistic groups and sequenced 439 complete mitochondrial genomes to an average coverage of 1,030×. The mtDNA data revealed that NWA populations have high genetic diversity with extensive sharing of haplotypes among groups. Moreover, groups who practice linguistic exogamy have higher genetic diversity, while the foraging Nukak have lower genetic diversity. We also find that rivers play a more important role than either geography or language affiliation in structuring the genetic relationships of populations. Contrary to the view of NWA as a pristine area inhabited by small human populations living in isolation, our data support a view of high diversity and contact among different ethnolinguistic groups, with movement along rivers probably facilitating this contact. Additionally, we provide evidence for the impact of cultural practices, such as linguistic exogamy, on patterns of genetic variation. Overall, this study provides new data and insights into a remote and little-studied region of the world. © 2017 Wiley Periodicals, Inc.

DNA-based identification and phylogeny of North American Armillaria species

Treesearch

Amy L. Ross-Davis; John W. Hanna; Ned B. Klopfenstein

2011-01-01

Because Armillaria species display different ecological behaviors across diverse forest ecosystems, it is critical to identify Armillaria species accurately for any assessment of forest health. To further develop DNA-based identification methods, partial sequences of the translation elongation factor-1 alpha (EF-1α) gene were used to examine the phylogenetic...

High-throughput sequencing of complete human mtDNA genomes from the Caucasus and West Asia: high diversity and demographic inferences.

PubMed

Schönberg, Anna; Theunert, Christoph; Li, Mingkun; Stoneking, Mark; Nasidze, Ivan

2011-09-01

To investigate the demographic history of human populations from the Caucasus and surrounding regions, we used high-throughput sequencing to generate 147 complete mtDNA genome sequences from random samples of individuals from three groups from the Caucasus (Armenians, Azeri and Georgians), and one group each from Iran and Turkey. Overall diversity is very high, with 144 different sequences that fall into 97 different haplogroups found among the 147 individuals. Bayesian skyline plots (BSPs) of population size change through time show a population expansion around 40-50 kya, followed by a constant population size, and then another expansion around 15-18 kya for the groups from the Caucasus and Iran. The BSP for Turkey differs the most from the others, with an increase from 35 to 50 kya followed by a prolonged period of constant population size, and no indication of a second period of growth. An approximate Bayesian computation approach was used to estimate divergence times between each pair of populations; the oldest divergence times were between Turkey and the other four groups from the South Caucasus and Iran (~400-600 generations), while the divergence time of the three Caucasus groups from each other was comparable to their divergence time from Iran (average of ~360 generations). These results illustrate the value of random sampling of complete mtDNA genome sequences that can be obtained with high-throughput sequencing platforms.

Barcoding of fresh water fishes from Pakistan.

PubMed

Karim, Asma; Iqbal, Asad; Akhtar, Rehan; Rizwan, Muhammad; Amar, Ali; Qamar, Usman; Jahan, Shah

2016-07-01

DNA bar-coding is a taxonomic method that uses small genetic markers in organisms' mitochondrial DNA (mt DNA) for identification of particular species. It uses sequence diversity in a 658-base pair fragment near the 5' end of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene as a tool for species identification. DNA barcoding is more accurate and reliable method as compared with the morphological identification. It is equally useful in juveniles as well as adult stages of fishes. The present study was conducted to identify three farm fish species of Pakistan (Cyprinus carpio, Cirrhinus mrigala, and Ctenopharyngodon idella) genetically. All of them belonged to family cyprinidae. CO1 gene was amplified. PCR products were sequenced and analyzed by bioinformatic software. Conspecific, congenric, and confamilial k2P nucleotide divergence was estimated. From these findings, it was concluded that the gene sequence, CO1, may serve as milestone for the identification of related species at molecular level.

EMPOP-quality mtDNA control region sequences from Kashmiri of Azad Jammu & Kashmir, Pakistan.

PubMed

Rakha, Allah; Peng, Min-Sheng; Bi, Rui; Song, Jiao-Jiao; Salahudin, Zeenat; Adan, Atif; Israr, Muhammad; Yao, Yong-Gang

2016-11-01

The mitochondrial DNA (mtDNA) control region (nucleotide position 16024-576) sequences were generated through Sanger sequencing method for 317 self-identified Kashmiris from all districts of Azad Jammu & Kashmir Pakistan. The population sample set showed a total of 251 haplotypes, with a relatively high haplotype diversity (0.9977) and a low random match probability (0.54%). The containing matrilineal lineages belonging to three different phylogeographic origins of Western Eurasian (48.9%), South Asian (47.0%) and East Asian (4.1%). The present study was compared to previous data from Pakistan and other worldwide populations (Central Asia, Western Asia, and East & Southeast Asia). The dataset is made available through EMPOP under accession number EMP00679 and will serve as an mtDNA reference database in forensic casework in Pakistan. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

An SRY mutation causing human sex reversal resolves a general mechanism of structure-specific DNA recognition: application to the four-way DNA junction.

PubMed

Peters, R; King, C Y; Ukiyama, E; Falsafi, S; Donahoe, P K; Weiss, M A

1995-04-11

SRY, a genetic "master switch" for male development in mammals, exhibits two biochemical activities: sequence-specific recognition of duplex DNA and sequence-independent binding to the sharp angles of four-way DNA junctions. Here, we distinguish between these activities by analysis of a mutant SRY associated with human sex reversal (46, XY female with pure gonadal dysgenesis). The substitution (168T in human SRY) alters a nonpolar side chain in the minor-groove DNA recognition alpha-helix of the HMG box [Haqq, C.M., King, C.-Y., Ukiyama, E., Haqq, T.N., Falsalfi, S., Donahoe, P.K., & Weiss, M.A. (1994) Science 266, 1494-1500]. The native (but not mutant) side chain inserts between specific base pairs in duplex DNA, interrupting base stacking at a site of induced DNA bending. Isotope-aided 1H-NMR spectroscopy demonstrates that analogous side-chain insertion occurs on binding of SRY to a four-way junction, establishing a shared mechanism of sequence- and structure-specific DNA binding. Although the mutant DNA-binding domain exhibits > 50-fold reduction in sequence-specific DNA recognition, near wild-type affinity for four-way junctions is retained. Our results (i) identify a shared SRY-DNA contact at a site of either induced or intrinsic DNA bending, (ii) demonstrate that this contact is not required to bind an intrinsically bent DNA target, and (iii) rationalize patterns of sequence conservation or diversity among HMG boxes. Clinical association of the I68T mutation with human sex reversal supports the hypothesis that specific DNA recognition by SRY is required for male sex determination.

Complete chloroplast genome and 45S nrDNA sequences of the medicinal plant species Glycyrrhiza glabra and Glycyrrhiza uralensis.

PubMed

Kang, Sang-Ho; Lee, Jeong-Hoon; Lee, Hyun Oh; Ahn, Byoung Ohg; Won, So Youn; Sohn, Seong-Han; Kim, Jung Sun

2017-10-06

Glycyrrhiza uralensis and G. glabra, members of the Fabaceae, are medicinally important species that are native to Asia and Europe. Extracts from these plants are widely used as natural sweeteners because of their much greater sweetness than sucrose. In this study, the three complete chloroplast genomes and five 45S nuclear ribosomal (nr)DNA sequences of these two licorice species and an interspecific hybrid are presented. The chloroplast genomes of G. glabra, G. uralensis and G. glabra × G. uralensis were 127,895 bp, 127,716 bp and 127,939 bp, respectively. The three chloroplast genomes harbored 110 annotated genes, including 76 protein-coding genes, 30 tRNA genes and 4 rRNA genes. The 45S nrDNA sequences were either 5,947 or 5,948 bp in length. Glycyrrhiza glabra and G. glabra × G. uralensis showed two types of nrDNA, while G. uralensis contained a single type. The complete 45S nrDNA sequence unit contains 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 26S rRNA. We identified simple sequence repeat and tandem repeat sequences. We also developed four reliable markers for analysis of Glycyrrhiza diversity authentication.

The Status, Quality, and Expansion of the NIH Full-Length cDNA Project: The Mammalian Gene Collection (MGC)

PubMed Central

2004-01-01

The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5′-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline. PMID:15489334

Normalization of environmental metagenomic DNA enhances the discovery of under-represented microbial community members.

PubMed

Ramond, J-B; Makhalanyane, T P; Tuffin, M I; Cowan, D A

2015-04-01

Normalization is a procedure classically employed to detect rare sequences in cellular expression profiles (i.e. cDNA libraries). Here, we present a normalization protocol involving the direct treatment of extracted environmental metagenomic DNA with S1 nuclease, referred to as normalization of metagenomic DNA: NmDNA. We demonstrate that NmDNA, prior to post hoc PCR-based experiments (16S rRNA gene T-RFLP fingerprinting and clone library), increased the diversity of sequences retrieved from environmental microbial communities by detection of rarer sequences. This approach could be used to enhance the resolution of detection of ecologically relevant rare members in environmental microbial assemblages and therefore is promising in enabling a better understanding of ecosystem functioning. This study is the first testing 'normalization' on environmental metagenomic DNA (mDNA). The aim of this procedure was to improve the identification of rare phylotypes in environmental communities. Using hypoliths as model systems, we present evidence that this post-mDNA extraction molecular procedure substantially enhances the detection of less common phylotypes and could even lead to the discovery of novel microbial genotypes within a given environment. © 2014 The Society for Applied Microbiology.

Decoding DNA labels by melting curve analysis using real-time PCR.

PubMed

Balog, József A; Fehér, Liliána Z; Puskás, László G

2017-12-01

Synthetic DNA has been used as an authentication code for a diverse number of applications. However, existing decoding approaches are based on either DNA sequencing or the determination of DNA length variations. Here, we present a simple alternative protocol for labeling different objects using a small number of short DNA sequences that differ in their melting points. Code amplification and decoding can be done in two steps using quantitative PCR (qPCR). To obtain a DNA barcode with high complexity, we defined 8 template groups, each having 4 different DNA templates, yielding 158 (>2.5 billion) combinations of different individual melting temperature (Tm) values and corresponding ID codes. The reproducibility and specificity of the decoding was confirmed by using the most complex template mixture, which had 32 different products in 8 groups with different Tm values. The industrial applicability of our protocol was also demonstrated by labeling a drone with an oil-based paint containing a predefined DNA code, which was then successfully decoded. The method presented here consists of a simple code system based on a small number of synthetic DNA sequences and a cost-effective, rapid decoding protocol using a few qPCR reactions, enabling a wide range of authentication applications.

Ancient DNA evidence for the loss of a highly divergent brown bear clade during historical times.

PubMed

Calvignac, Sebastien; Hughes, Sandrine; Tougard, Christelle; Michaux, Jacques; Thevenot, Michel; Philippe, Michel; Hamdine, Watik; Hänni, Catherine

2008-04-01

The genetic diversity of present-day brown bears (Ursus arctos) has been extensively studied over the years and appears to be geographically structured into five main clades. The question of the past diversity of the species has been recently addressed by ancient DNA studies that concluded to a relative genetic stability over the last 35,000 years. However, the post-last glacial maximum genetic diversity of the species still remains poorly documented, notably in the Old World. Here, we analyse Atlas brown bears, which became extinct during the Holocene period. A divergent brown bear mitochondrial DNA lineage not present in any of the previously studied modern or ancient bear samples was uncovered, suggesting that the diversity of U. arctos was larger in the past than it is now. Specifically, a significant portion (with respect to sequence divergence) of the intraspecific diversity of the brown bear was lost with the extinction of the Atlas brown bear after the Pleistocene/Holocene transition.

Metatranscriptomics of Soil Eukaryotic Communities.

PubMed

Yadav, Rajiv K; Bragalini, Claudia; Fraissinet-Tachet, Laurence; Marmeisse, Roland; Luis, Patricia

2016-01-01

Functions expressed by eukaryotic organisms in soil can be specifically studied by analyzing the pool of eukaryotic-specific polyadenylated mRNA directly extracted from environmental samples. In this chapter, we describe two alternative protocols for the extraction of high-quality RNA from soil samples. Total soil RNA or mRNA can be converted to cDNA for direct high-throughput sequencing. Polyadenylated mRNA-derived full-length cDNAs can also be cloned in expression plasmid vectors to constitute soil cDNA libraries, which can be subsequently screened for functional gene categories. Alternatively, the diversity of specific gene families can also be explored following cDNA sequence capture using exploratory oligonucleotide probes.

The punctilious RNA polymerase II core promoter

PubMed Central

Vo ngoc, Long; Wang, Yuan-Liang; Kassavetis, George A.; Kadonaga, James T.

2017-01-01

The signals that direct the initiation of transcription ultimately converge at the core promoter, which is the gateway to transcription. Here we provide an overview of the RNA polymerase II core promoter in bilateria (bilaterally symmetric animals). The core promoter is diverse in terms of its composition and function yet is also punctilious, as it acts with strict rules and precision. We additionally describe an expanded view of the core promoter that comprises the classical DNA sequence motifs, sequence-specific DNA-binding transcription factors, chromatin signals, and DNA structure. This model may eventually lead to a more unified conceptual understanding of the core promoter. PMID:28808065

Evaluation of haplotype diversity of Achatina fulica (Lissachatina) [Bowdich] from Indian sub-continent by means of 16S rDNA sequence and its phylogenetic relationships with other global populations.

PubMed

Ayyagari, Vijaya Sai; Sreerama, Krupanidhi

2017-08-01

Achatina fulica (Lissachatina fulica) is one of the most invasive species found across the globe causing a significant damage to crops, vegetables, and horticultural plants. This terrestrial snail is native to east Africa and spread to different parts of the world by introductions. India, a hot spot for biodiversity of several endemic gastropods, has witnessed an outburst of this snail population in several parts of the country posing a serious threat to crop loss and also to human health. With an objective to evaluate the genetic diversity of this snail, we have sampled this snail from different parts of India and analyzed its haplotype diversity by means of 16S rDNA sequence information. Apart from this, we have studied the phylogenetic relationships of the isolates sequenced in the present study in relation with other global populations by Bayesian and Maximum-likelihood approaches. Of the isolates sequenced, haplotype 'C' is the predominant one. A new haplotype 'S' from the state of Odisha was observed. The isolates sequenced in the present study clustered with its conspecifics from the Indian sub-continent. Haplotype network analyses were also carried out for studying the evolution of different haplotypes. It was observed that haplotype 'S' was associated with a Mauritius haplotype 'H', indicating the possibility of multiple introductions of A. fulica to India.

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile†

PubMed Central

Webster, Nicole S.; Wilson, Kate J.; Blackall, Linda L.; Hill, Russell T.

2001-01-01

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the β- and γ-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge. PMID:11133476

Impacts of Genome-Wide Analyses on Our Understanding of Human Herpesvirus Diversity and Evolution.

PubMed

Renner, Daniel W; Szpara, Moriah L

2018-01-01

Until fairly recently, genome-wide evolutionary dynamics and within-host diversity were more commonly examined in the context of small viruses than in the context of large double-stranded DNA viruses such as herpesviruses. The high mutation rates and more compact genomes of RNA viruses have inspired the investigation of population dynamics for these species, and recent data now suggest that herpesviruses might also be considered candidates for population modeling. High-throughput sequencing (HTS) and bioinformatics have expanded our understanding of herpesviruses through genome-wide comparisons of sequence diversity, recombination, allele frequency, and selective pressures. Here we discuss recent data on the mechanisms that generate herpesvirus genomic diversity and underlie the evolution of these virus families. We focus on human herpesviruses, with key insights drawn from veterinary herpesviruses and other large DNA virus families. We consider the impacts of cell culture on herpesvirus genomes and how to accurately describe the viral populations under study. The need for a strong foundation of high-quality genomes is also discussed, since it underlies all secondary genomic analyses such as RNA sequencing (RNA-Seq), chromatin immunoprecipitation, and ribosome profiling. Areas where we foresee future progress, such as the linking of viral genetic differences to phenotypic or clinical outcomes, are highlighted as well. Copyright © 2017 Renner and Szpara.

Non-B-Form DNA Is Enriched at Centromeres

PubMed Central

Henikoff, Steven

2018-01-01

Abstract Animal and plant centromeres are embedded in repetitive “satellite” DNA, but are thought to be epigenetically specified. To define genetic characteristics of centromeres, we surveyed satellite DNA from diverse eukaryotes and identified variation in <10-bp dyad symmetries predicted to adopt non-B-form conformations. Organisms lacking centromeric dyad symmetries had binding sites for sequence-specific DNA-binding proteins with DNA-bending activity. For example, human and mouse centromeres are depleted for dyad symmetries, but are enriched for non-B-form DNA and are associated with binding sites for the conserved DNA-binding protein CENP-B, which is required for artificial centromere function but is paradoxically nonessential. We also detected dyad symmetries and predicted non-B-form DNA structures at neocentromeres, which form at ectopic loci. We propose that centromeres form at non-B-form DNA because of dyad symmetries or are strengthened by sequence-specific DNA binding proteins. This may resolve the CENP-B paradox and provide a general basis for centromere specification. PMID:29365169

Multiple advanced logic gates made of DNA-Ag nanocluster and the application for intelligent detection of pathogenic bacterial genes† †Electronic supplementary information (ESI) available: Chemicals, materials and DNA sequences used in the investigation, the construction of YES, AND, OR, XOR and INH logic gates, CD and PAGE experimental results. See DOI: 10.1039/c7sc05246d

PubMed Central

Lin, Xiaodong; Deng, Jiankang; Lyu, Yanlong; Qian, Pengcheng; Li, Yunfei

2018-01-01

The integration of multiple DNA logic gates on a universal platform to implement advance logic functions is a critical challenge for DNA computing. Herein, a straightforward and powerful strategy in which a guanine-rich DNA sequence lighting up a silver nanocluster and fluorophore was developed to construct a library of logic gates on a simple DNA-templated silver nanoclusters (DNA-AgNCs) platform. This library included basic logic gates, YES, AND, OR, INHIBIT, and XOR, which were further integrated into complex logic circuits to implement diverse advanced arithmetic/non-arithmetic functions including half-adder, half-subtractor, multiplexer, and demultiplexer. Under UV irradiation, all the logic functions could be instantly visualized, confirming an excellent repeatability. The logic operations were entirely based on DNA hybridization in an enzyme-free and label-free condition, avoiding waste accumulation and reducing cost consumption. Interestingly, a DNA-AgNCs-based multiplexer was, for the first time, used as an intelligent biosensor to identify pathogenic genes, E. coli and S. aureus genes, with a high sensitivity. The investigation provides a prototype for the wireless integration of multiple devices on even the simplest single-strand DNA platform to perform diverse complex functions in a straightforward and cost-effective way. PMID:29675221

Patterns of diversity and recombination along chromosome 1 of maize (Zea mays ssp. mays L.).

PubMed Central

Tenaillon, Maud I; Sawkins, Mark C; Anderson, Lorinda K; Stack, Stephen M; Doebley, John; Gaut, Brandon S

2002-01-01

We investigate the interplay between genetic diversity and recombination in maize (Zea mays ssp. mays). Genetic diversity was measured in three types of markers: single-nucleotide polymorphisms, indels, and microsatellites. All three were examined in a sample of previously published DNA sequences from 21 loci on maize chromosome 1. Small indels (1-5 bp) were numerous and far more common than large indels. Furthermore, large indels (>100 bp) were infrequent in the population sample, suggesting they are slightly deleterious. The 21 loci also contained 47 microsatellites, of which 33 were polymorphic. Diversity in SNPs, indels, and microsatellites was compared to two measures of recombination: C (=4Nc) estimated from DNA sequence data and R based on a quantitative recombination nodule map of maize synaptonemal complex 1. SNP diversity was correlated with C (r = 0.65; P = 0.007) but not with R (r = -0.10; P = 0.69). Given the lack of correlation between R and SNP diversity, the correlation between SNP diversity and C may be driven by demography. In contrast to SNP diversity, microsatellite diversity was correlated with R (r = 0.45; P = 0.004) but not C (r = -0.025; P = 0.55). The correlation could arise if recombination is mutagenic for microsatellites, or it may be consistent with background selection that is apparent only in this class of rapidly evolving markers. PMID:12454083

Deep-branching Novel Lineages and High Diversity of Haptophytes in the Skagerrak (Norway) Uncovered by 454 Pyrosequencing

PubMed Central

Egge, Elianne S; Eikrem, Wenche; Edvardsen, Bente

2015-01-01

Microalgae in the division Haptophyta may be difficult to identify to species by microscopy because they are small and fragile. Here, we used high-throughput sequencing to explore the diversity of haptophytes in outer Oslofjorden, Skagerrak, and supplemented this with electron microscopy. Nano- and picoplanktonic subsurface samples were collected monthly for 2 yr, and the haptophytes were targeted by amplification of RNA/cDNA with Haptophyta-specific 18S ribosomal DNA V4 primers. Pyrosequencing revealed higher species richness of haptophytes than previously observed in the Skagerrak by microscopy. From ca. 400,000 reads we obtained 156 haptophyte operational taxonomic units (OTUs) after rigorous filtering and 99.5% clustering. The majority (84%) of the OTUs matched environmental sequences not linked to a morphological species, most of which were affiliated with the order Prymnesiales. Phylogenetic analyses including Oslofjorden OTUs and available cultured and environmental haptophyte sequences showed that several of the OTUs matched sequences forming deep-branching lineages, potentially representing novel haptophyte classes. Pyrosequencing also retrieved cultured species not previously reported by microscopy in the Skagerrak. Electron microscopy revealed species not yet genetically characterised and some potentially novel taxa. This study contributes to linking genotype to phenotype within this ubiquitous and ecologically important protist group, and reveals great, unknown diversity. PMID:25099994

Diversity and dynamics of the DNA- and cDNA-derived compost fungal communities throughout the commercial cultivation process for Agaricus bisporus.

PubMed

McGee, C F; Byrne, H; Irvine, A; Wilson, J

2017-01-01

Commercial cultivation of the button mushroom Agaricus bisporus is performed through the inoculation of a semipasteurized composted material. Pasteurization of the compost material prior to inoculation results in a substrate with a fungal community that becomes dominated by A. bisporus. However, little is known about the composition and activity in the wider fungal community beyond the presence of A. bisporus in compost throughout the mushroom cropping process. In this study, the fungal cropping compost community was characterized by sequencing nuc rDNA ITS1-5.8S-ITS2 amplified from extractable DNA and RNA. The fungal community generated from DNA extracts identified a diverse community containing 211 unique species, although only 51 were identified from cDNA. Agaricus bisporus was found to dominate in the DNA-derived fungal community for the duration of the cropping process. However, analysis of cDNA extracts found A. bisporus to dominate only up to the first crop flush, after which activity decreased sharply and a much broader fungal community became active. This study has highlighted the diverse fungal community that is present in mushroom compost during cropping.

Analyses of Methylomes Derived from Meso-American Common Bean (Phaseolus vulgaris L.) Using MeDIP-Seq and Whole Genome Sodium Bisulfite-Sequencing.

PubMed

Crampton, Mollee; Sripathi, Venkateswara R; Hossain, Khwaja; Kalavacharla, Venu

2016-01-01

Common bean (Phaseolus vulgaris L.) is economically important for its high protein, fiber, and micronutrient contents, with a relatively small genome size of ∼587 Mb. Common bean is genetically diverse with two major gene pools, Meso-American and Andean. The phenotypic variability within common bean is partly attributed to the genetic diversity and epigenetic changes that are largely influenced by environmental factors. It is well established that an important epigenetic regulator of gene expression is DNA methylation. Here, we present results generated from two high-throughput sequencing technologies, methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and whole genome bisulfite-sequencing (BS-Seq). Our analyses revealed that this Meso-American common bean displays similar methylation patterns as other previously published plant methylomes, with CG ∼50%, CHG ∼30%, and CHH ∼2.7% methylation, however, these differ from the common bean reference methylome of Andean origin. We identified higher CG methylation levels in both promoter and genic regions than CHG and CHH contexts. Moreover, we found relatively higher CG methylation levels in genes than in promoters. Conversely, the CHG and CHH methylation levels were highest in promoters than in genes. This is the first genome-wide DNA methylation profiling study in a Meso-American common bean cultivar ("Sierra") using NGS approaches. Our long-term goal is to generate genome-wide epigenomic maps in common bean focusing on chromatin accessibility, histone modifications, and DNA methylation.

Analyses of Methylomes Derived from Meso-American Common Bean (Phaseolus vulgaris L.) Using MeDIP-Seq and Whole Genome Sodium Bisulfite-Sequencing

PubMed Central

Crampton, Mollee; Sripathi, Venkateswara R.; Hossain, Khwaja; Kalavacharla, Venu

2016-01-01

Common bean (Phaseolus vulgaris L.) is economically important for its high protein, fiber, and micronutrient contents, with a relatively small genome size of ∼587 Mb. Common bean is genetically diverse with two major gene pools, Meso-American and Andean. The phenotypic variability within common bean is partly attributed to the genetic diversity and epigenetic changes that are largely influenced by environmental factors. It is well established that an important epigenetic regulator of gene expression is DNA methylation. Here, we present results generated from two high-throughput sequencing technologies, methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and whole genome bisulfite-sequencing (BS-Seq). Our analyses revealed that this Meso-American common bean displays similar methylation patterns as other previously published plant methylomes, with CG ∼50%, CHG ∼30%, and CHH ∼2.7% methylation, however, these differ from the common bean reference methylome of Andean origin. We identified higher CG methylation levels in both promoter and genic regions than CHG and CHH contexts. Moreover, we found relatively higher CG methylation levels in genes than in promoters. Conversely, the CHG and CHH methylation levels were highest in promoters than in genes. This is the first genome-wide DNA methylation profiling study in a Meso-American common bean cultivar (“Sierra”) using NGS approaches. Our long-term goal is to generate genome-wide epigenomic maps in common bean focusing on chromatin accessibility, histone modifications, and DNA methylation. PMID:27199997

On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires.

PubMed

Silas, Sukrit; Makarova, Kira S; Shmakov, Sergey; Páez-Espino, David; Mohr, Georg; Liu, Yi; Davison, Michelle; Roux, Simon; Krishnamurthy, Siddharth R; Fu, Becky Xu Hua; Hansen, Loren L; Wang, David; Sullivan, Matthew B; Millard, Andrew; Clokie, Martha R; Bhaya, Devaki; Lambowitz, Alan M; Kyrpides, Nikos C; Koonin, Eugene V; Fire, Andrew Z

2017-07-11

Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium- Arthrospira platensis Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown. IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis , a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic. Copyright © 2017 Silas et al.

Distinctive archaebacterial species associated with anaerobic rumen protozoan Entodinium caudatum.

PubMed

Tóthová, T; Piknová, M; Kisidayová, S; Javorský, P; Pristas, P

2008-01-01

The diversity of archaebacteria associated with anaerobic rumen protozoan Entodinium caudatum in long term in vitro culture was investigated by denaturing gradient gel electrophoresis (DGGE) analysis of hypervariable V3 region of archaebacterial 16S rRNA gene. PCR was accomplished directly from DNA extracted from a single protozoal cell and from total community genomic DNA and the obtained fingerprints were compared. The analysis indicated the presence of a solitary intensive band present in Entodinium caudatum single cell DNA, which had no counterparts in the profile from total DNA. The identity of archaebacterium represented by this band was determined by sequence analysis which showed that the sequence fell to the cluster of ciliate symbiotic methanogens identified recently by 16S gene library approach.

Things fall apart: biological species form unconnected parsimony networks.

PubMed

Hart, Michael W; Sunday, Jennifer

2007-10-22

The generality of operational species definitions is limited by problematic definitions of between-species divergence. A recent phylogenetic species concept based on a simple objective measure of statistically significant genetic differentiation uses between-species application of statistical parsimony networks that are typically used for population genetic analysis within species. Here we review recent phylogeographic studies and reanalyse several mtDNA barcoding studies using this method. We found that (i) alignments of DNA sequences typically fall apart into a separate subnetwork for each Linnean species (but with a higher rate of true positives for mtDNA data) and (ii) DNA sequences from single species typically stick together in a single haplotype network. Departures from these patterns are usually consistent with hybridization or cryptic species diversity.

Whole mitochondrial genome sequencing of domestic horses reveals incorporation of extensive wild horse diversity during domestication

PubMed Central

2011-01-01

Background DNA target enrichment by micro-array capture combined with high throughput sequencing technologies provides the possibility to obtain large amounts of sequence data (e.g. whole mitochondrial DNA genomes) from multiple individuals at relatively low costs. Previously, whole mitochondrial genome data for domestic horses (Equus caballus) were limited to only a few specimens and only short parts of the mtDNA genome (especially the hypervariable region) were investigated for larger sample sets. Results In this study we investigated whole mitochondrial genomes of 59 domestic horses from 44 breeds and a single Przewalski horse (Equus przewalski) using a recently described multiplex micro-array capture approach. We found 473 variable positions within the domestic horses, 292 of which are parsimony-informative, providing a well resolved phylogenetic tree. Our divergence time estimate suggests that the mitochondrial genomes of modern horse breeds shared a common ancestor around 93,000 years ago and no later than 38,000 years ago. A Bayesian skyline plot (BSP) reveals a significant population expansion beginning 6,000-8,000 years ago with an ongoing exponential growth until the present, similar to other domestic animal species. Our data further suggest that a large sample of wild horse diversity was incorporated into the domestic population; specifically, at least 46 of the mtDNA lineages observed in domestic horses (73%) already existed before the beginning of domestication about 5,000 years ago. Conclusions Our study provides a window into the maternal origins of extant domestic horses and confirms that modern domestic breeds present a wide sample of the mtDNA diversity found in ancestral, now extinct, wild horse populations. The data obtained allow us to detect a population expansion event coinciding with the beginning of domestication and to estimate both the minimum number of female horses incorporated into the domestic gene pool and the time depth of the domestic horse mtDNA gene pool. PMID:22082251

MULTILOCUS SEQUENCE TYPING OF BRUCELLA ISOLATES FROM THAILAND.

PubMed

Chawjiraphan, Wireeya; Sonthayanon, Piengchan; Chanket, Phanita; Benjathummarak, Surachet; Kerdsin, Anusak; Kalambhaheti, Thareerat

2016-11-01

Although brucellosis outbreaks in Thailand are rare, they cause abortions and infertility in animals, resulting in significant economic loss. Because Brucella spp display > 90% DNA homology, multilocus sequence typing (MLST) was employed to categorize local Brucella isolates into sequence types (STs) and to determine their genetic relatedness. Brucella samples were isolated from vaginal secretion of cows and goats, and from blood cultures of infected individuals. Brucella species were determined by multiplex PCR of eight loci, in addition to MLST based on partial DNA sequences of nine house-keeping genes. MLST analysis of 36 isolates revealed 78 distinct novel allele types and 34 novel STs, while two isolates possessed the known ST8. Sequence alignments identified polymorphic sites in each allele, ranging from 2-6%, while overall genetic diversity was 3.6%. MLST analysis of the 36 Brucella isolates classified them into three species, namely, B. melitensis, B. abortus and B. suis, in agreement with multiplex PCR results. Genetic relatedness among ST members of B. melitensis and B. abortus determined by eBURST program revealed ST2 as founder of B. abortus isolates and ST8 the founder of B. melitensis isolates. ST 36, 41 and 50 of Thai Brucella isolates were identified as single locus variants of clonal cluster (CC) 8, while the majority of STs were diverse. The genetic diversity and relatedness identified using MLST revealed hitherto unexpected diversity among Thai Brucella isolates. Genetic classification of isolates could reveal the route of brucellosis transmission among humans and farm animals and also reveal their relationship with other isolates in the region and other parts of the world.

Analysis of HIV using a high resolution melting (HRM) diversity assay: automation of HRM data analysis enhances the utility of the assay for analysis of HIV incidence.

PubMed

Cousins, Matthew M; Swan, David; Magaret, Craig A; Hoover, Donald R; Eshleman, Susan H

2012-01-01

HIV diversity may be a useful biomarker for discriminating between recent and non-recent HIV infection. The high resolution melting (HRM) diversity assay was developed to quantify HIV diversity in viral populations without sequencing. In this assay, HIV diversity is expressed as a single numeric HRM score that represents the width of a melting peak. HRM scores are highly associated with diversity measures obtained with next generation sequencing. In this report, a software package, the HRM Diversity Assay Analysis Tool (DivMelt), was developed to automate calculation of HRM scores from melting curve data. DivMelt uses computational algorithms to calculate HRM scores by identifying the start (T1) and end (T2) melting temperatures for a DNA sample and subtracting them (T2 - T1 =  HRM score). DivMelt contains many user-supplied analysis parameters to allow analyses to be tailored to different contexts. DivMelt analysis options were optimized to discriminate between recent and non-recent HIV infection and to maximize HRM score reproducibility. HRM scores calculated using DivMelt were compared to HRM scores obtained using a manual method that is based on visual inspection of DNA melting curves. HRM scores generated with DivMelt agreed with manually generated HRM scores obtained from the same DNA melting data. Optimal parameters for discriminating between recent and non-recent HIV infection were identified. DivMelt provided greater discrimination between recent and non-recent HIV infection than the manual method. DivMelt provides a rapid, accurate method of determining HRM scores from melting curve data, facilitating use of the HRM diversity assay for large-scale studies.

Ancient DNA from Giant Panda (Ailuropoda melanoleuca) of South-Western China Reveals Genetic Diversity Loss during the Holocene

PubMed Central

Barlow, Axel; Cooper, Alan; Hou, Xin-Dong; Ji, Xue-Ping; Zhong, Bo-Jian; Liu, Hong; Flynn, Lawrence J.; Yuan, Jun-Xia; Wang, Li-Rui; Basler, Nikolas; Westbury, Michael V.; Hofreiter, Michael; Lai, Xu-Long

2018-01-01

The giant panda was widely distributed in China and south-eastern Asia during the middle to late Pleistocene, prior to its habitat becoming rapidly reduced in the Holocene. While conservation reserves have been established and population numbers of the giant panda have recently increased, the interpretation of its genetic diversity remains controversial. Previous analyses, surprisingly, have indicated relatively high levels of genetic diversity raising issues concerning the efficiency and usefulness of reintroducing individuals from captive populations. However, due to a lack of DNA data from fossil specimens, it is unknown whether genetic diversity was even higher prior to the most recent population decline. We amplified complete cytb and 12s rRNA, partial 16s rRNA and ND1, and control region sequences from the mitochondrial genomes of two Holocene panda specimens. We estimated genetic diversity and population demography by analyzing the ancient mitochondrial DNA sequences alongside those from modern giant pandas, as well as from other members of the bear family (Ursidae). Phylogenetic analyses show that one of the ancient haplotypes is sister to all sampled modern pandas and the second ancient individual is nested among the modern haplotypes, suggesting that genetic diversity may indeed have been higher earlier during the Holocene. Bayesian skyline plot analysis supports this view and indicates a slight decline in female effective population size starting around 6000 years B.P., followed by a recovery around 2000 years ago. Therefore, while the genetic diversity of the giant panda has been affected by recent habitat contraction, it still harbors substantial genetic diversity. Moreover, while its still low population numbers require continued conservation efforts, there seem to be no immediate threats from the perspective of genetic evolutionary potential. PMID:29642393

Ancient DNA from Giant Panda (Ailuropoda melanoleuca) of South-Western China Reveals Genetic Diversity Loss during the Holocene.

PubMed

Sheng, Gui-Lian; Barlow, Axel; Cooper, Alan; Hou, Xin-Dong; Ji, Xue-Ping; Jablonski, Nina G; Zhong, Bo-Jian; Liu, Hong; Flynn, Lawrence J; Yuan, Jun-Xia; Wang, Li-Rui; Basler, Nikolas; Westbury, Michael V; Hofreiter, Michael; Lai, Xu-Long

2018-04-06

The giant panda was widely distributed in China and south-eastern Asia during the middle to late Pleistocene, prior to its habitat becoming rapidly reduced in the Holocene. While conservation reserves have been established and population numbers of the giant panda have recently increased, the interpretation of its genetic diversity remains controversial. Previous analyses, surprisingly, have indicated relatively high levels of genetic diversity raising issues concerning the efficiency and usefulness of reintroducing individuals from captive populations. However, due to a lack of DNA data from fossil specimens, it is unknown whether genetic diversity was even higher prior to the most recent population decline. We amplified complete cyt b and 12s rRNA, partial 16s rRNA and ND1 , and control region sequences from the mitochondrial genomes of two Holocene panda specimens. We estimated genetic diversity and population demography by analyzing the ancient mitochondrial DNA sequences alongside those from modern giant pandas, as well as from other members of the bear family (Ursidae). Phylogenetic analyses show that one of the ancient haplotypes is sister to all sampled modern pandas and the second ancient individual is nested among the modern haplotypes, suggesting that genetic diversity may indeed have been higher earlier during the Holocene. Bayesian skyline plot analysis supports this view and indicates a slight decline in female effective population size starting around 6000 years B.P., followed by a recovery around 2000 years ago. Therefore, while the genetic diversity of the giant panda has been affected by recent habitat contraction, it still harbors substantial genetic diversity. Moreover, while its still low population numbers require continued conservation efforts, there seem to be no immediate threats from the perspective of genetic evolutionary potential.

Analysis of HIV Using a High Resolution Melting (HRM) Diversity Assay: Automation of HRM Data Analysis Enhances the Utility of the Assay for Analysis of HIV Incidence

PubMed Central

Cousins, Matthew M.; Swan, David; Magaret, Craig A.; Hoover, Donald R.; Eshleman, Susan H.

2012-01-01

Background HIV diversity may be a useful biomarker for discriminating between recent and non-recent HIV infection. The high resolution melting (HRM) diversity assay was developed to quantify HIV diversity in viral populations without sequencing. In this assay, HIV diversity is expressed as a single numeric HRM score that represents the width of a melting peak. HRM scores are highly associated with diversity measures obtained with next generation sequencing. In this report, a software package, the HRM Diversity Assay Analysis Tool (DivMelt), was developed to automate calculation of HRM scores from melting curve data. Methods DivMelt uses computational algorithms to calculate HRM scores by identifying the start (T1) and end (T2) melting temperatures for a DNA sample and subtracting them (T2–T1 = HRM score). DivMelt contains many user-supplied analysis parameters to allow analyses to be tailored to different contexts. DivMelt analysis options were optimized to discriminate between recent and non-recent HIV infection and to maximize HRM score reproducibility. HRM scores calculated using DivMelt were compared to HRM scores obtained using a manual method that is based on visual inspection of DNA melting curves. Results HRM scores generated with DivMelt agreed with manually generated HRM scores obtained from the same DNA melting data. Optimal parameters for discriminating between recent and non-recent HIV infection were identified. DivMelt provided greater discrimination between recent and non-recent HIV infection than the manual method. Conclusion DivMelt provides a rapid, accurate method of determining HRM scores from melting curve data, facilitating use of the HRM diversity assay for large-scale studies. PMID:23240016

Assessing Species Diversity Using Metavirome Data: Methods and Challenges.

PubMed

Herath, Damayanthi; Jayasundara, Duleepa; Ackland, David; Saeed, Isaam; Tang, Sen-Lin; Halgamuge, Saman

2017-01-01

Assessing biodiversity is an important step in the study of microbial ecology associated with a given environment. Multiple indices have been used to quantify species diversity, which is a key biodiversity measure. Measuring species diversity of viruses in different environments remains a challenge relative to measuring the diversity of other microbial communities. Metagenomics has played an important role in elucidating viral diversity by conducting metavirome studies; however, metavirome data are of high complexity requiring robust data preprocessing and analysis methods. In this review, existing bioinformatics methods for measuring species diversity using metavirome data are categorised broadly as either sequence similarity-dependent methods or sequence similarity-independent methods. The former includes a comparison of DNA fragments or assemblies generated in the experiment against reference databases for quantifying species diversity, whereas estimates from the latter are independent of the knowledge of existing sequence data. Current methods and tools are discussed in detail, including their applications and limitations. Drawbacks of the state-of-the-art method are demonstrated through results from a simulation. In addition, alternative approaches are proposed to overcome the challenges in estimating species diversity measures using metavirome data.

Direct sequencing of hepatitis A virus and norovirus RT-PCR products from environmentally contaminated oyster using M13-tailed primers.

PubMed

Williams-Woods, Jacquelina; González-Escalona, Narjol; Burkhardt, William

2011-12-01

Human norovirus (HuNoV) and hepatitis A (HAV) are recognized as leading causes of non-bacterial foodborne associated illnesses in the United States. DNA sequencing is generally considered the standard for accurate viral genotyping in support of epidemiological investigations. Due to the genetic diversity of noroviruses (NoV), degenerate primer sets are often used in conventional reverse transcription (RT) PCR and real-time RT-quantitative PCR (RT-qPCR) for the detection of these viruses and cDNA fragments are generally cloned prior to sequencing. HAV detection methods that are sensitive and specific for real-time RT-qPCR yields small fragments sizes of 89-150bp, which can be difficult to sequence. In order to overcome these obstacles, norovirus and HAV primers were tailed with M13 forward and reverse primers. This modification increases the sequenced product size and allows for direct sequencing of the amplicons utilizing complementary M13 primers. HuNoV and HAV cDNA products from environmentally contaminated oysters were analyzed using this method. Alignments of the sequenced samples revealed ≥95% nucleotide identities. Tailing NoV and HAV primers with M13 sequence increases the cDNA product size, offers an alternative to cloning, and allows for rapid, accurate and direct sequencing of cDNA products produced by conventional or real time RT-qPCR assays. Published by Elsevier B.V.

Genetic diversity, virulence, and Meloidogyne incognita interactions of Fusarium oxysporum isolates causing cotton wilt in Georgia

USDA-ARS?s Scientific Manuscript database

Locally severe outbreaks of Fusarium wilt of cotton (Gossypium spp.) in South Georgia raised concerns about the genotypes of the causal pathogen, Fusarium oxysporum f. sp. vasinfectum. Vegetative complementation tests and DNA sequence analysis were used to determine genetic diversity among 492 F. ox...

Making sense of deep sequencing

PubMed Central

Goldman, D.; Domschke, K.

2016-01-01

This review, the first of an occasional series, tries to make sense of the concepts and uses of deep sequencing of polynucleic acids (DNA and RNA). Deep sequencing, synonymous with next-generation sequencing, high-throughput sequencing and massively parallel sequencing, includes whole genome sequencing but is more often and diversely applied to specific parts of the genome captured in different ways, for example the highly expressed portion of the genome known as the exome and portions of the genome that are epigenetically marked either by DNA methylation, the binding of proteins including histones, or that are in different configurations and thus more or less accessible to enzymes that cleave DNA. Deep sequencing of RNA (RNASeq) reverse-transcribed to complementary DNA is invaluable for measuring RNA expression and detecting changes in RNA structure. Important concepts in deep sequencing include the length and depth of sequence reads, mapping and assembly of reads, sequencing error, haplotypes, and the propensity of deep sequencing, as with other types of ‘big data’, to generate large numbers of errors, requiring monitoring for methodologic biases and strategies for replication and validation. Deep sequencing yields a unique genetic fingerprint that can be used to identify a person, and a trove of predictors of genetic medical diseases. Deep sequencing to identify epigenetic events including changes in DNA methylation and RNA expression can reveal the history and impact of environmental exposures. Because of the power of sequencing to identify and deliver biomedically significant information about a person and their blood relatives, it creates ethical dilemmas and practical challenges in research and clinical care, for example the decision and procedures to report incidental findings that will increasingly and frequently be discovered. PMID:24925306

Temporal dynamics of soil microbial communities under different moisture regimes: high-throughput sequencing and bioinformatics analysis

NASA Astrophysics Data System (ADS)

Semenov, Mikhail; Zhuravleva, Anna; Semenov, Vyacheslav; Yevdokimov, Ilya; Larionova, Alla

2017-04-01

Recent climate scenarios predict not only continued global warming but also an increased frequency and intensity of extreme climatic events such as strong changes in temperature and precipitation regimes. Microorganisms are well known to be more sensitive to changes in environmental conditions than to other soil chemical and physical parameters. In this study, we determined the shifts in soil microbial community structure as well as indicative taxa in soils under three moisture regimes using high-throughput Illumina sequencing and range of bioinformatics approaches for the assessment of sequence data. Incubation experiments were performed in soil-filled (Greyic Phaeozems Albic) rhizoboxes with maize and without plants. Three contrasting moisture regimes were being simulated: 1) optimal wetting (OW), a watering 2-3 times per week to maintain soil moisture of 20-25% by weight; 2) periodic wetting (PW), with alternating periods of wetting and drought; and 3) constant insufficient wetting (IW), while soil moisture of 12% by weight was permanently maintained. Sampled fresh soils were homogenized, and the total DNA of three replicates was extracted using the FastDNA® SPIN kit for Soil. DNA replicates were combined in a pooled sample and the DNA was used for PCR with specific primers for the 16S V3 and V4 regions. In order to compare variability between different samples and replicates within a single sample, some DNA replicates treated separately. The products were purified and submitted to Illumina MiSeq sequencing. Sequence data were evaluated by alpha-diversity (Chao1 and Shannon H' diversity indexes), beta-diversity (UniFrac and Bray-Curtis dissimilarity), heatmap, tagcloud, and plot-bar analyses using the MiSeq Reporter Metagenomics Workflow and R packages (phyloseq, vegan, tagcloud). Shannon index varied in a rather narrow range (4.4-4.9) with the lowest values for microbial communities under PW treatment. Chao1 index varied from 385 to 480, being a more flexible indicator than Shannon index. Chao1 had similar values for OW and IW communities, but alpha-diversity of microbial communities has sharply decreased under PW treatment. There was no visible difference in beta-diversity depending on sampling date and wetting regime, however, it could be possible to distinguish microbial communities in soils with maize and without plants. The presence of maize was acting as scattering agent, making microbial communities more distinguished. In all studied samples, the most dominant phyla were Proteobacteria, Firmicutes, Verrucomicrobia, Actinobacteria, and Acidobacteria. Chthoniobacter, Bacillus, Alicyclobacillus, Rhodoplanes, Cohnella, Kaistobacter, and Solibacter were the most abundant genera. Moreover, these genera were found as the most reactive and variable taxa in microbial community. Thus, DNA high-throughput sequencing revealed no dramatic shifts in bacterial community structure in soils under different moisture regimes. However, this technique allowed us to determine the effect of wetting regime and the presence of plants on soil microbial community which were adaptable to insufficient wetting, but lost diversity under periodic wetting. Furthermore, we detected the indicative taxa which dominate in microbial communities and at the same time strongly react to environmental changes.

Bacterivory by a Summer Assemblage of Nanoplankton in the Ross Sea, Antarctica: Mixotrophic Versus Heterotrophic Protists

NASA Astrophysics Data System (ADS)

Sanders, R. W.; Gast, R. J.

2016-02-01

Many protists traditionally described as phototrophic have recently been shown to have retained the primitive trait of phagotrophy, and thus function as mixotrophs. Mixotrophic nanoflagellates were identified in every sample examined from a summer cruise in the Ross Sea, Antarctica, where they often were more abundant than heterotrophic nanoflagellates that have previously been considered the major bacterivores in marine waters. Mixotrophs, identified by uptake of fluorescent tracers, comprised similar proportions (9-75%) of the total bacterivorous flagellates in summer as were previously determined for an earlier spring cruise in the Ross Sea. Protist diversity also was linked to functional bacterivores using a culture-independent method in which BrdU-labeled DNA of bacterial prey was incorporated into the DNA of eukaryotic grazers. Immunoprecipitation of the BrdU-labeld DNA was followed by high-throughput sequencing to identify a diverse group of bacterivores, including numerous uncultured eukaryotes. However, its utility for identification of mixotrophs was limited by the availability of sequences from known mixotrophs.

IM-TORNADO: A Tool for Comparison of 16S Reads from Paired-End Libraries

PubMed Central

Jeraldo, Patricio; Kalari, Krishna; Chen, Xianfeng; Bhavsar, Jaysheel; Mangalam, Ashutosh; White, Bryan; Nelson, Heidi; Kocher, Jean-Pierre; Chia, Nicholas

2014-01-01

Motivation 16S rDNA hypervariable tag sequencing has become the de facto method for accessing microbial diversity. Illumina paired-end sequencing, which produces two separate reads for each DNA fragment, has become the platform of choice for this application. However, when the two reads do not overlap, existing computational pipelines analyze data from read separately and underutilize the information contained in the paired-end reads. Results We created a workflow known as Illinois Mayo Taxon Organization from RNA Dataset Operations (IM-TORNADO) for processing non-overlapping reads while retaining maximal information content. Using synthetic mock datasets, we show that the use of both reads produced answers with greater correlation to those from full length 16S rDNA when looking at taxonomy, phylogeny, and beta-diversity. Availability and Implementation IM-TORNADO is freely available at http://sourceforge.net/projects/imtornado and produces BIOM format output for cross compatibility with other pipelines such as QIIME, mothur, and phyloseq. PMID:25506826

Analysis of Bacterial and Fungal Nucleic Acid in Canine Sterile Granulomatous and Pyogranulomatous Dermatitis and Panniculitis.

PubMed

Rosa, Fabio B; Older, Caitlin E; Meason-Smith, Courtney; Suchodolski, Jan S; Lingsweiler, Sonia; Mansell, Joanne E; Hoffmann, Aline Rodrigues

2018-01-01

Next generation sequencing (NGS) studies are revealing a diverse microbiota on the skin of dogs. The skin microbiota of canine sterile granulomatous and pyogranulomatous dermatitis (SGPD) has yet to be investigated using NGS techniques. NGS targeting the 16S rRNA and ITS-1 region of bacterial and fungal DNA, respectively, were used to investigate if bacterial and fungal DNA were associated with skin lesions in cases of canine SGPD. The study included 20 formalin-fixed paraffin-embedded (FFPE) skin samples and 12 fresh samples from SGPD-affected dogs, and 10 FFPE and 10 fresh samples from healthy dogs. DNA was extracted from deep dermis and panniculus, and microbial DNA was amplified using primers targeting the bacterial 16S rRNA V1-V3 and fungal ITS-1 regions. The amplified DNA was utilized for NGS on an Illumina MiSeq instrument. The sequences were processed using QIIME. No differences in fungal or bacterial alpha diversity were observed between the SGPD and control samples. Beta diversity analysis demonstrated differences in the bacterial communities between SGPD and control, but not in the fungal communities. Compared to controls, the family Erysipelotrichaceae and genus Staphylococcus were significantly more abundant in the SGPD FFPE samples, and genus Corynebacterium were more abundant in fresh samples. The bacteria found to be more abundant in SGPD are common inhabitants of skin surfaces, and likely secondary contaminants in SGPD cases. This study provides additional evidence that SGPD lesions are likely sterile.

Phylogenetic and physiological diversity of microorganisms isolated from a deep greenland glacier ice core

NASA Technical Reports Server (NTRS)

Miteva, V. I.; Sheridan, P. P.; Brenchley, J. E.

2004-01-01

We studied a sample from the GISP 2 (Greenland Ice Sheet Project) ice core to determine the diversity and survival of microorganisms trapped in the ice at least 120,000 years ago. Previously, we examined the phylogenetic relationships among 16S ribosomal DNA (rDNA) sequences in a clone library obtained by PCR amplification from genomic DNA extracted from anaerobic enrichments. Here we report the isolation of nearly 800 aerobic organisms that were grouped by morphology and amplified rDNA restriction analysis patterns to select isolates for further study. The phylogenetic analyses of 56 representative rDNA sequences showed that the isolates belonged to four major phylogenetic groups: the high-G+C gram-positives, low-G+C gram-positives, Proteobacteria, and the Cytophaga-Flavobacterium-Bacteroides group. The most abundant and diverse isolates were within the high-G+C gram-positive cluster that had not been represented in the clone library. The Jukes-Cantor evolutionary distance matrix results suggested that at least 7 isolates represent new species within characterized genera and that 49 are different strains of known species. The isolates were further categorized based on the isolation conditions, temperature range for growth, enzyme activity, antibiotic resistance, presence of plasmids, and strain-specific genomic variations. A significant observation with implications for the development of novel and more effective cultivation methods was that preliminary incubation in anaerobic and aerobic liquid prior to plating on agar media greatly increased the recovery of CFU from the ice core sample.

Cloning and analysis of DnaJ family members in the silkworm, Bombyx mori.

PubMed

Li, Yinü; Bu, Cuiyu; Li, Tiantian; Wang, Shibao; Jiang, Feng; Yi, Yongzhu; Yang, Huipeng; Zhang, Zhifang

2016-01-15

Heat shock proteins (Hsps) are involved in a variety of critical biological functions, including protein folding, degradation, and translocation and macromolecule assembly, act as molecular chaperones during periods of stress by binding to other proteins. Using expressed sequence tag (EST) and silkworm (Bombyx mori) transcriptome databases, we identified 27 cDNA sequences encoding the conserved J domain, which is found in DnaJ-type Hsps. Of the 27 J domain-containing sequences, 25 were complete cDNA sequences. We divided them into three types according to the number and presence of conserved domains. By analyzing the gene structures, intron numbers, and conserved domains and constructing a phylogenetic tree, we found that the DnaJ family had undergone convergent evolution, obtaining new domains to expand the diversity of its family members. The acquisition of the new DnaJ domains most likely occurred prior to the evolutionary divergence of prokaryotes and eukaryotes. The expression of DnaJ genes in the silkworm was generally higher in the fat body. The tissue distribution of DnaJ1 proteins was detected by western blotting, demonstrating that in the fifth-instar larvae, the DnaJ1 proteins were expressed at their highest levels in hemocytes, followed by the fat body and head. We also found that the DnaJ1 transcripts were likely differentially translated in different tissues. Using immunofluorescence cytochemistry, we revealed that in the blood cells, DnaJ1 was mainly localized in the cytoplasm. Copyright © 2015 Elsevier B.V. All rights reserved.

Effective DNA Inhibitors of Cathepsin G by In Vitro Selection

PubMed Central

Gatto, Barbara; Vianini, Elena; Lucatello, Lorena; Sissi, Claudia; Moltrasio, Danilo; Pescador, Rodolfo; Porta, Roberto; Palumbo, Manlio

2008-01-01

Cathepsin G (CatG) is a chymotrypsin-like protease released upon degranulation of neutrophils. In several inflammatory and ischaemic diseases the impaired balance between CatG and its physiological inhibitors leads to tissue destruction and platelet aggregation. Inhibitors of CatG are suitable for the treatment of inflammatory diseases and procoagulant conditions. DNA released upon the death of neutrophils at injury sites binds CatG. Moreover, short DNA fragments are more inhibitory than genomic DNA. Defibrotide, a single stranded polydeoxyribonucleotide with antithrombotic effect is also a potent CatG inhibitor. Given the above experimental evidences we employed a selection protocol to assess whether DNA inhibition of CatG may be ascribed to specific sequences present in defibrotide DNA. A Selex protocol was applied to identify the single-stranded DNA sequences exhibiting the highest affinity for CatG, the diversity of a combinatorial pool of oligodeoxyribonucleotides being a good representation of the complexity found in defibrotide. Biophysical and biochemical studies confirmed that the selected sequences bind tightly to the target enzyme and also efficiently inhibit its catalytic activity. Sequence analysis carried out to unveil a motif responsible for CatG recognition showed a recurrence of alternating TG repeats in the selected CatG binders, adopting an extended conformation that grants maximal interaction with the highly charged protein surface. This unprecedented finding is validated by our results showing high affinity and inhibition of CatG by specific DNA sequences of variable length designed to maximally reduce pairing/folding interactions. PMID:19325843

Genetic origin of goat populations in Oman revealed by mitochondrial DNA analysis.

PubMed

Al-Araimi, Nasser Ali; Gaafar, Osman Mahgoub; Costa, Vânia; Neira, Agusto Luzuriaga; Al-Atiyat, Raed Mahmoud; Beja-Pereira, Albano

2017-01-01

The Sultanate of Oman has a complex mosaic of livestock species and production systems, but the genetic diversity, demographic history or origins of these Omani animals has not been expensively studied. Goats might constitute one of the most abundant and important domestic livestock species since the Neolithic transition. Here, we examined the genetic diversity, origin, population structure and demographic history of Omani goats. Specifically, we analyzed a 525-bp fragment of the first hypervariable region of the mitochondrial DNA (mtDNA) control region from 69 Omani individuals and compared this fragment with 17 mtDNA sequences from Somalia and Yemen as well as 18 wild goat species and 1,198 previously published goat sequences from neighboring countries. The studied goat breeds show substantial diversity. The haplotype and nucleotide diversities of Omani goats were found equal to 0.983 ± 0.006 and 0.0284 ± 0.014, respectively. The phylogenetic analyses allowed us to classify Omani goats into three mtDNA haplogroups (A, B and G): haplogroup A was found to be predominant and widely distributed and accounted for 80% of all samples, and haplogroups B and G exhibited low frequencies. Phylogenetic comparisons with wild goats revealed that five of the native Omani goat populations originate from Capra aegagrus. Furthermore, most comparisons of pairwise population FST values within and between these five Omani goat breeds as well as between Omani goats and nine populations from nearby countries were not significant. These results suggest strong gene flow among goat populations caused by the extensive transport of goats and the frequent movements of human populations in ancient Arabia. The findings improve our understanding of the migration routes of modern goats from their region of domestication into southeastern Arabia and thereby shed light on human migratory and commercial networks during historical times.

Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons

PubMed Central

Haas, Brian J.; Gevers, Dirk; Earl, Ashlee M.; Feldgarden, Mike; Ward, Doyle V.; Giannoukos, Georgia; Ciulla, Dawn; Tabbaa, Diana; Highlander, Sarah K.; Sodergren, Erica; Methé, Barbara; DeSantis, Todd Z.; Petrosino, Joseph F.; Knight, Rob; Birren, Bruce W.

2011-01-01

Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys. PMID:21212162

Ancient DNA from marine mammals: studying long-lived species over ecological and evolutionary timescales.

PubMed

Foote, Andrew D; Hofreiter, Michael; Morin, Phillip A

2012-01-20

Marine mammals have long generation times and broad, difficult to sample distributions, which makes inferring evolutionary and demographic changes using field studies of extant populations challenging. However, molecular analyses from sub-fossil or historical materials of marine mammals such as bone, tooth, baleen, skin, fur, whiskers and scrimshaw using ancient DNA (aDNA) approaches provide an opportunity for investigating such changes over evolutionary and ecological timescales. Here, we review the application of aDNA techniques to the study of marine mammals. Most of the studies have focused on detecting changes in genetic diversity following periods of exploitation and environmental change. To date, these studies have shown that even small sample sizes can provide useful information on historical genetic diversity. Ancient DNA has also been used in investigations of changes in distribution and range of marine mammal species; we review these studies and discuss the limitations of such 'presence only' studies. Combining aDNA data with stable isotopes can provide further insights into changes in ecology and we review past studies and suggest future potential applications. We also discuss studies reconstructing inter- and intra-specific phylogenies from aDNA sequences and discuss how aDNA sequences could be used to estimate mutation rates. Finally, we highlight some of the problems of aDNA studies on marine mammals, such as obtaining sufficient sample sizes and calibrating for the marine reservoir effect when radiocarbon-dating such wide-ranging species. Copyright © 2011 Elsevier GmbH. All rights reserved.

MHC class II B diversity in blue tits: a preliminary study.

PubMed

Aguilar, Juan Rivero-de; Schut, Elske; Merino, Santiago; Martínez, Javier; Komdeur, Jan; Westerdahl, Helena

2013-07-01

In this study, we partly characterize major histocompatibility complex (MHC) class II B in the blue tit (Cyanistes caeruleus). A total of 22 individuals from three different European locations: Spain, The Netherlands, and Sweden were screened for MHC allelic diversity. The MHC genes were investigated using both PCR-based methods and unamplified genomic DNA with restriction fragment length polymorphism (RFLP) and southern blots. A total of 13 different exon 2 sequences were obtained independently from DNA and/or RNA, thus confirming gene transcription and likely functionality of the genes. Nine out of 13 alleles were found in more than one country, and two alleles appeared in all countries. Positive selection was detected in the region coding for the peptide binding region (PBR). A maximum of three alleles per individual was detected by sequencing and the RFLP pattern consisted of 4-7 fragments, indicating a minimum number of 2-4 loci per individual. A phylogenetic analysis, demonstrated that the blue tit sequences are divergent compared to sequences from other passerines resembling a different MHC lineage than those possessed by most passerines studied to date.

MHC class II B diversity in blue tits: a preliminary study

PubMed Central

Aguilar, Juan Rivero-de; Schut, Elske; Merino, Santiago; Martínez, Javier; Komdeur, Jan; Westerdahl, Helena

2013-01-01

In this study, we partly characterize major histocompatibility complex (MHC) class II B in the blue tit (Cyanistes caeruleus). A total of 22 individuals from three different European locations: Spain, The Netherlands, and Sweden were screened for MHC allelic diversity. The MHC genes were investigated using both PCR-based methods and unamplified genomic DNA with restriction fragment length polymorphism (RFLP) and southern blots. A total of 13 different exon 2 sequences were obtained independently from DNA and/or RNA, thus confirming gene transcription and likely functionality of the genes. Nine out of 13 alleles were found in more than one country, and two alleles appeared in all countries. Positive selection was detected in the region coding for the peptide binding region (PBR). A maximum of three alleles per individual was detected by sequencing and the RFLP pattern consisted of 4–7 fragments, indicating a minimum number of 2–4 loci per individual. A phylogenetic analysis, demonstrated that the blue tit sequences are divergent compared to sequences from other passerines resembling a different MHC lineage than those possessed by most passerines studied to date. PMID:23919136

Utility of DNA barcoding for rapid and accurate assessment of bat diversity in Malaysia in the absence of formally described species.

PubMed

Wilson, J-J; Sing, K-W; Halim, M R A; Ramli, R; Hashim, R; Sofian-Azirun, M

2014-02-19

Bats are important flagship species for biodiversity research; however, diversity in Southeast Asia is considerably underestimated in the current checklists and field guides. Incorporation of DNA barcoding into surveys has revealed numerous species-level taxa overlooked by conventional methods. Inclusion of these taxa in inventories provides a more informative record of diversity, but is problematic as these species lack formal description. We investigated how frequently documented, but undescribed, bat taxa are encountered in Peninsular Malaysia. We discuss whether a barcode library provides a means of recognizing and recording these taxa across biodiversity inventories. Tissue was sampled from bats trapped at Pasir Raja, Dungun Terengganu, Peninsular Malaysia. The DNA was extracted and the COI barcode region amplified and sequenced. We identified 9 species-level taxa within our samples, based on analysis of the DNA barcodes. Six specimens matched to four previously documented taxa considered candidate species but currently lacking formal taxonomic status. This study confirms the high diversity of bats within Peninsular Malaysia (9 species in 13 samples) and demonstrates how DNA barcoding allows for inventory and documentation of known taxa lacking formal taxonomic status.

DNA Barcoding of Bemisia tabaci Complex (Hemiptera: Aleyrodidae) Reveals Southerly Expansion of the Dominant Whitefly Species on Cotton in Pakistan

PubMed Central

Ashfaq, Muhammad; Hebert, Paul D. N.; Mirza, M. Sajjad; Khan, Arif M.; Mansoor, Shahid; Shah, Ghulam S.; Zafar, Yusuf

2014-01-01

Background Although whiteflies (Bemisia tabaci complex) are an important pest of cotton in Pakistan, its taxonomic diversity is poorly understood. As DNA barcoding is an effective tool for resolving species complexes and analyzing species distributions, we used this approach to analyze genetic diversity in the B. tabaci complex and map the distribution of B. tabaci lineages in cotton growing areas of Pakistan. Methods/Principal Findings Sequence diversity in the DNA barcode region (mtCOI-5′) was examined in 593 whiteflies from Pakistan to determine the number of whitefly species and their distributions in the cotton-growing areas of Punjab and Sindh provinces. These new records were integrated with another 173 barcode sequences for B. tabaci, most from India, to better understand regional whitefly diversity. The Barcode Index Number (BIN) System assigned the 766 sequences to 15 BINs, including nine from Pakistan. Representative specimens of each Pakistan BIN were analyzed for mtCOI-3′ to allow their assignment to one of the putative species in the B. tabaci complex recognized on the basis of sequence variation in this gene region. This analysis revealed the presence of Asia II 1, Middle East-Asia Minor 1, Asia 1, Asia II 5, Asia II 7, and a new lineage “Pakistan”. The first two taxa were found in both Punjab and Sindh, but Asia 1 was only detected in Sindh, while Asia II 5, Asia II 7 and “Pakistan” were only present in Punjab. The haplotype networks showed that most haplotypes of Asia II 1, a species implicated in transmission of the cotton leaf curl virus, occurred in both India and Pakistan. Conclusions DNA barcodes successfully discriminated cryptic species in B. tabaci complex. The dominant haplotypes in the B. tabaci complex were shared by India and Pakistan. Asia II 1 was previously restricted to Punjab, but is now the dominant lineage in southern Sindh; its southward spread may have serious implications for cotton plantations in this region. PMID:25099936

Investigating the Genome Diversity of B. cereus and Evolutionary Aspects of B. anthracis Emergence

PubMed Central

Papazisi, Leka; Rasko, David A.; Ratnayake, Shashikala; Bock, Geoff R.; Remortel, Brian G.; Appalla, Lakshmi; Liu, Jia; Dracheva, Tatiana; Braisted, John C.; Shallom, Shamira; Jarrahi, Benham; Snesrud, Erik; Ahn, Susie; Sun, Qiang; Rilstone, Jenifer; Økstad, Ole Andreas; Kolstø, Anne-Brit; Fleischmann, Robert D.; Peterson, Scott N.

2011-01-01

Here we report the use of a multi-genome DNA microarray to investigate the genome diversity of Bacillus cereus group members and elucidate the events associated with the emergence of B. anthracis the causative agent of anthrax–a lethal zoonotic disease. We initially performed directed genome sequencing of seven diverse B. cereus strains to identify novel sequences encoded in those genomes. The novel genes identified, combined with those publicly available, allowed the design of a “species” DNA microarray. Comparative genomic hybridization analyses of 41 strains indicates that substantial heterogeneity exists with respect to the genes comprising functional role categories. While the acquisition of the plasmid-encoded pathogenicity island (pXO1) and capsule genes (pXO2) represent a crucial landmark dictating the emergence of B. anthracis, the evolution of this species and its close relatives was associated with an overall a shift in the fraction of genes devoted to energy metabolism, cellular processes, transport, as well as virulence. PMID:21447378

Genetic Diversity of Sheep Breeds from Albania, Greece, and Italy Assessed by Mitochondrial DNA and Nuclear Polymorphisms (SNPs)

PubMed Central

Pariset, Lorraine; Mariotti, Marco; Gargani, Maria; Joost, Stephane; Negrini, Riccardo; Perez, Trinidad; Bruford, Michael; Ajmone Marsan, Paolo; Valentini, Alessio

2011-01-01

We employed mtDNA and nuclear SNPs to investigate the genetic diversity of sheep breeds of three countries of the Mediterranean basin: Albania, Greece, and Italy. In total, 154 unique mtDNA haplotypes were detected by means of D-loop sequence analysis. The major nucleotide diversity was observed in Albania. We identified haplogroups, A, B, and C in Albanian and Greek samples, while Italian individuals clustered in groups A and B. In general, the data show a pattern reflecting old migrations that occurred in postneolithic and historical times. PCA analysis on SNP data differentiated breeds with good correspondence to geographical locations. This could reflect geographical isolation, selection operated by local sheep farmers, and different flock management and breed admixture that occurred in the last centuries. PMID:22125424

Approach to determine the diversity of Legionella species by nested PCR-DGGE in aquatic environments.

PubMed

Huang, Wen-Chien; Tsai, Hsin-Chi; Tao, Chi-Wei; Chen, Jung-Sheng; Shih, Yi-Jia; Kao, Po-Min; Huang, Tung-Yi; Hsu, Bing-Mu

2017-01-01

In this study, we describe a nested PCR-DGGE strategy to detect Legionella communities from river water samples. The nearly full-length 16S rRNA gene was amplified using bacterial primer in the first step. After, the amplicons were employed as DNA templates in the second PCR using Legionella specific primer. The third round of gene amplification was conducted to gain PCR fragments apposite for DGGE analysis. Then the total numbers of amplified genes were observed in DGGE bands of products gained with primers specific for the diversity of Legionella species. The DGGE patterns are thus potential for a high-throughput preliminary determination of aquatic environmental Legionella species before sequencing. Comparative DNA sequence analysis of excised DGGE unique band patterns showed the identity of the Legionella community members, including a reference profile with two pathogenic species of Legionella strains. In addition, only members of Legionella pneumophila and uncultured Legionella sp. were detected. Development of three step nested PCR-DGGE tactic is seen as a useful method for studying the diversity of Legionella community. The method is rapid and provided sequence information for phylogenetic analysis.

Approach to determine the diversity of Legionella species by nested PCR-DGGE in aquatic environments

PubMed Central

Huang, Wen-Chien; Tsai, Hsin-Chi; Tao, Chi-Wei; Chen, Jung-Sheng; Shih, Yi-Jia; Kao, Po-Min; Huang, Tung-Yi; Hsu, Bing-Mu

2017-01-01

In this study, we describe a nested PCR-DGGE strategy to detect Legionella communities from river water samples. The nearly full-length 16S rRNA gene was amplified using bacterial primer in the first step. After, the amplicons were employed as DNA templates in the second PCR using Legionella specific primer. The third round of gene amplification was conducted to gain PCR fragments apposite for DGGE analysis. Then the total numbers of amplified genes were observed in DGGE bands of products gained with primers specific for the diversity of Legionella species. The DGGE patterns are thus potential for a high-throughput preliminary determination of aquatic environmental Legionella species before sequencing. Comparative DNA sequence analysis of excised DGGE unique band patterns showed the identity of the Legionella community members, including a reference profile with two pathogenic species of Legionella strains. In addition, only members of Legionella pneumophila and uncultured Legionella sp. were detected. Development of three step nested PCR-DGGE tactic is seen as a useful method for studying the diversity of Legionella community. The method is rapid and provided sequence information for phylogenetic analysis. PMID:28166249

Finding needles in haystacks: linking scientific names, reference specimens and molecular data for Fungi

Treesearch

C.L. Schoch; B. Robbertse; V. Robert; R.G. Haight; K. Kovacs; B. Leung; W. Meyer; R.H. Nilsson; K. Hughes; A.N. Miller; P.M. Kirk; K. Abarenkov; M.C. Aime; H.A. Ariyawansa; M. Bidartondo; T. Boekhout; B. Buyck; Q. Cai; J. Chen; A. Crespo; P.W. Crous; U. Damm; Z.W. De Beer; B.T.M. Dentinger; P.K. Divakar; M. Duenas; N. Feau; K. Fliegerova; M.A. Garcia; Z.-W. Ge; G.W. Griffith; J.Z. Groenewald; M. Groenewald; M. Grube; M. Gryzenhout; C. Gueidan; L. Guo; S. Hambleton; R. Hamelin; K. Hansen; V. Hofstetter; S.-B. Hong; J. Houbraken; K.D. Hyde; P. Inderbitzin; P.R. Johnston; S.C. Karunarathna; U. Koljalg; G.M. Kovacs; E. Kraichak; K. Krizsan; C.P. Kurtzman; K.-H. Larsson; S. Leavitt; P.M. Letcher; K. Liimatainen; J.-K. Liu; D.J. Lodge; J. Jennifer Luangsa-ard; H.T. Lumbsch; S.S.N. Maharachchikumbura; D. Manamgoda; M.P. Martin; A.M. Minnis; J.-M. Moncalvo; G. Mule; K.K. Nakasone; T. Niskanen; I. Olariaga; T. Papp; T. Petkovits; R. Pino-Bodas; M.J. Powell; H.A. Raja; D. Redecker; J.M. Sarmiento-Ramirez; K.A. Seifert; B. Shrestha; S. Stenroos; B. Stielow; S.-O. Suh; K. Tanaka; L. Tedersoo; M.T. Telleria; D. Udayanga; W.A. Untereiner; J. Dieguez Uribeondo; K.V. Subbarao; C. Vagvolgyi; C. Visagie; K. Voigt; D.M. Walker; B.S. Weir; M. Weiss; N.N. Wijayawardene; M.J. Wingfield; J.P. Xu; Z.L. Yang; N. Zhang; W.-Y. Zhuang; S. Federhen

2014-01-01

DNA phylogenetic comparisons have shown that morphology-based species recognition often underestimates fungal diversity. Therefore, the need for accurate DNA sequence data, tied to both correct taxonomic names and clearly annotated specimen data, has never been greater. Furthermore, the growing number of molecular ecology and microbiome projects using high-throughput...

DNA Barcode Analysis of Thrips (Thysanoptera) Diversity in Pakistan Reveals Cryptic Species Complexes.

PubMed

Iftikhar, Romana; Ashfaq, Muhammad; Rasool, Akhtar; Hebert, Paul D N

2016-01-01

Although thrips are globally important crop pests and vectors of viral disease, species identifications are difficult because of their small size and inconspicuous morphological differences. Sequence variation in the mitochondrial COI-5' (DNA barcode) region has proven effective for the identification of species in many groups of insect pests. We analyzed barcode sequence variation among 471 thrips from various plant hosts in north-central Pakistan. The Barcode Index Number (BIN) system assigned these sequences to 55 BINs, while the Automatic Barcode Gap Discovery detected 56 partitions, a count that coincided with the number of monophyletic lineages recognized by Neighbor-Joining analysis and Bayesian inference. Congeneric species showed an average of 19% sequence divergence (range = 5.6% - 27%) at COI, while intraspecific distances averaged 0.6% (range = 0.0% - 7.6%). BIN analysis suggested that all intraspecific divergence >3.0% actually involved a species complex. In fact, sequences for three major pest species (Haplothrips reuteri, Thrips palmi, Thrips tabaci), and one predatory thrips (Aeolothrips intermedius) showed deep intraspecific divergences, providing evidence that each is a cryptic species complex. The study compiles the first barcode reference library for the thrips of Pakistan, and examines global haplotype diversity in four important pest thrips.

Extensive gene conversion at the PMS2 DNA mismatch repair locus.

PubMed

Hayward, Bruce E; De Vos, Michel; Valleley, Elizabeth M A; Charlton, Ruth S; Taylor, Graham R; Sheridan, Eamonn; Bonthron, David T

2007-05-01

Mutations of the PMS2 DNA repair gene predispose to a characteristic range of malignancies, with either childhood onset (when both alleles are mutated) or a partially penetrant adult onset (if heterozygous). These mutations have been difficult to detect, due to interference from a family of pseudogenes located on chromosome 7. One of these, the PMS2CL pseudogene, lies within a 100-kb inverted duplication (inv dup), 700 kb centromeric to PMS2 itself on 7p22. Here, we show that the reference genomic sequences cannot be relied upon to distinguish PMS2 from PMS2CL, because of sequence transfer between the two loci. The 7p22 inv dup occurred prior to the divergence of modern ape species (15 million years ago [Mya]), but has undergone extensive sequence homogenization. This process appears to be ongoing, since there is considerable allelic diversity within the duplicated region, much of it derived from sequence exchange between PMS2 and PMS2CL. This sequence diversity can result in both false-positive and false-negative mutation analysis at this locus. Great caution is still needed in the design and interpretation of PMS2 mutation screens. 2007 Wiley-Liss, Inc.

Comparing Ecological and Genetic Diversity Within the Marine Diatom Genus Pseudo-nitzschia: A Multiregional Synthesis

NASA Astrophysics Data System (ADS)

Hubbard, K.; Bruzek, S.

2016-02-01

The globally distributed marine diatom genus Pseudo-nitzschia consists of approximately 40 species, more than half of which occur in US coastal waters. Here, sensitive genetic tools targeting a variable portion of the internal transcribed spacer 1 (ITS1) region of the rRNA gene were used to assess Pseudo-nitzschia spp. diversity in more than 600 environmental DNA samples collected from US Atlantic, Pacific, and Gulf of Mexico waters. Community-based approaches employed genus-specific primers for environmental DNA fingerprinting and targeted sequencing. For the Gulf of Mexico samples especially, a nested PCR approach (with or without degenerate primers) improved resolution of species diversity. To date, more than 40 unique ITS1 amplicon sizes have been repeatedly observed in ITS1 fingerprints. Targeted sequencing of environmental DNA as well as single chains isolated from live samples indicate that many of these represent novel and known inter- and intra-specific Pseudo-nitzschia diversity. A few species (e.g., P. pungens, P. cuspidata) occur across all three regions, whereas other species and intraspecific variants occurred at local to regional spatial scales only. Generally, species frequently co-occur in complex assemblages, and transitions in Pseudo-nitzschia community composition occur seasonally, prior to bloom initiation, and across (cross-shelf, latitudinal, and vertical) environmental gradients. These observations highlight the dynamic nature of diatom community composition in the marine environment and the importance of classifying diversity at relevant ecological and/or taxonomic scales.

Double-stranded telomeric DNA binding proteins: Diversity matters.

PubMed

Červenák, Filip; Juríková, Katarína; Sepšiová, Regina; Neboháčová, Martina; Nosek, Jozef; Tomáška, L'ubomír

2017-01-01

Telomeric sequences constitute only a small fraction of the whole genome yet they are crucial for ensuring genomic stability. This function is in large part mediated by protein complexes recruited to telomeric sequences by specific telomere-binding proteins (TBPs). Although the principal tasks of nuclear telomeres are the same in all eukaryotes, TBPs in various taxa exhibit a surprising diversity indicating their distinct evolutionary origin. This diversity is especially pronounced in ascomycetous yeasts where they must have co-evolved with rapidly diversifying sequences of telomeric repeats. In this article we (i) provide a historical overview of the discoveries leading to the current list of TBPs binding to double-stranded (ds) regions of telomeres, (ii) describe examples of dsTBPs highlighting their diversity in even closely related species, and (iii) speculate about possible evolutionary trajectories leading to a long list of various dsTBPs fulfilling the same general role(s) in their own unique ways.

Human beta-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old.

PubMed

Béraud-Colomb, E; Roubin, R; Martin, J; Maroc, N; Gardeisen, A; Trabuchet, G; Goosséns, M

1995-12-01

Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the beta-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for beta-globin frameworks by sequencing through two variable positions and for a polymorphic (AT) chi (T) gamma microsatellite 500 bp upstream of the beta-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible.

Skin microbiome: genomics-based insights into the diversity and role of skin microbes

PubMed Central

Kong, Heidi H.

2011-01-01

Recent advances in DNA sequencing methodology have enabled studies of human skin microbes that circumvent difficulties in isolating and characterizing fastidious microbes. Sequence-based approaches have identified greater diversity of cutaneous bacteria than studies using traditional cultivation techniques. However, improved sequencing technologies and analytical methods are needed to study all skin microbes, including bacteria, archaea, fungi, viruses, and mites, and how they interact with each other and their human hosts. This review discusses current skin microbiome research, with a primary focus on bacteria, and the challenges facing investigators striving to understand how skin micro-organisms contribute to health and disease. PMID:21376666

Escaping introns in COI through cDNA barcoding of mushrooms: Pleurotus as a test case.

PubMed

Avin, Farhat A; Subha, Bhassu; Tan, Yee-Shin; Braukmann, Thomas W A; Vikineswary, Sabaratnam; Hebert, Paul D N

2017-09-01

DNA barcoding involves the use of one or more short, standardized DNA fragments for the rapid identification of species. A 648-bp segment near the 5' terminus of the mitochondrial cytochrome c oxidase subunit I (COI) gene has been adopted as the universal DNA barcode for members of the animal kingdom, but its utility in mushrooms is complicated by the frequent occurrence of large introns. As a consequence, ITS has been adopted as the standard DNA barcode marker for mushrooms despite several shortcomings. This study employed newly designed primers coupled with cDNA analysis to examine COI sequence diversity in six species of Pleurotus and compared these results with those for ITS. The ability of the COI gene to discriminate six species of Pleurotus , the commonly cultivated oyster mushroom, was examined by analysis of cDNA. The amplification success, sequence variation within and among species, and the ability to design effective primers was tested. We compared ITS sequences to their COI cDNA counterparts for all isolates. ITS discriminated between all six species, but some sequence results were uninterpretable, because of length variation among ITS copies. By comparison, a complete COI sequences were recovered from all but three individuals of Pleurotus giganteus where only the 5' region was obtained. The COI sequences permitted the resolution of all species when partial data was excluded for P. giganteus . Our results suggest that COI can be a useful barcode marker for mushrooms when cDNA analysis is adopted, permitting identifications in cases where ITS cannot be recovered or where it offers higher resolution when fresh tissue is. The suitability of this approach remains to be confirmed for other mushrooms.

Analysis of mitochondrial DNA in Bolivian llama, alpaca and vicuna populations: a contribution to the phylogeny of the South American camelids.

PubMed

Barreta, J; Gutiérrez-Gil, B; Iñiguez, V; Saavedra, V; Chiri, R; Latorre, E; Arranz, J J

2013-04-01

The objectives of this work were to assess the mtDNA diversity of Bolivian South American camelid (SAC) populations and to shed light on the evolutionary relationships between the Bolivian camelids and other populations of SACs. We have analysed two different mtDNA regions: the complete coding region of the MT-CYB gene and 513 bp of the D-loop region. The populations sampled included Bolivian llamas, alpacas and vicunas, and Chilean guanacos. High levels of genetic diversity were observed in the studied populations. In general, MT-CYB was more variable than D-loop. On a species level, the vicunas showed the lowest genetic variability, followed by the guanacos, alpacas and llamas. Phylogenetic analyses performed by including additional available mtDNA sequences from the studied species confirmed the existence of the two monophyletic clades previously described by other authors for guanacos (G) and vicunas (V). Significant levels of mtDNA hybridization were found in the domestic species. Our sequence analyses revealed significant sequence divergence within clade G, and some of the Bolivian llamas grouped with the majority of the southern guanacos. This finding supports the existence of more than the one llama domestication centre in South America previously suggested on the basis of archaeozoological evidence. Additionally, analysis of D-loop sequences revealed two new matrilineal lineages that are distinct from the previously reported G and V clades. The results presented here represent the first report on the population structure and genetic variability of Bolivian camelids and may help to elucidate the complex and dynamic domestication process of SAC populations. © 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.

Genotyping of ancient Mycobacterium tuberculosis strains reveals historic genetic diversity.

PubMed

Müller, Romy; Roberts, Charlotte A; Brown, Terence A

2014-04-22

The evolutionary history of the Mycobacterium tuberculosis complex (MTBC) has previously been studied by analysis of sequence diversity in extant strains, but not addressed by direct examination of strain genotypes in archaeological remains. Here, we use ancient DNA sequencing to type 11 single nucleotide polymorphisms and two large sequence polymorphisms in the MTBC strains present in 10 archaeological samples from skeletons from Britain and Europe dating to the second-nineteenth centuries AD. The results enable us to assign the strains to groupings and lineages recognized in the extant MTBC. We show that at least during the eighteenth-nineteenth centuries AD, strains of M. tuberculosis belonging to different genetic groups were present in Britain at the same time, possibly even at a single location, and we present evidence for a mixed infection in at least one individual. Our study shows that ancient DNA typing applied to multiple samples can provide sufficiently detailed information to contribute to both archaeological and evolutionary knowledge of the history of tuberculosis.

Analysis of protein-coding genetic variation in 60,706 humans.

PubMed

Lek, Monkol; Karczewski, Konrad J; Minikel, Eric V; Samocha, Kaitlin E; Banks, Eric; Fennell, Timothy; O'Donnell-Luria, Anne H; Ware, James S; Hill, Andrew J; Cummings, Beryl B; Tukiainen, Taru; Birnbaum, Daniel P; Kosmicki, Jack A; Duncan, Laramie E; Estrada, Karol; Zhao, Fengmei; Zou, James; Pierce-Hoffman, Emma; Berghout, Joanne; Cooper, David N; Deflaux, Nicole; DePristo, Mark; Do, Ron; Flannick, Jason; Fromer, Menachem; Gauthier, Laura; Goldstein, Jackie; Gupta, Namrata; Howrigan, Daniel; Kiezun, Adam; Kurki, Mitja I; Moonshine, Ami Levy; Natarajan, Pradeep; Orozco, Lorena; Peloso, Gina M; Poplin, Ryan; Rivas, Manuel A; Ruano-Rubio, Valentin; Rose, Samuel A; Ruderfer, Douglas M; Shakir, Khalid; Stenson, Peter D; Stevens, Christine; Thomas, Brett P; Tiao, Grace; Tusie-Luna, Maria T; Weisburd, Ben; Won, Hong-Hee; Yu, Dongmei; Altshuler, David M; Ardissino, Diego; Boehnke, Michael; Danesh, John; Donnelly, Stacey; Elosua, Roberto; Florez, Jose C; Gabriel, Stacey B; Getz, Gad; Glatt, Stephen J; Hultman, Christina M; Kathiresan, Sekar; Laakso, Markku; McCarroll, Steven; McCarthy, Mark I; McGovern, Dermot; McPherson, Ruth; Neale, Benjamin M; Palotie, Aarno; Purcell, Shaun M; Saleheen, Danish; Scharf, Jeremiah M; Sklar, Pamela; Sullivan, Patrick F; Tuomilehto, Jaakko; Tsuang, Ming T; Watkins, Hugh C; Wilson, James G; Daly, Mark J; MacArthur, Daniel G

2016-08-18

Large-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) DNA sequence data for 60,706 individuals of diverse ancestries generated as part of the Exome Aggregation Consortium (ExAC). This catalogue of human genetic diversity contains an average of one variant every eight bases of the exome, and provides direct evidence for the presence of widespread mutational recurrence. We have used this catalogue to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; identifying 3,230 genes with near-complete depletion of predicted protein-truncating variants, with 72% of these genes having no currently established human disease phenotype. Finally, we demonstrate that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human 'knockout' variants in protein-coding genes.

Molecular diversity of some species belonging to the genus Daphnia O. F. Müller, 1785 (Crustacea: Cladocera) in Turkey.

PubMed

Özdemir, Ebru; Altındağ, Ahmet; Kandemir, İrfan

2017-05-01

Daphnia is a freshwater zooplankton species with controversial taxonomy due to its high morphological variation linked to environmental factors and inter-specific hybridization and polyploidy in some groups. The aim of the present study is to examine molecular diversity of some Daphnia species in Turkey and to establish DNA barcodes of Turkish Daphnia species. Sequence analysis was performed using 540 bp region of cytochrome oxidase subunit I gene of mitochondrial DNA. A total of 34 haplotypes have been identified for Turkey. Daphnia pulex complex was divided into two clades with 16.1% sequence divergence according to molecular taxonomy based on Kimura 2-parameter. The clade which was molecularly diverged from Daphnia pulex with 16.1% sequence divergence was found to show 99% similarity with Daphnia cf. pulicaria (sensu Alonso 1996) instead of Daphnia pulicaria Forbes, 1893. Furthermore, this study has contributed to Turkish zoogeography by demonstrating the distribution of Daphnia species in Turkey.

Five thousand years of tropical lake sediment DNA records from Benin

NASA Astrophysics Data System (ADS)

Bremond, L.; Favier, C.; Ficetola, G. F.; Tossou, M. G.; Akouégninou, A.; Gielly, L.; Giguet-Covex, C.; Oslisly, R.; Salzmann, U.

2017-08-01

Until now, sedimentary DNA (sedDNA) studies have only focused on cold and temperate regions were DNA is relatively well preserved. Consequently, the tropics, where vegetation is hyperdiverse and natural archives are rare, have been neglected and deserve attention. In this study, we used next-generation sequencing to barcode sedDNA from Lake Sele, localized in the tropical lowlands of Benin (Africa), and compared the taxonomic diversity detected by DNA analyses with pollen assemblages. Plant sedDNA was successfully amplified from 33 of the 34 successfully extracted samples. In total, 43 taxa were identified along the 5000 years spanned by the sediment: 22 taxa were identified at the family level and 21 at the genus level. The plant diversity recovered through sedDNA from Lake Sele showed a specific local signal and limited overlapping with pollen. Introduced plants, grown and cultivated close to the water, such as sweet potato, were also well recorded by sedDNA. It appears, therefore, to be a promising approach to studying past diversity in tropical regions, and could help in tracking the introduction and history of agriculture. This is the first time this method has been used in the field of domestication and dissemination of several specific crops, and the results are very encouraging.

Targeted Next-Generation Sequencing of Plasma DNA from Cancer Patients: Factors Influencing Consistency with Tumour DNA and Prospective Investigation of Its Utility for Diagnosis

PubMed Central

Kaisaki, Pamela J.; Cutts, Anthony; Popitsch, Niko; Camps, Carme; Pentony, Melissa M.; Wilson, Gareth; Page, Suzanne; Kaur, Kulvinder; Vavoulis, Dimitris; Henderson, Shirley; Gupta, Avinash; Middleton, Mark R.; Karydis, Ioannis; Talbot, Denis C.; Schuh, Anna; Taylor, Jenny C.

2016-01-01

Use of circulating tumour DNA (ctDNA) as a liquid biopsy has been proposed for potential identification and monitoring of solid tumours. We investigate a next-generation sequencing approach for mutation detection in ctDNA in two related studies using a targeted panel. The first study was retrospective, using blood samples taken from melanoma patients at diverse timepoints before or after treatment, aiming to evaluate correlation between mutations identified in biopsy and ctDNA, and to acquire a first impression of influencing factors. We found good concordance between ctDNA and tumour mutations of melanoma patients when blood samples were collected within one year of biopsy or before treatment. In contrast, when ctDNA was sequenced after targeted treatment in melanoma, mutations were no longer found in 9 out of 10 patients, suggesting the method might be useful for detecting treatment response. Building on these findings, we focused the second study on ctDNA obtained before biopsy in lung patients, i.e. when a tentative diagnosis of lung cancer had been made, but no treatment had started. The main objective of this prospective study was to evaluate use of ctDNA in diagnosis, investigating the concordance of biopsy and ctDNA-derived mutation detection. Here we also found positive correlation between diagnostic lung biopsy results and pre-biopsy ctDNA sequencing, providing support for using ctDNA as a cost-effective, non-invasive solution when the tumour is inaccessible or when biopsy poses significant risk to the patient. PMID:27626278

DNA Translator and Aligner: HyperCard utilities to aid phylogenetic analysis of molecules.

PubMed

Eernisse, D J

1992-04-01

DNA Translator and Aligner are molecular phylogenetics HyperCard stacks for Macintosh computers. They manipulate sequence data to provide graphical gene mapping, conversions, translations and manual multiple-sequence alignment editing. DNA Translator is able to convert documented GenBank or EMBL documented sequences into linearized, rescalable gene maps whose gene sequences are extractable by clicking on the corresponding map button or by selection from a scrolling list. Provided gene maps, complete with extractable sequences, consist of nine metazoan, one yeast, and one ciliate mitochondrial DNAs and three green plant chloroplast DNAs. Single or multiple sequences can be manipulated to aid in phylogenetic analysis. Sequences can be translated between nucleic acids and proteins in either direction with flexible support of alternate genetic codes and ambiguous nucleotide symbols. Multiple aligned sequence output from diverse sources can be converted to Nexus, Hennig86 or PHYLIP format for subsequent phylogenetic analysis. Input or output alignments can be examined with Aligner, a convenient accessory stack included in the DNA Translator package. Aligner is an editor for the manual alignment of up to 100 sequences that toggles between display of matched characters and normal unmatched sequences. DNA Translator also generates graphic displays of amino acid coding and codon usage frequency relative to all other, or only synonymous, codons for approximately 70 select organism-organelle combinations. Codon usage data is compatible with spreadsheet or UWGCG formats for incorporation of additional molecules of interest. The complete package is available via anonymous ftp and is free for non-commercial uses.

Genetic structure and diversity of Japanese kokanee Oncorhynchus nerka stocks as revealed by microsatellite and mitochondrial DNA markers.

PubMed

Yamamoto, S; Kitamura, S; Sakano, H; Morita, K

2011-11-01

Genetic structure and diversity of nine Japanese kokanee (landlocked) Oncorhynchus nerka stocks and anadromous O. nerka from the North Pacific and the Canadian Lake Cultus population were examined using microsatellite and mitochondrial DNA. Sequence analyses of the cytochrome b region of mtDNA for Japanese kokanee O. nerka stocks on Honshu and Hokkaido islands revealed that most Japanese stocks were monomorphic of one major haplotype, which was also dominant in the Lake Cultus population and anadromous O. nerka in the North Pacific. Assignment tests using microsatellite DNA revealed that there was no clear-cut population structure in Japanese kokanee O. nerka stocks. © 2011 The Authors. Journal of Fish Biology © 2011 The Fisheries Society of the British Isles.

Genetic Diversity of Bacterial Communities and Gene Transfer Agents in Northern South China Sea

PubMed Central

Sun, Fu-Lin; Wang, You-Shao; Wu, Mei-Lin; Jiang, Zhao-Yu; Sun, Cui-Ci; Cheng, Hao

2014-01-01

Pyrosequencing of the 16S ribosomal RNA gene (rDNA) amplicons was performed to investigate the unique distribution of bacterial communities in northern South China Sea (nSCS) and evaluate community structure and spatial differences of bacterial diversity. Cyanobacteria, Proteobacteria, Actinobacteria, and Bacteroidetes constitute the majority of bacteria. The taxonomic description of bacterial communities revealed that more Chroococcales, SAR11 clade, Acidimicrobiales, Rhodobacterales, and Flavobacteriales are present in the nSCS waters than other bacterial groups. Rhodobacterales were less abundant in tropical water (nSCS) than in temperate and cold waters. Furthermore, the diversity of Rhodobacterales based on the gene transfer agent (GTA) major capsid gene (g5) was investigated. Four g5 gene clone libraries were constructed from samples representing different regions and yielded diverse sequences. Fourteen g5 clusters could be identified among 197 nSCS clones. These clusters were also related to known g5 sequences derived from genome-sequenced Rhodobacterales. The composition of g5 sequences in surface water varied with the g5 sequences in the sampling sites; this result indicated that the Rhodobacterales population could be highly diverse in nSCS. Phylogenetic tree analysis result indicated distinguishable diversity patterns among tropical (nSCS), temperate, and cold waters, thereby supporting the niche adaptation of specific Rhodobacterales members in unique environments. PMID:25364820

Phylogenetic diversity and biogeography of the Mamiellophyceae lineage of eukaryotic phytoplankton across the oceans.

PubMed

Monier, Adam; Worden, Alexandra Z; Richards, Thomas A

2016-08-01

High-throughput diversity amplicon sequencing of marine microbial samples has revealed that members of the Mamiellophyceae lineage are successful phytoplankton in many oceanic habitats. Indeed, these eukaryotic green algae can dominate the picoplanktonic biomass, however, given the broad expanses of the oceans, their geographical distributions and the phylogenetic diversity of some groups remain poorly characterized. As these algae play a foundational role in marine food webs, it is crucial to assess their global distribution in order to better predict potential changes in abundance and community structure. To this end, we analyzed the V9-18S small subunit rDNA sequences deposited from the Tara Oceans expedition to evaluate the diversity and biogeography of these phytoplankton. Our results show that the phylogenetic composition of Mamiellophyceae communities is in part determined by geographical provenance, and do not appear to be influenced - in the samples recovered - by water depth, at least at the resolution possible with the V9-18S. Phylogenetic classification of Mamiellophyceae sequences revealed that the Dolichomastigales order encompasses more sequence diversity than other orders in this lineage. These results indicate that a large fraction of the Mamiellophyceae diversity has been hitherto overlooked, likely because of a combination of size fraction, sequencing and geographical limitations. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

High-quality mtDNA control region sequences from 680 individuals sampled across the Netherlands to establish a national forensic mtDNA reference database.

PubMed

Chaitanya, Lakshmi; van Oven, Mannis; Brauer, Silke; Zimmermann, Bettina; Huber, Gabriela; Xavier, Catarina; Parson, Walther; de Knijff, Peter; Kayser, Manfred

2016-03-01

The use of mitochondrial DNA (mtDNA) for maternal lineage identification often marks the last resort when investigating forensic and missing-person cases involving highly degraded biological materials. As with all comparative DNA testing, a match between evidence and reference sample requires a statistical interpretation, for which high-quality mtDNA population frequency data are crucial. Here, we determined, under high quality standards, the complete mtDNA control-region sequences of 680 individuals from across the Netherlands sampled at 54 sites, covering the entire country with 10 geographic sub-regions. The complete mtDNA control region (nucleotide positions 16,024-16,569 and 1-576) was amplified with two PCR primers and sequenced with ten different sequencing primers using the EMPOP protocol. Haplotype diversity of the entire sample set was very high at 99.63% and, accordingly, the random-match probability was 0.37%. No population substructure within the Netherlands was detected with our dataset. Phylogenetic analyses were performed to determine mtDNA haplogroups. Inclusion of these high-quality data in the EMPOP database (accession number: EMP00666) will improve its overall data content and geographic coverage in the interest of all EMPOP users worldwide. Moreover, this dataset will serve as (the start of) a national reference database for mtDNA applications in forensic and missing person casework in the Netherlands. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A comprehensive DNA barcode database for Central European beetles with a focus on Germany: adding more than 3500 identified species to BOLD.

PubMed

Hendrich, Lars; Morinière, Jérôme; Haszprunar, Gerhard; Hebert, Paul D N; Hausmann, Axel; Köhler, Frank; Balke, Michael

2015-07-01

Beetles are the most diverse group of animals and are crucial for ecosystem functioning. In many countries, they are well established for environmental impact assessment, but even in the well-studied Central European fauna, species identification can be very difficult. A comprehensive and taxonomically well-curated DNA barcode library could remedy this deficit and could also link hundreds of years of traditional knowledge with next generation sequencing technology. However, such a beetle library is missing to date. This study provides the globally largest DNA barcode reference library for Coleoptera for 15 948 individuals belonging to 3514 well-identified species (53% of the German fauna) with representatives from 97 of 103 families (94%). This study is the first comprehensive regional test of the efficiency of DNA barcoding for beetles with a focus on Germany. Sequences ≥500 bp were recovered from 63% of the specimens analysed (15 948 of 25 294) with short sequences from another 997 specimens. Whereas most specimens (92.2%) could be unambiguously assigned to a single known species by sequence diversity at CO1, 1089 specimens (6.8%) were assigned to more than one Barcode Index Number (BIN), creating 395 BINs which need further study to ascertain if they represent cryptic species, mitochondrial introgression, or simply regional variation in widespread species. We found 409 specimens (2.6%) that shared a BIN assignment with another species, most involving a pair of closely allied species as 43 BINs were involved. Most of these taxa were separated by barcodes although sequence divergences were low. Only 155 specimens (0.97%) show identical or overlapping clusters. © 2014 John Wiley & Sons Ltd.

Beyond the Colours: Discovering Hidden Diversity in the Nymphalidae of the Yucatan Peninsula in Mexico through DNA Barcoding

PubMed Central

Prado, Blanca R.; Pozo, Carmen; Valdez-Moreno, Martha; Hebert, Paul D. N.

2011-01-01

Background Recent studies have demonstrated the utility of DNA barcoding in the discovery of overlooked species and in the connection of immature and adult stages. In this study, we use DNA barcoding to examine diversity patterns in 121 species of Nymphalidae from the Yucatan Peninsula in Mexico. Our results suggest the presence of cryptic species in 8 of these 121 taxa. As well, the reference database derived from the analysis of adult specimens allowed the identification of nymphalid caterpillars providing new details on host plant use. Methodology/Principal Findings We gathered DNA barcode sequences from 857 adult Nymphalidae representing 121 different species. This total includes four species (Adelpha iphiclus, Adelpha malea, Hamadryas iphtime and Taygetis laches) that were initially overlooked because of their close morphological similarity to other species. The barcode results showed that each of the 121 species possessed a diagnostic array of barcode sequences. In addition, there was evidence of cryptic taxa; seven species included two barcode clusters showing more than 2% sequence divergence while one species included three clusters. All 71 nymphalid caterpillars were identified to a species level by their sequence congruence to adult sequences. These caterpillars represented 16 species, and included Hamadryas julitta, an endemic species from the Yucatan Peninsula whose larval stages and host plant (Dalechampia schottii, also endemic to the Yucatan Peninsula) were previously unknown. Conclusions/Significance This investigation has revealed overlooked species in a well-studied museum collection of nymphalid butterflies and suggests that there is a substantial incidence of cryptic species that await full characterization. The utility of barcoding in the rapid identification of caterpillars also promises to accelerate the assembly of information on life histories, a particularly important advance for hyperdiverse tropical insect assemblages. PMID:22132140

Ginkgo and Welwitschia Mitogenomes Reveal Extreme Contrasts in Gymnosperm Mitochondrial Evolution.

PubMed

Guo, Wenhu; Grewe, Felix; Fan, Weishu; Young, Gregory J; Knoop, Volker; Palmer, Jeffrey D; Mower, Jeffrey P

2016-06-01

Mitochondrial genomes (mitogenomes) of flowering plants are well known for their extreme diversity in size, structure, gene content, and rates of sequence evolution and recombination. In contrast, little is known about mitogenomic diversity and evolution within gymnosperms. Only a single complete genome sequence is available, from the cycad Cycas taitungensis, while limited information is available for the one draft sequence, from Norway spruce (Picea abies). To examine mitogenomic evolution in gymnosperms, we generated complete genome sequences for the ginkgo tree (Ginkgo biloba) and a gnetophyte (Welwitschia mirabilis). There is great disparity in size, sequence conservation, levels of shared DNA, and functional content among gymnosperm mitogenomes. The Cycas and Ginkgo mitogenomes are relatively small, have low substitution rates, and possess numerous genes, introns, and edit sites; we infer that these properties were present in the ancestral seed plant. By contrast, the Welwitschia mitogenome has an expanded size coupled with accelerated substitution rates and extensive loss of these functional features. The Picea genome has expanded further, to more than 4 Mb. With regard to structural evolution, the Cycas and Ginkgo mitogenomes share a remarkable amount of intergenic DNA, which may be related to the limited recombinational activity detected at repeats in Ginkgo Conversely, the Welwitschia mitogenome shares almost no intergenic DNA with any other seed plant. By conducting the first measurements of rates of DNA turnover in seed plant mitogenomes, we discovered that turnover rates vary by orders of magnitude among species. © The Author 2016. 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.

Parasitological Confirmation and Analysis of Leishmania Diversity in Asymptomatic and Subclinical Infection following Resolution of Cutaneous Leishmaniasis.

PubMed

Rosales-Chilama, Mariana; Gongora, Rafael E; Valderrama, Liliana; Jojoa, Jimena; Alexander, Neal; Rubiano, Luisa C; Cossio, Alexandra; Adams, Emily R; Saravia, Nancy G; Gomez, María Adelaida

2015-12-01

The contribution of individuals with subclinical infection to the transmission and endemicity of cutaneous leishmaniasis (CL) is unknown. Immunological evidence of exposure to Leishmania in residents of endemic areas has been the basis for defining the human population with asymptomatic infection. However, parasitological confirmation of subclinical infection is lacking. We investigated the presence and viability of Leishmania in blood and non-invasive mucosal tissue samples from individuals with immunological evidence of subclinical infection in endemic areas for CL caused by Leishmania (Viannia) in Colombia. Detection of Leishmania kDNA was conducted by PCR-Southern Blot, and parasite viability was confirmed by amplification of parasite 7SLRNA gene transcripts. A molecular tool for genetic diversity analysis of parasite populations causing persistent subclinical infection based on PCR amplification and sequence analysis of an 82bp region between kDNA conserved blocks 1 and 2 was developed. Persistent Leishmania infection was demonstrated in 40% (46 of 114) of leishmanin skin test (LST) positive individuals without active disease; parasite viability was established in 59% of these (27 of 46; 24% of total). Parasite burden quantified from circulating blood monocytes, nasal, conjunctival or tonsil mucosal swab samples was comparable, and ranged between 0.2 to 22 parasites per reaction. kDNA sequences were obtained from samples from 2 individuals with asymptomatic infection and from 26 with history of CL, allowing genetic distance analysis that revealed diversity among sequences and clustering within the L. (Viannia) subgenus. Our results provide parasitological confirmation of persistent infection among residents of endemic areas of L. (Viannia) transmission who have experienced asymptomatic infection or recovered from CL, revealing a reservoir of infection that potentially contributes to the endemicity and transmission of disease. kDNA genotyping establishes proof-of-principle of the feasibility of genetic diversity analysis in previously inaccessible and unexplored parasite populations in subclinically infected individuals.

Successful application of FTA Classic Card technology and use of bacteriophage phi29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes.

PubMed

Owor, Betty E; Shepherd, Dionne N; Taylor, Nigel J; Edema, Richard; Monjane, Adérito L; Thomson, Jennifer A; Martin, Darren P; Varsani, Arvind

2007-03-01

Leaf samples from 155 maize streak virus (MSV)-infected maize plants were collected from 155 farmers' fields in 23 districts in Uganda in May/June 2005 by leaf-pressing infected samples onto FTA Classic Cards. Viral DNA was successfully extracted from cards stored at room temperature for 9 months. The diversity of 127 MSV isolates was analysed by PCR-generated RFLPs. Six representative isolates having different RFLP patterns and causing either severe, moderate or mild disease symptoms, were chosen for amplification from FTA cards by bacteriophage phi29 DNA polymerase using the TempliPhi system. Full-length genomes were inserted into a cloning vector using a unique restriction enzyme site, and sequenced. The 1.3-kb PCR product amplified directly from FTA-eluted DNA and used for RFLP analysis was also cloned and sequenced. Comparison of cloned whole genome sequences with those of the original PCR products indicated that the correct virus genome had been cloned and that no errors were introduced by the phi29 polymerase. This is the first successful large-scale application of FTA card technology to the field, and illustrates the ease with which large numbers of infected samples can be collected and stored for downstream molecular applications such as diversity analysis and cloning of potentially new virus genomes.

Metagenomic analyses of the dominant bacterial community in the Fildes Peninsula, King George Island (South Shetland Islands)

NASA Astrophysics Data System (ADS)

Foong, Choon Pin; Wong Vui Ling, Clemente Michael; González, Marcelo

2010-08-01

There is little information on the bacterial diversity of the Fildes Peninsula, King George Island. Hence, this study was conducted to determine the bacterial population of sediments and soils from the lakes, river, glacier and an abandoned oil tank area in the Fildes Peninsula, using a metagenomic approach. DNA was extracted from the sediment and soil samples, and analyzed using the 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). A total of 299 DNA fragments resolved using the DGGE were sequenced. The results of the analysis provided an overview of the predominant groups of bacteria and the diversity of the bacterial communities. The most abundant phyla of bacteria in Fildes Peninsula were Bacteroidetes, Proteobacteria, Acidobacteria, Gemmatimonadetes, Nitrospira, Firmicutes, Actinobacteria, Chloroflexi, Cyanobacteria, Spirochaetes, Deinococcus-Thermus, WS3 and BRC1. All of the sediment samples from the lakes had different representatives of dominant bacterial species. Interestingly, 15% of the operational taxonomic units (OTUs) did not group into any of the existing phyla in the Ribosomal Database Project (RDP). One of the OTUs had a similarity of <0.90 when compared to the GenBank sequences and probably was a novel bacterium specific to that location. The majority of the bacterial 16S rDNA sequences were found to be closely related to those found elsewhere.

Nucleotide variation in the mitochondrial genome provides evidence for dual routes of postglacial recolonization and genetic recombination in the northeastern brook trout (Salvelinus fontinalis).

PubMed

Pilgrim, B L; Perry, R C; Barron, J L; Marshall, H D

2012-09-26

Levels and patterns of mitochondrial DNA (mtDNA) variation were examined to investigate the population structure and possible routes of postglacial recolonization of the world's northernmost native populations of brook trout (Salvelinus fontinalis), which are found in Labrador, Canada. We analyzed the sequence diversity of a 1960-bp portion of the mitochondrial genome (NADH dehydrogenase 1 gene and part of cytochrome oxidase 1) of 126 fish from 32 lakes distributed throughout seven regions of northeastern Canada. These populations were found to have low levels of mtDNA diversity, a characteristic trait of populations at northern extremes, with significant structuring at the level of the watershed. Upon comparison of northeastern brook trout sequences to the publicly available brook trout whole mitochondrial genome (GenBank AF154850), we infer that the GenBank sequence is from a fish whose mtDNA has recombined with that of Arctic charr (S. alpinus). The haplotype distribution provides evidence of two different postglacial founding groups contributing to present-day brook trout populations in the northernmost part of their range; the evolution of the majority of the haplotypes coincides with the timing of glacier retreat from Labrador. Our results exemplify the strong influence that historical processes such as glaciations have had on shaping the current genetic structure of northern species such as the brook trout.

Longitudinal evaluation of humoral immune response and merozoite surface antigen diversity in calves naturally infected with Babesia bovis, in São Paulo, Brazil.

PubMed

Matos, Carlos António; Gonçalves, Luiz Ricardo; Alvarez, Dasiel Obregón; Freschi, Carla Roberta; Silva, Jenevaldo Barbosa da; Val-Moraes, Silvana Pompeia; Mendes, Natalia Serra; André, Marcos Rogério; Machado, Rosangela Zacarias

2017-01-01

Babesiosis is an economically important infectious disease affecting cattle worldwide. In order to longitudinally evaluate the humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis among naturally infected calves in Taiaçu, Brazil, serum and DNA samples from 15 calves were obtained quarterly, from their birth to 12 months of age. Anti-B. bovis IgG antibodies were detected by means of the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). The polymerase chain reaction (PCR) was used to investigate the genetic diversity of B. bovis, based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 2, 4 and 5 sequences of the genes msa-1, msa-2b and msa-2c were obtained. The present study demonstrated that the msa-1 and msa-2b genes sequences amplified from blood DNA of calves positive to B. bovis from Taiaçu were genetically distinct, and that msa-2c was conserved. All animals were serologically positive to ELISA and IFAT, which used full repertoire of parasite antigens in despite of the genetic diversity of MSAs.

Rapid isolation of microsatellite DNAs and identification of polymorphic mitochondrial DNA regions in the fish rotan (Perccottus glenii) invading European Russia

USGS Publications Warehouse

King, Timothy L.; Eackles, Michael S.; Reshetnikov, Andrey N.

2015-01-01

Human-mediated translocations and subsequent large-scale colonization by the invasive fish rotan (Perccottus glenii Dybowski, 1877; Perciformes, Odontobutidae), also known as Amur or Chinese sleeper, has resulted in dramatic transformations of small lentic ecosystems. However, no detailed genetic information exists on population structure, levels of effective movement, or relatedness among geographic populations of P. glenii within the European part of the range. We used massively parallel genomic DNA shotgun sequencing on the semiconductor-based Ion Torrent Personal Genome Machine (PGM) sequencing platform to identify nuclear microsatellite and mitochondrial DNA sequences in P. glenii from European Russia. Here we describe the characterization of nine nuclear microsatellite loci, ascertain levels of allelic diversity, heterozygosity, and demographic status of P. glenii collected from Ilev, Russia, one of several initial introduction points in European Russia. In addition, we mapped sequence reads to the complete P. glenii mitochondrial DNA sequence to identify polymorphic regions. Nuclear microsatellite markers developed for P. glenii yielded sufficient genetic diversity to: (1) produce unique multilocus genotypes; (2) elucidate structure among geographic populations; and (3) provide unique perspectives for analysis of population sizes and historical demographics. Among 4.9 million filtered P. glenii Ion Torrent PGM sequence reads, 11,304 mapped to the mitochondrial genome (NC_020350). This resulted in 100 % coverage of this genome to a mean coverage depth of 102X. A total of 130 variable sites were observed between the publicly available genome from China and the studied composite mitochondrial genome. Among these, 82 were diagnostic and monomorphic between the mitochondrial genomes and distributed among 15 genome regions. The polymorphic sites (N = 48) were distributed among 11 mitochondrial genome regions. Our results also indicate that sequence reads generated from two three-hour runs on the Ion Torrent PGM can generate a sufficient number of nuclear and mitochondrial markers to improve understanding of the evolutionary and ecological dynamics of non-model and in particular, invasive species.

DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

DOE PAGES

None

2014-12-01

The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illuminamore » 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.« less

DNA extraction for streamlined metagenomics of diverse environmental samples.

PubMed

Marotz, Clarisse; Amir, Amnon; Humphrey, Greg; Gaffney, James; Gogul, Grant; Knight, Rob

2017-06-01

A major bottleneck for metagenomic sequencing is rapid and efficient DNA extraction. Here, we compare the extraction efficiencies of three magnetic bead-based platforms (KingFisher, epMotion, and Tecan) to a standardized column-based extraction platform across a variety of sample types, including feces, oral, skin, soil, and water. Replicate sample plates were extracted and prepared for 16S rRNA gene amplicon sequencing in parallel to assess extraction bias and DNA quality. The data demonstrate that any effect of extraction method on sequencing results was small compared with the variability across samples; however, the KingFisher platform produced the largest number of high-quality reads in the shortest amount of time. Based on these results, we have identified an extraction pipeline that dramatically reduces sample processing time without sacrificing bacterial taxonomic or abundance information.

Genome-wide comparison of medieval and modern Mycobacterium leprae.

PubMed

Schuenemann, Verena J; Singh, Pushpendra; Mendum, Thomas A; Krause-Kyora, Ben; Jäger, Günter; Bos, Kirsten I; Herbig, Alexander; Economou, Christos; Benjak, Andrej; Busso, Philippe; Nebel, Almut; Boldsen, Jesper L; Kjellström, Anna; Wu, Huihai; Stewart, Graham R; Taylor, G Michael; Bauer, Peter; Lee, Oona Y-C; Wu, Houdini H T; Minnikin, David E; Besra, Gurdyal S; Tucker, Katie; Roffey, Simon; Sow, Samba O; Cole, Stewart T; Nieselt, Kay; Krause, Johannes

2013-07-12

Leprosy was endemic in Europe until the Middle Ages. Using DNA array capture, we have obtained genome sequences of Mycobacterium leprae from skeletons of five medieval leprosy cases from the United Kingdom, Sweden, and Denmark. In one case, the DNA was so well preserved that full de novo assembly of the ancient bacterial genome could be achieved through shotgun sequencing alone. The ancient M. leprae sequences were compared with those of 11 modern strains, representing diverse genotypes and geographic origins. The comparisons revealed remarkable genomic conservation during the past 1000 years, a European origin for leprosy in the Americas, and the presence of an M. leprae genotype in medieval Europe now commonly associated with the Middle East. The exceptional preservation of M. leprae biomarkers, both DNA and mycolic acids, in ancient skeletons has major implications for palaeomicrobiology and human pathogen evolution.

Selecting Fully-Modified XNA Aptamers Using Synthetic Genetics.

PubMed

Taylor, Alexander I; Holliger, Philipp

2018-06-01

This unit describes the application of "synthetic genetics," i.e., the replication of xeno nucleic acids (XNAs), artificial analogs of DNA and RNA bearing alternative backbone or sugar congeners, to the directed evolution of synthetic oligonucleotide ligands (XNA aptamers) specific for target proteins or nucleic acid motifs, using a cross-chemistry selective exponential enrichment (X-SELEX) approach. Protocols are described for synthesis of diverse-sequence XNA repertoires (typically 10 14 molecules) using DNA templates, isolation and panning for functional XNA sequences using targets immobilized on solid phase or gel shift induced by target binding in solution, and XNA reverse transcription to allow cDNA amplification or sequencing. The method may be generally applied to select fully-modified XNA aptamers specific for a wide range of target molecules. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Genetic diversity and population structure of sickleweed (Falcaria vulgaris; Apiaceae) in the upper Midwest USA

Treesearch

Sarbottam Piya; Madhav P. Nepal; Jack L. Butler; Gary E. Larson; Achal Neupane

2014-01-01

Sickleweed (Falcaria vulgaris), an introduced species native to Europe and Asia, grows as an aggressive weed in some areas of the upper Midwest in the United States. We are reporting genetic diversity and population structure of sickleweed populations using microsatellite markers and nuclear and chloroplast DNA sequences. Populations showed high genetic differentiation...

Diversity of virophages in metagenomic data sets.

PubMed

Zhou, Jinglie; Zhang, Weijia; Yan, Shuling; Xiao, Jinzhou; Zhang, Yuanyuan; Li, Bailin; Pan, Yingjie; Wang, Yongjie

2013-04-01

Virophages, e.g., Sputnik, Mavirus, and Organic Lake virophage (OLV), are unusual parasites of giant double-stranded DNA (dsDNA) viruses, yet little is known about their diversity. Here, we describe the global distribution, abundance, and genetic diversity of virophages based on analyzing and mapping comprehensive metagenomic databases. The results reveal a distinct abundance and worldwide distribution of virophages, involving almost all geographical zones and a variety of unique environments. These environments ranged from deep ocean to inland, iced to hydrothermal lakes, and human gut- to animal-associated habitats. Four complete virophage genomic sequences (Yellowstone Lake virophages [YSLVs]) were obtained, as was one nearly complete sequence (Ace Lake Mavirus [ALM]). The genomes obtained were 27,849 bp long with 26 predicted open reading frames (ORFs) (YSLV1), 23,184 bp with 21 ORFs (YSLV2), 27,050 bp with 23 ORFs (YSLV3), 28,306 bp with 34 ORFs (YSLV4), and 17,767 bp with 22 ORFs (ALM). The homologous counterparts of five genes, including putative FtsK-HerA family DNA packaging ATPase and genes encoding DNA helicase/primase, cysteine protease, major capsid protein (MCP), and minor capsid protein (mCP), were present in all virophages studied thus far. They also shared a conserved gene cluster comprising the two core genes of MCP and mCP. Comparative genomic and phylogenetic analyses showed that YSLVs, having a closer relationship to each other than to the other virophages, were more closely related to OLV than to Sputnik but distantly related to Mavirus and ALM. These findings indicate that virophages appear to be widespread and genetically diverse, with at least 3 major lineages.

DIVERSITY OF BAT-ASSOCIATED LEPTOSPIRA IN THE PERUVIAN AMAZON INFERRED BY BAYESIAN PHYLOGENETIC ANALYSIS OF 16S RIBOSOMAL DNA SEQUENCES

PubMed Central

MATTHIAS, MICHAEL A.; DÍAZ, M. MÓNICA; CAMPOS, KALINA J.; CALDERON, MARITZA; WILLIG, MICHAEL R.; PACHECO, VICTOR; GOTUZZO, EDUARDO; GILMAN, ROBERT H.; VINETZ, JOSEPH M.

2008-01-01

The role of bats as potential sources of transmission to humans or as maintenance hosts of leptospires is poorly understood. We quantified the prevalence of leptospiral colonization in bats in the Peruvian Amazon in the vicinity of Iquitos, an area of high biologic diversity. Of 589 analyzed bats, culture (3 of 589) and molecular evidence (20 of 589) of leptospiral colonization was found in the kidneys, yielding an overall colonization rate of 3.4%. Infection rates differed with habitat and location, and among different bat species. Bayesian analysis was used to infer phylogenic relationships of leptospiral 16S ribosomal DNA sequences. Tree topologies were consistent with groupings based on DNA-DNA hybridization studies. A diverse group of leptospires was found in peri-Iquitos bat populations including Leptospira interrogans (5 clones), L. kirschneri (1), L. borgpetersenii (4), L. fainei (1), and two previously undescribed leptospiral species (8). Although L. kirschenri and L. interrogans have been previously isolated from bats, this report is the first to describe L. borgpetersenii and L. fainei infection of bats. A wild animal reservoir of L. fainei has not been previously described. The detection in bats of the L. interrogans serovar Icterohemorrhagiae, a leptospire typically maintained by peridomestic rats, suggests a rodent-bat infection cycle. Bats in Iquitos maintain a genetically diverse group of leptospires. These results provide a solid basis for pursuing molecular epidemiologic studies of bat-associated Leptospira, a potentially new epidemiologic reservoir of transmission of leptospirosis to humans. PMID:16282313

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

PubMed Central

de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

2014-01-01

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. PMID:24792163

High throughput SNP discovery and genotyping in grapevine (Vitis vinifera L.) by combining a re-sequencing approach and SNPlex technology

PubMed Central

Lijavetzky, Diego; Cabezas, José Antonio; Ibáñez, Ana; Rodríguez, Virginia; Martínez-Zapater, José M

2007-01-01

Background Single-nucleotide polymorphisms (SNPs) are the most abundant type of DNA sequence polymorphisms. Their higher availability and stability when compared to simple sequence repeats (SSRs) provide enhanced possibilities for genetic and breeding applications such as cultivar identification, construction of genetic maps, the assessment of genetic diversity, the detection of genotype/phenotype associations, or marker-assisted breeding. In addition, the efficiency of these activities can be improved thanks to the ease with which SNP genotyping can be automated. Expressed sequence tags (EST) sequencing projects in grapevine are allowing for the in silico detection of multiple putative sequence polymorphisms within and among a reduced number of cultivars. In parallel, the sequence of the grapevine cultivar Pinot Noir is also providing thousands of polymorphisms present in this highly heterozygous genome. Still the general application of those SNPs requires further validation since their use could be restricted to those specific genotypes. Results In order to develop a large SNP set of wide application in grapevine we followed a systematic re-sequencing approach in a group of 11 grape genotypes corresponding to ancient unrelated cultivars as well as wild plants. Using this approach, we have sequenced 230 gene fragments, what represents the analysis of over 1 Mb of grape DNA sequence. This analysis has allowed the discovery of 1573 SNPs with an average of one SNP every 64 bp (one SNP every 47 bp in non-coding regions and every 69 bp in coding regions). Nucleotide diversity in grape (π = 0.0051) was found to be similar to values observed in highly polymorphic plant species such as maize. The average number of haplotypes per gene sequence was estimated as six, with three haplotypes representing over 83% of the analyzed sequences. Short-range linkage disequilibrium (LD) studies within the analyzed sequences indicate the existence of a rapid decay of LD within the selected grapevine genotypes. To validate the use of the detected polymorphisms in genetic mapping, cultivar identification and genetic diversity studies we have used the SNPlex™ genotyping technology in a sample of grapevine genotypes and segregating progenies. Conclusion These results provide accurate values for nucleotide diversity in coding sequences and a first estimate of short-range LD in grapevine. Using SNPlex™ genotyping we have shown the application of a set of discovered SNPs as molecular markers for cultivar identification, linkage mapping and genetic diversity studies. Thus, the combination a highly efficient re-sequencing approach and the SNPlex™ high throughput genotyping technology provide a powerful tool for grapevine genetic analysis. PMID:18021442

[Using IRAP markers for analysis of genetic variability in populations of resource and rare species of plants].

PubMed

Boronnikova, S V; Kalendar', R N

2010-01-01

Species-specific LTR retrotransposons were first cloned in five rare relic species of drug plants located in the Perm' region. Sequences of LTR retrotransposons were used for PCR analysis based on amplification of repeated sequences from LTR or other sites of retrotransposons (IRAP). Genetic diversity was studied in six populations of rare relic species of plants Adonis vernalis L. by means of the IRAP method; 125 polymorphic IRAP-markers were analyzed. Parameters for DNA polymorphism and genetic diversity of A. vernalis populations were determined.

Binding to the DNA Minor Groove by Heterocyclic Dications: From AT Specific Monomers to GC Recognition with Dimers

PubMed Central

Nanjunda, Rupesh; Wilson, W. David

2012-01-01

Compounds that bind in the DNA minor groove have provided critical information on DNA molecular recognition, they have found extensive uses in biotechnology and they are providing clinically useful drugs against diseases as diverse as cancer and sleeping sickness. This review focuses on the development of clinically useful heterocyclic diamidine minor groove binders. These compounds have shown us that the classical model for minor groove binding in AT DNA sequences must be expanded in several ways: compounds with nonstandard shapes can bind strongly to the groove, water can be directly incorporated into the minor groove complex in an interfacial interaction, and the compounds can form cooperative stacked dimers to recognize GC and mixed AT/GC base pair sequences. PMID:23255206

DNA sequence-directed shape change of photopatterned hydrogels via high-degree swelling

NASA Astrophysics Data System (ADS)

Cangialosi, Angelo; Yoon, ChangKyu; Liu, Jiayu; Huang, Qi; Guo, Jingkai; Nguyen, Thao D.; Gracias, David H.; Schulman, Rebecca

2017-09-01

Shape-changing hydrogels that can bend, twist, or actuate in response to external stimuli are critical to soft robots, programmable matter, and smart medicine. Shape change in hydrogels has been induced by global cues, including temperature, light, or pH. Here we demonstrate that specific DNA molecules can induce 100-fold volumetric hydrogel expansion by successive extension of cross-links. We photopattern up to centimeter-sized gels containing multiple domains that undergo different shape changes in response to different DNA sequences. Experiments and simulations suggest a simple design rule for controlled shape change. Because DNA molecules can be coupled to molecular sensors, amplifiers, and logic circuits, this strategy introduces the possibility of building soft devices that respond to diverse biochemical inputs and autonomously implement chemical control programs.

Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments

PubMed Central

Hurt, Richard A.; Robeson, Michael S.; Shakya, Migun; Moberly, James G.; Vishnivetskaya, Tatiana A.; Gu, Baohua; Elias, Dwayne A.

2014-01-01

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N2 grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered. PMID:25033199

Genome Calligrapher: A Web Tool for Refactoring Bacterial Genome Sequences for de Novo DNA Synthesis.

PubMed

Christen, Matthias; Deutsch, Samuel; Christen, Beat

2015-08-21

Recent advances in synthetic biology have resulted in an increasing demand for the de novo synthesis of large-scale DNA constructs. Any process improvement that enables fast and cost-effective streamlining of digitized genetic information into fabricable DNA sequences holds great promise to study, mine, and engineer genomes. Here, we present Genome Calligrapher, a computer-aided design web tool intended for whole genome refactoring of bacterial chromosomes for de novo DNA synthesis. By applying a neutral recoding algorithm, Genome Calligrapher optimizes GC content and removes obstructive DNA features known to interfere with the synthesis of double-stranded DNA and the higher order assembly into large DNA constructs. Subsequent bioinformatics analysis revealed that synthesis constraints are prevalent among bacterial genomes. However, a low level of codon replacement is sufficient for refactoring bacterial genomes into easy-to-synthesize DNA sequences. To test the algorithm, 168 kb of synthetic DNA comprising approximately 20 percent of the synthetic essential genome of the cell-cycle bacterium Caulobacter crescentus was streamlined and then ordered from a commercial supplier of low-cost de novo DNA synthesis. The successful assembly into eight 20 kb segments indicates that Genome Calligrapher algorithm can be efficiently used to refactor difficult-to-synthesize DNA. Genome Calligrapher is broadly applicable to recode biosynthetic pathways, DNA sequences, and whole bacterial genomes, thus offering new opportunities to use synthetic biology tools to explore the functionality of microbial diversity. The Genome Calligrapher web tool can be accessed at https://christenlab.ethz.ch/GenomeCalligrapher  .

Molecular analysis of a 11 700-year-old rodent midden from the Atacama Desert, Chile

USGS Publications Warehouse

Kuch, M.; Rohland, N.; Betancourt, J.L.; Latorre, C.; Steppan, S.; Poinar, H.N.

2002-01-01

DNA was extracted from an 11 700-year-old rodent midden from the Atacama Desert, Chile and the chloroplast and animal mitochondrial DNA (mtDNA) gene sequences were analysed to investigate the floral environment surrounding the midden, and the identity of the midden agent. The plant sequences, together with the macroscopic identifications, suggest the presence of 13 plant families and three orders that no longer exist today at the midden locality, and thus point to a much more diverse and humid climate 11 700 years ago. The mtDNA sequences suggest the presence of at least four different vertebrates, which have been putatively identified as a camelid (vicuna), two rodents (Phyllotis and Abrocoma), and a cardinal bird (Passeriformes). To identify the midden agent, DNA was extracted from pooled faecal pellets, three small overlapping fragments of the mitochondrial cytochrome b gene were amplified and multiple clones were sequenced. These results were analysed along with complete cytochrome b sequences for several modern Phyllotis species to place the midden sequence phylogenetically. The results identified the midden agent as belonging to an ancestral P. limatus. Today, P. limatus is not found at the midden locality but it can be found 100 km to the north, indicating at least a small range shift. The more extensive sampling of modern Phyllotis reinforces the suggestion that P. limatus is recently derived from a peripheral isolate.

Phylogenetic Position of a Copper Age Sheep (Ovis aries) Mitochondrial DNA

PubMed Central

Olivieri, Cristina; Ermini, Luca; Rizzi, Ermanno; Corti, Giorgio; Luciani, Stefania; Marota, Isolina; De Bellis, Gianluca; Rollo, Franco

2012-01-01

Background Sheep (Ovis aries) were domesticated in the Fertile Crescent region about 9,000-8,000 years ago. Currently, few mitochondrial (mt) DNA studies are available on archaeological sheep. In particular, no data on archaeological European sheep are available. Methodology/Principal Findings Here we describe the first portion of mtDNA sequence of a Copper Age European sheep. DNA was extracted from hair shafts which were part of the clothes of the so-called Tyrolean Iceman or Ötzi (5,350 - 5,100 years before present). Mitochondrial DNA (a total of 2,429 base pairs, encompassing a portion of the control region, tRNAPhe, a portion of the 12S rRNA gene, and the whole cytochrome B gene) was sequenced using a mixed sequencing procedure based on PCR amplification and 454 sequencing of pooled amplification products. We have compared the sequence with the corresponding sequence of 334 extant lineages. Conclusions/Significance A phylogenetic network based on a new cladistic notation for the mitochondrial diversity of domestic sheep shows that the Ötzi's sheep falls within haplogroup B, thus demonstrating that sheep belonging to this haplogroup were already present in the Alps more than 5,000 years ago. On the other hand, the lineage of the Ötzi's sheep is defined by two transitions (16147, and 16440) which, assembled together, define a motif that has not yet been identified in modern sheep populations. PMID:22457789

A DNA Barcode Library for North American Pyraustinae (Lepidoptera: Pyraloidea: Crambidae).

PubMed

Yang, Zhaofu; Landry, Jean-François; Hebert, Paul D N

2016-01-01

Although members of the crambid subfamily Pyraustinae are frequently important crop pests, their identification is often difficult because many species lack conspicuous diagnostic morphological characters. DNA barcoding employs sequence diversity in a short standardized gene region to facilitate specimen identifications and species discovery. This study provides a DNA barcode reference library for North American pyraustines based upon the analysis of 1589 sequences recovered from 137 nominal species, 87% of the fauna. Data from 125 species were barcode compliant (>500bp, <1% n), and 99 of these taxa formed a distinct cluster that was assigned to a single BIN. The other 26 species were assigned to 56 BINs, reflecting frequent cases of deep intraspecific sequence divergence and a few instances of barcode sharing, creating a total of 155 BINs. Two systems for OTU designation, ABGD and BIN, were examined to check the correspondence between current taxonomy and sequence clusters. The BIN system performed better than ABGD in delimiting closely related species, while OTU counts with ABGD were influenced by the value employed for relative gap width. Different species with low or no interspecific divergence may represent cases of unrecognized synonymy, whereas those with high intraspecific divergence require further taxonomic scrutiny as they may involve cryptic diversity. The barcode library developed in this study will also help to advance understanding of relationships among species of Pyraustinae.

DNA breathing dynamics distinguish binding from nonbinding consensus sites for transcription factor YY1 in cells.

PubMed

Alexandrov, Boian S; Fukuyo, Yayoi; Lange, Martin; Horikoshi, Nobuo; Gelev, Vladimir; Rasmussen, Kim Ø; Bishop, Alan R; Usheva, Anny

2012-11-01

The genome-wide mapping of the major gene expression regulators, the transcription factors (TFs) and their DNA binding sites, is of great importance for describing cellular behavior and phenotypic diversity. Presently, the methods for prediction of genomic TF binding produce a large number of false positives, most likely due to insufficient description of the physiochemical mechanisms of protein-DNA binding. Growing evidence suggests that, in the cell, the double-stranded DNA (dsDNA) is subject to local transient strands separations (breathing) that contribute to genomic functions. By using site-specific chromatin immunopecipitations, gel shifts, BIOBASE data, and our model that accurately describes the melting behavior and breathing dynamics of dsDNA we report a specific DNA breathing profile found at YY1 binding sites in cells. We find that the genomic flanking sequence variations and SNPs, may exert long-range effects on DNA dynamics and predetermine YY1 binding. The ubiquitous TF YY1 has a fundamental role in essential biological processes by activating, initiating or repressing transcription depending upon the sequence context it binds. We anticipate that consensus binding sequences together with the related DNA dynamics profile may significantly improve the accuracy of genomic TF binding sites and TF binding-related functional SNPs.

Sources of PCR-induced distortions in high-throughput sequencing data sets

PubMed Central

Kebschull, Justus M.; Zador, Anthony M.

2015-01-01

PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error—bias, stochasticity, template switches and polymerase errors—on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules. PMID:26187991

Oligonucleotide fingerprinting of rRNA genes for analysis of fungal community composition.

PubMed

Valinsky, Lea; Della Vedova, Gianluca; Jiang, Tao; Borneman, James

2002-12-01

Thorough assessments of fungal diversity are currently hindered by technological limitations. Here we describe a new method for identifying fungi, oligonucleotide fingerprinting of rRNA genes (OFRG). ORFG sorts arrayed rRNA gene (ribosomal DNA [rDNA]) clones into taxonomic clusters through a series of hybridization experiments, each using a single oligonucleotide probe. A simulated annealing algorithm was used to design an OFRG probe set for fungal rDNA. Analysis of 1,536 fungal rDNA clones derived from soil generated 455 clusters. A pairwise sequence analysis showed that clones with average sequence identities of 99.2% were grouped into the same cluster. To examine the accuracy of the taxonomic identities produced by this OFRG experiment, we determined the nucleotide sequences for 117 clones distributed throughout the tree. For all but two of these clones, the taxonomic identities generated by this OFRG experiment were consistent with those generated by a nucleotide sequence analysis. Eighty-eight percent of the clones were affiliated with Ascomycota, while 12% belonged to BASIDIOMYCOTA: A large fraction of the clones were affiliated with the genera Fusarium (404 clones) and Raciborskiomyces (176 clones). Smaller assemblages of clones had high sequence identities to the Alternaria, Ascobolus, Chaetomium, Cryptococcus, and Rhizoctonia clades.

Caught in the act: the lifetime of synaptic intermediates during the search for homology on DNA

PubMed Central

Mani, Adam; Braslavsky, Ido; Arbel-Goren, Rinat; Stavans, Joel

2010-01-01

Homologous recombination plays pivotal roles in DNA repair and in the generation of genetic diversity. To locate homologous target sequences at which strand exchange can occur within a timescale that a cell’s biology demands, a single-stranded DNA-recombinase complex must search among a large number of sequences on a genome by forming synapses with chromosomal segments of DNA. A key element in the search is the time it takes for the two sequences of DNA to be compared, i.e. the synapse lifetime. Here, we visualize for the first time fluorescently tagged individual synapses formed by RecA, a prokaryotic recombinase, and measure their lifetime as a function of synapse length and differences in sequence between the participating DNAs. Surprisingly, lifetimes can be ∼10 s long when the DNAs are fully heterologous, and much longer for partial homology, consistently with ensemble FRET measurements. Synapse lifetime increases rapidly as the length of a region of full homology at either the 3′- or 5′-ends of the invading single-stranded DNA increases above 30 bases. A few mismatches can reduce dramatically the lifetime of synapses formed with nearly homologous DNAs. These results suggest the need for facilitated homology search mechanisms to locate homology successfully within the timescales observed in vivo. PMID:20044347

Genetic Diversity and Phylogenetic Evolution of Tibetan Sheep Based on mtDNA D-Loop Sequences

PubMed Central

Yue, Yaojing; Guo, Xian; Guo, Tingting; Chu, Min; Wang, Fan; Han, Jilong; Feng, Ruilin; Sun, Xiaoping; Niu, Chune; Yang, Bohui; Guo, Jian; Yuan, Chao

2016-01-01

The molecular and population genetic evidence of the phylogenetic status of the Tibetan sheep (Ovis aries) is not well understood, and little is known about this species’ genetic diversity. This knowledge gap is partly due to the difficulty of sample collection. This is the first work to address this question. Here, the genetic diversity and phylogenetic relationship of 636 individual Tibetan sheep from fifteen populations were assessed using 642 complete sequences of the mitochondrial DNA D-loop. Samples were collected from the Qinghai-Tibetan Plateau area in China, and reference data were obtained from the six reference breed sequences available in GenBank. The length of the sequences varied considerably, between 1031 and 1259 bp. The haplotype diversity and nucleotide diversity were 0.992±0.010 and 0.019±0.001, respectively. The average number of nucleotide differences was 19.635. The mean nucleotide composition of the 350 haplotypes was 32.961% A, 29.708% T, 22.892% C, 14.439% G, 62.669% A+T, and 37.331% G+C. Phylogenetic analysis showed that all four previously defined haplogroups (A, B, C, and D) were found in the 636 individuals of the fifteen Tibetan sheep populations but that only the D haplogroup was found in Linzhou sheep. Further, the clustering analysis divided the fifteen Tibetan sheep populations into at least two clusters. The estimation of the demographic parameters from the mismatch analyses showed that haplogroups A, B, and C had at least one demographic expansion in Tibetan sheep. These results contribute to the knowledge of Tibetan sheep populations and will help inform future conservation programs about the Tibetan sheep native to the Qinghai-Tibetan Plateau. PMID:27463976

Eukaryotic diversity in premise drinking water using 18S rDNA sequencing: implications for health risks

EPA Science Inventory

The goal of this study was to characterize microbial eukaryotes over a 12 month period, so as to provide insight into the occurrence of potentially important predators and bacterial hosts in hot and cold premise plumbing. Nearly 6,300 partial (600 bp) 18S rRNA gene sequences from...

Genetic variation patterns of American chestnut populations at EST-SSRs

Treesearch

Oliver Gailing; C. Dana Nelson

2017-01-01

The objective of this study is to analyze patterns of genetic variation at genic expressed sequence tag - simple sequence repeats (EST-SSRs) and at chloroplast DNA markers in populations of American chestnut (Castanea dentata Borkh.) to assist in conservation and breeding efforts. Allelic diversity at EST-SSRs decreased significantly from southwest to northeast along...

Analytical Framework for Identifying and Differentiating Recent Hitchhiking and Severe Bottleneck Effects from Multi-Locus DNA Sequence Data

DOE PAGES

Sargsyan, Ori

2012-05-25

Hitchhiking and severe bottleneck effects have impact on the dynamics of genetic diversity of a population by inducing homogenization at a single locus and at the genome-wide scale, respectively. As a result, identification and differentiation of the signatures of such events from DNA sequence data at a single locus is challenging. This study develops an analytical framework for identifying and differentiating recent homogenization events at multiple neutral loci in low recombination regions. The dynamics of genetic diversity at a locus after a recent homogenization event is modeled according to the infinite-sites mutation model and the Wright-Fisher model of reproduction withmore » constant population size. In this setting, I derive analytical expressions for the distribution, mean, and variance of the number of polymorphic sites in a random sample of DNA sequences from a locus affected by a recent homogenization event. Based on this framework, three likelihood-ratio based tests are presented for identifying and differentiating recent homogenization events at multiple loci. Lastly, I apply the framework to two data sets. First, I consider human DNA sequences from four non-coding loci on different chromosomes for inferring evolutionary history of modern human populations. The results suggest, in particular, that recent homogenization events at the loci are identifiable when the effective human population size is 50000 or greater in contrast to 10000, and the estimates of the recent homogenization events are agree with the “Out of Africa” hypothesis. Second, I use HIV DNA sequences from HIV-1-infected patients to infer the times of HIV seroconversions. The estimates are contrasted with other estimates derived as the mid-time point between the last HIV-negative and first HIV-positive screening tests. Finally, the results show that significant discrepancies can exist between the estimates.« less

Diversity of environmental single-stranded DNA phages revealed by PCR amplification of the partial major capsid protein

PubMed Central

Hopkins, Max; Kailasan, Shweta; Cohen, Allison; Roux, Simon; Tucker, Kimberly Pause; Shevenell, Amelia; Agbandje-McKenna, Mavis; Breitbart, Mya

2014-01-01

The small single-stranded DNA (ssDNA) bacteriophages of the subfamily Gokushovirinae were traditionally perceived as narrowly targeted, niche-specific viruses infecting obligate parasitic bacteria, such as Chlamydia. The advent of metagenomics revealed gokushoviruses to be widespread in global environmental samples. This study expands knowledge of gokushovirus diversity in the environment by developing a degenerate PCR assay to amplify a portion of the major capsid protein (MCP) gene of gokushoviruses. Over 500 amplicons were sequenced from 10 environmental samples (sediments, sewage, seawater and freshwater), revealing the ubiquity and high diversity of this understudied phage group. Residue-level conservation data generated from multiple alignments was combined with a predicted 3D structure, revealing a tendency for structurally internal residues to be more highly conserved than surface-presenting protein–protein or viral–host interaction domains. Aggregating this data set into a phylogenetic framework, many gokushovirus MCP clades contained samples from multiple environments, although distinct clades dominated the different samples. Antarctic sediment samples contained the most diverse gokushovirus communities, whereas freshwater springs from Florida were the least diverse. Whether the observed diversity is being driven by environmental factors or host-binding interactions remains an open question. The high environmental diversity of this previously overlooked ssDNA viral group necessitates further research elucidating their natural hosts and exploring their ecological roles. PMID:24694711

Assessing Date Palm Genetic Diversity Using Different Molecular Markers.

PubMed

Atia, Mohamed A M; Sakr, Mahmoud M; Adawy, Sami S

2017-01-01

Molecular marker technologies which rely on DNA analysis provide powerful tools to assess biodiversity at different levels, i.e., among and within species. A range of different molecular marker techniques have been developed and extensively applied for detecting variability in date palm at the DNA level. Recently, the employment of gene-targeting molecular marker approaches to study biodiversity and genetic variations in many plant species has increased the attention of researchers interested in date palm to carry out phylogenetic studies using these novel marker systems. Molecular markers are good indicators of genetic distances among accessions, because DNA-based markers are neutral in the face of selection. Here we describe the employment of multidisciplinary molecular marker approaches: amplified fragment length polymorphism (AFLP), start codon targeted (SCoT) polymorphism, conserved DNA-derived polymorphism (CDDP), intron-targeted amplified polymorphism (ITAP), simple sequence repeats (SSR), and random amplified polymorphic DNA (RAPD) to assess genetic diversity in date palm.

Identifications of captive and wild tilapia species existing in Hawaii by mitochondrial DNA control region sequence.

PubMed

Wu, Liang; Yang, Jinzeng

2012-01-01

The tilapia family of the Cichlidae includes many fish species, which live in freshwater and saltwater environments. Several species, such as O. niloticus, O. aureus, and O. mossambicus, are excellent for aquaculture because these fish are easily reproduced and readily adapt to diverse environments. Historically, tilapia species, including O. mossambicus, S. melanotheron, and O. aureus, were introduced to Hawaii many decades ago, and the state of Hawaii uses the import permit policy to prevent O. niloticus from coming into the islands. However, hybrids produced from O. niloticus may already be present in the freshwater and marine environments of the islands. The purpose of this study was to identify tilapia species that exist in Hawaii using mitochondrial DNA analysis. In this study, we analyzed 382 samples collected from 13 farm (captive) and wild tilapia populations in Oahu and the Hawaii Islands. Comparison of intraspecies variation between the mitochondrial DNA control region (mtDNA CR) and cytochrome c oxidase I (COI) gene from five populations indicated that mtDNA CR had higher nucleotide diversity than COI. A phylogenetic tree of all sampled tilapia was generated using mtDNA CR sequences. The neighbor-joining tree analysis identified seven distinctive tilapia species: O. aureus, O. mossambicus, O. niloticus, S. melanotheron, O. urolepies, T. redalli, and a hybrid of O. massambicus and O. niloticus. Of all the populations examined, 10 populations consisting of O. aureus, O. mossambicus, O. urolepis, and O. niloticus from the farmed sites were relatively pure, whereas three wild populations showed some degree of introgression and hybridization. This DNA-based tilapia species identification is the first report that confirmed tilapia species identities in the wild and captive populations in Hawaii. The DNA sequence comparisons of mtDNA CR appear to be a valid method for tilapia species identification. The suspected tilapia hybrids that consist of O. niloticus are present in captive and wild populations in Hawaii.

Identifications of Captive and Wild Tilapia Species Existing in Hawaii by Mitochondrial DNA Control Region Sequence

PubMed Central

Wu, Liang; Yang, Jinzeng

2012-01-01

Background The tilapia family of the Cichlidae includes many fish species, which live in freshwater and saltwater environments. Several species, such as O. niloticus, O. aureus, and O. mossambicus, are excellent for aquaculture because these fish are easily reproduced and readily adapt to diverse environments. Historically, tilapia species, including O. mossambicus, S. melanotheron, and O. aureus, were introduced to Hawaii many decades ago, and the state of Hawaii uses the import permit policy to prevent O. niloticus from coming into the islands. However, hybrids produced from O. niloticus may already be present in the freshwater and marine environments of the islands. The purpose of this study was to identify tilapia species that exist in Hawaii using mitochondrial DNA analysis. Methodology/Principal Findings In this study, we analyzed 382 samples collected from 13 farm (captive) and wild tilapia populations in Oahu and the Hawaii Islands. Comparison of intraspecies variation between the mitochondrial DNA control region (mtDNA CR) and cytochrome c oxidase I (COI) gene from five populations indicated that mtDNA CR had higher nucleotide diversity than COI. A phylogenetic tree of all sampled tilapia was generated using mtDNA CR sequences. The neighbor-joining tree analysis identified seven distinctive tilapia species: O. aureus, O. mossambicus, O. niloticus, S. melanotheron, O. urolepies, T. redalli, and a hybrid of O. massambicus and O. niloticus. Of all the populations examined, 10 populations consisting of O. aureus, O. mossambicus, O. urolepis, and O. niloticus from the farmed sites were relatively pure, whereas three wild populations showed some degree of introgression and hybridization. Conclusions/Significance This DNA-based tilapia species identification is the first report that confirmed tilapia species identities in the wild and captive populations in Hawaii. The DNA sequence comparisons of mtDNA CR appear to be a valid method for tilapia species identification. The suspected tilapia hybrids that consist of O. niloticus are present in captive and wild populations in Hawaii. PMID:23251613

Using mitochondrial DNA to test the hypothesis of a European post-glacial human recolonization from the Franco-Cantabrian refuge.

PubMed

García, O; Fregel, R; Larruga, J M; Álvarez, V; Yurrebaso, I; Cabrera, V M; González, A M

2011-01-01

It has been proposed that the distribution patterns and coalescence ages found in Europeans for mitochondrial DNA (mtDNA) haplogroups V, H1 and H3 are the result of a post-glacial expansion from a Franco-Cantabrian refuge that recolonized central and northern areas. In contrast, in this refined mtDNA study of the Cantabrian Cornice that contributes 413 partial and 9 complete new mtDNA sequences, including a large Basque sample and a sample of Asturians, no experimental evidence was found to support the human refuge-expansion theory. In fact, all measures of gene diversity point to the Cantabrian Cornice in general and the Basques in particular, as less polymorphic for V, H1 and H3 than other southern regions in Iberia or in Central Europe. Genetic distances show the Cantabrian Cornice is a very heterogeneous region with significant local differences. The analysis of several minor subhaplogroups, based on complete sequences, also suggests different focal expansions over a local and peninsular range that did not affect continental Europe. Furthermore, all detected clinal trends show stronger longitudinal than latitudinal profiles. In Northern Iberia, it seems that the highest diversity values for some haplogroups with Mesolithic coalescence ages are centred on the Mediterranean side, including Catalonia and South-eastern France.

Diversity, Bacterial Symbionts and Antibacterial Potential of Gut-Associated Fungi Isolated from the Pantala flavescens Larvae in China

PubMed Central

Shao, Ming-Wei; Lu, Yi-Hui; Miao, Shuang; Zhang, Yun; Chen, Ting-Ting; Zhang, Ying-Lao

2015-01-01

The diversity of fungi associated with the gut of Pantala flavescens larvae was investigated using a culture-dependent method and molecular identification based on an analysis of the internally transcribed spacer sequence. In total, 48 fungal isolates were obtained from P. flavescens larvae. Based on phylogenetic analyses, the fungal isolates were grouped in 5 classes and 12 different genera. Fourteen bacterial 16S rDNA sequences derived from total genomic DNA extractions of fungal mycelia were obtained. The majority of the sequences were associated with Proteobacteria (13/14), and one Bacillaceae (1/14) was included. Leclercia sp., Oceanobacillus oncorhynchi and Methylobacterium extorquens, were reported for the first time as bacterial endosymbionts in fungi. High-performance liquid chromatography (HPLC) analysis indicated that bacterial symbionts produced specific metabolites and also exerted an inhibitory effect on fungal metabolites. The biological activity of the fungal culture extracts against the pathogenic bacteria Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 8739) was investigated, and 20 extracts (42%) exhibited antibacterial activity against at least one of the tested bacterial strains. This study is the first report on the diversity and antibacterial activity of symbiotic fungi residing in the gut of P. flavescens larvae, and the results show that these fungi are highly diverse and could be exploited as a potential source of bioactive compounds. PMID:26221957

Diversity, Bacterial Symbionts and Antibacterial Potential of Gut-Associated Fungi Isolated from the Pantala flavescens Larvae in China.

PubMed

Shao, Ming-Wei; Lu, Yi-Hui; Miao, Shuang; Zhang, Yun; Chen, Ting-Ting; Zhang, Ying-Lao

2015-01-01

The diversity of fungi associated with the gut of Pantala flavescens larvae was investigated using a culture-dependent method and molecular identification based on an analysis of the internally transcribed spacer sequence. In total, 48 fungal isolates were obtained from P. flavescens larvae. Based on phylogenetic analyses, the fungal isolates were grouped in 5 classes and 12 different genera. Fourteen bacterial 16S rDNA sequences derived from total genomic DNA extractions of fungal mycelia were obtained. The majority of the sequences were associated with Proteobacteria (13/14), and one Bacillaceae (1/14) was included. Leclercia sp., Oceanobacillus oncorhynchi and Methylobacterium extorquens, were reported for the first time as bacterial endosymbionts in fungi. High-performance liquid chromatography (HPLC) analysis indicated that bacterial symbionts produced specific metabolites and also exerted an inhibitory effect on fungal metabolites. The biological activity of the fungal culture extracts against the pathogenic bacteria Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 8739) was investigated, and 20 extracts (42%) exhibited antibacterial activity against at least one of the tested bacterial strains. This study is the first report on the diversity and antibacterial activity of symbiotic fungi residing in the gut of P. flavescens larvae, and the results show that these fungi are highly diverse and could be exploited as a potential source of bioactive compounds.

Characterisation of culture-independent and -dependent microbial communities in a high-temperature offshore chalk petroleum reservoir.

PubMed

Kaster, Krista M; Bonaunet, Kristin; Berland, Harald; Kjeilen-Eilertsen, Grethe; Brakstad, Odd Gunnar

2009-11-01

Recent studies have indicated that oil reservoirs harbour diverse microbial communities. Culture-dependent and culture-independent methods were used to evaluate the microbial diversity in produced water samples of the Ekofisk oil field, a high temperature, and fractured chalk reservoir in the North Sea. DGGE analyses of 16S rRNA gene fragments were used to assess the microbial diversity of both archaeal and bacterial communities in produced water samples and enrichment cultures from 4 different wells (B-08, X-08, X-18 and X-25). Low diversity communities were found when 16S rDNA libraries of bacterial and archaeal assemblages were generated from total community DNA obtained from produced water samples and enrichment cultures. Sequence analysis of the clones indicated close matches to microbes associated with high-temperature oil reservoirs or other similar environments. Sequences were found to be similar to members of the genera Thermotoga, Caminicella, Thermoanaerobacter, Archaeoglobus, Thermococcus, and Methanobulbus. Enrichment cultures obtained from the produced water samples were dominated by sheathed rods. Sequence analyses of the cultures indicated predominance of the genera Petrotoga, Arcobacter, Archaeoglobus and Thermococcus. The communities of both produced water and enrichment cultures appeared to be dominated by thermophilic fermenters capable of reducing sulphur compounds. These results suggest that the biochemical processes in the Ekofisk chalk reservoir are similar to those observed in high-temperature sandstone reservoirs.

Diversity of Micromonospora strains isolated from nitrogen fixing nodules and rhizosphere of Pisum sativum analyzed by multilocus sequence analysis.

PubMed

Carro, Lorena; Spröer, Cathrin; Alonso, Pilar; Trujillo, Martha E

2012-03-01

It was recently reported that Micromonospora inhabits the intracellular tissues of nitrogen fixing nodules of the wild legume Lupinus angustifolius. To determine if Micromonospora populations are also present in nitrogen fixing nodules of cultivated legumes such as Pisum sativum, we carried out the isolation of this actinobacterium from P. sativum plants collected in two man-managed fields in the region of Castilla and León (Spain). In this work, we describe the isolation of 93 Micromonospora strains recovered from nitrogen fixing nodules and the rhizosphere of P. sativum. The genomic diversity of the strains was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). Forty-six isolates and 34 reference strains were further analyzed using a multilocus sequence analysis scheme developed to address the phylogeny of the genus Micromonospora and to evaluate the species distribution in the two studied habitats. The MLSA results were evaluated by DNA-DNA hybridization to determine their usefulness for the delineation of Micromonospora at the species level. In most cases, DDH values below 70% were obtained with strains that shared a sequence similarity of 98.5% or less. Thus, MLSA studies clearly supported the established taxonomy of the genus Micromonospora and indicated that genomic species could be delineated as groups of strains that share > 98.5% sequence similarity based on the 5 genes selected. The species diversity of the strains isolated from both the rhizosphere and nodules was very high and in many cases the new strains could not be related to any of the currently described species. Copyright © 2011 Elsevier GmbH. All rights reserved.

Genetic diversity of Leishmania donovani that causes cutaneous leishmaniasis in Sri Lanka: a cross sectional study with regional comparisons.

PubMed

Kariyawasam, Udeshika Lakmini; Selvapandiyan, Angamuthu; Rai, Keshav; Wani, Tasaduq Hussain; Ahuja, Kavita; Beg, Mizra Adil; Premathilake, Hasitha Upendra; Bhattarai, Narayan Raj; Siriwardena, Yamuna Deepani; Zhong, Daibin; Zhou, Guofa; Rijal, Suman; Nakhasi, Hira; Karunaweera, Nadira D

2017-12-22

Leishmania donovani is the etiological agent of visceral leishmaniasis (VL) in the Indian subcontinent. However, it is also known to cause cutaneous leishmaniasis (CL) in Sri Lanka. Sri Lankan L. donovani differs from other L. donovani strains, both at the molecular and biochemical level. To investigate the different species or strain-specific differences of L. donovani in Sri Lanka we evaluated sequence variation of the kinetoplastid DNA (kDNA). Parasites isolated from skin lesions of 34 CL patients and bone marrow aspirates from 4 VL patients were genotyped using the kDNA minicircle PCR analysis. A total of 301 minicircle sequences that included sequences from Sri Lanka, India, Nepal and six reference species of Leishmania were analyzed. Haplotype diversity of Sri Lankan isolates were high (H d  = 0.757) with strong inter-geographical genetic differentiation (F ST  > 0.25). In this study, L. donovani isolates clustered according to their geographic origin, while Sri Lankan isolates formed a separate cluster and were clearly distinct from other Leishmania species. Within the Sri Lankan group, there were three distinct sub-clusters formed, from CL patients who responded to standard antimony therapy, CL patients who responded poorly to antimony therapy and from VL patients. There was no specific clustering of sequences based on geographical origin within Sri Lanka. This study reveals high levels of haplotype diversity of L. donovani in Sri Lanka with a distinct genetic association with clinically relevant phenotypic characteristics. The use of genetic tools to identify clinically relevant features of Leishmania parasites has important therapeutic implications for leishmaniasis.

How good are indirect tests at detecting recombination in human mtDNA?

PubMed

White, Daniel James; Bryant, David; Gemmell, Neil John

2013-07-08

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D' and r(2), Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ(2)) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7-70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed.

Probing the electrostatics and pharmacologic modulation of sequence-specific binding by the DNA-binding domain of the ETS-family transcription factor PU.1: a binding affinity and kinetics investigation

PubMed Central

Munde, Manoj; Poon, Gregory M. K.; Wilson, W. David

2013-01-01

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally-conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤105 M−1 s−1), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt-dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>107 M−1 s−1). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes. PMID:23416556

How Good Are Indirect Tests at Detecting Recombination in Human mtDNA?

PubMed Central

White, Daniel James; Bryant, David; Gemmell, Neil John

2013-01-01

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D′ and r2, Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ2) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7−70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed. PMID:23665874

Integrative Clinical Genomics of Metastatic Cancer

PubMed Central

Robinson, Dan R.; Wu, Yi-Mi; Lonigro, Robert J.; Vats, Pankaj; Cobain, Erin; Everett, Jessica; Cao, Xuhong; Rabban, Erica; Kumar-Sinha, Chandan; Raymond, Victoria; Schuetze, Scott; Alva, Ajjai; Siddiqui, Javed; Chugh, Rashmi; Worden, Francis; Zalupski, Mark M.; Innis, Jeffrey; Mody, Rajen J.; Tomlins, Scott A.; Lucas, David; Baker, Laurence H.; Ramnath, Nithya; Schott, Ann F.; Hayes, Daniel F.; Vijai, Joseph; Offit, Kenneth; Stoffel, Elena M.; Roberts, J. Scott; Smith, David C.; Kunju, Lakshmi P.; Talpaz, Moshe; Cieslik, Marcin; Chinnaiyan, Arul M.

2017-01-01

SUMMARY Metastasis is the primary cause of cancer-related deaths. While The Cancer Genome Atlas (TCGA) has sequenced primary tumor types obtained from surgical resections, much less comprehensive molecular analysis is available from clinically acquired metastatic cancers. Here, we perform whole exome and transcriptome sequencing of 500 adult patients with metastatic solid tumors of diverse lineage and biopsy site. The most prevalent genes somatically altered in metastatic cancer included TP53, CDKN2A, PTEN, PIK3CA, and RB1. Putative pathogenic germline variants were present in 12.2% of cases of which 75% were related to defects in DNA repair. RNA sequencing complemented DNA sequencing for the identification of gene fusions, pathway activation, and immune profiling. Integrative sequence analysis provides a clinically relevant, multi-dimensional view of the complex molecular landscape and microenvironment of metastatic cancers. PMID:28783718

Accessing the Soil Metagenome for Studies of Microbial Diversity▿ †

PubMed Central

Delmont, Tom O.; Robe, Patrick; Cecillon, Sébastien; Clark, Ian M.; Constancias, Florentin; Simonet, Pascal; Hirsch, Penny R.; Vogel, Timothy M.

2011-01-01

Soil microbial communities contain the highest level of prokaryotic diversity of any environment, and metagenomic approaches involving the extraction of DNA from soil can improve our access to these communities. Most analyses of soil biodiversity and function assume that the DNA extracted represents the microbial community in the soil, but subsequent interpretations are limited by the DNA recovered from the soil. Unfortunately, extraction methods do not provide a uniform and unbiased subsample of metagenomic DNA, and as a consequence, accurate species distributions cannot be determined. Moreover, any bias will propagate errors in estimations of overall microbial diversity and may exclude some microbial classes from study and exploitation. To improve metagenomic approaches, investigate DNA extraction biases, and provide tools for assessing the relative abundances of different groups, we explored the biodiversity of the accessible community DNA by fractioning the metagenomic DNA as a function of (i) vertical soil sampling, (ii) density gradients (cell separation), (iii) cell lysis stringency, and (iv) DNA fragment size distribution. Each fraction had a unique genetic diversity, with different predominant and rare species (based on ribosomal intergenic spacer analysis [RISA] fingerprinting and phylochips). All fractions contributed to the number of bacterial groups uncovered in the metagenome, thus increasing the DNA pool for further applications. Indeed, we were able to access a more genetically diverse proportion of the metagenome (a gain of more than 80% compared to the best single extraction method), limit the predominance of a few genomes, and increase the species richness per sequencing effort. This work stresses the difference between extracted DNA pools and the currently inaccessible complete soil metagenome. PMID:21183646

Use of Simple Sequence Repeat (SSR) markers for DNA fingerprinting and diversity analysis of sugarcane (Saccharum spp.) cultivars resistant and susceptible to red rot

USDA-ARS?s Scientific Manuscript database

In recent years SSR markers have been used widely for the genetic analysis. The objective of present research was to use SSR markers to develop DNA-based genetic identification and analyze genetic relationship of sugarcane cultivars grown in Pakistan either resistant or susceptible to red rot. Twent...

Identification of the bacterial community of maple sap by using amplified ribosomal DNA (rDNA) restriction analysis and rDNA sequencing.

PubMed

Lagacé, L; Pitre, M; Jacques, M; Roy, D

2004-04-01

The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons. Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified by 16S rRNA gene fragment sequencing. Results showed a wide variety of organisms, with 22 different genera encountered. Pseudomonas and Ralstonia, of the gamma- and beta-Proteobacteria, respectively, were the most frequently encountered genera. Gram-positive bacteria were also observed, and Staphylococcus, Plantibacter, and Bacillus were the most highly represented genera. The sampling period corresponding to 50% of the cumulative sap flow percentage presented the greatest bacterial diversity according to its Shannon diversity index value (1.1). gamma-Proteobacteria were found to be dominant almost from the beginning of the season to the end. These results are providing interesting insights on maple sap microflora that will be useful for further investigation related to microbial contamination and quality of maple products and also for guiding new strategies on taphole contamination control.

Microbial diversity of an anoxic zone of a hydroelectric power station reservoir in Brazilian Amazonia.

PubMed

Graças, Diego A; Miranda, Paulo R; Baraúna, Rafael A; McCulloch, John A; Ghilardi, Rubens; Schneider, Maria Paula C; Silva, Artur

2011-11-01

Microbial diversity was evaluated in an anoxic zone of Tucuruí Hydroelectric Power Station reservoir in Brazilian Amazonia using a culture-independent approach by amplifying and sequencing fragments of the 16S rRNA gene using metagenomic DNA as a template. Samples obtained from the photic, aphotic (40 m) and sediment (60 m) layers were used to construct six 16S rDNA libraries containing a total of 1,152 clones. The sediment, aphotic and photic layers presented 64, 33 and 35 unique archaeal operational taxonomic units (OTUs). The estimated richness of these layers was evaluated to be 153, 106 and 79 archaeal OTUs, respectively, using the abundance-based coverage estimator (ACE) and 114, 83 and 77 OTUs using the Chao1 estimator. For bacterial sequences, 114, 69 and 57 OTUs were found in the sediment, aphotic and photic layers, which presented estimated richnesses of 1,414, 522 and 197 OTUs (ACE) and 1,059, 1,014 and 148 OTUs (Chao1), respectively. Phylogenetic analyses of the sequences obtained revealed a high richness of microorganisms which participate in the carbon cycle, namely, methanogenic archaea and methanotrophic proteobacteria. Most sequences obtained belong to non-culturable prokaryotes. The present study offers the first glimpse of the huge microbial diversity of an anoxic area of a man-made lacustrine environment in the tropics.

Investigation of the bottleneck leading to the domestication of maize

PubMed Central

Eyre-Walker, Adam; Gaut, Rebecca L.; Hilton, Holly; Feldman, Dawn L.; Gaut, Brandon S.

1998-01-01

Maize (Zea mays ssp. mays) is genetically diverse, yet it is also morphologically distinct from its wild relatives. These two observations are somewhat contradictory: the first observation is consistent with a large historical population size for maize, but the latter observation is consistent with strong, diversity-limiting selection during maize domestication. In this study, we sampled sequence diversity, coupled with simulations of the coalescent process, to study the dynamics of a population bottleneck during the domestication of maize. To do this, we determined the DNA sequence of a 1,400-bp region of the Adh1 locus from 19 individuals representing maize, its presumed progenitor (Z. mays ssp. parviglumis), and a more distant relative (Zea luxurians). The sequence data were used to guide coalescent simulations of population bottlenecks associated with domestication. Our study confirms high genetic diversity in maize—maize contains 75% of the variation found in its progenitor and is more diverse than its wild relative, Z. luxurians—but it also suggests that sequence diversity in maize can be explained by a bottleneck of short duration and very small size. For example, the breadth of genetic diversity in maize is consistent with a founding population of only 20 individuals when the domestication event is 10 generations in length. PMID:9539756

Comparative molecular cytogenetics of major repetitive sequence families of three Dendrobium species (Orchidaceae) from Bangladesh

PubMed Central

Begum, Rabeya; Alam, Sheikh Shamimul; Menzel, Gerhard; Schmidt, Thomas

2009-01-01

Background and Aims Dendrobium species show tremendous morphological diversity and have broad geographical distribution. As repetitive sequence analysis is a useful tool to investigate the evolution of chromosomes and genomes, the aim of the present study was the characterization of repetitive sequences from Dendrobium moschatum for comparative molecular and cytogenetic studies in the related species Dendrobium aphyllum, Dendrobium aggregatum and representatives from other orchid genera. Methods In order to isolate highly repetitive sequences, a c0t-1 DNA plasmid library was established. Repeats were sequenced and used as probes for Southern hybridization. Sequence divergence was analysed using bioinformatic tools. Repetitive sequences were localized along orchid chromosomes by fluorescence in situ hybridization (FISH). Key Results Characterization of the c0t-1 library resulted in the detection of repetitive sequences including the (GA)n dinucleotide DmoO11, numerous Arabidopsis-like telomeric repeats and the highly amplified dispersed repeat DmoF14. The DmoF14 repeat is conserved in six Dendrobium species but diversified in representative species of three other orchid genera. FISH analyses showed the genome-wide distribution of DmoF14 in D. moschatum, D. aphyllum and D. aggregatum. Hybridization with the telomeric repeats demonstrated Arabidopsis-like telomeres at the chromosome ends of Dendrobium species. However, FISH using the telomeric probe revealed two pairs of chromosomes with strong intercalary signals in D. aphyllum. FISH showed the terminal position of 5S and 18S–5·8S–25S rRNA genes and a characteristic number of rDNA sites in the three Dendrobium species. Conclusions The repeated sequences isolated from D. moschatum c0t-1 DNA constitute major DNA families of the D. moschatum, D. aphyllum and D. aggregatum genomes with DmoF14 representing an ancient component of orchid genomes. Large intercalary telomere-like arrays suggest chromosomal rearrangements in D. aphyllum while the number and localization of rRNA genes as well as the species-specific distribution pattern of an abundant microsatellite reflect the genomic diversity of the three Dendrobium species. PMID:19635741

Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications.

PubMed

Christen, Matthias; Del Medico, Luca; Christen, Heinz; Christen, Beat

2017-01-01

Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

Exploring bacterial epigenomics in the next-generation sequencing era: a new approach for an emerging frontier.

PubMed

Chen, Poyin; Jeannotte, Richard; Weimer, Bart C

2014-05-01

Epigenetics has an important role for the success of foodborne pathogen persistence in diverse host niches. Substantial challenges exist in determining DNA methylation to situation-specific phenotypic traits. DNA modification, mediated by restriction-modification systems, functions as an immune response against antagonistic external DNA, and bacteriophage-acquired methyltransferases (MTase) and orphan MTases - those lacking the cognate restriction endonuclease - facilitate evolution of new phenotypes via gene expression modulation via DNA and RNA modifications, including methylation and phosphorothioation. Recent establishment of large-scale genome sequencing projects will result in a significant increase in genome availability that will lead to new demands for data analysis including new predictive bioinformatics approaches that can be verified with traditional scientific rigor. Sequencing technologies that detect modification coupled with mass spectrometry to discover new adducts is a powerful tactic to study bacterial epigenetics, which is poised to make novel and far-reaching discoveries that link biological significance and the bacterial epigenome. Copyright © 2014 Elsevier Ltd. All rights reserved.

Bacteria of an anaerobic 1,2-dichloropropane-dechlorinating mixed culture are phylogenetically related to those of other anaerobic dechlorinating consortia.

PubMed

Schlötelburg, C; von Wintzingerode, F; Hauck, R; Hegemann, W; Göbel, U B

2000-07-01

A 16S-rDNA-based molecular study was performed to determine the bacterial diversity of an anaerobic, 1,2-dichloropropane-dechlorinating bioreactor consortium derived from sediment of the River Saale, Germany. Total community DNA was extracted and bacterial 16S rRNA genes were subsequently amplified using conserved primers. A clone library was constructed and analysed by sequencing the 16S rDNA inserts of randomly chosen clones followed by dot blot hybridization with labelled polynucleotide probes. The phylogenetic analysis revealed significant sequence similarities of several as yet uncultured bacterial species in the bioreactor to those found in other reductively dechlorinating freshwater consortia. In contrast, no close relationship was obtained with as yet uncultured bacteria found in reductively dechlorinating consortia derived from marine habitats. One rDNA clone showed >97% sequence similarity to Dehalobacter species, known for reductive dechlorination of tri- and tetrachloroethene. These results suggest that reductive dechlorination in microbial freshwater habitats depends upon a specific bacterial community structure.

Whole-Genome Sequencing and Variant Analysis of Human Papillomavirus 16 Infections.

PubMed

van der Weele, Pascal; Meijer, Chris J L M; King, Audrey J

2017-10-01

Human papillomavirus (HPV) is a strongly conserved DNA virus, high-risk types of which can cause cervical cancer in persistent infections. The most common type found in HPV-attributable cancer is HPV16, which can be subdivided into four lineages (A to D) with different carcinogenic properties. Studies have shown HPV16 sequence diversity in different geographical areas, but only limited information is available regarding HPV16 diversity within a population, especially at the whole-genome level. We analyzed HPV16 major variant diversity and conservation in persistent infections and performed a single nucleotide polymorphism (SNP) comparison between persistent and clearing infections. Materials were obtained in the Netherlands from a cohort study with longitudinal follow-up for up to 3 years. Our analysis shows a remarkably large variant diversity in the population. Whole-genome sequences were obtained for 57 persistent and 59 clearing HPV16 infections, resulting in 109 unique variants. Interestingly, persistent infections were completely conserved through time. One reinfection event was identified where the initial and follow-up samples clustered differently. Non-A1/A2 variants seemed to clear preferentially ( P = 0.02). Our analysis shows that population-wide HPV16 sequence diversity is very large. In persistent infections, the HPV16 sequence was fully conserved. Sequencing can identify HPV16 reinfections, although occurrence is rare. SNP comparison identified no strongly acting effect of the viral genome affecting HPV16 infection clearance or persistence in up to 3 years of follow-up. These findings suggest the progression of an early HPV16 infection could be host related. IMPORTANCE Human papillomavirus 16 (HPV16) is the predominant type found in cervical cancer. Progression of initial infection to cervical cancer has been linked to sequence properties; however, knowledge of variants circulating in European populations, especially with longitudinal follow-up, is limited. By sequencing a number of infections with known follow-up for up to 3 years, we gained initial insights into the genetic diversity of HPV16 and the effects of the viral genome on the persistence of infections. A SNP comparison between sequences obtained from clearing and persistent infections did not identify strongly acting DNA variations responsible for these infection outcomes. In addition, we identified an HPV16 reinfection event where sequencing of initial and follow-up samples showed different HPV16 variants. Based on conventional genotyping, this infection would incorrectly be considered a persistent HPV16 infection. In the context of vaccine efficacy and monitoring studies, such infections could potentially cause reduced reported efficacy or efficiency. Copyright © 2017 van der Weele et al.

Is ITS-2 rDNA suitable marker for genetic characterization of Sarcoptes mites from different wild animals in different geographic areas?

PubMed

Alasaad, S; Soglia, D; Spalenza, V; Maione, S; Soriguer, R C; Pérez, J M; Rasero, R; Degiorgis, M P Ryser; Nimmervoll, H; Zhu, X Q; Rossi, L

2009-02-05

The present study examined the relationship among individual Sarcoptes scabiei mites from 13 wild mammalian populations belonging to nine species in four European countries using the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) as genetic marker. The ITS-2 plus primer flanking 5.8S and 28S rDNA (ITS-2+) was amplified from individual mites by polymerase chain reaction (PCR) and the amplicons were sequenced directly. A total of 148 ITS-2+ sequences of 404bp in length were obtained and 67 variable sites were identified (16.59%). UPGMA analyses did not show any geographical or host-specific clustering, and a similar outcome was obtained using population pairwise Fst statistics. These results demonstrated that ITS-2 rDNA does not appear to be suitable for examining genetic diversity among mite populations.

Plasmodium vivax rhomboid-like protease 1 gene diversity in Thailand.

PubMed

Mataradchakul, Touchchapol; Uthaipibull, Chairat; Nosten, Francois; Vega-Rodriguez, Joel; Jacobs-Lorena, Marcelo; Lek-Uthai, Usa

2017-10-01

Plasmodium vivax infection remains a major public health problem, especially along the Thailand border regions. We examined the genetic diversity of this parasite by analyzing single-nucleotide polymorphisms (SNPs) of the P. vivax rhomboid-like protease 1 gene (Pvrom1) in parasites collected from western (Tak province, Thai-Myanmar border) and eastern (Chanthaburi province, Thai-Cambodia border) regions. Data were collected by a cross-sectional survey, consisting of 47 and 45 P. vivax-infected filter paper-spotted blood samples from the western and eastern regions of Thailand, respectively during September 2013 to May 2014. Extracted DNA was examined for presence of P. vivax using Plasmodium species-specific nested PCR. Pvrom1 gene was PCR amplified, sequenced and the SNP diversity was analyzed using F-STAT, DnaSP, MEGA and LIAN programs. Comparison of sequences of the 92 Pvrom1 831-base open reading frames with that of a reference sequence (GenBank acc. no. XM001615211) revealed 17 samples with a total of 8 polymorphic sites, consisting of singleton (exon 3, nt 645) and parsimony informative (exon 1, nt 22 and 39; exon 3, nt 336, 537 and 656; and exon 4, nt 719 and 748) sites, which resulted in six different deduced Pvrom1 variants. Non-synonymous to synonymous substitutions ratio estimated by the DnaSP program was 1.65 indicating positive selection, but the Z-tests of selection showed no significant deviations from neutrality for Pvrom1 samples from western region of Thailand. In addition McDonald Kreitman test (MK) showed not significant, and Fst values are not different between the two regions and the regions combined. Interestingly, only Pvrom1 exon 2 was the most conserved sequences among the four exons. The relatively high degree of Pvrom1 polymorphism suggests that the protein is important for parasite survival in face of changes in both insect vector and human populations. These polymorphisms could serve as a sensitive marker for studying plasmodial genetic diversity. The significance of Pvrom1 conserved exon 2 sequence remains to be investigated. Copyright © 2017 Mahidol University. Published by Elsevier Inc. All rights reserved.

Novel Method for High-Throughput Full-Length IGHV-D-J Sequencing of the Immune Repertoire from Bulk B-Cells with Single-Cell Resolution.

PubMed

Vergani, Stefano; Korsunsky, Ilya; Mazzarello, Andrea Nicola; Ferrer, Gerardo; Chiorazzi, Nicholas; Bagnara, Davide

2017-01-01

Efficient and accurate high-throughput DNA sequencing of the adaptive immune receptor repertoire (AIRR) is necessary to study immune diversity in healthy subjects and disease-related conditions. The high complexity and diversity of the AIRR coupled with the limited amount of starting material, which can compromise identification of the full biological diversity makes such sequencing particularly challenging. AIRR sequencing protocols often fail to fully capture the sampled AIRR diversity, especially for samples containing restricted numbers of B lymphocytes. Here, we describe a library preparation method for immunoglobulin sequencing that results in an exhaustive full-length repertoire where virtually every sampled B-cell is sequenced. This maximizes the likelihood of identifying and quantifying the entire IGHV-D-J repertoire of a sample, including the detection of rearrangements present in only one cell in the starting population. The methodology establishes the importance of circumventing genetic material dilution in the preamplification phases and incorporates the use of certain described concepts: (1) balancing the starting material amount and depth of sequencing, (2) avoiding IGHV gene-specific amplification, and (3) using Unique Molecular Identifier. Together, this methodology is highly efficient, in particular for detecting rare rearrangements in the sampled population and when only a limited amount of starting material is available.

Exploring the Impacts of Anthropogenic Disturbance on Seawater and Sediment Microbial Communities in Korean Coastal Waters Using Metagenomics Analysis

PubMed Central

Won, Nam-Il; Kim, Ki-Hwan; Kang, Ji Hyoun; Park, Sang Rul; Lee, Hyuk Je

2017-01-01

The coastal ecosystems are considered as one of the most dynamic and vulnerable environments under various anthropogenic developments and the effects of climate change. Variations in the composition and diversity of microbial communities may be a good indicator for determining whether the marine ecosystems are affected by complex forcing stressors. DNA sequence-based metagenomics has recently emerged as a promising tool for analyzing the structure and diversity of microbial communities based on environmental DNA (eDNA). However, few studies have so far been performed using this approach to assess the impacts of human activities on the microbial communities in marine systems. In this study, using metagenomic DNA sequencing (16S ribosomal RNA gene), we analyzed and compared seawater and sediment communities between sand mining and control (natural) sites in southern coastal waters of Korea to assess whether anthropogenic activities have significantly affected the microbial communities. The sand mining sites harbored considerably lower levels of microbial diversities in the surface seawater community during spring compared with control sites. Moreover, the sand mining areas had distinct microbial taxonomic group compositions, particularly during spring season. The microbial groups detected solely in the sediment load/dredging areas (e.g., Marinobacter, Alcanivorax, Novosphingobium) are known to be involved in degradation of toxic chemicals such as hydrocarbon, oil, and aromatic compounds, and they also contain potential pathogens. This study highlights the versatility of metagenomics in monitoring and diagnosing the impacts of human disturbance on the environmental health of marine ecosystems from eDNA. PMID:28134828

Exploring the Impacts of Anthropogenic Disturbance on Seawater and Sediment Microbial Communities in Korean Coastal Waters Using Metagenomics Analysis.

PubMed

Won, Nam-Il; Kim, Ki-Hwan; Kang, Ji Hyoun; Park, Sang Rul; Lee, Hyuk Je

2017-01-27

The coastal ecosystems are considered as one of the most dynamic and vulnerable environments under various anthropogenic developments and the effects of climate change. Variations in the composition and diversity of microbial communities may be a good indicator for determining whether the marine ecosystems are affected by complex forcing stressors. DNA sequence-based metagenomics has recently emerged as a promising tool for analyzing the structure and diversity of microbial communities based on environmental DNA (eDNA). However, few studies have so far been performed using this approach to assess the impacts of human activities on the microbial communities in marine systems. In this study, using metagenomic DNA sequencing (16S ribosomal RNA gene), we analyzed and compared seawater and sediment communities between sand mining and control (natural) sites in southern coastal waters of Korea to assess whether anthropogenic activities have significantly affected the microbial communities. The sand mining sites harbored considerably lower levels of microbial diversities in the surface seawater community during spring compared with control sites. Moreover, the sand mining areas had distinct microbial taxonomic group compositions, particularly during spring season. The microbial groups detected solely in the sediment load/dredging areas (e.g., Marinobacter, Alcanivorax, Novosphingobium) are known to be involved in degradation of toxic chemicals such as hydrocarbon, oil, and aromatic compounds, and they also contain potential pathogens. This study highlights the versatility of metagenomics in monitoring and diagnosing the impacts of human disturbance on the environmental health of marine ecosystems from eDNA.

mtDNA diversity in Azara's owl monkeys (Aotus azarai azarai) of the Argentinean Chaco.

PubMed

Babb, Paul L; Fernandez-Duque, Eduardo; Baiduc, Caitlin A; Gagneux, Pascal; Evans, Sian; Schurr, Theodore G

2011-10-01

Owl monkeys (Aotus spp.) inhabit much of South America yet represent an enigmatic evolutionary branch among primates. While morphological, cytogenetic, and immunological evidence suggest that owl monkey populations have undergone isolation and diversification since their emergence in the New World, problems with adjacent species ranges, and sample provenance have complicated efforts to characterize genetic variation within the genus. As a result, the phylogeographic history of owl monkey species and subspecies remains unclear, and the extent of genetic diversity at the population level is unknown. To explore these issues, we analyzed mitochondrial DNA (mt DNA) variation in a population of wild Azara's owl monkeys (Aotus azarai azarai) living in the Gran Chaco region of Argentina. We sequenced the complete mitochondrial genome from one individual (16,585 base pairs (bp)) and analyzed 1,099 bp of the hypervariable control region (CR) and 696 bp of the cytochrome oxidase II (COII) gene in 117 others. In addition, we sequenced the mitochondrial genome (16,472 bp) of one Nancy Ma's owl monkey (A. nancymaae). Based on the whole mtDNA and COII data, we observed an ancient phylogeographic discontinuity among Aotus species living north, south, and west of the Amazon River that began more than eight million years ago. Our population analyses identified three major CR lineages and detected a high level of haplotypic diversity within A. a. azarai. These data point to a recent expansion of Azara's owl monkeys into the Argentinean Chaco. Overall, we provide a detailed view of owl monkey mtDNA variation at genus, species, and population levels. Copyright © 2011 Wiley-Liss, Inc.

Association between Chloroplast and Mitochondrial DNA sequences in Chinese Prunus genotypes (Prunus persica, Prunus domestica, and Prunus avium).

PubMed

Pervaiz, Tariq; Sun, Xin; Zhang, Yanyi; Tao, Ran; Zhang, Junhuan; Fang, Jinggui

2015-01-16

The nuclear DNA is conventionally used to assess the diversity and relatedness among different species, but variations at the DNA genome level has also been used to study the relationship among different organisms. In most species, mitochondrial and chloroplast genomes are inherited maternally; therefore it is anticipated that organelle DNA remains completely associated. Many research studies were conducted simultaneously on organelle genome. The objectives of this study was to analyze the genetic relationship between chloroplast and mitochondrial DNA in three Chinese Prunus genotypes viz., Prunus persica, Prunus domestica, and Prunus avium. We investigated the genetic diversity of Prunus genotypes using simple sequence repeat (SSR) markers relevant to the chloroplast and mitochondria. Most of the genotypes were genetically similar as revealed by phylogenetic analysis. The Y2 Wu Xing (Cherry) and L2 Hong Xin Li (Plum) genotypes have a high similarity index (0.89), followed by Zi Ye Li (0.85), whereas; L1 Tai Yang Li (plum) has the lowest genetic similarity (0.35). In case of cpSSR, Hong Tao (Peach) and L1 Tai Yang Li (Plum) genotypes demonstrated similarity index of 0.85 and Huang Tao has the lowest similarity index of 0.50. The mtSSR nucleotide sequence analysis revealed that each genotype has similar amplicon length (509 bp) except M5Y1 i.e., 505 bp with CCB256 primer; while in case of NAD6 primer, all genotypes showed different sizes. The MEHO (Peach), MEY1 (Cherry), MEL2 (Plum) and MEL1 (Plum) have 586 bps; while MEY2 (Cherry), MEZI (Plum) and MEHU (Peach) have 585, 584 and 566 bp, respectively. The CCB256 primer showed highly conserved sequences and minute single polymorphic nucleotides with no deletion or mutation. The cpSSR (ARCP511) microsatellites showed the harmonious amplicon length. The CZI (Plum), CHO (Peach) and CL1 (Plum) showed 182 bp; whileCHU (Peach), CY2 (Cherry), CL2 (Plum) and CY1 (Cherry) showed 181 bp amplicon lengths. These results demonstrated high conservation in chloroplast and mitochondrial genome among Prunus species during the evolutionary process. These findings are valuable to study the organelle DNA diversity in different species and genotypes of Prunus to provide in depth insight in to the mitochondrial and chloroplast genomes.

Mimosa caesalpiniifolia rhizobial isolates from different origins of the Brazilian Northeast.

PubMed

Martins, Paulo Geovani Silva; Junior, Mario Andrade Lira; Fracetto, Giselle Gomes Monteiro; da Silva, Maria Luiza Ribeiro Bastos; Vincentin, Rayssa Pereira; de Lyra, Maria do Carmo Catanho Pereira

2015-04-01

Biological nitrogen fixation from the legume-rhizobia symbiosis is one of the main sources of fixed nitrogen on land environments. Diazotrophic bacteria taxonomy has been substantially modified by the joint use of phenotypic, physiological and molecular aspects. Among these molecular tools, sequencing and genotyping of genomic regions such as 16S rDNA and repetitive conserved DNA regions have boosted the accuracy of species identification. This research is a phylogenetic study of diazotrophic bacteria from sabiá (Mimosa caesalpiniifolia Benth.), inoculated with soils from five municipalities of the Brazilian Northeast. After bacterial isolation and morphophysiological characterization, genotyping was performed using REP, ERIC and BOX oligonucleotides and 16S rDNA sequencing for genetic diversity identification. A 1.5b Kb fragment of the 16S rDNA was amplified from each isolate. Morphophysiological characterization of the 47 isolates created a dendrogram, where isolate PE-GR02 formed a monophyletic branch. The fingerprinting conducted with BOX, ERIC and REP shows distinct patterns, and their compilation created a dendrogram with diverse groups and, after blasting in GenBank, resulted in genetic identities ranging from 77 to 99 % with Burkholderia strains. The 16S rDNA phylogenetic tree constructed with these isolates and GenBank deposits of strains recommended for inoculant production confirm these isolates are distinct from the previously deposited strains, whereas isolates PE-CR02, PE-CR4, PE-CR07, PE-CR09 and PE-GE06 were the most distinct within the group. Morphophysiological characterization and BOX, ERIC and REP compilation enhanced the discrimination of the isolates, and the 16S rDNA sequences compared with GenBank confirmed the preference of Mimosa for Burkholderia diazotrophic bacteria.

Predicting nuclear gene coalescence from mitochondrial data: the three-times rule.

PubMed

Palumbi, S R; Cipriano, F; Hare, M P

2001-05-01

Coalescence theory predicts when genetic drift at nuclear loci will result in fixation of sequence differences to produce monophyletic gene trees. However, the theory is difficult to apply to particular taxa because it hinges on genetically effective population size, which is generally unknown. Neutral theory also predicts that evolution of monophyly will be four times slower in nuclear than in mitochondrial genes primarily because genetic drift is slower at nuclear loci. Variation in mitochondrial DNA (mtDNA) within and between species has been studied extensively, but can these mtDNA data be used to predict coalescence in nuclear loci? Comparison of neutral theories of coalescence of mitochondrial and nuclear loci suggests a simple rule of thumb. The "three-times rule" states that, on average, most nuclear loci will be monophyletic when the branch length leading to the mtDNA sequences of a species is three times longer than the average mtDNA sequence diversity observed within that species. A test using mitochondrial and nuclear intron data from seven species of whales and dolphins suggests general agreement with predictions of the three-times rule. We define the coalescence ratio as the mitochondrial branch length for a species divided by intraspecific mtDNA diversity. We show that species with high coalescence ratios show nuclear monophyly, whereas species with low ratios have polyphyletic nuclear gene trees. As expected, species with intermediate coalescence ratios show a variety of patterns. Especially at very high or low coalescence ratios, the three-times rule predicts nuclear gene patterns that can help detect the action of selection. The three-times rule may be useful as an empirical benchmark for evaluating evolutionary processes occurring at multiple loci.

Diversity, expansion, and evolutionary novelty of plant DNA-binding transcription factor families.

PubMed

Lehti-Shiu, Melissa D; Panchy, Nicholas; Wang, Peipei; Uygun, Sahra; Shiu, Shin-Han

2017-01-01

Plant transcription factors (TFs) that interact with specific sequences via DNA-binding domains are crucial for regulating transcriptional initiation and are fundamental to plant development and environmental response. In addition, expansion of TF families has allowed functional divergence of duplicate copies, which has contributed to novel, and in some cases adaptive, traits in plants. Thus, TFs are central to the generation of the diverse plant species that we see today. Major plant agronomic traits, including those relevant to domestication, have also frequently arisen through changes in TF coding sequence or expression patterns. Here our goal is to provide an overview of plant TF evolution by first comparing the diversity of DNA-binding domains and the sizes of these domain families in plants and other eukaryotes. Because TFs are among the most highly expanded gene families in plants, the birth and death process of TFs as well as the mechanisms contributing to their retention are discussed. We also provide recent examples of how TFs have contributed to novel traits that are important in plant evolution and in agriculture.This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer. Copyright © 2016 Elsevier B.V. All rights reserved.

Diversity of indoor fungi as revealed by DNA metabarcoding.

PubMed

Korpelainen, Helena; Pietiläinen, Maria

2017-01-01

In the present study, we conducted DNA metabarcoding (the nuclear ITS2 region) for indoor fungal samples originating from two nursery schools with a suspected mould problem (sampling before and after renovation), from two university buildings, and from an old farmhouse. Good-quality sequences were obtained, and the results showed that DNA metabarcoding provides high resolution in fungal identification. The pooled proportions of sequences representing filamentous ascomycetes, filamentous basidiomycetes, yeasts, and other fungi equalled 62.3%, 8.0%, 28.3%, and 1.4%, respectively, and the total number of fungal genera found during the study was 585. When comparing fungal diversities and taxonomic composition between different types of buildings, no obvious pattern was detected. The average pairwise values of Sørensen Chao indices that were used to compare similarities for taxon composition between samples among the samples from the two university buildings, two nurseries, and farmhouse equaled 0.693, 0.736, 0.852, 0.928, and 0.981, respectively, while the mean similarity index for all samples was 0.864. We discovered that making explicit conclusions on the relationship between the indoor air quality and mycoflora is complicated by the lack of appropriate indicators for air quality and by the occurrence of wide spatial and temporal changes in diversity and compositions among samples.

Structural insight into the specificity of the B3 DNA-binding domains provided by the co-crystal structure of the C-terminal fragment of BfiI restriction enzyme

PubMed Central

Golovenko, Dmitrij; Manakova, Elena; Zakrys, Linas; Zaremba, Mindaugas; Sasnauskas, Giedrius; Gražulis, Saulius; Siksnys, Virginijus

2014-01-01

The B3 DNA-binding domains (DBDs) of plant transcription factors (TF) and DBDs of EcoRII and BfiI restriction endonucleases (EcoRII-N and BfiI-C) share a common structural fold, classified as the DNA-binding pseudobarrel. The B3 DBDs in the plant TFs recognize a diverse set of target sequences. The only available co-crystal structure of the B3-like DBD is that of EcoRII-N (recognition sequence 5′-CCTGG-3′). In order to understand the structural and molecular mechanisms of specificity of B3 DBDs, we have solved the crystal structure of BfiI-C (recognition sequence 5′-ACTGGG-3′) complexed with 12-bp cognate oligoduplex. Structural comparison of BfiI-C–DNA and EcoRII-N–DNA complexes reveals a conserved DNA-binding mode and a conserved pattern of interactions with the phosphodiester backbone. The determinants of the target specificity are located in the loops that emanate from the conserved structural core. The BfiI-C–DNA structure presented here expands a range of templates for modeling of the DNA-bound complexes of the B3 family of plant TFs. PMID:24423868

Deciphering amphibian diversity through DNA barcoding: chances and challenges.

PubMed

Vences, Miguel; Thomas, Meike; Bonett, Ronald M; Vieites, David R

2005-10-29

Amphibians globally are in decline, yet there is still a tremendous amount of unrecognized diversity, calling for an acceleration of taxonomic exploration. This process will be greatly facilitated by a DNA barcoding system; however, the mitochondrial population structure of many amphibian species presents numerous challenges to such a standardized, single locus, approach. Here we analyse intra- and interspecific patterns of mitochondrial variation in two distantly related groups of amphibians, mantellid frogs and salamanders, to determine the promise of DNA barcoding with cytochrome oxidase subunit I (cox1) sequences in this taxon. High intraspecific cox1 divergences of 7-14% were observed (18% in one case) within the whole set of amphibian sequences analysed. These high values are not caused by particularly high substitution rates of this gene but by generally deep mitochondrial divergences within and among amphibian species. Despite these high divergences, cox1 sequences were able to correctly identify species including disparate geographic variants. The main problems with cox1 barcoding of amphibians are (i) the high variability of priming sites that hinder the application of universal primers to all species and (ii) the observed distinct overlap of intraspecific and interspecific divergence values, which implies difficulties in the definition of threshold values to identify candidate species. Common discordances between geographical signatures of mitochondrial and nuclear markers in amphibians indicate that a single-locus approach can be problematic when high accuracy of DNA barcoding is required. We suggest that a number of mitochondrial and nuclear genes may be used as DNA barcoding markers to complement cox1.

Genetic diversity, morphological uniformity and polyketide production in dinoflagellates (Amphidinium, Dinoflagellata).

PubMed

Murray, Shauna A; Garby, Tamsyn; Hoppenrath, Mona; Neilan, Brett A

2012-01-01

Dinoflagellates are an intriguing group of eukaryotes, showing many unusual morphological and genetic features. Some groups of dinoflagellates are morphologically highly uniform, despite indications of genetic diversity. The species Amphidinium carterae is abundant and cosmopolitan in marine environments, grows easily in culture, and has therefore been used as a 'model' dinoflagellate in research into dinoflagellate genetics, polyketide production and photosynthesis. We have investigated the diversity of 'cryptic' species of Amphidinium that are morphologically similar to A. carterae, including the very similar species Amphidinium massartii, based on light and electron microscopy, two nuclear gene regions (LSU rDNA and ITS rDNA) and one mitochondrial gene region (cytochrome b). We found that six genetically distinct cryptic species (clades) exist within the species A. massartii and four within A. carterae, and that these clades differ from one another in molecular sequences at levels comparable to other dinoflagellate species, genera or even families. Using primers based on an alignment of alveolate ketosynthase sequences, we isolated partial ketosynthase genes from several Amphidinium species. We compared these genes to known dinoflagellate ketosynthase genes and investigated the evolution and diversity of the strains of Amphidinium that produce them.

Genetic Diversity, Morphological Uniformity and Polyketide Production in Dinoflagellates (Amphidinium, Dinoflagellata)

PubMed Central

Hoppenrath, Mona; Neilan, Brett A.

2012-01-01

Dinoflagellates are an intriguing group of eukaryotes, showing many unusual morphological and genetic features. Some groups of dinoflagellates are morphologically highly uniform, despite indications of genetic diversity. The species Amphidinium carterae is abundant and cosmopolitan in marine environments, grows easily in culture, and has therefore been used as a ‘model’ dinoflagellate in research into dinoflagellate genetics, polyketide production and photosynthesis. We have investigated the diversity of ‘cryptic’ species of Amphidinium that are morphologically similar to A. carterae, including the very similar species Amphidinium massartii, based on light and electron microscopy, two nuclear gene regions (LSU rDNA and ITS rDNA) and one mitochondrial gene region (cytochrome b). We found that six genetically distinct cryptic species (clades) exist within the species A. massartii and four within A. carterae, and that these clades differ from one another in molecular sequences at levels comparable to other dinoflagellate species, genera or even families. Using primers based on an alignment of alveolate ketosynthase sequences, we isolated partial ketosynthase genes from several Amphidinium species. We compared these genes to known dinoflagellate ketosynthase genes and investigated the evolution and diversity of the strains of Amphidinium that produce them. PMID:22675531

Trophic and Non-Trophic Interactions in a Biodiversity Experiment Assessed by Next-Generation Sequencing

PubMed Central

Tiede, Julia; Wemheuer, Bernd; Traugott, Michael; Daniel, Rolf; Tscharntke, Teja; Ebeling, Anne; Scherber, Christoph

2016-01-01

Plant diversity affects species richness and abundance of taxa at higher trophic levels. However, plant diversity effects on omnivores (feeding on multiple trophic levels) and their trophic and non-trophic interactions are not yet studied because appropriate methods were lacking. A promising approach is the DNA-based analysis of gut contents using next generation sequencing (NGS) technologies. Here, we integrate NGS-based analysis into the framework of a biodiversity experiment where plant taxonomic and functional diversity were manipulated to directly assess environmental interactions involving the omnivorous ground beetle Pterostichus melanarius. Beetle regurgitates were used for NGS-based analysis with universal 18S rDNA primers for eukaryotes. We detected a wide range of taxa with the NGS approach in regurgitates, including organisms representing trophic, phoretic, parasitic, and neutral interactions with P. melanarius. Our findings suggest that the frequency of (i) trophic interactions increased with plant diversity and vegetation cover; (ii) intraguild predation increased with vegetation cover, and (iii) neutral interactions with organisms such as fungi and protists increased with vegetation cover. Experimentally manipulated plant diversity likely affects multitrophic interactions involving omnivorous consumers. Our study therefore shows that trophic and non-trophic interactions can be assessed via NGS to address fundamental questions in biodiversity research. PMID:26859146

Strong spurious transcription likely contributes to DNA insert bias in typical metagenomic clone libraries.

PubMed

Lam, Kathy N; Charles, Trevor C

2015-01-01

Clone libraries provide researchers with a powerful resource to study nucleic acid from diverse sources. Metagenomic clone libraries in particular have aided in studies of microbial biodiversity and function, and allowed the mining of novel enzymes. Libraries are often constructed by cloning large inserts into cosmid or fosmid vectors. Recently, there have been reports of GC bias in fosmid metagenomic libraries, and it was speculated to be a result of fragmentation and loss of AT-rich sequences during cloning. However, evidence in the literature suggests that transcriptional activity or gene product toxicity may play a role. To explore possible mechanisms responsible for sequence bias in clone libraries, we constructed a cosmid library from a human microbiome sample and sequenced DNA from different steps during library construction: crude extract DNA, size-selected DNA, and cosmid library DNA. We confirmed a GC bias in the final cosmid library, and we provide evidence that the bias is not due to fragmentation and loss of AT-rich sequences but is likely occurring after DNA is introduced into Escherichia coli. To investigate the influence of strong constitutive transcription, we searched the sequence data for promoters and found that rpoD/σ(70) promoter sequences were underrepresented in the cosmid library. Furthermore, when we examined the genomes of taxa that were differentially abundant in the cosmid library relative to the original sample, we found the bias to be more correlated with the number of rpoD/σ(70) consensus sequences in the genome than with simple GC content. The GC bias of metagenomic libraries does not appear to be due to DNA fragmentation. Rather, analysis of promoter sequences provides support for the hypothesis that strong constitutive transcription from sequences recognized as rpoD/σ(70) consensus-like in E. coli may lead to instability, causing loss of the plasmid or loss of the insert DNA that gives rise to the transcription. Despite widespread use of E. coli to propagate foreign DNA in metagenomic libraries, the effects of in vivo transcriptional activity on clone stability are not well understood. Further work is required to tease apart the effects of transcription from those of gene product toxicity.

Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady's slipper orchid

PubMed Central

2011-01-01

Background Paphiopedilum is a horticulturally and ecologically important genus of ca. 80 species of lady's slipper orchids native to Southeast Asia. These plants have long been of interest regarding their chromosomal evolution, which involves a progressive aneuploid series based on either fission or fusion of centromeres. Chromosome number is positively correlated with genome size, so rearrangement processes must include either insertion or deletion of DNA segments. We have conducted Fluorescence In Situ Hybridization (FISH) studies using 5S and 25S ribosomal DNA (rDNA) probes to survey for rearrangements, duplications, and phylogenetically-correlated variation within Paphiopedilum. We further studied sequence variation of the non-transcribed spacers of 5S rDNA (5S-NTS) to examine their complex duplication history, including the possibility that concerted evolutionary forces may homogenize diversity. Results 5S and 25S rDNA loci among Paphiopedilum species, representing all key phylogenetic lineages, exhibit a considerable diversity that correlates well with recognized evolutionary groups. 25S rDNA signals range from 2 (representing 1 locus) to 9, the latter representing hemizygosity. 5S loci display extensive structural variation, and show from 2 specific signals to many, both major and minor and highly dispersed. The dispersed signals mainly occur at centromeric and subtelomeric positions, which are hotspots for chromosomal breakpoints. Phylogenetic analysis of cloned 5S rDNA non-transcribed spacer (5S-NTS) sequences showed evidence for both ancient and recent post-speciation duplication events, as well as interlocus and intralocus diversity. Conclusions Paphiopedilum species display many chromosomal rearrangements - for example, duplications, translocations, and inversions - but only weak concerted evolutionary forces among highly duplicated 5S arrays, which suggests that double-strand break repair processes are dynamic and ongoing. These results make the genus a model system for the study of complex chromosomal evolution in plants. PMID:21910890

Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady's slipper orchid.

PubMed

Lan, Tianying; Albert, Victor A

2011-09-12

Paphiopedilum is a horticulturally and ecologically important genus of ca. 80 species of lady's slipper orchids native to Southeast Asia. These plants have long been of interest regarding their chromosomal evolution, which involves a progressive aneuploid series based on either fission or fusion of centromeres. Chromosome number is positively correlated with genome size, so rearrangement processes must include either insertion or deletion of DNA segments. We have conducted Fluorescence In Situ Hybridization (FISH) studies using 5S and 25S ribosomal DNA (rDNA) probes to survey for rearrangements, duplications, and phylogenetically-correlated variation within Paphiopedilum. We further studied sequence variation of the non-transcribed spacers of 5S rDNA (5S-NTS) to examine their complex duplication history, including the possibility that concerted evolutionary forces may homogenize diversity. 5S and 25S rDNA loci among Paphiopedilum species, representing all key phylogenetic lineages, exhibit a considerable diversity that correlates well with recognized evolutionary groups. 25S rDNA signals range from 2 (representing 1 locus) to 9, the latter representing hemizygosity. 5S loci display extensive structural variation, and show from 2 specific signals to many, both major and minor and highly dispersed. The dispersed signals mainly occur at centromeric and subtelomeric positions, which are hotspots for chromosomal breakpoints. Phylogenetic analysis of cloned 5S rDNA non-transcribed spacer (5S-NTS) sequences showed evidence for both ancient and recent post-speciation duplication events, as well as interlocus and intralocus diversity. Paphiopedilum species display many chromosomal rearrangements--for example, duplications, translocations, and inversions--but only weak concerted evolutionary forces among highly duplicated 5S arrays, which suggests that double-strand break repair processes are dynamic and ongoing. These results make the genus a model system for the study of complex chromosomal evolution in plants.

Temporal fluctuation in North East Baltic Sea region cattle population revealed by mitochondrial and Y-chromosomal DNA analyses.

PubMed

Niemi, Marianna; Bläuer, Auli; Iso-Touru, Terhi; Harjula, Janne; Nyström Edmark, Veronica; Rannamäe, Eve; Lõugas, Lembi; Sajantila, Antti; Lidén, Kerstin; Taavitsainen, Jussi-Pekka

2015-01-01

Ancient DNA analysis offers a way to detect changes in populations over time. To date, most studies of ancient cattle have focused on their domestication in prehistory, while only a limited number of studies have analysed later periods. Conversely, the genetic structure of modern cattle populations is well known given the undertaking of several molecular and population genetic studies. Bones and teeth from ancient cattle populations from the North-East Baltic Sea region dated to the Prehistoric (Late Bronze and Iron Age, 5 samples), Medieval (14), and Post-Medieval (26) periods were investigated by sequencing 667 base pairs (bp) from the mitochondrial DNA (mtDNA) and 155 bp of intron 19 in the Y-chromosomal UTY gene. Comparison of maternal (mtDNA haplotypes) genetic diversity in ancient cattle (45 samples) with modern cattle populations in Europe and Asia (2094 samples) revealed 30 ancient mtDNA haplotypes, 24 of which were shared with modern breeds, while 6 were unique to the ancient samples. Of seven Y-chromosomal sequences determined from ancient samples, six were Y2 and one Y1 haplotype. Combined data including Swedish samples from the same periods (64 samples) was compared with the occurrence of Y-chromosomal haplotypes in modern cattle (1614 samples). The diversity of haplogroups was highest in the Prehistoric samples, where many haplotypes were unique. The Medieval and Post-Medieval samples also show a high diversity with new haplotypes. Some of these haplotypes have become frequent in modern breeds in the Nordic Countries and North-Western Russia while other haplotypes have remained in only a few local breeds or seem to have been lost. A temporal shift in Y-chromosomal haplotypes from Y2 to Y1 was detected that corresponds with the appearance of new mtDNA haplotypes in the Medieval and Post-Medieval period. This suggests a replacement of the Prehistoric mtDNA and Y chromosomal haplotypes by new types of cattle.

Temporal Fluctuation in North East Baltic Sea Region Cattle Population Revealed by Mitochondrial and Y-Chromosomal DNA Analyses

PubMed Central

Niemi, Marianna; Bläuer, Auli; Iso-Touru, Terhi; Harjula, Janne; Nyström Edmark, Veronica; Rannamäe, Eve; Lõugas, Lembi; Sajantila, Antti; Lidén, Kerstin; Taavitsainen, Jussi-Pekka

2015-01-01

Background Ancient DNA analysis offers a way to detect changes in populations over time. To date, most studies of ancient cattle have focused on their domestication in prehistory, while only a limited number of studies have analysed later periods. Conversely, the genetic structure of modern cattle populations is well known given the undertaking of several molecular and population genetic studies. Results Bones and teeth from ancient cattle populations from the North-East Baltic Sea region dated to the Prehistoric (Late Bronze and Iron Age, 5 samples), Medieval (14), and Post-Medieval (26) periods were investigated by sequencing 667 base pairs (bp) from the mitochondrial DNA (mtDNA) and 155 bp of intron 19 in the Y-chromosomal UTY gene. Comparison of maternal (mtDNA haplotypes) genetic diversity in ancient cattle (45 samples) with modern cattle populations in Europe and Asia (2094 samples) revealed 30 ancient mtDNA haplotypes, 24 of which were shared with modern breeds, while 6 were unique to the ancient samples. Of seven Y-chromosomal sequences determined from ancient samples, six were Y2 and one Y1 haplotype. Combined data including Swedish samples from the same periods (64 samples) was compared with the occurrence of Y-chromosomal haplotypes in modern cattle (1614 samples). Conclusions The diversity of haplogroups was highest in the Prehistoric samples, where many haplotypes were unique. The Medieval and Post-Medieval samples also show a high diversity with new haplotypes. Some of these haplotypes have become frequent in modern breeds in the Nordic Countries and North-Western Russia while other haplotypes have remained in only a few local breeds or seem to have been lost. A temporal shift in Y-chromosomal haplotypes from Y2 to Y1 was detected that corresponds with the appearance of new mtDNA haplotypes in the Medieval and Post-Medieval period. This suggests a replacement of the Prehistoric mtDNA and Y chromosomal haplotypes by new types of cattle. PMID:25992976

mtDNA variation predicts population size in humans and reveals a major Southern Asian chapter in human prehistory.

PubMed

Atkinson, Quentin D; Gray, Russell D; Drummond, Alexei J

2008-02-01

The relative timing and size of regional human population growth following our expansion from Africa remain unknown. Human mitochondrial DNA (mtDNA) diversity carries a legacy of our population history. Given a set of sequences, we can use coalescent theory to estimate past population size through time and draw inferences about human population history. However, recent work has challenged the validity of using mtDNA diversity to infer species population sizes. Here we use Bayesian coalescent inference methods, together with a global data set of 357 human mtDNA coding-region sequences, to infer human population sizes through time across 8 major geographic regions. Our estimates of relative population sizes show remarkable concordance with the contemporary regional distribution of humans across Africa, Eurasia, and the Americas, indicating that mtDNA diversity is a good predictor of population size in humans. Plots of population size through time show slow growth in sub-Saharan Africa beginning 143-193 kya, followed by a rapid expansion into Eurasia after the emergence of the first non-African mtDNA lineages 50-70 kya. Outside Africa, the earliest and fastest growth is inferred in Southern Asia approximately 52 kya, followed by a succession of growth phases in Northern and Central Asia (approximately 49 kya), Australia (approximately 48 kya), Europe (approximately 42 kya), the Middle East and North Africa (approximately 40 kya), New Guinea (approximately 39 kya), the Americas (approximately 18 kya), and a second expansion in Europe (approximately 10-15 kya). Comparisons of relative regional population sizes through time suggest that between approximately 45 and 20 kya most of humanity lived in Southern Asia. These findings not only support the use of mtDNA data for estimating human population size but also provide a unique picture of human prehistory and demonstrate the importance of Southern Asia to our recent evolutionary past.

Diversity of Streptomyces spp. in Eastern Himalayan region – computational RNomics approach to phylogeny

PubMed Central

Bhattacharjee, Kaushik; Banerjee, Subhro; Joshi, Santa Ram

2012-01-01

Isolation and characterization of actinomycetes from soil samples from altitudinal gradient of North-East India were investigated for computational RNomics based phylogeny. A total of 52 diverse isolates of Streptomyces from the soil samples were isolated on four different media and from these 6 isolates were selected on the basis of cultural characteristics, microscopic and biochemical studies. Sequencing of 16S rDNA of the selected isolates identified them to belong to six different species of Streptomyces. The molecular morphometric and physico-kinetic analysis of 16S rRNA sequences were performed to predict the diversity of the genus. The computational RNomics study revealed the significance of the structural RNA based phylogenetic analysis in a relatively diverse group of Streptomyces. PMID:22829729

Mitochondrial DNA haplotype distribution patterns in Pinus ponderosa (Pinaceae): range-wide evolutionary history and implications for conservation.

PubMed

Potter, Kevin M; Hipkins, Valerie D; Mahalovich, Mary F; Means, Robert E

2013-08-01

Ponderosa pine (Pinus ponderosa Douglas ex P. Lawson & C. Lawson) exhibits complicated patterns of morphological and genetic variation across its range in western North America. This study aims to clarify P. ponderosa evolutionary history and phylogeography using a highly polymorphic mitochondrial DNA marker, with results offering insights into how geographical and climatological processes drove the modern evolutionary structure of tree species in the region. We amplified the mtDNA nad1 second intron minisatellite region for 3,100 trees representing 104 populations, and sequenced all length variants. We estimated population-level haplotypic diversity and determined diversity partitioning among varieties, races and populations. After aligning sequences of minisatellite repeat motifs, we evaluated evolutionary relationships among haplotypes. The geographical structuring of the 10 haplotypes corresponded with division between Pacific and Rocky Mountain varieties. Pacific haplotypes clustered with high bootstrap support, and appear to have descended from Rocky Mountain haplotypes. A greater proportion of diversity was partitioned between Rocky Mountain races than between Pacific races. Areas of highest haplotypic diversity were the southern Sierra Nevada mountain range in California, northwestern California, and southern Nevada. Pinus ponderosa haplotype distribution patterns suggest a complex phylogeographic history not revealed by other genetic and morphological data, or by the sparse paleoecological record. The results appear consistent with long-term divergence between the Pacific and Rocky Mountain varieties, along with more recent divergences not well-associated with race. Pleistocene refugia may have existed in areas of high haplotypic diversity, as well as the Great Basin, Southwestern United States/northern Mexico, and the High Plains.

The Contribution of DNA Metabarcoding to Fungal Conservation: Diversity Assessment, Habitat Partitioning and Mapping Red-Listed Fungi in Protected Coastal Salix repens Communities in the Netherlands

PubMed Central

Geml, József; Gravendeel, Barbara; van der Gaag, Kristiaan J.; Neilen, Manon; Lammers, Youri; Raes, Niels; Semenova, Tatiana A.; de Knijff, Peter; Noordeloos, Machiel E.

2014-01-01

Western European coastal sand dunes are highly important for nature conservation. Communities of the creeping willow (Salix repens) represent one of the most characteristic and diverse vegetation types in the dunes. We report here the results of the first kingdom-wide fungal diversity assessment in S. repens coastal dune vegetation. We carried out massively parallel pyrosequencing of ITS rDNA from soil samples taken at ten sites in an extended area of joined nature reserves located along the North Sea coast of the Netherlands, representing habitats with varying soil pH and moisture levels. Fungal communities in Salix repens beds are highly diverse and we detected 1211 non-singleton fungal 97% sequence similarity OTUs after analyzing 688,434 ITS2 rDNA sequences. Our comparison along a north-south transect indicated strong correlation between soil pH and fungal community composition. The total fungal richness and the number OTUs of most fungal taxonomic groups negatively correlated with higher soil pH, with some exceptions. With regard to ecological groups, dark-septate endophytic fungi were more diverse in acidic soils, ectomycorrhizal fungi were represented by more OTUs in calcareous sites, while detected arbuscular mycorrhizal genera fungi showed opposing trends regarding pH. Furthermore, we detected numerous red listed species in our samples often from previously unknown locations, indicating that some of the fungal species currently considered rare may be more abundant in Dutch S. repens communities than previously thought. PMID:24937200

DNA barcoding and evaluation of genetic diversity in Cyprinidae fish in the midstream of the Yangtze River.

PubMed

Shen, Yanjun; Guan, Lihong; Wang, Dengqiang; Gan, Xiaoni

2016-05-01

The Yangtze River is the longest river in China and is divided into upstream and mid-downstream regions by the Three Gorges (the natural barriers of the Yangtze River), resulting in a complex distribution of fish. Dramatic changes to habitat environments may ultimately threaten fish survival; thus, it is necessary to evaluate the genetic diversity and propose protective measures. Species identification is the most significant task in many fields of biological research and in conservation efforts. DNA barcoding, which constitutes the analysis of a short fragment of the mitochondrial cytochrome c oxidase subunit I (COI) sequence, has been widely used for species identification. In this study, we collected 561 COI barcode sequences from 35 fish from the midstream of the Yangtze River. The intraspecific distances of all species were below 2% (with the exception of Acheilognathus macropterus and Hemibarbus maculatus). Nevertheless, all species could be unambiguously identified from the trees, barcoding gaps and taxonomic resolution ratio values. Furthermore, the COI barcode diversity was found to be low (≤0.5%), with the exception of H. maculatus (0.87%), A. macropterus (2.02%) and Saurogobio dabryi (0.82%). No or few shared haplotypes were detected between the upstream and downstream populations for ten species with overall nucleotide diversities greater than 0.00%, which indicated the likelihood of significant population genetic structuring. Our analyses indicated that DNA barcoding is an effective tool for the identification of cyprinidae fish in the midstream of the Yangtze River. It is vital that some protective measures be taken immediately because of the low COI barcode diversity.

A Personal Journey of Discovery: Developing Technology and Changing Biology

NASA Astrophysics Data System (ADS)

Hood, Lee

2008-07-01

This autobiographical article describes my experiences in developing chemically based, biological technologies for deciphering biological information: DNA, RNA, proteins, interactions, and networks. The instruments developed include protein and DNA sequencers and synthesizers, as well as ink-jet technology for synthesizing DNA chips. Diverse new strategies for doing biology also arose from novel applications of these instruments. The functioning of these instruments can be integrated to generate powerful new approaches to cloning and characterizing genes from a small amount of protein sequence or to using gene sequences to synthesize peptide fragments so as to characterize various properties of the proteins. I also discuss the five paradigm changes in which I have participated: the development and integration of biological instrumentation; the human genome project; cross-disciplinary biology; systems biology; and predictive, personalized, preventive, and participatory (P4) medicine. Finally, I discuss the origins, the philosophy, some accomplishments, and the future trajectories of the Institute for Systems Biology.

Intraspecific Genetic Variation and Phylogenetic Analysis of Dirofilaria immitis Samples from Western China Using Complete ND1 and 16S rDNA Gene Sequences

PubMed Central

Liu, Tianyu; Liang, Yinan; Zhong, Xiuqin; Wang, Ning; Hu, Dandan; Zhou, Xuan; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

2014-01-01

Dirofilaria immitis (heartworm) is the causative agent of an important zoonotic disease that is spread by mosquitoes. In this study, molecular and phylogenetic characterization of D. immitis were performed based on complete ND1 and 16S rDNA gene sequences, which provided the foundation for more advanced molecular diagnosis, prevention, and control of heartworm diseases. The mutation rate and evolutionary divergence in adult heartworm samples from seven dogs in western China were analyzed to obtain information on genetic diversity and variability. Phylogenetic relationships were inferred using both maximum parsimony (MP) and Bayes methods based on the complete gene sequences. The results suggest that D. immitis formed an independent monophyletic group in which the 16S rDNA gene has mutated more rapidly than has ND1. PMID:24639299

Transposable elements and G-quadruplexes.

PubMed

Kejnovsky, Eduard; Tokan, Viktor; Lexa, Matej

2015-09-01

A significant part of eukaryotic genomes is formed by transposable elements (TEs) containing not only genes but also regulatory sequences. Some of the regulatory sequences located within TEs can form secondary structures like hairpins or three-stranded (triplex DNA) and four-stranded (quadruplex DNA) conformations. This review focuses on recent evidence showing that G-quadruplex-forming sequences in particular are often present in specific parts of TEs in plants and humans. We discuss the potential role of these structures in the TE life cycle as well as the impact of G-quadruplexes on replication, transcription, translation, chromatin status, and recombination. The aim of this review is to emphasize that TEs may serve as vehicles for the genomic spread of G-quadruplexes. These non-canonical DNA structures and their conformational switches may constitute another regulatory system that, together with small and long non-coding RNA molecules and proteins, contribute to the complex cellular network resulting in the large diversity of eukaryotes.

PCR Conditions for 16S Primers for Analysis of Microbes in the Colon of Rats.

PubMed

Guillen, I A; Camacho, H; Tuero, A D; Bacardí, D; Palenzuela, D O; Aguilera, A; Silva, J A; Estrada, R; Gell, O; Suárez, J; Ancizar, J; Brown, E; Colarte, A B; Castro, J; Novoa, L I

2016-09-01

The study of the composition of the intestinal flora is important to the health of the host, playing a key role in maintaining intestinal homeostasis and the evolution of the immune system. For these studies, various universal primers of the 16S rDNA gene are used in microbial taxonomy. Here, we report an evaluation of 5 universal primers to explore the presence of microbial DNA in colon biopsies preserved in RNAlater solution. The DNA extracted was used for the amplification of PCR products containing the variable (V) regions of the microbial 16S rDNA gene. The PCR products were studied by restriction fragment length polymorphism (RFLP) analysis and DNA sequence, whose percent of homology with microbial sequences reported in GenBank was verified using bioinformatics tools. The presence of microbes in the colon of rats was quantified by the quantitative PCR (qPCR) technique. We obtained microbial DNA from rat, useful for PCR analysis with the universal primers for the bacteria 16S rDNA. The sequences of PCR products obtained from a colon biopsy of the animal showed homology with the classes bacilli (Lactobacillus spp) and proteobacteria, normally represented in the colon of rats. The proposed methodology allowed the attainment of DNA of bacteria with the quality and integrity for use in qPCR, sequencing, and PCR-RFLP analysis. The selected universal primers provided knowledge of the abundance of microorganisms and the formation of a preliminary test of bacterial diversity in rat colon biopsies.

Evolution in the block: common elements of 5S rDNA organization and evolutionary patterns in distant fish genera.

PubMed

Campo, Daniel; García-Vázquez, Eva

2012-01-01

The 5S rDNA is organized in the genome as tandemly repeated copies of a structural unit composed of a coding sequence plus a nontranscribed spacer (NTS). The coding region is highly conserved in the evolution, whereas the NTS vary in both length and sequence. It has been proposed that 5S rRNA genes are members of a gene family that have arisen through concerted evolution. In this study, we describe the molecular organization and evolution of the 5S rDNA in the genera Lepidorhombus and Scophthalmus (Scophthalmidae) and compared it with already known 5S rDNA of the very different genera Merluccius (Merluccidae) and Salmo (Salmoninae), to identify common structural elements or patterns for understanding 5S rDNA evolution in fish. High intra- and interspecific diversity within the 5S rDNA family in all the genera can be explained by a combination of duplications, deletions, and transposition events. Sequence blocks with high similarity in all the 5S rDNA members across species were identified for the four studied genera, with evidences of intense gene conversion within noncoding regions. We propose a model to explain the evolution of the 5S rDNA, in which the evolutionary units are blocks of nucleotides rather than the entire sequences or single nucleotides. This model implies a "two-speed" evolution: slow within blocks (homogenized by recombination) and fast within the gene family (diversified by duplications and deletions).

A Pelagic Microbiome (Viruses to Protists) from a Small Cup of Seawater.

PubMed

Flaviani, Flavia; Schroeder, Declan C; Balestreri, Cecilia; Schroeder, Joanna L; Moore, Karen; Paszkiewicz, Konrad; Pfaff, Maya C; Rybicki, Edward P

2017-03-17

The aquatic microbiome is composed of a multi-phylotype community of microbes, ranging from the numerically dominant viruses to the phylogenetically diverse unicellular phytoplankton. They influence key biogeochemical processes and form the base of marine food webs, becoming food for secondary consumers. Due to recent advances in next-generation sequencing, this previously overlooked component of our hydrosphere is starting to reveal its true diversity and biological complexity. We report here that 250 mL of seawater is sufficient to provide a comprehensive description of the microbial diversity in an oceanic environment. We found that there was a dominance of the order Caudovirales (59%), with the family Myoviridae being the most prevalent. The families Phycodnaviridae and Mimiviridae made up the remainder of pelagic double-stranded DNA (dsDNA) virome. Consistent with this analysis, the Cyanobacteria dominate (52%) the prokaryotic diversity. While the dinoflagellates and their endosymbionts, the superphylum Alveolata dominates (92%) the microbial eukaryotic diversity. A total of 834 prokaryotic, 346 eukaryotic and 254 unique virus phylotypes were recorded in this relatively small sample of water. We also provide evidence, through a metagenomic-barcoding comparative analysis, that viruses are the likely source of microbial environmental DNA (meDNA). This study opens the door to a more integrated approach to oceanographic sampling and data analysis.

The use of museum specimens with high-throughput DNA sequencers

PubMed Central

Burrell, Andrew S.; Disotell, Todd R.; Bergey, Christina M.

2015-01-01

Natural history collections have long been used by morphologists, anatomists, and taxonomists to probe the evolutionary process and describe biological diversity. These biological archives also offer great opportunities for genetic research in taxonomy, conservation, systematics, and population biology. They allow assays of past populations, including those of extinct species, giving context to present patterns of genetic variation and direct measures of evolutionary processes. Despite this potential, museum specimens are difficult to work with because natural postmortem processes and preservation methods fragment and damage DNA. These problems have restricted geneticists’ ability to use natural history collections primarily by limiting how much of the genome can be surveyed. Recent advances in DNA sequencing technology, however, have radically changed this, making truly genomic studies from museum specimens possible. We review the opportunities and drawbacks of the use of museum specimens, and suggest how to best execute projects when incorporating such samples. Several high-throughput (HT) sequencing methodologies, including whole genome shotgun sequencing, sequence capture, and restriction digests (demonstrated here), can be used with archived biomaterials. PMID:25532801

Evidence for Interspecies Gene Transfer in the Evolution of 2,4-Dichlorophenoxyacetic Acid Degraders

PubMed Central

McGowan, Catherine; Fulthorpe, Roberta; Wright, Alice; Tiedje, J. M.

1998-01-01

Small-subunit ribosomal DNA (SSU rDNA) from 20 phenotypically distinct strains of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was partially sequenced, yielding 18 unique strains belonging to members of the alpha, beta, and gamma subgroups of the class Proteobacteria. To understand the origin of 2,4-D degradation in this diverse collection, the first gene in the 2,4-D pathway, tfdA, was sequenced. The sequences fell into three unique classes found in various members of the beta and gamma subgroups of Proteobacteria. None of the α-Proteobacteria yielded tfdA PCR products. A comparison of the dendrogram of the tfdA genes with that of the SSU rDNA genes demonstrated incongruency in phylogenies, and hence 2,4-D degradation must have originated from gene transfer between species. Only those strains with tfdA sequences highly similar to the tfdA sequence of strain JMP134 (tfdA class I) transferred all the 2,4-D genes and conferred the 2,4-D degradation phenotype to a Burkholderia cepacia recipient. PMID:9758850

Short-read DNA sequencing yields microsatellite markers for Rheum

USDA-ARS?s Scientific Manuscript database

Identifying culinary rhubarb (Rheum ×hybridum Murray) cultivars using morphological characteristics is problematic due to variability within individual genotypes, variation caused by environmental factors, plant and leaf age, similarity between genetically diverse genotypes, multiple cultivar names ...

Molecular Technique to Understand Deep Microbial Diversity

NASA Technical Reports Server (NTRS)

Vaishampayan, Parag A.; Venkateswaran, Kasthuri J.

2012-01-01

Current sequencing-based and DNA microarray techniques to study microbial diversity are based on an initial PCR (polymerase chain reaction) amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of the minor template appears to be suppressed by the exponential amplification of the more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck has overlooked the presence of the less abundant minority population, and underestimated their role in the ecosystem maintenance. To generate PCR amplicons for subsequent diversity analysis, bacterial l6S rRNA genes are amplified by PCR using universal primers. Two distinct PCR regimes are employed in parallel: one using normal and the other using biotinlabeled universal primers. PCR products obtained with biotin-labeled primers are mixed with streptavidin-labeled magnetic beads and selectively captured in the presence of a magnetic field. Less-abundant DNA templates that fail to amplify in this first round of PCR amplification are subjected to a second round of PCR using normal universal primers. These PCR products are then subjected to downstream diversity analyses such as conventional cloning and sequencing. A second round of PCR amplified the minority population and completed the deep diversity picture of the environmental sample.

Bacterial diversity in the active stage of a bioremediation system for mineral oil hydrocarbon-contaminated soils.

PubMed

Popp, Nicole; Schlömann, Michael; Mau, Margit

2006-11-01

Soils contaminated with mineral oil hydrocarbons are often cleaned in off-site bioremediation systems. In order to find out which bacteria are active during the degradation phase in such systems, the diversity of the active microflora in a degrading soil remediation system was investigated by small-subunit (SSU) rRNA analysis. Two sequential RNA extracts from one soil sample were generated by a procedure incorporating bead beating. Both extracts were analysed separately by generating individual SSU rDNA clone libraries from cDNA of the two extracts. The sequencing results showed moderate diversity. The two clone libraries were dominated by Gammaproteobacteria, especially Pseudomonas spp. Alphaproteobacteria and Betaproteobacteria were two other large groups in the clone libraries. Actinobacteria, Firmicutes, Bacteroidetes and Epsilonproteobacteria were detected in lower numbers. The obtained sequences were predominantly related to genera for which cultivated representatives have been described, but were often clustered together in the phylogenetic tree, and the sequences that were most similar were originally obtained from soils and not from pure cultures. Most of the dominant genera in the clone libraries, e.g. Pseudomonas, Acinetobacter, Sphingomonas, Acidovorax and Thiobacillus, had already been detected in (mineral oil hydrocarbon) contaminated environmental samples. The occurrence of the genera Zymomonas and Rhodoferax was novel in mineral oil hydrocarbon-contaminated soil.

Molecular Phylogenetic Diversity and Spatial Distribution of Bacterial Communities in Cooling Stage during Swine Manure Composting

PubMed Central

Guo, Yan; Zhang, Jinliang; Yan, Yongfeng; Wu, Jian; Zhu, Nengwu; Deng, Changyan

2015-01-01

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and subsequent sub-cloning and sequencing were used in this study to analyze the molecular phylogenetic diversity and spatial distribution of bacterial communities in different spatial locations during the cooling stage of composted swine manure. Total microbial DNA was extracted, and bacterial near full-length 16S rRNA genes were subsequently amplified, cloned, RFLP-screened, and sequenced. A total of 420 positive clones were classified by RFLP and near-full-length 16S rDNA sequences. Approximately 48 operational taxonomic units (OTUs) were found among 139 positive clones from the superstratum sample; 26 among 149 were from the middle-level sample and 35 among 132 were from the substrate sample. Thermobifida fusca was common in the superstratum layer of the pile. Some Bacillus spp. were remarkable in the middle-level layer, and Clostridium sp. was dominant in the substrate layer. Among 109 OTUs, 99 displayed homology with those in the GenBank database. Ten OTUs were not closely related to any known species. The superstratum sample had the highest microbial diversity, and different and distinct bacterial communities were detected in the three different layers. This study demonstrated the spatial characteristics of the microbial community distribution in the cooling stage of swine manure compost. PMID:25925066

Trading genes along the silk road: mtDNA sequences and the origin of central Asian populations.

PubMed Central

Comas, D; Calafell, F; Mateu, E; Pérez-Lezaun, A; Bosch, E; Martínez-Arias, R; Clarimon, J; Facchini, F; Fiori, G; Luiselli, D; Pettener, D; Bertranpetit, J

1998-01-01

Central Asia is a vast region at the crossroads of different habitats, cultures, and trade routes. Little is known about the genetics and the history of the population of this region. We present the analysis of mtDNA control-region sequences in samples of the Kazakh, the Uighurs, the lowland Kirghiz, and the highland Kirghiz, which we have used to address both the population history of the region and the possible selective pressures that high altitude has on mtDNA genes. Central Asian mtDNA sequences present features intermediate between European and eastern Asian sequences, in several parameters-such as the frequencies of certain nucleotides, the levels of nucleotide diversity, mean pairwise differences, and genetic distances. Several hypotheses could explain the intermediate position of central Asia between Europe and eastern Asia, but the most plausible would involve extensive levels of admixture between Europeans and eastern Asians in central Asia, possibly enhanced during the Silk Road trade and clearly after the eastern and western Eurasian human groups had diverged. Lowland and highland Kirghiz mtDNA sequences are very similar, and the analysis of molecular variance has revealed that the fraction of mitochondrial genetic variance due to altitude is not significantly different from zero. Thus, it seems unlikely that altitude has exerted a major selective pressure on mitochondrial genes in central Asian populations. PMID:9837835

Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups.

PubMed

Herrnstadt, Corinna; Elson, Joanna L; Fahy, Eoin; Preston, Gwen; Turnbull, Douglass M; Anderson, Christen; Ghosh, Soumitra S; Olefsky, Jerrold M; Beal, M Flint; Davis, Robert E; Howell, Neil

2002-05-01

The evolution of the human mitochondrial genome is characterized by the emergence of ethnically distinct lineages or haplogroups. Nine European, seven Asian (including Native American), and three African mitochondrial DNA (mtDNA) haplogroups have been identified previously on the basis of the presence or absence of a relatively small number of restriction-enzyme recognition sites or on the basis of nucleotide sequences of the D-loop region. We have used reduced-median-network approaches to analyze 560 complete European, Asian, and African mtDNA coding-region sequences from unrelated individuals to develop a more complete understanding of sequence diversity both within and between haplogroups. A total of 497 haplogroup-associated polymorphisms were identified, 323 (65%) of which were associated with one haplogroup and 174 (35%) of which were associated with two or more haplogroups. Approximately one-half of these polymorphisms are reported for the first time here. Our results confirm and substantially extend the phylogenetic relationships among mitochondrial genomes described elsewhere from the major human ethnic groups. Another important result is that there were numerous instances both of parallel mutations at the same site and of reversion (i.e., homoplasy). It is likely that homoplasy in the coding region will confound evolutionary analysis of small sequence sets. By a linkage-disequilibrium approach, additional evidence for the absence of human mtDNA recombination is presented here.

Estimating Bacterial Diversity for Ecological Studies: Methods, Metrics, and Assumptions

PubMed Central

Birtel, Julia; Walser, Jean-Claude; Pichon, Samuel; Bürgmann, Helmut; Matthews, Blake

2015-01-01

Methods to estimate microbial diversity have developed rapidly in an effort to understand the distribution and diversity of microorganisms in natural environments. For bacterial communities, the 16S rRNA gene is the phylogenetic marker gene of choice, but most studies select only a specific region of the 16S rRNA to estimate bacterial diversity. Whereas biases derived from from DNA extraction, primer choice and PCR amplification are well documented, we here address how the choice of variable region can influence a wide range of standard ecological metrics, such as species richness, phylogenetic diversity, β-diversity and rank-abundance distributions. We have used Illumina paired-end sequencing to estimate the bacterial diversity of 20 natural lakes across Switzerland derived from three trimmed variable 16S rRNA regions (V3, V4, V5). Species richness, phylogenetic diversity, community composition, β-diversity, and rank-abundance distributions differed significantly between 16S rRNA regions. Overall, patterns of diversity quantified by the V3 and V5 regions were more similar to one another than those assessed by the V4 region. Similar results were obtained when analyzing the datasets with different sequence similarity thresholds used during sequences clustering and when the same analysis was used on a reference dataset of sequences from the Greengenes database. In addition we also measured species richness from the same lake samples using ARISA Fingerprinting, but did not find a strong relationship between species richness estimated by Illumina and ARISA. We conclude that the selection of 16S rRNA region significantly influences the estimation of bacterial diversity and species distributions and that caution is warranted when comparing data from different variable regions as well as when using different sequencing techniques. PMID:25915756

Location analysis for the estrogen receptor-α reveals binding to diverse ERE sequences and widespread binding within repetitive DNA elements

PubMed Central

Mason, Christopher E.; Shu, Feng-Jue; Wang, Cheng; Session, Ryan M.; Kallen, Roland G.; Sidell, Neil; Yu, Tianwei; Liu, Mei Hui; Cheung, Edwin; Kallen, Caleb B.

2010-01-01

Location analysis for estrogen receptor-α (ERα)-bound cis-regulatory elements was determined in MCF7 cells using chromatin immunoprecipitation (ChIP)-on-chip. Here, we present the estrogen response element (ERE) sequences that were identified at ERα-bound loci and quantify the incidence of ERE sequences under two stringencies of detection: <10% and 10–20% nucleotide deviation from the canonical ERE sequence. We demonstrate that ∼50% of all ERα-bound loci do not have a discernable ERE and show that most ERα-bound EREs are not perfect consensus EREs. Approximately one-third of all ERα-bound ERE sequences reside within repetitive DNA sequences, most commonly of the AluS family. In addition, the 3-bp spacer between the inverted ERE half-sites, rather than being random nucleotides, is C(A/T)G-enriched at bona fide receptor targets. Diverse ERα-bound loci were validated using electrophoretic mobility shift assay and ChIP-polymerase chain reaction (PCR). The functional significance of receptor-bound loci was demonstrated using luciferase reporter assays which proved that repetitive element ERE sequences contribute to enhancer function. ChIP-PCR demonstrated estrogen-dependent recruitment of the coactivator SRC3 to these loci in vivo. Our data demonstrate that ERα binds to widely variant EREs with less sequence specificity than had previously been suspected and that binding at repetitive and nonrepetitive genomic targets is favored by specific trinucleotide spacers. PMID:20047966

Location analysis for the estrogen receptor-alpha reveals binding to diverse ERE sequences and widespread binding within repetitive DNA elements.

PubMed

Mason, Christopher E; Shu, Feng-Jue; Wang, Cheng; Session, Ryan M; Kallen, Roland G; Sidell, Neil; Yu, Tianwei; Liu, Mei Hui; Cheung, Edwin; Kallen, Caleb B

2010-04-01

Location analysis for estrogen receptor-alpha (ERalpha)-bound cis-regulatory elements was determined in MCF7 cells using chromatin immunoprecipitation (ChIP)-on-chip. Here, we present the estrogen response element (ERE) sequences that were identified at ERalpha-bound loci and quantify the incidence of ERE sequences under two stringencies of detection: <10% and 10-20% nucleotide deviation from the canonical ERE sequence. We demonstrate that approximately 50% of all ERalpha-bound loci do not have a discernable ERE and show that most ERalpha-bound EREs are not perfect consensus EREs. Approximately one-third of all ERalpha-bound ERE sequences reside within repetitive DNA sequences, most commonly of the AluS family. In addition, the 3-bp spacer between the inverted ERE half-sites, rather than being random nucleotides, is C(A/T)G-enriched at bona fide receptor targets. Diverse ERalpha-bound loci were validated using electrophoretic mobility shift assay and ChIP-polymerase chain reaction (PCR). The functional significance of receptor-bound loci was demonstrated using luciferase reporter assays which proved that repetitive element ERE sequences contribute to enhancer function. ChIP-PCR demonstrated estrogen-dependent recruitment of the coactivator SRC3 to these loci in vivo. Our data demonstrate that ERalpha binds to widely variant EREs with less sequence specificity than had previously been suspected and that binding at repetitive and nonrepetitive genomic targets is favored by specific trinucleotide spacers.

A diverse family of serine proteinase genes expressed in cotton boll weevil (Anthonomus grandis): implications for the design of pest-resistant transgenic cotton plants.

PubMed

Oliveira-Neto, Osmundo B; Batista, João A N; Rigden, Daniel J; Fragoso, Rodrigo R; Silva, Rodrigo O; Gomes, Eliane A; Franco, Octávio L; Dias, Simoni C; Cordeiro, Célia M T; Monnerat, Rose G; Grossi-De-Sá, Maria F

2004-09-01

Fourteen different cDNA fragments encoding serine proteinases were isolated by reverse transcription-PCR from cotton boll weevil (Anthonomus grandis) larvae. A large diversity between the sequences was observed, with a mean pairwise identity of 22% in the amino acid sequence. The cDNAs encompassed 11 trypsin-like sequences classifiable into three families and three chymotrypsin-like sequences belonging to a single family. Using a combination of 5' and 3' RACE, the full-length sequence was obtained for five of the cDNAs, named Agser2, Agser5, Agser6, Agser10 and Agser21. The encoded proteins included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Southern blotting analysis suggested that one or two copies of these serine proteinase genes exist in the A. grandis genome. Northern blotting analysis of Agser2 and Agser5 showed that for both genes, expression is induced upon feeding and is concentrated in the gut of larvae and adult insects. Reverse northern analysis of the 14 cDNA fragments showed that only two trypsin-like and two chymotrypsin-like were expressed at detectable levels. Under the effect of the serine proteinase inhibitors soybean Kunitz trypsin inhibitor and black-eyed pea trypsin/chymotrypsin inhibitor, expression of one of the trypsin-like sequences was upregulated while expression of the two chymotrypsin-like sequences was downregulated. Copyright 2004 Elsevier Ltd.

Computational Design of DNA-Binding Proteins.

PubMed

Thyme, Summer; Song, Yifan

2016-01-01

Predicting the outcome of engineered and naturally occurring sequence perturbations to protein-DNA interfaces requires accurate computational modeling technologies. It has been well established that computational design to accommodate small numbers of DNA target site substitutions is possible. This chapter details the basic method of design used in the Rosetta macromolecular modeling program that has been successfully used to modulate the specificity of DNA-binding proteins. More recently, combining computational design and directed evolution has become a common approach for increasing the success rate of protein engineering projects. The power of such high-throughput screening depends on computational methods producing multiple potential solutions. Therefore, this chapter describes several protocols for increasing the diversity of designed output. Lastly, we describe an approach for building comparative models of protein-DNA complexes in order to utilize information from homologous sequences. These models can be used to explore how nature modulates specificity of protein-DNA interfaces and potentially can even be used as starting templates for further engineering.

Analyzing Mosquito (Diptera: Culicidae) Diversity in Pakistan by DNA Barcoding

PubMed Central

Ashfaq, Muhammad; Hebert, Paul D. N.; Mirza, Jawwad H.; Khan, Arif M.; Zafar, Yusuf; Mirza, M. Sajjad

2014-01-01

Background Although they are important disease vectors mosquito biodiversity in Pakistan is poorly known. Recent epidemics of dengue fever have revealed the need for more detailed understanding of the diversity and distributions of mosquito species in this region. DNA barcoding improves the accuracy of mosquito inventories because morphological differences between many species are subtle, leading to misidentifications. Methodology/Principal Findings Sequence variation in the barcode region of the mitochondrial COI gene was used to identify mosquito species, reveal genetic diversity, and map the distribution of the dengue-vector species in Pakistan. Analysis of 1684 mosquitoes from 491 sites in Punjab and Khyber Pakhtunkhwa during 2010–2013 revealed 32 species with the assemblage dominated by Culex quinquefasciatus (61% of the collection). The genus Aedes (Stegomyia) comprised 15% of the specimens, and was represented by six taxa with the two dengue vector species, Ae. albopictus and Ae. aegypti, dominant and broadly distributed. Anopheles made up another 6% of the catch with An. subpictus dominating. Barcode sequence divergence in conspecific specimens ranged from 0–2.4%, while congeneric species showed from 2.3–17.8% divergence. A global haplotype analysis of disease-vectors showed the presence of multiple haplotypes, although a single haplotype of each dengue-vector species was dominant in most countries. Geographic distribution of Ae. aegypti and Ae. albopictus showed the later species was dominant and found in both rural and urban environments. Conclusions As the first DNA-based analysis of mosquitoes in Pakistan, this study has begun the construction of a barcode reference library for the mosquitoes of this region. Levels of genetic diversity varied among species. Because of its capacity to differentiate species, even those with subtle morphological differences, DNA barcoding aids accurate tracking of vector populations. PMID:24827460

Analyzing mosquito (Diptera: culicidae) diversity in Pakistan by DNA barcoding.

PubMed

Ashfaq, Muhammad; Hebert, Paul D N; Mirza, Jawwad H; Khan, Arif M; Zafar, Yusuf; Mirza, M Sajjad

2014-01-01

Although they are important disease vectors mosquito biodiversity in Pakistan is poorly known. Recent epidemics of dengue fever have revealed the need for more detailed understanding of the diversity and distributions of mosquito species in this region. DNA barcoding improves the accuracy of mosquito inventories because morphological differences between many species are subtle, leading to misidentifications. Sequence variation in the barcode region of the mitochondrial COI gene was used to identify mosquito species, reveal genetic diversity, and map the distribution of the dengue-vector species in Pakistan. Analysis of 1684 mosquitoes from 491 sites in Punjab and Khyber Pakhtunkhwa during 2010-2013 revealed 32 species with the assemblage dominated by Culex quinquefasciatus (61% of the collection). The genus Aedes (Stegomyia) comprised 15% of the specimens, and was represented by six taxa with the two dengue vector species, Ae. albopictus and Ae. aegypti, dominant and broadly distributed. Anopheles made up another 6% of the catch with An. subpictus dominating. Barcode sequence divergence in conspecific specimens ranged from 0-2.4%, while congeneric species showed from 2.3-17.8% divergence. A global haplotype analysis of disease-vectors showed the presence of multiple haplotypes, although a single haplotype of each dengue-vector species was dominant in most countries. Geographic distribution of Ae. aegypti and Ae. albopictus showed the later species was dominant and found in both rural and urban environments. As the first DNA-based analysis of mosquitoes in Pakistan, this study has begun the construction of a barcode reference library for the mosquitoes of this region. Levels of genetic diversity varied among species. Because of its capacity to differentiate species, even those with subtle morphological differences, DNA barcoding aids accurate tracking of vector populations.

Diversity of Virophages in Metagenomic Data Sets

PubMed Central

Zhou, Jinglie; Zhang, Weijia; Yan, Shuling; Xiao, Jinzhou; Zhang, Yuanyuan; Li, Bailin; Pan, Yingjie

2013-01-01

Virophages, e.g., Sputnik, Mavirus, and Organic Lake virophage (OLV), are unusual parasites of giant double-stranded DNA (dsDNA) viruses, yet little is known about their diversity. Here, we describe the global distribution, abundance, and genetic diversity of virophages based on analyzing and mapping comprehensive metagenomic databases. The results reveal a distinct abundance and worldwide distribution of virophages, involving almost all geographical zones and a variety of unique environments. These environments ranged from deep ocean to inland, iced to hydrothermal lakes, and human gut- to animal-associated habitats. Four complete virophage genomic sequences (Yellowstone Lake virophages [YSLVs]) were obtained, as was one nearly complete sequence (Ace Lake Mavirus [ALM]). The genomes obtained were 27,849 bp long with 26 predicted open reading frames (ORFs) (YSLV1), 23,184 bp with 21 ORFs (YSLV2), 27,050 bp with 23 ORFs (YSLV3), 28,306 bp with 34 ORFs (YSLV4), and 17,767 bp with 22 ORFs (ALM). The homologous counterparts of five genes, including putative FtsK-HerA family DNA packaging ATPase and genes encoding DNA helicase/primase, cysteine protease, major capsid protein (MCP), and minor capsid protein (mCP), were present in all virophages studied thus far. They also shared a conserved gene cluster comprising the two core genes of MCP and mCP. Comparative genomic and phylogenetic analyses showed that YSLVs, having a closer relationship to each other than to the other virophages, were more closely related to OLV than to Sputnik but distantly related to Mavirus and ALM. These findings indicate that virophages appear to be widespread and genetically diverse, with at least 3 major lineages. PMID:23408616

Patterns of Adaptive and Neutral Diversity Identify the Xiaoxiangling Mountains as a Refuge for the Giant Panda

PubMed Central

Wan, Qiu-Hong; Lou, Ji-Kang; Li, Wen-Jing; Ge, Yun-Fa; Fang, Sheng-Guo

2013-01-01

Genetic variation plays a significant role in maintaining the evolutionary potential of a species. Comparing the patterns of adaptive and neutral diversity in extant populations is useful for understanding the local adaptations of a species. In this study, we determined the fine-scale genetic structure of 6 extant populations of the giant panda (Ailuropoda melanoleuca) using mtDNA and DNA fingerprints, and then overlaid adaptive variations in 6 functional Aime-MHC class II genes (DRA, DRB3, DQA1, DQA2, DQB1, and DQB2) on this framework. We found that: (1) analysis of the mtDNA and DNA fingerprint-based networks of the 6 populations identified the independent evolutionary histories of the 2 panda subspecies; (2) the basal (ancestral) branches of the fingerprint-based Sichuan-derived network all originated from the smallest Xiaoxiangling (XXL) population, suggesting the status of a glacial refuge in XXL; (3) the MHC variations among the tested populations showed that the XXL population exhibited extraordinary high levels of MHC diversity in allelic richness, which is consistent with the diversity characteristics of a glacial refuge; (4) the phylogenetic tree showed that the basal clades of giant panda DQB sequences were all occupied by XXL-specific sequences, providing evidence for the ancestor-resembling traits of XXL. Finally, we found that the giant panda had many more DQ alleles than DR alleles (33∶13), contrary to other mammals, and that the XXL refuge showed special characteristics in the DQB loci, with 7 DQB members of 9 XXL-unique alleles. Thus, this study identified XXL as a glacial refuge, specifically harboring the most number of primitive DQB alleles. PMID:23894623

Patterns of adaptive and neutral diversity identify the Xiaoxiangling mountains as a refuge for the giant panda.

PubMed

Chen, Yi-Yan; Zhu, Ying; Wan, Qiu-Hong; Lou, Ji-Kang; Li, Wen-Jing; Ge, Yun-Fa; Fang, Sheng-Guo

2013-01-01

Genetic variation plays a significant role in maintaining the evolutionary potential of a species. Comparing the patterns of adaptive and neutral diversity in extant populations is useful for understanding the local adaptations of a species. In this study, we determined the fine-scale genetic structure of 6 extant populations of the giant panda (Ailuropoda melanoleuca) using mtDNA and DNA fingerprints, and then overlaid adaptive variations in 6 functional Aime-MHC class II genes (DRA, DRB3, DQA1, DQA2, DQB1, and DQB2) on this framework. We found that: (1) analysis of the mtDNA and DNA fingerprint-based networks of the 6 populations identified the independent evolutionary histories of the 2 panda subspecies; (2) the basal (ancestral) branches of the fingerprint-based Sichuan-derived network all originated from the smallest Xiaoxiangling (XXL) population, suggesting the status of a glacial refuge in XXL; (3) the MHC variations among the tested populations showed that the XXL population exhibited extraordinary high levels of MHC diversity in allelic richness, which is consistent with the diversity characteristics of a glacial refuge; (4) the phylogenetic tree showed that the basal clades of giant panda DQB sequences were all occupied by XXL-specific sequences, providing evidence for the ancestor-resembling traits of XXL. Finally, we found that the giant panda had many more DQ alleles than DR alleles (33∶13), contrary to other mammals, and that the XXL refuge showed special characteristics in the DQB loci, with 7 DQB members of 9 XXL-unique alleles. Thus, this study identified XXL as a glacial refuge, specifically harboring the most number of primitive DQB alleles.

Diversity arrays technology: a generic genome profiling technology on open platforms.

PubMed

Kilian, Andrzej; Wenzl, Peter; Huttner, Eric; Carling, Jason; Xia, Ling; Blois, Hélène; Caig, Vanessa; Heller-Uszynska, Katarzyna; Jaccoud, Damian; Hopper, Colleen; Aschenbrenner-Kilian, Malgorzata; Evers, Margaret; Peng, Kaiman; Cayla, Cyril; Hok, Puthick; Uszynski, Grzegorz

2012-01-01

In the last 20 years, we have observed an exponential growth of the DNA sequence data and simular increase in the volume of DNA polymorphism data generated by numerous molecular marker technologies. Most of the investment, and therefore progress, concentrated on human genome and genomes of selected model species. Diversity Arrays Technology (DArT), developed over a decade ago, was among the first "democratizing" genotyping technologies, as its performance was primarily driven by the level of DNA sequence variation in the species rather than by the level of financial investment. DArT also proved more robust to genome size and ploidy-level differences among approximately 60 organisms for which DArT was developed to date compared to other high-throughput genotyping technologies. The success of DArT in a number of organisms, including a wide range of "orphan crops," can be attributed to the simplicity of underlying concepts: DArT combines genome complexity reduction methods enriching for genic regions with a highly parallel assay readout on a number of "open-access" microarray platforms. The quantitative nature of the assay enabled a number of applications in which allelic frequencies can be estimated from DArT arrays. A typical DArT assay tests for polymorphism tens of thousands of genomic loci with the final number of markers reported (hundreds to thousands) reflecting the level of DNA sequence variation in the tested loci. Detailed DArT methods, protocols, and a range of their application examples as well as DArT's evolution path are presented.

MtDNA genetic diversity and structure of Eurasian Collared Dove (Streptopelia decaocto).

PubMed

Bagi, Zoltán; Dimopoulos, Evangelos Antonis; Loukovitis, Dimitrios; Eraud, Cyril; Kusza, Szilvia

2018-01-01

The Eurasian Collared Dove (Streptopelia decaocto) is one of the most successful biological invaders among terrestrial vertebrates. However, little information is available on the genetic diversity of the species. A total of 134 Eurasian Collared Doves from Europe, Asia and the Caribbean (n = 20) were studied by sequencing a 658-bp length of mitochondrial DNA (mtDNA) cytochrome oxidase I (COI). Fifty-two different haplotypes and relatively high haplotype and nucleotide diversities (Hd±SD = 0.843±0.037 and π±SD = 0.026±0.013) were detected. Haplotype Ht1 was particularly dominant: it included 44.03% of the studied individuals, and contained sequences from 75% of the studied countries. Various analyses (FST, AMOVA, STRUCTURE) distinguished 2 groups on the genetic level, designated 'A' and 'B'. Two groups were also separated in the median-joining network and the maximum likelihood tree. The results of the neutrality tests were negative (Fu FS = -25.914; Tajima D = -2.606) and significantly different from zero (P≤0.001) for group A, whereas both values for group B were positive (Fu FS = 1.811; Tajima D = 0.674) and not significant (P>0.05). Statistically significant positive autocorrelation was revealed among individuals located up to 2000 km apart (r = 0.124; P = 0.001). The present results provide the first information on the genetic diversity and structure of the Eurasian Collared Dove, and can thereby serve as a factual and comparative basis for similar studies in the future.

Real-time DNA barcoding in a rainforest using nanopore sequencing: opportunities for rapid biodiversity assessments and local capacity building.

PubMed

Pomerantz, Aaron; Peñafiel, Nicolás; Arteaga, Alejandro; Bustamante, Lucas; Pichardo, Frank; Coloma, Luis A; Barrio-Amorós, César L; Salazar-Valenzuela, David; Prost, Stefan

2018-04-01

Advancements in portable scientific instruments provide promising avenues to expedite field work in order to understand the diverse array of organisms that inhabit our planet. Here, we tested the feasibility for in situ molecular analyses of endemic fauna using a portable laboratory fitting within a single backpack in one of the world's most imperiled biodiversity hotspots, the Ecuadorian Chocó rainforest. We used portable equipment, including the MinION nanopore sequencer (Oxford Nanopore Technologies) and the miniPCR (miniPCR), to perform DNA extraction, polymerase chain reaction amplification, and real-time DNA barcoding of reptile specimens in the field. We demonstrate that nanopore sequencing can be implemented in a remote tropical forest to quickly and accurately identify species using DNA barcoding, as we generated consensus sequences for species resolution with an accuracy of >99% in less than 24 hours after collecting specimens. The flexibility of our mobile laboratory further allowed us to generate sequence information at the Universidad Tecnológica Indoamérica in Quito for rare, endangered, and undescribed species. This includes the recently rediscovered Jambato toad, which was thought to be extinct for 28 years. Sequences generated on the MinION required as few as 30 reads to achieve high accuracy relative to Sanger sequencing, and with further multiplexing of samples, nanopore sequencing can become a cost-effective approach for rapid and portable DNA barcoding. Overall, we establish how mobile laboratories and nanopore sequencing can help to accelerate species identification in remote areas to aid in conservation efforts and be applied to research facilities in developing countries. This opens up possibilities for biodiversity studies by promoting local research capacity building, teaching nonspecialists and students about the environment, tackling wildlife crime, and promoting conservation via research-focused ecotourism.

Sequencing degraded DNA from non-destructively sampled museum specimens for RAD-tagging and low-coverage shotgun phylogenetics.

PubMed

Tin, Mandy Man-Ying; Economo, Evan Philip; Mikheyev, Alexander Sergeyevich

2014-01-01

Ancient and archival DNA samples are valuable resources for the study of diverse historical processes. In particular, museum specimens provide access to biotas distant in time and space, and can provide insights into ecological and evolutionary changes over time. However, archival specimens are difficult to handle; they are often fragile and irreplaceable, and typically contain only short segments of denatured DNA. Here we present a set of tools for processing such samples for state-of-the-art genetic analysis. First, we report a protocol for minimally destructive DNA extraction of insect museum specimens, which produced sequenceable DNA from all of the samples assayed. The 11 specimens analyzed had fragmented DNA, rarely exceeding 100 bp in length, and could not be amplified by conventional PCR targeting the mitochondrial cytochrome oxidase I gene. Our approach made these samples amenable to analysis with commonly used next-generation sequencing-based molecular analytic tools, including RAD-tagging and shotgun genome re-sequencing. First, we used museum ant specimens from three species, each with its own reference genome, for RAD-tag mapping. Were able to use the degraded DNA sequences, which were sequenced in full, to identify duplicate reads and filter them prior to base calling. Second, we re-sequenced six Hawaiian Drosophila species, with millions of years of divergence, but with only a single available reference genome. Despite a shallow coverage of 0.37 ± 0.42 per base, we could recover a sufficient number of overlapping SNPs to fully resolve the species tree, which was consistent with earlier karyotypic studies, and previous molecular studies, at least in the regions of the tree that these studies could resolve. Although developed for use with degraded DNA, all of these techniques are readily applicable to more recent tissue, and are suitable for liquid handling automation.

Distinct Circular Single-Stranded DNA Viruses Exist in Different Soil Types

PubMed Central

Swanson, Maud M.; Dawson, Lorna; Freitag, Thomas E.; Singh, Brajesh K.; Torrance, Lesley; Mushegian, Arcady R.

2015-01-01

The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors. PMID:25841004

The promise and challenge of high-throughput sequencing of the antibody repertoire

PubMed Central

Georgiou, George; Ippolito, Gregory C; Beausang, John; Busse, Christian E; Wardemann, Hedda; Quake, Stephen R

2014-01-01

Efforts to determine the antibody repertoire encoded by B cells in the blood or lymphoid organs using high-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and are transforming our understanding of humoral immune responses. Information gained from high-throughput DNA sequencing of immunoglobulin genes (Ig-seq) can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development and to understand autoimmunity. Rapid progress in the development of experimental protocols and informatics analysis tools is helping to reduce sequencing artifacts, to achieve more precise quantification of clonal diversity and to extract the most pertinent biological information. That said, broader application of Ig-seq, especially in clinical settings, will require the development of a standardized experimental design framework that will enable the sharing and meta-analysis of sequencing data generated by different laboratories. PMID:24441474

mtDNA sequence diversity in Africa.

PubMed Central

Watson, E.; Bauer, K.; Aman, R.; Weiss, G.; von Haeseler, A.; Pääbo, S.

1996-01-01

mtDNA sequences were determined from 241 individuals from nine ethnic groups in Africa. When they were compared with published data from other groups, it was found that the !Kung, Mbuti, and Biaka show on the order of 10 times more sequence differences between the three groups, as well as between those and the other groups (the Fulbe, Hausa, Tuareg, Songhai, Kanuri, Yoruba, Mandenka, Somali, Tukana, and Kikuyu), than these other groups do between one other. Furthermore, the pairwise sequence distributions, patterns of coalescence events, and numbers of variable positions relative to the mean sequence difference indicate that the former three groups have been of constant size over time, whereas the latter have expanded in size. We suggest that this reflects subsistence patterns in that the populations that have expanded in size are food producers whereas those that have not are hunters and gatherers. PMID:8755932

Application of Ion Torrent Sequencing to the Assessment of the Effect of Alkali Ballast Water Treatment on Microbial Community Diversity

PubMed Central

Fujimoto, Masanori; Moyerbrailean, Gregory A.; Noman, Sifat; Gizicki, Jason P.; Ram, Michal L.; Green, Phyllis A.; Ram, Jeffrey L.

2014-01-01

The impact of NaOH as a ballast water treatment (BWT) on microbial community diversity was assessed using the 16S rRNA gene based Ion Torrent sequencing with its new 400 base chemistry. Ballast water samples from a Great Lakes ship were collected from the intake and discharge of both control and NaOH (pH 12) treated tanks and were analyzed in duplicates. One set of duplicates was treated with the membrane-impermeable DNA cross-linking reagent propidium mono-azide (PMA) prior to PCR amplification to differentiate between live and dead microorganisms. Ion Torrent sequencing generated nearly 580,000 reads for 31 bar-coded samples and revealed alterations of the microbial community structure in ballast water that had been treated with NaOH. Rarefaction analysis of the Ion Torrent sequencing data showed that BWT using NaOH significantly decreased microbial community diversity relative to control discharge (p<0.001). UniFrac distance based principal coordinate analysis (PCoA) plots and UPGMA tree analysis revealed that NaOH-treated ballast water microbial communities differed from both intake communities and control discharge communities. After NaOH treatment, bacteria from the genus Alishewanella became dominant in the NaOH-treated samples, accounting for <0.5% of the total reads in intake samples but more than 50% of the reads in the treated discharge samples. The only apparent difference in microbial community structure between PMA-processed and non-PMA samples occurred in intake water samples, which exhibited a significantly higher amount of PMA-sensitive cyanobacteria/chloroplast 16S rRNA than their corresponding non-PMA total DNA samples. The community assembly obtained using Ion Torrent sequencing was comparable to that obtained from a subset of samples that were also subjected to 454 pyrosequencing. This study showed the efficacy of alkali ballast water treatment in reducing ballast water microbial diversity and demonstrated the application of new Ion Torrent sequencing techniques to microbial community studies. PMID:25222021

Application of ion torrent sequencing to the assessment of the effect of alkali ballast water treatment on microbial community diversity.

PubMed

Fujimoto, Masanori; Moyerbrailean, Gregory A; Noman, Sifat; Gizicki, Jason P; Ram, Michal L; Green, Phyllis A; Ram, Jeffrey L

2014-01-01

The impact of NaOH as a ballast water treatment (BWT) on microbial community diversity was assessed using the 16S rRNA gene based Ion Torrent sequencing with its new 400 base chemistry. Ballast water samples from a Great Lakes ship were collected from the intake and discharge of both control and NaOH (pH 12) treated tanks and were analyzed in duplicates. One set of duplicates was treated with the membrane-impermeable DNA cross-linking reagent propidium mono-azide (PMA) prior to PCR amplification to differentiate between live and dead microorganisms. Ion Torrent sequencing generated nearly 580,000 reads for 31 bar-coded samples and revealed alterations of the microbial community structure in ballast water that had been treated with NaOH. Rarefaction analysis of the Ion Torrent sequencing data showed that BWT using NaOH significantly decreased microbial community diversity relative to control discharge (p<0.001). UniFrac distance based principal coordinate analysis (PCoA) plots and UPGMA tree analysis revealed that NaOH-treated ballast water microbial communities differed from both intake communities and control discharge communities. After NaOH treatment, bacteria from the genus Alishewanella became dominant in the NaOH-treated samples, accounting for <0.5% of the total reads in intake samples but more than 50% of the reads in the treated discharge samples. The only apparent difference in microbial community structure between PMA-processed and non-PMA samples occurred in intake water samples, which exhibited a significantly higher amount of PMA-sensitive cyanobacteria/chloroplast 16S rRNA than their corresponding non-PMA total DNA samples. The community assembly obtained using Ion Torrent sequencing was comparable to that obtained from a subset of samples that were also subjected to 454 pyrosequencing. This study showed the efficacy of alkali ballast water treatment in reducing ballast water microbial diversity and demonstrated the application of new Ion Torrent sequencing techniques to microbial community studies.

Evaluation of genetic diversity in Chinese kale (Brassica oleracea L. var. alboglabra Bailey) by using rapid amplified polymorphic DNA and sequence-related amplified polymorphism markers.

PubMed

Zhang, J; Zhang, L G

2014-02-14

Chinese kale is an original Chinese vegetable of the Cruciferae family. To select suitable parents for hybrid breeding, we thoroughly analyzed the genetic diversity of Chinese kale. Random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) molecular markers were used to evaluate the genetic diversity across 21 Chinese kale accessions from AVRDC and Guangzhou in China. A total of 104 bands were detected by 11 RAPD primers, of which 66 (63.5%) were polymorphic, and 229 polymorphic bands (68.4%) were observed in 335 bands amplified by 17 SRAP primer combinations. The dendrogram showed the grouping of the 21 accessions into 4 main clusters based on RAPD data, and into 6 clusters based on SRAP and combined data (RAPD + SRAP). The clustering of accessions based on SRAP data was consistent with petal colors. The Mantel test indicated a poor fit for the RAPD and SRAP data (r = 0.16). These results have an important implication for Chinese kale germplasm characterization and improvement.

DNA barcoding survey of Trichoderma diversity in soil and litter of the Colombian lowland Amazonian rainforest reveals Trichoderma strigosellum sp. nov. and other species.

PubMed

López-Quintero, Carlos A; Atanasova, Lea; Franco-Molano, A Esperanza; Gams, Walter; Komon-Zelazowska, Monika; Theelen, Bart; Müller, Wally H; Boekhout, Teun; Druzhinina, Irina

2013-11-01

The diversity of Trichoderma (Hypocreales, Ascomycota) colonizing leaf litter as well as the rhizosphere of Garcinia macrophylla (Clusiaceae) was investigated in primary and secondary rain forests in Colombian Amazonia. DNA barcoding of 107 strains based on the internal transcribed spacers 1 and 2 (ITS1 and 2) of the ribosomal RNA gene cluster and the partial sequence of the translation elongation factor 1 alpha (tef1) gene revealed that the diversity of Trichoderma was dominated (71 %) by three common cosmopolitan species, namely Trichoderma harzianum sensu lato (41 %), Trichoderma spirale (17 %) and Trichoderma koningiopsis (13 %). Four ITS 1 and 2 phylotypes (13 strains) could not be identified with certainty. Multigene phylogenetic analysis and phenotype profiling of four strains with an ITS1 and 2 phylotype similar to Trichoderma strigosum revealed a new sister species of the latter that is described here as Trichoderma strigosellum sp. nov. Sequence similarity searches revealed that this species also occurs in soils of Malaysia and Cameroon, suggesting a pantropical distribution.

Exploring the uncultured microeukaryote majority in the oceans: reevaluation of ribogroups within stramenopiles.

PubMed

Massana, Ramon; del Campo, Javier; Sieracki, Michael E; Audic, Stéphane; Logares, Ramiro

2014-04-01

Molecular surveys in planktonic marine systems have unveiled a large novel diversity of small protists. A large part of this diversity belongs to basal heterotrophic stramenopiles and is distributed in a set of polyphyletic ribogroups (described from rDNA sequences) collectively named as MAST (MArine STramenopiles). In the few groups investigated, MAST cells are globally distributed and abundant bacterial grazers, therefore having a putatively large impact on marine ecosystem functioning. The main aim of this study is to reevaluate the MAST ribogroups described so far and to determine whether additional groups can be found. For this purpose, we used traditional and state-of-the-art molecular tools, combining 18S rDNA sequences from publicly available clone libraries, single amplified genomes (SAGs) of planktonic protists, and a pyrosequencing survey from coastal waters and sediments. Our analysis indicated a final set of 18 MAST groups plus 5 new ribogroups within Ochrophyta (named as MOCH). The MAST ribogroups were then analyzed in more detail. Seven were typical of anoxic systems and one of oxic sediments. The rest were clearly members of oxic marine picoplankton. We characterized the genetic diversity within each MAST group and defined subclades for the more diverse (46 subclades in 8 groups). The analyses of sequences within subclades revealed further ecological specializations. Our data provide a renovated framework for phylogenetic classification of the numerous MAST ribogroups and support the notion of a tight link between phylogeny and ecological distribution. These diverse and largely uncultured protists are widespread and ecologically relevant members of marine microbial assemblages.

Metabarcoding monitoring analysis: the pros and cons of using co-extracted environmental DNA and RNA data to assess offshore oil production impacts on benthic communities.

PubMed

Laroche, Olivier; Wood, Susanna A; Tremblay, Louis A; Lear, Gavin; Ellis, Joanne I; Pochon, Xavier

2017-01-01

Sequencing environmental DNA (eDNA) is increasingly being used as an alternative to traditional morphological-based identification to characterize biological assemblages and monitor anthropogenic impacts in marine environments. Most studies only assess eDNA which, compared to eRNA, can persist longer in the environment after cell death. Therefore, eRNA may provide a more immediate census of the environment due to its relatively weaker stability, leading some researchers to advocate for the use of eRNA as an additional, or perhaps superior proxy for portraying ecological changes. A variety of pre-treatment techniques for screening eDNA and eRNA derived operational taxonomic units (OTUs) have been employed prior to statistical analyses, including removing singleton taxa (i.e., OTUs found only once) and discarding those not present in both eDNA and eRNA datasets. In this study, we used bacterial (16S ribosomal RNA gene) and eukaryotic (18S ribosomal RNA gene) eDNA- and eRNA-derived data from benthic communities collected at increasing distances along a transect from an oil production platform (Taranaki, New Zealand). Macro-infauna (visual classification of benthic invertebrates) and physico-chemical data were analyzed in parallel. We tested the effect of removing singleton taxa, and removing taxa not present in the eDNA and eRNA libraries from the same environmental sample (trimmed by shared OTUs), by comparing the impact of the oil production platform on alpha- and beta-diversity of the eDNA/eRNA-based biological assemblages, and by correlating these to the morphologically identified macro-faunal communities and the physico-chemical data. When trimmed by singletons, presence/absence information from eRNA data represented the best proxy to detect changes on species diversity for both bacteria and eukaryotes. However, assessment of quantitative beta-diversity from read abundance information of bacteria eRNA did not, contrary to eDNA, reveal any impact from the oil production activity. Overall, the data appeared more robust when trimmed by shared OTUs, showing a greater effect of the platform on alpha- and beta-diversity. Trimming by shared OTUs likely removes taxa derived from legacy DNA and technical artefacts introduced through reverse transcriptase, polymerase-chain-reaction and sequencing. Findings from our scoping study suggest that metabarcoding-based biomonitoring surveys should, if funds, time and expertise allow, be assessed using both eDNA and eRNA products.

Metabarcoding monitoring analysis: the pros and cons of using co-extracted environmental DNA and RNA data to assess offshore oil production impacts on benthic communities

PubMed Central

Wood, Susanna A.; Tremblay, Louis A.; Lear, Gavin; Ellis, Joanne I.; Pochon, Xavier

2017-01-01

Sequencing environmental DNA (eDNA) is increasingly being used as an alternative to traditional morphological-based identification to characterize biological assemblages and monitor anthropogenic impacts in marine environments. Most studies only assess eDNA which, compared to eRNA, can persist longer in the environment after cell death. Therefore, eRNA may provide a more immediate census of the environment due to its relatively weaker stability, leading some researchers to advocate for the use of eRNA as an additional, or perhaps superior proxy for portraying ecological changes. A variety of pre-treatment techniques for screening eDNA and eRNA derived operational taxonomic units (OTUs) have been employed prior to statistical analyses, including removing singleton taxa (i.e., OTUs found only once) and discarding those not present in both eDNA and eRNA datasets. In this study, we used bacterial (16S ribosomal RNA gene) and eukaryotic (18S ribosomal RNA gene) eDNA- and eRNA-derived data from benthic communities collected at increasing distances along a transect from an oil production platform (Taranaki, New Zealand). Macro-infauna (visual classification of benthic invertebrates) and physico-chemical data were analyzed in parallel. We tested the effect of removing singleton taxa, and removing taxa not present in the eDNA and eRNA libraries from the same environmental sample (trimmed by shared OTUs), by comparing the impact of the oil production platform on alpha- and beta-diversity of the eDNA/eRNA-based biological assemblages, and by correlating these to the morphologically identified macro-faunal communities and the physico-chemical data. When trimmed by singletons, presence/absence information from eRNA data represented the best proxy to detect changes on species diversity for both bacteria and eukaryotes. However, assessment of quantitative beta-diversity from read abundance information of bacteria eRNA did not, contrary to eDNA, reveal any impact from the oil production activity. Overall, the data appeared more robust when trimmed by shared OTUs, showing a greater effect of the platform on alpha- and beta-diversity. Trimming by shared OTUs likely removes taxa derived from legacy DNA and technical artefacts introduced through reverse transcriptase, polymerase-chain-reaction and sequencing. Findings from our scoping study suggest that metabarcoding-based biomonitoring surveys should, if funds, time and expertise allow, be assessed using both eDNA and eRNA products. PMID:28533985

DNA Barcodes for the FIshes of the Narmada, One of India’s Longest Rivers

PubMed Central

Khedkar, Gulab Dattarao; Jamdade, Rahul; Naik, Suresh; David, Lior; Haymer, David

2014-01-01

This study describes the species diversity of fishes of the Narmada River in India. A total of 820 fish specimens were collected from 17 sampling locations across the whole river basin. Fish were taxonomically classified into one of 90 possible species based on morphological characters, and then DNA barcoding was employed using COI gene sequences as a supplemental identification method. A total of 314 different COI sequences were generated, and specimens were confirmed to belong to 85 species representing 63 genera, 34 families and 10 orders. Findings of this study include the identification of five putative cryptic or sibling species and 43 species not previously known from the Narmada River basin. Five species are endemic to India and three are introduced species that had not been previously reported to occur in the Narmada River. Conversely, 43 species previously reported to occur in the Narmada were not found. Genetic diversity and distance values were generated for all of the species within genera, families and orders using Kimura’s 2 parameter distance model followed by the construction of a Neighbor Joining tree. High resolution clusters generated in NJ trees aided the groupings of species corresponding to their genera and families which are in confirmation to the values generated by Automatic Barcode Gap Discovery bioinformatics platform. This aided to decide a threshold value for the discrimination of species boundary from the Narmada River. This study provides an important validation of the use of DNA barcode sequences for monitoring species diversity and changes within complex ecosystems such as the Narmada River. PMID:24991801

DNA barcodes for the fishes of the Narmada, one of India's longest rivers.

PubMed

Khedkar, Gulab Dattarao; Jamdade, Rahul; Naik, Suresh; David, Lior; Haymer, David

2014-01-01

This study describes the species diversity of fishes of the Narmada River in India. A total of 820 fish specimens were collected from 17 sampling locations across the whole river basin. Fish were taxonomically classified into one of 90 possible species based on morphological characters, and then DNA barcoding was employed using COI gene sequences as a supplemental identification method. A total of 314 different COI sequences were generated, and specimens were confirmed to belong to 85 species representing 63 genera, 34 families and 10 orders. Findings of this study include the identification of five putative cryptic or sibling species and 43 species not previously known from the Narmada River basin. Five species are endemic to India and three are introduced species that had not been previously reported to occur in the Narmada River. Conversely, 43 species previously reported to occur in the Narmada were not found. Genetic diversity and distance values were generated for all of the species within genera, families and orders using Kimura's 2 parameter distance model followed by the construction of a Neighbor Joining tree. High resolution clusters generated in NJ trees aided the groupings of species corresponding to their genera and families which are in confirmation to the values generated by Automatic Barcode Gap Discovery bioinformatics platform. This aided to decide a threshold value for the discrimination of species boundary from the Narmada River. This study provides an important validation of the use of DNA barcode sequences for monitoring species diversity and changes within complex ecosystems such as the Narmada River.

Strain-Level Diversity of Secondary Metabolism in Streptomyces albus

PubMed Central

Seipke, Ryan F.

2015-01-01

Streptomyces spp. are robust producers of medicinally-, industrially- and agriculturally-important small molecules. Increased resistance to antibacterial agents and the lack of new antibiotics in the pipeline have led to a renaissance in natural product discovery. This endeavor has benefited from inexpensive high quality DNA sequencing technology, which has generated more than 140 genome sequences for taxonomic type strains and environmental Streptomyces spp. isolates. Many of the sequenced streptomycetes belong to the same species. For instance, Streptomyces albus has been isolated from diverse environmental niches and seven strains have been sequenced, consequently this species has been sequenced more than any other streptomycete, allowing valuable analyses of strain-level diversity in secondary metabolism. Bioinformatics analyses identified a total of 48 unique biosynthetic gene clusters harboured by Streptomyces albus strains. Eighteen of these gene clusters specify the core secondary metabolome of the species. Fourteen of the gene clusters are contained by one or more strain and are considered auxiliary, while 16 of the gene clusters encode the production of putative strain-specific secondary metabolites. Analysis of Streptomyces albus strains suggests that each strain of a Streptomyces species likely harbours at least one strain-specific biosynthetic gene cluster. Importantly, this implies that deep sequencing of a species will not exhaust gene cluster diversity and will continue to yield novelty. PMID:25635820

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain.

PubMed

de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

2014-06-01

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Existing and emerging detection technologies for DNA (Deoxyribonucleic Acid) finger printing, sequencing, bio- and analytical chips: a multidisciplinary development unifying molecular biology, chemical and electronics engineering.

PubMed

Kumar Khanna, Vinod

2007-01-01

The current status and research trends of detection techniques for DNA-based analysis such as DNA finger printing, sequencing, biochips and allied fields are examined. An overview of main detectors is presented vis-à-vis these DNA operations. The biochip method is explained, the role of micro- and nanoelectronic technologies in biochip realization is highlighted, various optical and electrical detection principles employed in biochips are indicated, and the operational mechanisms of these detection devices are described. Although a diversity of biochips for diagnostic and therapeutic applications has been demonstrated in research laboratories worldwide, only some of these chips have entered the clinical market, and more chips are awaiting commercialization. The necessity of tagging is eliminated in refractive-index change based devices, but the basic flaw of indirect nature of most detection methodologies can only be overcome by generic and/or reagentless DNA sensors such as the conductance-based approach and the DNA-single electron transistor (DNA-SET) structure. Devices of the electrical detection-based category are expected to pave the pathway for the next-generation DNA chips. The review provides a comprehensive coverage of the detection technologies for DNA finger printing, sequencing and related techniques, encompassing a variety of methods from the primitive art to the state-of-the-art scenario as well as promising methods for the future.

Deep sequencing is an appropriate tool for the selection of unique Hepatitis C virus (HCV) variants after single genomic amplification.

PubMed

Guinoiseau, Thibault; Moreau, Alain; Hohnadel, Guillaume; Ngo-Giang-Huong, Nicole; Brulard, Celine; Vourc'h, Patrick; Goudeau, Alain; Gaudy-Graffin, Catherine

2017-01-01

Hepatitis C virus (HCV) evolves rapidly in a single host and circulates as a quasispecies wich is a complex mixture of genetically distinct virus's but closely related namely variants. To identify intra-individual diversity and investigate their functional properties in vitro, it is necessary to define their quasispecies composition and isolate the HCV variants. This is possible using single genome amplification (SGA). This technique, based on serially diluted cDNA to amplify a single cDNA molecule (clonal amplicon), has already been used to determine individual HCV diversity. In these studies, positive PCR reactions from SGA were directly sequenced using Sanger technology. The detection of non-clonal amplicons is necessary for excluding them to facilitate further functional analysis. Here, we compared Next Generation Sequencing (NGS) with De Novo assembly and Sanger sequencing for their ability to distinguish clonal and non-clonal amplicons after SGA on one plasma specimen. All amplicons (n = 42) classified as clonal by NGS were also classified as clonal by Sanger sequencing. No double peaks were seen on electropherograms for non-clonal amplicons with position-specific nucleotide variation below 15% by NGS. Altogether, NGS circumvented many of the difficulties encountered when using Sanger sequencing after SGA and is an appropriate tool to reliability select clonal amplicons for further functional studies.

Deep sequencing is an appropriate tool for the selection of unique Hepatitis C virus (HCV) variants after single genomic amplification

PubMed Central

Guinoiseau, Thibault; Moreau, Alain; Hohnadel, Guillaume; Ngo-Giang-Huong, Nicole; Brulard, Celine; Vourc’h, Patrick; Goudeau, Alain; Gaudy-Graffin, Catherine

2017-01-01

Hepatitis C virus (HCV) evolves rapidly in a single host and circulates as a quasispecies wich is a complex mixture of genetically distinct virus’s but closely related namely variants. To identify intra-individual diversity and investigate their functional properties in vitro, it is necessary to define their quasispecies composition and isolate the HCV variants. This is possible using single genome amplification (SGA). This technique, based on serially diluted cDNA to amplify a single cDNA molecule (clonal amplicon), has already been used to determine individual HCV diversity. In these studies, positive PCR reactions from SGA were directly sequenced using Sanger technology. The detection of non-clonal amplicons is necessary for excluding them to facilitate further functional analysis. Here, we compared Next Generation Sequencing (NGS) with De Novo assembly and Sanger sequencing for their ability to distinguish clonal and non-clonal amplicons after SGA on one plasma specimen. All amplicons (n = 42) classified as clonal by NGS were also classified as clonal by Sanger sequencing. No double peaks were seen on electropherograms for non-clonal amplicons with position-specific nucleotide variation below 15% by NGS. Altogether, NGS circumvented many of the difficulties encountered when using Sanger sequencing after SGA and is an appropriate tool to reliability select clonal amplicons for further functional studies. PMID:28362878

Population and forensic genetic analyses of mitochondrial DNA control region variation from six major provinces in the Korean population.

PubMed

Hong, Seung Beom; Kim, Ki Cheol; Kim, Wook

2015-07-01

We generated complete mitochondrial DNA (mtDNA) control region sequences from 704 unrelated individuals residing in six major provinces in Korea. In addition to our earlier survey of the distribution of mtDNA haplogroup variation, a total of 560 different haplotypes characterized by 271 polymorphic sites were identified, of which 473 haplotypes were unique. The gene diversity and random match probability were 0.9989 and 0.0025, respectively. According to the pairwise comparison of the 704 control region sequences, the mean number of pairwise differences between individuals was 13.47±6.06. Based on the result of mtDNA control region sequences, pairwise FST genetic distances revealed genetic homogeneity of the Korean provinces on a peninsular level, except in samples from Jeju Island. This result indicates there may be a need to formulate a local mtDNA database for Jeju Island, to avoid bias in forensic parameter estimates caused by genetic heterogeneity of the population. Thus, the present data may help not only in personal identification but also in determining maternal lineages to provide an expanded and reliable Korean mtDNA database. These data will be available on the EMPOP database via accession number EMP00661. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Genotyping and Molecular Identification of Date Palm Cultivars Using Inter-Simple Sequence Repeat (ISSR) Markers.

PubMed

Ayesh, Basim M

2017-01-01

Molecular markers are credible for the discrimination of genotypes and estimation of the extent of genetic diversity and relatedness in a set of genotypes. Inter-simple sequence repeat (ISSR) markers rapidly reveal high polymorphic fingerprints and have been used frequently to determine the genetic diversity among date palm cultivars. This chapter describes the application of ISSR markers for genotyping of date palm cultivars. The application involves extraction of genomic DNA from the target cultivars with reliable quality and quantity. Subsequently the extracted DNA serves as a template for amplification of genomic regions flanked by inverted simple sequence repeats using a single primer. The similarity of each pair of samples is measured by calculating the number of mono- and polymorphic bands revealed by gel electrophoresis. Matrices constructed for similarity and genetic distance are used to build a phylogenetic tree and cluster analysis, to determine the molecular relatedness of cultivars. The protocol describes 3 out of 9 tested primers consistently amplified 31 loci in 6 date palm cultivars, with 28 polymorphic loci.

Genome-Wide SNP Discovery and Analysis of Genetic Diversity in Farmed Sika Deer (Cervus nippon) in Northeast China Using Double-Digest Restriction Site-Associated DNA Sequencing.

PubMed

Ba, Hengxing; Jia, Boyin; Wang, Guiwu; Yang, Yifeng; Kedem, Gilead; Li, Chunyi

2017-09-07

Sika deer are an economically valuable species owing to their use in traditional Chinese medicine, particularly their velvet antlers. Sika deer in northeast China are mostly farmed in enclosure. Therefore, genetic management of farmed sika deer would benefit from detailed knowledge of their genetic diversity. In this study, we generated over 1.45 billion high-quality paired-end reads (288 Gbp) across 42 unrelated individuals using double-digest restriction site-associated DNA sequencing (ddRAD-seq). A total of 96,188 (29.63%) putative biallelic SNP loci were identified with an average sequencing depth of 23×. Based on the analysis, we found that the majority of the loci had a deficit of heterozygotes (F IS >0) and low values of H obs , which could be due to inbreeding and Wahlund effects. We also developed a collection of high-quality SNP probes that will likely be useful in a variety of applications in genotyping for cervid species in the future. Copyright © 2017 Ba et al.

Genome-Wide SNP Discovery and Analysis of Genetic Diversity in Farmed Sika Deer (Cervus nippon) in Northeast China Using Double-Digest Restriction Site-Associated DNA Sequencing

PubMed Central

Ba, Hengxing; Jia, Boyin; Wang, Guiwu; Yang, Yifeng; Kedem, Gilead; Li, Chunyi

2017-01-01

Sika deer are an economically valuable species owing to their use in traditional Chinese medicine, particularly their velvet antlers. Sika deer in northeast China are mostly farmed in enclosure. Therefore, genetic management of farmed sika deer would benefit from detailed knowledge of their genetic diversity. In this study, we generated over 1.45 billion high-quality paired-end reads (288 Gbp) across 42 unrelated individuals using double-digest restriction site-associated DNA sequencing (ddRAD-seq). A total of 96,188 (29.63%) putative biallelic SNP loci were identified with an average sequencing depth of 23×. Based on the analysis, we found that the majority of the loci had a deficit of heterozygotes (FIS >0) and low values of Hobs, which could be due to inbreeding and Wahlund effects. We also developed a collection of high-quality SNP probes that will likely be useful in a variety of applications in genotyping for cervid species in the future. PMID:28751500

DNA barcode analysis: a comparison of phylogenetic and statistical classification methods.

PubMed

Austerlitz, Frederic; David, Olivier; Schaeffer, Brigitte; Bleakley, Kevin; Olteanu, Madalina; Leblois, Raphael; Veuille, Michel; Laredo, Catherine

2009-11-10

DNA barcoding aims to assign individuals to given species according to their sequence at a small locus, generally part of the CO1 mitochondrial gene. Amongst other issues, this raises the question of how to deal with within-species genetic variability and potential transpecific polymorphism. In this context, we examine several assignation methods belonging to two main categories: (i) phylogenetic methods (neighbour-joining and PhyML) that attempt to account for the genealogical framework of DNA evolution and (ii) supervised classification methods (k-nearest neighbour, CART, random forest and kernel methods). These methods range from basic to elaborate. We investigated the ability of each method to correctly classify query sequences drawn from samples of related species using both simulated and real data. Simulated data sets were generated using coalescent simulations in which we varied the genealogical history, mutation parameter, sample size and number of species. No method was found to be the best in all cases. The simplest method of all, "one nearest neighbour", was found to be the most reliable with respect to changes in the parameters of the data sets. The parameter most influencing the performance of the various methods was molecular diversity of the data. Addition of genetically independent loci--nuclear genes--improved the predictive performance of most methods. The study implies that taxonomists can influence the quality of their analyses either by choosing a method best-adapted to the configuration of their sample, or, given a certain method, increasing the sample size or altering the amount of molecular diversity. This can be achieved either by sequencing more mtDNA or by sequencing additional nuclear genes. In the latter case, they may also have to modify their data analysis method.

Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles

PubMed Central

Nandi, Tannistha; Holden, Matthew T.G.; Didelot, Xavier; Mehershahi, Kurosh; Boddey, Justin A.; Beacham, Ifor; Peak, Ian; Harting, John; Baybayan, Primo; Guo, Yan; Wang, Susana; How, Lee Chee; Sim, Bernice; Essex-Lopresti, Angela; Sarkar-Tyson, Mitali; Nelson, Michelle; Smither, Sophie; Ong, Catherine; Aw, Lay Tin; Hoon, Chua Hui; Michell, Stephen; Studholme, David J.; Titball, Richard; Chen, Swaine L.; Parkhill, Julian

2015-01-01

Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity. PMID:25236617

Veterinary Fusarioses within the United States

USDA-ARS?s Scientific Manuscript database

Multilocus DNA sequence data was used to retrospectively assess the genetic diversity and evolutionary relationships of 67 Fusarium strains from veterinary sources, most of which were from the United States. Molecular phylogenetic analyses revealed that the strains comprised 23 phylogenetically dist...

Dasytricha dominance in Surti buffalo rumen revealed by 18S rRNA sequences and real-time PCR assay.

PubMed

Singh, K M; Tripathi, A K; Pandya, P R; Rank, D N; Kothari, R K; Joshi, C G

2011-09-01

The genetic diversity of protozoa in Surti buffalo rumen was studied by amplified ribosomal DNA restriction analysis, 18S rDNA sequence homology and phylogenetic and Real-time PCR analysis methods. Three animals were fed diet comprised green fodder Napier bajra 21 (Pennisetum purpureum), mature pasture grass (Dicanthium annulatum) and concentrate mixture (20% crude protein, 65% total digestible nutrients). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360 bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. A total of 91 clones were examined and identified 14 different 18S RNA sequences based on PCR-RFLP pattern. These 14 phylotypes were distributed into four genera-based 18S rDNA database sequences and identified as Dasytricha (57 clones), Isotricha (14 clones), Ostracodinium (11 clones) and Polyplastron (9 clones). Phylogenetic analyses were also used to infer the makeup of protozoa communities in the rumen of Surti buffalo. Out of 14 sequences, 8 sequences (69 clones) clustered with the Dasytricha ruminantium-like clone and 4 sequences (13 clones) were also phylogenetically placed with the Isotricha prostoma-like clone. Moreover, 2 phylotypes (9 clones) were related to Polyplastron multivesiculatum-like clone. In addition, the number of 18S rDNA gene copies of Dasytricha ruminantium (0.05% to ciliate protozoa) was higher than Entodinium sp. (2.0 × 10(5) vs. 1.3 × 10(4)) in per ml ruminal fluid.

Bacterial communities in Great Barrier Reef calcareous sediments: Contrasting 16S rDNA libraries from nearshore and outer shelf reefs

NASA Astrophysics Data System (ADS)

Uthicke, S.; McGuire, K.

2007-03-01

Bacterial communities in eight 16S rDNA clone libraries from calcareous sediments were investigated to provide an assessment of the bacterial diversity on sediments of the Great Barrier Reef (GBR) and to investigate differences due to decreased water quality. Sample effort was spread across two locations on each of four coral reefs, with two reefs located nearshore and two reefs on the outer shelf to allow robust statistical comparison of nearshore reefs (subjected to enhanced runoff) and outer shelf reefs (pristine conditions). Out of 221 non-chimeric sequences, 189 (85.5%) were unique and only one sequence occurred in more than one library. Rarefaction analyses and coverage calculations indicated that only a small fraction of the diversity was sampled. Cluster analyses and comparison to published sequences indicated that sequences retrieved belonged to the α, γ and δ subdivision of the Proteobacteria (6.8, 29.4 and 13.6% of the total, respectively), Cytophaga-Flavobacteria-Bacteroidetes (CFB) group (20.4%), Cyanobacteria (5.4%), Planctomycetaceae (7.7%), Verrucomicrobiaceae (6.8%), Acidobacteriaceae (2.7%). Analysis of Similarity (ANOSIM, based on grouping all retrieved sequences into 9 phylogenetic groups) indicated that subtle differences do exist in the community composition between nearshore and outer shelf reefs. Similarity percentage analysis (SIMPER) indicated that Acidobacteriaceae and Cyanobacteriaceae were the main contributors to the dissimilarity. A significant difference between bacteria on nearshore and outer shelf reefs also existed on the molecular level ( FST = 0.008, p = 0.007 for all samples, 0.006, p = 0.022 when repeated sequences within libraries were removed). Thus, bacterial communities on carbonate sediments investigated were highly diverse and differences in community composition may provide important leads for the search for indicator species or communities for water quality differences.

Compartmentalization of HIV-1 within the female genital tract is due to monotypic and low-diversity variants not distinct viral populations.

PubMed

Bull, Marta; Learn, Gerald; Genowati, Indira; McKernan, Jennifer; Hitti, Jane; Lockhart, David; Tapia, Kenneth; Holte, Sarah; Dragavon, Joan; Coombs, Robert; Mullins, James; Frenkel, Lisa

2009-09-22

Compartmentalization of HIV-1 between the genital tract and blood was noted in half of 57 women included in 12 studies primarily using cell-free virus. To further understand differences between genital tract and blood viruses of women with chronic HIV-1 infection cell-free and cell-associated virus populations were sequenced from these tissues, reasoning that integrated viral DNA includes variants archived from earlier in infection, and provides a greater array of genotypes for comparisons. Multiple sequences from single-genome-amplification of HIV-1 RNA and DNA from the genital tract and blood of each woman were compared in a cross-sectional study. Maximum likelihood phylogenies were evaluated for evidence of compartmentalization using four statistical tests. Genital tract and blood HIV-1 appears compartmentalized in 7/13 women by >/=2 statistical analyses. These subjects' phylograms were characterized by low diversity genital-specific viral clades interspersed between clades containing both genital and blood sequences. Many of the genital-specific clades contained monotypic HIV-1 sequences. In 2/7 women, HIV-1 populations were significantly compartmentalized across all four statistical tests; both had low diversity genital tract-only clades. Collapsing monotypic variants into a single sequence diminished the prevalence and extent of compartmentalization. Viral sequences did not demonstrate tissue-specific signature amino acid residues, differential immune selection, or co-receptor usage. In women with chronic HIV-1 infection multiple identical sequences suggest proliferation of HIV-1-infected cells, and low diversity tissue-specific phylogenetic clades are consistent with bursts of viral replication. These monotypic and tissue-specific viruses provide statistical support for compartmentalization of HIV-1 between the female genital tract and blood. However, the intermingling of these clades with clades comprised of both genital and blood sequences and the absence of tissue-specific genetic features suggests compartmentalization between blood and genital tract may be due to viral replication and proliferation of infected cells, and questions whether HIV-1 in the female genital tract is distinct from blood.

Genetic Diversity and Phylogenetic Analysis of South-East Asian Duck Populations Based on the mtDNA D-loop Sequences

PubMed Central

Sultana, H.; Seo, D. W.; Bhuiyan, M. S. A.; Choi, N. R.; Hoque, M. R.; Heo, K. N.; Lee, J. H.

2016-01-01

The maternally inherited mitochondrial DNA (mtDNA) D–loop region is widely used for exploring genetic relationships and for investigating the origin of various animal species. Currently, domestic ducks play an important role in animal protein supply. In this study, partial mtDNA D–loop sequences were obtained from 145 samples belonging to six South-East Asian duck populations and commercial duck population. All these populations were closely related to the mallard duck (Anas platyrhynchos), as indicated by their mean overall genetic distance. Sixteen nucleotide substitutions were identified in sequence analyses allowing the distinction of 28 haplotypes. Around 42.76% of the duck sequences were classified as Hap_02, which completely matched with Anas platyrhynchos duck species. The neighbor-joining phylogenetic tree also revealed that South-East Asian duck populations were closely related to Anas platyrhynchos. Network profiles were also traced using the 28 haplotypes. Overall, results showed that those duck populations D-loop haplotypes were shared between several duck breeds from Korea and Bangladesh sub continental regions. Therefore, these results confirmed that South-East Asian domestic duck populations have been domesticated from Anas platyrhynchos duck as the maternal origins. PMID:27004808

Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications

PubMed Central

Del Medico, Luca; Christen, Heinz; Christen, Beat

2017-01-01

Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner. PMID:28531174

Genetic origin of goat populations in Oman revealed by mitochondrial DNA analysis

PubMed Central

Gaafar, Osman Mahgoub; Costa, Vânia; Neira, Agusto Luzuriaga; Al-Atiyat, Raed Mahmoud; Beja-Pereira, Albano

2017-01-01

The Sultanate of Oman has a complex mosaic of livestock species and production systems, but the genetic diversity, demographic history or origins of these Omani animals has not been expensively studied. Goats might constitute one of the most abundant and important domestic livestock species since the Neolithic transition. Here, we examined the genetic diversity, origin, population structure and demographic history of Omani goats. Specifically, we analyzed a 525-bp fragment of the first hypervariable region of the mitochondrial DNA (mtDNA) control region from 69 Omani individuals and compared this fragment with 17 mtDNA sequences from Somalia and Yemen as well as 18 wild goat species and 1,198 previously published goat sequences from neighboring countries. The studied goat breeds show substantial diversity. The haplotype and nucleotide diversities of Omani goats were found equal to 0.983 ± 0.006 and 0.0284 ± 0.014, respectively. The phylogenetic analyses allowed us to classify Omani goats into three mtDNA haplogroups (A, B and G): haplogroup A was found to be predominant and widely distributed and accounted for 80% of all samples, and haplogroups B and G exhibited low frequencies. Phylogenetic comparisons with wild goats revealed that five of the native Omani goat populations originate from Capra aegagrus. Furthermore, most comparisons of pairwise population FST values within and between these five Omani goat breeds as well as between Omani goats and nine populations from nearby countries were not significant. These results suggest strong gene flow among goat populations caused by the extensive transport of goats and the frequent movements of human populations in ancient Arabia. The findings improve our understanding of the migration routes of modern goats from their region of domestication into southeastern Arabia and thereby shed light on human migratory and commercial networks during historical times. PMID:29281717

Detecting in situ copepod diet diversity using molecular technique: development of a copepod/symbiotic ciliate-excluding eukaryote-inclusive PCR protocol.

PubMed

Hu, Simin; Guo, Zhiling; Li, Tao; Carpenter, Edward J; Liu, Sheng; Lin, Senjie

2014-01-01

Knowledge of in situ copepod diet diversity is crucial for accurately describing pelagic food web structure but is challenging to achieve due to lack of an easily applicable methodology. To enable analysis with whole copepod-derived DNAs, we developed a copepod-excluding 18S rDNA-based PCR protocol. Although it is effective in depressing amplification of copepod 18S rDNA, its applicability to detect diverse eukaryotes in both mono- and mixed-species has not been demonstrated. Besides, the protocol suffers from the problem that sequences from symbiotic ciliates are overrepresented in the retrieved 18S rDNA libraries. In this study, we designed a blocking primer to make a combined primer set (copepod/symbiotic ciliate-excluding eukaryote-common: CEEC) to depress PCR amplification of symbiotic ciliate sequences while maximizing the range of eukaryotes amplified. We firstly examined the specificity and efficacy of CEEC by PCR-amplifying DNAs from 16 copepod species, 37 representative organisms that are potential prey of copepods and a natural microplankton sample, and then evaluated the efficiency in reconstructing diet composition by detecting the food of both lab-reared and field-collected copepods. Our results showed that the CEEC primer set can successfully amplify 18S rDNA from a wide range of isolated species and mixed-species samples while depressing amplification of that from copepod and targeted symbiotic ciliate, indicating the universality of CEEC in specifically detecting prey of copepods. All the predetermined food offered to copepods in the laboratory were successfully retrieved, suggesting that the CEEC-based protocol can accurately reconstruct the diets of copepods without interference of copepods and their associated ciliates present in the DNA samples. Our initial application to analyzing the food composition of field-collected copepods uncovered diverse prey species, including those currently known, and those that are unsuspected, as copepod prey. While testing is required, this protocol provides a useful strategy for depicting in situ dietary composition of copepods.

DIVERSITY OF THE TYPE 1 INTRON-ITS REGION OF THE 18S rRNA GENE IN PSEUDOGYMNOASCUS SPECIES FROM THE RED HILLS OF KANSAS.

PubMed

Chen, Xi; Crupper, Scott S

2016-09-01

Gypsum caves found throughout the Red Hills of Kansas have the state's most diverse and largest population of cave-roosting bats. White-nose syndrome (WNS), a disease caused by the fungus Pseudogymnoascus destructans, which threatens all temperate bat species, has not been previously detected in the gypsum caves as this disease moves westward from the eastern United States. Cave soil was obtained from the gypsum caves, and using the polymerase chain reaction, a 624-nucleotide DNA fragment specific to the Type 1 intron-internal transcribed spacer region of the 18S rRNA gene from Pseudogymnoascus species was amplified. Subsequent cloning and DNA sequencing indicated P. destructans DNA was present, along with 26 uncharacterized Pseudogymnoascus DNA variants. However, no evidence of WNS was observed in bat populations residing in these caves.

Rapidly expanding genetic diversity and host range of the Circoviridae viral family and other Rep encoding small circular ssDNA genomes.

PubMed

Delwart, Eric; Li, Linlin

2012-03-01

The genomes of numerous circoviruses and distantly related circular ssDNA viruses encoding a rolling circle replication initiator protein (Rep) have been characterized from the tissues of mammals, fish, insects, plants (geminivirus and nanovirus), in human and animal feces, in an algae cell, and in diverse environmental samples. We review the genome organization, phylogenetic relationships and initial prevalence studies of cycloviruses, a proposed new genus in the Circoviridae family. Viral fossil rep sequences were also recently identified integrated on the chromosomes of mammals, frogs, lancelets, crustaceans, mites, gastropods, roundworms, placozoans, hydrozoans, protozoans, land plants, fungi, algae, and phytoplasma bacterias and their plasmids, reflecting the very wide past host range of rep bearing viruses. An ancient origin for viruses with Rep-encoding small circular ssDNA genomes, predating the diversification of eukaryotes, is discussed. The cellular hosts and pathogenicity of many recently described rep-containing circular ssDNA genomes remain to be determined. Future studies of the virome of single cell and multi-cellular eukaryotes are likely to further extend the known diversity and host-range of small rep-containing circular ssDNA viral genomes. Copyright © 2011 Elsevier B.V. All rights reserved.

Genetics of Mitochondrial Disease.

PubMed

Saneto, Russell P

2017-01-01

Mitochondria are intracellular organelles responsible for adenosine triphosphate production. The strict control of intracellular energy needs require proper mitochondrial functioning. The mitochondria are under dual controls of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Mitochondrial dysfunction can arise from changes in either mtDNA or nDNA genes regulating function. There are an estimated ∼1500 proteins in the mitoproteome, whereas the mtDNA genome has 37 proteins. There are, to date, ∼275 genes shown to give rise to disease. The unique physiology of mitochondrial functioning contributes to diverse gene expression. The onset and range of phenotypic expression of disease is diverse, with onset from neonatal to seventh decade of life. The range of dysfunction is heterogeneous, ranging from single organ to multisystem involvement. The complexity of disease expression has severely limited gene discovery. Combining phenotypes with improvements in gene sequencing strategies are improving the diagnosis process. This chapter focuses on the interplay of the unique physiology and gene discovery in the current knowledge of genetically derived mitochondrial disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Genotypic and phenotypic diversity of Alicyclobacillus acidocaldarius isolates.

PubMed

Félix-Valenzuela, L; Guardiola-Avila, I; Burgara-Estrella, A; Ibarra-Zavala, M; Mata-Haro, V

2015-10-01

The fruit juice industry recognizes Alicyclobacillus as a major quality control target micro-organism. In this study, we analysed 19 bacterial isolates to identify Alicyclobacillus species by polymerase chain reaction (PCR) and sequencing analyses. Phenotypic and genomic diversity among isolates were investigated by API 50CHB system and ERIC-PCR (enterobacterial repetitive intergenic consensus-PCR) respectively. All bacterial isolates were identified as Alicyclobacillus acidocaldarius, and almost all showed identical DNA sequences according to their 16S rRNA (rDNA) gene partial sequences. Only few carbohydrates were fermented by A. acidocaldarius isolates, and there was little variability in the biochemical profile. Genotypic fingerprinting of the A. acidocaldarius isolates showed high diversity, and clusters by ERIC-PCR were distinct to those obtained from the 16S rRNA gene phylogenetic tree. There was no correlation between phenotypic and genotypic variability in the A. acidocaldarius isolates analysed in this study. Detection of Alicyclobacillus strains is imperative in fruit concentrates and juices due to the production of guaiacol. Identification of the genera originates rejection of the product by processing industry. However, not all the Alicyclobacillus species are deteriorative and hence the importance to differentiate among them. In this study, partial 16S ribosomal RNA sequence alignment allowed the differentiation of species. In addition, ERIC-PCR was introduced for the genotypic characterization of Alicyclobacillus, as an alternative for differentiation among isolates from the same species. © 2015 The Society for Applied Microbiology.

Molecular analysis of meso- and thermophilic microbiota associated with anaerobic biowaste degradation

PubMed Central

2012-01-01

Background Microbial anaerobic digestion (AD) is used as a waste treatment process to degrade complex organic compounds into methane. The archaeal and bacterial taxa involved in AD are well known, whereas composition of the fungal community in the process has been less studied. The present study aimed to reveal the composition of archaeal, bacterial and fungal communities in response to increasing organic loading in mesophilic and thermophilic AD processes by applying 454 amplicon sequencing technology. Furthermore, a DNA microarray method was evaluated in order to develop a tool for monitoring the microbiological status of AD. Results The 454 sequencing showed that the diversity and number of bacterial taxa decreased with increasing organic load, while archaeal i.e. methanogenic taxa remained more constant. The number and diversity of fungal taxa increased during the process and varied less in composition with process temperature than bacterial and archaeal taxa, even though the fungal diversity increased with temperature as well. Evaluation of the microarray using AD sample DNA showed correlation of signal intensities with sequence read numbers of corresponding target groups. The sensitivity of the test was found to be about 1%. Conclusions The fungal community survives in anoxic conditions and grows with increasing organic loading, suggesting that Fungi may contribute to the digestion by metabolising organic nutrients for bacterial and methanogenic groups. The microarray proof of principle tests suggest that the method has the potential for semiquantitative detection of target microbial groups given that comprehensive sequence data is available for probe design. PMID:22727142

DNA recombination protein-dependent mechanism of homoplasmy and its proposed functions.

PubMed

Shibata, Takehiko; Ling, Feng

2007-01-01

Homoplasmy is a basic genetic state of mitochondria, in which all of the hundreds to thousands of mitochondrial (mt)DNA copies within a cell or an individual have the same nucleotide-sequence. It was recently found that "vegetative segregation" to generate homoplasmic cells is an active process under genetic control. In the yeast Saccharomyces cerevisiae, the Mhr1 protein which catalyzes a key reaction in mtDNA homologous recombination, plays a pivotal role in vegetative segregation. Conversely, within the nuclear genome, homologous DNA recombination causes genetic diversity. Considering these contradictory roles of this key reaction in DNA recombination, possible functions of homoplasmy are discussed.

Archaeal Diversity in Waters from Deep South African Gold Mines

PubMed Central

Takai, Ken; Moser, Duane P.; DeFlaun, Mary; Onstott, Tullis C.; Fredrickson, James K.

2001-01-01

A culture-independent molecular analysis of archaeal communities in waters collected from deep South African gold mines was performed by performing a PCR-mediated terminal restriction fragment length polymorphism (T-RFLP) analysis of rRNA genes (rDNA) in conjunction with a sequencing analysis of archaeal rDNA clone libraries. The water samples used represented various environments, including deep fissure water, mine service water, and water from an overlying dolomite aquifer. T-RFLP analysis revealed that the ribotype distribution of archaea varied with the source of water. The archaeal communities in the deep gold mine environments exhibited great phylogenetic diversity; the majority of the members were most closely related to uncultivated species. Some archaeal rDNA clones obtained from mine service water and dolomite aquifer water samples were most closely related to environmental rDNA clones from surface soil (soil clones) and marine environments (marine group I [MGI]). Other clones exhibited intermediate phylogenetic affiliation between soil clones and MGI in the Crenarchaeota. Fissure water samples, derived from active or dormant geothermal environments, yielded archaeal sequences that exhibited novel phylogeny, including a novel lineage of Euryarchaeota. These results suggest that deep South African gold mines harbor novel archaeal communities distinct from those observed in other environments. Based on the phylogenetic analysis of archaeal strains and rDNA clones, including the newly discovered archaeal rDNA clones, the evolutionary relationship and the phylogenetic organization of the domain Archaea are reevaluated. PMID:11722932

Phylogenetic analysis of several Thermus strains from Rehai of Tengchong, Yunnan, China.

PubMed

Lin, Lianbing; Zhang, Jie; Wei, Yunlin; Chen, Chaoyin; Peng, Qian

2005-10-01

Several Thermus strains were isolated from 10 hot springs of the Rehai geothermal area in Tengchong, Yunnan province. The diversity of Thermus strains was examined by sequencing the 16S rRNA genes and comparing their sequences. Phylogenetic analysis showed that the 16S rDNA sequences from the Rehai geothermal isolates form four branches in the phylogenetic tree and had greater than 95.9% similarity in the phylogroup. Secondary structure comparison also indicated that the 16S rRNA from the Rehai geothermal isolates have unique secondary structure characteristics in helix 6, helix 9, and helix 10 (reference to Escherichia coli). This research is the first attempt to reveal the diversity of Thermus strains that are distributed in the Rehai geothermal area.