Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

PubMed

McMillan, Mary; Pereg, Lily

2014-01-01

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.

Selection and Evaluation of Potential Reference Genes for Gene Expression Analysis in the Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) Using Reverse-Transcription Quantitative PCR

PubMed Central

Zhu, Xun; Wan, Hu; Shakeel, Muhammad; Zhan, Sha; Jin, Byung-Rae; Li, Jianhong

2014-01-01

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects. PMID:24466124

Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reverse-transcription quantitative PCR.

PubMed

Yuan, Miao; Lu, Yanhui; Zhu, Xun; Wan, Hu; Shakeel, Muhammad; Zhan, Sha; Jin, Byung-Rae; Li, Jianhong

2014-01-01

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects.

Identifying the genes underlying quantitative traits: a rationale for the QTN programme.

PubMed

Lee, Young Wha; Gould, Billie A; Stinchcombe, John R

2014-01-01

The goal of identifying the genes or even nucleotides underlying quantitative and adaptive traits has been characterized as the 'QTN programme' and has recently come under severe criticism. Part of the reason for this criticism is that much of the QTN programme has asserted that finding the genes and nucleotides for adaptive and quantitative traits is a fundamental goal, without explaining why it is such a hallowed goal. Here we outline motivations for the QTN programme that offer general insight, regardless of whether QTNs are of large or small effect, and that aid our understanding of the mechanistic dynamics of adaptive evolution. We focus on five areas: (i) vertical integration of insight across different levels of biological organization, (ii) genetic parallelism and the role of pleiotropy in shaping evolutionary dynamics, (iii) understanding the forces maintaining genetic variation in populations, (iv) distinguishing between adaptation from standing variation and new mutation, and (v) the role of genomic architecture in facilitating adaptation. We argue that rather than abandoning the QTN programme, we should refocus our efforts on topics where molecular data will be the most effective for testing hypotheses about phenotypic evolution.

Identifying the genes underlying quantitative traits: a rationale for the QTN programme

PubMed Central

Lee, Young Wha; Gould, Billie A.; Stinchcombe, John R.

2014-01-01

The goal of identifying the genes or even nucleotides underlying quantitative and adaptive traits has been characterized as the ‘QTN programme’ and has recently come under severe criticism. Part of the reason for this criticism is that much of the QTN programme has asserted that finding the genes and nucleotides for adaptive and quantitative traits is a fundamental goal, without explaining why it is such a hallowed goal. Here we outline motivations for the QTN programme that offer general insight, regardless of whether QTNs are of large or small effect, and that aid our understanding of the mechanistic dynamics of adaptive evolution. We focus on five areas: (i) vertical integration of insight across different levels of biological organization, (ii) genetic parallelism and the role of pleiotropy in shaping evolutionary dynamics, (iii) understanding the forces maintaining genetic variation in populations, (iv) distinguishing between adaptation from standing variation and new mutation, and (v) the role of genomic architecture in facilitating adaptation. We argue that rather than abandoning the QTN programme, we should refocus our efforts on topics where molecular data will be the most effective for testing hypotheses about phenotypic evolution. PMID:24790125

Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan hulless barley.

PubMed

Cai, Jing; Li, Pengfei; Luo, Xiao; Chang, Tianliang; Li, Jiaxing; Zhao, Yuwei; Xu, Yao

2018-01-01

Hulless barley (Hordeum vulgare L. var. nudum. hook. f.) has been cultivated as a major crop in the Qinghai-Tibet plateau of China for thousands of years. Compared to other cereal crops, the Tibetan hulless barley has developed stronger endogenous resistances to survive in the severe environment of its habitat. To understand the unique resistant mechanisms of this plant, detailed genetic studies need to be performed. The quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method in detecting gene expression. However, the selection of stable reference genes under limited experimental conditions was considered to be an essential step for obtaining accurate results in qRT-PCR. In this study, 10 candidate reference genes-ACT (Actin), E2 (Ubiquitin conjugating enzyme 2), TUBα (Alpha-tubulin), TUBβ6 (Beta-tubulin 6), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), EF-1α (Elongation factor 1-alpha), SAMDC (S-adenosylmethionine decarboxylase), PKABA1 (Gene for protein kinase HvPKABA1), PGK (Phosphoglycerate kinase), and HSP90 (Heat shock protein 90)-were selected from the NCBI gene database of barley. Following qRT-PCR amplifications of all candidate reference genes in Tibetan hulless barley seedlings under various stressed conditions, the stabilities of these candidates were analyzed by three individual software packages including geNorm, NormFinder, and BestKeeper. The results demonstrated that TUBβ6, E2, TUBα, and HSP90 were generally the most suitable sets under all tested conditions; similarly, TUBα and HSP90 showed peak stability under salt stress, TUBα and EF-1α were the most suitable reference genes under cold stress, and ACT and E2 were the most stable under drought stress. Finally, a known circadian gene CCA1 was used to verify the service ability of chosen reference genes. The results confirmed that all recommended reference genes by the three software were suitable for gene expression analysis under tested stress conditions by the qRT-PCR method.

Selection and evaluation of reference genes for expression studies with quantitative PCR in the model fungus Neurospora crassa under different environmental conditions in continuous culture.

PubMed

Cusick, Kathleen D; Fitzgerald, Lisa A; Pirlo, Russell K; Cockrell, Allison L; Petersen, Emily R; Biffinger, Justin C

2014-01-01

Neurospora crassa has served as a model organism for studying circadian pathways and more recently has gained attention in the biofuel industry due to its enhanced capacity for cellulase production. However, in order to optimize N. crassa for biotechnological applications, metabolic pathways during growth under different environmental conditions must be addressed. Reverse-transcription quantitative PCR (RT-qPCR) is a technique that provides a high-throughput platform from which to measure the expression of a large set of genes over time. The selection of a suitable reference gene is critical for gene expression studies using relative quantification, as this strategy is based on normalization of target gene expression to a reference gene whose expression is stable under the experimental conditions. This study evaluated twelve candidate reference genes for use with N. crassa when grown in continuous culture bioreactors under different light and temperature conditions. Based on combined stability values from NormFinder and Best Keeper software packages, the following are the most appropriate reference genes under conditions of: (1) light/dark cycling: btl, asl, and vma1; (2) all-dark growth: btl, tbp, vma1, and vma2; (3) temperature flux: btl, vma1, act, and asl; (4) all conditions combined: vma1, vma2, tbp, and btl. Since N. crassa exists as different cell types (uni- or multi-nucleated), expression changes in a subset of the candidate genes was further assessed using absolute quantification. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes serve as a reliable reflection of transcript, and not gene copy number, fluctuations. The results of this study identified genes that are appropriate for use as reference genes in RT-qPCR studies with N. crassa and demonstrated that even with the presence of different cell types, relative quantification is an acceptable method for measuring gene expression changes during growth in bioreactors.

Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity

PubMed Central

Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A.; Bradford, William D.; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S.; Li, Rong

2015-01-01

Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein−based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. PMID:25823586

Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity.

PubMed

Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A; Bradford, William D; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S; Li, Rong

2015-03-30

Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein-based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. Copyright © 2015 Zhu et al.

Quantitative Resistance: More Than Just Perception of a Pathogen

PubMed Central

2017-01-01

Molecular plant pathology has focused on studying large-effect qualitative resistance loci that predominantly function in detecting pathogens and/or transmitting signals resulting from pathogen detection. By contrast, less is known about quantitative resistance loci, particularly the molecular mechanisms controlling variation in quantitative resistance. Recent studies have provided insight into these mechanisms, showing that genetic variation at hundreds of causal genes may underpin quantitative resistance. Loci controlling quantitative resistance contain some of the same causal genes that mediate qualitative resistance, but the predominant mechanisms of quantitative resistance extend beyond pathogen recognition. Indeed, most causal genes for quantitative resistance encode specific defense-related outputs such as strengthening of the cell wall or defense compound biosynthesis. Extending previous work on qualitative resistance to focus on the mechanisms of quantitative resistance, such as the link between perception of microbe-associated molecular patterns and growth, has shown that the mechanisms underlying these defense outputs are also highly polygenic. Studies that include genetic variation in the pathogen have begun to highlight a potential need to rethink how the field considers broad-spectrum resistance and how it is affected by genetic variation within pathogen species and between pathogen species. These studies are broadening our understanding of quantitative resistance and highlighting the potentially vast scale of the genetic basis of quantitative resistance. PMID:28302676

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.

PubMed

Trivedi, Vikas; Choi, Harry M T; Fraser, Scott E; Pierce, Niles A

2018-01-08

For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting and/or homogenization. Here, we eliminate the trade-off between quantitation and anatomical context, using quantitative in situ hybridization chain reaction (qHCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable bi-directional queries that open a new era for in situ hybridization. © 2018. Published by The Company of Biologists Ltd.

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

PubMed

Chapman, Joanne R; Waldenström, Jonas

2015-01-01

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

Selection of relatively exact reference genes for gene expression studies in goosegrass (Eleusine indica) under herbicide stress.

PubMed

Chen, Jingchao; Huang, Zhaofeng; Huang, Hongjuan; Wei, Shouhui; Liu, Yan; Jiang, Cuilan; Zhang, Jie; Zhang, Chaoxian

2017-04-21

Goosegrass (Eleusine indica) is one of the most serious annual grassy weeds worldwide, and its evolved herbicide-resistant populations are more difficult to control. Quantitative real-time PCR (qPCR) is a common technique for investigating the resistance mechanism; however, there is as yet no report on the systematic selection of stable reference genes for goosegrass. This study proposed to test the expression stability of 9 candidate reference genes in goosegrass in different tissues and developmental stages and under stress from three types of herbicide. The results show that for different developmental stages and organs (control), eukaryotic initiation factor 4 A (eIF-4) is the most stable reference gene. Chloroplast acetolactate synthase (ALS) is the most stable reference gene under glyphosate stress. Under glufosinate stress, eIF-4 is the best reference gene. Ubiquitin-conjugating enzyme (UCE) is the most stable reference gene under quizalofop-p-ethyl stress. The gene eIF-4 is the recommended reference gene for goosegrass under the stress of all three herbicides. Moreover, pairwise analysis showed that seven reference genes were sufficient to normalize the gene expression data under three herbicides treatment. This study provides a list of reliable reference genes for transcript normalization in goosegrass, which will facilitate resistance mechanism studies in this weed species.

Identification of internal control genes for quantitative expression analysis by real-time PCR in bovine peripheral lymphocytes.

PubMed

Spalenza, Veronica; Girolami, Flavia; Bevilacqua, Claudia; Riondato, Fulvio; Rasero, Roberto; Nebbia, Carlo; Sacchi, Paola; Martin, Patrice

2011-09-01

Gene expression studies in blood cells, particularly lymphocytes, are useful for monitoring potential exposure to toxicants or environmental pollutants in humans and livestock species. Quantitative PCR is the method of choice for obtaining accurate quantification of mRNA transcripts although variations in the amount of starting material, enzymatic efficiency, and the presence of inhibitors can lead to evaluation errors. As a result, normalization of data is of crucial importance. The most common approach is the use of endogenous reference genes as an internal control, whose expression should ideally not vary among individuals and under different experimental conditions. The accurate selection of reference genes is therefore an important step in interpreting quantitative PCR studies. Since no systematic investigation in bovine lymphocytes has been performed, the aim of the present study was to assess the expression stability of seven candidate reference genes in circulating lymphocytes collected from 15 dairy cows. Following the characterization by flow cytometric analysis of the cell populations obtained from blood through a density gradient procedure, three popular softwares were used to evaluate the gene expression data. The results showed that two genes are sufficient for normalization of quantitative PCR studies in cattle lymphocytes and that YWAHZ, S24 and PPIA are the most stable genes. Copyright © 2010 Elsevier Ltd. All rights reserved.

Selection and testing of reference genes for accurate RT-qPCR in rice seedlings under iron toxicity.

PubMed

Santos, Fabiane Igansi de Castro Dos; Marini, Naciele; Santos, Railson Schreinert Dos; Hoffman, Bianca Silva Fernandes; Alves-Ferreira, Marcio; de Oliveira, Antonio Costa

2018-01-01

Reverse Transcription quantitative PCR (RT-qPCR) is a technique for gene expression profiling with high sensibility and reproducibility. However, to obtain accurate results, it depends on data normalization by using endogenous reference genes whose expression is constitutive or invariable. Although the technique is widely used in plant stress analyzes, the stability of reference genes for iron toxicity in rice (Oryza sativa L.) has not been thoroughly investigated. Here, we tested a set of candidate reference genes for use in rice under this stressful condition. The test was performed using four distinct methods: NormFinder, BestKeeper, geNorm and the comparative ΔCt. To achieve reproducible and reliable results, Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were followed. Valid reference genes were found for shoot (P2, OsGAPDH and OsNABP), root (OsEF-1a, P8 and OsGAPDH) and root+shoot (OsNABP, OsGAPDH and P8) enabling us to perform further reliable studies for iron toxicity in both indica and japonica subspecies. The importance of the study of other than the traditional endogenous genes for use as normalizers is also shown here.

Selection and testing of reference genes for accurate RT-qPCR in rice seedlings under iron toxicity

PubMed Central

dos Santos, Fabiane Igansi de Castro; Marini, Naciele; dos Santos, Railson Schreinert; Hoffman, Bianca Silva Fernandes; Alves-Ferreira, Marcio

2018-01-01

Reverse Transcription quantitative PCR (RT-qPCR) is a technique for gene expression profiling with high sensibility and reproducibility. However, to obtain accurate results, it depends on data normalization by using endogenous reference genes whose expression is constitutive or invariable. Although the technique is widely used in plant stress analyzes, the stability of reference genes for iron toxicity in rice (Oryza sativa L.) has not been thoroughly investigated. Here, we tested a set of candidate reference genes for use in rice under this stressful condition. The test was performed using four distinct methods: NormFinder, BestKeeper, geNorm and the comparative ΔCt. To achieve reproducible and reliable results, Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were followed. Valid reference genes were found for shoot (P2, OsGAPDH and OsNABP), root (OsEF-1a, P8 and OsGAPDH) and root+shoot (OsNABP, OsGAPDH and P8) enabling us to perform further reliable studies for iron toxicity in both indica and japonica subspecies. The importance of the study of other than the traditional endogenous genes for use as normalizers is also shown here. PMID:29494624

Genomic approaches for the elucidation of genes and gene networks underlying cardiovascular traits.

PubMed

Adriaens, M E; Bezzina, C R

2018-06-22

Genome-wide association studies have shed light on the association between natural genetic variation and cardiovascular traits. However, linking a cardiovascular trait associated locus to a candidate gene or set of candidate genes for prioritization for follow-up mechanistic studies is all but straightforward. Genomic technologies based on next-generation sequencing technology nowadays offer multiple opportunities to dissect gene regulatory networks underlying genetic cardiovascular trait associations, thereby aiding in the identification of candidate genes at unprecedented scale. RNA sequencing in particular becomes a powerful tool when combined with genotyping to identify loci that modulate transcript abundance, known as expression quantitative trait loci (eQTL), or loci modulating transcript splicing known as splicing quantitative trait loci (sQTL). Additionally, the allele-specific resolution of RNA-sequencing technology enables estimation of allelic imbalance, a state where the two alleles of a gene are expressed at a ratio differing from the expected 1:1 ratio. When multiple high-throughput approaches are combined with deep phenotyping in a single study, a comprehensive elucidation of the relationship between genotype and phenotype comes into view, an approach known as systems genetics. In this review, we cover key applications of systems genetics in the broad cardiovascular field.

Identification of reference genes for RT-qPCR analysis in peach genotypes with contrasting chilling requirements.

PubMed

Marini, N; Bevilacqua, C B; Büttow, M V; Raseira, M C B; Bonow, S

2017-05-25

Selecting and validating reference genes are the first steps in studying gene expression by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). The present study aimed to evaluate the stability of five reference genes for the purpose of normalization when studying gene expression in various cultivars of Prunus persica with different chilling requirements. Flower bud tissues of nine peach genotypes from Embrapa's peach breeding program with different chilling requirements were used, and five candidate reference genes based on the RT-qPCR that were useful for studying the relative quantitative gene expression and stability were evaluated using geNorm, NormFinder, and bestKeeper software packages. The results indicated that among the genes tested, the most stable genes to be used as reference genes are Act and UBQ10. This study is the first survey of the stability of reference genes in peaches under chilling stress and provides guidelines for more accurate RT-qPCR results.

Selection of reliable reference genes for quantitative real-time PCR gene expression analysis in Jute (Corchorus capsularis) under stress treatments

PubMed Central

Niu, Xiaoping; Qi, Jianmin; Zhang, Gaoyang; Xu, Jiantang; Tao, Aifen; Fang, Pingping; Su, Jianguang

2015-01-01

To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR), reliable reference gene(s) are required for data normalization. Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as three different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC, and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α, and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions. PMID:26528312

Dynamic processes at stress promoters regulate the bimodal expression of HOG response genes

PubMed Central

2011-01-01

Osmotic stress triggers the activation of the HOG (high osmolarity glycerol) pathway in Saccharomyces cerevisiae. This signaling cascade culminates in the activation of the MAPK (mitogen-activated protein kinase) Hog1. Quantitative single cell measurements revealed a discrepancy between kinase- and transcriptional activities of Hog1. While kinase activity increases proportionally to stress stimulus, gene expression is inhibited under low stress conditions. Interestingly, a slow stochastic gene activation process is responsible for setting a tunable threshold for gene expression under basal or low stress conditions, which generates a bimodal expression pattern at intermediate stress levels. PMID:22446531

Quantitative Resistance: More Than Just Perception of a Pathogen.

PubMed

Corwin, Jason A; Kliebenstein, Daniel J

2017-04-01

Molecular plant pathology has focused on studying large-effect qualitative resistance loci that predominantly function in detecting pathogens and/or transmitting signals resulting from pathogen detection. By contrast, less is known about quantitative resistance loci, particularly the molecular mechanisms controlling variation in quantitative resistance. Recent studies have provided insight into these mechanisms, showing that genetic variation at hundreds of causal genes may underpin quantitative resistance. Loci controlling quantitative resistance contain some of the same causal genes that mediate qualitative resistance, but the predominant mechanisms of quantitative resistance extend beyond pathogen recognition. Indeed, most causal genes for quantitative resistance encode specific defense-related outputs such as strengthening of the cell wall or defense compound biosynthesis. Extending previous work on qualitative resistance to focus on the mechanisms of quantitative resistance, such as the link between perception of microbe-associated molecular patterns and growth, has shown that the mechanisms underlying these defense outputs are also highly polygenic. Studies that include genetic variation in the pathogen have begun to highlight a potential need to rethink how the field considers broad-spectrum resistance and how it is affected by genetic variation within pathogen species and between pathogen species. These studies are broadening our understanding of quantitative resistance and highlighting the potentially vast scale of the genetic basis of quantitative resistance. © 2017 American Society of Plant Biologists. All rights reserved.

tCRISPRi: tunable and reversible, one-step control of gene expression

NASA Astrophysics Data System (ADS)

Li, Xin-Tian; Jun, Yonggun; Erickstad, Michael J.; Brown, Steven D.; Parks, Adam; Court, Donald L.; Jun, Suckjoon

2016-12-01

The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study. Here, we report a tunable CRISPR-cas system, “tCRISPRi”, for precise and continuous titration of gene expression by more than 30-fold. Our tCRISPRi system employs various previous advancements into a single strain: (1) We constructed a new strain containing a tunable arabinose operon promoter PBAD to quantitatively control the expression of CRISPR-(d)Cas protein over two orders of magnitude in a plasmid-free system. (2) tCRISPRi is reversible, and gene expression is repressed under knockdown conditions. (3) tCRISPRi shows significantly less than 10% leaky expression. (4) Most important from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step of oligo recombineering. Our results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.

Evaluation and Selection of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Withania somnifera (L.) Dunal

PubMed Central

Singh, Varinder; Kaul, Sunil C.; Wadhwa, Renu; Pati, Pratap Kumar

2015-01-01

Quantitative real-time PCR (qRT-PCR) is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease), abiotic (wounding, salt, drought, heat and cold) stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid). The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ΔCt method) suggested that cyclophilin (CYP) is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species. PMID:25769035

Selection of relatively exact reference genes for gene expression studies in goosegrass (Eleusine indica) under herbicide stress

PubMed Central

Chen, Jingchao; Huang, Zhaofeng; Huang, Hongjuan; Wei, Shouhui; Liu, Yan; Jiang, Cuilan; Zhang, Jie; Zhang, Chaoxian

2017-01-01

Goosegrass (Eleusine indica) is one of the most serious annual grassy weeds worldwide, and its evolved herbicide-resistant populations are more difficult to control. Quantitative real-time PCR (qPCR) is a common technique for investigating the resistance mechanism; however, there is as yet no report on the systematic selection of stable reference genes for goosegrass. This study proposed to test the expression stability of 9 candidate reference genes in goosegrass in different tissues and developmental stages and under stress from three types of herbicide. The results show that for different developmental stages and organs (control), eukaryotic initiation factor 4 A (eIF-4) is the most stable reference gene. Chloroplast acetolactate synthase (ALS) is the most stable reference gene under glyphosate stress. Under glufosinate stress, eIF-4 is the best reference gene. Ubiquitin-conjugating enzyme (UCE) is the most stable reference gene under quizalofop-p-ethyl stress. The gene eIF-4 is the recommended reference gene for goosegrass under the stress of all three herbicides. Moreover, pairwise analysis showed that seven reference genes were sufficient to normalize the gene expression data under three herbicides treatment. This study provides a list of reliable reference genes for transcript normalization in goosegrass, which will facilitate resistance mechanism studies in this weed species. PMID:28429727

Identification and validation of reference genes for quantification of target gene expression with quantitative real-time PCR for tall fescue under four abiotic stresses.

PubMed

Yang, Zhimin; Chen, Yu; Hu, Baoyun; Tan, Zhiqun; Huang, Bingru

2015-01-01

Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

Quantitative trait loci from the host genetic background modulate the durability of a resistance gene: a rational basis for sustainable resistance breeding in plants.

PubMed

Quenouille, J; Paulhiac, E; Moury, B; Palloix, A

2014-06-01

The combination of major resistance genes with quantitative resistance factors is hypothesized as a promising breeding strategy to preserve the durability of resistant cultivar, as recently observed in different pathosystems. Using the pepper (Capsicum annuum)/Potato virus Y (PVY, genus Potyvirus) pathosystem, we aimed at identifying plant genetic factors directly affecting the frequency of virus adaptation to the major resistance gene pvr2(3) and at comparing them with genetic factors affecting quantitative resistance. The resistance breakdown frequency was a highly heritable trait (h(2)=0.87). Four loci including additive quantitative trait loci (QTLs) and epistatic interactions explained together 70% of the variance of pvr2(3) breakdown frequency. Three of the four QTLs controlling pvr2(3) breakdown frequency were also involved in quantitative resistance, strongly suggesting that QTLs controlling quantitative resistance have a pleiotropic effect on the durability of the major resistance gene. With the first mapping of QTLs directly affecting resistance durability, this study provides a rationale for sustainable resistance breeding. Surprisingly, a genetic trade-off was observed between the durability of PVY resistance controlled by pvr2(3) and the spectrum of the resistance against different potyviruses. This trade-off seemed to have been resolved by the combination of minor-effect durability QTLs under long-term farmer selection.

Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR.

PubMed

Zhang, Kun; Niu, Shaofang; Di, Dianping; Shi, Lindan; Liu, Deshui; Cao, Xiuling; Miao, Hongqin; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang

2013-10-10

Both genome-wide transcriptomic surveys of the mRNA expression profiles and virus-induced gene silencing-based molecular studies of target gene during virus-plant interaction involve the precise estimation of the transcript abundance. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Nevertheless, the stability of internal controls in virus-infected monocots needs to be fully explored. In this work, the suitability of ten housekeeping genes (ACT, EF1α, FBOX, GAPDH, GTPB, PP2A, SAND, TUBβ, UBC18 and UK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses including Barley stripe mosaic virus (BSMV), Brome mosaic virus (BMV), Rice black-streaked dwarf virus (RBSDV) and Sugarcane mosaic virus (SCMV). By using three different algorithms, the most appropriate reference genes or their combinations were identified for different experimental sets and their effectiveness for the normalisation of expression studies were further validated by quantitative analysis of a well-studied PR-1 gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots. Copyright © 2013 Elsevier B.V. All rights reserved.

Dissection of Insertion–Deletion Variants within Differentially Expressed Genes Involved in Wood Formation in Populus

PubMed Central

Gong, Chenrui; Du, Qingzhang; Xie, Jianbo; Quan, Mingyang; Chen, Beibei; Zhang, Deqiang

2018-01-01

Short insertions and deletions (InDels) are one of the major genetic variants and are distributed widely across the genome; however, few investigations of InDels have been conducted in long-lived perennial plants. Here, we employed a combination of RNA-seq and population resequencing to identify InDels within differentially expressed (DE) genes underlying wood formation in a natural population of Populus tomentosa (435 individuals) and utilized InDel-based association mapping to detect the causal variants under additive, dominance, and epistasis underlying growth and wood properties. In the present paper, 5,482 InDels detected from 629 DE genes showed uneven distributions throughout all 19 chromosomes, and 95.9% of these loci were diallelic InDels. Seventy-four InDels (positive false discovery rate q ≤ 0.10) from 68 genes exhibited significant additive/dominant effects on 10 growth and wood-properties, with an average of 14.7% phenotypic variance explained. Potential pleiotropy was observed in one-third of the InDels (representing 24 genes). Seven genes exhibited significantly differential expression among the genotypic classes of associated InDels, indicating possible important roles for these InDels. Epistasis analysis showed that overlapping interacting genes formed unique interconnected networks for each trait, supporting the putative biochemical links that control quantitative traits. Therefore, the identification and utilization of InDels in trees will be recognized as an effective marker system for molecular marker-assisted breeding applications, and further facilitate our understanding of quantitative genomics. PMID:29403506

Housekeeping gene expression during fetal brain development in the rat-validation by semi-quantitative RT-PCR.

PubMed

Al-Bader, Maie Dawoud; Al-Sarraf, Hameed Ali

2005-04-21

Mammalian gene expression is usually carried out at the level of mRNA where the amount of mRNA of interest is measured under different conditions such as growth and development. It is therefore important to use a "housekeeping gene", that does not change in relative abundance during the experimental conditions, as a standard or internal control. However, recent data suggest that expression of some housekeeping genes may vary with the extent of cell proliferation, differentiation and under various experimental conditions. In this study, the expression of various housekeeping genes (18S rRNA [18S], glyceraldehydes-3-phosphate dehydrogenase [G3PDH], beta-glucuronidase [BGLU], histone H4 [HH4], ribosomal protein L19 [RPL19] and cyclophilin [CY]) was investigated during fetal rat brain development using semi-quantitative RT-PCR at 16, 19 and 21 days gestation. It was found that all genes studied, with exception to G3PDH, did not show any change in their expression levels during development. G3PDH, on the other hand, showed increased expression with development. These results suggest that the choice of a housekeeping gene is critical to the interpretation of experimental results and should be modified according to the nature of the study.

An eQTL Analysis of Partial Resistance to Puccinia hordei in Barley

PubMed Central

Chen, Xinwei; Hackett, Christine A.; Niks, Rients E.; Hedley, Peter E.; Booth, Clare; Druka, Arnis; Marcel, Thierry C.; Vels, Anton; Bayer, Micha; Milne, Iain; Morris, Jenny; Ramsay, Luke; Marshall, David; Cardle, Linda; Waugh, Robbie

2010-01-01

Background Genetic resistance to barley leaf rust caused by Puccinia hordei involves both R genes and quantitative trait loci. The R genes provide higher but less durable resistance than the quantitative trait loci. Consequently, exploring quantitative or partial resistance has become a favorable alternative for controlling disease. Four quantitative trait loci for partial resistance to leaf rust have been identified in the doubled haploid Steptoe (St)/Morex (Mx) mapping population. Further investigations are required to study the molecular mechanisms underpinning partial resistance and ultimately identify the causal genes. Methodology/Principal Findings We explored partial resistance to barley leaf rust using a genetical genomics approach. We recorded RNA transcript abundance corresponding to each probe on a 15K Agilent custom barley microarray in seedlings from St and Mx and 144 doubled haploid lines of the St/Mx population. A total of 1154 and 1037 genes were, respectively, identified as being P. hordei-responsive among the St and Mx and differentially expressed between P. hordei-infected St and Mx. Normalized ratios from 72 distant-pair hybridisations were used to map the genetic determinants of variation in transcript abundance by expression quantitative trait locus (eQTL) mapping generating 15685 eQTL from 9557 genes. Correlation analysis identified 128 genes that were correlated with resistance, of which 89 had eQTL co-locating with the phenotypic quantitative trait loci (pQTL). Transcript abundance in the parents and conservation of synteny with rice allowed us to prioritise six genes as candidates for Rphq11, the pQTL of largest effect, and highlight one, a phospholipid hydroperoxide glutathione peroxidase (HvPHGPx) for detailed analysis. Conclusions/Significance The eQTL approach yielded information that led to the identification of strong candidate genes underlying pQTL for resistance to leaf rust in barley and on the general pathogen response pathway. The dataset will facilitate a systems appraisal of this host-pathogen interaction and, potentially, for other traits measured in this population. PMID:20066049

Gene-Based Testing of Interactions in Association Studies of Quantitative Traits

PubMed Central

Ma, Li; Clark, Andrew G.; Keinan, Alon

2013-01-01

Various methods have been developed for identifying gene–gene interactions in genome-wide association studies (GWAS). However, most methods focus on individual markers as the testing unit, and the large number of such tests drastically erodes statistical power. In this study, we propose novel interaction tests of quantitative traits that are gene-based and that confer advantage in both statistical power and biological interpretation. The framework of gene-based gene–gene interaction (GGG) tests combine marker-based interaction tests between all pairs of markers in two genes to produce a gene-level test for interaction between the two. The tests are based on an analytical formula we derive for the correlation between marker-based interaction tests due to linkage disequilibrium. We propose four GGG tests that extend the following P value combining methods: minimum P value, extended Simes procedure, truncated tail strength, and truncated P value product. Extensive simulations point to correct type I error rates of all tests and show that the two truncated tests are more powerful than the other tests in cases of markers involved in the underlying interaction not being directly genotyped and in cases of multiple underlying interactions. We applied our tests to pairs of genes that exhibit a protein–protein interaction to test for gene-level interactions underlying lipid levels using genotype data from the Atherosclerosis Risk in Communities study. We identified five novel interactions that are not evident from marker-based interaction testing and successfully replicated one of these interactions, between SMAD3 and NEDD9, in an independent sample from the Multi-Ethnic Study of Atherosclerosis. We conclude that our GGG tests show improved power to identify gene-level interactions in existing, as well as emerging, association studies. PMID:23468652

Selection of reliable reference genes for gene expression studies in Trichoderma afroharzianum LTR-2 under oxalic acid stress.

PubMed

Lyu, Yuping; Wu, Xiaoqing; Ren, He; Zhou, Fangyuan; Zhou, Hongzi; Zhang, Xinjian; Yang, Hetong

2017-10-01

An appropriate reference gene is required to get reliable results from gene expression analysis by quantitative real-time reverse transcription PCR (qRT-PCR). In order to identify stable and reliable reference genes in Trichoderma afroharzianum under oxalic acid (OA) stress, six commonly used housekeeping genes, i.e., elongation factor 1, ubiquitin, ubiquitin-conjugating enzyme, glyceraldehyde-3-phosphate dehydrogenase, α-tubulin, actin, from the effective biocontrol isolate T. afroharzianum strain LTR-2 were tested for their expression during growth in liquid culture amended with OA. Four in silico programs (comparative ΔCt, NormFinder, geNorm and BestKeeper) were used to evaluate the expression stabilities of six candidate reference genes. The elongation factor 1 gene EF-1 was identified as the most stably expressed reference gene, and was used as the normalizer to quantify the expression level of the oxalate decarboxylase coding gene OXDC in T. afroharzianum strain LTR-2 under OA stress. The result showed that the expression of OXDC was significantly up-regulated as expected. This study provides an effective method to quantify expression changes of target genes in T. afroharzianum under OA stress. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of Reference Genes and Analysis of Heat Shock Protein Gene Expression in Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum, after Exposure to Heat Stress.

PubMed

Liu, Yong-Nan; Lu, Xiao-Xiao; Ren, Ang; Shi, Liang; Jiang, Ai-Liang; Yu, Han-Shou; Zhao, Ming-Wen

2017-01-01

Ganoderma lucidum has been considered an emerging model species for studying how environmental factors regulate the growth, development, and secondary metabolism of Basidiomycetes. Heat stress, which is one of the most important environmental abiotic stresses, seriously affects the growth, development, and yield of microorganisms. Understanding the response to heat stress has gradually become a hotspot in microorganism research. But suitable reference genes for expression analysis under heat stress have not been reported in G. lucidum. In this study, we systematically identified 11 candidate reference genes that were measured using reverse transcriptase quantitative polymerase chain reaction, and the gene expression stability was analyzed under heat stress conditions using geNorm and NormFinder. The results show that 5 reference genes-CYP and TIF, followed by UCE2, ACTIN, and UBQ1-are the most stable genes under our experimental conditions. Moreover, the relative expression levels of 3 heat stress response genes (hsp17.4, hsp70, and hsp90) were analyzed under heat stress conditions with different normalization strategies. The results show that use of a gene with unstable expression (SAND) as the reference gene leads to biased data and misinterpretations of the target gene expression level under heat stress.

Inheritance of partial resistance against Colletotrichum lindemuthianum in Phaseolus vulgaris and co-localization of quantitative trait loci with genes involved in specific resistance.

PubMed

Geffroy, V; Sévignac, M; De Oliveira, J C; Fouilloux, G; Skroch, P; Thoquet, P; Gepts, P; Langin, T; Dron, M

2000-03-01

Anthracnose, one of the most important diseases of common bean (Phaseolus vulgaris), is caused by the fungus Colletotrichum lindemuthianum. A "candidate gene" approach was used to map anthracnose resistance quantitative trait loci (QTL). Candidate genes included genes for both pathogen recognition (resistance genes and resistance gene analogs [RGAs]) and general plant defense (defense response genes). Two strains of C. lindemuthianum, identified in a world collection of 177 strains, displayed a reproducible and differential aggressiveness toward BAT93 and JaloEEP558, two parental lines of P. vulgaris representing the two major gene pools of this crop. A reliable test was developed to score partial resistance in aerial organs of the plant (stem, leaf, petiole) under controlled growth chamber conditions. BAT93 was more resistant than JaloEEP558 regardless of the organ or strain tested. With a recombinant inbred line (RIL) population derived from a cross between these two parental lines, 10 QTL were located on a genetic map harboring 143 markers, including known defense response genes, anthracnose-specific resistance genes, and RGAs. Eight of the QTL displayed isolate specificity. Two were co-localized with known defense genes (phenylalanine ammonia-lyase and hydroxyproline-rich glycoprotein) and three with anthracnose-specific resistance genes and/or RGAs. Interestingly, two QTL, with different allelic contribution, mapped on linkage group B4 in a 5.0 cM interval containing Andean and Mesoamerican specific resistance genes against C. lindemuthianum and 11 polymorphic fragments revealed with a RGA probe. The possible relationship between genes underlying specific and partial resistance is discussed.

The IQ Quantitative Trait Loci Project: A Critique.

ERIC Educational Resources Information Center

King, David

1998-01-01

Describes the IQ Quantitative Trait Loci (QTL) project, an attempt to identify genes underlying IQ score variations using maps from the Human Genome Project. The essay argues against funding the IQ QTL project because it will end the debates about the genetic basis of intelligence and may lead directly to eugenic programs of genetic testing. (SLD)

qPCR for Second Year Undergraduates: A Short, Structured Inquiry to Illustrate Differential Gene Expression

ERIC Educational Resources Information Center

McCauslin, Christine Seitz; Gunn, Kathryn Elaine; Pirone, Dana; Staiger, Jennifer

2015-01-01

We describe a structured inquiry laboratory exercise that examines transcriptional regulation of the "NOS2" gene under conditions that simulate the inflammatory response in macrophages. Using quantitative PCR and the comparative C[subscript T] method, students are able determine whether transcriptional activation of "NOS2"…

WormQTLHD--a web database for linking human disease to natural variation data in C. elegans.

PubMed

van der Velde, K Joeri; de Haan, Mark; Zych, Konrad; Arends, Danny; Snoek, L Basten; Kammenga, Jan E; Jansen, Ritsert C; Swertz, Morris A; Li, Yang

2014-01-01

Interactions between proteins are highly conserved across species. As a result, the molecular basis of multiple diseases affecting humans can be studied in model organisms that offer many alternative experimental opportunities. One such organism-Caenorhabditis elegans-has been used to produce much molecular quantitative genetics and systems biology data over the past decade. We present WormQTL(HD) (Human Disease), a database that quantitatively and systematically links expression Quantitative Trait Loci (eQTL) findings in C. elegans to gene-disease associations in man. WormQTL(HD), available online at http://www.wormqtl-hd.org, is a user-friendly set of tools to reveal functionally coherent, evolutionary conserved gene networks. These can be used to predict novel gene-to-gene associations and the functions of genes underlying the disease of interest. We created a new database that links C. elegans eQTL data sets to human diseases (34 337 gene-disease associations from OMIM, DGA, GWAS Central and NHGRI GWAS Catalogue) based on overlapping sets of orthologous genes associated to phenotypes in these two species. We utilized QTL results, high-throughput molecular phenotypes, classical phenotypes and genotype data covering different developmental stages and environments from WormQTL database. All software is available as open source, built on MOLGENIS and xQTL workbench.

Selection and Validation of Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Studies in Mossy Maze Polypore, Cerrena unicolor (Higher Basidiomycetes).

PubMed

Yang, Jie; Lin, Qi; Lin, Juan; Ye, Xiuyun

2016-01-01

With its ability to produce ligninolytic enzymes such as laccases, white-rot basidiomycete Cerrena unicolor, a medicinal mushroom, has great potential in biotechnology. Elucidation of the expression profiles of genes encoding ligninolytic enzymes are important for increasing their production. Quantitative real-time polymerase chain reaction (qPCR) is a powerful tool to study transcriptional regulation of genes of interest. To ensure accuracy and reliability of qPCR analysis of C. unicolor, expression levels of seven candidate reference genes were studied at different growth phases, under various induction conditions, and with a range of carbon/nitrogen ratios and carbon and nitrogen sources. The stability of the genes were analyzed with five statistical approaches, namely geNorm, NormFinder, BestKeeper, the ΔCt method, and RefFinder. Our results indicated that the selection of reference genes varied with sample sets. A combination of four reference genes (Cyt-c, ATP6, TEF1, and β-tubulin) were recommended for normalizing gene expression at different growth phases. GAPDH and Cyt-c were the appropriate reference genes under different induction conditions. ATP6 and TEF1 were most stable in fermentation media with various carbon/nitrogen ratios. In the fermentation media with various carbon or nitrogen sources, 18S rRNA and GAPDH were the references of choice. The present study represents the first validation analysis of reference genes in C. unicolor and serves as a foundation for its qPCR analysis.

Complex Genetics of Behavior: BXDs in the Automated Home-Cage.

PubMed

Loos, Maarten; Verhage, Matthijs; Spijker, Sabine; Smit, August B

2017-01-01

This chapter describes a use case for the genetic dissection and automated analysis of complex behavioral traits using the genetically diverse panel of BXD mouse recombinant inbred strains. Strains of the BXD resource differ widely in terms of gene and protein expression in the brain, as well as in their behavioral repertoire. A large mouse resource opens the possibility for gene finding studies underlying distinct behavioral phenotypes, however, such a resource poses a challenge in behavioral phenotyping. To address the specifics of large-scale screening we describe how to investigate: (1) how to assess mouse behavior systematically in addressing a large genetic cohort, (2) how to dissect automation-derived longitudinal mouse behavior into quantitative parameters, and (3) how to map these quantitative traits to the genome, deriving loci underlying aspects of behavior.

An inducible HSP70 gene from the midge Chironomus dilutus: Characterization and transcription profile under environmental stress

USGS Publications Warehouse

Karouna-Renier, N. K.; Rao, K.R.

2009-01-01

In the present study, we identified and characterized an inducible heat shock protein 70 (HSP70) from the midge Chironomus dilutus and investigated the transcriptional profile of the gene under baseline and environmentally stressful conditions. Using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), we observed increased expression of CD-HSP70-1 in response to both heat shock and copper stress. We also investigated the expression of this gene during midge development. All C. dilutus developmental stages expressed CD-HSP70-1 under normal conditions, although at extremely low levels. Phylogenetic analysis of the amino acid sequence demonstrated distinct clustering of this gene with inducible HSP70s from other insect species. ?? 2008 The Authors.

Validation of Reference Genes for Gene Expression Studies in Virus-Infected Nicotiana benthamiana Using Quantitative Real-Time PCR

PubMed Central

Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang

2012-01-01

Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant–pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1α, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N. benthamiana under other biotic and abiotic stress conditions. PMID:23029521

The detection of large deletions or duplications in genomic DNA.

PubMed

Armour, J A L; Barton, D E; Cockburn, D J; Taylor, G R

2002-11-01

While methods for the detection of point mutations and small insertions or deletions in genomic DNA are well established, the detection of larger (>100 bp) genomic duplications or deletions can be more difficult. Most mutation scanning methods use PCR as a first step, but the subsequent analyses are usually qualitative rather than quantitative. Gene dosage methods based on PCR need to be quantitative (i.e., they should report molar quantities of starting material) or semi-quantitative (i.e., they should report gene dosage relative to an internal standard). Without some sort of quantitation, heterozygous deletions and duplications may be overlooked and therefore be under-ascertained. Gene dosage methods provide the additional benefit of reporting allele drop-out in the PCR. This could impact on SNP surveys, where large-scale genotyping may miss null alleles. Here we review recent developments in techniques for the detection of this type of mutation and compare their relative strengths and weaknesses. We emphasize that comprehensive mutation analysis should include scanning for large insertions and deletions and duplications. Copyright 2002 Wiley-Liss, Inc.

Exploring Valid Reference Genes for Quantitative Real-time PCR Analysis in Plutella xylostella (Lepidoptera: Plutellidae)

PubMed Central

Fu, Wei; Xie, Wen; Zhang, Zhuo; Wang, Shaoli; Wu, Qingjun; Liu, Yong; Zhou, Xiaomao; Zhou, Xuguo; Zhang, Youjun

2013-01-01

Abstract: Quantitative real-time PCR (qRT-PCR), a primary tool in gene expression analysis, requires an appropriate normalization strategy to control for variation among samples. The best option is to compare the mRNA level of a target gene with that of reference gene(s) whose expression level is stable across various experimental conditions. In this study, expression profiles of eight candidate reference genes from the diamondback moth, Plutella xylostella, were evaluated under diverse experimental conditions. RefFinder, a web-based analysis tool, integrates four major computational programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCt method to comprehensively rank the tested candidate genes. Elongation factor 1 (EF1) was the most suited reference gene for the biotic factors (development stage, tissue, and strain). In contrast, although appropriate reference gene(s) do exist for several abiotic factors (temperature, photoperiod, insecticide, and mechanical injury), we were not able to identify a single universal reference gene. Nevertheless, a suite of candidate reference genes were specifically recommended for selected experimental conditions. Our finding is the first step toward establishing a standardized qRT-PCR analysis of this agriculturally important insect pest. PMID:23983612

Practical applications of the bioinformatics toolbox for narrowing quantitative trait loci.

PubMed

Burgess-Herbert, Sarah L; Cox, Allison; Tsaih, Shirng-Wern; Paigen, Beverly

2008-12-01

Dissecting the genes involved in complex traits can be confounded by multiple factors, including extensive epistatic interactions among genes, the involvement of epigenetic regulators, and the variable expressivity of traits. Although quantitative trait locus (QTL) analysis has been a powerful tool for localizing the chromosomal regions underlying complex traits, systematically identifying the causal genes remains challenging. Here, through its application to plasma levels of high-density lipoprotein cholesterol (HDL) in mice, we demonstrate a strategy for narrowing QTL that utilizes comparative genomics and bioinformatics techniques. We show how QTL detected in multiple crosses are subjected to both combined cross analysis and haplotype block analysis; how QTL from one species are mapped to the concordant regions in another species; and how genomewide scans associating haplotype groups with their phenotypes can be used to prioritize the narrowed regions. Then we illustrate how these individual methods for narrowing QTL can be systematically integrated for mouse chromosomes 12 and 15, resulting in a significantly reduced number of candidate genes, often from hundreds to <10. Finally, we give an example of how additional bioinformatics resources can be combined with experiments to determine the most likely quantitative trait genes.

Monitoring the Single-Cell Stress Response of the Diatom Thalassiosira pseudonana by Quantitative Real-Time Reverse Transcription-PCR

PubMed Central

Shi, Xu; Gao, Weimin; Chao, Shih-hui

2013-01-01

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition. PMID:23315741

Monitoring the single-cell stress response of the diatom Thalassiosira pseudonana by quantitative real-time reverse transcription-PCR.

PubMed

Shi, Xu; Gao, Weimin; Chao, Shih-hui; Zhang, Weiwen; Meldrum, Deirdre R

2013-03-01

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition.

Selection of suitable reference genes for gene expression studies in Staphylococcus capitis during growth under erythromycin stress.

PubMed

Cui, Bintao; Smooker, Peter M; Rouch, Duncan A; Deighton, Margaret A

2016-08-01

Accurate and reproducible measurement of gene transcription requires appropriate reference genes, which are stably expressed under different experimental conditions to provide normalization. Staphylococcus capitis is a human pathogen that produces biofilm under stress, such as imposed by antimicrobial agents. In this study, a set of five commonly used staphylococcal reference genes (gyrB, sodA, recA, tuf and rpoB) were systematically evaluated in two clinical isolates of Staphylococcus capitis (S. capitis subspecies urealyticus and capitis, respectively) under erythromycin stress in mid-log and stationary phases. Two public software programs (geNorm and NormFinder) and two manual calculation methods, reference residue normalization (RRN) and relative quantitative (RQ), were applied. The potential reference genes selected by the four algorithms were further validated by comparing the expression of a well-studied biofilm gene (icaA) with phenotypic biofilm formation in S. capitis under four different experimental conditions. The four methods differed considerably in their ability to predict the most suitable reference gene or gene combination for comparing icaA expression under different conditions. Under the conditions used here, the RQ method provided better selection of reference genes than the other three algorithms; however, this finding needs to be confirmed with a larger number of isolates. This study reinforces the need to assess the stability of reference genes for analysis of target gene expression under different conditions and the use of more than one algorithm in such studies. Although this work was conducted using a specific human pathogen, it emphasizes the importance of selecting suitable reference genes for accurate normalization of gene expression more generally.

Fine-Mapping and Selective Sweep Analysis of QTL for Cold Tolerance in Drosophila melanogaster

PubMed Central

Wilches, Ricardo; Voigt, Susanne; Duchen, Pablo; Laurent, Stefan; Stephan, Wolfgang

2014-01-01

There is a growing interest in investigating the relationship between genes with signatures of natural selection and genes identified in QTL mapping studies using combined population and quantitative genetics approaches. We dissected an X-linked interval of 6.2 Mb, which contains two QTL underlying variation in chill coma recovery time (CCRT) in Drosophila melanogaster from temperate (European) and tropical (African) regions. This resulted in two relatively small regions of 131 kb and 124 kb. The latter one co-localizes with a very strong selective sweep in the European population. We examined the genes within and near the sweep region individually using gene expression analysis and P-element insertion lines. Of the genes overlapping with the sweep, none appears to be related to CCRT. However, we have identified a new candidate gene of CCRT, brinker, which is located just outside the sweep region and is inducible by cold stress. We discuss these results in light of recent population genetics theories on quantitative traits. PMID:24970882

Expression of a chitin deacetylase gene, up-regulated in Cryptococcus laurentii strain RY1, under nitrogen limitation.

PubMed

Chakraborty, Writachit; Sarkar, Soumyadev; Chakravorty, Somnath; Bhattacharya, Semantee; Bhattacharya, Debanjana; Gachhui, Ratan

2016-05-01

This study reports the identification of a chitin deacetylase gene in Cryptococcus laurentii strain RY1 over-expressing under nitrogen limitation by differential display. The up-regulation took place in robustly growing cells rather than in starving quiescent autophagic cells. Quantitative Real Time-PCR, enzyme activity in cell lysate and cell wall analysis corroborated the up-regulation of chitin deacetylase under nitrogen limitation. These results suggest chitin deacetylase might play a significant role in nitrogen limiting growth of Cryptococcus laurentii strain RY1. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reference Gene Selection for Quantitative Real-Time Reverse-Transcriptase PCR in Annual Ryegrass (Lolium multiflorum) Subjected to Various Abiotic Stresses.

PubMed

Liu, Qiuxu; Qi, Xiao; Yan, Haidong; Huang, Linkai; Nie, Gang; Zhang, Xinquan

2018-01-16

To select the most stable reference genes in annual ryegrass ( Lolium multiflorum ), we studied annual ryegrass leaf tissues exposed to various abiotic stresses by qRT-PCR and selected 11 candidate reference genes, i.e., 18S rRNA, E2, GAPDH, eIF4A, HIS3, SAMDC, TBP-1, Unigene71, Unigene77, Unigene755, and Unigene14912. We then used GeNorm, NormFinder, and BestKeeper to analyze the expression stability of these 11 genes, and used RefFinder to comprehensively rank genes according to stability. Under different stress conditions, the most suitable reference genes for studies of leaf tissues of annual ryegrass were different. The expression of the eIF4A gene was the most stable under drought stress. Under saline-alkali stress, Unigene14912 has the highest expression stability. Under acidic aluminum stress, SAMDC expression stability was highest. Under heavy metal stress, Unigene71 expression had the highest stability. According to the software analyses, Unigene14912, HIS3, and eIF4A were the most suitable for analyses of abiotic stress in tissues of annual ryegrass. GAPDH was the least suitable reference gene. In conclusion, selecting appropriate reference genes under abiotic stress not only improves the accuracy of annual ryegrass gene expression analyses, but also provides a theoretical reference for the development of reference genes in plants of the genus Lolium .

Quantitative high-resolution genomic analysis of single cancer cells.

PubMed

Hannemann, Juliane; Meyer-Staeckling, Sönke; Kemming, Dirk; Alpers, Iris; Joosse, Simon A; Pospisil, Heike; Kurtz, Stefan; Görndt, Jennifer; Püschel, Klaus; Riethdorf, Sabine; Pantel, Klaus; Brandt, Burkhard

2011-01-01

During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.

Discovering hidden relationships between renal diseases and regulated genes through 3D network visualizations

PubMed Central

2010-01-01

Background In a recent study, two-dimensional (2D) network layouts were used to visualize and quantitatively analyze the relationship between chronic renal diseases and regulated genes. The results revealed complex relationships between disease type, gene specificity, and gene regulation type, which led to important insights about the underlying biological pathways. Here we describe an attempt to extend our understanding of these complex relationships by reanalyzing the data using three-dimensional (3D) network layouts, displayed through 2D and 3D viewing methods. Findings The 3D network layout (displayed through the 3D viewing method) revealed that genes implicated in many diseases (non-specific genes) tended to be predominantly down-regulated, whereas genes regulated in a few diseases (disease-specific genes) tended to be up-regulated. This new global relationship was quantitatively validated through comparison to 1000 random permutations of networks of the same size and distribution. Our new finding appeared to be the result of using specific features of the 3D viewing method to analyze the 3D renal network. Conclusions The global relationship between gene regulation and gene specificity is the first clue from human studies that there exist common mechanisms across several renal diseases, which suggest hypotheses for the underlying mechanisms. Furthermore, the study suggests hypotheses for why the 3D visualization helped to make salient a new regularity that was difficult to detect in 2D. Future research that tests these hypotheses should enable a more systematic understanding of when and how to use 3D network visualizations to reveal complex regularities in biological networks. PMID:21070623

Reference Gene Selection for qPCR Normalization of Kosteletzkya virginica under Salt Stress

PubMed Central

Tang, Xiaoli; Wang, Hongyan; Shao, Chuyang; Shao, Hongbo

2015-01-01

Kosteletzkya virginica (L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene in K. virginica which showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA), β-actin (ACT), α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and 18SrRNA was assessed to be the most stable reference gene in this study. However, TUA was identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies in K. virginica. PMID:26581422

A gene encoding maize caffeoyl-CoA O-methyltransferase confers quantitative resistance to multiple pathogens.

PubMed

Yang, Qin; He, Yijian; Kabahuma, Mercy; Chaya, Timothy; Kelly, Amy; Borrego, Eli; Bian, Yang; El Kasmi, Farid; Yang, Li; Teixeira, Paulo; Kolkman, Judith; Nelson, Rebecca; Kolomiets, Michael; L Dangl, Jeffery; Wisser, Randall; Caplan, Jeffrey; Li, Xu; Lauter, Nick; Balint-Kurti, Peter

2017-09-01

Alleles that confer multiple disease resistance (MDR) are valuable in crop improvement, although the molecular mechanisms underlying their functions remain largely unknown. A quantitative trait locus, qMdr 9.02 , associated with resistance to three important foliar maize diseases-southern leaf blight, gray leaf spot and northern leaf blight-has been identified on maize chromosome 9. Through fine-mapping, association analysis, expression analysis, insertional mutagenesis and transgenic validation, we demonstrate that ZmCCoAOMT2, which encodes a caffeoyl-CoA O-methyltransferase associated with the phenylpropanoid pathway and lignin production, is the gene within qMdr 9.02 conferring quantitative resistance to both southern leaf blight and gray leaf spot. We suggest that resistance might be caused by allelic variation at the level of both gene expression and amino acid sequence, thus resulting in differences in levels of lignin and other metabolites of the phenylpropanoid pathway and regulation of programmed cell death.

Identification of reference genes for quantitative expression analysis using large-scale RNA-seq data of Arabidopsis thaliana and model crop plants.

PubMed

Kudo, Toru; Sasaki, Yohei; Terashima, Shin; Matsuda-Imai, Noriko; Takano, Tomoyuki; Saito, Misa; Kanno, Maasa; Ozaki, Soichi; Suwabe, Keita; Suzuki, Go; Watanabe, Masao; Matsuoka, Makoto; Takayama, Seiji; Yano, Kentaro

2016-10-13

In quantitative gene expression analysis, normalization using a reference gene as an internal control is frequently performed for appropriate interpretation of the results. Efforts have been devoted to exploring superior novel reference genes using microarray transcriptomic data and to evaluating commonly used reference genes by targeting analysis. However, because the number of specifically detectable genes is totally dependent on probe design in the microarray analysis, exploration using microarray data may miss some of the best choices for the reference genes. Recently emerging RNA sequencing (RNA-seq) provides an ideal resource for comprehensive exploration of reference genes since this method is capable of detecting all expressed genes, in principle including even unknown genes. We report the results of a comprehensive exploration of reference genes using public RNA-seq data from plants such as Arabidopsis thaliana (Arabidopsis), Glycine max (soybean), Solanum lycopersicum (tomato) and Oryza sativa (rice). To select reference genes suitable for the broadest experimental conditions possible, candidates were surveyed by the following four steps: (1) evaluation of the basal expression level of each gene in each experiment; (2) evaluation of the expression stability of each gene in each experiment; (3) evaluation of the expression stability of each gene across the experiments; and (4) selection of top-ranked genes, after ranking according to the number of experiments in which the gene was expressed stably. Employing this procedure, 13, 10, 12 and 21 top candidates for reference genes were proposed in Arabidopsis, soybean, tomato and rice, respectively. Microarray expression data confirmed that the expression of the proposed reference genes under broad experimental conditions was more stable than that of commonly used reference genes. These novel reference genes will be useful for analyzing gene expression profiles across experiments carried out under various experimental conditions.

Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence

PubMed Central

2017-01-01

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies. PMID:28591185

Apparent polyploidization after gamma irradiation: pitfalls in the use of quantitative polymerase chain reaction (qPCR) for the estimation of mitochondrial and nuclear DNA gene copy numbers.

PubMed

Kam, Winnie W Y; Lake, Vanessa; Banos, Connie; Davies, Justin; Banati, Richard

2013-05-30

Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization.

Allele Mining in Barley Genetic Resources Reveals Genes of Race-Non-Specific Powdery Mildew Resistance

PubMed Central

Spies, Annika; Korzun, Viktor; Bayles, Rosemary; Rajaraman, Jeyaraman; Himmelbach, Axel; Hedley, Pete E.; Schweizer, Patrick

2012-01-01

Race-non-specific, or quantitative, pathogen resistance is of high importance to plant breeders due to its expected durability. However, it is usually controlled by multiple quantitative trait loci (QTL) and therefore difficult to handle in practice. Knowing the genes that underlie race-non-specific resistance (NR) would allow its exploitation in a more targeted manner. Here, we performed an association-genetic study in a customized worldwide collection of spring barley accessions for candidate genes of race-NR to the powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) and combined data with results from QTL mapping as well as functional-genomics approaches. This led to the identification of 11 associated genes with converging evidence for an important role in race-NR in the presence of the Mlo gene for basal susceptibility. Outstanding in this respect was the gene encoding the transcription factor WRKY2. The results suggest that unlocking plant genetic resources and integrating functional-genomic with genetic approaches can accelerate the discovery of genes underlying race-NR in barley and other crop plants. PMID:22629270

Studies on the factors modulating indole-3-acetic acid production in endophytic bacterial isolates from Piper nigrum and molecular analysis of ipdc gene.

PubMed

Jasim, B; Jimtha John, C; Shimil, V; Jyothis, M; Radhakrishnan, E K

2014-09-01

The study mainly aimed quantitative analysis of IAA produced by endophytic bacteria under various conditions including the presence of extract from Piper nigrum. Analysis of genetic basis of IAA production was also conducted by studying the presence and diversity of the ipdc gene among the selected isolates. Five endophytic bacteria isolated previously from P. nigrum were used for the study. The effect of temperature, pH, agitation, tryptophan concentration and plant extract on modulating IAA production of selected isolates was analysed by colorimetric method. Comparative and quantitative analysis of IAA production by colorimetric isolates under optimal culture condition was analysed by HPTLC method. Presence of ipdc gene and thereby biosynthetic basis of IAA production among the selected isolates were studied by PCR-based amplification and subsequent insilico analysis of sequence obtained. Among the selected bacterial isolates from P. nigrum, isolate PnB 8 (Klebsiella pneumoniae) was found to have the maximum yield of IAA under various conditions optimized and was confirmed by colorimetric, HPLC and HPTLC analysis. Very interestingly, the study showed stimulating effect of phytochemicals from P. nigrum on IAA production by endophytic bacteria isolated from same plant. This study is unique because of the selection of endophytes from same source for comparative and quantitative analysis of IAA production under various conditions. Study on stimulatory effect of phytochemicals on bacterial IAA production as explained in the study is a novel approach. Studies on molecular basis of IAA production which was confirmed by sequence analysis of ipdc gene make the study scientifically attractive. Even though microbial production of IAA is well known, current report on detailed optimization, effect of plant extract and molecular confirmation of IAA biosynthesis is comparatively novel in its approach. © 2014 The Society for Applied Microbiology.

Population structure and strong divergent selection shape phenotypic diversification in maize landraces.

PubMed

Pressoir, G; Berthaud, J

2004-02-01

To conserve the long-term selection potential of maize, it is necessary to investigate past and present evolutionary processes that have shaped quantitative trait variation. Understanding the dynamics of quantitative trait evolution is crucial to future crop breeding. We characterized population differentiation of maize landraces from the State of Oaxaca, Mexico for quantitative traits and molecular markers. Qst values were much higher than Fst values obtained for molecular markers. While low values of Fst (0.011 within-village and 0.003 among-villages) suggest that considerable gene flow occurred among the studied populations, high levels of population differentiation for quantitative traits were observed (ie an among-village Qst value of 0.535 for kernel weight). Our results suggest that although quantitative traits appear to be under strong divergent selection, a considerable amount of gene flow occurs among populations. Furthermore, we characterized nonproportional changes in the G matrix structure both within and among villages that are consequences of farmer selection. As a consequence of these differences in the G matrix structure, the response to multivariate selection will be different from one population to another. Large changes in the G matrix structure could indicate that farmers select for genes of major and pleiotropic effect. Farmers' decision and selection strategies have a great impact on phenotypic diversification in maize landraces.

QTLs for Seed Vigor-Related Traits Identified in Maize Seeds Germinated under Artificial Aging Conditions

PubMed Central

Han, Zanping; Ku, Lixia; Zhang, Zhenzhen; Zhang, Jun; Guo, ShuLei; Liu, Haiying; Zhao, Ruifang; Ren, Zhenzhen; Zhang, Liangkun; Su, Huihui; Dong, Lei; Chen, Yanhui

2014-01-01

High seed vigor is important for agricultural production due to the associated potential for increased growth and productivity. However, a better understanding of the underlying molecular mechanisms is required because the genetic basis for seed vigor remains unknown. We used single-nucleotide polymorphism (SNP) markers to map quantitative trait loci (QTLs) for four seed vigor traits in two connected recombinant inbred line (RIL) maize populations under four treatment conditions during seed germination. Sixty-five QTLs distributed between the two populations were identified and a meta-analysis was used to integrate genetic maps. Sixty-one initially identified QTLs were integrated into 18 meta-QTLs (mQTLs). Initial QTLs with contribution to phenotypic variation values of R2>10% were integrated into mQTLs. Twenty-three candidate genes for association with seed vigor traits coincided with 13 mQTLs. The candidate genes had functions in the glycolytic pathway and in protein metabolism. QTLs with major effects (R2>10%) were identified under at least one treatment condition for mQTL2, mQTL3-2, and mQTL3-4. Candidate genes included a calcium-dependent protein kinase gene (302810918) involved in signal transduction that mapped in the mQTL3-2 interval associated with germination energy (GE) and germination percentage (GP), and an hsp20/alpha crystallin family protein gene (At5g51440) that mapped in the mQTL3-4 interval associated with GE and GP. Two initial QTLs with a major effect under at least two treatment conditions were identified for mQTL5-2. A cucumisin-like Ser protease gene (At5g67360) mapped in the mQTL5-2 interval associated with GP. The chromosome regions for mQTL2, mQTL3-2, mQTL3-4, and mQTL5-2 may be hot spots for QTLs related to seed vigor traits. The mQTLs and candidate genes identified in this study provide valuable information for the identification of additional quantitative trait genes. PMID:24651614

QTLs for seed vigor-related traits identified in maize seeds germinated under artificial aging conditions.

PubMed

Han, Zanping; Ku, Lixia; Zhang, Zhenzhen; Zhang, Jun; Guo, Shulei; Liu, Haiying; Zhao, Ruifang; Ren, Zhenzhen; Zhang, Liangkun; Su, Huihui; Dong, Lei; Chen, Yanhui

2014-01-01

High seed vigor is important for agricultural production due to the associated potential for increased growth and productivity. However, a better understanding of the underlying molecular mechanisms is required because the genetic basis for seed vigor remains unknown. We used single-nucleotide polymorphism (SNP) markers to map quantitative trait loci (QTLs) for four seed vigor traits in two connected recombinant inbred line (RIL) maize populations under four treatment conditions during seed germination. Sixty-five QTLs distributed between the two populations were identified and a meta-analysis was used to integrate genetic maps. Sixty-one initially identified QTLs were integrated into 18 meta-QTLs (mQTLs). Initial QTLs with contribution to phenotypic variation values of R(2)>10% were integrated into mQTLs. Twenty-three candidate genes for association with seed vigor traits coincided with 13 mQTLs. The candidate genes had functions in the glycolytic pathway and in protein metabolism. QTLs with major effects (R(2)>10%) were identified under at least one treatment condition for mQTL2, mQTL3-2, and mQTL3-4. Candidate genes included a calcium-dependent protein kinase gene (302810918) involved in signal transduction that mapped in the mQTL3-2 interval associated with germination energy (GE) and germination percentage (GP), and an hsp20/alpha crystallin family protein gene (At5g51440) that mapped in the mQTL3-4 interval associated with GE and GP. Two initial QTLs with a major effect under at least two treatment conditions were identified for mQTL5-2. A cucumisin-like Ser protease gene (At5g67360) mapped in the mQTL5-2 interval associated with GP. The chromosome regions for mQTL2, mQTL3-2, mQTL3-4, and mQTL5-2 may be hot spots for QTLs related to seed vigor traits. The mQTLs and candidate genes identified in this study provide valuable information for the identification of additional quantitative trait genes.

Identification of Reference Genes for Normalizing Quantitative Real-Time PCR in Urechis unicinctus

NASA Astrophysics Data System (ADS)

Bai, Yajiao; Zhou, Di; Wei, Maokai; Xie, Yueyang; Gao, Beibei; Qin, Zhenkui; Zhang, Zhifeng

2018-06-01

The reverse transcription quantitative real-time PCR (RT-qPCR) has become one of the most important techniques of studying gene expression. A set of valid reference genes are essential for the accurate normalization of data. In this study, five candidate genes were analyzed with geNorm, NormFinder, BestKeeper and ΔCt methods to identify the genes stably expressed in echiuran Urechis unicinctus, an important commercial marine benthic worm, under abiotic (sulfide stress) and normal (adult tissues, embryos and larvae at different development stages) conditions. The comprehensive results indicated that the expression of TBP was the most stable at sulfide stress and in developmental process, while the expression of EF- 1- α was the most stable at sulfide stress and in various tissues. TBP and EF- 1- α were recommended as a suitable reference gene combination to accurately normalize the expression of target genes at sulfide stress; and EF- 1- α, TBP and TUB were considered as a potential reference gene combination for normalizing the expression of target genes in different tissues. No suitable gene combination was obtained among these five candidate genes for normalizing the expression of target genes for developmental process of U. unicinctus. Our results provided a valuable support for quantifying gene expression using RT-qPCR in U. unicinctus.

Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis1[w

PubMed Central

Czechowski, Tomasz; Stitt, Mark; Altmann, Thomas; Udvardi, Michael K.; Scheible, Wolf-Rüdiger

2005-01-01

Gene transcripts with invariant abundance during development and in the face of environmental stimuli are essential reference points for accurate gene expression analyses, such as RNA gel-blot analysis or quantitative reverse transcription-polymerase chain reaction (PCR). An exceptionally large set of data from Affymetrix ATH1 whole-genome GeneChip studies provided the means to identify a new generation of reference genes with very stable expression levels in the model plant species Arabidopsis (Arabidopsis thaliana). Hundreds of Arabidopsis genes were found that outperform traditional reference genes in terms of expression stability throughout development and under a range of environmental conditions. Most of these were expressed at much lower levels than traditional reference genes, making them very suitable for normalization of gene expression over a wide range of transcript levels. Specific and efficient primers were developed for 22 genes and tested on a diverse set of 20 cDNA samples. Quantitative reverse transcription-PCR confirmed superior expression stability and lower absolute expression levels for many of these genes, including genes encoding a protein phosphatase 2A subunit, a coatomer subunit, and an ubiquitin-conjugating enzyme. The developed PCR primers or hybridization probes for the novel reference genes will enable better normalization and quantification of transcript levels in Arabidopsis in the future. PMID:16166256

In-depth analysis of internal control genes for quantitative real-time PCR in Brassica oleracea var. botrytis.

PubMed

Sheng, X G; Zhao, Z Q; Yu, H F; Wang, J S; Zheng, C F; Gu, H H

2016-07-15

Quantitative reverse-transcription PCR (qRT-PCR) is a versatile technique for the analysis of gene expression. The selection of stable reference genes is essential for the application of this technique. Cauliflower (Brassica oleracea L. var. botrytis) is a commonly consumed vegetable that is rich in vitamin, calcium, and iron. Thus far, to our knowledge, there have been no reports on the validation of suitable reference genes for the data normalization of qRT-PCR in cauliflower. In the present study, we analyzed 12 candidate housekeeping genes in cauliflower subjected to different abiotic stresses, hormone treatment conditions, and accessions. geNorm and NormFinder algorithms were used to assess the expression stability of these genes. ACT2 and TIP41 were selected as suitable reference genes across all experimental samples in this study. When different accessions were compared, ACT2 and UNK3 were found to be the most suitable reference genes. In the hormone and abiotic stress treatments, ACT2, TIP41, and UNK2 were the most stably expressed. Our study also provided guidelines for selecting the best reference genes under various experimental conditions.

Differentially expressed genes of Tetrahymena thermophila in response to tributyltin (TBT) identified by suppression subtractive hybridization and real time quantitative PCR.

PubMed

Feng, Lifang; Miao, Wei; Wu, Yuxuan

2007-02-15

Tributyltin (TBT) is widely used as antifouling paints, agriculture biocides, and plastic stabilizers around the world, resulting in great pollution problem in aquatic environments. However, it has been short of the biomonitor to detect TBT in freshwater. We constructed the suppression subtractive hybridization library of Tetrahymena thermophila exposed to TBT, and screened out 101 Expressed Sequence Tags whose expressions were significantly up- or down-regulated with TBT treatment. From this, a series of genes related to the TBT toxicity were discovered, such as glutathione-S-transferase gene (down-regulated), plasma membrane Ca2+ ATPase isoforms 3 gene (up-regulated) and NgoA (up-regulated). Furthermore, their expressions under different concentrations of TBT treatment (0.5-40 ppb) were detected by real time fluorescent quantitative PCR. The differentially expressed genes of T. thermophila in response to TBT were identified, which provide the basic to make Tetrahymena as a sensitive, rapid and convenient TBT biomonitor in freshwater based on rDNA inducible expression system.

Genetic Mapping of Quantitative Trait Loci for Grain Yield under Drought in Rice under Controlled Greenhouse Conditions

NASA Astrophysics Data System (ADS)

Solis, Julio; Gutierrez, Andres; Mangu, Venkata; Sanchez, Eduardo; Bedre, Renesh; Linscombe, Steve; Baisakh, Niranjan

2017-12-01

Drought stress is a constant threat to rice production worldwide. Most Mmodern rice cultivars are sensitive to drought, and the effect is severe at the reproductive stage. Conventional breeding for drought resistant (DR) rice varieties is slow and limited due to the quantitative nature of the DR traits. Identification of genes (QTLs)/markers associated with DR traits is a prerequisite for marker-assisted breeding. Grain yield is the most important trait and to this end drought yield QTLs have been identified under field conditions. The present study reports identification of drought yield QTLs under controlled conditions without confounding effects of other factors prevalent under natural conditions. A linkage map covering 1,781.5 cM with an average resolution of 9.76 cM was constructed using an F2 population from a cross between two Japonica cultivars, Cocodrie (drought sensitive) and Vandana (drought tolerant) with 213 markers distributed over 12 rice chromosomes. A subset of 59 markers (22 genic SSRs and 37 SNPs) derived from the transcriptome of the parents were also placed in the map. Single marker analysis using 187 F2:3 progeny identified 6 markers distributed on chromosomes 1, 5, and 8 to be associated with grain yield under drought (GYD). Composite interval mapping identified six genomic regions/quantitative trait loci (QTL) on chromosome 1, 5, 8, and 9 to be associated with GYD. QTLs located on chromosome 1 (qGYD1.2, qGYD1.3), chromosome 5 (qGYD5.1) and chromosome 8 (qGYD8.1) were contributed by Vandana alleles, whereas the QTLs, qGYD1.1 and qQYD9.1 were contributed by Cocodrie alelles. The additive positive phenotypic variance explained by the QTLs ranged from 30.0% to 34.0%. Candidate genes annotation within QTLs suggested the role of transcription factors and genes involved in osmotic potential regulation through catalytic/metabolic pathways in drought resistance tolerance mechanism contributing to yield.

Identification of Differentially Expressed Genes in Blood Cells of Narcolepsy Patients

PubMed Central

Tanaka, Susumu; Honda, Yutaka; Honda, Makoto

2007-01-01

Study Objective: A close association between the human leukocyte antigen (HLA)-DRB1*1501/DQB1*0602 and abnormalities in some inflammatory cytokines have been demonstrated in narcolepsy. Specific alterations in the immune system have been suggested to occur in this disorder. We attempted to identify alterations in gene expression underlying the abnormalities in the blood cells of narcoleptic patients. Designs: Total RNA from 12 narcolepsy-cataplexy patients and from 12 age- and sex-matched healthy controls were pooled. The pooled samples were initially screened for candidate genes for narcolepsy by differential display analysis using annealing control primers (ACP). The second screening of the samples was carried out by semiquantitative PCR using gene-specific primers. Finally, the expression levels of the candidate genes were further confirmed by quantitative real-time PCR using a new set of samples (20 narcolepsy-cataplexy patients and 20 healthy controls). Results: The second screening revealed differential expression of 4 candidate genes. Among them, MX2 was confirmed as a significantly down-regulated gene in the white blood cells of narcoleptic patients by quantitative real-time PCR. Conclusion: We found the MX2 gene to be significantly less expressed in comparison with normal subjects in the white blood cells of narcoleptic patients. This gene is relevant to the immune system. Although differential display analysis using ACP technology has a limitation in that it does not help in determining the functional mechanism underlying sleep/wakefulness dysregulation, it is useful for identifying novel genetic factors related to narcolepsy, such as HLA molecules. Further studies are required to explore the functional relationship between the MX2 gene and narcolepsy pathophysiology. Citation: Tanaka S; Honda Y; Honda M. Identification of differentially expressed genes in blood cells of narcolepsy patients. SLEEP 2007;30(8):974-979. PMID:17702266

Predicting epidermal growth factor receptor gene amplification status in glioblastoma multiforme by quantitative enhancement and necrosis features deriving from conventional magnetic resonance imaging.

PubMed

Dong, Fei; Zeng, Qiang; Jiang, Biao; Yu, Xinfeng; Wang, Weiwei; Xu, Jingjing; Yu, Jinna; Li, Qian; Zhang, Minming

2018-05-01

To study whether some of the quantitative enhancement and necrosis features in preoperative conventional MRI (cMRI) had a predictive value for epidermal growth factor receptor (EGFR) gene amplification status in glioblastoma multiforme (GBM).Fifty-five patients with pathologically determined GBMs who underwent cMRI were retrospectively reviewed. The following cMRI features were quantitatively measured and recorded: long and short diameters of the enhanced portion (LDE and SDE), maximum and minimum thickness of the enhanced portion (MaxTE and MinTE), and long and short diameters of the necrotic portion (LDN and SDN). Univariate analysis of each feature and a decision tree model fed with all the features were performed. Area under the receiver operating characteristic (ROC) curve (AUC) was used to assess the performance of features, and predictive accuracy was used to assess the performance of the model.For single feature, MinTE showed the best performance in differentiating EGFR gene amplification negative (wild-type) (nEGFR) GBM from EGFR gene amplification positive (pEGFR) GBM, and it got an AUC of 0.68 with a cut-off value of 2.6 mm. The decision tree model included 2 features MinTE and SDN, and got an accuracy of 0.83 in validation dataset.Our results suggest that quantitative measurement of the features MinTE and SDN in preoperative cMRI had a high accuracy for predicting EGFR gene amplification status in GBM.

Intelligence: shared genetic basis between Mendelian disorders and a polygenic trait.

PubMed

Franić, Sanja; Groen-Blokhuis, Maria M; Dolan, Conor V; Kattenberg, Mathijs V; Pool, René; Xiao, Xiangjun; Scheet, Paul A; Ehli, Erik A; Davies, Gareth E; van der Sluis, Sophie; Abdellaoui, Abdel; Hansell, Narelle K; Martin, Nicholas G; Hudziak, James J; van Beijsterveldt, Catherina E M; Swagerman, Suzanne C; Hulshoff Pol, Hilleke E; de Geus, Eco J C; Bartels, Meike; Ropers, H Hilger; Hottenga, Jouke-Jan; Boomsma, Dorret I

2015-10-01

Multiple inquiries into the genetic etiology of human traits indicated an overlap between genes underlying monogenic disorders (eg, skeletal growth defects) and those affecting continuous variability of related quantitative traits (eg, height). Extending the idea of a shared genetic basis between a Mendelian disorder and a classic polygenic trait, we performed an association study to examine the effect of 43 genes implicated in autosomal recessive cognitive disorders on intelligence in an unselected Dutch population (N=1316). Using both single-nucleotide polymorphism (SNP)- and gene-based association testing, we detected an association between intelligence and the genes of interest, with genes ELP2, TMEM135, PRMT10, and RGS7 showing the strongest associations. This is a demonstration of the relevance of genes implicated in monogenic disorders of intelligence to normal-range intelligence, and a corroboration of the utility of employing knowledge on monogenic disorders in identifying the genetic variability underlying complex traits.

Integrative strategies to identify candidate genes in rodent models of human alcoholism.

PubMed

Treadwell, Julie A

2006-01-01

The search for genes underlying alcohol-related behaviours in rodent models of human alcoholism has been ongoing for many years with only limited success. Recently, new strategies that integrate several of the traditional approaches have provided new insights into the molecular mechanisms underlying ethanol's actions in the brain. We have used alcohol-preferring C57BL/6J (B6) and alcohol-avoiding DBA/2J (D2) genetic strains of mice in an integrative strategy combining high-throughput gene expression screening, genetic segregation analysis, and mapping to previously published quantitative trait loci to uncover candidate genes for the ethanol-preference phenotype. In our study, 2 genes, retinaldehyde binding protein 1 (Rlbp1) and syntaxin 12 (Stx12), were found to be strong candidates for ethanol preference. Such experimental approaches have the power and the potential to greatly speed up the laborious process of identifying candidate genes for the animal models of human alcoholism.

WormQTLHD—a web database for linking human disease to natural variation data in C. elegans

PubMed Central

van der Velde, K. Joeri; de Haan, Mark; Zych, Konrad; Arends, Danny; Snoek, L. Basten; Kammenga, Jan E.; Jansen, Ritsert C.; Swertz, Morris A.; Li, Yang

2014-01-01

Interactions between proteins are highly conserved across species. As a result, the molecular basis of multiple diseases affecting humans can be studied in model organisms that offer many alternative experimental opportunities. One such organism—Caenorhabditis elegans—has been used to produce much molecular quantitative genetics and systems biology data over the past decade. We present WormQTLHD (Human Disease), a database that quantitatively and systematically links expression Quantitative Trait Loci (eQTL) findings in C. elegans to gene–disease associations in man. WormQTLHD, available online at http://www.wormqtl-hd.org, is a user-friendly set of tools to reveal functionally coherent, evolutionary conserved gene networks. These can be used to predict novel gene-to-gene associations and the functions of genes underlying the disease of interest. We created a new database that links C. elegans eQTL data sets to human diseases (34 337 gene–disease associations from OMIM, DGA, GWAS Central and NHGRI GWAS Catalogue) based on overlapping sets of orthologous genes associated to phenotypes in these two species. We utilized QTL results, high-throughput molecular phenotypes, classical phenotypes and genotype data covering different developmental stages and environments from WormQTL database. All software is available as open source, built on MOLGENIS and xQTL workbench. PMID:24217915

Quantitative High-Resolution Genomic Analysis of Single Cancer Cells

PubMed Central

Hannemann, Juliane; Meyer-Staeckling, Sönke; Kemming, Dirk; Alpers, Iris; Joosse, Simon A.; Pospisil, Heike; Kurtz, Stefan; Görndt, Jennifer; Püschel, Klaus; Riethdorf, Sabine; Pantel, Klaus; Brandt, Burkhard

2011-01-01

During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics. PMID:22140428

Reference genes for normalization of qPCR assays in sugarcane plants under water deficit.

PubMed

de Andrade, Larissa Mara; Dos Santos Brito, Michael; Fávero Peixoto Junior, Rafael; Marchiori, Paulo Eduardo Ribeiro; Nóbile, Paula Macedo; Martins, Alexandre Palma Boer; Ribeiro, Rafael Vasconcelos; Creste, Silvana

2017-01-01

Sugarcane ( Saccharum spp.) is the main raw material for sugar and ethanol production. Among the abiotic stress, drought is the main one that negatively impact sugarcane yield. Although gene expression analysis through quantitative PCR (qPCR) has increased our knowledge about biological processes related to drought, gene network that mediates sugarcane responses to water deficit remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving qPCR. In this study, candidate genes were tested for their suitable as reference genes for qPCR analyses in two sugarcane cultivars with varying drought tolerance. Eight candidate reference genes were evaluated in leaves sampled in plants subjected to water deficit in both field and greenhouse conditions. In addition, five genes were evaluated in shoot roots of plants subjected to water deficit by adding PEG8000 to the nutrient solution. NormFinder and RefFinder algorithms were used to identify the most stable gene(s) among genotypes and under different experimental conditions. Both algorithms revealed that in leaf samples, UBQ1 and GAPDH genes were more suitable as reference genes, whereas GAPDH was the best reference one in shoot roots. Reference genes suitable for sugarcane under water deficit were identified, which would lead to a more accurate and reliable analysis of qPCR. Thus, results obtained in this study may guide future research on gene expression in sugarcane under varying water conditions.

Genetic dissection of growth, wood basic density and gene expression in interspecific backcrosses of Eucalyptus grandis and E. urophylla

PubMed Central

2012-01-01

Background F1 hybrid clones of Eucalyptus grandis and E. urophylla are widely grown for pulp and paper production in tropical and subtropical regions. Volume growth and wood quality are priority objectives in Eucalyptus tree improvement. The molecular basis of quantitative variation and trait expression in eucalypt hybrids, however, remains largely unknown. The recent availability of a draft genome sequence (http://www.phytozome.net) and genome-wide genotyping platforms, combined with high levels of genetic variation and high linkage disequilibrium in hybrid crosses, greatly facilitate the detection of quantitative trait loci (QTLs) as well as underlying candidate genes for growth and wood property traits. In this study, we used Diversity Arrays Technology markers to assess the genetic architecture of volume growth (diameter at breast height, DBH) and wood basic density in four-year-old progeny of an interspecific backcross pedigree of E. grandis and E. urophylla. In addition, we used Illumina RNA-Seq expression profiling in the E. urophylla backcross family to identify cis- and trans-acting polymorphisms (eQTLs) affecting transcript abundance of genes underlying QTLs for wood basic density. Results A total of five QTLs for DBH and 12 for wood basic density were identified in the two backcross families. Individual QTLs for DBH and wood basic density explained 3.1 to 12.2% of phenotypic variation. Candidate genes underlying QTLs for wood basic density on linkage groups 8 and 9 were found to share trans-acting eQTLs located on linkage groups 4 and 10, which in turn coincided with QTLs for wood basic density suggesting that these QTLs represent segregating components of an underlying transcriptional network. Conclusion This is the first demonstration of the use of next-generation expression profiling to quantify transcript abundance in a segregating tree population and identify candidate genes potentially affecting wood property variation. The QTLs identified in this study provide a resource for identifying candidate genes and developing molecular markers for marker-assisted breeding of volume growth and wood basic density. Our results suggest that integrated analysis of transcript and trait variation in eucalypt hybrids can be used to dissect the molecular basis of quantitative variation in wood property traits. PMID:22817272

TRAM (Transcriptome Mapper): database-driven creation and analysis of transcriptome maps from multiple sources

PubMed Central

2011-01-01

Background Several tools have been developed to perform global gene expression profile data analysis, to search for specific chromosomal regions whose features meet defined criteria as well as to study neighbouring gene expression. However, most of these tools are tailored for a specific use in a particular context (e.g. they are species-specific, or limited to a particular data format) and they typically accept only gene lists as input. Results TRAM (Transcriptome Mapper) is a new general tool that allows the simple generation and analysis of quantitative transcriptome maps, starting from any source listing gene expression values for a given gene set (e.g. expression microarrays), implemented as a relational database. It includes a parser able to assign univocal and updated gene symbols to gene identifiers from different data sources. Moreover, TRAM is able to perform intra-sample and inter-sample data normalization, including an original variant of quantile normalization (scaled quantile), useful to normalize data from platforms with highly different numbers of investigated genes. When in 'Map' mode, the software generates a quantitative representation of the transcriptome of a sample (or of a pool of samples) and identifies if segments of defined lengths are over/under-expressed compared to the desired threshold. When in 'Cluster' mode, the software searches for a set of over/under-expressed consecutive genes. Statistical significance for all results is calculated with respect to genes localized on the same chromosome or to all genome genes. Transcriptome maps, showing differential expression between two sample groups, relative to two different biological conditions, may be easily generated. We present the results of a biological model test, based on a meta-analysis comparison between a sample pool of human CD34+ hematopoietic progenitor cells and a sample pool of megakaryocytic cells. Biologically relevant chromosomal segments and gene clusters with differential expression during the differentiation toward megakaryocyte were identified. Conclusions TRAM is designed to create, and statistically analyze, quantitative transcriptome maps, based on gene expression data from multiple sources. The release includes FileMaker Pro database management runtime application and it is freely available at http://apollo11.isto.unibo.it/software/, along with preconfigured implementations for mapping of human, mouse and zebrafish transcriptomes. PMID:21333005

Computerized image analysis for quantitative neuronal phenotyping in zebrafish.

PubMed

Liu, Tianming; Lu, Jianfeng; Wang, Ye; Campbell, William A; Huang, Ling; Zhu, Jinmin; Xia, Weiming; Wong, Stephen T C

2006-06-15

An integrated microscope image analysis pipeline is developed for automatic analysis and quantification of phenotypes in zebrafish with altered expression of Alzheimer's disease (AD)-linked genes. We hypothesize that a slight impairment of neuronal integrity in a large number of zebrafish carrying the mutant genotype can be detected through the computerized image analysis method. Key functionalities of our zebrafish image processing pipeline include quantification of neuron loss in zebrafish embryos due to knockdown of AD-linked genes, automatic detection of defective somites, and quantitative measurement of gene expression levels in zebrafish with altered expression of AD-linked genes or treatment with a chemical compound. These quantitative measurements enable the archival of analyzed results and relevant meta-data. The structured database is organized for statistical analysis and data modeling to better understand neuronal integrity and phenotypic changes of zebrafish under different perturbations. Our results show that the computerized analysis is comparable to manual counting with equivalent accuracy and improved efficacy and consistency. Development of such an automated data analysis pipeline represents a significant step forward to achieve accurate and reproducible quantification of neuronal phenotypes in large scale or high-throughput zebrafish imaging studies.

Scanning the genome for gene single nucleotide polymorphisms involved in adaptive population differentiation in white spruce

PubMed Central

Namroud, Marie-Claire; Beaulieu, Jean; Juge, Nicolas; Laroche, Jérôme; Bousquet, Jean

2008-01-01

Conifers are characterized by a large genome size and a rapid decay of linkage disequilibrium, most often within gene limits. Genome scans based on noncoding markers are less likely to detect molecular adaptation linked to genes in these species. In this study, we assessed the effectiveness of a genome-wide single nucleotide polymorphism (SNP) scan focused on expressed genes in detecting local adaptation in a conifer species. Samples were collected from six natural populations of white spruce (Picea glauca) moderately differentiated for several quantitative characters. A total of 534 SNPs representing 345 expressed genes were analysed. Genes potentially under natural selection were identified by estimating the differentiation in SNP frequencies among populations (FST) and identifying outliers, and by estimating local differentiation using a Bayesian approach. Both average expected heterozygosity and population differentiation estimates (HE = 0.270 and FST = 0.006) were comparable to those obtained with other genetic markers. Of all genes, 5.5% were identified as outliers with FST at the 95% confidence level, while 14% were identified as candidates for local adaptation with the Bayesian method. There was some overlap between the two gene sets. More than half of the candidate genes for local adaptation were specific to the warmest population, about 20% to the most arid population, and 15% to the coldest and most humid higher altitude population. These adaptive trends were consistent with the genes’ putative functions and the divergence in quantitative traits noted among the populations. The results suggest that an approach separating the locus and population effects is useful to identify genes potentially under selection. These candidates are worth exploring in more details at the physiological and ecological levels. PMID:18662225

Detection of susceptibility genes as modifiers due to subgroup differences in complex disease.

PubMed

Bergen, Sarah E; Maher, Brion S; Fanous, Ayman H; Kendler, Kenneth S

2010-08-01

Complex diseases invariably involve multiple genes and often exhibit variable symptom profiles. The extent to which disease symptoms, course, and severity differ between affected individuals may result from underlying genetic heterogeneity. Genes with modifier effects may or may not also influence disease susceptibility. In this study, we have simulated data in which a subset of cases differ by some effect size (ES) on a quantitative trait and are also enriched for a risk allele. Power to detect this 'pseudo-modifier' gene in case-only and case-control designs was explored blind to case substructure. Simulations involved 1000 iterations and calculations for 80% power at P<0.01 while varying the risk allele frequency (RAF), sample size (SS), ES, odds ratio (OR), and proportions of the case subgroups. With realistic values for the RAF (0.20), SS (3000) and ES (1), an OR of 1.7 is necessary to detect a pseudo-modifier gene. Unequal numbers of subjects in the case groups result in little decrement in power until the group enriched for the risk allele is <30% or >70% of the total case population. In practice, greater numbers of subjects and selection of a quantitative trait with a large range will provide researchers with greater power to detect a pseudo-modifier gene. However, even under ideal conditions, studies involving alleles with low frequencies or low ORs are usually underpowered for detection of a modifier or susceptibility gene. This may explain some of the inconsistent association results for many candidate gene studies of complex diseases.

Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions.

PubMed

Chen, Lei; Zhong, Hai-ying; Kuang, Jian-fei; Li, Jian-guo; Lu, Wang-jin; Chen, Jian-ye

2011-08-01

Reverse transcription quantitative real-time PCR (RT-qPCR) is a sensitive technique for quantifying gene expression, but its success depends on the stability of the reference gene(s) used for data normalization. Only a few studies on validation of reference genes have been conducted in fruit trees and none in banana yet. In the present work, 20 candidate reference genes were selected, and their expression stability in 144 banana samples were evaluated and analyzed using two algorithms, geNorm and NormFinder. The samples consisted of eight sample sets collected under different experimental conditions, including various tissues, developmental stages, postharvest ripening, stresses (chilling, high temperature, and pathogen), and hormone treatments. Our results showed that different suitable reference gene(s) or combination of reference genes for normalization should be selected depending on the experimental conditions. The RPS2 and UBQ2 genes were validated as the most suitable reference genes across all tested samples. More importantly, our data further showed that the widely used reference genes, ACT and GAPDH, were not the most suitable reference genes in many banana sample sets. In addition, the expression of MaEBF1, a gene of interest that plays an important role in regulating fruit ripening, under different experimental conditions was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene(s) selection under different experimental conditions and a foundation for more accurate and widespread use of RT-qPCR in banana.

Transient structural variations have strong effects on quantitative traits and reproductive isolation in fission yeast

PubMed Central

Jeffares, Daniel C.; Jolly, Clemency; Hoti, Mimoza; Speed, Doug; Shaw, Liam; Rallis, Charalampos; Balloux, Francois; Dessimoz, Christophe; Bähler, Jürg; Sedlazeck, Fritz J.

2017-01-01

Large structural variations (SVs) within genomes are more challenging to identify than smaller genetic variants but may substantially contribute to phenotypic diversity and evolution. We analyse the effects of SVs on gene expression, quantitative traits and intrinsic reproductive isolation in the yeast Schizosaccharomyces pombe. We establish a high-quality curated catalogue of SVs in the genomes of a worldwide library of S. pombe strains, including duplications, deletions, inversions and translocations. We show that copy number variants (CNVs) show a variety of genetic signals consistent with rapid turnover. These transient CNVs produce stoichiometric effects on gene expression both within and outside the duplicated regions. CNVs make substantial contributions to quantitative traits, most notably intracellular amino acid concentrations, growth under stress and sugar utilization in winemaking, whereas rearrangements are strongly associated with reproductive isolation. Collectively, these findings have broad implications for evolution and for our understanding of quantitative traits including complex human diseases. PMID:28117401

Expression analysis in response to drought stress in soybean: Shedding light on the regulation of metabolic pathway genes.

PubMed

Guimarães-Dias, Fábia; Neves-Borges, Anna Cristina; Viana, Antonio Americo Barbosa; Mesquita, Rosilene Oliveira; Romano, Eduardo; de Fátima Grossi-de-Sá, Maria; Nepomuceno, Alexandre Lima; Loureiro, Marcelo Ehlers; Alves-Ferreira, Márcio

2012-06-01

Metabolomics analysis of wild type Arabidopsis thaliana plants, under control and drought stress conditions revealed several metabolic pathways that are induced under water deficit. The metabolic response to drought stress is also associated with ABA dependent and independent pathways, allowing a better understanding of the molecular mechanisms in this model plant. Through combining an in silico approach and gene expression analysis by quantitative real-time PCR, the present work aims at identifying genes of soybean metabolic pathways potentially associated with water deficit. Digital expression patterns of Arabidopsis genes, which were selected based on the basis of literature reports, were evaluated under drought stress condition by Genevestigator. Genes that showed strong induction under drought stress were selected and used as bait to identify orthologs in the soybean genome. This allowed us to select 354 genes of putative soybean orthologs of 79 Arabidopsis genes belonging to 38 distinct metabolic pathways. The expression pattern of the selected genes was verified in the subtractive libraries available in the GENOSOJA project. Subsequently, 13 genes from different metabolic pathways were selected for validation by qPCR experiments. The expression of six genes was validated in plants undergoing drought stress in both pot-based and hydroponic cultivation systems. The results suggest that the metabolic response to drought stress is conserved in Arabidopsis and soybean plants.

Behavioral and molecular studies of quantitative differences in hygienic behavior in honeybees.

PubMed

Gempe, Tanja; Stach, Silke; Bienefeld, Kaspar; Otte, Marianne; Beye, Martin

2016-10-21

Hygienic behavior (HB) enables honeybees to tolerate parasites, including infection with the parasitic mite Varroa destructor, and it is a well-known example of a quantitative genetic trait. The understanding of the molecular processes underpinning the quantitative differences in this behavior remains limited. We performed gene expression studies in worker bees that displayed quantitative genetic differences in HB. We established a high and low genetic source of HB performance and studied the engagements into HB of single worker bees under the same environmental conditions. We found that the percentage of worker bees that engaged in a hygienic behavioral task tripled in the high versus low HB sources, thus suggesting that genetic differences may mediate differences in stimulated states to perform HB. We found 501 differently expressed genes (DEGs) in the brains of hygienic and non-hygienic performing workers in the high HB source bees, and 342 DEGs in the brains of hygienic performing worker bees, relative to the gene expression in non-hygienic worker bees from the low HB source group. "Cell surface receptor ligand signal transduction" in the high and "negative regulation of cell communication" in the low HB source were overrepresented molecular processes, suggesting that these molecular processes in the brain may play a role in the regulation of quantitative differences in HB. Moreover, only 21 HB-associated DEGs were common between the high and low HB sources. The better HB colony performance is primarily achieved by a high number of bees engaging in the hygienic tasks that associate with distinct molecular processes in the brain. We propose that different gene products and pathways may mediate the quantitative genetic differences of HB.

Gene Expression Biomarkers Provide Sensitive Indicators of in Planta Nitrogen Status in Maize[W][OA

PubMed Central

Yang, Xiaofeng S.; Wu, Jingrui; Ziegler, Todd E.; Yang, Xiao; Zayed, Adel; Rajani, M.S.; Zhou, Dafeng; Basra, Amarjit S.; Schachtman, Daniel P.; Peng, Mingsheng; Armstrong, Charles L.; Caldo, Rico A.; Morrell, James A.; Lacy, Michelle; Staub, Jeffrey M.

2011-01-01

Over the last several decades, increased agricultural production has been driven by improved agronomic practices and a dramatic increase in the use of nitrogen-containing fertilizers to maximize the yield potential of crops. To reduce input costs and to minimize the potential environmental impacts of nitrogen fertilizer that has been used to optimize yield, an increased understanding of the molecular responses to nitrogen under field conditions is critical for our ability to further improve agricultural sustainability. Using maize (Zea mays) as a model, we have characterized the transcriptional response of plants grown under limiting and sufficient nitrogen conditions and during the recovery of nitrogen-starved plants. We show that a large percentage (approximately 7%) of the maize transcriptome is nitrogen responsive, similar to previous observations in other plant species. Furthermore, we have used statistical approaches to identify a small set of genes whose expression profiles can quantitatively assess the response of plants to varying nitrogen conditions. Using a composite gene expression scoring system, this single set of biomarker genes can accurately assess nitrogen responses independently of genotype, developmental stage, tissue type, or environment, including in plants grown under controlled environments or in the field. Importantly, the biomarker composite expression response is much more rapid and quantitative than phenotypic observations. Consequently, we have successfully used these biomarkers to monitor nitrogen status in real-time assays of field-grown maize plants under typical production conditions. Our results suggest that biomarkers have the potential to be used as agronomic tools to monitor and optimize nitrogen fertilizer usage to help achieve maximal crop yields. PMID:21980173

Gene expression biomarkers provide sensitive indicators of in planta nitrogen status in maize.

PubMed

Yang, Xiaofeng S; Wu, Jingrui; Ziegler, Todd E; Yang, Xiao; Zayed, Adel; Rajani, M S; Zhou, Dafeng; Basra, Amarjit S; Schachtman, Daniel P; Peng, Mingsheng; Armstrong, Charles L; Caldo, Rico A; Morrell, James A; Lacy, Michelle; Staub, Jeffrey M

2011-12-01

Over the last several decades, increased agricultural production has been driven by improved agronomic practices and a dramatic increase in the use of nitrogen-containing fertilizers to maximize the yield potential of crops. To reduce input costs and to minimize the potential environmental impacts of nitrogen fertilizer that has been used to optimize yield, an increased understanding of the molecular responses to nitrogen under field conditions is critical for our ability to further improve agricultural sustainability. Using maize (Zea mays) as a model, we have characterized the transcriptional response of plants grown under limiting and sufficient nitrogen conditions and during the recovery of nitrogen-starved plants. We show that a large percentage (approximately 7%) of the maize transcriptome is nitrogen responsive, similar to previous observations in other plant species. Furthermore, we have used statistical approaches to identify a small set of genes whose expression profiles can quantitatively assess the response of plants to varying nitrogen conditions. Using a composite gene expression scoring system, this single set of biomarker genes can accurately assess nitrogen responses independently of genotype, developmental stage, tissue type, or environment, including in plants grown under controlled environments or in the field. Importantly, the biomarker composite expression response is much more rapid and quantitative than phenotypic observations. Consequently, we have successfully used these biomarkers to monitor nitrogen status in real-time assays of field-grown maize plants under typical production conditions. Our results suggest that biomarkers have the potential to be used as agronomic tools to monitor and optimize nitrogen fertilizer usage to help achieve maximal crop yields.

A genome scan for selection signatures comparing farmed Atlantic salmon with two wild populations: Testing colocalization among outlier markers, candidate genes, and quantitative trait loci for production traits.

PubMed

Liu, Lei; Ang, Keng Pee; Elliott, J A K; Kent, Matthew Peter; Lien, Sigbjørn; MacDonald, Danielle; Boulding, Elizabeth Grace

2017-03-01

Comparative genome scans can be used to identify chromosome regions, but not traits, that are putatively under selection. Identification of targeted traits may be more likely in recently domesticated populations under strong artificial selection for increased production. We used a North American Atlantic salmon 6K SNP dataset to locate genome regions of an aquaculture strain (Saint John River) that were highly diverged from that of its putative wild founder population (Tobique River). First, admixed individuals with partial European ancestry were detected using STRUCTURE and removed from the dataset. Outlier loci were then identified as those showing extreme differentiation between the aquaculture population and the founder population. All Arlequin methods identified an overlapping subset of 17 outlier loci, three of which were also identified by BayeScan. Many outlier loci were near candidate genes and some were near published quantitative trait loci (QTLs) for growth, appetite, maturity, or disease resistance. Parallel comparisons using a wild, nonfounder population (Stewiacke River) yielded only one overlapping outlier locus as well as a known maturity QTL. We conclude that genome scans comparing a recently domesticated strain with its wild founder population can facilitate identification of candidate genes for traits known to have been under strong artificial selection.

Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions

PubMed Central

Barsalobres-Cavallari, Carla F; Severino, Fábio E; Maluf, Mirian P; Maia, Ivan G

2009-01-01

Background Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including Coffea arabica. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress. Results The expression levels of five frequently used housekeeping genes (reference genes), namely alcohol dehydrogenase (adh), 14-3-3, polyubiquitin (poly), β-actin (actin) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of Coffea arabica plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (cys), a caffeine synthase (ccs) and the 60S ribosomal protein L7 (rpl7) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of gapdh, which showed no significant changes in expression among the investigated experimental conditions. Conclusion Our data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, gapdh, followed by 14-3-3 and rpl7 were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/organ samples. Gapdh is therefore the recommended reference gene for measuring gene expression in Coffea arabica. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant. PMID:19126214

Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR.

PubMed

Fang, Peng; Lu, Rongfei; Sun, Feng; Lan, Ying; Shen, Wenbiao; Du, Linlin; Zhou, Yijun; Zhou, Tong

2015-10-24

Stably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), particularly for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia. The expression of fourteen common used reference genes of Oryza sativa L. were evaluated by RT-qPCR in RSV and RBSDV infected rice plants. Suitable normalization reference gene(s) were identified by geNorm and NormFinder algorithms. UBQ 10 + GAPDH and UBC + Actin1 were identified as suitable reference genes for RT-qPCR normalization under RSV and RBSDV infection, respectively. When using multiple reference genes, the expression patterns of OsPRIb and OsWRKY, two virus resistance genes, were approximately similar with that reported previously. Comparatively, by using single reference gene (TIP41-Like), a weaker inducible response was observed. We proposed that the combination of two reference genes could obtain more accurate and reliable normalization of RT-qPCR results in RSV- and RBSDV-infected plants. This work therefore sheds light on establishing a standardized RT-qPCR procedure in RSV- and RBSDV-infected rice plants, and might serve as an important point for discovering complex regulatory networks and identifying genes relevant to biological processes or implicated in virus.

Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.

PubMed Central

Schena, M; Shalon, D; Heller, R; Chai, A; Brown, P O; Davis, R W

1996-01-01

Microarrays containing 1046 human cDNAs of unknown sequence were printed on glass with high-speed robotics. These 1.0-cm2 DNA "chips" were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. Array elements that displayed differential expression patterns under given experimental conditions were characterized by sequencing. The identification of known and novel heat shock and phorbol ester-regulated genes in human T cells demonstrates the sensitivity of the assay. Parallel gene analysis with microarrays provides a rapid and efficient method for large-scale human gene discovery. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855227

Optimization of Saanen sperm genes amplification: evaluation of standardized protocols in genetically uncharacterized rural goats reared under a subtropical environment.

PubMed

Barbour, Elie K; Saade, Maya F; Sleiman, Fawwak T; Hamadeh, Shady K; Mouneimne, Youssef; Kassaifi, Zeina; Kayali, Ghazi; Harakeh, Steve; Jaber, Lina S; Shaib, Houssam A

2012-10-01

The purpose of this research is to optimize quantitatively the amplification of specific sperm genes in reference genomically characterized Saanen goat and to evaluate the standardized protocols applicability on sperms of uncharacterized genome of rural goats reared under subtropical environment for inclusion in future selection programs. The optimization of the protocols in Saanen sperms included three production genes (growth hormone (GH) exons 2, 3, and 4, αS1-casein (CSN1S1), and α-lactalbumin) and two health genes (MHC class II DRB and prion (PrP)). The optimization was based on varying the primers concentrations and the inclusion of a PCR cosolvent (Triton X). The impact of the studied variables on statistically significant increase in the yield of amplicons was noticed in four out of five (80%) optimized protocols, namely in those related to GH, CSN1S1, α-lactalbumin, and PrP genes (P < 0.05). There was no significant difference in the yield of amplicons related to MHC class II DRB gene, regardless of the variables used (P > 0.05). The applicability of the optimized protocols of Saanen sperm genes on amplification of uncharacterized rural goat sperms revealed a 100% success in tested individuals for amplification of GH, CSN1S1, α-lactalbumin, and MHC class II DRB genes and a 75% success for the PrP gene. The significant success in applicability of the Saanen quantitatively optimized protocols to other uncharacterized genome of rural goats allows for their inclusion in future selection, targeting the sustainability of this farming system in a subtropical environment and the improvement of the farmers livelihood.

Variants in TTC25 affect autistic trait in patients with autism spectrum disorder and general population.

PubMed

Vojinovic, Dina; Brison, Nathalie; Ahmad, Shahzad; Noens, Ilse; Pappa, Irene; Karssen, Lennart C; Tiemeier, Henning; van Duijn, Cornelia M; Peeters, Hilde; Amin, Najaf

2017-08-01

Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder with a complex genetic architecture. To identify genetic variants underlying ASD, we performed single-variant and gene-based genome-wide association studies using a dense genotyping array containing over 2.3 million single-nucleotide variants in a discovery sample of 160 families with at least one child affected with non-syndromic ASD using a binary (ASD yes/no) phenotype and a quantitative autistic trait. Replication of the top findings was performed in Psychiatric Genomics Consortium and Erasmus Rucphen Family (ERF) cohort study. Significant association of quantitative autistic trait was observed with the TTC25 gene at 17q21.2 (effect size=10.2, P-value=3.4 × 10 -7 ) in the gene-based analysis. The gene also showed nominally significant association in the cohort-based ERF study (effect=1.75, P-value=0.05). Meta-analysis of discovery and replication improved the association signal (P-value meta =1.5 × 10 -8 ). No genome-wide significant signal was observed in the single-variant analysis of either the binary ASD phenotype or the quantitative autistic trait. Our study has identified a novel gene TTC25 to be associated with quantitative autistic trait in patients with ASD. The replication of association in a cohort-based study and the effect estimate suggest that variants in TTC25 may also be relevant for broader ASD phenotype in the general population. TTC25 is overexpressed in frontal cortex and testis and is known to be involved in cilium movement and thus an interesting candidate gene for autistic trait.

Expression patterns of WRKY genes in di-haploid Populus simonii × P. nigra in response to salinity stress revealed by quantitative real-time PCR and RNA sequencing.

PubMed

Wang, Shengji; Wang, Jiying; Yao, Wenjing; Zhou, Boru; Li, Renhua; Jiang, Tingbo

2014-10-01

Spatio-temporal expression patterns of 13 out of 119 poplar WRKY genes indicated dynamic and tissue-specific roles of WRKY family proteins in salinity stress tolerance. To understand the expression patterns of poplar WRKY genes under salinity stress, 51 of the 119 WRKY genes were selected from di-haploid Populus simonii × P. nigra by quantitative real-time PCR (qRT-PCR). We used qRT-PCR to profile the expression of the top 13 genes under salinity stress across seven time points, and employed RNA-Seq platforms to cross-validate it. Results demonstrated that all the 13 WRKY genes were expressed in root, stem, and leaf tissues, but their expression levels and overall patterns varied notably in these tissues. Regarding overall gene expression in roots, the 13 genes were significantly highly expressed at all six time points after the treatment, reaching the plateau of expression at hour 9. In leaves, the 13 genes were similarly up-regulated from 3 to 12 h in response to NaCl treatment. In stems, however, expression levels of the 13 genes did not show significant changes after the NaCl treatment. Regarding individual gene expression across the time points and the three tissues, the 13 genes can be classified into three clusters: the lowly expressed Cluster 1 containing PthWRKY28, 45 and 105; intermediately expressed Clusters 2 including PthWRKY56, 88 and 116; and highly expressed Cluster 3 consisting of PthWRKY41, 44, 51, 61, 62, 75 and 106. In general, genes in Cluster 2 and 3 displayed a dynamic pattern of "induced amplification-recovering", suggesting that these WRKY genes and corresponding pathways may play a critical role in mediating salt response and tolerance in a dynamic and tissue-specific manner.

Selection of Suitable Reference Genes for RT-qPCR Normalization under Abiotic Stresses and Hormone Stimulation in Persimmon (Diospyros kaki Thunb)

PubMed Central

Wang, Peihong; Xiong, Aisheng; Gao, Zhihong; Yu, Xinyi; Li, Man; Hou, Yingjun; Sun, Chao; Qu, Shenchun

2016-01-01

The success of quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) to quantify gene expression depends on the stability of the reference genes used for data normalization. To date, systematic screening for reference genes in persimmon (Diospyros kaki Thunb) has never been reported. In this study, 13 candidate reference genes were cloned from 'Nantongxiaofangshi' using information available in the transcriptome database. Their expression stability was assessed by geNorm and NormFinder algorithms under abiotic stress and hormone stimulation. Our results showed that the most suitable reference genes across all samples were UBC and GAPDH, and not the commonly used persimmon reference gene ACT. In addition, UBC combined with RPII or TUA were found to be appropriate for the "abiotic stress" group and α-TUB combined with PP2A were found to be appropriate for the "hormone stimuli" group. For further validation, the transcript level of the DkDREB2C homologue under heat stress was studied with the selected genes (CYP, GAPDH, TUA, UBC, α-TUB, and EF1-α). The results suggested that it is necessary to choose appropriate reference genes according to the test materials or experimental conditions. Our study will be useful for future studies on gene expression in persimmon. PMID:27513755

Suppression Subtractive Hybridization Analysis of Genes Regulated by Application of Exogenous Abscisic Acid in Pepper Plant (Capsicum annuum L.) Leaves under Chilling Stress.

PubMed

Guo, Wei-Li; Chen, Ru-Gang; Gong, Zhen-Hui; Yin, Yan-Xu; Li, Da-Wei

2013-01-01

Low temperature is one of the major factors limiting pepper (Capsicum annuum L.) production during winter and early spring in non-tropical regions. Application of exogenous abscisic acid (ABA) effectively alleviates the symptoms of chilling injury, such as wilting and formation of necrotic lesions on pepper leaves; however, the underlying molecular mechanism is not understood. The aim of this study was to identify genes that are differentially up- or downregulated in ABA-pretreated hot pepper seedlings incubated at 6°C for 48 h, using a suppression subtractive hybridization (SSH) method. A total of 235 high-quality ESTs were isolated, clustered and assembled into a collection of 73 unigenes including 18 contigs and 55 singletons. A total of 37 unigenes (50.68%) showed similarities to genes with known functions in the non-redundant database; the other 36 unigenes (49.32%) showed low similarities or unknown functions. Gene ontology analysis revealed that the 37 unigenes could be classified into nine functional categories. The expression profiles of 18 selected genes were analyzed using quantitative RT-PCR; the expression levels of 10 of these genes were at least two-fold higher in the ABA-pretreated seedlings under chilling stress than water-pretreated (control) plants under chilling stress. In contrast, the other eight genes were downregulated in ABA-pretreated seedlings under chilling stress, with expression levels that were one-third or less of the levels observed in control seedlings under chilling stress. These results suggest that ABA can positively and negatively regulate genes in pepper plants under chilling stress.

Hd6, a rice quantitative trait locus involved in photoperiod sensitivity, encodes the α subunit of protein kinase CK2

PubMed Central

Takahashi, Yuji; Shomura, Ayahiko; Sasaki, Takuji; Yano, Masahiro

2001-01-01

Hd6 is a quantitative trait locus involved in rice photoperiod sensitivity. It was detected in backcross progeny derived from a cross between the japonica variety Nipponbare and the indica variety Kasalath. To isolate a gene at Hd6, we used a large segregating population for the high-resolution and fine-scale mapping of Hd6 and constructed genomic clone contigs around the Hd6 region. Linkage analysis with P1-derived artificial chromosome clone-derived DNA markers delimited Hd6 to a 26.4-kb genomic region. We identified a gene encoding the α subunit of protein kinase CK2 (CK2α) in this region. The Nipponbare allele of CK2α contains a premature stop codon, and the resulting truncated product is undoubtedly nonfunctional. Genetic complementation analysis revealed that the Kasalath allele of CK2α increases days-to-heading. Map-based cloning with advanced backcross progeny enabled us to identify a gene underlying a quantitative trait locus even though it exhibited a relatively small effect on the phenotype. PMID:11416158

Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L.)

PubMed Central

Moazzam Jazi, Maryam; Ghadirzadeh Khorzoghi, Effat; Botanga, Christopher; Seyedi, Seyed Mahdi

2016-01-01

The tree species, Pistacia vera (P. vera) is an important commercial product that is salt-tolerant and long-lived, with a possible lifespan of over one thousand years. Gene expression analysis is an efficient method to explore the possible regulatory mechanisms underlying these characteristics. Therefore, having the most suitable set of reference genes is required for transcript level normalization under different conditions in P. vera. In the present study, we selected eight widely used reference genes, ACT, EF1α, α-TUB, β-TUB, GAPDH, CYP2, UBQ10, and 18S rRNA. Using qRT-PCR their expression was assessed in 54 different samples of three cultivars of P. vera. The samples were collected from different organs under various abiotic treatments (cold, drought, and salt) across three time points. Several statistical programs (geNorm, NormFinder, and BestKeeper) were applied to estimate the expression stability of candidate reference genes. Results obtained from the statistical analysis were then exposed to Rank aggregation package to generate a consensus gene rank. Based on our results, EF1α was found to be the superior reference gene in all samples under all abiotic treatments. In addition to EF1α, ACT and β-TUB were the second best reference genes for gene expression analysis in leaf and root. We recommended β-TUB as the second most stable gene for samples under the cold and drought treatments, while ACT holds the same position in samples analyzed under salt treatment. This report will benefit future research on the expression profiling of P. vera and other members of the Anacardiaceae family. PMID:27308855

Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L.).

PubMed

Moazzam Jazi, Maryam; Ghadirzadeh Khorzoghi, Effat; Botanga, Christopher; Seyedi, Seyed Mahdi

2016-01-01

The tree species, Pistacia vera (P. vera) is an important commercial product that is salt-tolerant and long-lived, with a possible lifespan of over one thousand years. Gene expression analysis is an efficient method to explore the possible regulatory mechanisms underlying these characteristics. Therefore, having the most suitable set of reference genes is required for transcript level normalization under different conditions in P. vera. In the present study, we selected eight widely used reference genes, ACT, EF1α, α-TUB, β-TUB, GAPDH, CYP2, UBQ10, and 18S rRNA. Using qRT-PCR their expression was assessed in 54 different samples of three cultivars of P. vera. The samples were collected from different organs under various abiotic treatments (cold, drought, and salt) across three time points. Several statistical programs (geNorm, NormFinder, and BestKeeper) were applied to estimate the expression stability of candidate reference genes. Results obtained from the statistical analysis were then exposed to Rank aggregation package to generate a consensus gene rank. Based on our results, EF1α was found to be the superior reference gene in all samples under all abiotic treatments. In addition to EF1α, ACT and β-TUB were the second best reference genes for gene expression analysis in leaf and root. We recommended β-TUB as the second most stable gene for samples under the cold and drought treatments, while ACT holds the same position in samples analyzed under salt treatment. This report will benefit future research on the expression profiling of P. vera and other members of the Anacardiaceae family.

Real-time quantification of xeroderma pigmentosum mRNA from the mammalian cochlea

PubMed Central

Guthrie, O'neil W.; Carrero-Martínez, Franklin A.

2010-01-01

Short Summary Xeroderma pigmentosum (XP) is a genetic disease that results in poor genomic defense from endogenous and exogenous stressors. Patients with mutations in the XPC and XPA genes exhibit cochlear hearing loss and to date, the underlying molecular mechanism is unknown. However, recent evidence suggests that the cochlea experiences persistent oxidative stress under normal conditions. In the current study, XPC and XPA gene products were purified from the cochlea and quantitative polymerase chain reaction was used to study the kinetics and magnitude of their expression. Additionally, immunohistochemistry was used to locate their respective polypeptides in the cochlea. The results revealed a significant demand for XP genes in the mammalian cochlea, which suggest that XP genomic defenses contribute in counterbalancing endogenous stress in the peripheral end-organ under normal conditions. PMID:20539233

Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.)

PubMed Central

2010-01-01

Background Perennial ryegrass (Lolium perenne L.) is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use. Results Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2) were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L.) samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h), a moderately, but stably expressed eEF1A (s), and combined expression of multigene eEF1A (m). NormFinder identified eEF1A (s) and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field. Conclusions This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples, helping pave the way to conduct gene expression studies in perennial biomass crops under field-conditions. From our study several stably expressed reference genes have been validated. This provides useful candidates for reference gene selection in perennial ryegrass under conditions other than those tested here. PMID:20089196

Gene expression profile of steroid-induced necrosis of femoral head of rats.

PubMed

Tong, Peijian; Wu, Chengliang; Jin, Hongting; Mao, Qiang; Yu, Nanze; Holz, Jonathan D; Shan, Letian; Liu, Hui; Xiao, Luwei

2011-10-01

The key to treating steroid-induced necrosis of femoral heads (SINFH) is early diagnosis. Dramatic improvements in diagnosis could be made if the pathogenesis of SINFH was more fully understood; however, the underlying mechanism of this disease is currently unknown. To explore the potential mechanism of SINFH, we performed gene array analysis on a rat model of the disease and compare the expression profile with that of normal rats. A quantitative RT-PCR and immunohistochemistry (IHC) assays were used to confirm the microarray results. Compared to the control group, 190 genes in the experimental group were differentially expressed, with 52 up-regulated and 138 down-regulated. Of these genes, 102 are known (deposited in GenBank), while 88 of them are unknown. The known genes can be divided into several families according to their biological functions, such as oxidative stress, apoptosis, signal transduction, angiogenesis, extracellular matrix, lipid metabolism, and transcription related genes. The results of quantitative RT-PCR and IHC were consistent with gene chip results. Our findings indicate that many genes involved in diverse signaling pathways were differentially expressed between SINFH rats and normal rats. Furthermore, our findings suggest that the development of SINFH is a complicated and dynamic process affected by multiple factors and signaling pathways and regulated by various genes.

[Discovery of the target genes inhibited by formic acid in Candida shehatae].

PubMed

Cai, Peng; Xiong, Xujie; Xu, Yong; Yong, Qiang; Zhu, Junjun; Shiyuan, Yu

2014-01-04

At transcriptional level, the inhibitory effects of formic acid was investigated on Candida shehatae, a model yeast strain capable of fermenting xylose to ethanol. Thereby, the target genes were regulated by formic acid and the transcript profiles were discovered. On the basis of the transcriptome data of C. shehatae metabolizing glucose and xylose, the genes responsible for ethanol fermentation were chosen as candidates by the combined method of yeast metabolic pathway analysis and manual gene BLAST search. These candidates were then quantitatively detected by RQ-PCR technique to find the regulating genes under gradient doses of formic acid. By quantitative analysis of 42 candidate genes, we finally identified 10 and 5 genes as markedly down-regulated and up-regulated targets by formic acid, respectively. With regard to gene transcripts regulated by formic acid in C. shehatae, the markedly down-regulated genes ranking declines as follows: xylitol dehydrogenase (XYL2), acetyl-CoA synthetase (ACS), ribose-5-phosphate isomerase (RKI), transaldolase (TAL), phosphogluconate dehydrogenase (GND1), transketolase (TKL), glucose-6-phosphate dehydrogenase (ZWF1), xylose reductase (XYL1), pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PDC); and a declining rank for up-regulated gens as follows: fructose-bisphosphate aldolase (ALD), glucokinase (GLK), malate dehydrogenase (MDH), 6-phosphofructokinase (PFK) and alcohol dehydrogenase (ADH).

Phenotypic Signatures Arising from Unbalanced Bacterial Growth

PubMed Central

Tan, Cheemeng; Smith, Robert Phillip; Tsai, Ming-Chi; Schwartz, Russell; You, Lingchong

2014-01-01

Fluctuations in the growth rate of a bacterial culture during unbalanced growth are generally considered undesirable in quantitative studies of bacterial physiology. Under well-controlled experimental conditions, however, these fluctuations are not random but instead reflect the interplay between intra-cellular networks underlying bacterial growth and the growth environment. Therefore, these fluctuations could be considered quantitative phenotypes of the bacteria under a specific growth condition. Here, we present a method to identify “phenotypic signatures” by time-frequency analysis of unbalanced growth curves measured with high temporal resolution. The signatures are then applied to differentiate amongst different bacterial strains or the same strain under different growth conditions, and to identify the essential architecture of the gene network underlying the observed growth dynamics. Our method has implications for both basic understanding of bacterial physiology and for the classification of bacterial strains. PMID:25101949

Phenotypic signatures arising from unbalanced bacterial growth.

PubMed

Tan, Cheemeng; Smith, Robert Phillip; Tsai, Ming-Chi; Schwartz, Russell; You, Lingchong

2014-08-01

Fluctuations in the growth rate of a bacterial culture during unbalanced growth are generally considered undesirable in quantitative studies of bacterial physiology. Under well-controlled experimental conditions, however, these fluctuations are not random but instead reflect the interplay between intra-cellular networks underlying bacterial growth and the growth environment. Therefore, these fluctuations could be considered quantitative phenotypes of the bacteria under a specific growth condition. Here, we present a method to identify "phenotypic signatures" by time-frequency analysis of unbalanced growth curves measured with high temporal resolution. The signatures are then applied to differentiate amongst different bacterial strains or the same strain under different growth conditions, and to identify the essential architecture of the gene network underlying the observed growth dynamics. Our method has implications for both basic understanding of bacterial physiology and for the classification of bacterial strains.

Systems genetics: a paradigm to improve discovery of candidate genes and mechanisms underlying complex traits.

PubMed

Feltus, F Alex

2014-06-01

Understanding the control of any trait optimally requires the detection of causal genes, gene interaction, and mechanism of action to discover and model the biochemical pathways underlying the expressed phenotype. Functional genomics techniques, including RNA expression profiling via microarray and high-throughput DNA sequencing, allow for the precise genome localization of biological information. Powerful genetic approaches, including quantitative trait locus (QTL) and genome-wide association study mapping, link phenotype with genome positions, yet genetics is less precise in localizing the relevant mechanistic information encoded in DNA. The coupling of salient functional genomic signals with genetically mapped positions is an appealing approach to discover meaningful gene-phenotype relationships. Techniques used to define this genetic-genomic convergence comprise the field of systems genetics. This short review will address an application of systems genetics where RNA profiles are associated with genetically mapped genome positions of individual genes (eQTL mapping) or as gene sets (co-expression network modules). Both approaches can be applied for knowledge independent selection of candidate genes (and possible control mechanisms) underlying complex traits where multiple, likely unlinked, genomic regions might control specific complex traits. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Bacterial cellulose synthesis mechanism of facultative anaerobe Enterobacter sp. FY-07.

PubMed

Ji, Kaihua; Wang, Wei; Zeng, Bing; Chen, Sibin; Zhao, Qianqian; Chen, Yueqing; Li, Guoqiang; Ma, Ting

2016-02-25

Enterobacter sp. FY-07 can produce bacterial cellulose (BC) under aerobic and anaerobic conditions. Three potential BC synthesis gene clusters (bcsI, bcsII and bcsIII) of Enterobacter sp. FY-07 have been predicted using genome sequencing and comparative genome analysis, in which bcsIII was confirmed as the main contributor to BC synthesis by gene knockout and functional reconstitution methods. Protein homology, gene arrangement and gene constitution analysis indicated that bcsIII had high identity to the bcsI operon of Enterobacter sp. 638; however, its arrangement and composition were same as those of BC synthesizing operon of G. xylinum ATCC53582 except for the flanking sequences. According to the BC biosynthesizing process, oxygen is not directly involved in the reactions of BC synthesis, however, energy is required to activate intermediate metabolites and synthesize the activator, c-di-GMP. Comparative transcriptome and metabolite quantitative analysis demonstrated that under anaerobic conditions genes involved in the TCA cycle were downregulated, however, genes in the nitrate reduction and gluconeogenesis pathways were upregulated, especially, genes in three pyruvate metabolism pathways. These results suggested that Enterobacter sp. FY-07 could produce energy efficiently under anaerobic conditions to meet the requirement of BC biosynthesis.

Bacterial cellulose synthesis mechanism of facultative anaerobe Enterobacter sp. FY-07

PubMed Central

Ji, Kaihua; Wang, Wei; Zeng, Bing; Chen, Sibin; Zhao, Qianqian; Chen, Yueqing; Li, Guoqiang; Ma, Ting

2016-01-01

Enterobacter sp. FY-07 can produce bacterial cellulose (BC) under aerobic and anaerobic conditions. Three potential BC synthesis gene clusters (bcsI, bcsII and bcsIII) of Enterobacter sp. FY-07 have been predicted using genome sequencing and comparative genome analysis, in which bcsIII was confirmed as the main contributor to BC synthesis by gene knockout and functional reconstitution methods. Protein homology, gene arrangement and gene constitution analysis indicated that bcsIII had high identity to the bcsI operon of Enterobacter sp. 638; however, its arrangement and composition were same as those of BC synthesizing operon of G. xylinum ATCC53582 except for the flanking sequences. According to the BC biosynthesizing process, oxygen is not directly involved in the reactions of BC synthesis, however, energy is required to activate intermediate metabolites and synthesize the activator, c-di-GMP. Comparative transcriptome and metabolite quantitative analysis demonstrated that under anaerobic conditions genes involved in the TCA cycle were downregulated, however, genes in the nitrate reduction and gluconeogenesis pathways were upregulated, especially, genes in three pyruvate metabolism pathways. These results suggested that Enterobacter sp. FY-07 could produce energy efficiently under anaerobic conditions to meet the requirement of BC biosynthesis. PMID:26911736

Identifying gene networks underlying the neurobiology of ethanol and alcoholism.

PubMed

Wolen, Aaron R; Miles, Michael F

2012-01-01

For complex disorders such as alcoholism, identifying the genes linked to these diseases and their specific roles is difficult. Traditional genetic approaches, such as genetic association studies (including genome-wide association studies) and analyses of quantitative trait loci (QTLs) in both humans and laboratory animals already have helped identify some candidate genes. However, because of technical obstacles, such as the small impact of any individual gene, these approaches only have limited effectiveness in identifying specific genes that contribute to complex diseases. The emerging field of systems biology, which allows for analyses of entire gene networks, may help researchers better elucidate the genetic basis of alcoholism, both in humans and in animal models. Such networks can be identified using approaches such as high-throughput molecular profiling (e.g., through microarray-based gene expression analyses) or strategies referred to as genetical genomics, such as the mapping of expression QTLs (eQTLs). Characterization of gene networks can shed light on the biological pathways underlying complex traits and provide the functional context for identifying those genes that contribute to disease development.

Deciphering principles of transcription regulation in eukaryotic genomes

PubMed Central

Nguyen, Dat H; D'haeseleer, Patrik

2006-01-01

Transcription regulation has been responsible for organismal complexity and diversity in the course of biological evolution and adaptation, and it is determined largely by the context-dependent behavior of cis-regulatory elements (CREs). Therefore, understanding principles underlying CRE behavior in regulating transcription constitutes a fundamental objective of quantitative biology, yet these remain poorly understood. Here we present a deterministic mathematical strategy, the motif expression decomposition (MED) method, for deriving principles of transcription regulation at the single-gene resolution level. MED operates on all genes in a genome without requiring any a priori knowledge of gene cluster membership, or manual tuning of parameters. Applying MED to Saccharomyces cerevisiae transcriptional networks, we identified four functions describing four different ways that CREs can quantitatively affect gene expression levels. These functions, three of which have extrema in different positions in the gene promoter (short-, mid-, and long-range) whereas the other depends on the motif orientation, are validated by expression data. We illustrate how nature could use these principles as an additional dimension to amplify the combinatorial power of a small set of CREs in regulating transcription. PMID:16738557

Selection of reference genes for miRNA qRT-PCR under abiotic stress in grapevine.

PubMed

Luo, Meng; Gao, Zhen; Li, Hui; Li, Qin; Zhang, Caixi; Xu, Wenping; Song, Shiren; Ma, Chao; Wang, Shiping

2018-03-13

Grapevine is among the fruit crops with high economic value, and because of the economic losses caused by abiotic stresses, the stress resistance of Vitis vinifera has become an increasingly important research area. Among the mechanisms responding to environmental stresses, the role of miRNA has received much attention recently. qRT-PCR is a powerful method for miRNA quantitation, but the accuracy of the method strongly depends on the appropriate reference genes. To determine the most suitable reference genes for grapevine miRNA qRT-PCR, 15 genes were chosen as candidate reference genes. After eliminating 6 candidate reference genes with unsatisfactory amplification efficiency, the expression stability of the remaining candidate reference genes under salinity, cold and drought was analysed using four algorithms, geNorm, NormFinder, deltaCt and Bestkeeper. The results indicated that U6 snRNA was the most suitable reference gene under salinity and cold stresses; whereas miR168 was the best for drought stress. The best reference gene sets for salinity, cold and drought stresses were miR160e + miR164a, miR160e + miR168 and ACT + UBQ + GAPDH, respectively. The selected reference genes or gene sets were verified using miR319 or miR408 as the target gene.

Evaluation and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) Under Drought Stress Conditions

PubMed Central

Sinha, Pallavi; Singh, Vikas K.; Suryanarayana, V.; Krishnamurthy, L.; Saxena, Rachit K.; Varshney, Rajeev K.

2015-01-01

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions. PMID:25849964

Evaluation and validation of housekeeping genes as reference for gene expression studies in pigeonpea (Cajanus cajan) under drought stress conditions.

PubMed

Sinha, Pallavi; Singh, Vikas K; Suryanarayana, V; Krishnamurthy, L; Saxena, Rachit K; Varshney, Rajeev K

2015-01-01

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions.

Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia.

PubMed

Sacramento, C B; Moraes, J Z; Denapolis, P M A; Han, S W

2010-08-01

The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2) and hypoxia (less than 5% O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

Integrated genomic approaches to identification of candidate genes underlying metabolic and cardiovascular phenotypes in the spontaneously hypertensive rat.

PubMed

Morrissey, Catherine; Grieve, Ian C; Heinig, Matthias; Atanur, Santosh; Petretto, Enrico; Pravenec, Michal; Hubner, Norbert; Aitman, Timothy J

2011-11-07

The spontaneously hypertensive rat (SHR) is a widely used rodent model of hypertension and metabolic syndrome. Previously we identified thousands of cis-regulated expression quantitative trait loci (eQTLs) across multiple tissues using a panel of rat recombinant inbred (RI) strains derived from Brown Norway and SHR progenitors. These cis-eQTLs represent potential susceptibility loci underlying physiological and pathophysiological traits manifested in SHR. We have prioritized 60 cis-eQTLs and confirmed differential expression between the parental strains by quantitative PCR in 43 (72%) of the eQTL transcripts. Quantitative trait transcript (QTT) analysis in the RI strains showed highly significant correlation between cis-eQTL transcript abundance and clinically relevant traits such as systolic blood pressure and blood glucose, with the physical location of a subset of the cis-eQTLs colocalizing with "physiological" QTLs (pQTLs) for these same traits. These colocalizing correlated cis-eQTLs (c3-eQTLs) are highly attractive as primary susceptibility loci for the colocalizing pQTLs. Furthermore, sequence analysis of the c3-eQTL genes identified single nucleotide polymorphisms (SNPs) that are predicted to affect transcription factor binding affinity, splicing and protein function. These SNPs, which potentially alter transcript abundance and stability, represent strong candidate factors underlying not just eQTL expression phenotypes, but also the correlated metabolic and physiological traits. In conclusion, by integration of genomic sequence, eQTL and QTT datasets we have identified several genes that are strong positional candidates for pathophysiological traits observed in the SHR strain. These findings provide a basis for the functional testing and ultimate elucidation of the molecular basis of these metabolic and cardiovascular phenotypes.

Utilization of urea and expression profiles of related genes in the dinoflagellate Prorocentrum donghaiense

PubMed Central

Jing, Xiaoli; Lin, Senjie; Zhang, Huan; Koerting, Claudia; Yu, Zhigang

2017-01-01

Urea has been shown to contribute more than half of total nitrogen (N) required by phytoplankton in some estuaries and coastal waters and to provide a substantial portion of the N demand for many harmful algal blooms (HABs) of dinoflagellates. In this study, we investigated the physiological and transcriptional responses in Prorocentrum donghaiense to changes in nitrate and urea availability. We found that this species could efficiently utilize urea as sole N source and achieve comparable growth rate and photosynthesis capability as it did under nitrate. These physiological parameters were markedly lower in cultures grown under nitrate- or urea-limited conditions. P. donghaiense N content was similarly low under nitrate- or urea-limited culture condition, but was markedly higher under urea-replete condition than under nitrate-replete condition. Carbon (C) content was consistently elevated under N-limited condition. Consequently, the C:N ratio was as high as 21:1 under nitrate- or urea-limitation, but 7:1 under urea-replete condition and 9:1 to 10:1 under nitrate-replete condition. Using quantitative reverse transcription PCR, we investigated the expression pattern for four genes involved in N transport and assimilation. The results indicated that genes encoding nitrate transport, urea hydrolysis, and nickel transporter gene were sensitive to changes in general N nutrient availability whereas the urea transporter gene responded much more strongly to changes in urea concentration. Taken together, our study shows the high bioavailability of urea, its impact on C:N stoichiometry, and the sensitivity of urea transporter gene expression to urea availability. PMID:29117255

Utilization of urea and expression profiles of related genes in the dinoflagellate Prorocentrum donghaiense.

PubMed

Jing, Xiaoli; Lin, Senjie; Zhang, Huan; Koerting, Claudia; Yu, Zhigang

2017-01-01

Urea has been shown to contribute more than half of total nitrogen (N) required by phytoplankton in some estuaries and coastal waters and to provide a substantial portion of the N demand for many harmful algal blooms (HABs) of dinoflagellates. In this study, we investigated the physiological and transcriptional responses in Prorocentrum donghaiense to changes in nitrate and urea availability. We found that this species could efficiently utilize urea as sole N source and achieve comparable growth rate and photosynthesis capability as it did under nitrate. These physiological parameters were markedly lower in cultures grown under nitrate- or urea-limited conditions. P. donghaiense N content was similarly low under nitrate- or urea-limited culture condition, but was markedly higher under urea-replete condition than under nitrate-replete condition. Carbon (C) content was consistently elevated under N-limited condition. Consequently, the C:N ratio was as high as 21:1 under nitrate- or urea-limitation, but 7:1 under urea-replete condition and 9:1 to 10:1 under nitrate-replete condition. Using quantitative reverse transcription PCR, we investigated the expression pattern for four genes involved in N transport and assimilation. The results indicated that genes encoding nitrate transport, urea hydrolysis, and nickel transporter gene were sensitive to changes in general N nutrient availability whereas the urea transporter gene responded much more strongly to changes in urea concentration. Taken together, our study shows the high bioavailability of urea, its impact on C:N stoichiometry, and the sensitivity of urea transporter gene expression to urea availability.

Pathogenic diversity of Phytophthora sojae and breeding strategies to develop Phytophthora-resistant soybeans

PubMed Central

Sugimoto, Takuma; Kato, Masayasu; Yoshida, Shinya; Matsumoto, Isao; Kobayashi, Tamotsu; Kaga, Akito; Hajika, Makita; Yamamoto, Ryo; Watanabe, Kazuhiko; Aino, Masataka; Matoh, Toru; Walker, David R.; Biggs, Alan R.; Ishimoto, Masao

2012-01-01

Phytophthora stem and root rot, caused by Phytophthora sojae, is one of the most destructive diseases of soybean [Glycine max (L.) Merr.], and the incidence of this disease has been increasing in several soybean-producing areas around the world. This presents serious limitations for soybean production, with yield losses from 4 to 100%. The most effective method to reduce damage would be to grow Phytophthora-resistant soybean cultivars, and two types of host resistance have been described. Race-specific resistance conditioned by single dominant Rps (“resistance to Phytophthora sojae”) genes and quantitatively inherited partial resistance conferred by multiple genes could both provide protection from the pathogen. Molecular markers linked to Rps genes or quantitative trait loci (QTLs) underlying partial resistance have been identified on several molecular linkage groups corresponding to chromosomes. These markers can be used to screen for Phytophthora-resistant plants rapidly and efficiently, and to combine multiple resistance genes in the same background. This paper reviews what is currently known about pathogenic races of P. sojae in the USA and Japan, selection of sources of Rps genes or minor genes providing partial resistance, and the current state and future scope of breeding Phytophthora-resistant soybean cultivars. PMID:23136490

Environmental and Microbial Features Affecting Denitrification and Anammox Hotspots in an Estuarine Ecosystem

NASA Astrophysics Data System (ADS)

Lisa, J.; Song, B.; Lefcheck, J. S.; Tobias, C. R.

2016-02-01

Biogeochemical hotspots are characterized as a few sites that exhibit extremely high reaction rates relative to surrounding area, and often account for a high percentage of the overall reaction rates in an ecosystem. Criteria for quantitatively identifying these sites have not been well established. Further, the underlying mechanisms of hotspots have been described in terms of environmental conditions, with little attention paid to the microbial community. The objectives of this study were to establish quantitative criteria to identify denitrification and anammox hotspots, and determine the underlying microbial and environmental factors responsible for elevated N2 production. We used 15N isotope pairing incubation experiments to measure denitrification and anammox rates in the New River Estuary, NC. Quantitative PCR assays of nitrous oxide reductase (nosZ Clades I and II) and hydrazine oxidoreductase (hzo) genes were conducted to estimate denitrifier and anammox abundance. Structural Equation Modeling (SEM) was used to elucidate complex causal relationships between environmental and biological variables. Denitrification hotspots, quantitatively defined as statistical outliers, accounted for 35.6% total denitrification while comprising only 7.3% of the sites. Anammox hotspots,10.6% of the sites, accounted for 60.9% of total anammox. SEM revealed increased sediment organics at lower salinities supported higher functional gene abundance, which in turn resulted in higher N2 production. Surprisingly, denitrification rates were significantly and positively correlated with nosZ Clade II gene abundance, after accounting for the non-significant contributions of the naturally more abundant nosZ Clade I, and other environmental covariates. This is the first time that a quantitative definition of biogeochemical hotspots was put forth and used to determine the importance of anammox and denitrification hotspots in estuarine nitrogen removal capacity. Despite the low area coverage, denitrification and anammox hotspots are major nitrogen removal components and may play a significant role in mitigating eutrophication.

High-resolution mapping of a fruit firmness-related quantitative trait locus in tomato reveals epistatic interactions associated with a complex combinatorial locus.

PubMed

Chapman, Natalie H; Bonnet, Julien; Grivet, Laurent; Lynn, James; Graham, Neil; Smith, Rebecca; Sun, Guiping; Walley, Peter G; Poole, Mervin; Causse, Mathilde; King, Graham J; Baxter, Charles; Seymour, Graham B

2012-08-01

Fruit firmness in tomato (Solanum lycopersicum) is determined by a number of factors including cell wall structure, turgor, and cuticle properties. Firmness is a complex polygenic trait involving the coregulation of many genes and has proved especially challenging to unravel. In this study, a quantitative trait locus (QTL) for fruit firmness was mapped to tomato chromosome 2 using the Zamir Solanum pennellii interspecific introgression lines (ILs) and fine-mapped in a population consisting of 7,500 F2 and F3 lines from IL 2-3 and IL 2-4. This firmness QTL contained five distinct subpeaks, Fir(s.p.)QTL2.1 to Fir(s.p.)QTL2.5, and an effect on a distal region of IL 2-4 that was nonoverlapping with IL 2-3. All these effects were located within an 8.6-Mb region. Using genetic markers, each subpeak within this combinatorial locus was mapped to a physical location within the genome, and an ethylene response factor (ERF) underlying Fir(s.p.)QTL2.2 and a region containing three pectin methylesterase (PME) genes underlying Fir(s.p.)QTL2.5 were nominated as QTL candidate genes. Statistical models used to explain the observed variability between lines indicated that these candidates and the nonoverlapping portion of IL 2-4 were sufficient to account for the majority of the fruit firmness effects. Quantitative reverse transcription-polymerase chain reaction was used to quantify the expression of each candidate gene. ERF showed increased expression associated with soft fruit texture in the mapping population. In contrast, PME expression was tightly linked with firm fruit texture. Analysis of a range of recombinant lines revealed evidence for an epistatic interaction that was associated with this combinatorial locus.

Suppression Subtractive Hybridization Analysis of Genes Regulated by Application of Exogenous Abscisic Acid in Pepper Plant (Capsicum annuum L.) Leaves under Chilling Stress

PubMed Central

Gong, Zhen-Hui; Yin, Yan-Xu; Li, Da-Wei

2013-01-01

Low temperature is one of the major factors limiting pepper (Capsicum annuum L.) production during winter and early spring in non-tropical regions. Application of exogenous abscisic acid (ABA) effectively alleviates the symptoms of chilling injury, such as wilting and formation of necrotic lesions on pepper leaves; however, the underlying molecular mechanism is not understood. The aim of this study was to identify genes that are differentially up- or downregulated in ABA-pretreated hot pepper seedlings incubated at 6°C for 48 h, using a suppression subtractive hybridization (SSH) method. A total of 235 high-quality ESTs were isolated, clustered and assembled into a collection of 73 unigenes including 18 contigs and 55 singletons. A total of 37 unigenes (50.68%) showed similarities to genes with known functions in the non-redundant database; the other 36 unigenes (49.32%) showed low similarities or unknown functions. Gene ontology analysis revealed that the 37 unigenes could be classified into nine functional categories. The expression profiles of 18 selected genes were analyzed using quantitative RT-PCR; the expression levels of 10 of these genes were at least two-fold higher in the ABA-pretreated seedlings under chilling stress than water-pretreated (control) plants under chilling stress. In contrast, the other eight genes were downregulated in ABA-pretreated seedlings under chilling stress, with expression levels that were one-third or less of the levels observed in control seedlings under chilling stress. These results suggest that ABA can positively and negatively regulate genes in pepper plants under chilling stress. PMID:23825555

Stability evaluation of reference genes for gene expression analysis by RT-qPCR in soybean under different conditions.

PubMed

Wan, Qiao; Chen, Shuilian; Shan, Zhihui; Yang, Zhonglu; Chen, Limiao; Zhang, Chanjuan; Yuan, Songli; Hao, Qinnan; Zhang, Xiaojuan; Qiu, Dezhen; Chen, Haifeng; Zhou, Xinan

2017-01-01

Real-time quantitative reverse transcription PCR is a sensitive and widely used technique to quantify gene expression. To achieve a reliable result, appropriate reference genes are highly required for normalization of transcripts in different samples. In this study, 9 previously published reference genes (60S, Fbox, ELF1A, ELF1B, ACT11, TUA5, UBC4, G6PD, CYP2) of soybean [Glycine max (L.) Merr.] were selected. The expression stability of the 9 genes was evaluated under conditions of biotic stress caused by infection with soybean mosaic virus, nitrogen stress, across different cultivars and developmental stages. ΔCt and geNorm algorithms were used to evaluate and rank the expression stability of the 9 reference genes. Results obtained from two algorithms showed high consistency. Moreover, results of pairwise variation showed that two reference genes were sufficient to normalize the expression levels of target genes under each experimental setting. For virus infection, ELF1A and ELF1B were the most stable reference genes for accurate normalization. For different developmental stages, Fbox and G6PD had the highest expression stability between two soybean cultivars (Tanlong No. 1 and Tanlong No. 2). ELF1B and ACT11 were identified as the most stably expressed reference genes both under nitrogen stress and among different cultivars. The results showed that none of the candidate reference genes were uniformly expressed at different conditions, and selecting appropriate reference genes was pivotal for gene expression studies with particular condition and tissue. The most stable combination of genes identified in this study will help to achieve more accurate and reliable results in a wide variety of samples in soybean.

Dominant Epistasis Between Two Quantitative Trait Loci Governing Sporulation Efficiency in Yeast Saccharomyces cerevisiae

PubMed Central

Bergman, Juraj; Mitrikeski, Petar T.

2015-01-01

Summary Sporulation efficiency in the yeast Saccharomyces cerevisiae is a well-established model for studying quantitative traits. A variety of genes and nucleotides causing different sporulation efficiencies in laboratory, as well as in wild strains, has already been extensively characterised (mainly by reciprocal hemizygosity analysis and nucleotide exchange methods). We applied a different strategy in order to analyze the variation in sporulation efficiency of laboratory yeast strains. Coupling classical quantitative genetic analysis with simulations of phenotypic distributions (a method we call phenotype modelling) enabled us to obtain a detailed picture of the quantitative trait loci (QTLs) relationships underlying the phenotypic variation of this trait. Using this approach, we were able to uncover a dominant epistatic inheritance of loci governing the phenotype. Moreover, a molecular analysis of known causative quantitative trait genes and nucleotides allowed for the detection of novel alleles, potentially responsible for the observed phenotypic variation. Based on the molecular data, we hypothesise that the observed dominant epistatic relationship could be caused by the interaction of multiple quantitative trait nucleotides distributed across a 60--kb QTL region located on chromosome XIV and the RME1 locus on chromosome VII. Furthermore, we propose a model of molecular pathways which possibly underlie the phenotypic variation of this trait. PMID:27904371

Fidgetin-Like1 Is a Strong Candidate for a Dynamic Impairment of Male Meiosis Leading to Reduced Testis Weight in Mice

PubMed Central

L'Hôte, David; Vatin, Magalie; Auer, Jana; Castille, Johan; Passet, Bruno; Montagutelli, Xavier

2011-01-01

Background In a previous work, using an interspecific recombinant congenic mouse model, we reported a genomic region of 23 Mb on mouse chromosome 11 implicated in testis weight decrease and moderate teratozoospermia (∼20–30%), a Quantitative Trait Locus (QTL) called Ltw1. The objective of the present study is to identify the gene underlying this phenotype. Results In the present study, we refined the QTL position to a 5 Mb fragment encompassing only 11 genes. We showed that the low testis weight phenotype was due to kinetic alterations occurring during the first wave of the spermatogenesis where we could point out to an abnormal lengthening of spermatocyte prophase. We identify Fidgetin-like 1 (Fignl1) as the gene underlying the phenotype, since if fulfilled both the physiological and molecular characteristics required. Indeed, amongst the 11 positional candidates it is the only gene that is expressed during meiosis at the spermatocyte stage, and that presents with non-synonymous coding variations differentiating the two mouse strains at the origin of the cross. Conclusions This work prompted us to propose Fignl1 as a novel actor in mammal's male meiosis dynamics which has fundamental interest. Besides, this gene is a new potential candidate for human infertilities caused by teratozoospermia and blockades of spermatogenesis. In addition this study demonstrates that interspecific models may be useful for understanding complex quantitative traits. PMID:22110678

Genome-wide analysis of soybean HD-Zip gene family and expression profiling under salinity and drought treatments.

PubMed

Chen, Xue; Chen, Zhu; Zhao, Hualin; Zhao, Yang; Cheng, Beijiu; Xiang, Yan

2014-01-01

Homeodomain-leucine zipper (HD-Zip) proteins, a group of homeobox transcription factors, participate in various aspects of normal plant growth and developmental processes as well as environmental responses. To date, no overall analysis or expression profiling of the HD-Zip gene family in soybean (Glycine max) has been reported. An investigation of the soybean genome revealed 88 putative HD-Zip genes. These genes were classified into four subfamilies, I to IV, based on phylogenetic analysis. In each subfamily, the constituent parts of gene structure and motif were relatively conserved. A total of 87 out of 88 genes were distributed unequally on 20 chromosomes with 36 segmental duplication events, indicating that segmental duplication is important for the expansion of the HD-Zip family. Analysis of the Ka/Ks ratios showed that the duplicated genes of the HD-Zip family basically underwent purifying selection with restrictive functional divergence after the duplication events. Analysis of expression profiles showed that 80 genes differentially expressed across 14 tissues, and 59 HD-Zip genes are differentially expressed under salinity and drought stress, with 20 paralogous pairs showing nearly identical expression patterns and three paralogous pairs diversifying significantly under drought stress. Quantitative real-time RT-PCR (qRT-PCR) analysis of six paralogous pairs of 12 selected soybean HD-Zip genes under both drought and salinity stress confirmed their stress-inducible expression patterns. This study presents a thorough overview of the soybean HD-Zip gene family and provides a new perspective on the evolution of this gene family. The results indicate that HD-Zip family genes may be involved in many plant responses to stress conditions. Additionally, this study provides a solid foundation for uncovering the biological roles of HD-Zip genes in soybean growth and development.

Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis.

PubMed

Chen, Chun; Xie, Tingna; Ye, Sudan; Jensen, Annette Bruun; Eilenberg, Jørgen

2016-01-01

The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host-pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S), 28S rRNA(28S) and elongation factor 1 alpha-like protein (EF1), were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae), and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189), 28S (1.414) and EF1 (3). The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis. Copyright © 2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Identification of suitable internal controls to study expression of a Staphylococcus aureus multidrug resistance system by quantitative real-time PCR.

PubMed

Theis, Torsten; Skurray, Ronald A; Brown, Melissa H

2007-08-01

Quantitative real-time PCR (qRT-PCR) has become a routine technique for gene expression analysis. Housekeeping genes are customarily used as endogenous references for the relative quantification of genes of interest. The aim of this study was to develop a quantitative real-time PCR assay to analyze gene expression in multidrug resistant Staphylococcus aureus in the presence of cationic lipophilic substrates of multidrug transport proteins. Eleven different housekeeping genes were analyzed for their expression stability in the presence of a range of concentrations of four structurally different antimicrobial compounds. This analysis demonstrated that the genes rho, pyk and proC were least affected by rhodamine 6G and crystal violet, whereas fabD, tpiA and gyrA or fabD, proC and pyk were stably expressed in cultures grown in the presence of ethidium or berberine, respectively. Subsequently, these housekeeping genes were used as internal controls to analyze expression of the multidrug transport protein QacA and its transcriptional regulator QacR in the presence of the aforementioned compounds. Expression of qacA was induced by all four compounds, whereas qacR expression was found to be unaffected, reduced or enhanced. This study demonstrates that staphylococcal gene expression, including housekeeping genes previously used to normalize qRT-PCR data, is affected by growth in the presence of different antimicrobial compounds. Thus, identification of suitable genes usable as a control set requires rigorous testing. Identification of a such a set enabled them to be utilized as internal standards for accurate quantification of transcripts of the qac multidrug resistance system from S. aureus grown under different inducing conditions. Moreover, the qRT-PCR assay presented in this study may also be applied to gene expression studies of other multidrug transporters from S. aureus.

Validation of reference genes for quantitative gene expression analysis in experimental epilepsy.

PubMed

Sadangi, Chinmaya; Rosenow, Felix; Norwood, Braxton A

2017-12-01

To grasp the molecular mechanisms and pathophysiology underlying epilepsy development (epileptogenesis) and epilepsy itself, it is important to understand the gene expression changes that occur during these phases. Quantitative real-time polymerase chain reaction (qPCR) is a technique that rapidly and accurately determines gene expression changes. It is crucial, however, that stable reference genes are selected for each experimental condition to ensure that accurate values are obtained for genes of interest. If reference genes are unstably expressed, this can lead to inaccurate data and erroneous conclusions. To date, epilepsy studies have used mostly single, nonvalidated reference genes. This is the first study to systematically evaluate reference genes in male Sprague-Dawley rat models of epilepsy. We assessed 15 potential reference genes in hippocampal tissue obtained from 2 different models during epileptogenesis, 1 model during chronic epilepsy, and a model of noninjurious seizures. Reference gene ranking varied between models and also differed between epileptogenesis and chronic epilepsy time points. There was also some variance between the four mathematical models used to rank reference genes. Notably, we found novel reference genes to be more stably expressed than those most often used in experimental epilepsy studies. The consequence of these findings is that reference genes suitable for one epilepsy model may not be appropriate for others and that reference genes can change over time. It is, therefore, critically important to validate potential reference genes before using them as normalizing factors in expression analysis in order to ensure accurate, valid results. © 2017 Wiley Periodicals, Inc.

Quantitative Proteomics of the Root of Transgenic Wheat Expressing TaBWPR-1.2 Genes in Response to Waterlogging.

PubMed

Haque, Emdadul; Abe, Fumitaka; Mori, Masahiko; Nanjo, Yohei; Komatsu, Setsuko; Oyanagi, Atsushi; Kawaguchi, Kentaro

2014-11-04

Once candidate genes are available, the application of genetic transformation plays a major part to study their function in plants for adaptation to respective environmental stresses, including waterlogging (WL). The introduction of stress-inducible genes into wheat remains difficult because of low transformation and plant regeneration efficiencies and expression variability and instability. Earlier, we found two cDNAs encoding WL stress-responsive wheat pathogenesis-related proteins 1.2 ( TaBWPR-1.2 ), TaBWPR-1.2#2 and TaBWPR-1.2# 13. Using microprojectile bombardment, both cDNAs were introduced into "Bobwhite". Despite low transformation efficiency, four independent T₂ homozygous lines for each gene were isolated, where transgenes were ubiquitously and variously expressed. The highest transgene expression was obtained in Ubi: TaBWPR-1.2#2 L#11a and Ubi:TaBWPR-1.2#13 L#4a. Using quantitative proteomics, the root proteins of L#11a were analyzed to explore possible physiological pathways regulated by TaBWPR-1.2 under normal and waterlogged conditions. In L#11a, the abundance of proteasome subunit alpha type-3 decreased under normal conditions, whereas that of ferredoxin precursor and elongation factor-2 increased under waterlogged conditions in comparison with normal plants. Proteomic results suggest that L#11a is one of the engineered wheat plants where TaBWPR-1.2#2 is most probably involved in proteolysis, protein synthesis and alteration in the energy pathway in root tissues via the above proteins in order to gain metabolic adjustment to WL.

Root-tip-mediated inhibition of hydrotropism is accompanied with the suppression of asymmetric expression of auxin-inducible genes in response to moisture gradients in cucumber roots

PubMed Central

Miyabayashi, Sachiko; Sugita, Tomoki; Kobayashi, Akie; Yamazaki, Chiaki; Miyazawa, Yutaka; Kamada, Motoshi; Kasahara, Haruo; Osada, Ikuko; Shimazu, Toru; Fusejima, Yasuo; Higashibata, Akira; Yamazaki, Takashi; Ishioka, Noriaki; Takahashi, Hideyuki

2018-01-01

In cucumber seedlings, gravitropism interferes with hydrotropism, which results in the nearly complete inhibition of hydrotropism under stationary conditions. However, hydrotropic responses are induced when the gravitropic response in the root is nullified by clinorotation. Columella cells in the root cap sense gravity, which induces the gravitropic response. In this study, we found that removing the root tip induced hydrotropism in cucumber roots under stationary conditions. The application of auxin transport inhibitors to cucumber seedlings under stationary conditions suppressed the hydrotropic response induced by the removal of the root tip. To investigate the expression of genes related to hydrotropism in de-tipped cucumber roots, we conducted transcriptome analysis of gene expression by RNA-Seq using seedlings exhibiting hydrotropic and gravitropic responses. Of the 21 and 45 genes asymmetrically expressed during hydrotropic and gravitropic responses, respectively, five genes were identical. Gene ontology (GO) analysis indicated that the category auxin-inducible genes was significantly enriched among genes that were more highly expressed in the concave side of the root than the convex side during hydrotropic or gravitropic responses. Reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) analysis revealed that root hydrotropism induced under stationary conditions (by removing the root tip) was accompanied by the asymmetric expression of several auxin-inducible genes. However, intact roots did not exhibit the asymmetric expression patterns of auxin-inducible genes under stationary conditions, even in the presence of a moisture gradient. These results suggest that the root tip inhibits hydrotropism by suppressing the induction of asymmetric auxin distribution. Auxin transport and distribution not mediated by the root tip might play a role in hydrotropism in cucumber roots. PMID:29324818

Identification of Appropriate Reference Genes for Normalization of miRNA Expression in Grafted Watermelon Plants under Different Nutrient Stresses

PubMed Central

Wu, Weifang; Deng, Qin; Shi, Pibiao; Yang, Jinghua; Hu, Zhongyuan; Zhang, Mingfang

2016-01-01

Watermelon (Citrullus lanatus) is a globally important crop belonging to the family Cucurbitaceae. The grafting technique is commonly used to improve its tolerance to stress, as well as to enhance its nutrient uptake and utilization. It is believed that miRNA is most likely involved in its nutrient-starvation response as a graft-transportable signal. The quantitative real-time reverse transcriptase polymerase chain reaction is the preferred method for miRNA functional analysis, in which reliable reference genes for normalization are crucial to ensure the accuracy. The purpose of this study was to select appropriate reference genes in scion (watermelon) and rootstocks (squash and bottle gourd) of grafted watermelon plants under normal growth conditions and nutrient stresses (nitrogen and phosphorus starvation). Under nutrient starvation, geNorm identified miR167c and miR167f as two most stable genes in both watermelon leaves and squash roots. miR166b was recommended by both geNorm and NormFinder as the best reference in bottle gourd roots under nutrient limitation. Expression of a new Cucurbitaceae miRNA, miR85, was used to validate the reliability of candidate reference genes under nutrient starvation. Moreover, by comparing several target genes expression in qRT-PCR analysis with those in RNA-seq data, miR166b and miR167c were proved to be the most suitable reference genes to normalize miRNA expression under normal growth condition in scion and rootstock tissues, respectively. This study represents the first comprehensive survey of the stability of miRNA reference genes in Cucurbitaceae and provides valuable information for investigating more accurate miRNA expression involving grafted watermelon plants. PMID:27749935

Identification of Appropriate Reference Genes for Normalization of miRNA Expression in Grafted Watermelon Plants under Different Nutrient Stresses.

PubMed

Wu, Weifang; Deng, Qin; Shi, Pibiao; Yang, Jinghua; Hu, Zhongyuan; Zhang, Mingfang

2016-01-01

Watermelon (Citrullus lanatus) is a globally important crop belonging to the family Cucurbitaceae. The grafting technique is commonly used to improve its tolerance to stress, as well as to enhance its nutrient uptake and utilization. It is believed that miRNA is most likely involved in its nutrient-starvation response as a graft-transportable signal. The quantitative real-time reverse transcriptase polymerase chain reaction is the preferred method for miRNA functional analysis, in which reliable reference genes for normalization are crucial to ensure the accuracy. The purpose of this study was to select appropriate reference genes in scion (watermelon) and rootstocks (squash and bottle gourd) of grafted watermelon plants under normal growth conditions and nutrient stresses (nitrogen and phosphorus starvation). Under nutrient starvation, geNorm identified miR167c and miR167f as two most stable genes in both watermelon leaves and squash roots. miR166b was recommended by both geNorm and NormFinder as the best reference in bottle gourd roots under nutrient limitation. Expression of a new Cucurbitaceae miRNA, miR85, was used to validate the reliability of candidate reference genes under nutrient starvation. Moreover, by comparing several target genes expression in qRT-PCR analysis with those in RNA-seq data, miR166b and miR167c were proved to be the most suitable reference genes to normalize miRNA expression under normal growth condition in scion and rootstock tissues, respectively. This study represents the first comprehensive survey of the stability of miRNA reference genes in Cucurbitaceae and provides valuable information for investigating more accurate miRNA expression involving grafted watermelon plants.

Methylation analysis of plasma cell-free DNA for breast cancer early detection using bisulfite next-generation sequencing.

PubMed

Li, Zibo; Guo, Xinwu; Tang, Lili; Peng, Limin; Chen, Ming; Luo, Xipeng; Wang, Shouman; Xiao, Zhi; Deng, Zhongping; Dai, Lizhong; Xia, Kun; Wang, Jun

2016-10-01

Circulating cell-free DNA (cfDNA) has been considered as a potential biomarker for non-invasive cancer detection. To evaluate the methylation levels of six candidate genes (EGFR, GREM1, PDGFRB, PPM1E, SOX17, and WRN) in plasma cfDNA as biomarkers for breast cancer early detection, quantitative analysis of the promoter methylation of these genes from 86 breast cancer patients and 67 healthy controls was performed by using microfluidic-PCR-based target enrichment and next-generation bisulfite sequencing technology. The predictive performance of different logistic models based on methylation status of candidate genes was investigated by means of the area under the ROC curve (AUC) and odds ratio (OR) analysis. Results revealed that EGFR, PPM1E, and 8 gene-specific CpG sites showed significantly hypermethylation in cancer patients' plasma and significantly associated with breast cancer (OR ranging from 2.51 to 9.88). The AUC values for these biomarkers were ranging from 0.66 to 0.75. Combinations of multiple hypermethylated genes or CpG sites substantially improved the predictive performance for breast cancer detection. Our study demonstrated the feasibility of quantitative measurement of candidate gene methylation in cfDNA by using microfluidic-PCR-based target enrichment and bisulfite next-generation sequencing, which is worthy of further validation and potentially benefits a broad range of applications in clinical oncology practice. Quantitative analysis of methylation pattern of plasma cfDNA by next-generation sequencing might be a valuable non-invasive tool for early detection of breast cancer.

Screening for diverse PDGFRA or PDGFRB fusion genes is facilitated by generic quantitative reverse transcriptase polymerase chain reaction analysis

PubMed Central

Erben, Philipp; Gosenca, Darko; Müller, Martin C.; Reinhard, Jelena; Score, Joannah; del Valle, Francesco; Walz, Christoph; Mix, Jürgen; Metzgeroth, Georgia; Ernst, Thomas; Haferlach, Claudia; Cross, Nicholas C.P.; Hochhaus, Andreas; Reiter, Andreas

2010-01-01

Background Rapid identification of diverse fusion genes with involvement of PDGFRA or PDGFRB in eosinophilia-associated myeloproliferative neoplasms is essential for adequate clinical management but is complicated by the multitude and heterogeneity of partner genes and breakpoints. Design and Methods We established a generic quantitative reverse transcriptase polymerase chain reaction to detect overexpression of the 3′-regions of PDGFRA or PDGFRB as a possible indicator of an underlying fusion. Results At diagnosis, all patients with known fusion genes involving PDGFRA (n=5; 51 patients) or PDGFRB (n=5; 7 patients) showed significantly increased normalized expression levels compared to 191 patients with fusion gene-negative eosinophilia or healthy individuals (PDGFRA/ABL: 0.73 versus 0.0066 versus 0.0064, P<0.0001; PDGFRB/ABL: 196 versus 3.8 versus 5.85, P<0.0001). The sensitivity and specificity of the activation screening test were, respectively, 100% and 88.4% for PDGFRA and 100% and 94% for PDGFRB. Furthermore, significant overexpression of PDGFRB was found in a patient with an eosinophilia-associated myeloproliferative neoplasm with uninformative cytogenetics and an excellent response to imatinib. Subsequently, a new SART3-PDGFRB fusion gene was identified by 5′-rapid amplification of cDNA ends polymerase chain reaction (5′-RACE-PCR). Conclusions Quantitative reverse transcriptase polymerase chain reaction analysis is a simple and useful adjunct to standard diagnostic assays to detect clinically significant overexpression of PDGFRA and PDGFRB in eosinophilia-associated myeloproliferative neoplasms or related disorders. PMID:20107158

Determination of internal controls for quantitative real time RT-PCR analysis of the effect of Edwardsiella tarda infection on gene expression in turbot (Scophthalmus maximus).

PubMed

Dang, Wei; Sun, Li

2011-02-01

In recent years, quantitative real time reverse transcriptase-PCR (qRT-PCR) has been used frequently in the study of gene expression in turbot (Scophthalmus maximus) in relation to bacterial infection. However, no investigations on appropriate qRT-PCR reference genes have been documented. In this report, we determined the potential of eight housekeeping genes, i.e. β-actin (ACTB), ribosomal protein L17 (RPL17), α-tubulin (TUBA), elongation factor-1-α(EF1A), β-2-Microglobulin (B2M), RNA polymerase II subunit D (RPSD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S ribosomal RNA (18S rRNA), as internal standards for qRT-PCR analysis of gene expression in turbot as a function of bacterial infection. For this purpose, the expression of the eight housekeeping genes in seven turbot tissues was determined by qRT-PCR before and after bacterial challenge, and the data were analyzed with the geNorm and NormFinder algorisms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations both before and after bacterial challenge. Before bacterial challenge, geNorm and NormFinder identified RPSD as the gene that showed least tissue specific expression. At 12 h post-bacterial infection, geNorm ranked ACTB/GAPDH, 18S rRNA/ACTB, ACTB/GAPDH, 18S rRNA/ACTB, RPL17/TUBA, RPSD/GAPDH, and RPSD/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 24 h post-bacterial infection. Taken together, these results indicate that RPSD is the most stable gene across tissue types under normal physiological conditions and that, during bacterial infection, ACTB might be used as an internal standard for the normalization of gene expression in immune relevant organs; however, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types under the condition of bacterial infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions

PubMed Central

Storch, Tatiane Timm; Pegoraro, Camila; Finatto, Taciane; Quecini, Vera; Rombaldi, Cesar Valmor; Girardi, César Luis

2015-01-01

Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference—ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)—along with two novel candidates—HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest. PMID:25774904

Identification of a novel reference gene for apple transcriptional profiling under postharvest conditions.

PubMed

Storch, Tatiane Timm; Pegoraro, Camila; Finatto, Taciane; Quecini, Vera; Rombaldi, Cesar Valmor; Girardi, César Luis

2015-01-01

Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference--ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)--along with two novel candidates--HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest.

Quantitative trait loci analyses and RNA-seq identify genes affecting stress response in rainbow trout

USDA-ARS?s Scientific Manuscript database

Genomic analyses have the potential to impact aquaculture production traits by identifying markers as proxies for traits which are expensive or difficult to measure and characterizing genetic variation and biochemical mechanisms underlying phenotypic variation. One such trait is the response of rai...

A maize caffeoyl-CoA O-methyltransferase gene confers quantitative resistance to multiple pathogens

USDA-ARS?s Scientific Manuscript database

Alleles that confer multiple disease resistance (MDR) are valuable in crop improvement though molecular mechanisms underlying their functions remain largely unknown. A QTL, qMdr9.02, associated with resistance to three important foliar maize diseases, southern leaf blight (SLB), gray leaf spot (GLS)...

Comparison of growth and metabolic regulation between wild, domesticated and transgenic salmonids.

USDA-ARS?s Scientific Manuscript database

To gain a better understanding of the aspects underlying normal and growth hormone enhanced growth in salmonids, quantitative expression analysis was performed for a number of genes related to muscle growth, metabolism, immunology and energy regulation. This analysis was performed in liver and musc...

Cloning of quantitative trait genes from rice reveals conservation and divergence of photoperiod flowering pathways in Arabidopsis and rice

PubMed Central

Matsubara, Kazuki; Hori, Kiyosumi; Ogiso-Tanaka, Eri; Yano, Masahiro

2014-01-01

Flowering time in rice (Oryza sativa L.) is determined primarily by daylength (photoperiod), and natural variation in flowering time is due to quantitative trait loci involved in photoperiodic flowering. To date, genetic analysis of natural variants in rice flowering time has resulted in the positional cloning of at least 12 quantitative trait genes (QTGs), including our recently cloned QTGs, Hd17, and Hd16. The QTGs have been assigned to specific photoperiodic flowering pathways. Among them, 9 have homologs in the Arabidopsis genome, whereas it was evident that there are differences in the pathways between rice and Arabidopsis, such that the rice Ghd7–Ehd1–Hd3a/RFT1 pathway modulated by Hd16 is not present in Arabidopsis. In this review, we describe QTGs underlying natural variation in rice flowering time. Additionally, we discuss the implications of the variation in adaptive divergence and its importance in rice breeding. PMID:24860584

rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum.

PubMed

Jacob, Tiago R; Peres, Nalu T A; Persinoti, Gabriela F; Silva, Larissa G; Mazucato, Mendelson; Rossi, Antonio; Martinez-Rossi, Nilce M

2012-05-01

The selection of reference genes used for data normalization to quantify gene expression by real-time PCR amplifications (qRT-PCR) is crucial for the accuracy of this technique. In spite of this, little information regarding such genes for qRT-PCR is available for gene expression analyses in pathogenic fungi. Thus, we investigated the suitability of eight candidate reference genes in isolates of the human dermatophyte Trichophyton rubrum subjected to several environmental challenges, such as drug exposure, interaction with human nail and skin, and heat stress. The stability of these genes was determined by geNorm, NormFinder and Best-Keeper programs. The gene with the most stable expression in the majority of the conditions tested was rpb2 (DNA-dependent RNA polymerase II), which was validated in three T. rubrum strains. Moreover, the combination of rpb2 and chs1 (chitin synthase) genes provided for the most reliable qRT-PCR data normalization in T. rubrum under a broad range of biological conditions. To the best of our knowledge this is the first report on the selection of reference genes for qRT-PCR data normalization in dermatophytes and the results of these studies should permit further analysis of gene expression under several experimental conditions, with improved accuracy and reliability.

Selection of reference genes for expression analyses of red-fleshed sweet orange (Citrus sinensis).

PubMed

Pinheiro, T T; Nishimura, D S; De Nadai, F B; Figueira, A; Latado, R R

2015-12-28

Red-fleshed oranges (Citrus sinensis) contain high levels of carotenoids and lycopene. The growing consumer demand for products with health benefits has increased interest in these types of Citrus cultivars as a potential source of nutraceuticals. However, little is known about the physiology of these cultivars under Brazilian conditions. Transcriptome and gene expression analyses are important tools in the breeding and management of red-fleshed sweet orange cultivars. Reverse transcription quantitative polymerase chain reaction is a method of quantifying gene expression, but various standardizations are required to obtain precise, accurate, and specific results. Among the standardizations required, the choice of suitable stable reference genes is fundamental. The objective of this study was to evaluate the stability of 11 candidate genes using various tissue and organ samples from healthy plants or leaves from citrus greening disease (Huanglongbing)-symptomatic plants of a Brazilian red-fleshed cultivar ('Sanguínea de Mombuca'), in order to select the most suitable reference gene for investigating gene expression under these conditions. geNorm and NormFinder identified genes that encoded translation initiation factor 3, ribosomal protein L35, and translation initiation factor 5A as the most stable genes under the biological conditions tested, and genes coding actin (ACT) and the subunit of the PSI reaction center subunit III were the least stable. Phosphatase, malate dehydrogenase, and ACT were the most stable genes in the leaf samples of infected plants.

Challenges and opportunities in bioanalytical support for gene therapy medicinal product development.

PubMed

Ma, Mark; Balasubramanian, Nanda; Dodge, Robert; Zhang, Yan

2017-09-01

Gene and nucleic acid therapies have demonstrated patient benefits to address unmet medical needs. Beside considerations regarding the biological nature of the gene therapy, the quality of bioanalytical methods plays an important role in ensuring the success of these novel therapies. Inconsistent approaches among bioanalytical labs during preclinical and clinical phases have been observed. There are many underlying reasons for this inconsistency. Various platforms and reagents used in quantitative methods, lacking of detailed regulatory guidance on method validation and uncertainty of immunogenicity strategy in supporting gene therapy may all be influential. This review summarizes recent practices and considerations in bioanalytical support of pharmacokinetics/pharmacodynamics and immunogenicity evaluations in gene therapy development with insight into method design, development and validations.

[Molecular mechanisms in sex determination: from gene regulation to pathology].

PubMed

Ravel, C; Chantot-Bastaraud, S; Siffroi, J-P

2004-01-01

Testis determination is the complex process by which the bipotential gonad becomes a normal testis during embryo development. As a consequence, this process leads to sexual differentiation corresponding to the masculinization of both genital track and external genitalia. The whole phenomenon is under genetic control and is particularly driven by the presence of the Y chromosome and by the SRY gene, which acts as the key initiator of the early steps of testis determination. However, many other autosomal genes, present in both males and females, are expressed during testis formation in a gene activation pathway, which is far to be totally elucidated. All these genes act in a dosage-sensitive manner by which quantitative gene abnormalities, due to chromosomal deletions, duplications or mosaicism, may lead to testis determination failure and sex reversal.

PGMapper: a web-based tool linking phenotype to genes.

PubMed

Xiong, Qing; Qiu, Yuhui; Gu, Weikuan

2008-04-01

With the availability of whole genome sequence in many species, linkage analysis, positional cloning and microarray are gradually becoming powerful tools for investigating the links between phenotype and genotype or genes. However, in these methods, causative genes underlying a quantitative trait locus, or a disease, are usually located within a large genomic region or a large set of genes. Examining the function of every gene is very time consuming and needs to retrieve and integrate the information from multiple databases or genome resources. PGMapper is a software tool for automatically matching phenotype to genes from a defined genome region or a group of given genes by combining the mapping information from the Ensembl database and gene function information from the OMIM and PubMed databases. PGMapper is currently available for candidate gene search of human, mouse, rat, zebrafish and 12 other species. Available online at http://www.genediscovery.org/pgmapper/index.jsp.

Identification of Suitable Reference Genes for Gene Expression Normalization in qRT-PCR Analysis in Watermelon

PubMed Central

Gao, Lingyun; Zhao, Shuang; Jiang, Wei; Huang, Yuan; Bie, Zhilong

2014-01-01

Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT–PCR) is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT–PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1α and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT–PCR analyses involving watermelon. PMID:24587403

Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon.

PubMed

Kong, Qiusheng; Yuan, Jingxian; Gao, Lingyun; Zhao, Shuang; Jiang, Wei; Huang, Yuan; Bie, Zhilong

2014-01-01

Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT-PCR) is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT-PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1α and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT-PCR analyses involving watermelon.

Reference genes for gene expression studies in wheat flag leaves grown under different farming conditions

PubMed Central

2011-01-01

Background Internal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from Triticum aestivum cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR) reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways. Findings Individual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus: TaFNRII (ferredoxin-NADP(H) oxidoreductase; AJ457980.1), ACT2 (actin 2; TC234027), and rrn26 (a putative homologue to RNA 26S gene; AL827977.1). In addition of these three genes that were also top-ranked by NormFinder, two extra genes: CYP18-2 (Cyclophilin A, AY456122.1) and TaWIN1 (14-3-3 like protein, AB042193) were most consistently stably expressed. Furthermore, we showed that TaFNRII, ACT2, and CYP18-2 are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire) grown under three treatments (organic, conventional and no nitrogen) and a different environment than the one tested with cv. Cubus. Conclusions This study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production. PMID:21951810

Identification and validation of reference genes for quantitative real-time PCR normalization and its applications in lycium.

PubMed

Zeng, Shaohua; Liu, Yongliang; Wu, Min; Liu, Xiaomin; Shen, Xiaofei; Liu, Chunzhao; Wang, Ying

2014-01-01

Lycium barbarum and L. ruthenicum are extensively used as traditional Chinese medicinal plants. Next generation sequencing technology provides a powerful tool for analyzing transcriptomic profiles of gene expression in non-model species. Such gene expression can then be confirmed with quantitative real-time polymerase chain reaction (qRT-PCR). Therefore, use of systematically identified suitable reference genes is a prerequisite for obtaining reliable gene expression data. Here, we calculated the expression stability of 18 candidate reference genes across samples from different tissues and grown under salt stress using geNorm and NormFinder procedures. The geNorm-determined rank of reference genes was similar to those defined by NormFinder with some differences. Both procedures confirmed that the single most stable reference gene was ACNTIN1 for L. barbarum fruits, H2B1 for L. barbarum roots, and EF1α for L. ruthenicum fruits. PGK3, H2B2, and PGK3 were identified as the best stable reference genes for salt-treated L. ruthenicum leaves, roots, and stems, respectively. H2B1 and GAPDH1+PGK1 for L. ruthenicum and SAMDC2+H2B1 for L. barbarum were the best single and/or combined reference genes across all samples. Finally, expression of salt-responsive gene NAC, fruit ripening candidate gene LrPG, and anthocyanin genes were investigated to confirm the validity of the selected reference genes. Suitable reference genes identified in this study provide a foundation for accurately assessing gene expression and further better understanding of novel gene function to elucidate molecular mechanisms behind particular biological/physiological processes in Lycium.

Dynamics of Bacterial Gene Regulatory Networks.

PubMed

Shis, David L; Bennett, Matthew R; Igoshin, Oleg A

2018-05-20

The ability of bacterial cells to adjust their gene expression program in response to environmental perturbation is often critical for their survival. Recent experimental advances allowing us to quantitatively record gene expression dynamics in single cells and in populations coupled with mathematical modeling enable mechanistic understanding on how these responses are shaped by the underlying regulatory networks. Here, we review how the combination of local and global factors affect dynamical responses of gene regulatory networks. Our goal is to discuss the general principles that allow extrapolation from a few model bacteria to less understood microbes. We emphasize that, in addition to well-studied effects of network architecture, network dynamics are shaped by global pleiotropic effects and cell physiology.

Transcriptome profiling of anthocyanin-related genes reveals effects of light intensity on anthocyanin biosynthesis in red leaf lettuce.

PubMed

Zhang, Yanzhao; Xu, Shuzhen; Cheng, Yanwei; Peng, Zhengfeng; Han, Jianming

2018-01-01

Red leaf lettuce ( Lactuca sativa L.) is popular due to its high anthocyanin content, but poor leaf coloring often occurs under low light intensity. In order to reveal the mechanisms of anthocyanins affected by light intensity, we compared the transcriptome of L. sativa L. var. capitata under light intensities of 40 and 100 μmol m -2 s -1 . A total of 62,111 unigenes were de novo assembled with an N50 of 1,681 bp, and 48,435 unigenes were functionally annotated in public databases. A total of 3,899 differentially expressed genes (DEGs) were detected, of which 1,377 unigenes were up-regulated and 2,552 unigenes were down-regulated in the high light samples. By Kyoto Encyclopedia of Genes and Genomes enrichment analysis, the DEGs were significantly enriched in 14 pathways. Using gene annotation and phylogenetic analysis, we identified seven anthocyanin structural genes, including CHS , CHI , F3H , F3'H , DFR , ANS , and 3GT , and two anthocyanin transport genes, GST and MATE . In terms of anthocyanin regulatory genes, five MYBs and one bHLH gene were identified. An HY5 gene was discovered, which may respond to light-signaling and regulate anthocyanin structural genes. These genes showed a log2FC of 2.7-9.0 under high irradiance, and were validated using quantitative real-time-PCR. In conclusion, our results indicated transcriptome variance in red leaf lettuce under low and high light intensity, and observed a anthocyanin biosynthesis and regulation pattern. The data should further help to unravel the molecular mechanisms of anthocyanins influenced by light intensity.

LOD score exclusion analyses for candidate QTLs using random population samples.

PubMed

Deng, Hong-Wen

2003-11-01

While extensive analyses have been conducted to test for, no formal analyses have been conducted to test against, the importance of candidate genes as putative QTLs using random population samples. Previously, we developed an LOD score exclusion mapping approach for candidate genes for complex diseases. Here, we extend this LOD score approach for exclusion analyses of candidate genes for quantitative traits. Under this approach, specific genetic effects (as reflected by heritability) and inheritance models at candidate QTLs can be analyzed and if an LOD score is < or = -2.0, the locus can be excluded from having a heritability larger than that specified. Simulations show that this approach has high power to exclude a candidate gene from having moderate genetic effects if it is not a QTL and is robust to population admixture. Our exclusion analysis complements association analysis for candidate genes as putative QTLs in random population samples. The approach is applied to test the importance of Vitamin D receptor (VDR) gene as a potential QTL underlying the variation of bone mass, an important determinant of osteoporosis.

Quantitative genetic analysis of brain copper and zinc in BXD recombinant inbred mice.

PubMed

Jones, Leslie C; McCarthy, Kristin A; Beard, John L; Keen, Carl L; Jones, Byron C

2006-01-01

Copper and zinc are trace nutrients essential for normal brain function, yet an excess of these elements can be toxic. It is important therefore that these metals be closely regulated. We recently conducted a quantitative trait loci (QTL) analysis to identify chromosomal regions in the mouse containing possible regulatory genes. The animals came from 15 strains of the BXD/Ty recombinant inbred (RI) strain panel and the brain regions analyzed were frontal cortex, caudate-putamen, nucleus accumbens and ventral midbrain. Several QTL were identified for copper and/or zinc, most notably on chromosomes 1, 8, 16 and 17. Genetic correlational analysis also revealed associations between these metals and dopamine, cocaine responses, saccharine preference, immune response and seizure susceptibility. Notably, the QTL on chromosome 17 is also associated with seizure susceptibility and contains the histocompatibility H2 complex. This work shows that regulation of zinc and copper is under polygenic influence and is intimately related to CNS function. Future work will reveal genes underlying the QTL and how they interact with other genes and the environment. More importantly, revelation of the genetic underpinnings of copper and zinc brain homeostasis will aid our understanding of neurological diseases that are related to copper and zinc imbalance.

Genome-wide survey and characterization of the WRKY gene family in Populus trichocarpa.

PubMed

He, Hongsheng; Dong, Qing; Shao, Yuanhua; Jiang, Haiyang; Zhu, Suwen; Cheng, Beijiu; Xiang, Yan

2012-07-01

WRKY transcription factors participate in diverse physiological and developmental processes in plants. They have highly conserved WRKYGQK amino acid sequences in their N-termini, followed by the novel zinc-finger-like motifs, Cys₂His₂ or Cys₂HisCys. To date, numerous WRKY genes have been identified and characterized in a number of herbaceous species. Survey and characterization of WRKY genes in a ligneous species would facilitate a better understanding of the evolutionary processes and functions of this gene family. In this study, 104 poplar WRKY genes (PtWRKY) were identified in the latest poplar genome sequence. According to their structural features, the predicted members were divided into the previously defined groups I-III, as described in rice. In addition, chromosomal localization of the genes demonstrated that there might be WRKY gene hot spots in 2.3 Mb regions on chromosome 14. Furthermore, approximately 83% (86 out of 104) WRKY genes participated in gene duplication events, including 69% (29 out of 42) gene pairs which exhibited segmental duplication. Using semi-quantitative RT-PCR, the expression patterns of subgroup III genes were investigated under different stresses [cold, drought, salinity and salicylic acid (SA)]. The data revealed that these genes presented different expression levels in response to various stress conditions. Expression analysis exhibited PtWRKY76 gene induced markedly in 0.1 mM SA or 25% PEG-6000 treatment. The results presented here provide a fundamental clue for cloning specific function genes in further studies and applications. This study identified 104 poplar WRKY genes and demonstrated WRKY gene hot spots on chromosome 14. Furthermore, semi-quantitative RT-PCR showed variable stress responses in subgroup III.

Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Lycoris aurea

PubMed Central

Ma, Rui; Xu, Sheng; Zhao, Yucheng; Xia, Bing; Wang, Ren

2016-01-01

Lycoris aurea (L' Hér.) Herb, a perennial grass species, produces a unique variety of pharmacologically active Amaryllidaceae alkaloids. However, the key enzymes and their expression pattern involved in the biosynthesis of Amaryllidaceae alkaloids (especially for galanthamine) are far from being fully understood. Quantitative real-time polymerase chain reaction (qRT-PCR), a commonly used method for quantifying gene expression, requires stable reference genes to normalize its data. In this study, to choose the appropriate reference genes under different experimental conditions, 14 genes including YLS8 (mitosis protein YLS8), CYP2 (Cyclophilin 2), CYP 1 (Cyclophilin 1), TIP41 (TIP41-like protein), EXP2 (Expressed protein 2), PTBP1 (Polypyrimidine tract-binding protein 1), EXP1 (Expressed protein 1), PP2A (Serine/threonine-protein phosphatase 2A), β-TUB (β-tubulin), α-TUB (α-tubulin), EF1-α (Elongation factor 1-α), UBC (Ubiquitin-conjugating enzyme), ACT (Actin) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were selected from the transcriptome datasets of L. aurea. And then, expressions of these genes were assessed by qRT-PCR in various tissues and the roots under different treatments. The expression stability of the 14 candidates was analyzed by three commonly used software programs (geNorm, NormFinder, and BestKeeper), and their results were further integrated into a comprehensive ranking based on the geometric mean. The results show the relatively stable genes for each subset as follows: (1) EXP1 and TIP41 for all samples; (2) UBC and EXP1 for NaCl stress; (3) PTBP1 and EXP1 for heat stress, polyethylene glycol (PEG) stress and ABA treatment; (4) UBC and CYP2 for cold stress; (5) PTBP1 and PP2A for sodium nitroprusside (SNP) treatment; (6) CYP1 and TIP41 for methyl jasmonate (MeJA) treatment; and (7) EXP1 and TIP41 for various tissues. The reliability of these results was further enhanced through comparison between part qRT-PCR result and RNA sequencing (RNA-seq) data. In summary, our results identified appropriate reference genes for qRT-PCR in L. aurea, and will facilitate gene expression studies under these conditions. PMID:27200013

Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells

PubMed Central

Kampmann, Martin; Bassik, Michael C.; Weissman, Jonathan S.

2013-01-01

A major challenge of the postgenomic era is to understand how human genes function together in normal and disease states. In microorganisms, high-density genetic interaction (GI) maps are a powerful tool to elucidate gene functions and pathways. We have developed an integrated methodology based on pooled shRNA screening in mammalian cells for genome-wide identification of genes with relevant phenotypes and systematic mapping of all GIs among them. We recently demonstrated the potential of this approach in an application to pathways controlling the susceptibility of human cells to the toxin ricin. Here we present the complete quantitative framework underlying our strategy, including experimental design, derivation of quantitative phenotypes from pooled screens, robust identification of hit genes using ultra-complex shRNA libraries, parallel measurement of tens of thousands of GIs from a single double-shRNA experiment, and construction of GI maps. We describe the general applicability of our strategy. Our pooled approach enables rapid screening of the same shRNA library in different cell lines and under different conditions to determine a range of different phenotypes. We illustrate this strategy here for single- and double-shRNA libraries. We compare the roles of genes for susceptibility to ricin and Shiga toxin in different human cell lines and reveal both toxin-specific and cell line-specific pathways. We also present GI maps based on growth and ricin-resistance phenotypes, and we demonstrate how such a comparative GI mapping strategy enables functional dissection of physical complexes and context-dependent pathways. PMID:23739767

Simulating the yield impacts of organ-level quantitative trait loci associated with drought response in maize: a "gene-to-phenotype" modeling approach.

PubMed

Chenu, Karine; Chapman, Scott C; Tardieu, François; McLean, Greg; Welcker, Claude; Hammer, Graeme L

2009-12-01

Under drought, substantial genotype-environment (G x E) interactions impede breeding progress for yield. Identifying genetic controls associated with yield response is confounded by poor genetic correlations across testing environments. Part of this problem is related to our inability to account for the interplay of genetic controls, physiological traits, and environmental conditions throughout the crop cycle. We propose a modeling approach to bridge this "gene-to-phenotype" gap. For maize under drought, we simulated the impact of quantitative trait loci (QTL) controlling two key processes (leaf and silk elongation) that influence crop growth, water use, and grain yield. Substantial G x E interaction for yield was simulated for hypothetical recombinant inbred lines (RILs) across different seasonal patterns of drought. QTL that accelerated leaf elongation caused an increase in crop leaf area and yield in well-watered or preflowering water deficit conditions, but a reduction in yield under terminal stresses (as such "leafy" genotypes prematurely exhausted the water supply). The QTL impact on yield was substantially enhanced by including pleiotropic effects of these QTL on silk elongation and on consequent grain set. The simulations obtained illustrated the difficulty of interpreting the genetic control of yield for genotypes influenced only by the additive effects of QTL associated with leaf and silk growth. The results highlight the potential of integrative simulation modeling for gene-to-phenotype prediction and for exploiting G x E interactions for complex traits such as drought tolerance.

Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species

PubMed Central

Reddy, Palakolanu Sudhakar; Sri Cindhuri, Katamreddy; Sivaji Ganesh, Adusumalli; Sharma, Kiran Kumar

2016-01-01

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses. PMID:26863232

Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species.

PubMed

Reddy, Dumbala Srinivas; Bhatnagar-Mathur, Pooja; Reddy, Palakolanu Sudhakar; Sri Cindhuri, Katamreddy; Sivaji Ganesh, Adusumalli; Sharma, Kiran Kumar

2016-01-01

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.

Genome-wide identification and expression profile analysis of the NAC transcription factor family during abiotic and biotic stress in woodland strawberry

PubMed Central

Qi, Yanxiang; Liu, Xiaomei; Pu, Jinji

2018-01-01

The NAC transcription factors involved plant development and response to various stress stimuli. However, little information is available concerning the NAC family in the woodland strawberry. Herein, 37 NAC genes were identified from the woodland strawberry genome and were classified into 13 groups based on phylogenetic analysis. And further analyses of gene structure and conserved motifs showed closer relationship of them in every subgroup. Quantitative real-time PCR evaluation different tissues revealed distinct spatial expression profiles of the FvNAC genes. The comprehensive expression of FvNAC genes revealed under abiotic stress (cold, heat, drought, salt), signal molecule treatments (H2O2, ABA, melatonin, rapamycin), biotic stress (Colletotrichum gloeosporioides and Ralstonia solanacearum). Expression profiles derived from quantitative real-time PCR suggested that 5 FvNAC genes responded dramatically to the various abiotic and biotic stresses, indicating their contribution to abiotic and biotic stresses resistance in woodland strawberry. Interestingly, FvNAC genes showed greater extent responded to the cold treatment than other abiotic stress, and H2O2 exhibited a greater response than ABA, melatonin, and rapamycin. For biotic stresses, 3 FvNAC genes were up-regulated during infection with C. gloeosporioides, while 6 FvNAC genes were down-regulated during infection with R. solanacearum. In conclusion, this study identified candidate FvNAC genes to be used for the genetic improvement of abiotic and biotic stress tolerance in woodland strawberry. PMID:29897926

Reference gene selection for quantitative real-time PCR in Solanum lycopersicum L. inoculated with the mycorrhizal fungus Rhizophagus irregularis.

PubMed

Fuentes, Alejandra; Ortiz, Javier; Saavedra, Nicolás; Salazar, Luis A; Meneses, Claudio; Arriagada, Cesar

2016-04-01

The gene expression stability of candidate reference genes in the roots and leaves of Solanum lycopersicum inoculated with arbuscular mycorrhizal fungi was investigated. Eight candidate reference genes including elongation factor 1 α (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), protein phosphatase 2A (PP2Acs), ribosomal protein L2 (RPL2), β-tubulin (TUB), ubiquitin (UBI) and actin (ACT) were selected, and their expression stability was assessed to determine the most stable internal reference for quantitative PCR normalization in S. lycopersicum inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. The stability of each gene was analysed in leaves and roots together and separated using the geNorm and NormFinder algorithms. Differences were detected between leaves and roots, varying among the best-ranked genes depending on the algorithm used and the tissue analysed. PGK, TUB and EF1 genes showed higher stability in roots, while EF1 and UBI had higher stability in leaves. Statistical algorithms indicated that the GAPDH gene was the least stable under the experimental conditions assayed. Then, we analysed the expression levels of the LePT4 gene, a phosphate transporter whose expression is induced by fungal colonization in host plant roots. No differences were observed when the most stable genes were used as reference genes. However, when GAPDH was used as the reference gene, we observed an overestimation of LePT4 expression. In summary, our results revealed that candidate reference genes present variable stability in S. lycopersicum arbuscular mycorrhizal symbiosis depending on the algorithm and tissue analysed. Thus, reference gene selection is an important issue for obtaining reliable results in gene expression quantification. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Quantitative trait loci and metabolic pathways

PubMed Central

McMullen, M. D.; Byrne, P. F.; Snook, M. E.; Wiseman, B. R.; Lee, E. A.; Widstrom, N. W.; Coe, E. H.

1998-01-01

The interpretation of quantitative trait locus (QTL) studies is limited by the lack of information on metabolic pathways leading to most economic traits. Inferences about the roles of the underlying genes with a pathway or the nature of their interaction with other loci are generally not possible. An exception is resistance to the corn earworm Helicoverpa zea (Boddie) in maize (Zea mays L.) because of maysin, a C-glycosyl flavone synthesized in silks via a branch of the well characterized flavonoid pathway. Our results using flavone synthesis as a model QTL system indicate: (i) the importance of regulatory loci as QTLs, (ii) the importance of interconnecting biochemical pathways on product levels, (iii) evidence for “channeling” of intermediates, allowing independent synthesis of related compounds, (iv) the utility of QTL analysis in clarifying the role of specific genes in a biochemical pathway, and (v) identification of a previously unknown locus on chromosome 9S affecting flavone level. A greater understanding of the genetic basis of maysin synthesis and associated corn earworm resistance should lead to improved breeding strategies. More broadly, the insights gained in relating a defined genetic and biochemical pathway affecting a quantitative trait should enhance interpretation of the biological basis of variation for other quantitative traits. PMID:9482823

Mutation spectrum and differential gene expression in cystic and solid vestibular schwannoma.

PubMed

Zhang, Zhihua; Wang, Zhaoyan; Sun, Lianhua; Li, Xiaohua; Huang, Qi; Yang, Tao; Wu, Hao

2014-03-01

We sought to characterize the mutation spectrum of NF2 and the differential gene expression in cystic and solid vestibular schwannomas. We collected tumor tissue and blood samples of 31 cystic vestibular schwannomas and 114 solid vestibular schwannomas. Mutation screening of NF2 was performed in both tumor and blood DNA samples of all patients. cDNA microarray was used to analyze the differential gene expression between 11 cystic vestibular schwannomas and 6 solid vestibular schwannomas. Expression levels of top candidate genes were verified by quantitative reverse transcription PCR. NF2 mutations were identified in 34.5% of sporadic vestibular schwannomas, with all mutations being exclusively somatic. No significant difference was found between the mutation detection rates of cystic vestibular schwannoma (35.5%) and solid vestibular schwannoma (34.2%). cDNA microarray analysis detected a total of 46 differentially expressed genes between the cystic vestibular schwannoma and solid vestibular schwannoma samples. The significantly decreased expression of four top candidate genes, C1orf130, CNTF, COL4A3, and COL4A4, was verified by quantitative reverse transcription PCR. NF2 mutations are not directly involved in the cystic formation of vestibular schwannoma. In addition, the differential gene expression of cystic vestibular schwannoma reported in our study may provide useful insights into the molecular mechanism underlying this process.

Genetic, physiological and comparative genomic studies of hypertension and insulin resistance in the spontaneously hypertensive rat.

PubMed

Coan, Philip M; Hummel, Oliver; Garcia Diaz, Ana; Barrier, Marjorie; Alfazema, Neza; Norsworthy, Penny J; Pravenec, Michal; Petretto, Enrico; Hübner, Norbert; Aitman, Timothy J

2017-03-01

We previously mapped hypertension-related insulin resistance quantitative trait loci (QTLs) to rat chromosomes 4, 12 and 16 using adipocytes from F2 crosses between spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats, and subsequently identified Cd36 as the gene underlying the chromosome 4 locus. The identity of the chromosome 12 and 16 genes remains unknown. To identify whole-body phenotypes associated with the chromosome 12 and 16 linkage regions, we generated and characterised new congenic strains, with WKY donor segments introgressed onto an SHR genetic background, for the chromosome 12 and 16 linkage regions. We found a >50% increase in insulin sensitivity in both the chromosome 12 and 16 strains. Blood pressure and left ventricular mass were reduced in the two congenic strains consistent with the congenic segments harbouring SHR genes for insulin resistance, hypertension and cardiac hypertrophy. Integrated genomic analysis, using physiological and whole-genome sequence data across 42 rat strains, identified variants within the congenic regions in Upk3bl , RGD1565131 and AABR06087018.1 that were associated with blood pressure, cardiac mass and insulin sensitivity. Quantitative trait transcript analysis across 29 recombinant inbred strains showed correlation between expression of Hspb1 , Zkscan5 and Pdgfrl with adipocyte volume, systolic blood pressure and cardiac mass, respectively. Comparative genome analysis showed a marked enrichment of orthologues for human GWAS-associated genes for insulin resistance within the syntenic regions of both the chromosome 12 and 16 congenic intervals. Our study defines whole-body phenotypes associated with the SHR chromosome 12 and 16 insulin-resistance QTLs, identifies candidate genes for these SHR QTLs and finds human orthologues of rat genes in these regions that associate with related human traits. Further study of these genes in the congenic strains will lead to robust identification of the underlying genes and cellular mechanisms. © 2017. Published by The Company of Biologists Ltd.

Genetic, physiological and comparative genomic studies of hypertension and insulin resistance in the spontaneously hypertensive rat

PubMed Central

Hummel, Oliver; Garcia Diaz, Ana; Barrier, Marjorie; Alfazema, Neza; Norsworthy, Penny J.; Pravenec, Michal; Petretto, Enrico; Hübner, Norbert

2017-01-01

ABSTRACT We previously mapped hypertension-related insulin resistance quantitative trait loci (QTLs) to rat chromosomes 4, 12 and 16 using adipocytes from F2 crosses between spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats, and subsequently identified Cd36 as the gene underlying the chromosome 4 locus. The identity of the chromosome 12 and 16 genes remains unknown. To identify whole-body phenotypes associated with the chromosome 12 and 16 linkage regions, we generated and characterised new congenic strains, with WKY donor segments introgressed onto an SHR genetic background, for the chromosome 12 and 16 linkage regions. We found a >50% increase in insulin sensitivity in both the chromosome 12 and 16 strains. Blood pressure and left ventricular mass were reduced in the two congenic strains consistent with the congenic segments harbouring SHR genes for insulin resistance, hypertension and cardiac hypertrophy. Integrated genomic analysis, using physiological and whole-genome sequence data across 42 rat strains, identified variants within the congenic regions in Upk3bl, RGD1565131 and AABR06087018.1 that were associated with blood pressure, cardiac mass and insulin sensitivity. Quantitative trait transcript analysis across 29 recombinant inbred strains showed correlation between expression of Hspb1, Zkscan5 and Pdgfrl with adipocyte volume, systolic blood pressure and cardiac mass, respectively. Comparative genome analysis showed a marked enrichment of orthologues for human GWAS-associated genes for insulin resistance within the syntenic regions of both the chromosome 12 and 16 congenic intervals. Our study defines whole-body phenotypes associated with the SHR chromosome 12 and 16 insulin-resistance QTLs, identifies candidate genes for these SHR QTLs and finds human orthologues of rat genes in these regions that associate with related human traits. Further study of these genes in the congenic strains will lead to robust identification of the underlying genes and cellular mechanisms. PMID:28130354

Characterization of Novel Gene Yr79 and Four Additional Quantitative Trait Loci for All-Stage and High-Temperature Adult-Plant Resistance to Stripe Rust in Spring Wheat PI 182103.

PubMed

Feng, Junyan; Wang, Meinan; See, Deven R; Chao, Shiaoman; Zheng, Youliang; Chen, Xianming

2018-06-01

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important disease of wheat worldwide. Exploring new resistance genes is essential for breeding resistant wheat cultivars. PI 182103, a spring wheat landrace originally from Pakistan, has shown a high level of resistance to stripe rust in fields for many years, but genes for resistance to stripe rust in the variety have not been studied. To map the resistance gene(s) in PI 182103, 185 recombinant inbred lines (RILs) were developed from a cross with Avocet Susceptible (AvS). The RIL population was genotyped with simple sequence repeat (SSR) and single nucleotide polymorphism markers and tested with races PST-100 and PST-114 at the seedling stage under controlled greenhouse conditions and at the adult-plant stage in fields at Pullman and Mt. Vernon, Washington under natural infection by the stripe rust pathogen in 2011, 2012, and 2013. A total of five quantitative trait loci (QTL) were detected. QyrPI182103.wgp-2AS and QyrPI182103.wgp-3AL were detected at the seedling stage, QyrPI182103.wgp-4DL was detected only in Mt. Vernon field tests, and QyrPI182103.wgp-5BS was detected in both seedling and field tests. QyrPI182103.wgp-7BL was identified as a high-temperature adult-plant resistance gene and detected in all field tests. Interactions among the QTL were mostly additive, but some negative interactions were detected. The 7BL QTL was mapped in chromosomal bin 7BL 0.40 to 0.45 and identified as a new gene, permanently designated as Yr79. SSR markers Xbarc72 and Xwmc335 flanking the Yr79 locus were highly polymorphic in various wheat genotypes, indicating that the molecular markers are useful for incorporating the new gene for potentially durable stripe rust resistance into new wheat cultivars.

Optimal Reference Gene Selection for Expression Studies in Human Reticulocytes.

PubMed

Aggarwal, Anu; Jamwal, Manu; Viswanathan, Ganesh K; Sharma, Prashant; Sachdeva, ManUpdesh S; Bansal, Deepak; Malhotra, Pankaj; Das, Reena

2018-05-01

Reference genes are indispensable for normalizing mRNA levels across samples in real-time quantitative PCR. Their expression levels vary under different experimental conditions and because of several inherent characteristics. Appropriate reference gene selection is thus critical for gene-expression studies. This study aimed at selecting optimal reference genes for gene-expression analysis of reticulocytes and at validating them in hereditary spherocytosis (HS) and β-thalassemia intermedia (βTI) patients. Seven reference genes (PGK1, MPP1, HPRT1, ACTB, GAPDH, RN18S1, and SDHA) were selected because of published reports. Real-time quantitative PCR was performed on reticulocytes in 20 healthy volunteers, 15 HS patients, and 10 βTI patients. Threshold cycle values were compared with fold-change method and RefFinder software. The stable reference genes recommended by RefFinder were validated with SLC4A1 and flow cytometric eosin-5'-maleimide binding assay values in HS patients and HBG2 and high performance liquid chromatography-derived percentage of hemoglobin F in βTI. Comprehensive ranking predicted MPP1 and GAPDH as optimal reference genes for reticulocytes that were not affected in HS and βTI. This was further confirmed on validation with eosin-5'-maleimide results and percentage of hemoglobin F in HS and βTI patients, respectively. Hence, MPP1 and GAPDH are good reference genes for reticulocyte expression studies compared with ACTB and RN18S1, the two most commonly used reference genes. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Quantitative real time RT-PCR study of pathogen-induced gene expression in rock bream (Oplegnathus fasciatus): internal controls for data normalization.

PubMed

Zhang, Bao-cun; Sun, Li; Xiao, Zhi-zhong; Hu, Yong-hua

2014-06-01

Rock bream Oplegnathus fasciatus is an important economic fish species. In this study, we evaluated the appropriateness of six housekeeping genes as internal controls for quantitative real-time PCR (RT-qPCR) analysis of gene expression in rock bream before and after pathogen infection. The expression of the selected genes in eight tissues infected with Vibrio alginolyticus or megalocytivirus was determined by RT-qPCR, and the PCR data were analyzed with geNorm and NormFinder algorithms. The results showed that before pathogen infection, mediator of RNA polymerase II transcription subunit 8 and β-actin were ranked as the most stable genes across the examined tissues. After bacterial or viral infection, the stabilities of the housekeeping genes varied to significant extents in tissue-dependent manners, and no single pair of genes was identified as suitable references for all tissues for either of the pathogen stimuli. In addition, for the majority of tissues, the most stable genes during bacterial infection differed from those during viral infection. Nevertheless, optimum reference genes were identified for each tissue under different conditions. Taken together, these results indicate that tissue type and the nature of the infectious agent used in the study can all influence the choice of normalization factors, and that the optimum reference genes identified in this study will provide a useful guidance for the selection of internal controls in future RT-PCR study of gene expression in rock bream. Copyright © 2014 Elsevier B.V. All rights reserved.

Microarray-assisted fine-mapping of quantitative trait loci for cold tolerance in rice.

PubMed

Liu, Fengxia; Xu, Wenying; Song, Qian; Tan, Lubin; Liu, Jiayong; Zhu, Zuofeng; Fu, Yongcai; Su, Zhen; Sun, Chuanqing

2013-05-01

Many important agronomic traits, including cold stress resistance, are complex and controlled by quantitative trait loci (QTLs). Isolation of these QTLs will greatly benefit the agricultural industry but it is a challenging task. This study explored an integrated strategy by combining microarray with QTL-mapping in order to identify cold-tolerant QTLs from a cold-tolerant variety IL112 at early-seedling stage. All the early seedlings of IL112 survived normally for 9 d at 4-5°C, while Guichao2 (GC2), an indica cultivar, died after 4 d under the same conditions. Using the F2:3 population derived from the progeny of GC2 and IL112, we identified seven QTLs for cold tolerance. Furthermore, we performed Affymetrix rice whole-genome array hybridization and obtained the expression profiles of IL112 and GC2 under both low-temperature and normal conditions. Four genes were selected as cold QTL-related candidates, based on microarray data mining and QTL-mapping. One candidate gene, LOC_Os07g22494, was shown to be highly associated with cold tolerance in a number of rice varieties and in the F2:3 population, and its overexpression transgenic rice plants displayed strong tolerance to low temperature at early-seedling stage. The results indicated that overexpression of this gene (LOC_Os07g22494) could increase cold tolerance in rice seedlings. Therefore, this study provides a promising strategy for identifying candidate genes in defined QTL regions.

Regulation of melanin biosynthesis via the dihydroxynaphthalene pathway is dependent on sexual development in the ascomycete Sordaria macrospora.

PubMed

Engh, Ines; Nowrousian, Minou; Kück, Ulrich

2007-10-01

The filamentous ascomycete Sordaria macrospora accumulates melanin during sexual development. The four melanin biosynthesis genes pks, teh, sdh and tih were isolated and their homology to genes involved in 1,8 dihydroxynaphthalene (DHN) melanin biosynthesis was shown. The presence of DHN melanin in S. macrospora was further confirmed by disrupting the pks gene encoding a putative polyketide synthase and by RNA interference-mediated silencing of the sdh gene encoding a putative scytalone dehydratase. Because melanin occurs in fruiting bodies that develop through several intermediate stages within 7 days of growth, a Northern analysis of a developmental time-course was conducted. These data revealed a time-dependent regulation of teh and sdh transcript levels. Comparing the transcriptional expression by real-time PCR of melanin biosynthesis genes in the wild type under conditions allowing or repressing sexual development, a significant downregulation during vegetative growth was detected. Quantitative real-time PCR and Northern blot analysis of melanin biosynthesis gene expression in different developmental mutants confirmed that melanin biosynthesis is linked to fruiting body development and is under the control of specific regulatory genes that participate in sexual differentiation.

[Expression of plant antimicrobial peptide pro-SmAMP2 gene increases resistance of transgenic potato plants to Alternaria and Fusarium pathogens].

PubMed

Vetchinkina, E M; Komakhina, V V; Vysotskii, D A; Zaitsev, D V; Smirnov, A N; Babakov, A V; Komakhin, R A

2016-09-01

The chickweed (Stellaria media L.) pro-SmAMP2 gene encodes the hevein-like peptides that have in vitro antimicrobial activity against certain harmful microorganisms. These peptides play an important role in protecting the chickweed plants from infection, and the pro-SmAMP2 gene was previously used to protect transgenic tobacco and Arabidopsis plants from phytopathogens. In this study, the pro-SmAMP2 gene under control of viral CaMV35S promoter or under control of its own pro-SmAMP2 promoter was transformed into cultivated potato plants of two cultivars, differing in the resistance to Alternaria: Yubiley Zhukova (resistant) and Skoroplodny (susceptible). With the help of quantitative real-time PCR, it was demonstrated that transgenic potato plants expressed the pro-SmAMP2 gene under control of both promoters at the level comparable to or exceeding the level of the potato actin gene. Assessment of the immune status of the transformants demonstrated that expression of antimicrobial peptide pro-SmAMP2 gene was able to increase the resistance to a complex of Alternaria sp. and Fusarium sp. phytopathogens only in potato plants of the Yubiley Zhukova cultivar. The possible role of the pro-SmAMP2 products in protecting potatoes from Alternaria sp. and Fusarium sp. is discussed.

Restriction fragment length polymorphisms in dairy and beef cattle at the growth hormone and prolactin loci.

PubMed

Hallerman, E M; Nave, A; Kashi, Y; Holzer, Z; Soller, M; Beckmann, J S

1987-01-01

Two bovine populations, a Holstein-Friesian dairy stock and a synthetic (Baladi X Hereford X Simmental X Charolais) beef stock, were screened for restriction fragment length polymorphisms (RFLPs) at the growth hormone and prolactin genes. Most RFLPs at the growth hormone gene are apparently the consequence of an insertion/deletion event which was localized to a region downstream of the structural gene. The restriction map for the genomic region including the growth hormone gene was extended. Two HindIII RFLPs at the growth hormone locus, as well as several RFLPs at the prolactin gene, seemed to be the consequence of a series of point mutations. The results are discussed in terms of the possibility that minor genomic variability underlies quantitative genetic variation.

Genes are differentially expressed at transcriptional level of Neocaridina denticulata following short-term exposure to nonylphenol.

PubMed

Liu, Chang-Lun; Sung, Hung-Hung

2011-09-01

To assess the toxicity of nonylphenol towards aquatic crustaceans, Neocaridina denticulata were exposed short-term to sublethal concentration (0.001-0.5 mg/L). Following treatment, differentially expressed genes were identified using suppression subtractive hybridization on samples prepared from whole specimens. There were 20 differentially expressed sequence tags that corresponded to known genes and could be divided into six functional classes: defence, translation, metabolism, ribosomal gene expression, respiration, and genes involved in the stress response. Using semi-quantitative RT-PCR, we found that 14 of the differentially expressed sequence tags significantly responded to nonylphenol, including six at a nominal concentration of 0.01 mg/L; among them, 12 genes were down-regulated. These results suggest that under non-lethal concentrations of nonylphenol, the polluted aquatic environment may still present a potential risk to N. denticulata.

Initial leukemic gene expression profiles of patients with poor in vivo prednisone response are similar to those of blasts persisting under prednisone treatment in childhood acute lymphoblastic leukemia.

PubMed

Cario, Gunnar; Fetz, Andrea; Bretscher, Christian; Möricke, Anja; Schrauder, Andre; Stanulla, Martin; Schrappe, Martin

2008-09-01

Response to initial glucocorticoid (GC) treatment is a strong prognostic factor in childhood acute lymphoblastic leukemia (ALL). Patients with a poor prednisone response (PPR) have a poor event-free survival as compared to those with a good prednisone response (PGR). Causes of prednisone resistance are still not well understood. We hypothesized that GC resistance is an intrinsic feature of ALL cells which is reflected in the gene expression pattern and analyzed genome-wide gene expression using microarrays. A case-control study was performed comparing gene expression profiles from initial ALL samples of 20 patients with PPR and those of 20 patients with PGR. Differential gene expression of a subset of genes was confirmed by real-time quantitative polymerase chain reaction analysis and validation was performed in a second independent patient sample (n=20). We identified 121 genes that clearly distinguished prednisone-resistant from sensitive ALL samples (FDR<5%, fold change>or=1.5). Differential gene expression of 21 of these genes could be validated in a second independent set. Of importance, there was a remarkable concordance of genes identified by comparing expression signatures of PPR and PGR cells at diagnosis and those previously described to be up- or downregulated in leukemic cells persisting under GC treatment. Thus, GC resistance seems at least in part to be an intrinsic feature of leukemic cells. Leukemic cells of patients with PPR are characterized by gene expression pattern which are similar to those of resistant cells persisting under glucocorticoid treatment.

Identification and expression profiling analysis of calmodulin-binding transcription activator genes in maize (Zea mays L.) under abiotic and biotic stresses

PubMed Central

Yue, Runqing; Lu, Caixia; Sun, Tao; Peng, Tingting; Han, Xiaohua; Qi, Jianshuang; Yan, Shufeng; Tie, Shuanggui

2015-01-01

The calmodulin-binding transcription activators (CAMTA) play critical roles in plant growth and responses to environmental stimuli. However, how CAMTAs function in responses to abiotic and biotic stresses in maize (Zea mays L.) is largely unknown. In this study, we first identified all the CAMTA homologous genes in the whole genome of maize. The results showed that nine ZmCAMTA genes showed highly diversified gene structures and tissue-specific expression patterns. Many ZmCAMTA genes displayed high expression levels in the roots. We then surveyed the distribution of stress-related cis-regulatory elements in the −1.5 kb promoter regions of ZmCAMTA genes. Notably, a large number of stress-related elements present in the promoter regions of some ZmCAMTA genes, indicating a genetic basis of stress expression regulation of these genes. Quantitative real-time PCR was used to test the expression of ZmCAMTA genes under several abiotic stresses (drought, salt, and cold), various stress-related hormones [abscisic acid, auxin, salicylic acid (SA), and jasmonic acid] and biotic stress [rice black-streaked dwarf virus (RBSDV) infection]. Furthermore, the expression pattern of ZmCAMTA genes under RBSDV infection was analyzed to investigate their potential roles in responses of different maize cultivated varieties to RBSDV. The expression of most ZmCAMTA genes responded to both abiotic and biotic stresses. The data will help us to understand the roles of CAMTA-mediated Ca2+ signaling in maize tolerance to environmental stresses. PMID:26284092

Genome-Wide Analysis of Soybean HD-Zip Gene Family and Expression Profiling under Salinity and Drought Treatments

PubMed Central

Chen, Xue; Chen, Zhu; Zhao, Hualin; Zhao, Yang; Cheng, Beijiu; Xiang, Yan

2014-01-01

Background Homeodomain-leucine zipper (HD-Zip) proteins, a group of homeobox transcription factors, participate in various aspects of normal plant growth and developmental processes as well as environmental responses. To date, no overall analysis or expression profiling of the HD-Zip gene family in soybean (Glycine max) has been reported. Methods and Findings An investigation of the soybean genome revealed 88 putative HD-Zip genes. These genes were classified into four subfamilies, I to IV, based on phylogenetic analysis. In each subfamily, the constituent parts of gene structure and motif were relatively conserved. A total of 87 out of 88 genes were distributed unequally on 20 chromosomes with 36 segmental duplication events, indicating that segmental duplication is important for the expansion of the HD-Zip family. Analysis of the Ka/Ks ratios showed that the duplicated genes of the HD-Zip family basically underwent purifying selection with restrictive functional divergence after the duplication events. Analysis of expression profiles showed that 80 genes differentially expressed across 14 tissues, and 59 HD-Zip genes are differentially expressed under salinity and drought stress, with 20 paralogous pairs showing nearly identical expression patterns and three paralogous pairs diversifying significantly under drought stress. Quantitative real-time RT-PCR (qRT-PCR) analysis of six paralogous pairs of 12 selected soybean HD-Zip genes under both drought and salinity stress confirmed their stress-inducible expression patterns. Conclusions This study presents a thorough overview of the soybean HD-Zip gene family and provides a new perspective on the evolution of this gene family. The results indicate that HD-Zip family genes may be involved in many plant responses to stress conditions. Additionally, this study provides a solid foundation for uncovering the biological roles of HD-Zip genes in soybean growth and development. PMID:24498296

A public platform for the verification of the phenotypic effect of candidate genes for resistance to aflatoxin accumulation and Aspergillus flavus infection in maize.

PubMed

Warburton, Marilyn L; Williams, William Paul; Hawkins, Leigh; Bridges, Susan; Gresham, Cathy; Harper, Jonathan; Ozkan, Seval; Mylroie, J Erik; Shan, Xueyan

2011-07-01

A public candidate gene testing pipeline for resistance to aflatoxin accumulation or Aspergillus flavus infection in maize is presented here. The pipeline consists of steps for identifying, testing, and verifying the association of selected maize gene sequences with resistance under field conditions. Resources include a database of genetic and protein sequences associated with the reduction in aflatoxin contamination from previous studies; eight diverse inbred maize lines for polymorphism identification within any maize gene sequence; four Quantitative Trait Loci (QTL) mapping populations and one association mapping panel, all phenotyped for aflatoxin accumulation resistance and associated phenotypes; and capacity for Insertion/Deletion (InDel) and SNP genotyping in the population(s) for mapping. To date, ten genes have been identified as possible candidate genes and put through the candidate gene testing pipeline, and results are presented here to demonstrate the utility of the pipeline.

Differential gene expression of wheat progeny with contrasting levels of transpiration efficiency.

PubMed

Xue, Gang-Ping; McIntyre, C Lynne; Chapman, Scott; Bower, Neil I; Way, Heather; Reverter, Antonio; Clarke, Bryan; Shorter, Ray

2006-08-01

High water use efficiency or transpiration efficiency (TE) in wheat is a desirable physiological trait for increasing grain yield under water-limited environments. The identification of genes associated with this trait would facilitate the selection for genotypes with higher TE using molecular markers. We performed an expression profiling (microarray) analysis of approximately 16,000 unique wheat ESTs to identify genes that were differentially expressed between wheat progeny lines with contrasting TE levels from a cross between Quarrion (high TE) and Genaro 81 (low TE). We also conducted a second microarray analysis to identify genes responsive to drought stress in wheat leaves. Ninety-three genes that were differentially expressed between high and low TE progeny lines were identified. One fifth of these genes were markedly responsive to drought stress. Several potential growth-related regulatory genes, which were down-regulated by drought, were expressed at a higher level in the high TE lines than the low TE lines and are potentially associated with a biomass production component of the Quarrion-derived high TE trait. Eighteen of the TE differentially expressed genes were further analysed using quantitative RT-PCR on a separate set of plant samples from those used for microarray analysis. The expression levels of 11 of the 18 genes were positively correlated with the high TE trait, measured as carbon isotope discrimination (Delta(13)C). These data indicate that some of these TE differentially expressed genes are candidates for investigating processes that underlie the high TE trait or for use as expression quantitative trait loci (eQTLs) for TE.

Interdependence of cell growth and gene expression: origins and consequences.

PubMed

Scott, Matthew; Gunderson, Carl W; Mateescu, Eduard M; Zhang, Zhongge; Hwa, Terence

2010-11-19

In bacteria, the rate of cell proliferation and the level of gene expression are intimately intertwined. Elucidating these relations is important both for understanding the physiological functions of endogenous genetic circuits and for designing robust synthetic systems. We describe a phenomenological study that reveals intrinsic constraints governing the allocation of resources toward protein synthesis and other aspects of cell growth. A theory incorporating these constraints can accurately predict how cell proliferation and gene expression affect one another, quantitatively accounting for the effect of translation-inhibiting antibiotics on gene expression and the effect of gratuitous protein expression on cell growth. The use of such empirical relations, analogous to phenomenological laws, may facilitate our understanding and manipulation of complex biological systems before underlying regulatory circuits are elucidated.

Toll-like receptor 6 and connective tissue growth factor are significantly upregulated in mitomycin-C-treated urothelial carcinoma cells under hydrostatic pressure stimulation.

PubMed

Chen, Shao-Kuan; Chung, Chih-Ang; Cheng, Yu-Che; Huang, Chi-Jung; Chen, Wen-Yih; Ruaan, Ruoh-Chyu; Li, Chuan; Tsao, Chia-Wen; Hu, Wei-Wen; Chien, Chih-Cheng

2014-06-01

Urothelial carcinoma (UC) is the most common histologic subtype of bladder cancer. The administration of mitomycin C (MMC) into the bladder after transurethral resection of the bladder tumor (TURBT) is a common treatment strategy for preventing recurrence after surgery. We previously applied hydrostatic pressure combined with MMC in UC cells and found that hydrostatic pressure synergistically enhanced MMC-induced UC cell apoptosis through the Fas/FasL pathways. To understand the alteration of gene expressions in UC cells caused by hydrostatic pressure and MMC, oligonucleotide microarray was used to explore all the differentially expressed genes. After bioinformatics analysis and gene annotation, Toll-like receptor 6 (TLR6) and connective tissue growth factor (CTGF) showed significant upregulation among altered genes, and their gene and protein expressions with each treatment of UC cells were validated by quantitative real-time PCR and immunoblotting. Under treatment with MMC and hydrostatic pressure, UC cells showed increasing apoptosis using extrinsic pathways through upregulation of TLR6 and CTGF.

Allelic-based gene-gene interaction associated with quantitative traits.

PubMed

Jung, Jeesun; Sun, Bin; Kwon, Deukwoo; Koller, Daniel L; Foroud, Tatiana M

2009-05-01

Recent studies have shown that quantitative phenotypes may be influenced not only by multiple single nucleotide polymorphisms (SNPs) within a gene but also by the interaction between SNPs at unlinked genes. We propose a new statistical approach that can detect gene-gene interactions at the allelic level which contribute to the phenotypic variation in a quantitative trait. By testing for the association of allelic combinations at multiple unlinked loci with a quantitative trait, we can detect the SNP allelic interaction whether or not it can be detected as a main effect. Our proposed method assigns a score to unrelated subjects according to their allelic combination inferred from observed genotypes at two or more unlinked SNPs, and then tests for the association of the allelic score with a quantitative trait. To investigate the statistical properties of the proposed method, we performed a simulation study to estimate type I error rates and power and demonstrated that this allelic approach achieves greater power than the more commonly used genotypic approach to test for gene-gene interaction. As an example, the proposed method was applied to data obtained as part of a candidate gene study of sodium retention by the kidney. We found that this method detects an interaction between the calcium-sensing receptor gene (CaSR), the chloride channel gene (CLCNKB) and the Na, K, 2Cl cotransporter gene (CLC12A1) that contributes to variation in diastolic blood pressure.

Genome-wide identification of expression quantitative trait loci for human telomerase.

PubMed

Kim, Hanseol; Ryu, Jihye; Lee, Chaeyoung

2016-10-01

A genome-wide association study was conducted to identify expression quantitative trait loci (eQTL) for human telomerase.We tested the genetic associations of nucleotide variants with expression of the genes encoding human telomerase reverse transcriptase (hTERT) and telomerase RNA components (TERC) in lymphoblastoid cell lines derived from 373 Europeans.Our results revealed 6 eQTLs associated with hTERT (P < 5 × 10). One eQTL (rs17755753) was located in the intron 1 of the gene encoding R-spondin-3 (RSPO3), a well-known Wnt signaling regulator. Transcriptome-wide association analysis for these eQTLs revealed their additional associations with the expression of 29 genes (P < 4.75 × 10), including prickle planar cell polarity protein 2 (PRICKLE2) gene important for the Wnt signaling pathway. This concurs with previous studies in which significant expressional relationships between hTERT and some genes (β-catenin and Wnt-3a) in the Wnt signaling pathway have been observed.This study suggested 6 novel eQTLs for hTERT and the association of hTERT with the Wnt signaling pathway. Further studies are needed to understand their underlying mechanisms to improve our understanding of the role of hTERT in cancer.

Selection and validation of reference genes for qRT-PCR analysis during biological invasions: The thermal adaptability of Bemisia tabaci MED.

PubMed

Dai, Tian-Mei; Lü, Zhi-Chuang; Liu, Wan-Xue; Wan, Fang-Hao

2017-01-01

The Bemisia tabaci Mediterranean (MED) cryptic species has been rapidly invading to most parts of the world owing to its strong ecological adaptability, which is considered as a model insect for stress tolerance studies under rapidly changing environments. Selection of a suitable reference gene for quantitative stress-responsive gene expression analysis based on qRT-PCR is critical for elaborating the molecular mechanisms of thermotolerance. To obtain accurate and reliable normalization data in MED, eight candidate reference genes (β-act, GAPDH, β-tub, EF1-α, GST, 18S, RPL13A and α-tub) were examined under various thermal stresses for varied time periods by using geNorm, NormFinder and BestKeeper algorithms, respectively. Our results revealed that β-tub and EF1-α were the best reference genes across all sample sets. On the other hand, 18S and GADPH showed the least stability for all the samples studied. β-act was proved to be highly stable only in case of short-term thermal stresses. To our knowledge this was the first comprehensive report on validation of reference genes under varying temperature stresses in MED. The study could expedite particular discovery of thermotolerance genes in MED. Further, the present results can form the basis of further research on suitable reference genes in this invasive insect and will facilitate transcript profiling in other invasive insects.

The banana E2 gene family: Genomic identification, characterization, expression profiling analysis.

PubMed

Dong, Chen; Hu, Huigang; Jue, Dengwei; Zhao, Qiufang; Chen, Hongliang; Xie, Jianghui; Jia, Liqiang

2016-04-01

The E2 is at the center of a cascade of Ub1 transfers, and it links activation of the Ub1 by E1 to its eventual E3-catalyzed attachment to substrate. Although the genome-wide analysis of this family has been performed in some species, little is known about analysis of E2 genes in banana. In this study, 74 E2 genes of banana were identified and phylogenetically clustered into thirteen subgroups. The predicted banana E2 genes were distributed across all 11 chromosomes at different densities. Additionally, the E2 domain, gene structure and motif compositions were analyzed. The expression of all of the banana E2 genes was analyzed in the root, stem, leaf, flower organs, five stages of fruit development and under abiotic stresses. All of the banana E2 genes, with the exception of few genes in each group, were expressed in at least one of the organs and fruit developments, which indicated that the E2 genes might involve in various aspects of the physiological and developmental processes of the banana. Quantitative RT-PCR (qRT-PCR) analysis identified that 45 E2s under drought and 33 E2s under salt were induced. To the best of our knowledge, this report describes the first genome-wide analysis of the banana E2 gene family, and the results should provide valuable information for understanding the classification, cloning and putative functions of this family. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Deciphering molecular circuits from genetic variation underlying transcriptional responsiveness to stimuli.

PubMed

Gat-Viks, Irit; Chevrier, Nicolas; Wilentzik, Roni; Eisenhaure, Thomas; Raychowdhury, Raktima; Steuerman, Yael; Shalek, Alex K; Hacohen, Nir; Amit, Ido; Regev, Aviv

2013-04-01

Individual genetic variation affects gene responsiveness to stimuli, often by influencing complex molecular circuits. Here we combine genomic and intermediate-scale transcriptional profiling with computational methods to identify variants that affect the responsiveness of genes to stimuli (responsiveness quantitative trait loci or reQTLs) and to position these variants in molecular circuit diagrams. We apply this approach to study variation in transcriptional responsiveness to pathogen components in dendritic cells from recombinant inbred mouse strains. We identify reQTLs that correlate with particular stimuli and position them in known pathways. For example, in response to a virus-like stimulus, a trans-acting variant responds as an activator of the antiviral response; using RNA interference, we identify Rgs16 as the likely causal gene. Our approach charts an experimental and analytic path to decipher the mechanisms underlying genetic variation in circuits that control responses to stimuli.

System-level modeling of acetone-butanol-ethanol fermentation.

PubMed

Liao, Chen; Seo, Seung-Oh; Lu, Ting

2016-05-01

Acetone-butanol-ethanol (ABE) fermentation is a metabolic process of clostridia that produces bio-based solvents including butanol. It is enabled by an underlying metabolic reaction network and modulated by cellular gene regulation and environmental cues. Mathematical modeling has served as a valuable strategy to facilitate the understanding, characterization and optimization of this process. In this review, we highlight recent advances in system-level, quantitative modeling of ABE fermentation. We begin with an overview of integrative processes underlying the fermentation. Next we survey modeling efforts including early simple models, models with a systematic metabolic description, and those incorporating metabolism through simple gene regulation. Particular focus is given to a recent system-level model that integrates the metabolic reactions, gene regulation and environmental cues. We conclude by discussing the remaining challenges and future directions towards predictive understanding of ABE fermentation. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Selection of suitable soybean EF1α genes as internal controls for real-time PCR analyses of tissues during plant development and under stress conditions.

PubMed

Saraiva, Kátia D C; Fernandes de Melo, Dirce; Morais, Vanessa D; Vasconcelos, Ilka M; Costa, José H

2014-09-01

The EF1α genes were stable in the large majority of soybean tissues during development and in specific tissues/conditions under stress. Quantitative real-time PCR (qPCR) analysis strongly depends on transcript normalization using stable reference genes. Reference genes are generally encoded by multigene families and are used in qPCR normalization; however, little effort has been made to verify the stability of different gene members within a family. Here, the expression stability of members of the soybean EF1α gene family (named EF1α 1a1, 1a2, 1b, 2a, 2b and 3) was evaluated in different tissues during plant development and stress exposure (SA and PEG). Four genes (UKN1, SKIP 16, EF1β and MTP) already established as stably expressed were also used in the comparative analysis. GeNorm analyses revealed different combinations of reference genes as stable in soybean tissues during development. The EF1α genes were the most stable in cotyledons (EF1α 3 and EF1α 1b), epicotyls (EF1α 1a2, EF1α 2b and EF1α 1a1), hypocotyls (EF1α 1a1 and EF1β), pods (EF1α 2a and EF1α 2b) and roots (EF1α 2a and UKN1) and less stable in tissues such as trifoliate and unifoliate leaves and germinating seeds. Under stress conditions, no suitable combination including only EF1α genes was found; however, some genes were relatively stable in leaves (EF1α 1a2) and roots (EF1α 1a1) treated with SA as well as in roots treated with PEG (EF1α 2b). EF1α 2a was the most stably expressed EF1α gene in all soybean tissues under stress. Taken together, our data provide guidelines for the selection of EF1α genes for use as reference genes in qPCR expression analyses during plant development and under stress conditions.

Computational Identification and Analysis of Signaling Subnetworks with Distinct Functional Roles in the Regulation of TNF Production

DTIC Science & Technology

2016-01-04

extract four distinct quantitative features of response timing and intensity: the trajectory peak height, the peak time, the area under the curve, and...J., 2007, 21, 325–332. 16 S. L. Werner, J. D. Kearns, V. Zadorozhnaya, C. Lynch, E. O’Dea, M. P. Boldin , A. Ma, D. Baltimore and A. Hoffmann, Genes...Werner, J. D. Kearns, V. Zadorozhnaya, C. Lynch, E. O’Dea, M. P. Boldin , A. Ma, D. Baltimore and A. Hoffmann, Genes Dev., 2008, 22, 2093–2101. 58 J

Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae).

PubMed

Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

2015-01-01

The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

A R2R3-MYB transcription factor gene in common wheat (namely TaMYBsm1) involved in enhancement of drought tolerance in transgenic Arabidopsis.

PubMed

Li, Meng-Jun; Qiao, Yu; Li, Ya-Qing; Shi, Zhan-Liang; Zhang, Nan; Bi, Cai-Li; Guo, Jin-Kao

2016-11-01

We isolated the TaMYBsm1 genes, encoding R2R3-type MYB proteins in common wheat, aimed to uncover the possible molecular mechanisms related to drought response. The TaMYBsm1 genes, TaMYBsm1-A, TaMYBsm1-B and TaMYBsm1-D, were isolated and analyzed from the common wheat cultivar Shimai 15. Their expression patterns under PEG 6000 and mannitol were monitored by semi-quantitative RT-PCR and β-glucuronidase (Gus) assay. The function of TaMYBsm1-D under drought stress in transgenic Arabidopsis plants was investigated, and the germination rate, water loss rate, as well as the proline and malondialdehyde (MDA) content were compared with that in wild type (WT) plants. The expression of three downstream genes (DREB2A, P5CS1 and RD29A) in TaMYBsm1-D transgenic plants was analyzed. The R2R3-MYB domains of the MYBsm1 proteins were highly conserved in plants. In addition, the TaMYBsm1 proteins were targeted to the nucleus and contained transcriptional activation domains (TADs). Gus assay and semi-quantitative RT-PCR analysis demonstrated that the TaMYBsm1 genes were up-regulated when the wheat was treated by PEG and mannitol. Compared with WT plants, the germination rates were much higher, but the water loss rates were much lower in TaMYBsm1-D overexpression plants. TaMYBsm1-D transgenic plants showed distinct higher proline contents but a lower MDA content than the WT plants. The three downstream genes were highly expressed in TaMYBsm1-D transgenic plants. We concluded from these results that TaMYBsm1 genes play an important role in plant drought stress tolerance through up-regulation of DREB2A, P5CS1 and RD29A. The increase of proline content and decrease of MDA content may also be involved in the drought response.

Widespread genetic linkage of mating signals and preferences in the Hawaiian cricket Laupala

PubMed Central

Wiley, Chris; Ellison, Christopher K.; Shaw, Kerry L.

2012-01-01

The evolution of novel sexual communication systems is integral to the process of speciation, as it discourages gene flow between incipient species. Physical linkage between genes underlying male–female communication (i.e. sexual signals and preferences for them) facilitates both rapid and coordinated divergence of sexual communication systems between populations and reduces recombination in the face of occasional hybridization between diverging populations. Despite these ramifications of the genetic architecture of sexual communication for sexual selection and speciation, few studies have examined this relationship empirically. Previous studies of the closely related Hawaiian crickets Laupala paranigra and Laupala kohalensis have indirectly suggested that many of the genes underlying the difference in pulse rate of male song are physically linked with genes underlying the difference in female preference for pulse rate. Using marker-assisted introgression, we moved ‘slow pulse rate’ alleles from L. paranigra at five known quantitative trait loci (QTL) underlying male pulse rate into the ‘fast pulse rate’ genetic background of L. kohalensis and assessed the effect of these loci on female preference. An astounding four out of five song QTL predicted the preferences of female fourth-generation backcrosses, providing direct evidence for the extensive genetic linkage of song and preference in one of the fastest diversifying genera currently known. PMID:21957135

Mapping, fine mapping, and molecular dissection of quantitative trait Loci in domestic animals.

PubMed

Georges, Michel

2007-01-01

Artificial selection has created myriad breeds of domestic animals, each characterized by unique phenotypes pertaining to behavior, morphology, physiology, and disease. Most domestic animal populations share features with isolated founder populations, making them well suited for positional cloning. Genome sequences are now available for most domestic species, and with them a panoply of tools including high-density single-nucleotide polymorphism panels. As a result, domestic animal populations are becoming invaluable resources for studying the molecular architecture of complex traits and of adaptation. Here we review recent progress and issues in the positional identification of genes underlying complex traits in domestic animals. As many phenotypes studied in animals are quantitative, we focus on mapping, fine mapping, and cloning of quantitative trait loci.

Hypoxia triggers angiogenesis by increasing expression of LOX genes in 3-D culture of ASCs and ECs.

PubMed

Xie, Qiang; Xie, Jiamin; Tian, Taoran; Ma, Quanquan; Zhang, Qi; Zhu, Bofeng; Cai, Xiaoxiao

2017-03-01

This study aimed to investigate the expression changes of LOX (lysyl oxidase) family genes, VEGFA, and VEGFB under hypoxic conditions in a co-culture system of ASCs (adipose-derived stromal cells) and ECs (endothelial cells). ASCs and ECs were co-cultured under hypoxic and normal oxygen conditions in a 1:1 ratio, and the formation of vessel-like was detected at 7 days. The transwell co-culture system was used and cell lysates were collected at 7 days after co-culture in hypoxic and normal oxygen condition. Semi-quantitative PCR was performed to quantify the mRNA expression of VEGFA, VEGFB, and the LOX genes (LOX, LOXL-1, LOXL-2, LOXL-3, and LOXL-4). Expression changes were determined by densitomery. Enhanced angiogenesis was detected in the co-culture of ASCs and ECs under hypoxic conditions. Among the genes screened, VEGFA, VEGFB, LOXL-1, and LOXL-3 in ECs, both mono-cultured and co-cultured, were significantly enhanced after culturing under hypoxic conditions. In ASCs, VEGFB, LOXL-1, and LOXL-3 were upregulated. Contact co-culture between ASCs and ECs promotes angiogenesis under hypoxia. LOXL-1 and LOXL-3 expressions were increased in both ASCs and ECs and might play important roles in the enhanced angiogenesis promoted by hypoxia. Copyright © 2017 Elsevier Inc. All rights reserved.

Genome-Wide Identification and Expression Profiling Analysis of ZmPIN, ZmPILS, ZmLAX and ZmABCB Auxin Transporter Gene Families in Maize (Zea mays L.) under Various Abiotic Stresses

PubMed Central

Sun, Tao; Zhang, Lei; Yang, Yanjun; Qi, Jianshuang; Yan, Shufeng; Han, Xiaohua; Wang, Huizhong; Shen, Chenjia

2015-01-01

The auxin influx carriers auxin resistant 1/like aux 1 (AUX/LAX), efflux carriers pin-formed (PIN) (together with PIN-like proteins) and efflux/conditional P-glycoprotein (ABCB) are major protein families involved in auxin polar transport. However, how they function in responses to exogenous auxin and abiotic stresses in maize is largely unknown. In this work, the latest updated maize (Zea mays L.) reference genome sequence was used to characterize and analyze the ZmLAX, ZmPIN, ZmPILS and ZmABCB family genes from maize. The results showed that five ZmLAXs, fifteen ZmPINs, nine ZmPILSs and thirty-five ZmABCBs were mapped on all ten maize chromosomes. Highly diversified gene structures, nonconservative transmembrane helices and tissue-specific expression patterns suggested the possibility of function diversification for these genes. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the expression patterns of ZmLAX, ZmPIN, ZmPILS and ZmABCB genes under exogenous auxin and different environmental stresses. The expression levels of most ZmPIN, ZmPILS, ZmLAX and ZmABCB genes were induced in shoots and were reduced in roots by various abiotic stresses (drought, salt and cold stresses). The opposite expression response patterns indicated the dynamic auxin transport between shoots and roots under abiotic stresses. Analysis of the expression patterns of ZmPIN, ZmPILS, ZmLAX and ZmABCB genes under drought, salt and cold treatment may help us to understand the possible roles of maize auxin transporter genes in responses and tolerance to environmental stresses. PMID:25742625

Simulating the Yield Impacts of Organ-Level Quantitative Trait Loci Associated With Drought Response in Maize: A “Gene-to-Phenotype” Modeling Approach

PubMed Central

Chenu, Karine; Chapman, Scott C.; Tardieu, François; McLean, Greg; Welcker, Claude; Hammer, Graeme L.

2009-01-01

Under drought, substantial genotype–environment (G × E) interactions impede breeding progress for yield. Identifying genetic controls associated with yield response is confounded by poor genetic correlations across testing environments. Part of this problem is related to our inability to account for the interplay of genetic controls, physiological traits, and environmental conditions throughout the crop cycle. We propose a modeling approach to bridge this “gene-to-phenotype” gap. For maize under drought, we simulated the impact of quantitative trait loci (QTL) controlling two key processes (leaf and silk elongation) that influence crop growth, water use, and grain yield. Substantial G × E interaction for yield was simulated for hypothetical recombinant inbred lines (RILs) across different seasonal patterns of drought. QTL that accelerated leaf elongation caused an increase in crop leaf area and yield in well-watered or preflowering water deficit conditions, but a reduction in yield under terminal stresses (as such “leafy” genotypes prematurely exhausted the water supply). The QTL impact on yield was substantially enhanced by including pleiotropic effects of these QTL on silk elongation and on consequent grain set. The simulations obtained illustrated the difficulty of interpreting the genetic control of yield for genotypes influenced only by the additive effects of QTL associated with leaf and silk growth. The results highlight the potential of integrative simulation modeling for gene-to-phenotype prediction and for exploiting G × E interactions for complex traits such as drought tolerance. PMID:19786622

New animal models reveal that coenzyme Q2 (Coq2) and placenta-specific 8 (Plac8) are candidate genes for the onset of type 2 diabetes associated with obesity in rats.

PubMed

Sasaki, Daiki; Kotoh, Jun; Watadani, Risa; Matsumoto, Kozo

2015-12-01

Obesity is a major risk factor for the onset of type 2 diabetes; however, little is known about the gene(s) involved. Therefore, we developed new animal models of obesity to search for diabetogenic genes associated with obesity. We generated double congenic rat strains with a hyperglycaemic quantitative trait locus (QTL) derived from the Otsuka Long-Evans Tokushima Fatty rat and a fa/fa (Lepr-/-) locus derived from the Zucker Fatty rat; phenotypic analysis for plasma glucose and insulin levels and RNA and protein levels were determined using reverse transcription quantitative PCR and Western blotting analyses, respectively. The double congenic strain F344-fa-nidd2 (Lepr-/- and Nidd2/of) exhibited significantly higher glucose levels and significantly lower hypoglycaemic response to insulin than the obese control strain F344-fa (Lepr-/-). These phenotypes were clearly observed in the obese strains but not in the lean strains. These results indicate that the Nidd2/of locus harbours a diabetogenic gene associated with obesity. We measured the expression of 60 genes in the Nidd2/of QTL region between the strains and found that the mRNA expression levels of five genes were significantly different between the strains under the condition of obesity. However, three of the five genes were differentially expressed in both obese and lean rats, indicating that these genes are not specific for the condition of obesity. Conversely, the other two genes, coenzyme Q2 (Coq2) and placenta-specific 8 (Plac8), were differentially expressed only in the obese rats, suggesting that these two genes are candidates for the onset of type 2 diabetes associated with obesity in rats.

EPS-LASSO: Test for High-Dimensional Regression Under Extreme Phenotype Sampling of Continuous Traits.

PubMed

Xu, Chao; Fang, Jian; Shen, Hui; Wang, Yu-Ping; Deng, Hong-Wen

2018-01-25

Extreme phenotype sampling (EPS) is a broadly-used design to identify candidate genetic factors contributing to the variation of quantitative traits. By enriching the signals in extreme phenotypic samples, EPS can boost the association power compared to random sampling. Most existing statistical methods for EPS examine the genetic factors individually, despite many quantitative traits have multiple genetic factors underlying their variation. It is desirable to model the joint effects of genetic factors, which may increase the power and identify novel quantitative trait loci under EPS. The joint analysis of genetic data in high-dimensional situations requires specialized techniques, e.g., the least absolute shrinkage and selection operator (LASSO). Although there are extensive research and application related to LASSO, the statistical inference and testing for the sparse model under EPS remain unknown. We propose a novel sparse model (EPS-LASSO) with hypothesis test for high-dimensional regression under EPS based on a decorrelated score function. The comprehensive simulation shows EPS-LASSO outperforms existing methods with stable type I error and FDR control. EPS-LASSO can provide a consistent power for both low- and high-dimensional situations compared with the other methods dealing with high-dimensional situations. The power of EPS-LASSO is close to other low-dimensional methods when the causal effect sizes are small and is superior when the effects are large. Applying EPS-LASSO to a transcriptome-wide gene expression study for obesity reveals 10 significant body mass index associated genes. Our results indicate that EPS-LASSO is an effective method for EPS data analysis, which can account for correlated predictors. The source code is available at https://github.com/xu1912/EPSLASSO. hdeng2@tulane.edu. Supplementary data are available at Bioinformatics online. © The Author (2018). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Deploying QTL-seq for rapid delineation of a potential candidate gene underlying major trait-associated QTL in chickpea

PubMed Central

Das, Shouvik; Upadhyaya, Hari D.; Bajaj, Deepak; Kujur, Alice; Badoni, Saurabh; Laxmi; Kumar, Vinod; Tripathi, Shailesh; Gowda, C. L. Laxmipathi; Sharma, Shivali; Singh, Sube; Tyagi, Akhilesh K.; Parida, Swarup K.

2015-01-01

A rapid high-resolution genome-wide strategy for molecular mapping of major QTL(s)/gene(s) regulating important agronomic traits is vital for in-depth dissection of complex quantitative traits and genetic enhancement in chickpea. The present study for the first time employed a NGS-based whole-genome QTL-seq strategy to identify one major genomic region harbouring a robust 100-seed weight QTL using an intra-specific 221 chickpea mapping population (desi cv. ICC 7184 × desi cv. ICC 15061). The QTL-seq-derived major SW QTL (CaqSW1.1) was further validated by single-nucleotide polymorphism (SNP) and simple sequence repeat (SSR) marker-based traditional QTL mapping (47.6% R2 at higher LOD >19). This reflects the reliability and efficacy of QTL-seq as a strategy for rapid genome-wide scanning and fine mapping of major trait regulatory QTLs in chickpea. The use of QTL-seq and classical QTL mapping in combination narrowed down the 1.37 Mb (comprising 177 genes) major SW QTL (CaqSW1.1) region into a 35 kb genomic interval on desi chickpea chromosome 1 containing six genes. One coding SNP (G/A)-carrying constitutive photomorphogenic9 (COP9) signalosome complex subunit 8 (CSN8) gene of these exhibited seed-specific expression, including pronounced differential up-/down-regulation in low and high seed weight mapping parents and homozygous individuals during seed development. The coding SNP mined in this potential seed weight-governing candidate CSN8 gene was found to be present exclusively in all cultivated species/genotypes, but not in any wild species/genotypes of primary, secondary and tertiary gene pools. This indicates the effect of strong artificial and/or natural selection pressure on target SW locus during chickpea domestication. The proposed QTL-seq-driven integrated genome-wide strategy has potential to delineate major candidate gene(s) harbouring a robust trait regulatory QTL rapidly with optimal use of resources. This will further assist us to extrapolate the molecular mechanism underlying complex quantitative traits at a genome-wide scale leading to fast-paced marker-assisted genetic improvement in diverse crop plants, including chickpea. PMID:25922536

[Quantitative changes of main components of erythrocyte membranes which define architectonics of cells under pttg gene knockout].

PubMed

Kaniuka, O P; Filiak, Ie Z; Kulachkovs'kyĭ, O R; Osyp, Iu L; Sybirna, N O

2014-01-01

A pttg gene knockout affects the functional state of erythron in mice which could be associated with structural changes in the structure of erythrocyte membranes. The pttg gene knockout causes a significant modification of fatty acids composition of erythrocyte membrane lipids by reducing the content of palmitic acid and increasing of polyunsaturated fatty acids amount by 18%. Analyzing the erythrocyte surface architectonics of mice under pttg gene knockout, it was found that on the background of reduction of the functionally complete biconcave discs population one could observe an increase of the number of transformed cells at different degeneration stages. Researches have shown that in mice with a pttg gene knockout compared with a control group of animals cytoskeletal protein--beta-spectrin was reduced by 17.03%. However, there is a reduction of membrane protein band 3 by 33.04%, simultaneously the content of anion transport protein band 4.5 increases by 35.2% and protein band 4.2 by 32.1%. The lectin blot analysis has helped to reveal changes in the structure of the carbohydrate determinants of erythrocyte membrane glycoproteins under conditions of directed pttg gene inactivation, accompanied by changes in the type of communication, which joins the terminal residue in carbohydrate determinant of glycoproteins. Thus, a significant redistribution of protein and fatty acids contents in erythrocyte membranes that manifested in the increase of the deformed shape of red blood cells is observed underpttg gene knockout.

Genome-Wide Association Studies of Quantitatively Measured Skin, Hair, and Eye Pigmentation in Four European Populations

PubMed Central

Candille, Sophie I.; Absher, Devin M.; Beleza, Sandra; Bauchet, Marc; McEvoy, Brian; Garrison, Nanibaa’ A.; Li, Jun Z.; Myers, Richard M.; Barsh, Gregory S.; Tang, Hua; Shriver, Mark D.

2012-01-01

Pigmentation of the skin, hair, and eyes varies both within and between human populations. Identifying the genes and alleles underlying this variation has been the goal of many candidate gene and several genome-wide association studies (GWAS). Most GWAS for pigmentary traits to date have been based on subjective phenotypes using categorical scales. But skin, hair, and eye pigmentation vary continuously. Here, we seek to characterize quantitative variation in these traits objectively and accurately and to determine their genetic basis. Objective and quantitative measures of skin, hair, and eye color were made using reflectance or digital spectroscopy in Europeans from Ireland, Poland, Italy, and Portugal. A GWAS was conducted for the three quantitative pigmentation phenotypes in 176 women across 313,763 SNP loci, and replication of the most significant associations was attempted in a sample of 294 European men and women from the same countries. We find that the pigmentation phenotypes are highly stratified along axes of European genetic differentiation. The country of sampling explains approximately 35% of the variation in skin pigmentation, 31% of the variation in hair pigmentation, and 40% of the variation in eye pigmentation. All three quantitative phenotypes are correlated with each other. In our two-stage association study, we reproduce the association of rs1667394 at the OCA2/HERC2 locus with eye color but we do not identify new genetic determinants of skin and hair pigmentation supporting the lack of major genes affecting skin and hair color variation within Europe and suggesting that not only careful phenotyping but also larger cohorts are required to understand the genetic architecture of these complex quantitative traits. Interestingly, we also see that in each of these four populations, men are more lightly pigmented in the unexposed skin of the inner arm than women, a fact that is underappreciated and may vary across the world. PMID:23118974

Identification and Evaluation of Reliable Reference Genes in the Medicinal Fungus Shiraia bambusicola.

PubMed

Song, Liang; Li, Tong; Fan, Li; Shen, Xiao-Ye; Hou, Cheng-Lin

2016-04-01

The stability of reference genes plays a vital role in real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, which is generally regarded as a convenient and sensitive tool for the analysis of gene expression. A well-known medicinal fungus, Shiraia bambusicola, has great potential in the pharmaceutical, agricultural and food industries, but its suitable reference genes have not yet been determined. In the present study, 11 candidate reference genes in S. bambusicola were first evaluated and validated comprehensively. To identify the suitable reference genes for qRT-PCR analysis, three software-based algorithms, geNorm, NormFinder and Best Keeper, were applied to rank the tested genes. RNA samples were collected from seven fermentation stages using different media (potato dextrose or Czapek medium) and under different light conditions (12-h light/12-h dark and all-dark). The three most appropriate reference genes, ubi, tfc and ags, were able to normalize the qRT-PCR results under the culturing conditions of 12-h light/12-h dark, whereas the other three genes, vac, gke and acyl, performed better in the culturing conditions of all-dark growth. Therefore, under different light conditions, at least two reference genes (ubi and vac) could be employed to assure the reliability of qRT-PCR results. For both the natural culture medium (the most appropriate genes of this group: ubi, tfc and ags) and the chemically defined synthetic medium (the most stable genes of this group: tfc, vac and ef), the tfc gene remained the best gene used for normalizing the gene expression found with qRT-PCR. It is anticipated that these results would improve the selection of suitable reference genes for qRT-PCR assays and lay the foundation for an accurate analysis of gene expression in S. bambusicola.

A comparative study of quantitative immunohistochemistry and quantum dot immunohistochemistry for mutation carrier identification in Lynch syndrome.

PubMed

Barrow, Emma; Evans, D Gareth; McMahon, Ray; Hill, James; Byers, Richard

2011-03-01

Lynch Syndrome is caused by mutations in DNA mismatch repair (MMR) genes. Mutation carrier identification is facilitated by immunohistochemical detection of the MMR proteins MHL1 and MSH2 in tumour tissue and is desirable as colonoscopic screening reduces mortality. However, protein detection by conventional immunohistochemistry (IHC) is subjective, and quantitative techniques are required. Quantum dots (QDs) are novel fluorescent labels that enable quantitative multiplex staining. This study compared their use with quantitative 3,3'-diaminobenzidine (DAB) IHC for the diagnosis of Lynch Syndrome. Tumour sections from 36 mutation carriers and six controls were obtained. These were stained with DAB on an automated platform using antibodies against MLH1 and MSH2. Multiplex QD immunofluorescent staining of the sections was performed using antibodies against MLH1, MSH2 and smooth muscle actin (SMA). Multispectral analysis of the slides was performed. The staining intensity of DAB and QDs was measured in multiple colonic crypts, and the mean intensity scores calculated. Receiver operating characteristic (ROC) curves of staining performance for the identification of mutation carriers were evaluated. For quantitative DAB IHC, the area under the MLH1 ROC curve was 0.872 (95% CI 0.763 to 0.981), and the area under the MSH2 ROC curve was 0.832 (95% CI 0.704 to 0.960). For quantitative QD IHC, the area under the MLH1 ROC curve was 0.812 (95% CI 0.681 to 0.943), and the area under the MSH2 ROC curve was 0.598 (95% CI 0.418 to 0.777). Despite the advantage of QD staining to enable several markers to be measured simultaneously, it is of lower utility than DAB IHC for the identification of MMR mutation carriers. Automated DAB IHC staining and quantitative slide analysis may enable high-throughput IHC.

Selection of Reference Genes for Expression Studies of Xenobiotic Adaptation in Tetranychus urticae.

PubMed

Morales, Mariany Ashanty; Mendoza, Bianca Marie; Lavine, Laura Corley; Lavine, Mark Daniel; Walsh, Douglas Bruce; Zhu, Fang

2016-01-01

Quantitative real-time PCR (qRT-PCR) is an extensively used, high-throughput method to analyze transcriptional expression of genes of interest. An appropriate normalization strategy with reliable reference genes is required for calculating gene expression across diverse experimental conditions. In this study, we aim to identify the most stable reference genes for expression studies of xenobiotic adaptation in Tetranychus urticae, an extremely polyphagous herbivore causing significant yield reduction of agriculture. We chose eight commonly used housekeeping genes as candidates. The qRT-PCR expression data for these genes were evaluated from seven populations: a susceptible and three acaricide resistant populations feeding on lima beans, and three other susceptible populations which had been shifted host from lima beans to three other plant species. The stability of the candidate reference genes was then assessed using four different algorithms (comparative ΔCt method, geNorm, NormFinder, and BestKeeper). Additionally, we used an online web-based tool (RefFinder) to assign an overall final rank for each candidate gene. Our study found that CycA and Rp49 are best for investigating gene expression in acaricide susceptible and resistant populations. GAPDH, Rp49, and Rpl18 are best for host plant shift studies. And GAPDH and Rp49 were the most stable reference genes when investigating gene expression under changes in both experimental conditions. These results will facilitate research in revealing molecular mechanisms underlying the xenobiotic adaptation of this notorious agricultural pest.

Selection of Reference Genes for Expression Studies of Xenobiotic Adaptation in Tetranychus urticae

PubMed Central

Morales, Mariany Ashanty; Mendoza, Bianca Marie; Lavine, Laura Corley; Lavine, Mark Daniel; Walsh, Douglas Bruce; Zhu, Fang

2016-01-01

Quantitative real-time PCR (qRT-PCR) is an extensively used, high-throughput method to analyze transcriptional expression of genes of interest. An appropriate normalization strategy with reliable reference genes is required for calculating gene expression across diverse experimental conditions. In this study, we aim to identify the most stable reference genes for expression studies of xenobiotic adaptation in Tetranychus urticae, an extremely polyphagous herbivore causing significant yield reduction of agriculture. We chose eight commonly used housekeeping genes as candidates. The qRT-PCR expression data for these genes were evaluated from seven populations: a susceptible and three acaricide resistant populations feeding on lima beans, and three other susceptible populations which had been shifted host from lima beans to three other plant species. The stability of the candidate reference genes was then assessed using four different algorithms (comparative ΔCt method, geNorm, NormFinder, and BestKeeper). Additionally, we used an online web-based tool (RefFinder) to assign an overall final rank for each candidate gene. Our study found that CycA and Rp49 are best for investigating gene expression in acaricide susceptible and resistant populations. GAPDH, Rp49, and Rpl18 are best for host plant shift studies. And GAPDH and Rp49 were the most stable reference genes when investigating gene expression under changes in both experimental conditions. These results will facilitate research in revealing molecular mechanisms underlying the xenobiotic adaptation of this notorious agricultural pest. PMID:27570487

A microarray analysis of potential genes underlying the neurosensitivity of mice to propofol.

PubMed

Lowes, Damon A; Galley, Helen F; Lowe, Peter R; Rikke, Brad A; Johnson, Thomas E; Webster, Nigel R

2005-09-01

Establishing the mechanism of action of general anesthetics at the molecular level is difficult because of the multiple targets with which these drugs are associated. Inbred short sleep (ISS) and long sleep (ILS) mice are differentially sensitive in response to ethanol and other sedative hypnotics and contain a single quantitative trait locus (Lorp1) that accounts for the genetic variance of loss-of-righting reflex in response to propofol (LORP). In this study, we used high-density oligonucleotide microarrays to identify global gene expression and candidate genes differentially expressed within the Lorp1 region that may give insight into the molecular mechanism underlying LORP. Microarray analysis was performed using Affymetrix MG-U74Av2 Genechips and a selection of differentially expressed genes was confirmed by semiquantitative reverse transcription-polymerase chain reaction. Global expression in the brains of ILS and ISS mice revealed 3423 genes that were significantly expressed, of which 139 (4%) were differentially expressed. Analysis of genes located within the Lorp1 region showed that 26 genes were significantly expressed and that just 2 genes (7%) were differentially expressed. These genes encoded for the proteins AWP1 (associated with protein kinase 1) and "BTB (POZ) domain containing 1," whose functions are largely uncharacterized. Genes differentially expressed outside Lorp1 included seven genes with previously characterized neuronal functions and thus stand out as additional candidate genes that may be involved in mediating the neurosensitivity differences between ISS and ILS.

Selection of reference genes for expression analysis of Kumamoto and Portuguese oysters and their hybrid

NASA Astrophysics Data System (ADS)

Yan, Lulu; Su, Jiaqi; Wang, Zhaoping; Yan, Xiwu; Yu, Ruihai

2017-12-01

Quantitative real-time polymerase chain reaction (qRT-PCR) is a rapid and reliable technique which has been widely used to quantifying gene transcripts (expression analysis). It is also employed for studying heterosis, hybridization breeding and hybrid tolerability of oysters, an ecologically and economically important taxonomic group. For these studies, selection of a suitable set of housekeeping genes as references is crucial for correct interpretation of qRT-PCR data. To identify suitable reference genes for oysters during low temperature and low salinity stresses, we analyzed twelve genes from the gill tissue of Crassostrea sikamea (SS), Crassostrea angulata (AA) and their hybrid (SA), which included three ribosomal genes, 28S ribosomal protein S5 ( RPS5), ribosomal protein L35 ( RPL35), and 60S ribosomal protein L29 ( RPL29); three structural genes, tubulin gamma ( TUBγ), annexin A6 and A7 ( AA6 and AA7); three metabolic pathway genes, ornithine decarboxylase ( OD), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) and glutathione S-transferase P1 ( GSP); two transcription factors, elongation factor 1 alpha and beta ( EF1α and EF1β); and one protein synthesis gene (ubiquitin ( UBQ). Primers specific for these genes were successfully developed for the three groups of oysters. Three different algorithms, geNorm, NormFinder and BestKeeper, were used to evaluate the expression stability of these candidate genes. BestKeeper program was found to be the most reliable. Based on our analysis, we found that the expression of RPL35 and EF1α was stable under low salinity stress, and the expression of OD, GAPDH and EF1α was stable under low temperature stress in hybrid (SA) oyster; the expression of RPS5 and GAPDH was stable under low salinity stress, and the expression of RPS5, UBQ, GAPDH was stable under low temperature stress in SS oyster; the expression of RPS5, GAPDH, EF1β and AA7 was stable under low salinity stress, and the expression of RPL35, EF1α, GAPDH and EF1β was stable under low temperature stress in AA oyster. Furthermore, to evaluate their suitability, the reference genes were used to quantify six target genes. In conclusion, we have successfully developed primers appropriate for the expression analysis in SS, SA and AA.

[Differential gene expression profile in ischemic myocardium of Wistar rats with acute myocardial infarction: the study on gene construction, identification and function].

PubMed

Guo, Chun Yu; Yin, Hui Jun; Jiang, Yue Rong; Xue, Mei; Zhang, Lu; Shi, Da Zhuo

2008-06-18

To construct the differential genes expressed profile in the ischemic myocardium tissue reduced from acute myocardial infarction(AMI), and determine the biological functions of target genes. AMI model was generated by ligation of the left anterior descending coronary artery in Wistar rats. Total RNA was extracted from the normal and the ischemic heart tissues under the ligation point 7 days after the operation. Differential gene expression profiles of the two samples were constructed using Long Serial Analysis of Gene Expression(LongSAGE). Real time fluorescence quantitative PCR was used to verify gene expression profile and to identify the expression of 2 functional genes. The activities of enzymes from functional genes were determined by histochemistry. A total of 15,966 tags were screened from the normal and the ischemic LongSAGE maps. The similarities of the sequences were compared using the BLAST algebra in NCBI and 7,665 novel tags were found. In the ischemic tissue 142 genes were significantly changed compared with those in the normal tissue (P<0.05). These differentially expressed genes represented the proteins which might play important roles in the pathways of oxidation and phosphorylation, ATP synthesis and glycolysis. The partial genes identified by LongSAGE were confirmed using real time fluorescence quantitative PCR. Two genes related to energy metabolism, COX5a and ATP5e, were screened and quantified. Expression of two functional genes down-regulated at their mRNA levels and the activities of correlative functional enzymes decreased compared with those in the normal tissue. AMI causes a series of changes in gene expression, in which the abnormal expression of genes related to energy metabolism could be one of the molecular mechanisms of AMI. The intervention of the expressions of COX5a and ATP5e may be a new target for AMI therapy.

Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction.

PubMed

Tetteh, Antonia Y; Sun, Katherine H; Hung, Chiu-Yueh; Kittur, Farooqahmed S; Ibeanu, Gordon C; Williams, Daniel; Xie, Jiahua

2014-01-01

Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se(0)), but the mechanism on how bacterial cells reduce selenite at molecular level is still not clear. We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3) treatment and then used quantitative real-time PCR (qRT-PCR) to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys) biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼ 50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼ 30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes (fdnG, fdoG, and fdhF) whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S) metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process.

Biochemical characteristics and gene expression profiles of two paralogous luciferases from the Japanese firefly Pyrocoelia atripennis (Coleoptera, Lampyridae, Lampyrinae): insight into the evolution of firefly luciferase genes.

PubMed

Bessho-Uehara, Manabu; Konishi, Kaori; Oba, Yuichi

2017-08-09

Two paralogous genes of firefly luciferase, Luc1 and Luc2, have been isolated from the species in two subfamilies, Luciolinae and Photurinae, of the family Lampyridae. The gene expression profiles have previously been examined only in the species of Luciolinae. Here we isolated Luc1 and Luc2 genes from the Japanese firefly Pyrocoelia atripennis. This is the first report of the presence of both Luc1 and Luc2 genes in the species of the subfamily Lampyrinae and of the exon-intron structure of Luc2 in the family Lampyridae. The luminescence of both gene products peaked at 547 nm under neutral buffer conditions, and the spectrum of Luc1, but not Luc2, was red-shifted under acidic conditions, as observed for Luc2 in the Luciolinae species. The semi-quantitative reverse transcription-polymerase chain reaction suggested that Luc1 was expressed in lanterns of all the stages except eggs, while Luc2 was expressed in the non-lantern bodies of eggs, prepupae, pupae, and female adults. These expression profiles are consistent with those in the Luciolinae species. Considering the distant phylogenetic relationship between Lampyrinae and Luciolinae in Lampyridae, we propose that fireflies generally possess two different luciferase genes and the biochemical properties and gene expression profiles for each paralog are conserved among lampyrid species.

Quantitative trait locus mapping of drought and salt tolerance in as introgressed recombinant inbred line population of upland cotton under the greenhouse and feild conditions

USDA-ARS?s Scientific Manuscript database

Drought and salt tolerances are complex traits and controlled by multiple genes, environmental factors and their interactions. Drought and salt stresses can result in more than 50% yield loss in Upland cotton (Gossypium hirsutum L.). G. barbadense L. (the source of Pima cotton) carries desirable tra...

HpQTL: a geometric morphometric platform to compute the genetic architecture of heterophylly.

PubMed

Sun, Lidan; Wang, Jing; Zhu, Xuli; Jiang, Libo; Gosik, Kirk; Sang, Mengmeng; Sun, Fengsuo; Cheng, Tangren; Zhang, Qixiang; Wu, Rongling

2017-02-15

Heterophylly, i.e. morphological changes in leaves along the axis of an individual plant, is regarded as a strategy used by plants to cope with environmental change. However, little is known of the extent to which heterophylly is controlled by genes and how each underlying gene exerts its effect on heterophyllous variation. We described a geometric morphometric model that can quantify heterophylly in plants and further constructed an R-based computing platform by integrating this model into a genetic mapping and association setting. The platform, named HpQTL, allows specific quantitative trait loci mediating heterophyllous variation to be mapped throughout the genome. The statistical properties of HpQTL were examined and validated via computer simulation. Its biological relevance was demonstrated by results from a real data analysis of heterophylly in a wood plant, mei (Prunus mume). HpQTL provides a powerful tool to analyze heterophylly and its underlying genetic architecture in a quantitative manner. It also contributes a new approach for genome-wide association studies aimed to dissect the programmed regulation of plant development and evolution. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Correlations of Genotype with Climate Parameters Suggest Caenorhabditis elegans Niche Adaptations

PubMed Central

Evans, Kathryn S.; Zhao, Yuehui; Brady, Shannon C.; Long, Lijiang; McGrath, Patrick T.; Andersen, Erik C.

2016-01-01

Species inhabit a variety of environmental niches, and the adaptation to a particular niche is often controlled by genetic factors, including gene-by-environment interactions. The genes that vary in order to regulate the ability to colonize a niche are often difficult to identify, especially in the context of complex ecological systems and in experimentally uncontrolled natural environments. Quantitative genetic approaches provide an opportunity to investigate correlations between genetic factors and environmental parameters that might define a niche. Previously, we have shown how a collection of 208 whole-genome sequenced wild Caenorhabditis elegans can facilitate association mapping approaches. To correlate climate parameters with the variation found in this collection of wild strains, we used geographic data to exhaustively curate daily weather measurements in short-term (3 month), middle-term (one year), and long-term (three year) durations surrounding the date of strain isolation. These climate parameters were used as quantitative traits in association mapping approaches, where we identified 11 quantitative trait loci (QTL) for three climatic variables: elevation, relative humidity, and average temperature. We then narrowed the genomic interval of interest to identify gene candidates with variants potentially underlying phenotypic differences. Additionally, we performed two-strain competition assays at high and low temperatures to validate a QTL that could underlie adaptation to temperature and found suggestive evidence supporting that hypothesis. PMID:27866149

A high-throughput quantitative expression analysis of cancer-related genes in human HepG2 cells in response to limonene, a potential anticancer agent.

PubMed

Hafidh, Rand R; Hussein, Saba Z; MalAllah, Mohammed Q; Abdulamir, Ahmed S; Abu Bakar, Fatimah

2017-11-14

Citrus bioactive compounds, as active anticancer agent, have been under focus by several studies worldwide. However, the underlying genes responsible for the anticancer potential have not been sufficiently highlighted. The current study investigated the gene expression profile of hepatocellular carcinoma, HepG2, cells after treatment with Limonene. The concentration that killed 50% of HepG2 cells was used to elucidate the genetic mechanisms of limonene anticancer activity. The apoptotic induction was detected by flow cytometry and confocal fluorescence microscope. Two of pro-apoptotic events, caspase-3 activation and phosphatidylserine translocation were manifested by confocal fluorescence microscopy. High-throughput real-time PCR was used to profile 1023 cancer-related genes in 16 different gene families related to the cancer development. In comparison to untreated cells, limonene increased the percentage of apoptotic cells up to 89.61%, by flow cytometry, and 48.2% by fluorescence microscopy. There was a significant limonene-driven differential gene expression of HepG2 cells in 15 different gene families. Limonene was shown to significantly (>2log) up-regulate and down-regulate 14 and 59 genes, respectively. The affected gene families, from most to least affected, were apoptosis induction, signal transduction, cancer genes augmentation, alteration in kinases expression, inflammation, DNA damage repair, and cell cycle proteins. The current study reveals that limonene could be a promising, cheap, and effective anticancer compound. The broad spectrum of limonene anticancer activity is interesting for anticancer drug development. Further research is needed to confirm the current findings and to examine the anticancer potential of limonene along with underlying mechanisms on different cell lines. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Expression Characterization of Stress Genes Under High and Low Temperature Stresses in the Pacific Oyster, Crassostrea gigas.

PubMed

Zhu, Qihui; Zhang, Linlin; Li, Li; Que, Huayong; Zhang, Guofan

2016-04-01

As a characteristic sessile inhabitant of the intertidal zone, the Pacific oyster Crassostrea gigas occupies one of the most physically stressful environments on earth. With high exposure to terrestrial conditions, oysters must tolerate broad fluctuations in temperature range. However, oysters' cellular and molecular responses to temperature stresses have not been fully characterized. Here, we analyzed oyster transcriptome data under high and low temperatures. We also identified over 30 key temperature stress-responsive candidate genes, which encoded stress proteins such as heat shock proteins and apoptosis-associated proteins. The expression characterization of these genes under short-term cold and hot environments (5 and 35 °C) and long-term cold environments (5 °C) was detected by quantitative real-time PCR. Most of these genes reached expression peaks during the recovery stage after 24 h of heat stress, and these genes were greatly induced around day 3 in long-term cold stress while responded little to short-term cold stress. In addition, in the second heat stress after 2 days of recovery, oysters showed milder expression in these genes and a lower mortality rate, which indicated the existence of plasticity in the oyster's response to heat stress. We confirmed that homeostatic flexibility and anti-apoptosis might be crucial centers of temperature stress responses in oysters. Furthermore, we analyzed stress gene families in 11 different species and found that the linage-specific expansion of stress genes might be implicated in adaptive evolution. These results indicated that both plasticity and evolution played an important role in the stress response adaptation of oysters.

A Strategy for Identifying Quantitative Trait Genes Using Gene Expression Analysis and Causal Analysis.

PubMed

Ishikawa, Akira

2017-11-27

Large numbers of quantitative trait loci (QTL) affecting complex diseases and other quantitative traits have been reported in humans and model animals. However, the genetic architecture of these traits remains elusive due to the difficulty in identifying causal quantitative trait genes (QTGs) for common QTL with relatively small phenotypic effects. A traditional strategy based on techniques such as positional cloning does not always enable identification of a single candidate gene for a QTL of interest because it is difficult to narrow down a target genomic interval of the QTL to a very small interval harboring only one gene. A combination of gene expression analysis and statistical causal analysis can greatly reduce the number of candidate genes. This integrated approach provides causal evidence that one of the candidate genes is a putative QTG for the QTL. Using this approach, I have recently succeeded in identifying a single putative QTG for resistance to obesity in mice. Here, I outline the integration approach and discuss its usefulness using my studies as an example.

Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells.

PubMed

Ede, Christopher; Chen, Ximin; Lin, Meng-Yin; Chen, Yvonne Y

2016-05-20

Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning 2 orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality.

Impact of Hypoxia on Drug Resistance and Growth Characteristics of Mycobacterium tuberculosis Clinical Isolates.

PubMed

Liu, Zhonghua; Gao, Yulu; Yang, Hua; Bao, Haiyang; Qin, Lianhua; Zhu, Changtai; Chen, Yawen; Hu, Zhongyi

2016-01-01

Mycobacterium tuberculosis (MTB) is a specific aerobic bacterium, but can survive under hypoxic conditions, such as those in lung cheese necrosis, granulomas, or macrophages. It is not clear whether the drug sensitivity and growth characteristics of MTB under hypoxic conditions are different from those under aerobic conditions. In this study, we examined the drug resistance and growth characteristics of MTB clinical isolates by a large sample of in vitro drug susceptibility tests, using an automatic growth instrument. Under hypoxic conditions, variance in drug resistance was observed in nearly one-third of the MTB strains and was defined as MTB strains with changed drug sensitivity (MTB-CDS). Among these strains, resistance in a considerable proportion of clinical strains was significantly increased, and some strains emerged as multi-drug resistant. Growth test results revealed a high growth rate and large survival number in macrophages under hypoxia in MTB-CDS. According to the results of fluorescence quantitative PCR, the expression of some genes, including RegX3 (involving RIF resistance), Rv0194 (efflux pump gene), four genes related to transcription regulation (KstR, DosR, Rv0081 and WhiB3) and gene related to translation regulation (DATIN), were upregulated significantly under hypoxic conditions compared to that under aerobic conditions (p < 0.05). Thus, we concluded that some MTB clinical isolates can survive under hypoxic conditions and their resistance could change. As for poor clinical outcomes in patients, based on routine drug susceptibility testing, drug susceptibility tests for tuberculosis under hypoxic conditions should also be recommended. However, the detailed mechanisms of the effect of hypoxia on drug sensitivity and growth characteristics of MTB clinical isolates still requires further study.

Archaeal amoA and ureC genes and their transcriptional activity in the Arctic Ocean

NASA Astrophysics Data System (ADS)

Pedneault, Estelle; Galand, Pierre E.; Potvin, Marianne; Tremblay, Jean-Éric; Lovejoy, Connie

2014-04-01

Thaumarchaeota and the gene encoding for a subunit of ammonia monooxygenase (amoA) are ubiquitous in Polar Seas, and some Thaumarchaeota also have a gene coding for ureC, diagnostic for urease. Using quantitative PCR we investigated the occurrence of genes and transcripts of ureC and amoA in Arctic samples from winter, spring and summer. AmoA genes, ureC genes and amoA transcripts were always present, but ureC transcripts were rarely detected. Over a 48 h light manipulation experiment amoA transcripts persisted under light and dark conditions, but not ureC transcripts. In addition, maxima for amoA transcript were nearer the surface compared to amoA genes. Clone libraries using DNA template recovered shallow and deep amoA clades but only the shallow clade was recovered from cDNA (from RNA). These results imply environmental control of amoA expression with direct or indirect light effects, and rare ureC expression despite its widespread occurrence in the Arctic Ocean.

Short-term exposure of Arabidopsis cell culures to hyper-G: Short-term changes in transcription regulation expression

NASA Astrophysics Data System (ADS)

Babbick, Maren; Hampp, Rudiger

2005-08-01

Callus cultures of Arabidopsis thaliana (cv. Columbia) were used to screen for early changes in gene expression in response to altered gravitational fields. In a recent microarray study we found hyper- g dependent changes in gene expression which indicated the involvement of WRKY genes [Martzivanou M. and Hampp R., Physiol. Plant., 118, 221-231,2003]. WRKY genes code for a family of plant-specific regulators of gene expression. In this study we report on the exposure of Arabidopsis callus cultures to 8g for up to 30 min. Quantitative analysis by real time RT-PCR of the amount of transcripts of WRKYs 3, 6, 22, 46, 65 and 70 showed individual changes in expression. As far as their function is known, these WRKY proteins are mainly involved in stress responses. As most alterations in transcript amount occurred within 10 min of treatment, such genes can be used for the investigation of microgravity-related effects on gene expression under sounding rocket conditions (TEXUS, MAXUS).

Oligonucleotide microarray analysis reveals dysregulation of energy-related metabolism in insulin-sensitive tissues of type 2 diabetes patients.

PubMed

Wang, M; Wang, X C; Zhao, L; Zhang, Y; Yao, L L; Lin, Y; Peng, Y D; Hu, R M

2014-06-17

Impaired insulin action within skeletal muscle, adipose tissue, and the liver is an important characteristic of type 2 diabetes (T2D). In order to identify common underlying defects in insulin-sensitive tissues that may be involved in the pathogenesis of T2D, the gene expression profiles of skeletal muscle, visceral adipose tissue, and liver from autopsy donors with or without T2D were examined using oligonucleotide microarrays and quantitative reverse transcriptase-PCR. Compared with controls, 691 genes were commonly dysregulated in these three insulin-sensitive tissues of humans with T2D. These co-expressed genes were enriched within the mitochondrion, with suggested involvement in energy metabolic processes such as glycolysis and gluconeogenesis, fatty acid beta oxidative, tricarboxylic acid cycle, and electron transport. Genes related to energy metabolism were mostly downregulated in diabetic skeletal muscle and visceral adipose tissue, while they were upregulated in the diabetic liver. This observed dysregulation in energy-related metabolism may be the underlying factor leading to the molecular mechanisms responsible for the insulin resistance of patients with T2D.

RatMap--rat genome tools and data.

PubMed

Petersen, Greta; Johnson, Per; Andersson, Lars; Klinga-Levan, Karin; Gómez-Fabre, Pedro M; Ståhl, Fredrik

2005-01-01

The rat genome database RatMap (http://ratmap.org or http://ratmap.gen.gu.se) has been one of the main resources for rat genome information since 1994. The database is maintained by CMB-Genetics at Goteborg University in Sweden and provides information on rat genes, polymorphic rat DNA-markers and rat quantitative trait loci (QTLs), all curated at RatMap. The database is under the supervision of the Rat Gene and Nomenclature Committee (RGNC); thus much attention is paid to rat gene nomenclature. RatMap presents information on rat idiograms, karyotypes and provides a unified presentation of the rat genome sequence and integrated rat linkage maps. A set of tools is also available to facilitate the identification and characterization of rat QTLs, as well as the estimation of exon/intron number and sizes in individual rat genes. Furthermore, comparative gene maps of rat in regard to mouse and human are provided.

RatMap—rat genome tools and data

PubMed Central

Petersen, Greta; Johnson, Per; Andersson, Lars; Klinga-Levan, Karin; Gómez-Fabre, Pedro M.; Ståhl, Fredrik

2005-01-01

The rat genome database RatMap (http://ratmap.org or http://ratmap.gen.gu.se) has been one of the main resources for rat genome information since 1994. The database is maintained by CMB–Genetics at Göteborg University in Sweden and provides information on rat genes, polymorphic rat DNA-markers and rat quantitative trait loci (QTLs), all curated at RatMap. The database is under the supervision of the Rat Gene and Nomenclature Committee (RGNC); thus much attention is paid to rat gene nomenclature. RatMap presents information on rat idiograms, karyotypes and provides a unified presentation of the rat genome sequence and integrated rat linkage maps. A set of tools is also available to facilitate the identification and characterization of rat QTLs, as well as the estimation of exon/intron number and sizes in individual rat genes. Furthermore, comparative gene maps of rat in regard to mouse and human are provided. PMID:15608244

Mapping and characterization of the major quantitative trait locus qSS7 associated with increased length and decreased width of rice seeds.

PubMed

Qiu, Xianjin; Gong, Rong; Tan, Youbin; Yu, Sibin

2012-12-01

Seed shape in rice (Oryza sativa) is an important factor that determines grain appearance, cooking quality and grain yield. Here, we report a major quantitative trait locus qSS7 on the long arm of chromosome 7 for seed length, seed width and the ratio of seed length to width, identified using a segregating population derived from a cross between an indica variety Zhenshan97 and a chromosomal segment substitution line of a japonica variety Cypress within the genetic background of Zhenshan97. The Cypress allele at qSS7 contributes to an increase in seed length and the ratio of length to width, but a decrease in seed width, without significantly changing seed weight, plant height, heading date or number of spikelets per panicle. Using a large F(2) population generated from a substitution line that carries only a heterozygous single segment surrounding qSS7, we delimited the QTL to a 23-kb region containing two annotated genes. Progeny testing of the informative recombinants suggested that this qSS7 region is a composite QTL in which at least two genes contribute to seed length and width. Sequence comparison and expression analysis of two probable candidate genes revealed differences between the parental lines. These results will facilitate cloning of the gene(s) underlying qSS7 as well as marker-assisted transfer of desirable genes for seed shape in rice improvement.

Transcriptome profiling reveals the immune response of goose T cells under selenium stimuli.

PubMed

Cao, Nan; Li, Wanyan; Li, Bingxin; Tian, Yunbo; Xu, Danning

2017-12-01

The goose is an economically important poultry species and a principal natural host of avian viruses. This study aimed to determine the effects of selenium on the immune response of geese. Under selenium stimulation, gene expression profiling was investigated using transcriptome sequencing. The selenoproteins were promoted by selenium stimulation, while the heat shock proteins, interleukin and interferons were mainly down-regulated. After comparison, 2228 differentially expressed genes were primarily involved in immune and environmental response, and infectious disease and genetic information processing related pathways were identified. Specifically, the enzymes of the lysosomes which acted as a safeguard in preventing pathogens were mostly up-regulated and six randomly selected differentially expressed genes were validated by quantitative polymerase chain reaction. In addition, the most proportional increased transcription factor family basic helix-loop-helix (bHLH) located in the 5' flank of selenoprotein P-like protein for selenium metabolism was identified by response to the selenium stimulation in this study. These analyses show that selenium can promote immune function by activating selenoproteins, transcript factors and lysosome pathway related genes, while weakening cytokine content genes in geese. © 2017 Japanese Society of Animal Science.

Overexpression of an aquaglyceroporin gene from Trichoderma harzianum improves water-use efficiency and drought tolerance in Nicotiana tabacum.

PubMed

Vieira, Pabline Marinho; Santos, Mirella Pupo; Andrade, Cristiana Moura; Souza-Neto, Otacílio Antônio; Ulhoa, Cirano José; Aragão, Francisco José Lima

2017-12-01

Aquaporins (AQPs) and aquaglyceroporins (AQGPs) are integral membrane proteins that mediate the transport of water and solutes, such as glycerol and urea, across membranes. AQP and AQGP genes represent a valuable tool for biotechnological improvement of plant tolerance to environmental stresses. We previously isolated a gene encoding for an aquaglyceroporin (ThAQGP), which was up-regulated in Trichoderma harzianum during interaction with the plant pathogen Fusarium solani. This gene was introduced into Nicotiana tabacum and plants were physiologically characterized. Under favorable growth conditions, transgenic progenies did not had differences in both germination and growth rates when compared to wild type. However, physiological responses under drought stress revealed that transgenic plants presented significantly higher transpiration rate, stomatal conductance, photosynthetic efficiency and faster turgor recovery than wild type. Quantitative RT-PCR analysis demonstrated the presence of ThAQGP transcripts in transgenic lines, showing the cause-effect relationship between the observed phenotype and the expression of the transgene. Our results underscore the high potential of T. harzianum as a source of genes with promising applications in transgenic plants tolerant to drought stress. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Haplotype Analysis in Multiple Crosses to Identify a QTL Gene

PubMed Central

Wang, Xiaosong; Korstanje, Ron; Higgins, David; Paigen, Beverly

2004-01-01

Identifying quantitative trait locus (QTL) genes is a challenging task. Herein, we report using a two-step process to identify Apoa2 as the gene underlying Hdlq5, a QTL for plasma high-density lipoprotein cholesterol (HDL) levels on mouse chromosome 1. First, we performed a sequence analysis of the Apoa2 coding region in 46 genetically diverse mouse strains and found five different APOA2 protein variants, which we named APOA2a to APOA2e. Second, we conducted a haplotype analysis of the strains in 21 crosses that have so far detected HDL QTLs; we found that Hdlq5 was detected only in the nine crosses where one parent had the APOA2b protein variant characterized by an Ala61-to-Val61 substitution. We then found that strains with the APOA2b variant had significantly higher (P ≤ 0.002) plasma HDL levels than those with either the APOA2a or the APOA2c variant. These findings support Apoa2 as the underlying Hdlq5 gene and suggest the Apoa2 polymorphisms responsible for the Hdlq5 phenotype. Therefore, haplotype analysis in multiple crosses can be used to support a candidate QTL gene. PMID:15310659

Haplotype analysis in multiple crosses to identify a QTL gene.

PubMed

Wang, Xiaosong; Korstanje, Ron; Higgins, David; Paigen, Beverly

2004-09-01

Identifying quantitative trait locus (QTL) genes is a challenging task. Herein, we report using a two-step process to identify Apoa2 as the gene underlying Hdlq5, a QTL for plasma high-density lipoprotein cholesterol (HDL) levels on mouse chromosome 1. First, we performed a sequence analysis of the Apoa2 coding region in 46 genetically diverse mouse strains and found five different APOA2 protein variants, which we named APOA2a to APOA2e. Second, we conducted a haplotype analysis of the strains in 21 crosses that have so far detected HDL QTLs; we found that Hdlq5 was detected only in the nine crosses where one parent had the APOA2b protein variant characterized by an Ala61-to-Val61 substitution. We then found that strains with the APOA2b variant had significantly higher (P < or = 0.002) plasma HDL levels than those with either the APOA2a or the APOA2c variant. These findings support Apoa2 as the underlying Hdlq5 gene and suggest the Apoa2 polymorphisms responsible for the Hdlq5 phenotype. Therefore, haplotype analysis in multiple crosses can be used to support a candidate QTL gene.

A Novel Role for Banana MaASR in the Regulation of Flowering Time in Transgenic Arabidopsis

PubMed Central

Yu, Xiaomeng; Jia, Caihong; Liu, Juhua; Zhang, Jianbin; Wang, Jingyi; Wang, Zhuo; Wang, Anbang; Xu, Biyu; Jin, Zhiqiang

2016-01-01

The abscisic acid (ABA)-, stress-, and ripening-induced (ASR) protein is a plant-specific hydrophilic transcriptional factor involved in fruit ripening and the abiotic stress response. To date, there have been no studies on the role of ASR genes in delayed flowering time. Here, we found that the ASR from banana, designated as MaASR, was preferentially expressed in the banana female flowers from the eighth, fourth, and first cluster of the inflorescence. MaASR transgenic lines (L14 and L38) had a clear delayed-flowering phenotype. The number of rosette leaves, sepals, and pedicel trichomes in L14 and L38 was greater than in the wild type (WT) under long day (LD) conditions. The period of buds, mid-flowers, and full bloom of L14 and L38 appeared later than the WT. cDNA microarray and quantitative real-time PCR (qRT-PCR) analyses revealed that overexpression of MaASR delays flowering through reduced expression of several genes, including photoperiod pathway genes, vernalization pathway genes, gibberellic acid pathway genes, and floral integrator genes, under short days (SD) for 28 d (from vegetative to reproductive transition stage); however, the expression of the autonomous pathway genes was not affected. This study provides the first evidence of a role for ASR genes in delayed flowering time in plants. PMID:27486844

A Novel Role for Banana MaASR in the Regulation of Flowering Time in Transgenic Arabidopsis.

PubMed

Sun, Peiguang; Miao, Hongxia; Yu, Xiaomeng; Jia, Caihong; Liu, Juhua; Zhang, Jianbin; Wang, Jingyi; Wang, Zhuo; Wang, Anbang; Xu, Biyu; Jin, Zhiqiang

2016-01-01

The abscisic acid (ABA)-, stress-, and ripening-induced (ASR) protein is a plant-specific hydrophilic transcriptional factor involved in fruit ripening and the abiotic stress response. To date, there have been no studies on the role of ASR genes in delayed flowering time. Here, we found that the ASR from banana, designated as MaASR, was preferentially expressed in the banana female flowers from the eighth, fourth, and first cluster of the inflorescence. MaASR transgenic lines (L14 and L38) had a clear delayed-flowering phenotype. The number of rosette leaves, sepals, and pedicel trichomes in L14 and L38 was greater than in the wild type (WT) under long day (LD) conditions. The period of buds, mid-flowers, and full bloom of L14 and L38 appeared later than the WT. cDNA microarray and quantitative real-time PCR (qRT-PCR) analyses revealed that overexpression of MaASR delays flowering through reduced expression of several genes, including photoperiod pathway genes, vernalization pathway genes, gibberellic acid pathway genes, and floral integrator genes, under short days (SD) for 28 d (from vegetative to reproductive transition stage); however, the expression of the autonomous pathway genes was not affected. This study provides the first evidence of a role for ASR genes in delayed flowering time in plants.

An allele of an ancestral transcription factor dependent on a horizontally acquired gene product.

PubMed

Chen, H Deborah; Jewett, Mollie W; Groisman, Eduardo A

2012-01-01

Changes in gene regulatory circuits often give rise to phenotypic differences among closely related organisms. In bacteria, these changes can result from alterations in the ancestral genome and/or be brought about by genes acquired by horizontal transfer. Here, we identify an allele of the ancestral transcription factor PmrA that requires the horizontally acquired pmrD gene product to promote gene expression. We determined that a single amino acid difference between the PmrA proteins from the human adapted Salmonella enterica serovar Paratyphi B and the broad host range S. enterica serovar Typhimurium rendered transcription of PmrA-activated genes dependent on the PmrD protein in the former but not the latter serovar. Bacteria harboring the serovar Typhimurium allele exhibited polymyxin B resistance under PmrA- or under PmrA- and PmrD-inducing conditions. By contrast, isogenic strains with the serovar Paratyphi B allele displayed PmrA-regulated polymyxin B resistance only when experiencing activating conditions for both PmrA and PmrD. We establish that the two PmrA orthologs display quantitative differences in several biochemical properties. Strains harboring the serovar Paratyphi B allele showed enhanced biofilm formation, a property that might promote serovar Paratyphi B's chronic infection of the gallbladder. Our findings illustrate how subtle differences in ancestral genes can impact the ability of horizontally acquired genes to confer new properties.

Gene expression profile analysis of rat cerebellum under acute alcohol intoxication.

PubMed

Zhang, Yu; Wei, Guangkuan; Wang, Yuehong; Jing, Ling; Zhao, Qingjie

2015-02-25

Acute alcohol intoxication, a common disease causing damage to the central nervous system (CNS) has been primarily studied on the aspects of alcohol addiction and chronic alcohol exposure. The understanding of gene expression change in the CNS during acute alcohol intoxication is still lacking. We established a model for acute alcohol intoxication in SD rats by oral gavage. A rat cDNA microarray was used to profile mRNA expression in the cerebella of alcohol-intoxicated rats (experimental group) and saline-treated rats (control group). A total of 251 differentially expressed genes were identified in response to acute alcohol intoxication, in which 208 of them were up-regulated and 43 were down-regulated. Gene ontology (GO) term enrichment analysis and pathway analysis revealed that the genes involved in the biological processes of immune response and endothelial integrity are among the most severely affected in response to acute alcohol intoxication. We discovered five transcription factors whose consensus binding motifs are overrepresented in the promoter region of differentially expressed genes. Additionally, we identified 20 highly connected hub genes by co-expression analysis, and validated the differential expression of these genes by real-time quantitative PCR. By determining novel biological pathways and transcription factors that have functional implication to acute alcohol intoxication, our study substantially contributes to the understanding of the molecular mechanism underlying the pathology of acute alcoholism. Copyright © 2014 Elsevier B.V. All rights reserved.

Deletion in a quantitative trait gene qPE9-1 associated with panicle erectness improves plant architecture during rice domestication.

PubMed

Zhou, Yong; Zhu, Jinyan; Li, Zhengyi; Yi, Chuandeng; Liu, Jun; Zhang, Honggen; Tang, Shuzhu; Gu, Minghong; Liang, Guohua

2009-09-01

Rice plant architecture is an important agronomic trait and a major determinant in high productivity. Panicle erectness is the preferred plant architecture in japonica rice, but the molecular mechanism underlying domestication of the erect panicle remains elusive. Here we report the map-based cloning of a major quantitative trait locus, qPE9-1, which plays an integral role in regulation of rice plant architecture including panicle erectness. The R6547 qPE9-1 gene encodes a 426-amino-acid protein, homologous to the keratin-associated protein 5-4 family. The gene is composed of three Von Willebrand factor type C domains, one transmembrane domain, and one 4-disulfide-core domain. Phenotypic comparisons of a set of near-isogenic lines and transgenic lines reveal that the functional allele (qPE9-1) results in drooping panicles, and the loss-of-function mutation (qpe9-1) leads to more erect panicles. In addition, the qPE9-1 locus regulates panicle and grain length, grain weight, and consequently grain yield. We propose that the panicle erectness trait resulted from a natural random loss-of-function mutation for the qPE9-1 gene and has subsequently been the target of artificial selection during japonica rice breeding.

Genome-wide Association Study of a Quantitative Disordered Gambling Trait

PubMed Central

Lind, Penelope A.; Zhu, Gu; Montgomery, Grant W; Madden, Pamela A.F.; Heath, Andrew C.; Martin, Nicholas G.; Slutske, Wendy S.

2012-01-01

Disordered gambling is a moderately heritable trait, but the underlying genetic basis is largely unknown. We performed a genome-wide association study (GWAS) for disordered gambling using a quantitative factor score in 1,312 twins from 894 Australian families. Association was conducted for 2,381,914 single nucleotide polymorphisms (SNPs) using the family-based association test in Merlin followed by gene and pathway enrichment analyses. Although no SNP reached genome-wide significance, six achieved P-values < 1 × 10−5 with variants in three genes (MT1X, ATXN1 and VLDLR) implicated in disordered gambling. Secondary case-control analyses found two SNPs on chromosome 9 (rs1106076 and rs12305135 near VLDLR) and rs10812227 near FZD10 on chromosome 12 to be significantly associated with lifetime DSM-IV pathological gambling and SOGS classified probable pathological gambling status. Furthermore, several addiction-related pathways were enriched for SNPs associated with disordered gambling. Finally, gene-based analysis of 24 candidate genes for dopamine agonist induced gambling in individuals with Parkinson’s disease suggested an enrichment of SNPs associated with disordered gambling. We report the first GWAS of disordered gambling. While further replication is required, the identification of susceptibility loci and biological pathways will be important in characterizing the biological mechanisms that underpin disordered gambling. PMID:22780124

Selection of reliable reference genes for normalization of quantitative RT-PCR from different developmental stages and tissues in amphioxus

PubMed Central

Zhang, Qi-Lin; Zhu, Qian-Hua; Liao, Xin; Wang, Xiu-Qiang; Chen, Tao; Xu, Han-Ting; Wang, Juan; Yuan, Ming-Long; Chen, Jun-Yuan

2016-01-01

Amphioxus is a closest living proxy to the ancestor of cephalochordates with vertebrates, and key animal for novel understanding in the evolutionary origin of vertebrate body plan, genome, tissues and immune system. Reliable analyses using quantitative real-time PCR (qRT-PCR) for answering these scientific questions is heavily dependent on reliable reference genes (RGs). In this study, we evaluated stability of thirteen candidate RGs in qRT-PCR for different developmental stages and tissues of amphioxus by four independent (geNorm, NormFinder, BestKeeper and deltaCt) and one comparative algorithms (RefFinder). The results showed that the top two stable RGs were the following: (1) S20 and 18 S in thirteen developmental stages, (2) EF1A and ACT in seven normal tissues, (3) S20 and L13 in both intestine and hepatic caecum challenged with lipopolysaccharide (LPS), and (4) S20 and EF1A in gill challenged with LPS. The expression profiles of two target genes (EYA and HHEX) in thirteen developmental stages were used to confirm the reliability of chosen RGs. This study identified optimal RGs that can be used to accurately measure gene expression under these conditions, which will benefit evolutionary and functional genomics studies in amphioxus. PMID:27869224

Evaluation of Reference Genes for RT qPCR Analyses of Structure-Specific and Hormone Regulated Gene Expression in Physcomitrella patens Gametophytes

PubMed Central

Le Bail, Aude; Scholz, Sebastian; Kost, Benedikt

2013-01-01

The use of the moss Physcomitrella patens as a model system to study plant development and physiology is rapidly expanding. The strategic position of P. patens within the green lineage between algae and vascular plants, the high efficiency with which transgenes are incorporated by homologous recombination, advantages associated with the haploid gametophyte representing the dominant phase of the P. patens life cycle, the simple structure of protonemata, leafy shoots and rhizoids that constitute the haploid gametophyte, as well as a readily accessible high-quality genome sequence make this moss a very attractive experimental system. The investigation of the genetic and hormonal control of P. patens development heavily depends on the analysis of gene expression patterns by real time quantitative PCR (RT qPCR). This technique requires well characterized sets of reference genes, which display minimal expression level variations under all analyzed conditions, for data normalization. Sets of suitable reference genes have been described for most widely used model systems including e.g. Arabidopsis thaliana, but not for P. patens. Here, we present a RT qPCR based comparison of transcript levels of 12 selected candidate reference genes in a range of gametophytic P. patens structures at different developmental stages, and in P. patens protonemata treated with hormones or hormone transport inhibitors. Analysis of these RT qPCR data using GeNorm and NormFinder software resulted in the identification of sets of P. patens reference genes suitable for gene expression analysis under all tested conditions, and suggested that the two best reference genes are sufficient for effective data normalization under each of these conditions. PMID:23951063

Identification and evaluation of reference genes for qRT-PCR studies in Lentinula edodes

PubMed Central

Qin, Peng; He, Maolan; Yu, Xiumei; Zhao, Ke; Zhang, Xiaoping; Ma, Menggen; Chen, Qiang; Chen, Xiaoqiong; Zeng, Xianfu; Gu, Yunfu

2018-01-01

Lentinula edodes (shiitake mushroom) is a common edible mushroom with a number of potential therapeutic and nutritional applications. It contains various medically important molecules, such as polysaccharides, terpenoids, sterols, and lipids, were contained in this mushroom. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to analyze the mechanisms underlying the biosynthetic pathways of these substances. qRT-PCR is used for accurate analyses of transcript levels owing to its rapidity, sensitivity, and reliability. However, its accuracy and reliability for the quantification of transcripts rely on the expression stability of the reference genes used for data normalization. To ensure the reliability of gene expression analyses using qRT-PCR in L. edodes molecular biology research, it is necessary to systematically evaluate reference genes. In the current study, ten potential reference genes were selected from L. edodes genomic data and their expression levels were measured by qRT-PCR using various samples. The expression stability of each candidate gene was analyzed by three commonly used software packages: geNorm, NormFinder, and BestKeeper. Base on the results, Rpl4 was the most stable reference gene across all experimental conditions, and Atu was the most stable gene among strains. 18S was found to be the best reference gene for different development stages, and Rpl4 was the most stably expressed gene under various nutrient conditions. The present work will contribute to qRT-PCR studies in L. edodes. PMID:29293626

Identification and evaluation of reference genes for qRT-PCR studies in Lentinula edodes.

PubMed

Xiang, Quanju; Li, Jin; Qin, Peng; He, Maolan; Yu, Xiumei; Zhao, Ke; Zhang, Xiaoping; Ma, Menggen; Chen, Qiang; Chen, Xiaoqiong; Zeng, Xianfu; Gu, Yunfu

2018-01-01

Lentinula edodes (shiitake mushroom) is a common edible mushroom with a number of potential therapeutic and nutritional applications. It contains various medically important molecules, such as polysaccharides, terpenoids, sterols, and lipids, were contained in this mushroom. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to analyze the mechanisms underlying the biosynthetic pathways of these substances. qRT-PCR is used for accurate analyses of transcript levels owing to its rapidity, sensitivity, and reliability. However, its accuracy and reliability for the quantification of transcripts rely on the expression stability of the reference genes used for data normalization. To ensure the reliability of gene expression analyses using qRT-PCR in L. edodes molecular biology research, it is necessary to systematically evaluate reference genes. In the current study, ten potential reference genes were selected from L. edodes genomic data and their expression levels were measured by qRT-PCR using various samples. The expression stability of each candidate gene was analyzed by three commonly used software packages: geNorm, NormFinder, and BestKeeper. Base on the results, Rpl4 was the most stable reference gene across all experimental conditions, and Atu was the most stable gene among strains. 18S was found to be the best reference gene for different development stages, and Rpl4 was the most stably expressed gene under various nutrient conditions. The present work will contribute to qRT-PCR studies in L. edodes.

Selection and evaluation of novel reference genes for quantitative reverse transcription PCR (qRT-PCR) based on genome and transcriptome data in Brassica napus L.

PubMed

Yang, Hongli; Liu, Jing; Huang, Shunmou; Guo, Tingting; Deng, Linbin; Hua, Wei

2014-03-15

Selection of reference genes in Brassica napus, a tetraploid (4×) species, is a very difficult task without information on genome and transcriptome. By now, only several traditional reference genes which show significant expression differentiation under different conditions are used in B. napus. In the present study, based on genome and transcriptome data of the rapeseed Zhongshuang-11 cultivar, 14 candidate reference genes were screened for investigation in different tissues, cultivars, and treated conditions of B. napus. These genes were as follows: ELF5, ENTH, F-BOX7, F-BOX2, FYPP1, GDI1, GYF, MCP2d, OTP80, PPR, SPOC, Unknown1, Unknown2 and UBA. Among them, excluding GYF and FYPP1, another 12 genes, were identified to perform better than traditional reference genes ACTIN7 and GAPDH. To further validate the accuracy of the newly developed reference genes in normalization, expression levels of BnCAT1 (B. napus catalase 1) in different rapeseed tissues and seedlings under stress conditions were normalized by the three most stable reference genes PPR, GDI1, and ENTH and little difference existed in normalization results. To the best of our knowledge, this is the first time B. napus reference genes have been provided with the help of complete genome and transcriptome information. The new reference genes provided in this study are more accurate than previously reported reference genes in quantifying expression levels of B. napus genes. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

Validation of Reference Genes for RT-qPCR Studies of Gene Expression in Preharvest and Postharvest Longan Fruits under Different Experimental Conditions

PubMed Central

Wu, Jianyang; Zhang, Hongna; Liu, Liqin; Li, Weicai; Wei, Yongzan; Shi, Shengyou

2016-01-01

Reverse transcription quantitative PCR (RT-qPCR) as the accurate and sensitive method is use for gene expression analysis, but the veracity and reliability result depends on whether select appropriate reference gene or not. To date, several reliable reference gene validations have been reported in fruits trees, but none have been done on preharvest and postharvest longan fruits. In this study, 12 candidate reference genes, namely, CYP, RPL, GAPDH, TUA, TUB, Fe-SOD, Mn-SOD, Cu/Zn-SOD, 18SrRNA, Actin, Histone H3, and EF-1a, were selected. Expression stability of these genes in 150 longan samples was evaluated and analyzed using geNorm and NormFinder algorithms. Preharvest samples consisted of seven experimental sets, including different developmental stages, organs, hormone stimuli (NAA, 2,4-D, and ethephon) and abiotic stresses (bagging and girdling with defoliation). Postharvest samples consisted of different temperature treatments (4 and 22°C) and varieties. Our findings indicate that appropriate reference gene(s) should be picked for each experimental condition. Our data further showed that the commonly used reference gene Actin does not exhibit stable expression across experimental conditions in longan. Expression levels of the DlACO gene, which is a key gene involved in regulating fruit abscission under girdling with defoliation treatment, was evaluated to validate our findings. In conclusion, our data provide a useful framework for choice of suitable reference genes across different experimental conditions for RT-qPCR analysis of preharvest and postharvest longan fruits. PMID:27375640

Mapping QTLs for water-use efficiency reveals the potential candidate genes involved in regulating the trait in apple under drought stress.

PubMed

Wang, Haibo; Zhao, Shuang; Mao, Ke; Dong, Qinglong; Liang, Bowen; Li, Chao; Wei, Zhiwei; Li, Mingjun; Ma, Fengwang

2018-06-26

Improvement of water-use efficiency (WUE) can effectively reduce production losses caused by drought stress. A better understanding of the genetic determination of WUE in crops under drought stress has great potential value for developing cultivars adapted to arid regions. To identify the genetic loci associated with WUE and reveal genes responsible for the trait in apple, we aim to map the quantitative trait loci (QTLs) for carbon isotope composition, the proxy for WUE, applying two contrasting irrigating regimes over the two-year experiment and search for the candidate genes encompassed in the mapped QTLs. We constructed a high-density genetic linkage map with 10,172 markers of apple, using single nucleotide polymorphism (SNP) markers obtained through restriction site-associated DNA sequencing (RADseq) and a final segregating population of 350 seedlings from the cross of Honeycrisp and Qinguan. In total, 33 QTLs were identified for carbon isotope composition in apple under both well-watered and drought-stressed conditions. Three QTLs were stable over 2 years under drought stress on linkage groups LG8, LG15 and LG16, as validated by Kompetitive Allele-Specific PCR (KASP) assays. In those validated QTLs, 258 genes were screened according to their Gene Ontology functional annotations. Among them, 28 genes were identified, which exhibited significant responses to drought stress in 'Honeycrisp' and/or 'Qinguan'. These genes are involved in signaling, photosynthesis, response to stresses, carbohydrate metabolism, protein metabolism and modification, hormone metabolism and transport, transport, respiration, transcriptional regulation, and development regulation. They, especially those for photoprotection and relevant signal transduction, are potential candidate genes connected with WUE regulation in drought-stressed apple. We detected three stable QTLs for carbon isotope composition in apple under drought stress over 2 years, and validated them by KASP assay. Twenty-eight candidate genes encompassed in these QTLs were identified. These stable genetic loci and series of genes provided here serve as a foundation for further studies on marker-assisted selection of high WUE and regulatory mechanism of WUE in apple exposed to drought conditions, respectively.

A genome-wide expression profile of salt-responsive genes in the apple rootstock Malus zumi.

PubMed

Li, Qingtian; Liu, Jia; Tan, Dunxian; Allan, Andrew C; Jiang, Yuzhuang; Xu, Xuefeng; Han, Zhenhai; Kong, Jin

2013-10-18

In some areas of cultivation, a lack of salt tolerance severely affects plant productivity. Apple, Malus x domestica Borkh., is sensitive to salt, and, as a perennial woody plant the mechanism of salt stress adaption will be different from that of annual herbal model plants, such as Arabidopsis. Malus zumi is a salt tolerant apple rootstock, which survives high salinity (up to 0.6% NaCl). To examine the mechanism underlying this tolerance, a genome-wide expression analysis was performed, using a cDNA library constructed from salt-treated seedlings of Malus zumi. A total of 15,000 cDNA clones were selected for microarray analysis. In total a group of 576 cDNAs, of which expression changed more than four-fold, were sequenced and 18 genes were selected to verify their expression pattern under salt stress by semi-quantitative RT-PCR. Our genome-wide expression analysis resulted in the isolation of 50 novel Malus genes and the elucidation of a new apple-specific mechanism of salt tolerance, including the stabilization of photosynthesis under stress, involvement of phenolic compounds, and sorbitol in ROS scavenging and osmoprotection. The promoter regions of 111 genes were analyzed by PlantCARE, suggesting an intensive cross-talking of abiotic stress in Malus zumi. An interaction network of salt responsive genes was constructed and molecular regulatory pathways of apple were deduced. Our research will contribute to gene function analysis and further the understanding of salt-tolerance mechanisms in fruit trees.

A Genome-Wide Expression Profile of Salt-Responsive Genes in the Apple Rootstock Malus zumi

PubMed Central

Li, Qingtian; Liu, Jia; Tan, Dunxian; Allan, Andrew C.; Jiang, Yuzhuang; Xu, Xuefeng; Han, Zhenhai; Kong, Jin

2013-01-01

In some areas of cultivation, a lack of salt tolerance severely affects plant productivity. Apple, Malus x domestica Borkh., is sensitive to salt, and, as a perennial woody plant the mechanism of salt stress adaption will be different from that of annual herbal model plants, such as Arabidopsis. Malus zumi is a salt tolerant apple rootstock, which survives high salinity (up to 0.6% NaCl). To examine the mechanism underlying this tolerance, a genome-wide expression analysis was performed, using a cDNA library constructed from salt-treated seedlings of Malus zumi. A total of 15,000 cDNA clones were selected for microarray analysis. In total a group of 576 cDNAs, of which expression changed more than four-fold, were sequenced and 18 genes were selected to verify their expression pattern under salt stress by semi-quantitative RT-PCR. Our genome-wide expression analysis resulted in the isolation of 50 novel Malus genes and the elucidation of a new apple-specific mechanism of salt tolerance, including the stabilization of photosynthesis under stress, involvement of phenolic compounds, and sorbitol in ROS scavenging and osmoprotection. The promoter regions of 111 genes were analyzed by PlantCARE, suggesting an intensive cross-talking of abiotic stress in Malus zumi. An interaction network of salt responsive genes was constructed and molecular regulatory pathways of apple were deduced. Our research will contribute to gene function analysis and further the understanding of salt-tolerance mechanisms in fruit trees. PMID:24145753

The quantitative and condition-dependent Escherichia coli proteome

PubMed Central

Schmidt, Alexander; Kochanowski, Karl; Vedelaar, Silke; Ahrné, Erik; Volkmer, Benjamin; Callipo, Luciano; Knoops, Kèvin; Bauer, Manuel; Aebersold, Ruedi; Heinemann, Matthias

2016-01-01

Measuring precise concentrations of proteins can provide insights into biological processes. Here, we use efficient protein extraction and sample fractionation and state-of-the-art quantitative mass spectrometry techniques to generate a comprehensive, condition-dependent protein abundance map of Escherichia coli. We measure cellular protein concentrations for 55% of predicted E. coli genes (>2300 proteins) under 22 different experimental conditions and identify methylation and N-terminal protein acetylations previously not known to be prevalent in bacteria. We uncover system-wide proteome allocation, expression regulation, and post-translational adaptations. These data provide a valuable resource for the systems biology and broader E. coli research communities. PMID:26641532

Identification of Reference Genes for Quantitative Real-Time PCR in Date Palm (Phoenix dactylifera L.) Subjected to Drought and Salinity.

PubMed

V Patankar, Himanshu; M Assaha, Dekoum V; Al-Yahyai, Rashid; Sunkar, Ramanjulu; Yaish, Mahmoud W

2016-01-01

Date palm is an important crop plant in the arid and semi-arid regions supporting human population in the Middle East and North Africa. These areas have been largely affected by drought and salinity due to insufficient rainfall and improper irrigation practices. Date palm is a relatively salt- and drought-tolerant plant and more recently efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Quantitative real-time PCR (qPCR) is a promising technique for the analysis of stress-induced differential gene expression, which involves the use of stable reference genes for normalizing gene expression. In an attempt to find the best reference genes for date palm's drought and salinity research, we evaluated the stability of 12 most commonly used reference genes using the geNorm, NormFinder, BestKeeper statistical algorithms and the comparative ΔCT method. The comprehensive results revealed that HEAT SHOCK PROTEIN (HSP), UBIQUITIN (UBQ) and YTH domain-containing family protein (YT521) were stable in drought-stressed leaves whereas GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH), ACTIN and TUBULIN were stable in drought-stressed roots. On the other hand, SMALL SUBUNIT RIBOSOMAL RNA (25S), YT521 and 18S ribosomal RNA (18S); and UBQ, ACTIN and ELONGATION FACTOR 1-ALPHA (eEF1a) were stable in leaves and roots, respectively, under salt stress. The stability of these reference genes was verified by using the abiotic stress-responsive CYTOSOLIC Cu/Zn SUPEROXIDE DISMUTASE (Cyt-Cu/Zn SOD), an ABA RECEPTOR, and a PROLINE TRANSPORTER 2 (PRO) genes. A combination of top 2 or 3 stable reference genes were found to be suitable for normalization of the target gene expression and will facilitate gene expression analysis studies aimed at identifying functional genes associated with drought and salinity tolerance in date palm.

Quantitative analysis of bristle number in Drosophila mutants identifies genes involved in neural development

NASA Technical Reports Server (NTRS)

Norga, Koenraad K.; Gurganus, Marjorie C.; Dilda, Christy L.; Yamamoto, Akihiko; Lyman, Richard F.; Patel, Prajal H.; Rubin, Gerald M.; Hoskins, Roger A.; Mackay, Trudy F.; Bellen, Hugo J.

2003-01-01

BACKGROUND: The identification of the function of all genes that contribute to specific biological processes and complex traits is one of the major challenges in the postgenomic era. One approach is to employ forward genetic screens in genetically tractable model organisms. In Drosophila melanogaster, P element-mediated insertional mutagenesis is a versatile tool for the dissection of molecular pathways, and there is an ongoing effort to tag every gene with a P element insertion. However, the vast majority of P element insertion lines are viable and fertile as homozygotes and do not exhibit obvious phenotypic defects, perhaps because of the tendency for P elements to insert 5' of transcription units. Quantitative genetic analysis of subtle effects of P element mutations that have been induced in an isogenic background may be a highly efficient method for functional genome annotation. RESULTS: Here, we have tested the efficacy of this strategy by assessing the extent to which screening for quantitative effects of P elements on sensory bristle number can identify genes affecting neural development. We find that such quantitative screens uncover an unusually large number of genes that are known to function in neural development, as well as genes with yet uncharacterized effects on neural development, and novel loci. CONCLUSIONS: Our findings establish the use of quantitative trait analysis for functional genome annotation through forward genetics. Similar analyses of quantitative effects of P element insertions will facilitate our understanding of the genes affecting many other complex traits in Drosophila.

Regulatory Architecture of Gene Expression Variation in the Threespine Stickleback Gasterosteus aculeatus.

PubMed

Pritchard, Victoria L; Viitaniemi, Heidi M; McCairns, R J Scott; Merilä, Juha; Nikinmaa, Mikko; Primmer, Craig R; Leder, Erica H

2017-01-05

Much adaptive evolutionary change is underlain by mutational variation in regions of the genome that regulate gene expression rather than in the coding regions of the genes themselves. An understanding of the role of gene expression variation in facilitating local adaptation will be aided by an understanding of underlying regulatory networks. Here, we characterize the genetic architecture of gene expression variation in the threespine stickleback (Gasterosteus aculeatus), an important model in the study of adaptive evolution. We collected transcriptomic and genomic data from 60 half-sib families using an expression microarray and genotyping-by-sequencing, and located expression quantitative trait loci (eQTL) underlying the variation in gene expression in liver tissue using an interval mapping approach. We identified eQTL for several thousand expression traits. Expression was influenced by polymorphism in both cis- and trans-regulatory regions. Trans-eQTL clustered into hotspots. We did not identify master transcriptional regulators in hotspot locations: rather, the presence of hotspots may be driven by complex interactions between multiple transcription factors. One observed hotspot colocated with a QTL recently found to underlie salinity tolerance in the threespine stickleback. However, most other observed hotspots did not colocate with regions of the genome known to be involved in adaptive divergence between marine and freshwater habitats. Copyright © 2017 Pritchard et al.

Toxicity and Transcriptome Sequencing (RNA-seq) Analyses of Adult Zebrafish in Response to Exposure Carboxymethyl Cellulose Stabilized Iron Sulfide Nanoparticles.

PubMed

Zheng, Min; Lu, Jianguo; Zhao, Dongye

2018-05-24

Increasing utilization of stabilized iron sulfides (FeS) nanoparticles implies an elevated release of the materials into the environment. To understand potential impacts and underlying mechanisms of nanoparticle-induced stress, we used the transcriptome sequencing (RNA-seq) technique to characterize the transcriptomes from adult zebrafish exposed to 10 mg/L carboxymethyl cellulose (CMC) stabilized FeS nanoparticles for 96 h, demonstrating striking differences in the gene expression profiles in liver. The exposure caused significant expression alterations in genes related to immune and inflammatory responses, detoxification, oxidative stress and DNA damage/repair. The complement and coagulation cascades Kyoto encyclopedia of genes and genomes (KEGG) pathway was found significantly up-regulated under nanoparticle exposure. The quantitative real-time polymerase chain reaction using twelve genes confirmed the RNA-seq results. We identified several candidate genes commonly regulated in liver, which may serve as gene indicators when exposed to the nanoparticles. Hepatic inflammation was further confirmed by histological observation of pyknotic nuclei, and vacuole formation upon exposure. Tissue accumulation tests showed a 2.2 times higher iron concentration in the fish tissue upon exposure. This study provides preliminary mechanistic insights into potential toxic effects of organic matter stabilized FeS nanoparticles, which will improve our understanding of the genotoxicity caused by stabilized nanoparticles.

Regulatory Architecture of Gene Expression Variation in the Threespine Stickleback Gasterosteus aculeatus

PubMed Central

Pritchard, Victoria L.; Viitaniemi, Heidi M.; McCairns, R. J. Scott; Merilä, Juha; Nikinmaa, Mikko; Primmer, Craig R.; Leder, Erica H.

2016-01-01

Much adaptive evolutionary change is underlain by mutational variation in regions of the genome that regulate gene expression rather than in the coding regions of the genes themselves. An understanding of the role of gene expression variation in facilitating local adaptation will be aided by an understanding of underlying regulatory networks. Here, we characterize the genetic architecture of gene expression variation in the threespine stickleback (Gasterosteus aculeatus), an important model in the study of adaptive evolution. We collected transcriptomic and genomic data from 60 half-sib families using an expression microarray and genotyping-by-sequencing, and located expression quantitative trait loci (eQTL) underlying the variation in gene expression in liver tissue using an interval mapping approach. We identified eQTL for several thousand expression traits. Expression was influenced by polymorphism in both cis- and trans-regulatory regions. Trans-eQTL clustered into hotspots. We did not identify master transcriptional regulators in hotspot locations: rather, the presence of hotspots may be driven by complex interactions between multiple transcription factors. One observed hotspot colocated with a QTL recently found to underlie salinity tolerance in the threespine stickleback. However, most other observed hotspots did not colocate with regions of the genome known to be involved in adaptive divergence between marine and freshwater habitats. PMID:27836907

Novel genes on rat chromosome 10 are linked to body fat mass, preadipocyte number and adipocyte size.

PubMed

Weingarten, A; Turchetti, L; Krohn, K; Klöting, I; Kern, M; Kovacs, P; Stumvoll, M; Blüher, M; Klöting, N

2016-12-01

The genetic architecture of obesity is multifactorial. We have previously identified a quantitative trait locus (QTL) on rat chromosome 10 in a F2 cross of Wistar Ottawa Karlsburg (WOKW) and Dark Agouti (DA) rats responsible for obesity-related traits. The QTL was confirmed in congenic DA.WOKW10 rats. To pinpoint the region carrying causal genes, we established two new subcongenic lines, L1 and L2, with smaller refined segments of chromosome 10 to identify novel candidate genes. All lines were extensively characterized under different diet conditions. We employed transcriptome analysis in visceral adipose tissue (VAT) by RNA-Seq technology to identify potential underlying genes in the segregating regions. Three candidate genes were measured in human paired samples of VAT and subcutaneous (SC) AT (SAT) (N=304) individuals with a wide range of body weight and glucose homeostasis parameters. DA.WOKW and L1 subcongenic lines were protected against body fat gain under high-fat diet (HFD), whereas L2 and DA had significantly more body fat after high-fat feeding. Interestingly, adipocyte size distribution in SAT and epigonadal AT of L1 subcongenic rats did not undergo typical ballooning under HFD and the number of preadipocytes in AT was significantly elevated in L2 compared with L1 and parental rats. Transcriptome analysis identified three candidate genes in VAT on rat chromosome 10. In humans, these candidate genes were differentially expressed between SAT and VAT. Moreover, HID1 mRNA significantly correlates with parameters of obesity and glucose metabolism. Our data suggest novel candidate genes for obesity that map on rat chromosome 10 in an interval 102.2-104.7 Mb and are strongly associated with body fat mass regulation, preadipocyte number and adipocyte size in rats. Among those genes, AT head involution defective (HID1) mRNA expression may be relevant for human fat distribution and glucose homeostasis.

Salt Stress Induced Variation in DNA Methylation Pattern and Its Influence on Gene Expression in Contrasting Rice Genotypes

PubMed Central

Karan, Ratna; DeLeon, Teresa; Biradar, Hanamareddy; Subudhi, Prasanta K.

2012-01-01

Background Salinity is a major environmental factor limiting productivity of crop plants including rice in which wide range of natural variability exists. Although recent evidences implicate epigenetic mechanisms for modulating the gene expression in plants under environmental stresses, epigenetic changes and their functional consequences under salinity stress in rice are underexplored. DNA methylation is one of the epigenetic mechanisms regulating gene expression in plant’s responses to environmental stresses. Better understanding of epigenetic regulation of plant growth and response to environmental stresses may create novel heritable variation for crop improvement. Methodology/Principal Findings Methylation sensitive amplification polymorphism (MSAP) technique was used to assess the effect of salt stress on extent and patterns of DNA methylation in four genotypes of rice differing in the degree of salinity tolerance. Overall, the amount of DNA methylation was more in shoot compared to root and the contribution of fully methylated loci was always more than hemi-methylated loci. Sequencing of ten randomly selected MSAP fragments indicated gene-body specific DNA methylation of retrotransposons, stress responsive genes, and chromatin modification genes, distributed on different rice chromosomes. Bisulphite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied with genotypes and tissue types irrespective of the level of salinity tolerance of rice genotypes. Conclusions/Significance The gene body methylation may have an important role in regulating gene expression in organ and genotype specific manner under salinity stress. Association between salt tolerance and methylation changes observed in some cases suggested that many methylation changes are not “directed”. The natural genetic variation for salt tolerance observed in rice germplasm may be independent of the extent and pattern of DNA methylation which may have been induced by abiotic stress followed by accumulation through the natural selection process. PMID:22761959

Detection of medically important Candida species by absolute quantitation real-time polymerase chain reaction.

PubMed

Than, Leslie Thian Lung; Chong, Pei Pei; Ng, Kee Peng; Seow, Heng Fong

2015-01-01

The number of invasive candidiasis cases has risen especially with an increase in the number of immunosuppressed and immunocom promised patients. The early detection of Candida species which is specific and sensitive is important in determining the correct administration of antifungal drugs to patients. This study aims to develop a method for the detection, identification and quantitation of medically important Candida species through quantitative polymerase chain reaction (qPCR). The isocitrate lyase (ICL) gene which is not found in mammals was chosen as the target gene of real-time PCR. Absolute quantitation of the gene copy number was achieved by constructing the plasmid containing the ICL gene which is used to generate standard curve. Twenty fungal species, two bacterial species and human DNA were tested to check the specificity of the detection method. All eight Candida species were successfully detected, identified and quantitated based on the ICL gene. A seven-log range of the gene copy number and a minimum detection limit of 10(3) copies were achieved. A one-tube absolute quantification real-time PCR that differentiates medically important Candida species via individual unique melting temperature was achieved. Analytical sensitivity and specificity were not compromised.

Metabolite profiling and quantitative genetics of natural variation for flavonoids in Arabidopsis

PubMed Central

Routaboul, Jean-Marc; Dubos, Christian; Beck, Gilles; Marquis, Catherine; Bidzinski, Przemyslaw; Loudet, Olivier; Lepiniec, Loïc

2012-01-01

Little is known about the range and the genetic bases of naturally occurring variation for flavonoids. Using Arabidopsis thaliana seed as a model, the flavonoid content of 41 accessions and two recombinant inbred line (RIL) sets derived from divergent accessions (Cvi-0×Col-0 and Bay-0×Shahdara) were analysed. These accessions and RILs showed mainly quantitative rather than qualitative changes. To dissect the genetic architecture underlying these differences, a quantitative trait locus (QTL) analysis was performed on the two segregating populations. Twenty-two flavonoid QTLs were detected that accounted for 11–64% of the observed trait variations, only one QTL being common to both RIL sets. Sixteen of these QTLs were confirmed and coarsely mapped using heterogeneous inbred families (HIFs). Three genes, namely TRANSPARENT TESTA (TT)7, TT15, and MYB12, were proposed to underlie their variations since the corresponding mutants and QTLs displayed similar specific flavonoid changes. Interestingly, most loci did not co-localize with any gene known to be involved in flavonoid metabolism. This latter result shows that novel functions have yet to be characterized and paves the way for their isolation. PMID:22442426

Highly Tissue Substructure-Specific Effects of Human Papilloma Virus in Mucosa of HIV-Infected Patients Revealed by Laser-Dissection Microscopy-Assisted Gene Expression Profiling

PubMed Central

Baumgarth, Nicole; Szubin, Richard; Dolganov, Greg M.; Watnik, Mitchell R.; Greenspan, Deborah; Da Costa, Maria; Palefsky, Joel M.; Jordan, Richard; Roederer, Mario; Greenspan, John S.

2004-01-01

Human papilloma virus (HPV) causes focal infections of epithelial layers in skin and mucosa. HIV-infected patients on highly active antiretroviral therapy (HAART) appear to be at increased risk of developing HPV-induced oral warts. To identify the mechanisms that allow long-term infection of oral epithelial cells in these patients, we used a combination of laser-dissection microscopy (LDM) and highly sensitive and quantitative, non-biased, two-step multiplex real-time RT-PCR to study pathogen-induced alterations of specific tissue subcompartments. Expression of 166 genes was compared in three distinct epithelial and subepithelial compartments isolated from biopsies of normal mucosa from HIV-infected and non-infected patients and of HPV32-induced oral warts from HIV-infected patients. In contrast to the underlying HIV infection and/or HAART, which did not significantly elaborate tissue substructure-specific effects, changes in oral warts were strongly tissue substructure-specific. HPV 32 seems to establish infection by selectively enhancing epithelial cell growth and differentiation in the stratum spinosum and to evade the immune system by actively suppressing inflammatory responses in adjacent underlying tissues. With this highly sensitive and quantitative method tissue-specific expression of hundreds of genes can be studied simultaneously in a few cells. Because of its large dynamic measurement range it could also become a method of choice to confirm and better quantify results obtained by microarray analysis. PMID:15331396

Transcriptomic analysis of Crassostrea sikamea × Crassostrea angulata hybrids in response to low salinity stress.

PubMed

Yan, Lulu; Su, Jiaqi; Wang, Zhaoping; Yan, Xiwu; Yu, Ruihai; Ma, Peizhen; Li, Yangchun; Du, Junpeng

2017-01-01

Hybrid oysters often show heterosis in growth rate, weight, survival and adaptability to extremes of salinity. Oysters have also been used as model organisms to study the evolution of host-defense system. To gain comprehensive knowledge about various physiological processes in hybrid oysters under low salinity stress, we performed transcriptomic analysis of gill tissue of Crassostrea sikamea ♀ × Crassostrea angulata♂ hybrid using the deep-sequencing platform Illumina HiSeq. We exploited the high-throughput technique to delineate differentially expressed genes (DEGs) in oysters maintained in hypotonic conditions. A total of 199,391 high quality unigenes, with average length of 644 bp, were generated. Of these 35 and 31 genes showed up- and down-regulation, respectively. Functional categorization and pathway analysis of these DEGs revealed enrichment for immune mechanism, apoptosis, energy metabolism and osmoregulation under low salinity stress. The expression patterns of 41 DEGs in hybrids and their parental species were further analyzed by quantitative real-time PCR (qRT-PCR). This study will serve as a platform for subsequent gene expression analysis regarding environmental stress. Our findings will also provide valuable information about gene expression to better understand the immune mechanism, apoptosis, energy metabolism and osmoregulation in hybrid oysters under low salinity stress.

Transcriptomic analysis of Crassostrea sikamea × Crassostrea angulata hybrids in response to low salinity stress

PubMed Central

Yan, Lulu; Su, Jiaqi; Wang, Zhaoping; Yan, Xiwu; Yu, Ruihai; Ma, Peizhen; Li, Yangchun; Du, Junpeng

2017-01-01

Hybrid oysters often show heterosis in growth rate, weight, survival and adaptability to extremes of salinity. Oysters have also been used as model organisms to study the evolution of host-defense system. To gain comprehensive knowledge about various physiological processes in hybrid oysters under low salinity stress, we performed transcriptomic analysis of gill tissue of Crassostrea sikamea ♀ × Crassostrea angulata♂ hybrid using the deep-sequencing platform Illumina HiSeq. We exploited the high-throughput technique to delineate differentially expressed genes (DEGs) in oysters maintained in hypotonic conditions. A total of 199,391 high quality unigenes, with average length of 644 bp, were generated. Of these 35 and 31 genes showed up- and down-regulation, respectively. Functional categorization and pathway analysis of these DEGs revealed enrichment for immune mechanism, apoptosis, energy metabolism and osmoregulation under low salinity stress. The expression patterns of 41 DEGs in hybrids and their parental species were further analyzed by quantitative real-time PCR (qRT-PCR). This study will serve as a platform for subsequent gene expression analysis regarding environmental stress. Our findings will also provide valuable information about gene expression to better understand the immune mechanism, apoptosis, energy metabolism and osmoregulation in hybrid oysters under low salinity stress. PMID:28182701

Expression profiling of Chrysanthemum crassum under salinity stress and the initiation of morphological changes

PubMed Central

Guan, Zhiyong; Feng, Yitong; Song, Aiping; Shi, Xiaomeng; Mao, Yachao; Chen, Sumei; Jiang, Jiafu; Ding, Lian; Chen, Fadi

2017-01-01

Chrysanthemum crassum is a decaploid species of Chrysanthemum with high stress tolerance that allows survival under salinity stress while maintaining a relatively ideal growth rate. We previously recorded morphological changes after salt treatment, such as the expansion of leaf cells. To explore the underlying salinity tolerance mechanisms, we used an Illumina platform and obtained three sequencing libraries from samples collected after 0 h, 12 h and 24 h of salt treatment. Following de novo assembly, 154,944 transcripts were generated, and 97,833 (63.14%) transcripts were annotated, including 55 Gene Ontology (GO) terms and 128 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression profile of C. crassum was globally altered after salt treatment. We selected functional genes and pathways that may contribute to salinity tolerance and identified some factors involved in the salinity tolerance strategies of C. crassum, such as signal transduction, transcription factors and plant hormone regulation, enhancement of energy metabolism, functional proteins and osmolyte synthesis, reactive oxygen species (ROS) scavenging, photosystem protection and recovery, and cell wall protein modifications. Forty-six genes were selected for quantitative real-time polymerase chain reaction detection, and their expression patterns were shown to be consistent with the changes in their transcript abundance determined by RNA sequencing. PMID:28437448

Mapping DNA damage-dependent genetic interactions in yeast via party mating and barcode fusion genetics.

PubMed

Díaz-Mejía, J Javier; Celaj, Albi; Mellor, Joseph C; Coté, Atina; Balint, Attila; Ho, Brandon; Bansal, Pritpal; Shaeri, Fatemeh; Gebbia, Marinella; Weile, Jochen; Verby, Marta; Karkhanina, Anna; Zhang, YiFan; Wong, Cassandra; Rich, Justin; Prendergast, D'Arcy; Gupta, Gaurav; Öztürk, Sedide; Durocher, Daniel; Brown, Grant W; Roth, Frederick P

2018-05-28

Condition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double-mutant strains, does not scale readily to multi-condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG-GI), by which double-mutant strains generated via en masse "party" mating can also be monitored en masse for growth to detect genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1 , SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Transcripts of Anthocyanidin Reductase and Leucoanthocyanidin Reductase and Measurement of Catechin and Epicatechin in Tartary Buckwheat

PubMed Central

Kim, Yeon Bok; Thwe, Aye Aye; Kim, YeJi; Li, Xiaohua; Cho, Jin Woong; Park, Phun Bum; Valan Arasu, Mariadhas; Abdullah Al-Dhabi, Naif; Kim, Sun-Ju; Suzuki, Tastsuro; Hyun Jho, Kwang; Park, Sang Un

2014-01-01

Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs) such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions. PMID:24605062

Comparative Transcriptomic Analysis in Paddy Rice under Storage and Identification of Differentially Regulated Genes in Response to High Temperature and Humidity.

PubMed

Zhao, Chanjuan; Xie, Junqi; Li, Li; Cao, Chongjiang

2017-09-20

The transcriptomes of paddy rice in response to high temperature and humidity were studied using a high-throughput RNA sequencing approach. Effects of high temperature and humidity on the sucrose and starch contents and α/β-amylase activity were also investigated. Results showed that 6876 differentially expressed genes (DEGs) were identified in paddy rice under high temperature and humidity storage. Importantly, 12 DEGs that were downregulated fell into the "starch and sucrose pathway". The quantitative real-time polymerase chain reaction assays indicated that expression of these 12 DEGs was significantly decreased, which was in parallel with the reduced level of enzyme activities and the contents of sucrose and starch in paddy rice stored at high temperature and humidity conditions compared to the control group. Taken together, high temperature and humidity influence the quality of paddy rice at least partially by downregulating the expression of genes encoding sucrose transferases and hydrolases, which might result in the decrease of starch and sucrose contents.

Transcriptome-wide selection of a reliable set of reference genes for gene expression studies in potato cyst nematodes (Globodera spp.).

PubMed

Sabeh, Michael; Duceppe, Marc-Olivier; St-Arnaud, Marc; Mimee, Benjamin

2018-01-01

Relative gene expression analyses by qRT-PCR (quantitative reverse transcription PCR) require an internal control to normalize the expression data of genes of interest and eliminate the unwanted variation introduced by sample preparation. A perfect reference gene should have a constant expression level under all the experimental conditions. However, the same few housekeeping genes selected from the literature or successfully used in previous unrelated experiments are often routinely used in new conditions without proper validation of their stability across treatments. The advent of RNA-Seq and the availability of public datasets for numerous organisms are opening the way to finding better reference genes for expression studies. Globodera rostochiensis is a plant-parasitic nematode that is particularly yield-limiting for potato. The aim of our study was to identify a reliable set of reference genes to study G. rostochiensis gene expression. Gene expression levels from an RNA-Seq database were used to identify putative reference genes and were validated with qRT-PCR analysis. Three genes, GR, PMP-3, and aaRS, were found to be very stable within the experimental conditions of this study and are proposed as reference genes for future work.

Topological analysis of metabolic networks integrating co-segregating transcriptomes and metabolomes in type 2 diabetic rat congenic series.

PubMed

Dumas, Marc-Emmanuel; Domange, Céline; Calderari, Sophie; Martínez, Andrea Rodríguez; Ayala, Rafael; Wilder, Steven P; Suárez-Zamorano, Nicolas; Collins, Stephan C; Wallis, Robert H; Gu, Quan; Wang, Yulan; Hue, Christophe; Otto, Georg W; Argoud, Karène; Navratil, Vincent; Mitchell, Steve C; Lindon, John C; Holmes, Elaine; Cazier, Jean-Baptiste; Nicholson, Jeremy K; Gauguier, Dominique

2016-09-30

The genetic regulation of metabolic phenotypes (i.e., metabotypes) in type 2 diabetes mellitus occurs through complex organ-specific cellular mechanisms and networks contributing to impaired insulin secretion and insulin resistance. Genome-wide gene expression profiling systems can dissect the genetic contributions to metabolome and transcriptome regulations. The integrative analysis of multiple gene expression traits and metabolic phenotypes (i.e., metabotypes) together with their underlying genetic regulation remains a challenge. Here, we introduce a systems genetics approach based on the topological analysis of a combined molecular network made of genes and metabolites identified through expression and metabotype quantitative trait locus mapping (i.e., eQTL and mQTL) to prioritise biological characterisation of candidate genes and traits. We used systematic metabotyping by 1 H NMR spectroscopy and genome-wide gene expression in white adipose tissue to map molecular phenotypes to genomic blocks associated with obesity and insulin secretion in a series of rat congenic strains derived from spontaneously diabetic Goto-Kakizaki (GK) and normoglycemic Brown-Norway (BN) rats. We implemented a network biology strategy approach to visualize the shortest paths between metabolites and genes significantly associated with each genomic block. Despite strong genomic similarities (95-99 %) among congenics, each strain exhibited specific patterns of gene expression and metabotypes, reflecting the metabolic consequences of series of linked genetic polymorphisms in the congenic intervals. We subsequently used the congenic panel to map quantitative trait loci underlying specific mQTLs and genome-wide eQTLs. Variation in key metabolites like glucose, succinate, lactate, or 3-hydroxybutyrate and second messenger precursors like inositol was associated with several independent genomic intervals, indicating functional redundancy in these regions. To navigate through the complexity of these association networks we mapped candidate genes and metabolites onto metabolic pathways and implemented a shortest path strategy to highlight potential mechanistic links between metabolites and transcripts at colocalized mQTLs and eQTLs. Minimizing the shortest path length drove prioritization of biological validations by gene silencing. These results underline the importance of network-based integration of multilevel systems genetics datasets to improve understanding of the genetic architecture of metabotype and transcriptomic regulation and to characterize novel functional roles for genes determining tissue-specific metabolism.

Gene Expression Profile Analysis is Directly Affected by the Selected Reference Gene: The Case of Leaf-Cutting Atta Sexdens

PubMed Central

Máximo, Wesley P. F.; Zanetti, Ronald; Paiva, Luciano V.

2018-01-01

Although several ant species are important targets for the development of molecular control strategies, only a few studies focus on identifying and validating reference genes for quantitative reverse transcription polymerase chain reaction (RT-qPCR) data normalization. We provide here an extensive study to identify and validate suitable reference genes for gene expression analysis in the ant Atta sexdens, a threatening agricultural pest in South America. The optimal number of reference genes varies according to each sample and the result generated by RefFinder differed about which is the most suitable reference gene. Results suggest that the RPS16, NADH and SDHB genes were the best reference genes in the sample pool according to stability values. The SNF7 gene expression pattern was stable in all evaluated sample set. In contrast, when using less stable reference genes for normalization a large variability in SNF7 gene expression was recorded. There is no universal reference gene suitable for all conditions under analysis, since these genes can also participate in different cellular functions, thus requiring a systematic validation of possible reference genes for each specific condition. The choice of reference genes on SNF7 gene normalization confirmed that unstable reference genes might drastically change the expression profile analysis of target candidate genes. PMID:29419794

Four out of eight genes in a mouse chromosome 7 congenic donor region are candidate obesity genes.

PubMed

Sarahan, Kari A; Fisler, Janis S; Warden, Craig H

2011-09-22

We previously identified a region of mouse chromosome 7 that influences body fat mass in F2 littermates of congenic × background intercrosses. Current analyses revealed that alleles in the donor region of the subcongenic B6.C-D7Mit318 (318) promoted a twofold increase in adiposity in homozygous lines of 318 compared with background C57BL/6ByJ (B6By) mice. Parent-of-origin effects were discounted through cross-fostering studies and an F1 reciprocal cross. Mapping of the donor region revealed that it has a maximal size of 2.8 Mb (minimum 1.8 Mb) and contains a maximum of eight protein coding genes. Quantitative PCR in whole brain, liver, and gonadal white adipose tissue (GWAT) revealed differential expression between genotypes for three genes in females and two genes in males. Alpha-2,8-sialyltransferase 8B (St8sia2) showed reduced 318 mRNA levels in brain for females and males and in GWAT for females only. Both sexes of 318 mice had reduced Repulsive guidance molecule-a (Rgma) expression in GWAT. In brain, Family with sequence similarity 174 member b (Fam174b) had increased expression in 318 females, whereas Chromodomain helicase DNA binding protein 2 (Chd2-2) had reduced expression in 318 males. No donor region genes were differentially expressed in liver. Sequence analysis of coding exons for all genes in the 318 donor region revealed only one single nucleotide polymorphism that produced a nonsynonymous missense mutation, Gln7Pro, in Fam174b. Our findings highlight the difficulty of using expression and sequence to identify quantitative trait genes underlying obesity even in small genomic regions.

Selection and assessment of reference genes for quantitative PCR normalization in migratory locust Locusta migratoria (Orthoptera: Acrididae).

PubMed

Yang, Qingpo; Li, Zhen; Cao, Jinjun; Zhang, Songdou; Zhang, Huaijiang; Wu, Xiaoyun; Zhang, Qingwen; Liu, Xiaoxia

2014-01-01

Locusta migratoria is a classic hemimetamorphosis insect and has caused widespread economic damage to crops as a migratory pest. Researches on the expression pattern of functional genes in L. migratoria have drawn focus in recent years, especially with the release of genome information. Real-time quantitative PCR is the most reproducible and sensitive approach for detecting transcript expression levels of target genes, but optimal internal standards are key factors for its accuracy and reliability. Therefore, it's necessary to provide a systematic stability assessment of internal control for well-performed tests of target gene expression profile. In this study, twelve candidate genes (Ach, Act, Cht2, EF1α, RPL32, Hsp70, Tub, RP49, SDH, GAPDH, 18S, and His) were analyzed with four statistical methods: the delta Ct approach, geNorm, Bestkeeper and NormFinder. The results from these analyses aimed to choose the best suitable reference gene across different experimental situations for gene profile study in L. migratoria. The result demonstrated that for different developmental stages, EF1α, Hsp70 and RPL32 exhibited the most stable expression status for all samples; EF1α and RPL32 were selected as the best reference genes for studies involving embryo and larvae stages, while SDH and RP49 were identified for adult stage. The best-ranked reference genes across different tissues are RPL32, Hsp70 and RP49. For abiotic treatments, the most appropriate genes we identified were as follows: Act and SDH for larvae subjected to different insecticides; RPL32 and Ach for larvae exposed to different temperature treatments; and Act and Ach for larvae suffering from starvation. The present report should facilitate future researches on gene expression in L. migratoria with accessibly optimal reference genes under different experimental contexts.

Exploring Valid Reference Genes for Quantitative Real-Time PCR Analysis in Sesamia inferens (Lepidoptera: Noctuidae)

PubMed Central

Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

2015-01-01

The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (−8, −6, −4, −2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens. PMID:25585250

Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations

PubMed Central

Brodsky, Arthur Nathan; Caldwell, Mary; Bae, Sooneon; Harcum, Sarah W.

2014-01-01

NS0 and Chinese hamster ovary (CHO) cell lines are used to produce recombinant proteins for human therapeutics; however, ammonium accumulation can negatively impact cell growth, recombinant protein production, and protein glycosylation. To improve product quality and decrease costs, the relationship between ammonium and protein glycosylation needs to be elucidated. While ammonium has been shown to adversely affect glycosylation-related gene expression in CHO cells, NS0 studies have not been performed. Therefore, this study sought to determine if glycosylation in NS0 cells were ammonium-sensitive at the gene expression level. Using a DNA microarray that contained mouse glycosylation-related and housekeeping genes, the of these genes was analysed in response to various culture conditions – elevated ammonium, elevated salt, and elevated ammonium with proline. Surprisingly, no significant differences in gene expression levels were observed between the control and these conditions. Further, the elevated ammonium cultures were analysed using real-time quantitative reverse transcriptase PCR (qRT-PCR) for key glycosylation genes, and the qRT-PCR results corroborated the DNA microarray results, demonstrating that NS0 cells are ammonium-insensitive at the gene expression level. Since NS0 are known to have elevated nucleotide sugar pools under ammonium stress, and none of the genes directly responsible for these metabolic pools were changed, consequently cellular control at the translational or substrate-level must be responsible for the universally observed decreased glycosylation quality under elevated ammonium. PMID:25062658

Identification and evaluation of reference genes for qRT-PCR normalization in Ganoderma lucidum.

PubMed

Xu, Jiang; Xu, ZhiChao; Zhu, YingJie; Luo, HongMei; Qian, Jun; Ji, AiJia; Hu, YuanLei; Sun, Wei; Wang, Bo; Song, JingYuan; Sun, Chao; Chen, ShiLin

2014-01-01

Quantitative real-time reverse transcription PCR (qRT-PCR) is a rapid, sensitive, and reliable technique for gene expression studies. The accuracy and reliability of qRT-PCR results depend on the stability of the reference genes used for gene normalization. Therefore, a systematic process of reference gene evaluation is needed. Ganoderma lucidum is a famous medicinal mushroom in East Asia. In the current study, 10 potential reference genes were selected from the G. lucidum genomic data. The sequences of these genes were manually curated, and primers were designed following strict criteria. The experiment was conducted using qRT-PCR, and the stability of each candidate gene was assessed using four commonly used statistical programs-geNorm, NormFinder, BestKeeper, and RefFinder. According to our results, PP2A was expressed at the most stable levels under different fermentation conditions, and RPL4 was the most stably expressed gene in different tissues. RPL4, PP2A, and β-tubulin are the most commonly recommended reference genes for normalizing gene expression in the entire sample set. The current study provides a foundation for the further use of qRT-PCR in G. lucidum gene analysis.

Interactions between genetic background, insulin resistance and β-cell function.

PubMed

Kahn, S E; Suvag, S; Wright, L A; Utzschneider, K M

2012-10-01

An interaction between genes and the environment is a critical component underlying the pathogenesis of the hyperglycaemia of type 2 diabetes. The development of more sophisticated techniques for studying gene variants and for analysing genetic data has led to the discovery of some 40 genes associated with type 2 diabetes. Most of these genes are related to changes in β-cell function, with a few associated with decreased insulin sensitivity and obesity. Interestingly, using quantitative traits based on continuous measures rather than dichotomous ones, it has become evident that not all genes associated with changes in fasting or post-prandial glucose are also associated with a diagnosis of type 2 diabetes. Identification of these gene variants has provided novel insights into the physiology and pathophysiology of the β-cell, including the identification of molecules involved in β-cell function that were not previously recognized as playing a role in this critical cell. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

Gene chips and arrays revealed: a primer on their power and their uses.

PubMed

Watson, S J; Akil, H

1999-03-01

This article provides an overview and general explanation of the rapidly developing area of gene chips and expression array technology. These are methods targeted at allowing the simultaneous study of thousands of genes or messenger RNAs under various physiological and pathological states. Their technical basis grows from the Human Genome Project. Both methods place DNA strands on glass computer chips (or microscope slides). Expression arrays start with complementary DNA (cDNA) clones derived from the EST data base, whereas Gene Chips synthesize oligonucleotides directly on the chip itself. Both are analyzed using image analysis systems, are capable of reading values from two different individuals at any one site, and can yield quantitative data for thousands of genes or mRNAs per slide. These methods promise to revolutionize molecular biology, cell biology, neuroscience and psychiatry. It is likely that this technology will radically open up our ability to study the actions and structure of the multiple genes involved in the complex genetics of brain disorders.

Evaluation of reference genes in Vibrio parahaemolyticus for gene expression analysis using quantitative RT-PCR

USDA-ARS?s Scientific Manuscript database

Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize the viru...

RNA-Seq and UHPLC-Q-TOF/MS Based Lipidomics Study in Lysiphlebia japonica.

PubMed

Gao, Xueke; Luo, Junyu; Lü, Limin; Zhang, LiJuan; Zhang, Shuai; Cui, Jinjie

2018-05-17

Lipids play an important role in energy storage, membrane structure stabilization and signaling. Parasitoids are excellent models to study lipidomics because a majority of them do not accumulate during their free-living life-stage. Studies on parasitoids have mostly focused on the changes in the lipids and gene transcripts in hosts and little attention has been devoted to lipidomics and transcriptomics changes in parasitoids. In this study, a relative quantitative analysis of lipids and their gene transcripts in 3-days-old Lysiphlebia japonica larva (3 days after spawning) and pupae were performed using liquid chromatography, mass spectrometry and RNA-seq. Thirty-three glycerolipids and 250 glycerophospholipids were identified in this study; all triglycerides and the vast majority of phospholipids accumulated in the pupal stage. This was accompanied by differentially regulated lipid uptake and remolding. Furthermore, our data showed that gene transcription was up-regulated in key nutrient metabolic pathways involved in lipid synthesis in 3-days-old larvae. Finally, our data suggests that larva and pupa of L. japonica may lack the ability for fatty acids synthesis. A comprehensive, quantitative, and expandable resource was provided for further studies of metabolic regulation and molecular mechanisms underlying parasitic response to hosts defense.

Evaluation and Validation of Reference Genes for qRT-PCR Normalization in Frankliniella occidentalis (Thysanoptera:Thripidae)

PubMed Central

Zheng, Yu-Tao; Li, Hong-Bo; Lu, Ming-Xing; Du, Yu-Zhou

2014-01-01

Quantitative real time PCR (qRT-PCR) has emerged as a reliable and reproducible technique for studying gene expression analysis. For accurate results, the normalization of data with reference genes is particularly essential. Once the transcriptome sequencing of Frankliniella occidentalis was completed, numerous unigenes were identified and annotated. Unfortunately, there are no studies on the stability of reference genes used in F. occidentalis. In this work, seven candidate reference genes, including actin, 18S rRNA, H3, tubulin, GAPDH, EF-1 and RPL32, were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper, geNorm, Normfinder and the comparative ΔCt method. Because the rankings of the reference genes provided by each of the four programs were different, we chose a user-friendly web-based comprehensive tool RefFinder to get the final ranking. The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures. In this study, we validated the suitable reference genes in F. occidentalis for gene expression profiling under different experimental conditions. The choice of internal standard is very important in the normalization of the target gene expression levels, thus validating and selecting the best genes will help improve the quality of gene expression data of F. occidentalis. What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects. PMID:25356721

Evaluation and validation of reference genes for qRT-PCR normalization in Frankliniella occidentalis (Thysanoptera: Thripidae).

PubMed

Zheng, Yu-Tao; Li, Hong-Bo; Lu, Ming-Xing; Du, Yu-Zhou

2014-01-01

Quantitative real time PCR (qRT-PCR) has emerged as a reliable and reproducible technique for studying gene expression analysis. For accurate results, the normalization of data with reference genes is particularly essential. Once the transcriptome sequencing of Frankliniella occidentalis was completed, numerous unigenes were identified and annotated. Unfortunately, there are no studies on the stability of reference genes used in F. occidentalis. In this work, seven candidate reference genes, including actin, 18S rRNA, H3, tubulin, GAPDH, EF-1 and RPL32, were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper, geNorm, Normfinder and the comparative ΔCt method. Because the rankings of the reference genes provided by each of the four programs were different, we chose a user-friendly web-based comprehensive tool RefFinder to get the final ranking. The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures. In this study, we validated the suitable reference genes in F. occidentalis for gene expression profiling under different experimental conditions. The choice of internal standard is very important in the normalization of the target gene expression levels, thus validating and selecting the best genes will help improve the quality of gene expression data of F. occidentalis. What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects.

Molecular biology of Homo sapiens: Abstracts of papers presented at the 51st Cold Spring Harbor symposium on quantitative biology

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

Watson, J.D.; Siniscalco, M.

This volume contains abstracts of papers presented at the 51st Cold Springs Harbor Symposium on Quantitative Biology. The topic for this meeting was the ''Molecular Biology of Homo sapiens.'' Sessions were entitled Human Gene Map, Human Cancer Genes, Genetic Diagnosis, Human Evolution, Drugs Made Off Human Genes, Receptors, and Gene Therapy. (DT)

Identification of suitable qPCR reference genes in leaves of Brassica oleracea under abiotic stresses.

PubMed

Brulle, Franck; Bernard, Fabien; Vandenbulcke, Franck; Cuny, Damien; Dumez, Sylvain

2014-04-01

Real-time quantitative PCR is nowadays a standard method to study gene expression variations in various samples and experimental conditions. However, to interpret results accurately, data normalization with appropriate reference genes appears to be crucial. The present study describes the identification and the validation of suitable reference genes in Brassica oleracea leaves. Expression stability of eight candidates was tested following drought and cold abiotic stresses by using three different softwares (BestKeeper, NormFinder and geNorm). Four genes (BolC.TUB6, BolC.SAND1, BolC.UBQ2 and BolC.TBP1) emerged as the most stable across the tested conditions. Further gene expression analysis of a drought- and a cold-responsive gene (BolC.DREB2A and BolC.ELIP, respectively), confirmed the stability and the reliability of the identified reference genes when used for normalization in the leaves of B. oleracea. These four genes were finally tested upon a benzene exposure and all appeared to be useful reference genes along this toxicological condition. These results provide a good starting point for future studies involving gene expression measurement on leaves of B. oleracea exposed to environmental modifications.

A Checklist for Successful Quantitative Live Cell Imaging in Systems Biology

PubMed Central

Sung, Myong-Hee

2013-01-01

Mathematical modeling of signaling and gene regulatory networks has provided unique insights about systems behaviors for many cell biological problems of medical importance. Quantitative single cell monitoring has a crucial role in advancing systems modeling of molecular networks. However, due to the multidisciplinary techniques that are necessary for adaptation of such systems biology approaches, dissemination to a wide research community has been relatively slow. In this essay, I focus on some technical aspects that are often under-appreciated, yet critical in harnessing live cell imaging methods to achieve single-cell-level understanding and quantitative modeling of molecular networks. The importance of these technical considerations will be elaborated with examples of successes and shortcomings. Future efforts will benefit by avoiding some pitfalls and by utilizing the lessons collectively learned from recent applications of imaging in systems biology. PMID:24709701

The effects of dominance, regular inbreeding and sampling design on Q(ST), an estimator of population differentiation for quantitative traits.

PubMed

Goudet, Jérôme; Büchi, Lucie

2006-02-01

To test whether quantitative traits are under directional or homogenizing selection, it is common practice to compare population differentiation estimates at molecular markers (F(ST)) and quantitative traits (Q(ST)). If the trait is neutral and its determinism is additive, then theory predicts that Q(ST) = F(ST), while Q(ST) > F(ST) is predicted under directional selection for different local optima, and Q(ST) < F(ST) is predicted under homogenizing selection. However, nonadditive effects can alter these predictions. Here, we investigate the influence of dominance on the relation between Q(ST) and F(ST) for neutral traits. Using analytical results and computer simulations, we show that dominance generally deflates Q(ST) relative to F(ST). Under inbreeding, the effect of dominance vanishes, and we show that for selfing species, a better estimate of Q(ST) is obtained from selfed families than from half-sib families. We also compare several sampling designs and find that it is always best to sample many populations (>20) with few families (five) rather than few populations with many families. Provided that estimates of Q(ST) are derived from individuals originating from many populations, we conclude that the pattern Q(ST) > F(ST), and hence the inference of directional selection for different local optima, is robust to the effect of nonadditive gene actions.

A hot topic: the genetics of adaptation to geothermal vents in Mimulus guttatus.

PubMed

Ferris, Kathleen G

2016-11-01

Identifying the individual loci and mutations that underlie adaptation to extreme environments has long been a goal of evolutionary biology. However, finding the genes that underlie adaptive traits is difficult for several reasons. First, because many traits and genes evolve simultaneously as populations diverge, it is difficult to disentangle adaptation from neutral demographic processes. Second, finding the individual loci involved in any trait is challenging given the respective limitations of quantitative and population genetic methods. In this issue of Molecular Ecology, Hendrick et al. (2016) overcome these difficulties and determine the genetic basis of microgeographic adaptation between geothermal vent and nonthermal populations of Mimulus guttatus in Yellowstone National Park. The authors accomplish this by combining population and quantitative genetic techniques, a powerful, but labour-intensive, strategy for identifying individual causative adaptive loci that few studies have used (Stinchcombe & Hoekstra ). In a previous common garden experiment (Lekberg et al. 2012), thermal M. guttatus populations were found to differ from their closely related nonthermal neighbours in various adaptive phenotypes including trichome density. Hendrick et al. (2016) combine quantitative trait loci (QTL) mapping, population genomic scans for selection and admixture mapping to identify a single genetic locus underlying differences in trichome density between thermal and nonthermal M. guttatus. The candidate gene, R2R3 MYB, is homologous to genes involved in trichome development across flowering plants. The major trichome QTL, Tr14, is also involved in trichome density differences in an independent M. guttatus population comparison (Holeski et al. 2010) making this an example of parallel genetic evolution. © 2016 John Wiley & Sons Ltd.

Real-time quantitative reverse transcription-PCR assay for renal cell carcinoma-associated antigen G250.

PubMed

Chuanzhong, Ye; Ming, Guan; Fanglin, Zhang; Haijiao, Chen; Zhen, Lin; Shiping, Chen; YongKang, Zhang

2002-04-01

Gene amplification/expression of G250 is a major event in human renal tumorigenesis. G250-based therapeutic agents and G250-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen G250 alterations in renal cell cancer (RCC) patients and investigate the relationship between G250 mRNA expression and RCC. We developed a quantitative RT-PCR assay for the measurement of G250 mRNA expression using a real-time procedure based on the use of fluorogenic probes and the ABI PRISM 7700 Sequence Detector System. The method has been applied to the measurement of quantitative mRNA level of G250 in 31 cases RCC and 6 normal renal tissues. The dynamic range was 10(3)-10(8). The relationship between Ct and log starting concentration was linear (r=0.99). G250 expression was present in all RCCs with G250 amplification but was absent in normal ones. G250 mRNA expression ranged from 2.9 x 10(3) to 6.5 x 10(7) copy/microg RNA, with a mean value of 3.5 x 10(6) copy/microg RNA. The expression of G250 revealed an inverse correlation to tumor grade. G250 mRNA level did not correlate with the cell types and clinical stages (P>0.05). G250 has the potential to be used as a marker of diagnosis and increasing proliferation in RCC. This new simple, rapid, semi-automated assay was a major alternative to competitive PCR and Northern blot analysis for gene alteration analysis in human tumors and might be a powerful tool for large randomized, prospective cooperative group trials and supporting future G250-based biological and gene therapy approaches.

Effect of carbon monoxide on gene expression in cerebrocortical astrocytes: Validation of reference genes for quantitative real-time PCR.

PubMed

Oliveira, Sara R; Vieira, Helena L A; Duarte, Carlos B

2015-09-15

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a widely used technique to characterize changes in gene expression in complex cellular and tissue processes, such as cytoprotection or inflammation. The accurate assessment of changes in gene expression depends on the selection of adequate internal reference gene(s). Carbon monoxide (CO) affects several metabolic pathways and de novo protein synthesis is crucial in the cellular responses to this gasotransmitter. Herein a selection of commonly used reference genes was analyzed to identify the most suitable internal control genes to evaluate the effect of CO on gene expression in cultured cerebrocortical astrocytes. The cells were exposed to CO by treatment with CORM-A1 (CO releasing molecule A1) and four different algorithms (geNorm, NormFinder, Delta Ct and BestKeeper) were applied to evaluate the stability of eight putative reference genes. Our results indicate that Gapdh (glyceraldehyde-3-phosphate dehydrogenase) together with Ppia (peptidylpropyl isomerase A) is the most suitable gene pair for normalization of qRT-PCR results under the experimental conditions used. Pgk1 (phosphoglycerate kinase 1), Hprt1 (hypoxanthine guanine phosphoribosyl transferase I), Sdha (Succinate Dehydrogenase Complex, Subunit A), Tbp (TATA box binding protein), Actg1 (actin gamma 1) and Rn18s (18S rRNA) genes presented less stable expression profiles in cultured cortical astrocytes exposed to CORM-A1 for up to 60 min. For validation, we analyzed the effect of CO on the expression of Bdnf and bcl-2. Different results were obtained, depending on the reference genes used. A significant increase in the expression of both genes was found when the results were normalized with Gapdh and Ppia, in contrast with the results obtained when the other genes were used as reference. These findings highlight the need for a proper and accurate selection of the reference genes used in the quantification of qRT-PCR results in studies on the effect of CO in gene expression. Copyright © 2015 Elsevier Inc. All rights reserved.

Berry and phenology-related traits in grapevine (Vitis vinifera L.): From Quantitative Trait Loci to underlying genes

PubMed Central

Costantini, Laura; Battilana, Juri; Lamaj, Flutura; Fanizza, Girolamo; Grando, Maria Stella

2008-01-01

Background The timing of grape ripening initiation, length of maturation period, berry size and seed content are target traits in viticulture. The availability of early and late ripening varieties is desirable for staggering harvest along growing season, expanding production towards periods when the fruit gets a higher value in the market and ensuring an optimal plant adaptation to climatic and geographic conditions. Berry size determines grape productivity; seedlessness is especially demanded in the table grape market and is negatively correlated to fruit size. These traits result from complex developmental processes modified by genetic, physiological and environmental factors. In order to elucidate their genetic determinism we carried out a quantitative analysis in a 163 individuals-F1 segregating progeny obtained by crossing two table grape cultivars. Results Molecular linkage maps covering most of the genome (2n = 38 for Vitis vinifera) were generated for each parent. Eighteen pairs of homologous groups were integrated into a consensus map spanning over 1426 cM with 341 markers (mainly microsatellite, AFLP and EST-derived markers) and an average map distance between loci of 4.2 cM. Segregating traits were evaluated in three growing seasons by recording flowering, veraison and ripening dates and by measuring berry size, seed number and weight. QTL (Quantitative Trait Loci) analysis was carried out based on single marker and interval mapping methods. QTLs were identified for all but one of the studied traits, a number of them steadily over more than one year. Clusters of QTLs for different characters were detected, suggesting linkage or pleiotropic effects of loci, as well as regions affecting specific traits. The most interesting QTLs were investigated at the gene level through a bioinformatic analysis of the underlying Pinot noir genomic sequence. Conclusion Our results revealed novel insights into the genetic control of relevant grapevine features. They provide a basis for performing marker-assisted selection and testing the role of specific genes in trait variation. PMID:18419811

Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects.

PubMed

Shakeel, Muhammad; Rodriguez, Alicia; Tahir, Urfa Bin; Jin, Fengliang

2018-02-01

Whenever gene expression is being examined, it is essential that a normalization process is carried out to eliminate non-biological variations. The use of reference genes, such as glyceraldehyde-3-phosphate dehydrogenase, actin, and ribosomal protein genes, is the usual method of choice for normalizing gene expression. Although reference genes are used to normalize target gene expression, a major problem is that the stability of these genes differs among tissues, developmental stages, species, and responses to abiotic factors. Therefore, the use and validation of multiple reference genes are required. This review discusses the reasons that why RT-qPCR has become the preferred method for validating results of gene expression profiles, the use of specific and non-specific dyes and the importance of use of primers and probes for qPCR as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. The conflicts arising in the use of classical reference genes in gene normalization and their replacement with novel references are also discussed by citing the high stability and low stability of classical and novel reference genes under various biotic and abiotic experimental conditions by employing various methods applied for the reference genes amplification.

Identification of optimal reference genes for RT-qPCR in the rat hypothalamus and intestine for the study of obesity.

PubMed

Li, B; Matter, E K; Hoppert, H T; Grayson, B E; Seeley, R J; Sandoval, D A

2014-02-01

Obesity has a complicated metabolic pathology, and defining the underlying mechanisms of obesity requires integrative studies with molecular end points. Real-time quantitative PCR (RT-qPCR) is a powerful tool that has been widely utilized. However, the importance of using carefully validated reference genes in RT-qPCR seems to have been overlooked in obesity-related research. The objective of this study was to select a set of reference genes with stable expressions to be used for RT-qPCR normalization in rats under fasted vs re-fed and chow vs high-fat diet (HFD) conditions. Male long-Evans rats were treated under four conditions: chow/fasted, chow/re-fed, HFD/fasted and HFD/re-fed. Expression stabilities of 13 candidate reference genes were evaluated in the rat hypothalamus, duodenum, jejunum and ileum using the ReFinder software program. The optimal number of reference genes needed for RT-qPCR analyses was determined using geNorm. Using geNorm analysis, we found that it was sufficient to use the two most stably expressed genes as references in RT-qPCR analyses for each tissue under specific experimental conditions. B2M and RPLP0 in the hypothalamus, RPS18 and HMBS in the duodenum, RPLP2 and RPLP0 in the jejunum and RPS18 and YWHAZ in the ileum were the most suitable pairs for a normalization study when the four aforementioned experimental conditions were considered. Our study demonstrates that gene expression levels of reference genes commonly used in obesity-related studies, such as ACTB or RPS18, are altered by changes in acute or chronic energy status. These findings underline the importance of using reference genes that are stable in expression across experimental conditions when studying the rat hypothalamus and intestine, because these tissues have an integral role in the regulation of energy homeostasis. It is our hope that this study will raise awareness among obesity researchers on the essential need for reference gene validation in gene expression studies.

clusterProfiler: an R package for comparing biological themes among gene clusters.

PubMed

Yu, Guangchuang; Wang, Li-Gen; Han, Yanyan; He, Qing-Yu

2012-05-01

Increasing quantitative data generated from transcriptomics and proteomics require integrative strategies for analysis. Here, we present an R package, clusterProfiler that automates the process of biological-term classification and the enrichment analysis of gene clusters. The analysis module and visualization module were combined into a reusable workflow. Currently, clusterProfiler supports three species, including humans, mice, and yeast. Methods provided in this package can be easily extended to other species and ontologies. The clusterProfiler package is released under Artistic-2.0 License within Bioconductor project. The source code and vignette are freely available at http://bioconductor.org/packages/release/bioc/html/clusterProfiler.html.

Validation of Reference Genes for Robust qRT-PCR Gene Expression Analysis in the Rice Blast Fungus Magnaporthe oryzae.

PubMed

Che Omar, Sarena; Bentley, Michael A; Morieri, Giulia; Preston, Gail M; Gurr, Sarah J

2016-01-01

The rice blast fungus causes significant annual harvest losses. It also serves as a genetically-tractable model to study fungal ingress. Whilst pathogenicity determinants have been unmasked and changes in global gene expression described, we know little about Magnaporthe oryzae cell wall remodelling. Our interests, in wall remodelling genes expressed during infection, vegetative growth and under exogenous wall stress, demand robust choice of reference genes for quantitative Real Time-PCR (qRT-PCR) data normalisation. We describe the expression stability of nine candidate reference genes profiled by qRT-PCR with cDNAs derived during asexual germling development, from sexual stage perithecia and from vegetative mycelium grown under various exogenous stressors. Our Minimum Information for Publication of qRT-PCR Experiments (MIQE) compliant analysis reveals a set of robust reference genes used to track changes in the expression of the cell wall remodelling gene MGG_Crh2 (MGG_00592). We ranked nine candidate reference genes by their expression stability (M) and report the best gene combination needed for reliable gene expression normalisation, when assayed in three tissue groups (Infective, Vegetative, and Global) frequently used in M. oryzae expression studies. We found that MGG_Actin (MGG_03982) and the 40S 27a ribosomal subunit MGG_40s (MGG_02872) proved to be robust reference genes for the Infection group and MGG_40s and MGG_Ef1 (Elongation Factor1-α) for both Vegetative and Global groups. Using the above validated reference genes, M. oryzae MGG_Crh2 expression was found to be significantly (p<0.05) elevated three-fold during vegetative growth as compared with dormant spores and two fold higher under cell wall stress (Congo Red) compared to growth under optimal conditions. We recommend the combinatorial use of two reference genes, belonging to the cytoskeleton and ribosomal synthesis functional groups, MGG_Actin, MGG_40s, MGG_S8 (Ribosomal subunit 40S S8) or MGG_Ef1, which demonstrated low M values across heterogeneous tissues. By contrast, metabolic pathway genes MGG_Fad (FAD binding domain-containing protein) and MGG_Gapdh (Glyceraldehyde-3-phosphate dehydrogenase) performed poorly, due to their lack of expression stability across samples.

Dissection of Host Susceptibility to Bacterial Infections and Its Toxins.

PubMed

Nashef, Aysar; Agbaria, Mahmoud; Shusterman, Ariel; Lorè, Nicola Ivan; Bragonzi, Alessandra; Wiess, Ervin; Houri-Haddad, Yael; Iraqi, Fuad A

2017-01-01

Infection is one of the leading causes of human mortality and morbidity. Exposure to microbial agents is obviously required. However, also non-microbial environmental and host factors play a key role in the onset, development and outcome of infectious disease, resulting in large of clinical variability between individuals in a population infected with the same microbe. Controlled and standardized investigations of the genetics of susceptibility to infectious disease are almost impossible to perform in humans whereas mouse models allow application of powerful genomic techniques to identify and validate causative genes underlying human diseases with complex etiologies. Most of current animal models used in complex traits diseases genetic mapping have limited genetic diversity. This limitation impedes the ability to create incorporated network using genetic interactions, epigenetics, environmental factors, microbiota, and other phenotypes. A novel mouse genetic reference population for high-resolution mapping and subsequently identifying genes underlying the QTL, namely the Collaborative Cross (CC) mouse genetic reference population (GRP) was recently developed. In this chapter, we discuss a variety of approaches using CC mice for mapping genes underlying quantitative trait loci (QTL) to dissect the host response to polygenic traits, including infectious disease caused by bacterial agents and its toxins.

Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

EPA Science Inventory

Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

Identification of downy mildew resistance gene candidates by positional cloning in maize (Zea mays subsp. mays; Poaceae)1

PubMed Central

Kim, Jae Yoon; Moon, Jun-Cheol; Kim, Hyo Chul; Shin, Seungho; Song, Kitae; Kim, Kyung-Hee; Lee, Byung-Moo

2017-01-01

Premise of the study: Positional cloning in combination with phenotyping is a general approach to identify disease-resistance gene candidates in plants; however, it requires several time-consuming steps including population or fine mapping. Therefore, in the present study, we suggest a new combined strategy to improve the identification of disease-resistance gene candidates. Methods and Results: Downy mildew (DM)–resistant maize was selected from five cultivars using a spreader row technique. Positional cloning and bioinformatics tools were used to identify the DM-resistance quantitative trait locus marker (bnlg1702) and 47 protein-coding gene annotations. Eventually, five DM-resistance gene candidates, including bZIP34, Bak1, and Ppr, were identified by quantitative reverse-transcription PCR (RT-PCR) without fine mapping of the bnlg1702 locus. Conclusions: The combined protocol with the spreader row technique, quantitative trait locus positional cloning, and quantitative RT-PCR was effective for identifying DM-resistance candidate genes. This cloning approach may be applied to other whole-genome-sequenced crops or resistance to other diseases. PMID:28224059

Identification of stable reference genes in differentiating human pluripotent stem cells.

PubMed

Holmgren, Gustav; Ghosheh, Nidal; Zeng, Xianmin; Bogestål, Yalda; Sartipy, Peter; Synnergren, Jane

2015-06-01

Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs. Copyright © 2015 the American Physiological Society.

Gene-expression profiles of epithelial cells treated with EMD in vitro: analysis using complementary DNA arrays.

PubMed

Kapferer, I; Schmidt, S; Gstir, R; Durstberger, G; Huber, L A; Vietor, I

2011-02-01

During surgical periodontal treatment, EMD is topically applied in order to facilitate regeneration of the periodontal ligament, acellular cementum and alveolar bone. Suppresion of epithelial down-growth is essential for successful periodontal regeneration; however, the underlying mechanisms of how EMD influences epithelial wound healing are poorly understood. In the present study, the effects of EMD on gene-expression profiling in an epithelial cell line (HSC-2) model were investigated. Gene-expression modifications, determined using a comparative genome-wide expression-profiling strategy, were independently validated by quantitative real-time RT-PCR. Additionally, cell cycle, cell growth and in vitro wound-healing assays were conducted. A set of 43 EMD-regulated genes was defined, which may be responsible for the reduced epithelial down-growth upon EMD application. Gene ontology analysis revealed genes that could be attributed to pathways of locomotion, developmental processes and associated processes such as regulation of cell size and cell growth. Additionally, eight regulated genes have previously been reported to take part in the process of epithelial-to-mesenchymal transition. Several independent experimental assays revealed significant inhibition of cell migration, growth and cell cycle by EMD. The set of EMD-regulated genes identified in this study offers the opportunity to clarify mechanisms underlying the effects of EMD on epithelial cells. Reduced epithelial repopulation of the dental root upon periodontal surgery may be the consequence of reduced migration and cell growth, as well as epithelial-to-mesenchymal transition. © 2010 John Wiley & Sons A/S.

qPCR for second year undergraduates: A short, structured inquiry to illustrate differential gene expression.

PubMed

McCauslin, Christine Seitz; Gunn, Kathryn Elaine; Pirone, Dana; Staiger, Jennifer

2015-01-01

We describe a structured inquiry laboratory exercise that examines transcriptional regulation of the NOS2 gene under conditions that simulate the inflammatory response in macrophages. Using quantitative PCR and the comparative CT method, students are able determine whether transcriptional activation of NOS2 occurs and to what degree. The exercise is aimed at second year undergraduates who possess basic knowledge of gene expression events. It requires only 4-5 hr of dedicated laboratory time and focuses on use of the primary literature, data analysis, and interpretation. Importantly, this exercise provides a mechanism to introduce the concept of differential gene expression and provides a starting point for development of more complex guided or open inquiry projects for students moving into upper level molecular biology, immunology, and biochemistry course work. © 2015 The International Union of Biochemistry and Molecular Biology.

Differential regulation of mnp2, a new manganese peroxidase-encoding gene from the ligninolytic fungus Trametes versicolor PRL 572.

PubMed

Johansson, Tomas; Nyman, Per Olof; Cullen, Daniel

2002-04-01

A peroxidase-encoding gene, mnp2, and its corresponding cDNA were characterized from the white-rot basidiomycete Trametes versicolor PRL 572. We used quantitative reverse transcriptase-mediated PCR to identify mnp2 transcripts in nutrient-limited stationary cultures. Although mnp2 lacks upstream metal response elements (MREs), addition of MnSO(4) to cultures increased mnp2 transcript levels 250-fold. In contrast, transcript levels of an MRE-containing gene of T. versicolor, mnp1, increased only eightfold under the same conditions. Thus, the manganese peroxidase genes in T. versicolor are differentially regulated, and upstream MREs are not necessarily involved. Our results support the hypothesis that fungal and plant peroxidases arose through an ancient duplication and folding of two structural domains, since we found the mnp1 and mnp2 polypeptides to have internal homology.

Differential Regulation of mnp2, a New Manganese Peroxidase-Encoding Gene from the Ligninolytic Fungus Trametes versicolor PRL 572

PubMed Central

Johansson, Tomas; Nyman, Per Olof; Cullen, Daniel

2002-01-01

A peroxidase-encoding gene, mnp2, and its corresponding cDNA were characterized from the white-rot basidiomycete Trametes versicolor PRL 572. We used quantitative reverse transcriptase-mediated PCR to identify mnp2 transcripts in nutrient-limited stationary cultures. Although mnp2 lacks upstream metal response elements (MREs), addition of MnSO4 to cultures increased mnp2 transcript levels 250-fold. In contrast, transcript levels of an MRE-containing gene of T. versicolor, mnp1, increased only eightfold under the same conditions. Thus, the manganese peroxidase genes in T. versicolor are differentially regulated, and upstream MREs are not necessarily involved. Our results support the hypothesis that fungal and plant peroxidases arose through an ancient duplication and folding of two structural domains, since we found the mnp1 and mnp2 polypeptides to have internal homology. PMID:11916737

A highly sensitive and accurate gene expression analysis by sequencing ("bead-seq") for a single cell.

PubMed

Matsunaga, Hiroko; Goto, Mari; Arikawa, Koji; Shirai, Masataka; Tsunoda, Hiroyuki; Huang, Huan; Kambara, Hideki

2015-02-15

Analyses of gene expressions in single cells are important for understanding detailed biological phenomena. Here, a highly sensitive and accurate method by sequencing (called "bead-seq") to obtain a whole gene expression profile for a single cell is proposed. A key feature of the method is to use a complementary DNA (cDNA) library on magnetic beads, which enables adding washing steps to remove residual reagents in a sample preparation process. By adding the washing steps, the next steps can be carried out under the optimal conditions without losing cDNAs. Error sources were carefully evaluated to conclude that the first several steps were the key steps. It is demonstrated that bead-seq is superior to the conventional methods for single-cell gene expression analyses in terms of reproducibility, quantitative accuracy, and biases caused during sample preparation and sequencing processes. Copyright © 2014 Elsevier Inc. All rights reserved.

Thermodynamics-based models of transcriptional regulation with gene sequence.

PubMed

Wang, Shuqiang; Shen, Yanyan; Hu, Jinxing

2015-12-01

Quantitative models of gene regulatory activity have the potential to improve our mechanistic understanding of transcriptional regulation. However, the few models available today have been based on simplistic assumptions about the sequences being modeled or heuristic approximations of the underlying regulatory mechanisms. In this work, we have developed a thermodynamics-based model to predict gene expression driven by any DNA sequence. The proposed model relies on a continuous time, differential equation description of transcriptional dynamics. The sequence features of the promoter are exploited to derive the binding affinity which is derived based on statistical molecular thermodynamics. Experimental results show that the proposed model can effectively identify the activity levels of transcription factors and the regulatory parameters. Comparing with the previous models, the proposed model can reveal more biological sense.

Oxidative stress provokes distinct transcriptional responses in the stress-tolerant atr7 and stress-sensitive loh2 Arabidopsis thaliana mutants as revealed by multi-parallel quantitative real-time PCR analysis of ROS marker and antioxidant genes.

PubMed

Mehterov, Nikolay; Balazadeh, Salma; Hille, Jacques; Toneva, Valentina; Mueller-Roeber, Bernd; Gechev, Tsanko

2012-10-01

The Arabidopsis thaliana atr7 mutant is tolerant to oxidative stress induced by paraquat (PQ) or the catalase inhibitor aminotriazole (AT), while its original background loh2 and wild-type plants are sensitive. Both, AT and PQ, which stimulate the intracellular formation of H₂O₂ or superoxide anions, respectively, trigger cell death in loh2 but do not lead to visible damage in atr7. To study gene expression during oxidative stress and ROS-induced programmed cell death, two platforms for multi-parallel quantitative real-time PCR (qRT-PCR) analysis of 217 antioxidant and 180 ROS marker genes were employed. The qRT-PCR analyses revealed AT- and PQ-induced expression of many ROS-responsive genes mainly in loh2, confirming that an oxidative burst plays a role in the activation of the cell death in this mutant. Some of the genes were specifically regulated by either AT or PQ, serving as markers for particular types of ROS. Genes significantly induced by both AT and PQ in loh2 included transcription factors (ANAC042/JUB1, ANAC102, DREB19, HSFA2, RRTF1, ZAT10, ZAT12, ethylene-responsive factors), signaling compounds, ferritins, alternative oxidases, and antioxidant enzymes. Many of these genes were upregulated in atr7 compared to loh2 under non-stress conditions at the first time point, indicating that higher basal levels of ROS and higher antioxidant capacity in atr7 are responsible for the enhanced tolerance to oxidative stress and suggesting a possible tolerance against multiple stresses of this mutant. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Evaluation of reference genes in mouse preimplantation embryos for gene expression studies using real-time quantitative RT-PCR (RT-qPCR).

PubMed

Jeong, Jae-Kyo; Kang, Min-Hee; Gurunathan, Sangiliyandi; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Kim, Jin-Hoi

2014-09-25

Real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) is the most sensitive, and valuable technique for rare mRNA detection. However, the expression profiles of reference genes under different experimental conditions, such as different mouse strains, developmental stage, and culture conditions have been poorly studied. mRNA stability of the actb, gapdh, sdha, ablim, ywhaz, sptbn, h2afz, tgfb1, 18 s and wrnip genes was analyzed. Using the NormFinder program, the most stable genes are as follows: h2afz for the B6D2F-1 and C57BL/6 strains; sptbn for ICR; h2afz for KOSOM and CZB cultures of B6D2F-1 and C57BL/6 strain-derived embryos; wrnip for M16 culture of B6D2F-1 and C57BL/6 strain-derived embryos; ywhaz, tgfb1, 18 s, 18 s, ywhaz, and h2afz for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst embryonic stages cultured in KSOM medium, respectively; h2afz, wrnip, wrnip, h2afz, ywhaz, and ablim for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in CZB medium, respectively; 18 s, h2afz, h2afz, actb, h2afz, and wrnip for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in M16 medium, respectively. These results demonstrated that candidate reference genes for normalization of target gene expression using RT-qPCR should be selected according to mouse strains, developmental stage, and culture conditions.

PGPR-mediated expression of salt tolerance gene in soybean through volatiles under sodium nitroprusside.

PubMed

Vaishnav, Anukool; Kumari, Sarita; Jain, Shekhar; Varma, Ajit; Tuteja, Narendra; Choudhary, Devendra Kumar

2016-11-01

Increasing evidence shows that nitric oxide (NO), a typical signaling molecule plays important role in development of plant and in bacteria-plant interaction. In the present study, we tested the effect of sodium nitroprusside (SNP)-a nitric oxide donor, on bacterial metabolism and its role in establishment of PGPR-plant interaction under salinity condition. In the present study, we adopted methods namely, biofilm formation assay, GC-MS analysis of bacterial volatiles, chemotaxis assay of root exudates (REs), measurement of electrolyte leakage and lipid peroxidation, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for gene expression. GC-MS analysis revealed that three new volatile organic compounds (VOCs) were expressed after treatment with SNP. Two VOCs namely, 4-nitroguaiacol and quinoline were found to promote soybean seed germination under 100 mM NaCl stress. Chemotaxis assay revealed that SNP treatment, altered root exudates profiling (SS-RE), found more attracted to Pseudomonas simiae bacterial cells as compared to non-treated root exudates (S-RE) under salt stress. Expression of Peroxidase (POX), catalase (CAT), vegetative storage protein (VSP), and nitrite reductase (NR) genes were up-regulated in T6 treatment seedlings, whereas, high affinity K + transporter (HKT1), lipoxygenase (LOX), polyphenol oxidase (PPO), and pyrroline-5-carboxylate synthase (P5CS) genes were down-regulated under salt stress. The findings suggest that NO improves the efficiency and establishment of PGPR strain in the plant environment during salt condition. This strategy may be applied on soybean plants to increase their growth during salinity stress. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gene expression analysis of disabled and re-induced isoprene emission by the tropical tree Ficus septica before and after cold ambient temperature exposure.

PubMed

Mutanda, Ishmael; Saitoh, Seikoh; Inafuku, Masashi; Aoyama, Hiroaki; Takamine, Tomonori; Satou, Kazuhito; Akutsu, Masako; Teruya, Kuniko; Tamotsu, Hinako; Shimoji, Makiko; Sunagawa, Haruki; Oku, Hirosuke

2016-07-01

Isoprene is the most abundant type of nonmethane, biogenic volatile organic compound in the atmosphere, and it is produced mainly by terrestrial plants. The tropical tree species Ficus septica Burm. F. (Rosales: Moraceae) has been shown to cease isoprene emissions when exposed to temperatures of 12 °C or lower and to re-induce isoprene synthesis upon subsequent exposure to temperatures of 30 °C or higher for 24 h. To elucidate the regulation of genes underlying the disabling and then induction of isoprene emission during acclimatization to ambient temperature, we conducted gene expression analyses of F. septica plants under changing temperature using quantitative real-time polymerase chain reaction and western blotting. Transcription levels were analyzed for 17 genes that are involved in metabolic pathways potentially associated with isoprene biosynthesis, including isoprene synthase (ispS). The protein levels of ispS were also measured. Changes in transcription and protein levels of the ispS gene, but not in the other assessed genes, showed identical temporal patterns to isoprene emission capacity under the changing temperature regime. The ispS protein levels strongly and positively correlated with isoprene emission capacity (R(2) = 0.92). These results suggest that transcriptional regulation of ispS gave rise to the temporal variation in isoprene emission capacity in response to changing temperature. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Bacterial growth laws and their applications

PubMed Central

SCOTT, Matthew; HWA, Terence

2011-01-01

Quantitative empirical relationships between cell composition and growth rate played an important role in the early days of microbiology. Gradually, the focus of the field began to shift from growth physiology to the ever more elaborate molecular mechanisms of regulation employed by the organisms. Advances in systems biology and biotechnology have renewed interest in the physiology of the cell as a whole. Furthermore, gene expression is known to be intimately coupled to the growth state of the cell. Here, we review recent efforts in characterizing such couplings, particularly the quantitative phenomenological approaches exploiting bacterial `growth laws.' These approaches point toward underlying design principles that can guide the predictive manipulation of cell behavior in the absence of molecular details. PMID:21592775

Comparative transcriptome profiling of chilling stress responsiveness in grafted watermelon seedlings.

PubMed

Xu, Jinhua; Zhang, Man; Liu, Guang; Yang, Xingping; Hou, Xilin

2016-12-01

Rootstock grafting may improve the resistance of watermelon plants to low temperatures. However, information regarding the molecular responses of rootstock grafted plants to chilling stress is limited. To elucidate the molecular mechanisms of chilling tolerance in grafted plants, the transcriptomic responses of grafted watermelon under chilling stress were analyzed using RNA-seq analysis. Sequencing data were used for digital gene expression (DGE) analysis to characterize the transcriptomic responses in grafted watermelon seedlings. A total of 702 differentially-expressed genes (DEGs) were found in rootstock grafted (RG) watermelon relative to self-grafted (SG) watermelon; among these genes, 522 genes were up-regulated and 180 were down-regulated. Additionally, 164 and 953 genes were found to specifically expressed in RG and SG seedlings under chilling stress, respectively. Functional annotations revealed that up-regulated DEGs are involved in protein processing, plant-pathogen interaction and the spliceosome, whereas down-regulated DEGs are associated with photosynthesis. Moreover, 13 DEGs were randomly selected for quantitative real time PCR (qRT-PCR) analysis. The expression profiles of these 13 DEGs were consistent with those detected by the DGE analysis, supporting the reliability of the DGE data. This work provides additional insight into the molecular basis of grafted watermelon responses to chilling stress. Copyright © 2016. Published by Elsevier Masson SAS.

Identification of differentially expressed genes in cucumber (Cucumis sativus L.) root under waterlogging stress by digital gene expression profile.

PubMed

Qi, Xiao-Hua; Xu, Xue-Wen; Lin, Xiao-Jian; Zhang, Wen-Jie; Chen, Xue-Hao

2012-03-01

High-throughput tag-sequencing (Tag-seq) analysis based on the Solexa Genome Analyzer platform was applied to analyze the gene expression profiling of cucumber plant at 5 time points over a 24h period of waterlogging treatment. Approximately 5.8 million total clean sequence tags per library were obtained with 143013 distinct clean tag sequences. Approximately 23.69%-29.61% of the distinct clean tags were mapped unambiguously to the unigene database, and 53.78%-60.66% of the distinct clean tags were mapped to the cucumber genome database. Analysis of the differentially expressed genes revealed that most of the genes were down-regulated in the waterlogging stages, and the differentially expressed genes mainly linked to carbon metabolism, photosynthesis, reactive oxygen species generation/scavenging, and hormone synthesis/signaling. Finally, quantitative real-time polymerase chain reaction using nine genes independently verified the tag-mapped results. This present study reveals the comprehensive mechanisms of waterlogging-responsive transcription in cucumber. Copyright © 2011 Elsevier Inc. All rights reserved.

Reverse-engineering of gene networks for regulating early blood development from single-cell measurements.

PubMed

Wei, Jiangyong; Hu, Xiaohua; Zou, Xiufen; Tian, Tianhai

2017-12-28

Recent advances in omics technologies have raised great opportunities to study large-scale regulatory networks inside the cell. In addition, single-cell experiments have measured the gene and protein activities in a large number of cells under the same experimental conditions. However, a significant challenge in computational biology and bioinformatics is how to derive quantitative information from the single-cell observations and how to develop sophisticated mathematical models to describe the dynamic properties of regulatory networks using the derived quantitative information. This work designs an integrated approach to reverse-engineer gene networks for regulating early blood development based on singel-cell experimental observations. The wanderlust algorithm is initially used to develop the pseudo-trajectory for the activities of a number of genes. Since the gene expression data in the developed pseudo-trajectory show large fluctuations, we then use Gaussian process regression methods to smooth the gene express data in order to obtain pseudo-trajectories with much less fluctuations. The proposed integrated framework consists of both bioinformatics algorithms to reconstruct the regulatory network and mathematical models using differential equations to describe the dynamics of gene expression. The developed approach is applied to study the network regulating early blood cell development. A graphic model is constructed for a regulatory network with forty genes and a dynamic model using differential equations is developed for a network of nine genes. Numerical results suggests that the proposed model is able to match experimental data very well. We also examine the networks with more regulatory relations and numerical results show that more regulations may exist. We test the possibility of auto-regulation but numerical simulations do not support the positive auto-regulation. In addition, robustness is used as an importantly additional criterion to select candidate networks. The research results in this work shows that the developed approach is an efficient and effective method to reverse-engineer gene networks using single-cell experimental observations.

A genome-wide association study of a global rice panel reveals resistance in Oryza sativa to root-knot nematodes

PubMed Central

Dimkpa, Stanley O. N.; Lahari, Zobaida; Shrestha, Roshi; Douglas, Alex; Gheysen, Godelieve; Price, Adam H.

2016-01-01

The root-knot nematode Meloidogyne graminicola is one of the most serious nematode pests worldwide and represents a major constraint on rice production. While variation in the susceptibility of Asian rice (Oryza sativa) exists, so far no strong and reliable resistance has been reported. Quantitative trait loci for partial resistance have been reported but no underlying genes have been tagged or cloned. Here, 332 accessions of the Rice Diversity Panel 1 were assessed for gall formation, revealing large variation across all subpopulations of rice and higher susceptibility in temperate japonica accessions. Accessions Khao Pahk Maw and LD 24 appeared to be resistant, which was confirmed in large pot experiments where no galls were observed. Detailed observations on these two accessions revealed no nematodes inside the roots 2 days after inoculation and very few females after 17 days (5 in Khao Pahk Maw and <1 in LD 24, in comparison with >100 in the susceptible controls). These two cultivars appear ideal donors for breeding root-knot nematode resistance. A genome-wide association study revealed 11 quantitative trait loci, two of which are close to epistatic loci detected in the Bala x Azucena population. The discussion highlights a small number of candidate genes worth exploring further, in particular many genes with lectin domains and genes on chromosome 11 with homology to the Hordeum Mla locus. PMID:26552884

Wheat TaSP gene improves salt tolerance in transgenic Arabidopsis thaliana.

PubMed

Ma, Xiaoli; Cui, Weina; Liang, Wenji; Huang, Zhanjing

2015-12-01

A novel salt-induced gene with unknown functions was cloned through analysis of gene expression profile of a salt-tolerant wheat mutant RH8706-49 under salt stress. The gene was named Triticum aestivum salt-related protein (TaSP) and deposited in GenBank (Accession No. KF307326). Quantitative polymerase chain reaction (qPCR) results showed that TaSP expression was induced under salt, abscisic acid (ABA), and polyethylene glycol (PEG) stresses. Subcellular localization revealed that TaSP was mainly localized in cell membrane. Overexpression of TaSP in Arabidopsis could improve salt tolerance of 35S::TaSP transgenic Arabidopsis. 35S::TaSP transgenic Arabidopsis lines after salt stress presented better physiological indexes than the control group. In the non-invasive micro-test (NMT), an evident Na(+) excretion was observed at the root tip of salt-stressed 35S::TaSP transgenic Arabidopsis. TaSP promoter was cloned, and its beta-glucuronidase (GUS) activities before and after ABA, salt, cold, heat, and salicylic acid (SA) stresses were determined. Full-length TaSP promoter contained ABA and salt response elements. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Serine Proteases-Like Genes in the Asian Rice Gall Midge Show Differential Expression in Compatible and Incompatible Interactions with Rice

PubMed Central

Sinha, Deepak Kumar; Lakshmi, Mulagondla; Anuradha, Ghanta; Rahman, Shaik J.; Siddiq, Ebrahimali A.; Bentur, Jagadish S.; Nair, Suresh

2011-01-01

The Asian rice gall midge, Orseolia oryzae (Wood-Mason), is a serious pest of rice. Investigations into the gall midge-rice interaction will unveil the underlying molecular mechanisms which, in turn, can be used as a tool to assist in developing suitable integrated pest management strategies. The insect gut is known to be involved in various physiological and biological processes including digestion, detoxification and interaction with the host. We have cloned and identified two genes, OoprotI and OoprotII, homologous to serine proteases with the conserved His87, Asp136 and Ser241 residues. OoProtI shared 52.26% identity with mosquito-type trypsin from Hessian fly whereas OoProtII showed 52.49% identity to complement component activated C1s from the Hessian fly. Quantitative real time PCR analysis revealed that both the genes were significantly upregulated in larvae feeding on resistant cultivar than in those feeding on susceptible cultivar. These results provide an opportunity to understand the gut physiology of the insect under compatible or incompatible interactions with the host. Phylogenetic analysis grouped these genes in the clade containing proteases of phytophagous insects away from hematophagous insects. PMID:21686154

Systematic profiling of Caenorhabditis elegans locomotive behaviors reveals additional components in G-protein Gαq signaling.

PubMed

Yu, Hui; Aleman-Meza, Boanerges; Gharib, Shahla; Labocha, Marta K; Cronin, Christopher J; Sternberg, Paul W; Zhong, Weiwei

2013-07-16

Genetic screens have been widely applied to uncover genetic mechanisms of movement disorders. However, most screens rely on human observations of qualitative differences. Here we demonstrate the application of an automatic imaging system to conduct a quantitative screen for genes regulating the locomotive behavior in Caenorhabditis elegans. Two hundred twenty-seven neuronal signaling genes with viable homozygous mutants were selected for this study. We tracked and recorded each animal for 4 min and analyzed over 4,400 animals of 239 genotypes to obtain a quantitative, 10-parameter behavioral profile for each genotype. We discovered 87 genes whose inactivation causes movement defects, including 50 genes that had never been associated with locomotive defects. Computational analysis of the high-content behavioral profiles predicted 370 genetic interactions among these genes. Network partition revealed several functional modules regulating locomotive behaviors, including sensory genes that detect environmental conditions, genes that function in multiple types of excitable cells, and genes in the signaling pathway of the G protein Gαq, a protein that is essential for animal life and behavior. We developed quantitative epistasis analysis methods to analyze the locomotive profiles and validated the prediction of the γ isoform of phospholipase C as a component in the Gαq pathway. These results provided a system-level understanding of how neuronal signaling genes coordinate locomotive behaviors. This study also demonstrated the power of quantitative approaches in genetic studies.

Expression of stress/defense-related genes in barley grown under space environment

NASA Astrophysics Data System (ADS)

Sugimoto, Manabu; Shagimardanova, Elena; Gusev, Oleg; Bingham, Gail; Levinskikh, Margarita; Sychev, Vladimir

Plants are exposed to the extreme environment in space, especially space radiation is suspected to induce oxidative stress by generating high-energy free radicals and microgravity would enhance the effect of space radiation, however, current understandings of plant growth and responses on this synergistic effect of radiation and microgravity is limited to a few experiments. In this study, expression of stress/defense-related genes in barley grown under space environment was analyzed by RT-PCR and DNA microarray experiments to understand plant responses and adaptation to space environment and to develop the space stress-tolerant plants. The seeds of barley, Hordeum vulgare L. cv. Haruna nijo, kept in the international space station (ISS) over 4 months, were germinated after 3 days of irrigation in LADA plant growth chamber onboard Russian segment of ISS and the final germination ratio was over 90 %. The height of plants was about 50 to 60 cm and flag leaf has been opened after 26 days of irrigation under 24 hr lighting, showing the similar growth to ground-grown barley. Expression levels of stress/defense-related genes in space-grown barley were compared to those in ground-grown barley by semi-quantitative RT-PCR. In 17 stress/defense-related genes that are up-regulated by oxidative stress or other abiotic stress, only catalase, pathogenesis-related protein 13, chalcone synthase, and phenylalanine ammonia-lyase genes were increased in space-grown barley. DNA microarrya analysis with the GeneChip Barley Genome Array showed the similar expression profiles of the stress/defense-related genes to those by RT-PCR experiment, suggesting that the barley germinated and grown in LADA onboard ISS is not damaged by space environment, especially oxidative stress induced by space radiation and microgravity.

Genome-wide identification and characterisation of F-box family in maize.

PubMed

Jia, Fengjuan; Wu, Bingjiang; Li, Hui; Huang, Jinguang; Zheng, Chengchao

2013-11-01

F-box-containing proteins, as the key components of the protein degradation machinery, are widely distributed in higher plants and are considered as one of the largest known families of regulatory proteins. The F-box protein family plays a crucial role in plant growth and development and in response to biotic and abiotic stresses. However, systematic analysis of the F-box family in maize (Zea mays) has not been reported yet. In this paper, we identified and characterised the maize F-box genes in a genome-wide scale, including phylogenetic analysis, chromosome distribution, gene structure, promoter analysis and gene expression profiles. A total of 359 F-box genes were identified and divided into 15 subgroups by phylogenetic analysis. The F-box domain was relatively conserved, whereas additional motifs outside the F-box domain may indicate the functional diversification of maize F-box genes. These genes were unevenly distributed in ten maize chromosomes, suggesting that they expanded in the maize genome because of tandem and segmental duplication events. The expression profiles suggested that the maize F-box genes had temporal and spatial expression patterns. Putative cis-acting regulatory DNA elements involved in abiotic stresses were observed in maize F-box gene promoters. The gene expression profiles under abiotic stresses also suggested that some genes participated in stress responsive pathways. Furthermore, ten genes were chosen for quantitative real-time PCR analysis under drought stress and the results were consistent with the microarray data. This study has produced a comparative genomics analysis of the maize ZmFBX gene family that can be used in further studies to uncover their roles in maize growth and development.

Maternal Diets Trigger Sex-Specific Divergent Trajectories of Gene Expression and Epigenetic Systems in Mouse Placenta

PubMed Central

Gabory, Anne; Ferry, Laure; Fajardy, Isabelle; Jouneau, Luc; Gothié, Jean-David; Vigé, Alexandre; Fleur, Cécile; Mayeur, Sylvain; Gallou-Kabani, Catherine; Gross, Marie-Sylvie; Attig, Linda; Vambergue, Anne; Lesage, Jean; Reusens, Brigitte; Vieau, Didier; Remacle, Claude; Jais, Jean-Philippe; Junien, Claudine

2012-01-01

Males and females responses to gestational overnutrition set the stage for subsequent sex-specific differences in adult onset non communicable diseases. Placenta, as a widely recognized programming agent, contibutes to the underlying processes. According to our previous findings, a high-fat diet during gestation triggers sex-specific epigenetic alterations within CpG and throughout the genome, together with the deregulation of clusters of imprinted genes. We further investigated the impact of diet and sex on placental histology, transcriptomic and epigenetic signatures in mice. Both basal gene expression and response to maternal high-fat diet were sexually dimorphic in whole placentas. Numerous genes showed sexually dimorphic expression, but only 11 genes regardless of the diet. In line with the key role of genes belonging to the sex chromosomes, 3 of these genes were Y-specific and 3 were X-specific. Amongst all the genes that were differentially expressed under a high-fat diet, only 16 genes were consistently affected in both males and females. The differences were not only quantitative but remarkably qualitative. The biological functions and networks of genes dysregulated differed markedly between the sexes. Seven genes of the epigenetic machinery were dysregulated, due to effects of diet, sex or both, including the Y- and X-linked histone demethylase paralogues Kdm5c and Kdm5d, which could mark differently male and female epigenomes. The DNA methyltransferase cofactor Dnmt3l gene expression was affected, reminiscent of our previous observation of changes in global DNA methylation. Overall, this striking sexual dimorphism of programming trajectories impose a considerable revision of the current dietary interventions protocols. PMID:23144842

Functional relevance for type 1 diabetes mellitus-associated genetic variants by using integrative analyses.

PubMed

Qiu, Ying-Hua; Deng, Fei-Yan; Tang, Zai-Xiang; Jiang, Zhen-Huan; Lei, Shu-Feng

2015-10-01

Type 1 diabetes mellitus (type 1 DM) is an autoimmune disease. Although genome-wide association studies (GWAS) and meta-analyses have successfully identified numerous type 1 DM-associated susceptibility loci, the underlying mechanisms for these susceptibility loci are currently largely unclear. Based on publicly available datasets, we performed integrative analyses (i.e., integrated gene relationships among implicated loci, differential gene expression analysis, functional prediction and functional annotation clustering analysis) and combined with expression quantitative trait loci (eQTL) results to further explore function mechanisms underlying the associations between genetic variants and type 1 DM. Among a total of 183 type 1 DM-associated SNPs, eQTL analysis showed that 17 SNPs with cis-regulated eQTL effects on 9 genes. All the 9 eQTL genes enrich in immune-related pathways or Gene Ontology (GO) terms. Functional prediction analysis identified 5 SNPs located in transcription factor (TF) binding sites. Of the 9 eQTL genes, 6 (TAP2, HLA-DOB, HLA-DQB1, HLA-DQA1, HLA-DRB5 and CTSH) were differentially expressed in type 1 DM-associated related cells. Especially, rs3825932 in CTSH has integrative functional evidence supporting the association with type 1 DM. These findings indicated that integrative analyses can yield important functional information to link genetic variants and type 1 DM. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Construction of a high-density genetic map by specific locus amplified fragment sequencing (SLAF-seq) and its application to Quantitative Trait Loci (QTL) analysis for boll weight in upland cotton (Gossypium hirsutum.).

PubMed

Zhang, Zhen; Shang, Haihong; Shi, Yuzhen; Huang, Long; Li, Junwen; Ge, Qun; Gong, Juwu; Liu, Aiying; Chen, Tingting; Wang, Dan; Wang, Yanling; Palanga, Koffi Kibalou; Muhammad, Jamshed; Li, Weijie; Lu, Quanwei; Deng, Xiaoying; Tan, Yunna; Song, Weiwu; Cai, Juan; Li, Pengtao; Rashid, Harun or; Gong, Wankui; Yuan, Youlu

2016-04-11

Upland Cotton (Gossypium hirsutum) is one of the most important worldwide crops it provides natural high-quality fiber for the industrial production and everyday use. Next-generation sequencing is a powerful method to identify single nucleotide polymorphism markers on a large scale for the construction of a high-density genetic map for quantitative trait loci mapping. In this research, a recombinant inbred lines population developed from two upland cotton cultivars 0-153 and sGK9708 was used to construct a high-density genetic map through the specific locus amplified fragment sequencing method. The high-density genetic map harbored 5521 single nucleotide polymorphism markers which covered a total distance of 3259.37 cM with an average marker interval of 0.78 cM without gaps larger than 10 cM. In total 18 quantitative trait loci of boll weight were identified as stable quantitative trait loci and were detected in at least three out of 11 environments and explained 4.15-16.70 % of the observed phenotypic variation. In total, 344 candidate genes were identified within the confidence intervals of these stable quantitative trait loci based on the cotton genome sequence. These genes were categorized based on their function through gene ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis and eukaryotic orthologous groups analysis. This research reported the first high-density genetic map for Upland Cotton (Gossypium hirsutum) with a recombinant inbred line population using single nucleotide polymorphism markers developed by specific locus amplified fragment sequencing. We also identified quantitative trait loci of boll weight across 11 environments and identified candidate genes within the quantitative trait loci confidence intervals. The results of this research would provide useful information for the next-step work including fine mapping, gene functional analysis, pyramiding breeding of functional genes as well as marker-assisted selection.

Quantitative Resistance to Plant Pathogens in Pyramiding Strategies for Durable Crop Protection.

PubMed

Pilet-Nayel, Marie-Laure; Moury, Benoît; Caffier, Valérie; Montarry, Josselin; Kerlan, Marie-Claire; Fournet, Sylvain; Durel, Charles-Eric; Delourme, Régine

2017-01-01

Quantitative resistance has gained interest in plant breeding for pathogen control in low-input cropping systems. Although quantitative resistance frequently has only a partial effect and is difficult to select, it is considered more durable than major resistance (R) genes. With the exponential development of molecular markers over the past 20 years, resistance QTL have been more accurately detected and better integrated into breeding strategies for resistant varieties with increased potential for durability. This review summarizes current knowledge on the genetic inheritance, molecular basis, and durability of quantitative resistance. Based on this knowledge, we discuss how strategies that combine major R genes and QTL in crops can maintain the effectiveness of plant resistance to pathogens. Combining resistance QTL with complementary modes of action appears to be an interesting strategy for breeding effective and potentially durable resistance. Combining quantitative resistance with major R genes has proven to be a valuable approach for extending the effectiveness of major genes. In the plant genomics era, improved tools and methods are becoming available to better integrate quantitative resistance into breeding strategies. Nevertheless, optimal combinations of resistance loci will still have to be identified to preserve resistance effectiveness over time for durable crop protection.

CMV induces HERV-K and HERV-W expression in kidney transplant recipients.

PubMed

Bergallo, Massimiliano; Galliano, Ilaria; Montanari, Paola; Gambarino, Stefano; Mareschi, Katia; Ferro, Francesca; Fagioli, Franca; Tovo, Pier-Angelo; Ravanini, Paolo

2015-07-01

Human endogenous retrovirus (HERVs) constitute approximately 8% of the human genome. Induction of HERV transcription is possible under certain circumstances, and may have a possible role in some pathological conditions. The aim of this study was to evaluate HERV-K and -W pol gene expression in kidney transplant recipients and to investigate the possible relationship between HERVs gene expression and CMV infection in these patients. Thirty-three samples of kidney transplant patients and twenty healthy blood donors were used to analyze, HERV-K and -W pol gene RNA expression by relative quantitative relative Real-Time PCR. We demonstrated that HERVs pol gene expression levels were higher in kidney transplant recipients than in healthy subjects. Moreover, HERV-K and -W pol gene expression was significantly higher in the group of kidney transplant recipients with high CMV viral load than in the groups with no or moderate CMV viral load. Our data suggest that CMV may facilitate in vivo HERV activation. Published by Elsevier B.V.

Differences in Brain Transcriptomes of Closely Related Baikal Coregonid Species

PubMed Central

Bychenko, Oksana S.; Sukhanova, Lyubov V.; Azhikina, Tatyana L.; Skvortsov, Timofey A.; Belomestnykh, Tuyana V.; Sverdlov, Eugene D.

2014-01-01

The aim of this work was to get deeper insight into genetic factors involved in the adaptive divergence of closely related species, specifically two representatives of Baikal coregonids—Baikal whitefish (Coregonus baicalensis Dybowski) and Baikal omul (Coregonus migratorius Georgi)—that diverged from a common ancestor as recently as 10–20 thousand years ago. Using the Serial Analysis of Gene Expression method, we obtained libraries of short representative cDNA sequences (tags) from the brains of Baikal whitefish and omul. A comparative analysis of the libraries revealed quantitative differences among ~4% tags of the fishes under study. Based on the similarity of these tags with cDNA of known organisms, we identified candidate genes taking part in adaptive divergence. The most important candidate genes related to the adaptation of Baikal whitefish and Baikal omul, identified in this work, belong to the genes of cell metabolism, nervous and immune systems, protein synthesis, and regulatory genes as well as to DTSsa4 Tc1-like transposons which are widespread among fishes. PMID:24719892

Accumulation of Flavonoid Glycosides and UFGT Gene Expression in Mulberry Leaves (Morus alba L.) before and after Frost.

PubMed

Yu, Xiaofeng; Zhu, Yiling; Fan, Jingyi; Wang, Dujun; Gong, Xiaohui; Ouyang, Zhen

2017-08-01

In order to determine the molecular mechanism underlying the influence of frost on chemical changes in mulberry leaves, the UFGT activity, expression level, and accumulation of flavonoid glycosides in mulberry leaves (Morus alba L.) were studied. The expression of UFGT gene was investigated by quantitative real-time PCR (qRT-PCR) and the UFGT activity, accumulation of flavonoid glycosides was studied by high performance liquid chromatography. Then, the correlation between the expression level of UFGT, the UFGT activity, and the flavonoid glycosides accumulation with temperature was explored. The accumulation of isoquercitrin and astragalin is significantly positively correlated with UFGT gene expression and UFGT activity. On the contrary, the average temperature was significantly negatively correlated with the level of UFGT gene expression and UFGT activity. The results show that after frost, low temperature can induce the expression of UFGT gene in mulberry leaves, resulting in the accumulation of flavonoid glycosides. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

Determination of Bifidobacterium and Lactobacillus in breast milk of healthy women by digital PCR.

PubMed

Qian, L; Song, H; Cai, W

2016-09-01

Breast milk is one of the most important sources of postnatal microbes. Quantitative real-time polymerase chain reaction (qRT-PCR) is currently used for the quantitative analysis of bacterial 16S rRNA genes in breast milk. However, this method relies on the use of standard curves and is imprecise when quantitating target DNA of low abundance. In contrast, droplet digital PCR (DD-PCR) provides an absolute quantitation without the need for calibration curves. A comparison between DD-PCR and qRT-PCR was conducted for the quantitation of Bifidobacterium and Lactobacillus 16S RNA genes in human breast milk, and the impacts of selected maternal factors were studied on the composition of these two bacteria in breast milk. From this study, DD-PCR reported between 0-34,460 16S rRNA gene copies of Bifidobacterium genera and between 1,108-634,000 16S rRNA gene copies of Lactobacillus genera in 1 ml breast milk. The 16S rRNA gene copy number of Lactobacillus genera was much greater than that of Bifidobacterium genera in breast milk. DD-PCR showed a 10-fold lower limit of quantitation as compared to qRT-PCR. A higher correlation and agreement was observed between qRT-PCR and DD-PCR in Lactobacillus quantitation as compared to Bifidobacterium quantitation. Based on our DD-PCR quantitation, a low abundance of Bifidobacterium bacteria in breast milk was correlated to higher pre-pregnancy body mass index (BMI). However, no significant difference was observed for these two bacteria in breast milk between mothers who had vaginal deliveries and caesarean deliveries. This study suggests that DD-PCR is a better tool to quantitate the bacterial load of breast milk compared to the conventional qRT-PCR method. The number of breast milk Bifidobacterium bacteria is influenced by maternal pre-pregnancy BMI.

Quantitative and functional dynamics of Dehalococcoides spp. and its tceA and vcrA genes under TCE exposure.

PubMed

Doğan-Subaşi, Eylem; Bastiaens, Leen; Leys, Natalie; Boon, Nico; Dejonghe, Winnie

2014-07-01

This study aimed at monitoring the dynamics of phylogenetic and catabolic genes of a dechlorinating enrichment culture before, during, and after complete dechlorination of chlorinated compounds. More specifically, the effect of 40 μM trichloroethene (TCE) and 5.6 mM lactate on the gene abundance and activity of an enrichment culture was investigated for 40 days. Although tceA and vcrA gene copy numbers were relatively stable in DNA extracts over time, tceA and vcrA mRNA abundances were upregulated from undetectable levels to 2.96 × and 6.33 × 10⁴ transcripts/mL, respectively, only after exposure to TCE and lactate. While tceA gene transcripts decreased over time with TCE dechlorination, the vcrA gene was expressed steadily even when the concentration of vinyl chloride was at undetectable levels. In addition, ratios between catabolic and phylogenetic genes indicated that tceA and vcrA gene carrying organisms dechlorinated TCE and its produced daughter products, while vcrA gene was mainly responsible for the dechlorination of the lower VC concentrations in a later stage of degradation.

An Integrative Genetics Approach to Identify Candidate Genes Regulating BMD: Combining Linkage, Gene Expression, and Association

PubMed Central

Farber, Charles R; van Nas, Atila; Ghazalpour, Anatole; Aten, Jason E; Doss, Sudheer; Sos, Brandon; Schadt, Eric E; Ingram-Drake, Leslie; Davis, Richard C; Horvath, Steve; Smith, Desmond J; Drake, Thomas A; Lusis, Aldons J

2009-01-01

Numerous quantitative trait loci (QTLs) affecting bone traits have been identified in the mouse; however, few of the underlying genes have been discovered. To improve the process of transitioning from QTL to gene, we describe an integrative genetics approach, which combines linkage analysis, expression QTL (eQTL) mapping, causality modeling, and genetic association in outbred mice. In C57BL/6J × C3H/HeJ (BXH) F2 mice, nine QTLs regulating femoral BMD were identified. To select candidate genes from within each QTL region, microarray gene expression profiles from individual F2 mice were used to identify 148 genes whose expression was correlated with BMD and regulated by local eQTLs. Many of the genes that were the most highly correlated with BMD have been previously shown to modulate bone mass or skeletal development. Candidates were further prioritized by determining whether their expression was predicted to underlie variation in BMD. Using network edge orienting (NEO), a causality modeling algorithm, 18 of the 148 candidates were predicted to be causally related to differences in BMD. To fine-map QTLs, markers in outbred MF1 mice were tested for association with BMD. Three chromosome 11 SNPs were identified that were associated with BMD within the Bmd11 QTL. Finally, our approach provides strong support for Wnt9a, Rasd1, or both underlying Bmd11. Integration of multiple genetic and genomic data sets can substantially improve the efficiency of QTL fine-mapping and candidate gene identification. PMID:18767929

Linkage disequilibrium interval mapping of quantitative trait loci.

PubMed

Boitard, Simon; Abdallah, Jihad; de Rochambeau, Hubert; Cierco-Ayrolles, Christine; Mangin, Brigitte

2006-03-16

For many years gene mapping studies have been performed through linkage analyses based on pedigree data. Recently, linkage disequilibrium methods based on unrelated individuals have been advocated as powerful tools to refine estimates of gene location. Many strategies have been proposed to deal with simply inherited disease traits. However, locating quantitative trait loci is statistically more challenging and considerable research is needed to provide robust and computationally efficient methods. Under a three-locus Wright-Fisher model, we derived approximate expressions for the expected haplotype frequencies in a population. We considered haplotypes comprising one trait locus and two flanking markers. Using these theoretical expressions, we built a likelihood-maximization method, called HAPim, for estimating the location of a quantitative trait locus. For each postulated position, the method only requires information from the two flanking markers. Over a wide range of simulation scenarios it was found to be more accurate than a two-marker composite likelihood method. It also performed as well as identity by descent methods, whilst being valuable in a wider range of populations. Our method makes efficient use of marker information, and can be valuable for fine mapping purposes. Its performance is increased if multiallelic markers are available. Several improvements can be developed to account for more complex evolution scenarios or provide robust confidence intervals for the location estimates.

Transcriptome analysis of Brassica napus pod using RNA-Seq and identification of lipid-related candidate genes.

PubMed

Xu, Hai-Ming; Kong, Xiang-Dong; Chen, Fei; Huang, Ji-Xiang; Lou, Xiang-Yang; Zhao, Jian-Yi

2015-10-24

Brassica napus is an important oilseed crop. Dissection of the genetic architecture underlying oil-related biological processes will greatly facilitates the genetic improvement of rapeseed. The differential gene expression during pod development offers a snapshot on the genes responsible for oil accumulation in. To identify candidate genes in the linkage peaks reported previously, we used RNA sequencing (RNA-Seq) technology to analyze the pod transcriptomes of German cultivar Sollux and Chinese inbred line Gaoyou. The RNA samples were collected for RNA-Seq at 5-7, 15-17 and 25-27 days after flowering (DAF). Bioinformatics analysis was performed to investigate differentially expressed genes (DEGs). Gene annotation analysis was integrated with QTL mapping and Brassica napus pod transcriptome profiling to detect potential candidate genes in oilseed. Four hundred sixty five and two thousand, one hundred fourteen candidate DEGs were identified, respectively, between two varieties at the same stages and across different periods of each variety. Then, 33 DEGs between Sollux and Gaoyou were identified as the candidate genes affecting seed oil content by combining those DEGs with the quantitative trait locus (QTL) mapping results, of which, one was found to be homologous to Arabidopsis thaliana lipid-related genes. Intervarietal DEGs of lipid pathways in QTL regions represent important candidate genes for oil-related traits. Integrated analysis of transcriptome profiling, QTL mapping and comparative genomics with other relative species leads to efficient identification of most plausible functional genes underlying oil-content related characters, offering valuable resources for bettering breeding program of Brassica napus. This study provided a comprehensive overview on the pod transcriptomes of two varieties with different oil-contents at the three developmental stages.

Selection of housekeeping genes and demonstration of RNAi in cotton leafhopper, Amrasca biguttula biguttula (Ishida)

PubMed Central

Gupta, Mridula; Pandher, Suneet; Kaur, Gurmeet; Rathore, Pankaj; Palli, Subba Reddy

2018-01-01

Amrasca biguttula biguttula (Ishida) commonly known as cotton leafhopper is a severe pest of cotton and okra. Not much is known on this insect at molecular level due to lack of genomic and transcriptomic data. To prepare for functional genomic studies in this insect, we evaluated 15 common housekeeping genes (Tub, B-Tub, EF alpha, GADPH, UbiCF, RP13, Ubiq, G3PD, VATPase, Actin, 18s, 28s, TATA, ETF, SOD and Cytolytic actin) during different developmental stages and under starvation stress. We selected early (1st and 2nd), late (3rd and 4th) stage nymphs and adults for identification of stable housekeeping genes using geNorm, NormFinder, BestKeeper and RefFinder software. Based on the different algorithms, RP13 and VATPase are identified as the most suitable reference genes for quantification of gene expression by reverse transcriptase quantitative PCR (RT-qPCR). Based on RefFinder which comprehended the results of three algorithms, RP13 in adults, Tubulin (Tub) in late nymphs, 28S in early nymph and UbiCF under starvation stress were identified as the most stable genes. We also developed methods for feeding double-stranded RNA (dsRNA) incorporated in the diet. Feeding dsRNA targeting Snf7, IAP, AQP1, and VATPase caused 56.17–77.12% knockdown of targeted genes compared to control and 16 to 48% mortality of treated insects when compared to control. PMID:29329327

Gene Profiling Technique to Accelerate Stem Cell Therapies for Eye Diseases

MedlinePlus

... like RPE. They also use a technique called quantitative RT-PCR to measure the expression of genes ... higher in iPS cells than mature RPE. But quantitative RT-PCR only permits the simultaneous measurement of ...

Genetic dissection of the maize (Zea mays L.) MAMP response.

PubMed

Zhang, Xinye; Valdés-López, Oswaldo; Arellano, Consuelo; Stacey, Gary; Balint-Kurti, Peter

2017-06-01

Loci associated with variation in maize responses to two microbe-associated molecular patterns (MAMPs) were identified. MAMP responses were correlated. No relationship between MAMP responses and quantitative disease resistance was identified. Microbe-associated molecular patterns (MAMPs) are highly conserved molecules commonly found in microbes which can be recognized by plant pattern recognition receptors. Recognition triggers a suite of responses including production of reactive oxygen species (ROS) and nitric oxide (NO) and expression changes of defense-related genes. In this study, we used two well-studied MAMPs (flg22 and chitooctaose) to challenge different maize lines to determine whether there was variation in the level of responses to these MAMPs, to dissect the genetic basis underlying that variation and to understand the relationship between MAMP response and quantitative disease resistance (QDR). Naturally occurring quantitative variation in ROS, NO production, and defense genes expression levels triggered by MAMPs was observed. A major quantitative traits locus (QTL) associated with variation in the ROS production response to both flg22 and chitooctaose was identified on chromosome 2 in a recombinant inbred line (RIL) population derived from the maize inbred lines B73 and CML228. Minor QTL associated with variation in the flg22 ROS response was identified on chromosomes 1 and 4. Comparison of these results with data previously obtained for variation in QDR and the defense response in the same RIL population did not provide any evidence for a common genetic basis controlling variation in these traits.

Using the candidate gene approach for detecting genes underlying seed oil concentration and yield in soybean.

PubMed

Eskandari, Mehrzad; Cober, Elroy R; Rajcan, Istvan

2013-07-01

Increasing the oil concentration in soybean seeds has been given more attention in recent years because of demand for both edible oil and biodiesel production. Oil concentration in soybean is a complex quantitative trait regulated by many genes as well as environmental conditions. To identify genes governing seed oil concentration in soybean, 16 putative candidate genes of three important gene families (GPAT: acyl-CoA:sn-glycerol-3-phosphate acyltransferase, DGAT: acyl-CoA:diacylglycerol acyltransferase, and PDAT: phospholipid:diacylglycerol acyltransferase) involved in triacylglycerol (TAG) biosynthesis pathways were selected and their sequences retrieved from the soybean database ( http://www.phytozome.net/soybean ). Three sequence mutations were discovered in either coding or noncoding regions of three DGAT soybean isoforms when comparing the parents of a 203 recombinant inbreed line (RIL) population; OAC Wallace and OAC Glencoe. The RIL population was used to study the effects of these mutations on seed oil concentration and other important agronomic and seed composition traits, including seed yield and protein concentration across three field locations in Ontario, Canada, in 2009 and 2010. An insertion/deletion (indel) mutation in the GmDGAT2B gene in OAC Wallace was significantly associated with reduced seed oil concentration across three environments and reduced seed yield at Woodstock in 2010. A mutation in the 3' untranslated (3'UTR) region of GmDGAT2C was associated with seed yield at Woodstock in 2009. A mutation in the intronic region of GmDGAR1B was associated with seed yield and protein concentration at Ottawa in 2010. The genes identified in this study had minor effects on either seed yield or oil concentration, which was in agreement with the quantitative nature of the traits. However, the novel gene-specific markers designed in the present study can be used in soybean breeding for marker-assisted selection aimed at increasing seed yield and oil concentration with no significant impact on seed protein concentration.

Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies.

PubMed

Tricarico, Carmela; Pinzani, Pamela; Bianchi, Simonetta; Paglierani, Milena; Distante, Vito; Pazzagli, Mario; Bustin, Stephen A; Orlando, Claudio

2002-10-15

Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a housekeeping gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used housekeeping genes and 18S rRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single housekeeping genes or rRNA is inappropriate for studies involving tissue biopsies.

A Dual-Color Reporter Assay of Cohesin-Mediated Gene Regulation in Budding Yeast Meiosis.

PubMed

Fan, Jinbo; Jin, Hui; Yu, Hong-Guo

2017-01-01

In this chapter, we describe a quantitative fluorescence-based assay of gene expression using the ratio of the reporter green fluorescence protein (GFP) to the internal red fluorescence protein (RFP) control. With this dual-color heterologous reporter assay, we have revealed cohesin-regulated genes and discovered a cis-acting DNA element, the Ty1-LTR, which interacts with cohesin and regulates gene expression during yeast meiosis. The method described here provides an effective cytological approach for quantitative analysis of global gene expression in budding yeast meiosis.

Quantitative proteomics reveals the importance of nitrogen source to control glucosinolate metabolism in Arabidopsis thaliana and Brassica oleracea

PubMed Central

Marino, Daniel; Ariz, Idoia; Lasa, Berta; Santamaría, Enrique; Fernández-Irigoyen, Joaquín; González-Murua, Carmen; Aparicio Tejo, Pedro M.

2016-01-01

Accessing different nitrogen (N) sources involves a profound adaptation of plant metabolism. In this study, a quantitative proteomic approach was used to further understand how the model plant Arabidopsis thaliana adjusts to different N sources when grown exclusively under nitrate or ammonium nutrition. Proteome data evidenced that glucosinolate metabolism was differentially regulated by the N source and that both TGG1 and TGG2 myrosinases were more abundant under ammonium nutrition, which is generally considered to be a stressful situation. Moreover, Arabidopsis plants displayed glucosinolate accumulation and induced myrosinase activity under ammonium nutrition. Interestingly, these results were also confirmed in the economically important crop broccoli (Brassica oleracea var. italica). Moreover, these metabolic changes were correlated in Arabidopsis with the differential expression of genes from the aliphatic glucosinolate metabolic pathway. This study underlines the importance of nitrogen nutrition and the potential of using ammonium as the N source in order to stimulate glucosinolate metabolism, which may have important applications not only in terms of reducing pesticide use, but also for increasing plants’ nutritional value. PMID:27085186

Application of Genome Wide Association and Genomic Prediction for Improvement of Cacao Productivity and Resistance to Black and Frosty Pod Diseases

PubMed Central

Romero Navarro, J. Alberto; Phillips-Mora, Wilbert; Arciniegas-Leal, Adriana; Mata-Quirós, Allan; Haiminen, Niina; Mustiga, Guiliana; Livingstone III, Donald; van Bakel, Harm; Kuhn, David N.; Parida, Laxmi; Kasarskis, Andrew; Motamayor, Juan C.

2017-01-01

Chocolate is a highly valued and palatable confectionery product. Chocolate is primarily made from the processed seeds of the tree species Theobroma cacao. Cacao cultivation is highly relevant for small-holder farmers throughout the tropics, yet its productivity remains limited by low yields and widespread pathogens. A panel of 148 improved cacao clones was assembled based on productivity and disease resistance, and phenotypic single-tree replicated clonal evaluation was performed for 8 years. Using high-density markers, the diversity of clones was expressed relative to 10 known ancestral cacao populations, and significant effects of ancestry were observed in productivity and disease resistance. Genome-wide association (GWA) was performed, and six markers were significantly associated with frosty pod disease resistance. In addition, genomic selection was performed, and consistent with the observed extensive linkage disequilibrium, high predictive ability was observed at low marker densities for all traits. Finally, quantitative trait locus mapping and differential expression analysis of two cultivars with contrasting disease phenotypes were performed to identify genes underlying frosty pod disease resistance, identifying a significant quantitative trait locus and 35 differentially expressed genes using two independent differential expression analyses. These results indicate that in breeding populations of heterozygous and recently admixed individuals, mapping approaches can be used for low complexity traits like pod color cacao, or in other species single gene disease resistance, however genomic selection for quantitative traits remains highly effective relative to mapping. Our results can help guide the breeding process for sustainable improved cacao productivity. PMID:29184558

Applicability of the chymopapain gene used as endogenous reference gene for transgenic huanong no. 1 papaya detection.

PubMed

Guo, Jinchao; Yang, Litao; Liu, Xin; Zhang, Haibo; Qian, Bingjun; Zhang, Dabing

2009-08-12

The virus-resistant papaya (Carica papaya L.), Huanong no. 1, was the genetically modified (GM) fruit approved for growing in China in 2006. To implement the labeling regulation of GM papaya and its derivates, the development of papaya endogenous reference gene is very necessary for GM papaya detection. Herein, we reported one papaya specific gene, Chymopapain (CHY), as one suitable endogenous reference gene, used for GM papaya identification. Thereafter, we established the conventional and real-time quantitative PCR assays of the CHY gene. In the CHY conventional PCR assay, the limit of detection (LOD) was 25 copies of haploid papaya genome. In the CHY real-time quantitative PCR assay, both the LOD and the limit of quantification (LOQ) were as low as 12.5 copies of haploid papaya genome. Furthermore, we revealed the construct-specific sequence of Chinese GM papaya Huanong no. 1 and developed its conventional and quantitative PCR systems employing the CHY gene as endogenous reference gene. This work is useful for papaya specific identification and GM papaya detection.

Gene expression profiling of single cells on large-scale oligonucleotide arrays

PubMed Central

Hartmann, Claudia H.; Klein, Christoph A.

2006-01-01

Over the last decade, important insights into the regulation of cellular responses to various stimuli were gained by global gene expression analyses of cell populations. More recently, specific cell functions and underlying regulatory networks of rare cells isolated from their natural environment moved to the center of attention. However, low cell numbers still hinder gene expression profiling of rare ex vivo material in biomedical research. Therefore, we developed a robust method for gene expression profiling of single cells on high-density oligonucleotide arrays with excellent coverage of low abundance transcripts. The protocol was extensively tested with freshly isolated single cells of very low mRNA content including single epithelial, mature and immature dendritic cells and hematopoietic stem cells. Quantitative PCR confirmed that the PCR-based global amplification method did not change the relative ratios of transcript abundance and unsupervised hierarchical cluster analysis revealed that the histogenetic origin of an individual cell is correctly reflected by the gene expression profile. Moreover, the gene expression data from dendritic cells demonstrate that cellular differentiation and pathway activation can be monitored in individual cells. PMID:17071717

Functional diversification of the dehydrin gene family in apple and its contribution to cold acclimation during dormancy.

PubMed

Falavigna, Vítor da Silveira; Miotto, Yohanna Evelyn; Porto, Diogo Denardi; Anzanello, Rafael; Santos, Henrique Pessoa dos; Fialho, Flávio Bello; Margis-Pinheiro, Márcia; Pasquali, Giancarlo; Revers, Luís Fernando

2015-11-01

Dehydrins (DHN) are proteins involved in plant adaptive responses to abiotic stresses, mainly dehydration. Several studies in perennial crops have linked bud dormancy progression, a process characterized by the inability to initiate growth from meristems under favorable conditions, with DHN gene expression. However, an in-depth characterization of DHNs during bud dormancy progression is still missing. An extensive in silico characterization of the apple DHN gene family was performed. Additionally, we used five different experiments that generated samples with different dormancy status, including genotypes with contrasting dormancy traits, to analyze how DHN genes are being regulated during bud dormancy progression in apple by real-time quantitative polymerase chain reaction (RT-qPCR). Duplication events took place in the diversification of apple DHN family. Additionally, MdDHN genes presented tissue- and bud dormant-specific expression patterns. Our results indicate that MdDHN genes are highly divergent in function, with overlapping levels, and that their expressions are fine-tuned by the environment during the dormancy process in apple. © 2015 Scandinavian Plant Physiology Society.

Strategies to regulate transcription factor-mediated gene positioning and interchromosomal clustering at the nuclear periphery.

PubMed

Randise-Hinchliff, Carlo; Coukos, Robert; Sood, Varun; Sumner, Michael Chas; Zdraljevic, Stefan; Meldi Sholl, Lauren; Garvey Brickner, Donna; Ahmed, Sara; Watchmaker, Lauren; Brickner, Jason H

2016-03-14

In budding yeast, targeting of active genes to the nuclear pore complex (NPC) and interchromosomal clustering is mediated by transcription factor (TF) binding sites in the gene promoters. For example, the binding sites for the TFs Put3, Ste12, and Gcn4 are necessary and sufficient to promote positioning at the nuclear periphery and interchromosomal clustering. However, in all three cases, gene positioning and interchromosomal clustering are regulated. Under uninducing conditions, local recruitment of the Rpd3(L) histone deacetylase by transcriptional repressors blocks Put3 DNA binding. This is a general function of yeast repressors: 16 of 21 repressors blocked Put3-mediated subnuclear positioning; 11 of these required Rpd3. In contrast, Ste12-mediated gene positioning is regulated independently of DNA binding by mitogen-activated protein kinase phosphorylation of the Dig2 inhibitor, and Gcn4-dependent targeting is up-regulated by increasing Gcn4 protein levels. These different regulatory strategies provide either qualitative switch-like control or quantitative control of gene positioning over different time scales. © 2016 Randise-Hinchliff et al.

Quantitative real-time PCR normalization for gene expression studies in the plant pathogenic fungi Lasiodiplodia theobromae.

PubMed

Paolinelli-Alfonso, Marcos; Galindo-Sánchez, Clara Elizabeth; Hernandez-Martinez, Rufina

2016-08-01

Lasiodiplodia theobromae is a highly virulent plant pathogen. It has been suggested that heat stress increases its virulence. The aim of this work was to evaluate, compare, and recommend normalization strategies for gene expression analysis of the fungus growing with grapevine wood under heat stress. Using RT-qPCR-derived data, reference gene stability was evaluated through geNorm, NormFinder and Bestkeeper applications. Based on the geometric mean using the ranking position obtained for each independent analysis, genes were ranked from least to most stable as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), β-tubulin (TUB) and elongation factor-1α (EF1α). Using RNAseq-derived data based on the calculated tagwise dispersion these genes were ordered by increasing stability as follows: GAPDH, ACT, TUB, and EF1α. The correlation between RNAseq and RTqPCR results was used as criteria to identify the best RT-qPCR normalization approach. The gene TUB is recommended as the best option for normalization among the commonly used reference genes, but alternative fungal reference genes are also suggested. Copyright © 2016 Elsevier B.V. All rights reserved.

REVIEW ARTICLE: Oscillations and temporal signalling in cells

NASA Astrophysics Data System (ADS)

Tiana, G.; Krishna, S.; Pigolotti, S.; Jensen, M. H.; Sneppen, K.

2007-06-01

The development of new techniques to quantitatively measure gene expression in cells has shed light on a number of systems that display oscillations in protein concentration. Here we review the different mechanisms which can produce oscillations in gene expression or protein concentration using a framework of simple mathematical models. We focus on three eukaryotic genetic regulatory networks which show 'ultradian' oscillations, with a time period of the order of hours, and involve, respectively, proteins important for development (Hes1), apoptosis (p53) and immune response (NF-κB). We argue that underlying all three is a common design consisting of a negative feedback loop with time delay which is responsible for the oscillatory behaviour.

RefPrimeCouch—a reference gene primer CouchApp

PubMed Central

Silbermann, Jascha; Wernicke, Catrin; Pospisil, Heike; Frohme, Marcus

2013-01-01

To support a quantitative real-time polymerase chain reaction standardization project, a new reference gene database application was required. The new database application was built with the explicit goal of simplifying not only the development process but also making the user interface more responsive and intuitive. To this end, CouchDB was used as the backend with a lightweight dynamic user interface implemented client-side as a one-page web application. Data entry and curation processes were streamlined using an OpenRefine-based workflow. The new RefPrimeCouch database application provides its data online under an Open Database License. Database URL: http://hpclife.th-wildau.de:5984/rpc/_design/rpc/view.html PMID:24368831

RefPrimeCouch--a reference gene primer CouchApp.

PubMed

Silbermann, Jascha; Wernicke, Catrin; Pospisil, Heike; Frohme, Marcus

2013-01-01

To support a quantitative real-time polymerase chain reaction standardization project, a new reference gene database application was required. The new database application was built with the explicit goal of simplifying not only the development process but also making the user interface more responsive and intuitive. To this end, CouchDB was used as the backend with a lightweight dynamic user interface implemented client-side as a one-page web application. Data entry and curation processes were streamlined using an OpenRefine-based workflow. The new RefPrimeCouch database application provides its data online under an Open Database License. Database URL: http://hpclife.th-wildau.de:5984/rpc/_design/rpc/view.html.

Phylogeny and expression profiling of CAD and CAD-like genes in hybrid Populus (P. deltoides × P. nigra): evidence from herbivore damage for subfunctionalization and functional divergence

PubMed Central

2010-01-01

Background Cinnamyl Alcohol Dehydrogenase (CAD) proteins function in lignin biosynthesis and play a critical role in wood development and plant defense against stresses. Previous phylogenetic studies did not include genes from seedless plants and did not reflect the deep evolutionary history of this gene family. We reanalyzed the phylogeny of CAD and CAD-like genes using a representative dataset including lycophyte and bryophyte sequences. Many CAD/CAD-like genes do not seem to be associated with wood development under normal growth conditions. To gain insight into the functional evolution of CAD/CAD-like genes, we analyzed their expression in Populus plant tissues in response to feeding damage by gypsy moth larvae (Lymantria dispar L.). Expression of CAD/CAD-like genes in Populus tissues (xylem, leaves, and barks) was analyzed in herbivore-treated and non-treated plants by real time quantitative RT-PCR. Results CAD family genes were distributed in three classes based on sequence conservation. All the three classes are represented by seedless as well as seed plants, including the class of bona fide lignin pathway genes. The expression of some CAD/CAD-like genes that are not associated with xylem development were induced following herbivore damage in leaves, while other genes were induced in only bark or xylem tissues. Five of the CAD/CAD-like genes, however, showed a shift in expression from one tissue to another between non-treated and herbivore-treated plants. Systemic expression of the CAD/CAD-like genes was generally suppressed. Conclusions Our results indicated a correlation between the evolution of the CAD gene family and lignin and that the three classes of genes may have evolved in the ancestor of land plants. Our results also suggest that the CAD/CAD-like genes have evolved a diversity of expression profiles and potentially different functions, but that they are nonetheless co-regulated under stress conditions. PMID:20509918

Predicting gene regulatory networks of soybean nodulation from RNA-Seq transcriptome data.

PubMed

Zhu, Mingzhu; Dahmen, Jeremy L; Stacey, Gary; Cheng, Jianlin

2013-09-22

High-throughput RNA sequencing (RNA-Seq) is a revolutionary technique to study the transcriptome of a cell under various conditions at a systems level. Despite the wide application of RNA-Seq techniques to generate experimental data in the last few years, few computational methods are available to analyze this huge amount of transcription data. The computational methods for constructing gene regulatory networks from RNA-Seq expression data of hundreds or even thousands of genes are particularly lacking and urgently needed. We developed an automated bioinformatics method to predict gene regulatory networks from the quantitative expression values of differentially expressed genes based on RNA-Seq transcriptome data of a cell in different stages and conditions, integrating transcriptional, genomic and gene function data. We applied the method to the RNA-Seq transcriptome data generated for soybean root hair cells in three different development stages of nodulation after rhizobium infection. The method predicted a soybean nodulation-related gene regulatory network consisting of 10 regulatory modules common for all three stages, and 24, 49 and 70 modules separately for the first, second and third stage, each containing both a group of co-expressed genes and several transcription factors collaboratively controlling their expression under different conditions. 8 of 10 common regulatory modules were validated by at least two kinds of validations, such as independent DNA binding motif analysis, gene function enrichment test, and previous experimental data in the literature. We developed a computational method to reliably reconstruct gene regulatory networks from RNA-Seq transcriptome data. The method can generate valuable hypotheses for interpreting biological data and designing biological experiments such as ChIP-Seq, RNA interference, and yeast two hybrid experiments.

Combining laser microdissection and RNA-seq to chart the transcriptional landscape of fungal development

PubMed Central

2012-01-01

Background During sexual development, filamentous ascomycetes form complex, three-dimensional fruiting bodies for the protection and dispersal of sexual spores. Fruiting bodies contain a number of cell types not found in vegetative mycelium, and these morphological differences are thought to be mediated by changes in gene expression. However, little is known about the spatial distribution of gene expression in fungal development. Here, we used laser microdissection (LM) and RNA-seq to determine gene expression patterns in young fruiting bodies (protoperithecia) and non-reproductive mycelia of the ascomycete Sordaria macrospora. Results Quantitative analysis showed major differences in the gene expression patterns between protoperithecia and total mycelium. Among the genes strongly up-regulated in protoperithecia were the pheromone precursor genes ppg1 and ppg2. The up-regulation was confirmed by fluorescence microscopy of egfp expression under the control of ppg1 regulatory sequences. RNA-seq analysis of protoperithecia from the sterile mutant pro1 showed that many genes that are differentially regulated in these structures are under the genetic control of transcription factor PRO1. Conclusions We have generated transcriptional profiles of young fungal sexual structures using a combination of LM and RNA-seq. This allowed a high spatial resolution and sensitivity, and yielded a detailed picture of gene expression during development. Our data revealed significant differences in gene expression between protoperithecia and non-reproductive mycelia, and showed that the transcription factor PRO1 is involved in the regulation of many genes expressed specifically in sexual structures. The LM/RNA-seq approach will also be relevant to other eukaryotic systems in which multicellular development is investigated. PMID:23016559

Quantitative genome re-sequencing defines multiple mutations conferring chloroquine resistance in rodent malaria

PubMed Central

2012-01-01

Background Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance. A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions. Results Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter. Conclusions Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum. PMID:22435897

Genome-wide study of the adaptation of Saccharomyces cerevisiae to the early stages of wine fermentation.

PubMed

Novo, Maite; Mangado, Ana; Quirós, Manuel; Morales, Pilar; Salvadó, Zoel; Gonzalez, Ramon

2013-01-01

This work was designed to identify yeast cellular functions specifically affected by the stress factors predominating during the early stages of wine fermentation, and genes required for optimal growth under these conditions. The main experimental method was quantitative fitness analysis by means of competition experiments in continuous culture of whole genome barcoded yeast knockout collections. This methodology allowed the identification of haploinsufficient genes, and homozygous deletions resulting in growth impairment in synthetic must. However, genes identified as haploproficient, or homozygous deletions resulting in fitness advantage, were of little predictive power concerning optimal growth in this medium. The relevance of these functions for enological performance of yeast was assessed in batch cultures with single strains. Previous studies addressing yeast adaptation to winemaking conditions by quantitative fitness analysis were not specifically focused on the proliferative stages. In some instances our results highlight the importance of genes not previously linked to winemaking. In other cases they are complementary to those reported in previous studies concerning, for example, the relevance of some genes involved in vacuolar, peroxisomal, or ribosomal functions. Our results indicate that adaptation to the quickly changing growth conditions during grape must fermentation require the function of different gene sets in different moments of the process. Transport processes and glucose signaling seem to be negatively affected by the stress factors encountered by yeast in synthetic must. Vacuolar activity is important for continued growth during the transition to stationary phase. Finally, reduced biogenesis of peroxisomes also seems to be advantageous. However, in contrast to what was described for later stages, reduced protein synthesis is not advantageous for the early (proliferative) stages of the fermentation process. Finally, we found adenine and lysine to be in short supply for yeast growth in some natural grape musts.

A quantitative validated model reveals two phases of transcriptional regulation for the gap gene giant in Drosophila.

PubMed

Hoermann, Astrid; Cicin-Sain, Damjan; Jaeger, Johannes

2016-03-15

Understanding eukaryotic transcriptional regulation and its role in development and pattern formation is one of the big challenges in biology today. Most attempts at tackling this problem either focus on the molecular details of transcription factor binding, or aim at genome-wide prediction of expression patterns from sequence through bioinformatics and mathematical modelling. Here we bridge the gap between these two complementary approaches by providing an integrative model of cis-regulatory elements governing the expression of the gap gene giant (gt) in the blastoderm embryo of Drosophila melanogaster. We use a reverse-engineering method, where mathematical models are fit to quantitative spatio-temporal reporter gene expression data to infer the regulatory mechanisms underlying gt expression in its anterior and posterior domains. These models are validated through prediction of gene expression in mutant backgrounds. A detailed analysis of our data and models reveals that gt is regulated by domain-specific CREs at early stages, while a late element drives expression in both the anterior and the posterior domains. Initial gt expression depends exclusively on inputs from maternal factors. Later, gap gene cross-repression and gt auto-activation become increasingly important. We show that auto-regulation creates a positive feedback, which mediates the transition from early to late stages of regulation. We confirm the existence and role of gt auto-activation through targeted mutagenesis of Gt transcription factor binding sites. In summary, our analysis provides a comprehensive picture of spatio-temporal gene regulation by different interacting enhancer elements for an important developmental regulator. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Context-specific metabolic networks are consistent with experiments.

PubMed

Becker, Scott A; Palsson, Bernhard O

2008-05-16

Reconstructions of cellular metabolism are publicly available for a variety of different microorganisms and some mammalian genomes. To date, these reconstructions are "genome-scale" and strive to include all reactions implied by the genome annotation, as well as those with direct experimental evidence. Clearly, many of the reactions in a genome-scale reconstruction will not be active under particular conditions or in a particular cell type. Methods to tailor these comprehensive genome-scale reconstructions into context-specific networks will aid predictive in silico modeling for a particular situation. We present a method called Gene Inactivity Moderated by Metabolism and Expression (GIMME) to achieve this goal. The GIMME algorithm uses quantitative gene expression data and one or more presupposed metabolic objectives to produce the context-specific reconstruction that is most consistent with the available data. Furthermore, the algorithm provides a quantitative inconsistency score indicating how consistent a set of gene expression data is with a particular metabolic objective. We show that this algorithm produces results consistent with biological experiments and intuition for adaptive evolution of bacteria, rational design of metabolic engineering strains, and human skeletal muscle cells. This work represents progress towards producing constraint-based models of metabolism that are specific to the conditions where the expression profiling data is available.

Identification of quantitative trait loci and candidate genes for cadmium tolerance in Populus

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

Induri, Brahma R; Ellis, Danielle R; Slavov, Goncho T.

2012-01-01

Understanding genetic variation for the response of Populus to heavy metals like cadmium (Cd) is an important step in elucidating the underlying mechanisms of tolerance. In this study, a pseudo-backcross pedigree of Populus trichocarpa Torr. & Gray and Populus deltoides Bart. was characterized for growth and performance traits after Cd exposure. A total of 16 quantitative trait loci (QTL) at logarithm of odds (LOD) ratio 2.5 were detected for total dry weight, its components and root volume. Major QTL for Cd responses were mapped to two different linkage groups and the relative allelic effects were in opposing directions on themore » two chromosomes, suggesting differential mechanisms at these two loci. The phenotypic variance explained by Cd QTL ranged from 5.9 to 11.6% and averaged 8.2% across all QTL. A whole-genome microarray study led to the identification of nine Cd-responsive genes from these QTL. Promising candidates for Cd tolerance include an NHL repeat membrane-spanning protein, a metal transporter and a putative transcription factor. Additional candidates in the QTL intervals include a putative homolog of a glutamate cysteine ligase, and a glutathione-S-transferase. Functional characterization of these candidate genes should enhance our understanding of Cd metabolism and transport and phytoremediation capabilities of Populus.« less

Digital gene expression analysis in mice lung with coinfection of influenza and streptococcus pneumoniae.

PubMed

Luo, Jun; Zhou, Linlin; Wang, Hongren; Qin, Zhen; Xiang, Li; Zhu, Jie; Huang, Xiaojun; Yang, Yuan; Li, Wanyi; Wang, Baoning; Li, Mingyuan

2017-12-22

Influenza A virus (IAV) and Streptococcus pneumoniae (SP) are two major upper respiratory tract pathogens that can also cause infection in polarized bronchial epithelial cells to exacerbate disease in coinfected individuals which may result in significant morbidity. However, the underlying molecular mechanism is poorly understood. Here, we employed BALB/c ByJ mice inflected with SP, IAV, IAV followed by SP (IAV+SP) and PBS (Control) as models to survey the global gene expression using digital gene expression (DGE) profiling. We attempt to gain insights into the underlying genetic basis of this synergy at the expression level. Gene expression profiles were obtain using the Illimina/Hisseq sequencing technique, and further analyzed by enrichment analysis of Gene Ontology (GO) and Pathway function. The hematoxylin-eosin (HE) staining revealed different tissue changes in groups during which IAV+SP group showed the most severe cell apoptosis. Compared with Control, a total of 2731, 3221 and 3946 differentially expressed genes (DEGs) were detected in SP, IAV and IAV+SP respectively. Besides, sixty-two GO terms were identified by Gene Ontology functional enrichment analysis, such as cell killing, biological regulation, response to stimulus, signaling, biological adhesion, enzyme regulator activity, receptor regulator activity and translation regulator activity. Pathway significant enrichment analysis indicated the dysregulation of multiple pathways, including apoptosis pathway. Among these, five selected genes were further verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). This study shows that infection with SP, IAV or IAV+SP induces apoptosis with different degrees which might provide insights into the molecular mechanisms to facilitate further research.

Physiological and Molecular Analysis of Aluminium-Induced Organic Acid Anion Secretion from Grain Amaranth (Amaranthus hypochondriacus L.) Roots

PubMed Central

Fan, Wei; Xu, Jia-Meng; Lou, He-Qiang; Xiao, Chuan; Chen, Wei-Wei; Yang, Jian-Li

2016-01-01

Grain amaranth (Amaranthus hypochondriacus L.) is abundant in oxalate and can secrete oxalate under aluminium (Al) stress. However, the features of Al-induced secretion of organic acid anions (OA) and potential genes responsible for OA secretion are poorly understood. Here, Al-induced OA secretion in grain amaranth roots was characterized by ion charomatography and enzymology methods, and suppression subtractive hybridization (SSH) together with quantitative real-time PCR (qRT-PCR) was used to identify up-regulated genes that are potentially involved in OA secretion. The results showed that grain amaranth roots secrete both oxalate and citrate in response to Al stress. The secretion pattern, however, differs between oxalate and citrate. Neither lanthanum chloride (La) nor cadmium chloride (Cd) induced OA secretion. A total of 84 genes were identified as up-regulated by Al, in which six genes were considered as being potentially involved in OA secretion. The expression pattern of a gene belonging to multidrug and toxic compound extrusion (MATE) family, AhMATE1, was in close agreement with that of citrate secretion. The expression of a gene encoding tonoplast dicarboxylate transporter and four genes encoding ATP-binding cassette transporters was differentially regulated by Al stress, but the expression pattern was not correlated well with that of oxalate secretion. Our results not only reveal the secretion pattern of oxalate and citrate from grain amaranth roots under Al stress, but also provide some genetic information that will be useful for further characterization of genes involved in Al toxicity and tolerance mechanisms. PMID:27144562

Physiological and Molecular Analysis of Aluminium-Induced Organic Acid Anion Secretion from Grain Amaranth (Amaranthus hypochondriacus L.) Roots.

PubMed

Fan, Wei; Xu, Jia-Meng; Lou, He-Qiang; Xiao, Chuan; Chen, Wei-Wei; Yang, Jian-Li

2016-04-30

Grain amaranth (Amaranthus hypochondriacus L.) is abundant in oxalate and can secrete oxalate under aluminium (Al) stress. However, the features of Al-induced secretion of organic acid anions (OA) and potential genes responsible for OA secretion are poorly understood. Here, Al-induced OA secretion in grain amaranth roots was characterized by ion charomatography and enzymology methods, and suppression subtractive hybridization (SSH) together with quantitative real-time PCR (qRT-PCR) was used to identify up-regulated genes that are potentially involved in OA secretion. The results showed that grain amaranth roots secrete both oxalate and citrate in response to Al stress. The secretion pattern, however, differs between oxalate and citrate. Neither lanthanum chloride (La) nor cadmium chloride (Cd) induced OA secretion. A total of 84 genes were identified as up-regulated by Al, in which six genes were considered as being potentially involved in OA secretion. The expression pattern of a gene belonging to multidrug and toxic compound extrusion (MATE) family, AhMATE1, was in close agreement with that of citrate secretion. The expression of a gene encoding tonoplast dicarboxylate transporter and four genes encoding ATP-binding cassette transporters was differentially regulated by Al stress, but the expression pattern was not correlated well with that of oxalate secretion. Our results not only reveal the secretion pattern of oxalate and citrate from grain amaranth roots under Al stress, but also provide some genetic information that will be useful for further characterization of genes involved in Al toxicity and tolerance mechanisms.

Line differences in Cor/Lea and fructan biosynthesis-related gene transcript accumulation are related to distinct freezing tolerance levels in synthetic wheat hexaploids.

PubMed

Yokota, Hirokazu; Iehisa, Julio C M; Shimosaka, Etsuo; Takumi, Shigeo

2015-03-15

In common wheat, cultivar differences in freezing tolerance are considered to be mainly due to allelic differences at two major loci controlling freezing tolerance. One of the two loci, Fr-2, is coincident with a cluster of genes encoding C-repeat binding factors (CBFs), which induce downstream Cor/Lea genes during cold acclimation. Here, we conducted microarray analysis to study comprehensive changes in gene expression profile under long-term low-temperature (LT) treatment and to identify other LT-responsive genes related to cold acclimation in leaves of seedlings and crown tissues of a synthetic hexaploid wheat line. The microarray analysis revealed marked up-regulation of a number of Cor/Lea genes and fructan biosynthesis-related genes under the long-term LT treatment. For validation of the microarray data, we selected four synthetic wheat lines that contain the A and B genomes from the tetraploid wheat cultivar Langdon and the diverse D genomes originating from different Aegilops tauschii accessions with distinct levels of freezing tolerance after cold acclimation. Quantitative RT-PCR showed increased transcript levels of the Cor/Lea, CBF, and fructan biosynthesis-related genes in more freezing-tolerant lines than in sensitive lines. After a 14-day LT treatment, a significant difference in fructan accumulation was observed among the four lines. Therefore, the fructan biosynthetic pathway is associated with cold acclimation in development of wheat freezing tolerance and is another pathway related to diversity in freezing tolerance, in addition to the CBF-mediated Cor/Lea expression pathway. Copyright © 2014 Elsevier GmbH. All rights reserved.

Characteristics and expression patterns of the aldehyde dehydrogenase (ALDH) gene superfamily of foxtail millet (Setaria italica L.).

PubMed

Chen, Zhu; Chen, Ming; Xu, Zhao-shi; Li, Lian-cheng; Chen, Xue-ping; Ma, You-zhi

2014-01-01

Recent genomic sequencing of the foxtail millet, an abiotic, stress-tolerant crop, has provided a great opportunity for novel gene discovery and functional analysis of this popularly-grown grass. However, few stress-mediated gene families have been studied. Aldehyde dehydrogenases (ALDHs) comprise a gene superfamily encoding NAD (P) +-dependent enzymes that play the role of "aldehyde scavengers", which indirectly detoxify cellular ROS and reduce the effect of lipid peroxidation meditated cellular toxicity under various environmental stresses. In the current paper, we identified a total of 20 ALDH genes in the foxtail millet genome using a homology search and a phylogenetic analysis and grouped them into ten distinct families based on their amino acid sequence identity. Furthermore, evolutionary analysis of foxtail millet reveals that both tandem and segmental duplication contributed significantly to the expansion of its ALDH genes. The exon-intron structures of members of the same family in foxtail millet or the orthologous genes in rice display highly diverse distributions of their exonic and intronic regions. Also, synteny analysis shows that the majority of foxtail millet and rice ALDH gene homologs exist in the syntenic blocks between the two, implying that these ALDH genes arose before the divergence of cereals. Semi-quantitative and real-time quantitative PCR data reveals that a few SiALDH genes are expressed in an organ-specific manner and that the expression of a number of foxtail millet ALDH genes, such as, SiALDH7B1, SiALDH12A1 and SiALDH18B2 are up-regulated by osmotic stress, cold, H2O2, and phytohormone abscisic acid (ABA). Furthermore, the transformation of SiALDH2B2, SiALDH10A2, SiALDH5F1, SiALDH22A1, and SiALDH3E2 into Escherichia coli (E.coli) was able to improve their salt tolerance. Taken together, our results show that genome-wide identification characteristics and expression analyses provide unique opportunities for assessing the functional roles of foxtail millet ALDH genes in stress responses.

Frequent silencing of RASSF1A by DNA methylation in thymic neuroendocrine tumours.

PubMed

Kajiura, Koichiro; Takizawa, Hiromitsu; Morimoto, Yuki; Masuda, Kiyoshi; Tsuboi, Mitsuhiro; Kishibuchi, Reina; Wusiman, Nuliamina; Sawada, Toru; Kawakita, Naoya; Toba, Hiroaki; Yoshida, Mitsuteru; Kawakami, Yukikiyo; Naruto, Takuya; Imoto, Issei; Tangoku, Akira; Kondo, Kazuya

2017-09-01

Aberrant methylation of promoter CpG islands (CGIs) of tumour suppressor genes is a common epigenetic mechanism underlying cancer pathogenesis. The methylation patterns of thymic tumours have not been studied in detail since such tumours are rare. Herein, we sought to identify genes that could serve as epigenetic targets for thymic neuroendocrine tumour (NET) therapy. Genome-wide screening for aberrantly methylated CGIs was performed in three NET samples, seven thymic carcinoma (TC) samples, and eight type-B3 thymoma samples. The methylation status of thymic epithelial tumours (TETs) samples was validated by pyrosequencing in a larger cohort. The expression status was analysed by quantitative polymerase chain reaction (PCR) and immunohistochemistry. We identified a CGI on a novel gene, RASSF1A, which was strongly hypermethylated in NET, but not in thymic carcinoma or B3 thymoma. RASSF1A was identified as a candidate gene statistically and bibliographically, as it showed frequent CGI hypermethylation in NET by genome-wide screening. Pyrosequencing confirmed significant hypermethylation of a RASSF1A CGI in NET. Low-grade NET tissue was more strongly methylated than high-grade NET. Quantitative PCR and immunohistochemical staining revealed that RASSF1A mRNA and protein expression levels were negatively regulated by DNA methylation. RASSF1A is a tumour suppressor gene epigenetically dysregulated in NET. Aberrant methylation of RASSF1A has been reported in various tumours, but this is the first report of RASSF1A hypermethylation in TETs. RASSF1A may represent an epigenetic therapeutic target in thymic NET. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecularly tagged genes and quantitative trait loci in cucumber

USDA-ARS?s Scientific Manuscript database

Since the release of the cucumber draft genome, significant progress has been made in molecular mapping, tagging or cloning of horticulturally important genes and quantitative trait loci (QTLs) in cucumber, which provides the foundation for practicing marker-assisted selection in cucumber breeding. ...

Quantitative analysis of ChIP-seq data uncovers dynamic and sustained H3K4me3 and H3K27me3 modulation in cancer cells under hypoxia.

PubMed

Adriaens, Michiel E; Prickaerts, Peggy; Chan-Seng-Yue, Michelle; van den Beucken, Twan; Dahlmans, Vivian E H; Eijssen, Lars M; Beck, Timothy; Wouters, Bradly G; Voncken, Jan Willem; Evelo, Chris T A

2016-01-01

A comprehensive assessment of the epigenetic dynamics in cancer cells is the key to understanding the molecular mechanisms underlying cancer and to improving cancer diagnostics, prognostics and treatment. By combining genome-wide ChIP-seq epigenomics and microarray transcriptomics, we studied the effects of oxygen deprivation and subsequent reoxygenation on histone 3 trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in a breast cancer cell line, serving as a model for abnormal oxygenation in solid tumors. A priori, epigenetic markings and gene expression levels not only are expected to vary greatly between hypoxic and normoxic conditions, but also display a large degree of heterogeneity across the cell population. Where traditionally ChIP-seq data are often treated as dichotomous data, the model and experiment here necessitate a quantitative, data-driven analysis of both datasets. We first identified genomic regions with sustained epigenetic markings, which provided a sample-specific reference enabling quantitative ChIP-seq data analysis. Sustained H3K27me3 marking was located around centromeres and intergenic regions, while sustained H3K4me3 marking is associated with genes involved in RNA binding, translation and protein transport and localization. Dynamic marking with both H3K4me3 and H3K27me3 (hypoxia-induced bivalency) was found in CpG-rich regions at loci encoding factors that control developmental processes, congruent with observations in embryonic stem cells. In silico -identified epigenetically sustained and dynamic genomic regions were confirmed through ChIP-PCR in vitro, and obtained results are corroborated by published data and current insights regarding epigenetic regulation.

Targeting GPR30 in Abiraterone and MDV3100 Resistant Prostate Cancer

DTIC Science & Technology

2017-12-01

ID Labs, London, ON, Canada) following the manufacturer’s protocols. Quantitative real- time PCR Total RNA was treated with RNase-free DNase (Qiagen...99-gene panel for confirmation based on a literature search showing their relatedness to cell-mediated immune responses. Quantitative real- time PCR...mouse neutrophils (Geiser et al. 1993, Schaider et al. 2003), we analyzed murine neutrophil-related cytokine genes using quantitative real- time PCR

Genotype by watering regime interaction in cultivated tomato: lessons from linkage mapping and gene expression.

PubMed

Albert, Elise; Gricourt, Justine; Bertin, Nadia; Bonnefoi, Julien; Pateyron, Stéphanie; Tamby, Jean-Philippe; Bitton, Frédérique; Causse, Mathilde

2016-02-01

In tomato, genotype by watering interaction resulted from genotype re-ranking more than scale changes. Interactive QTLs according to watering regime were detected. Differentially expressed genes were identified in some intervals. As a result of climate change, drought will increasingly limit crop production in the future. Studying genotype by watering regime interactions is necessary to improve plant adaptation to low water availability. In cultivated tomato (Solanum lycopersicum L.), extensively grown in dry areas, well-mastered water deficits can stimulate metabolite production, increasing plant defenses and concentration of compounds involved in fruit quality, at the same time. However, few tomato Quantitative Trait Loci (QTLs) and genes involved in response to drought are identified or only in wild species. In this study, we phenotyped a population of 119 recombinant inbred lines derived from a cross between a cherry tomato and a large fruit tomato, grown in greenhouse under two watering regimes, in two locations. A large genetic variability was measured for 19 plant and fruit traits, under the two watering treatments. Highly significant genotype by watering regime interactions were detected and resulted from re-ranking more than scale changes. The population was genotyped for 679 SNP markers to develop a genetic map. In total, 56 QTLs were identified among which 11 were interactive between watering regimes. These later mainly exhibited antagonist effects according to watering treatment. Variation in gene expression in leaves of parental accessions revealed 2259 differentially expressed genes, among which candidate genes presenting sequence polymorphisms were identified under two main interactive QTLs. Our results provide knowledge about the genetic control of genotype by watering regime interactions in cultivated tomato and the possible use of deficit irrigation to improve tomato quality.

A Novel Dwarfism with Gonadal Dysfunction Due to Loss-of-Function Allele of the Collagen Receptor Gene, Ddr2, in the Mouse

PubMed Central

Kano, Kiyoshi; Marín de Evsikova, C.; Young, James; Wnek, Christopher; Maddatu, Terry P.; Nishina, Patsy M.; Naggert, Jürgen K.

2008-01-01

Smallie (slie), a spontaneous, autosomal-recessive mutation causes dwarfing and infertility in mice. The purpose of this study was to determine and characterize the underlying molecular genetic basis for its phenotype. The slie locus was mapped to chromosome 1, and fine-structure mapping narrowed the slie allele within 2 Mb between genetic markers D1Mit36 and Mpz. To pinpoint the underlying mutation quantitative real-time PCR was used to measure the relative expression levels for the genes residing within this region. Expression of one gene, Ddr2, which encodes discoidin domain receptor 2 (DDR2), was absent in slie homozygote mice. Genomic sequencing analysis detected a 150-kb deletion that extended into the Ddr2 gene transcript. Detailed phenotype analysis revealed that gonadal dysregulation underlies infertility in slie mice because all females were anovulatory and most adult males lacked spermatogenesis. The pituitary gland of prepubertal slie mice was smaller than in wild-type mice. The basal levels and gene expression for pituitary and hypothalamic hormones, and gene expression for hypothalamic-releasing hormones, were not significantly different between slie and wild-type mice. Circulating levels of IGF-1 did not differ in slie mice despite lower Igf-1 mRNA expression in the liver. After exogenous gonadotropin administration, the levels of secreted steroid hormones in both male and female adult slie mice were blunted compared to adult wild-type, but was similar to prepubertal wild-type mice. Taken together, our results indicate that the absence of DDR2 leads to growth retardation and gonadal dysfunction due to peripheral defects in hormonal-responsive pathways in slie mice. PMID:18483174

[Genetic characteristics associated with drought tolerance of plant height and thousand-grain mass of recombinant inbred lines of wheat].

PubMed

Yang, De-Long; Zhang, Guo-Hong; Li, Xing-Mao; Xing, Hua; Cheng, Hong-Bo; Ni, Sheng-Li; Chen, Xiao-Ping

2012-06-01

A total of 120 recombinant inbred lines (RIL) derived from Chinese winter wheat cultivars Longjian 19xQ9086 and the two parents were taken as test materials to study the quantitative genetics characteristics of their plant height at different development stages, thousand-grain mass, as well as the correlations between the two traits under rainfed (drought stress) and well-watered conditions, and evaluate the genetic variation of the RIL. Under the two water conditions, the target traits of the RIL showed substantial transgressive segregation and great sensitivity to water condition. The drought stress coefficient of the plant height was higher at jointing stage, being up to 0.851. There was a significant positive correlation between the plant height at different development stages and the thousand-grain mass, and comparing with that at other growth stages, the plant height at jointing stage had a higher correlation coefficient with the thousand-grain mass (R2DS = 0.32, R2WW = 0.28). The plant height at both jointing and flowering stages had significant positive and direct effect but negative and indirect gross effect on the thousand-grain mass, while the plant height at heading and maturing stages was in adverse. The target traits showed a lower heritability ranged from 0.27 to 0.60. The numbers of the gene pairs controlling the thousand-grain mass were 10 under rainfed and 13 under well-watered conditions, while those of the gene pairs controlling the plant height at different development stages were 3-7 under rainfed and 4-14 under well-watered conditions, respectively. According to the clustering of the drought stress coefficient of plant height, the RIL could be classified into five subgroups, showing the abundant variation of the RIL in their phe- notypes and in the sensitivity to water condition. It was considered that the test RIL were appropriate for the study of the quantitative genetics of wheat drought resistance.

Bacterial growth laws and their applications.

PubMed

Scott, Matthew; Hwa, Terence

2011-08-01

Quantitative empirical relationships between cell composition and growth rate played an important role in the early days of microbiology. Gradually, the focus of the field began to shift from growth physiology to the ever more elaborate molecular mechanisms of regulation employed by the organisms. Advances in systems biology and biotechnology have renewed interest in the physiology of the cell as a whole. Furthermore, gene expression is known to be intimately coupled to the growth state of the cell. Here, we review recent efforts in characterizing such couplings, particularly the quantitative phenomenological approaches exploiting bacterial 'growth laws.' These approaches point toward underlying design principles that can guide the predictive manipulation of cell behavior in the absence of molecular details. Copyright © 2011 Elsevier Ltd. All rights reserved.

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

PubMed

Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

2015-11-20

The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood.

PubMed

Zheng, Zhi; Luo, Yuling; McMaster, Gary K

2006-07-01

Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format. We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection. The single- and multiplex assays could quantitatively measure as few as 6000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 microL of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood. Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

A systems-genetics approach and data mining tool to assist in the discovery of genes underlying complex traits in Oryza sativa.

PubMed

Ficklin, Stephen P; Feltus, Frank Alex

2013-01-01

Many traits of biological and agronomic significance in plants are controlled in a complex manner where multiple genes and environmental signals affect the expression of the phenotype. In Oryza sativa (rice), thousands of quantitative genetic signals have been mapped to the rice genome. In parallel, thousands of gene expression profiles have been generated across many experimental conditions. Through the discovery of networks with real gene co-expression relationships, it is possible to identify co-localized genetic and gene expression signals that implicate complex genotype-phenotype relationships. In this work, we used a knowledge-independent, systems genetics approach, to discover a high-quality set of co-expression networks, termed Gene Interaction Layers (GILs). Twenty-two GILs were constructed from 1,306 Affymetrix microarray rice expression profiles that were pre-clustered to allow for improved capture of gene co-expression relationships. Functional genomic and genetic data, including over 8,000 QTLs and 766 phenotype-tagged SNPs (p-value < = 0.001) from genome-wide association studies, both covering over 230 different rice traits were integrated with the GILs. An online systems genetics data-mining resource, the GeneNet Engine, was constructed to enable dynamic discovery of gene sets (i.e. network modules) that overlap with genetic traits. GeneNet Engine does not provide the exact set of genes underlying a given complex trait, but through the evidence of gene-marker correspondence, co-expression, and functional enrichment, site visitors can identify genes with potential shared causality for a trait which could then be used for experimental validation. A set of 2 million SNPs was incorporated into the database and serve as a potential set of testable biomarkers for genes in modules that overlap with genetic traits. Herein, we describe two modules found using GeneNet Engine, one with significant overlap with the trait amylose content and another with significant overlap with blast disease resistance.

A Systems-Genetics Approach and Data Mining Tool to Assist in the Discovery of Genes Underlying Complex Traits in Oryza sativa

PubMed Central

Ficklin, Stephen P.; Feltus, Frank Alex

2013-01-01

Many traits of biological and agronomic significance in plants are controlled in a complex manner where multiple genes and environmental signals affect the expression of the phenotype. In Oryza sativa (rice), thousands of quantitative genetic signals have been mapped to the rice genome. In parallel, thousands of gene expression profiles have been generated across many experimental conditions. Through the discovery of networks with real gene co-expression relationships, it is possible to identify co-localized genetic and gene expression signals that implicate complex genotype-phenotype relationships. In this work, we used a knowledge-independent, systems genetics approach, to discover a high-quality set of co-expression networks, termed Gene Interaction Layers (GILs). Twenty-two GILs were constructed from 1,306 Affymetrix microarray rice expression profiles that were pre-clustered to allow for improved capture of gene co-expression relationships. Functional genomic and genetic data, including over 8,000 QTLs and 766 phenotype-tagged SNPs (p-value < = 0.001) from genome-wide association studies, both covering over 230 different rice traits were integrated with the GILs. An online systems genetics data-mining resource, the GeneNet Engine, was constructed to enable dynamic discovery of gene sets (i.e. network modules) that overlap with genetic traits. GeneNet Engine does not provide the exact set of genes underlying a given complex trait, but through the evidence of gene-marker correspondence, co-expression, and functional enrichment, site visitors can identify genes with potential shared causality for a trait which could then be used for experimental validation. A set of 2 million SNPs was incorporated into the database and serve as a potential set of testable biomarkers for genes in modules that overlap with genetic traits. Herein, we describe two modules found using GeneNet Engine, one with significant overlap with the trait amylose content and another with significant overlap with blast disease resistance. PMID:23874666

Evaluation of Candidate Reference Genes for Quantitative Gene Expression Analysis in Spodoptera exigu a after Long-time Exposure to Cadmium.

PubMed

Płachetka-Bożek, Anna; Augustyniak, Maria

2017-08-21

Studies on the transcriptional control of gene expression play an important role in many areas of biology. Reference genes, which are often referred to as housekeeping genes, such as GAPDH, G3PDH, EF2, RpL7A, RpL10, TUBα and Actin, have traditionally been assumed to be stably expressed in all conditions, and they are frequently used to normalize mRNA levels between different samples in qPCR analysis. However, it is known that the expression of these genes is influenced by numerous factors, such as experimental conditions. The difference in gene expression underlies a range of biological processes, including development, reproduction and behavior. The aim of this study was to show the problems associated with using reference genes in the qPCR technique, in a study on inbred strains of Spodoptera exigua selected toward cadmium resistance. We present and discuss our results and observations, and give some recommendations concerning the use and limitations of housekeeping genes as internal standards, especially in research on insects. Our results suggest that holometabolism and poikilothermia, as well as time since metamorphosis and the level of exposure to the selective factor (cadmium in this case), have a significant effect on the expression of reference genes.

Reference genes for quantitative real-time PCR analysis in symbiont Entomomyces delphacidicola of Nilaparvata lugens (Stål)

PubMed Central

Wan, Pin-Jun; Tang, Yao-Hua; Yuan, San-Yue; He, Jia-Chun; Wang, Wei-Xia; Lai, Feng-Xiang; Fu, Qiang

2017-01-01

Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) is a major rice pest that harbors an endosymbiont ascomycete fungus, Entomomyces delphacidicola str. NLU (also known as yeast-like symbiont, YLS). Driving by demand of novel population management tactics (e.g. RNAi), the importance of YLS has been studied and revealed, which greatly boosts the interest of molecular level studies related to YLS. The current study focuses on reference genes for RT-qPCR studies related to YLS. Eight previously unreported YLS genes were cloned, and their expressions were evaluated for N. lugens samples of different developmental stages and sexes, and under different nutritional conditions and temperatures. Expression stabilities were analyzed by BestKeeper, geNorm, NormFinder, ΔCt method and RefFinder. Furthermore, the selected reference genes for RT-qPCR of YLS genes were validated using targeted YLS genes that respond to different nutritional conditions (amino acid deprivation) and RNAi. The results suggest that ylsRPS15p/ylsACT are the most suitable reference genes for temporal gene expression profiling, while ylsTUB/ylsACT and ylsRPS15e/ylsGADPH are the most suitable reference gene choices for evaluating nutrition and temperature effects. Validation studies demonstrated the advantage of using endogenous YLS reference genes for YLS studies. PMID:28198810

Genome-Wide Survey of Cold Stress Regulated Alternative Splicing in Arabidopsis thaliana with Tiling Microarray

PubMed Central

Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

2013-01-01

Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression. PMID:23776682

Mechanisms underlying Actinobacillus pleuropneumoniae exotoxin ApxI induced expression of IL-1β, IL-8 and TNF-α in porcine alveolar macrophages

PubMed Central

2011-01-01

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes fibrino-hemorrhagic necrotizing pleuropneumonia in pigs. Production of proinflammatory mediators in the lungs is an important feature of A. pleuropneumoniae infection. However, bacterial components other than lipopolysaccharide involved in this process remain unidentified. The goals of this study were to determine the role of A. pleuropneumoniae exotoxin ApxI in cytokine induction and to delineate the underlying mechanisms. Using real-time quantitative PCR analysis, we found native ApxI stimulated porcine alveolar macrophages (PAMs) to transcribe mRNAs of IL-1β, IL-8 and TNF-α in a concentration- and time-dependent manner. Heat-inactivation or pre-incubation of ApxI with a neutralizing antiserum attenuated ApxI bioactivity to induce cytokine gene expression. The secretion of IL-1β, IL-8 and TNF-α protein from PAMs stimulated with ApxI was also confirmed by quantitative ELISA. In delineating the underlying signaling pathways contributing to cytokine expression, we observed mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK) were activated upon ApxI stimulation. Administration of an inhibitor specific to p38 or JNK resulted in varying degrees of attenuation on ApxI-induced cytokine expression, suggesting the differential regulatory roles of p38 and JNK in IL-1β, IL-8 and TNF-α production. Further, pre-incubation of PAMs with a CD18-blocking antibody prior to ApxI stimulation significantly reduced the activation of p38 and JNK, and subsequent expression of IL-1β, IL-8 or TNF-α gene, indicating a pivotal role of β2 integrins in the ApxI-mediated effect. Collectively, this study demonstrated ApxI induces gene expression of IL-1β, IL-8 and TNF-α in PAMs that involves β2 integrins and downstream MAPKs. PMID:21314908

A Simple Test Identifies Selection on Complex Traits.

PubMed

Beissinger, Tim; Kruppa, Jochen; Cavero, David; Ha, Ngoc-Thuy; Erbe, Malena; Simianer, Henner

2018-05-01

Important traits in agricultural, natural, and human populations are increasingly being shown to be under the control of many genes that individually contribute only a small proportion of genetic variation. However, the majority of modern tools in quantitative and population genetics, including genome-wide association studies and selection-mapping protocols, are designed to identify individual genes with large effects. We have developed an approach to identify traits that have been under selection and are controlled by large numbers of loci. In contrast to existing methods, our technique uses additive-effects estimates from all available markers, and relates these estimates to allele-frequency change over time. Using this information, we generate a composite statistic, denoted [Formula: see text] which can be used to test for significant evidence of selection on a trait. Our test requires pre- and postselection genotypic data but only a single time point with phenotypic information. Simulations demonstrate that [Formula: see text] is powerful for identifying selection, particularly in situations where the trait being tested is controlled by many genes, which is precisely the scenario where classical approaches for selection mapping are least powerful. We apply this test to breeding populations of maize and chickens, where we demonstrate the successful identification of selection on traits that are documented to have been under selection. Copyright © 2018 Beissinger et al.

A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays.

PubMed

Hampf, Mathias; Gossen, Manfred

2006-09-01

We established a quantitative reporter gene protocol, the P/Rluc assay system, allowing the sequential measurement of Photinus and Renilla luciferase activities from the same extract. Other than comparable commercial reporter assay systems and their noncommercial counterparts, the P/Rluc assay system was formulated under the aspect of full compatibility with standard methods for protein assays. This feature greatly expands the range of applications for assay systems quantifying the expression of multiple luciferase reporters.

Increased pericarp cell length underlies a major quantitative trait locus for grain weight in hexaploid wheat.

PubMed

Brinton, Jemima; Simmonds, James; Minter, Francesca; Leverington-Waite, Michelle; Snape, John; Uauy, Cristobal

2017-08-01

Crop yields must increase to address food insecurity. Grain weight, determined by grain length and width, is an important yield component, but our understanding of the underlying genes and mechanisms is limited. We used genetic mapping and near isogenic lines (NILs) to identify, validate and fine-map a major quantitative trait locus (QTL) on wheat chromosome 5A associated with grain weight. Detailed phenotypic characterisation of developing and mature grains from the NILs was performed. We identified a stable and robust QTL associated with a 6.9% increase in grain weight. The positive interval leads to 4.0% longer grains, with differences first visible 12 d after fertilization. This grain length effect was fine-mapped to a 4.3 cM interval. The locus also has a pleiotropic effect on grain width (1.5%) during late grain development that determines the relative magnitude of the grain weight increase. Positive NILs have increased maternal pericarp cell length, an effect which is independent of absolute grain length. These results provide direct genetic evidence that pericarp cell length affects final grain size and weight in polyploid wheat. We propose that combining genes that control distinct biological mechanisms, such as cell expansion and proliferation, will enhance crop yields. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

High-resolution gene expression data from blastoderm embryos of the scuttle fly Megaselia abdita

PubMed Central

Wotton, Karl R; Jiménez-Guri, Eva; Crombach, Anton; Cicin-Sain, Damjan; Jaeger, Johannes

2015-01-01

Gap genes are involved in segment determination during early development in dipteran insects (flies, midges, and mosquitoes). We carried out a systematic quantitative comparative analysis of the gap gene network across different dipteran species. Our work provides mechanistic insights into the evolution of this pattern-forming network. As a central component of our project, we created a high-resolution quantitative spatio-temporal data set of gap and maternal co-ordinate gene expression in the blastoderm embryo of the non-drosophilid scuttle fly, Megaselia abdita. Our data include expression patterns in both wild-type and RNAi-treated embryos. The data—covering 10 genes, 10 time points, and over 1,000 individual embryos—consist of original embryo images, quantified expression profiles, extracted positions of expression boundaries, and integrated expression patterns, plus metadata and intermediate processing steps. These data provide a valuable resource for researchers interested in the comparative study of gene regulatory networks and pattern formation, an essential step towards a more quantitative and mechanistic understanding of developmental evolution. PMID:25977812

Expression of root-related transcription factors associated with flooding tolerance of soybean (Glycine max).

PubMed

Valliyodan, Babu; Van Toai, Tara T; Alves, Jose Donizeti; de Fátima P Goulart, Patricia; Lee, Jeong Dong; Fritschi, Felix B; Rahman, Mohammed Atiqur; Islam, Rafiq; Shannon, J Grover; Nguyen, Henry T

2014-09-29

Much research has been conducted on the changes in gene expression of the model plant Arabidopsis to low-oxygen stress. Flooding results in a low oxygen environment in the root zone. However, there is ample evidence that tolerance to soil flooding is more than tolerance to low oxygen alone. In this study, we investigated the physiological response and differential expression of root-related transcription factors (TFs) associated with the tolerance of soybean plants to soil flooding. Differential responses of PI408105A and S99-2281 plants to ten days of soil flooding were evaluated at physiological, morphological and anatomical levels. Gene expression underlying the tolerance response was investigated using qRT-PCR of root-related TFs, known anaerobic genes, and housekeeping genes. Biomass of flood-sensitive S99-2281 roots remained unchanged during the entire 10 days of flooding. Flood-tolerant PI408105A plants exhibited recovery of root growth after 3 days of flooding. Flooding induced the development of aerenchyma and adventitious roots more rapidly in the flood-tolerant than the flood-sensitive genotype. Roots of tolerant plants also contained more ATP than roots of sensitive plants at the 7th and 10th days of flooding. Quantitative transcript analysis identified 132 genes differentially expressed between the two genotypes at one or more time points of flooding. Expression of genes related to the ethylene biosynthesis pathway and formation of adventitious roots was induced earlier and to higher levels in roots of the flood-tolerant genotype. Three potential flood-tolerance TFs which were differentially expressed between the two genotypes during the entire 10-day flooding duration were identified. This study confirmed the expression of anaerobic genes in response to soil flooding. Additionally, the differential expression of TFs associated with soil flooding tolerance was not qualitative but quantitative and temporal. Functional analyses of these genes will be necessary to reveal their potential to enhance flooding tolerance of soybean cultivars.

Expression of Root-Related Transcription Factors Associated with Flooding Tolerance of Soybean (Glycine max)

PubMed Central

Valliyodan, Babu; Van Toai, Tara T.; Alves, Jose Donizeti; de Fátima P. Goulart, Patricia; Lee, Jeong Dong; Fritschi, Felix B.; Rahman, Mohammed Atiqur; Islam, Rafiq; Shannon, J. Grover; Nguyen, Henry T.

2014-01-01

Much research has been conducted on the changes in gene expression of the model plant Arabidopsis to low-oxygen stress. Flooding results in a low oxygen environment in the root zone. However, there is ample evidence that tolerance to soil flooding is more than tolerance to low oxygen alone. In this study, we investigated the physiological response and differential expression of root-related transcription factors (TFs) associated with the tolerance of soybean plants to soil flooding. Differential responses of PI408105A and S99-2281 plants to ten days of soil flooding were evaluated at physiological, morphological and anatomical levels. Gene expression underlying the tolerance response was investigated using qRT-PCR of root-related TFs, known anaerobic genes, and housekeeping genes. Biomass of flood-sensitive S99-2281 roots remained unchanged during the entire 10 days of flooding. Flood-tolerant PI408105A plants exhibited recovery of root growth after 3 days of flooding. Flooding induced the development of aerenchyma and adventitious roots more rapidly in the flood-tolerant than the flood-sensitive genotype. Roots of tolerant plants also contained more ATP than roots of sensitive plants at the 7th and 10th days of flooding. Quantitative transcript analysis identified 132 genes differentially expressed between the two genotypes at one or more time points of flooding. Expression of genes related to the ethylene biosynthesis pathway and formation of adventitious roots was induced earlier and to higher levels in roots of the flood-tolerant genotype. Three potential flood-tolerance TFs which were differentially expressed between the two genotypes during the entire 10-day flooding duration were identified. This study confirmed the expression of anaerobic genes in response to soil flooding. Additionally, the differential expression of TFs associated with soil flooding tolerance was not qualitative but quantitative and temporal. Functional analyses of these genes will be necessary to reveal their potential to enhance flooding tolerance of soybean cultivars. PMID:25268626

Expression quantitative trait loci (eQTL) mapping in Puerto Rican children.

PubMed

Chen, Wei; Brehm, John M; Lin, Jerome; Wang, Ting; Forno, Erick; Acosta-Pérez, Edna; Boutaoui, Nadia; Canino, Glorisa; Celedón, Juan C

2015-01-01

Expression quantitative trait loci (eQTL) have been identified using tissue or cell samples from diverse human populations, thus enhancing our understanding of regulation of gene expression. However, few studies have attempted to identify eQTL in racially admixed populations such as Hispanics. We performed a systematic eQTL study to identify regulatory variants of gene expression in whole blood from 121 Puerto Rican children with (n = 63) and without (n = 58) asthma. Genome-wide genotyping was conducted using the Illumina Omni2.5M Bead Chip, and gene expression was assessed using the Illumina HT-12 microarray. After completing quality control, we performed a pair-wise genome analysis of ~15 K transcripts and ~1.3 M SNPs for both local and distal effects. This analysis was conducted under a regression framework adjusting for age, gender and principal components derived from both genotypic and mRNA data. We used a false discovery rate (FDR) approach to identify significant eQTL signals, which were next compared to top eQTL signals from existing eQTL databases. We then performed a pathway analysis for our top genes. We identified 36,720 local pairs in 3,391 unique genes and 1,851 distal pairs in 446 unique genes at FDR <0.05, corresponding to unadjusted P values lower than 1.5x10-4 and 4.5x10-9, respectively. A significant proportion of genes identified in our study overlapped with those identified in previous studies. We also found an enrichment of disease-related genes in our eQTL list. We present results from the first eQTL study in Puerto Rican children, who are members of a unique Hispanic cohort disproportionately affected with asthma, prematurity, obesity and other common diseases. Our study confirmed eQTL signals identified in other ethnic groups, while also detecting additional eQTLs unique to our study population. The identified eQTLs will help prioritize findings from future genome-wide association studies in Puerto Ricans.

Deep Conservation of Genes Required for Both Drosophila melanogaster and Caenorhabditis elegans Sleep Includes a Role for Dopaminergic Signaling

PubMed Central

Singh, Komudi; Ju, Jennifer Y.; Walsh, Melissa B.; DiIorio, Michael A.; Hart, Anne C.

2014-01-01

Objectives: Cross-species conservation of sleep-like behaviors predicts the presence of conserved molecular mechanisms underlying sleep. However, limited experimental evidence of conservation exists. Here, this prediction is tested directly. Measurements and Results: During lethargus, Caenorhabditis elegans spontaneously sleep in short bouts that are interspersed with bouts of spontaneous locomotion. We identified 26 genes required for Drosophila melanogaster sleep. Twenty orthologous C. elegans genes were selected based on similarity. Their effect on C. elegans sleep and arousal during the last larval lethargus was assessed. The 20 most similar genes altered both the quantity of sleep and arousal thresholds. In 18 cases, the direction of change was concordant with Drosophila studies published previously. Additionally, we delineated a conserved genetic pathway by which dopamine regulates sleep and arousal. In C. elegans neurons, G-alpha S, adenylyl cyclase, and protein kinase A act downstream of D1 dopamine receptors to regulate these behaviors. Finally, a quantitative analysis of genes examined herein revealed that C. elegans arousal thresholds were directly correlated with amount of sleep during lethargus. However, bout duration varies little and was not correlated with arousal thresholds. Conclusions: The comprehensive analysis presented here suggests that conserved genes and pathways are required for sleep in invertebrates and, likely, across the entire animal kingdom. The genetic pathway delineated in this study implicates G-alpha S and previously known genes downstream of dopamine signaling in sleep. Quantitative analysis of various components of quiescence suggests that interdependent or identical cellular and molecular mechanisms are likely to regulate both arousal and sleep entry. Citation: Singh K, Ju JY, Walsh MB, Dilorio MA, Hart AC. Deep conservation of genes required for both Drosophila melanogaster and Caenorhabditis elegans sleep includes a role for dopaminergic signaling. SLEEP 2014;37(9):1439-1451. PMID:25142568

Validating internal controls for quantitative plant gene expression studies.

PubMed

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-08-18

Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.

Detecting Genetic Interactions for Quantitative Traits Using m-Spacing Entropy Measure

PubMed Central

Yee, Jaeyong; Kwon, Min-Seok; Park, Taesung; Park, Mira

2015-01-01

A number of statistical methods for detecting gene-gene interactions have been developed in genetic association studies with binary traits. However, many phenotype measures are intrinsically quantitative and categorizing continuous traits may not always be straightforward and meaningful. Association of gene-gene interactions with an observed distribution of such phenotypes needs to be investigated directly without categorization. Information gain based on entropy measure has previously been successful in identifying genetic associations with binary traits. We extend the usefulness of this information gain by proposing a nonparametric evaluation method of conditional entropy of a quantitative phenotype associated with a given genotype. Hence, the information gain can be obtained for any phenotype distribution. Because any functional form, such as Gaussian, is not assumed for the entire distribution of a trait or a given genotype, this method is expected to be robust enough to be applied to any phenotypic association data. Here, we show its use to successfully identify the main effect, as well as the genetic interactions, associated with a quantitative trait. PMID:26339620

Four Linked Genes Participate in Controlling Sporulation Efficiency in Budding Yeast

PubMed Central

Ben-Ari, Giora; Zenvirth, Drora; Sherman, Amir; David, Lior; Klutstein, Michael; Lavi, Uri; Hillel, Jossi; Simchen, Giora

2006-01-01

Quantitative traits are conditioned by several genetic determinants. Since such genes influence many important complex traits in various organisms, the identification of quantitative trait loci (QTLs) is of major interest, but still encounters serious difficulties. We detected four linked genes within one QTL, which participate in controlling sporulation efficiency in Saccharomyces cerevisiae. Following the identification of single nucleotide polymorphisms by comparing the sequences of 145 genes between the parental strains SK1 and S288c, we analyzed the segregating progeny of the cross between them. Through reciprocal hemizygosity analysis, four genes, RAS2, PMS1, SWS2, and FKH2, located in a region of 60 kilobases on Chromosome 14, were found to be associated with sporulation efficiency. Three of the four “high” sporulation alleles are derived from the “low” sporulating strain. Two of these sporulation-related genes were verified through allele replacements. For RAS2, the causative variation was suggested to be a single nucleotide difference in the upstream region of the gene. This quantitative trait nucleotide accounts for sporulation variability among a set of ten closely related winery yeast strains. Our results provide a detailed view of genetic complexity in one “QTL region” that controls a quantitative trait and reports a single nucleotide polymorphism-trait association in wild strains. Moreover, these findings have implications on QTL identification in higher eukaryotes. PMID:17112318

Association mapping reveals the genetic architecture of tomato response to water deficit: focus on major fruit quality traits

PubMed Central

Albert, Elise; Segura, Vincent; Gricourt, Justine; Bonnefoi, Julien; Derivot, Laurent; Causse, Mathilde

2016-01-01

Water scarcity constitutes a crucial constraint for agriculture productivity. High-throughput approaches in model plant species identified hundreds of genes potentially involved in survival under drought, but few having beneficial effects on quality and yield. Nonetheless, controlled water deficit may improve fruit quality through higher concentration of flavor compounds. The underlying genetic determinants are still poorly known. In this study, we phenotyped 141 highly diverse small fruit tomato accessions for 27 traits under two contrasting watering conditions. A subset of 55 accessions exhibited increased metabolite contents and maintained yield under water deficit. Using 6100 single nucleotide polymorphisms (SNPs), association mapping revealed 31, 41, and 44 quantitative trait loci (QTLs) under drought, control, and both conditions, respectively. Twenty-five additional QTLs were interactive between conditions, emphasizing the interest in accounting for QTLs by watering regime interactions in fruit quality improvement. Combining our results with the loci previously identified in a biparental progeny resulted in 11 common QTLs and contributed to a first detailed characterization of the genetic determinants of response to water deficit in tomato. Major QTLs for fruit quality traits were dissected and candidate genes were proposed using expression and polymorphism data. The outcomes provide a basis for fruit quality improvement under deficit irrigation while limiting yield losses. PMID:27856709

Effects of Leucine Supplementation and Serum Withdrawal on Branched-Chain Amino Acid Pathway Gene and Protein Expression in Mouse Adipocytes

PubMed Central

Vivar, Juan C.; Knight, Megan S.; Pointer, Mildred A.; Gwathmey, Judith K.; Ghosh, Sujoy

2014-01-01

The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of the transcriptomic response of this pathway to variations in nutrient availability. PMID:25050624

Serratia entomophila bet gene induction and the impact of glycine betaine accumulation on desiccation tolerance.

PubMed

Sheen, T R; O'Callaghan, M; Smalley, D J; Ronson, C W; Hurst, M R H

2013-02-01

The genes involved in choline transport and oxidation to glycine betaine in the biopesticidal bacterium Serratia entomophila were characterized, and the potential of osmoprotectants, coupled with increased NaCl concentrations, to improve the desiccation tolerance of this species was investigated. Serratia entomophila carries sequences similar to the Escherichia coli betTIBA genes encoding a choline transporter and dehydrogenase, a betaine aldehyde dehydrogenase and a regulatory protein. Disruption of betA abolished the ability of Ser. entomophila to utilize choline as a carbon source. Quantitative reverse-transcriptase PCR analysis revealed that betA transcription was reduced compared to that of the upstream genes in the operon, and that NaCl and choline induced bet gene expression. Glycine betaine and choline increased the NaCl tolerance of Ser. entomophila, and osmotically preconditioned cultures survived better than control cultures following desiccation and immediately after application to agricultural soil. Addition of glycine betaine and NaCl to growth medium can greatly enhance the desiccation survival of Ser. entomophila, and its initial survival in soil. Serratia entomophila is sensitive to desiccation and does not persist under low soil moisture conditions. Techniques described here for enhancing the desiccation survival of Ser. entomophila can be used to improve formulations of this bacterium, and allow its application under a wider range of environmental conditions. © 2012 AgResearch.

Time-Series Analyses of Transcriptomes and Proteomes Reveal Molecular Networks Underlying Oil Accumulation in Canola.

PubMed

Wan, Huafang; Cui, Yixin; Ding, Yijuan; Mei, Jiaqin; Dong, Hongli; Zhang, Wenxin; Wu, Shiqi; Liang, Ying; Zhang, Chunyu; Li, Jiana; Xiong, Qing; Qian, Wei

2016-01-01

Understanding the regulation of lipid metabolism is vital for genetic engineering of canola ( Brassica napus L.) to increase oil yield or modify oil composition. We conducted time-series analyses of transcriptomes and proteomes to uncover the molecular networks associated with oil accumulation and dynamic changes in these networks in canola. The expression levels of genes and proteins were measured at 2, 4, 6, and 8 weeks after pollination (WAP). Our results show that the biosynthesis of fatty acids is a dominant cellular process from 2 to 6 WAP, while the degradation mainly happens after 6 WAP. We found that genes in almost every node of fatty acid synthesis pathway were significantly up-regulated during oil accumulation. Moreover, significant expression changes of two genes, acetyl-CoA carboxylase and acyl-ACP desaturase, were detected on both transcriptomic and proteomic levels. We confirmed the temporal expression patterns revealed by the transcriptomic analyses using quantitative real-time PCR experiments. The gene set association analysis show that the biosynthesis of fatty acids and unsaturated fatty acids are the most significant biological processes from 2-4 WAP and 4-6 WAP, respectively, which is consistent with the results of time-series analyses. These results not only provide insight into the mechanisms underlying lipid metabolism, but also reveal novel candidate genes that are worth further investigation for their values in the genetic engineering of canola.

A genome-wide association study of a global rice panel reveals resistance in Oryza sativa to root-knot nematodes.

PubMed

Dimkpa, Stanley O N; Lahari, Zobaida; Shrestha, Roshi; Douglas, Alex; Gheysen, Godelieve; Price, Adam H

2016-02-01

The root-knot nematode Meloidogyne graminicola is one of the most serious nematode pests worldwide and represents a major constraint on rice production. While variation in the susceptibility of Asian rice (Oryza sativa) exists, so far no strong and reliable resistance has been reported. Quantitative trait loci for partial resistance have been reported but no underlying genes have been tagged or cloned. Here, 332 accessions of the Rice Diversity Panel 1 were assessed for gall formation, revealing large variation across all subpopulations of rice and higher susceptibility in temperate japonica accessions. Accessions Khao Pahk Maw and LD 24 appeared to be resistant, which was confirmed in large pot experiments where no galls were observed. Detailed observations on these two accessions revealed no nematodes inside the roots 2 days after inoculation and very few females after 17 days (5 in Khao Pahk Maw and <1 in LD 24, in comparison with >100 in the susceptible controls). These two cultivars appear ideal donors for breeding root-knot nematode resistance. A genome-wide association study revealed 11 quantitative trait loci, two of which are close to epistatic loci detected in the Bala x Azucena population. The discussion highlights a small number of candidate genes worth exploring further, in particular many genes with lectin domains and genes on chromosome 11 with homology to the Hordeum Mla locus. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Neutral mutation as the source of genetic variation in life history traits.

PubMed

Brcić-Kostić, Krunoslav

2005-08-01

The mechanism underlying the maintenance of adaptive genetic variation is a long-standing question in evolutionary genetics. There are two concepts (mutation-selection balance and balancing selection) which are based on the phenotypic differences between alleles. Mutation - selection balance and balancing selection cannot properly explain the process of gene substitution, i.e. the molecular evolution of quantitative trait loci affecting fitness. I assume that such loci have non-essential functions (small effects on fitness), and that they have the potential to evolve into new functions and acquire new adaptations. Here I show that a high amount of neutral polymorphism at these loci can exist in real populations. Consistent with this, I propose a hypothesis for the maintenance of genetic variation in life history traits which can be efficient for the fixation of alleles with very small selective advantage. The hypothesis is based on neutral polymorphism at quantitative trait loci and both neutral and adaptive gene substitutions. The model of neutral - adaptive conversion (NAC) assumes that neutral alleles are not neutral indefinitely, and that in specific and very rare situations phenotypic (relative fitness) differences between them can appear. In this paper I focus on NAC due to phenotypic plasticity of neutral alleles. The important evolutionary consequence of NAC could be the increased adaptive potential of a population. Loci responsible for adaptation should be fast evolving genes with minimally discernible phenotypic effects, and the recent discovery of genes with such characteristics implicates them as suitable candidates for loci involved in adaptation.

Normalization of Reverse Transcription Quantitative PCR Data During Ageing in Distinct Cerebral Structures.

PubMed

Bruckert, G; Vivien, D; Docagne, F; Roussel, B D

2016-04-01

Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a routine method in many laboratories. Normalization of data from experimental conditions is critical for data processing and is usually achieved by the use of a single reference gene. Nevertheless, as pointed by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, several reference genes should be used for reliable normalization. Ageing is a physiological process that results in a decline of many expressed genes. Reliable normalization of RT-qPCR data becomes crucial when studying ageing. Here, we propose a RT-qPCR study from four mouse brain regions (cortex, hippocampus, striatum and cerebellum) at different ages (from 8 weeks to 22 months) in which we studied the expression of nine commonly used reference genes. With the use of two different algorithms, we found that all brain structures need at least two genes for a good normalization step. We propose specific pairs of gene for efficient data normalization in the four brain regions studied. These results underline the importance of reliable reference genes for specific brain regions in ageing.

A quantitative study of the benefits of co-regulation using the spoIIA operon as an example

PubMed Central

Iber, Dagmar

2006-01-01

The distribution of most genes is not random, and functionally linked genes are often found in clusters. Several theories have been put forward to explain the emergence and persistence of operons in bacteria. Careful analysis of genomic data favours the co-regulation model, where gene organization into operons is driven by the benefits of coordinated gene expression and regulation. Direct evidence that coexpression increases the individual's fitness enough to ensure operon formation and maintenance is, however, still lacking. Here, a previously described quantitative model of the network that controls the transcription factor σF during sporulation in Bacillus subtilis is employed to quantify the benefits arising from both organization of the sporulation genes into the spoIIA operon and from translational coupling. The analysis shows that operon organization, together with translational coupling, is important because of the inherent stochastic nature of gene expression, which skews the ratios between protein concentrations in the absence of co-regulation. The predicted impact of different forms of gene regulation on fitness and survival agrees quantitatively with published sporulation efficiencies. PMID:16924264

A quantitative study of the benefits of co-regulation using the spoIIA operon as an example.

PubMed

Iber, Dagmar

2006-01-01

The distribution of most genes is not random, and functionally linked genes are often found in clusters. Several theories have been put forward to explain the emergence and persistence of operons in bacteria. Careful analysis of genomic data favours the co-regulation model, where gene organization into operons is driven by the benefits of coordinated gene expression and regulation. Direct evidence that coexpression increases the individual's fitness enough to ensure operon formation and maintenance is, however, still lacking. Here, a previously described quantitative model of the network that controls the transcription factor sigma(F) during sporulation in Bacillus subtilis is employed to quantify the benefits arising from both organization of the sporulation genes into the spoIIA operon and from translational coupling. The analysis shows that operon organization, together with translational coupling, is important because of the inherent stochastic nature of gene expression, which skews the ratios between protein concentrations in the absence of co-regulation. The predicted impact of different forms of gene regulation on fitness and survival agrees quantitatively with published sporulation efficiencies.

Quantitative PCR for Tracking the Megaplasmid-Borne Biodegradation Potential of a Model Sphingomonad

PubMed Central

Hartmann, Erica M.; Badalamenti, Jonathan P.; Krajmalnik-Brown, Rosa

2012-01-01

We developed a quantitative PCR method for tracking the dxnA1 gene, the initial, megaplasmid-borne gene in Sphingomonas wittichii RW1's dibenzo-p-dioxin degradation pathway. We used this method on complex environmental samples and report on growth of S. wittichii RW1 in landfill leachate, thus furnishing a novel tool for monitoring megaplasmid-borne, dioxygenase-encoding genes. PMID:22492441

Dynamics of the Streptococcus gordonii Transcriptome in Response to Medium, Salivary α-Amylase, and Starch

PubMed Central

Haase, Elaine M.; Feng, Xianghui; Pan, Jiachuan; Miecznikowski, Jeffrey C.

2015-01-01

Streptococcus gordonii, a primary colonizer of the tooth surface, interacts with salivary α-amylase via amylase-binding protein A (AbpA). This enzyme hydrolyzes starch to glucose, maltose, and maltodextrins that can be utilized by various oral bacteria for nutrition. Microarray studies demonstrated that AbpA modulates gene expression in response to amylase, suggesting that the amylase-streptococcal interaction may function in ways other than nutrition. The goal of this study was to explore the role of AbpA in gene regulation through comparative transcriptional profiling of wild-type KS1 and AbpA− mutant KS1ΩabpA under various environmental conditions. A portion of the total RNA isolated from mid-log-phase cells grown in 5% CO2 in (i) complex medium with or without amylase, (ii) defined medium (DM) containing 0.8% glucose with/without amylase, and (iii) DM containing 0.2% glucose and amylase with or without starch was reverse transcribed to cDNA and the rest used for RNA sequencing. Changes in the expression of selected genes were validated by quantitative reverse transcription-PCR. Maltodextrin-associated genes, fatty acid synthesis genes and competence genes were differentially expressed in a medium-dependent manner. Genes in another cluster containing a putative histidine kinase/response regulator, peptide methionine sulfoxide reductase, thioredoxin protein, lipoprotein, and cytochrome c-type protein were downregulated in KS1ΩabpA under all of the environmental conditions tested. Thus, AbpA appears to modulate genes associated with maltodextrin utilization/transport and fatty acid synthesis. Importantly, in all growth conditions AbpA was associated with increased expression of a potential two-component signaling system associated with genes involved in reducing oxidative stress, suggesting a role in signal transduction and stress tolerance. PMID:26025889

The WRKY transcription factors in the diploid woodland strawberry Fragaria vesca: Identification and expression analysis under biotic and abiotic stresses.

PubMed

Wei, Wei; Hu, Yang; Han, Yong-Tao; Zhang, Kai; Zhao, Feng-Li; Feng, Jia-Yue

2016-08-01

WRKY proteins comprise a large family of transcription factors that play important roles in response to biotic and abiotic stresses and in plant growth and development. To date, little is known about the WRKY gene family in strawberry. In this study, we identified 62 WRKY genes (FvWRKYs) in the wild diploid woodland strawberry (Fragaria vesca, 2n = 2x = 14) accession Heilongjiang-3. According to the phylogenetic analysis and structural features, these identified strawberry FvWRKY genes were classified into three main groups. In addition, eight FvWRKY-GFP fusion proteins showed distinct subcellular localizations in Arabidopsis mesophyll protoplasts. Furthermore, we examined the expression of the 62 FvWRKY genes in 'Heilongjiang-3' under various conditions, including biotic stress (Podosphaera aphanis), abiotic stresses (drought, salt, cold, and heat), and hormone treatments (abscisic acid, ethephon, methyl jasmonate, and salicylic acid). The expression levels of 33 FvWRKY genes were upregulated, while 12 FvWRKY genes were downregulated during powdery mildew infection. FvWRKY genes responded to drought and salt treatment to a greater extent than to temperature stress. Expression profiles derived from quantitative real-time PCR suggested that 11 FvWRKY genes responded dramatically to various stimuli at the transcriptional level, indicating versatile roles in responses to biotic and abiotic stresses. Interaction networks revealed that the crucial pathways controlled by WRKY proteins may be involved in the differential response to biotic stress. Taken together, the present work may provide the basis for future studies of the genetic modification of WRKY genes for pathogen resistance and stress tolerance in strawberry. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Transcript Assembly and Quantification by RNA-Seq Reveals Differentially Expressed Genes between Soft-Endocarp and Hard-Endocarp Hawthorns

PubMed Central

Zhang, Feng; Liu, Zhongchi; Li, Xiaoming; Li, Wenran; Ma, Yue; Li, He; Liu, Yuexue; Zhang, Zhihong

2013-01-01

Hawthorn (Crataegus spp.) is an important pome with a long history as a fruit, an ornamental, and a source of medicine. Fruits of hawthorn are marked by hard stony endocarps, but a hawthorn germplasm with soft and thin endocarp was found in Liaoning province of China. To elucidate the molecular mechanism underlying the soft endocarp of hawthorn, we conducted a de novo assembly of the fruit transcriptome of Crataegus pinnatifida and compared gene expression profiles between the soft-endocarp and the hard-endocarp hawthorn varieties. De novo assembly yielded 52,673 putative unigenes, 20.4% of which are longer than 1,000 bp. Among the high-quality unique sequences, 35,979 (68.3%) had at least one significant match to an existing gene model. A total of 1,218 genes, represented 2.31% total putative unigenes, were differentially expressed between the soft-endocarp hawthorn and the hard-endocarp hawthorn. Among these differentially expressed genes, a number of lignin biosynthetic pathway genes were down-regulated while almost all the flavonoid biosynthetic pathway genes were strongly up-regulated, concomitant with the formation of soft endocarp. In addition, we have identified some MYB and NAC transcription factors that could potentially control lignin and flavonoid biosynthesis. The altered expression levels of the genes encoding lignin biosynthetic enzymes, MYB and NAC transcription factors were confirmed by quantitative RT-PCR. This is the first transcriptome analysis of Crataegus genus. The high quality ESTs generated in this study will aid future gene cloning from hawthorn. Our study provides important insights into the molecular mechanisms underlying soft endocarp formation in hawthorn. PMID:24039819

Marine Microbial Gene Abundance and Community Composition in Response to Ocean Acidification and Elevated Temperature in Two Contrasting Coastal Marine Sediments.

PubMed

Currie, Ashleigh R; Tait, Karen; Parry, Helen; de Francisco-Mora, Beatriz; Hicks, Natalie; Osborn, A Mark; Widdicombe, Steve; Stahl, Henrik

2017-01-01

Marine ecosystems are exposed to a range of human-induced climate stressors, in particular changing carbonate chemistry and elevated sea surface temperatures as a consequence of climate change. More research effort is needed to reduce uncertainties about the effects of global-scale warming and acidification for benthic microbial communities, which drive sedimentary biogeochemical cycles. In this research, mesocosm experiments were set up using muddy and sandy coastal sediments to investigate the independent and interactive effects of elevated carbon dioxide concentrations (750 ppm CO 2 ) and elevated temperature (ambient +4°C) on the abundance of taxonomic and functional microbial genes. Specific quantitative PCR primers were used to target archaeal, bacterial, and cyanobacterial/chloroplast 16S rRNA in both sediment types. Nitrogen cycling genes archaeal and bacterial ammonia monooxygenase ( amoA ) and bacterial nitrite reductase ( nirS ) were specifically targeted to identify changes in microbial gene abundance and potential impacts on nitrogen cycling. In muddy sediment, microbial gene abundance, including amoA and nirS genes, increased under elevated temperature and reduced under elevated CO 2 after 28 days, accompanied by shifts in community composition. In contrast, the combined stressor treatment showed a non-additive effect with lower microbial gene abundance throughout the experiment. The response of microbial communities in the sandy sediment was less pronounced, with the most noticeable response seen in the archaeal gene abundances in response to environmental stressors over time. 16S rRNA genes ( amoA and nirS ) were lower in abundance in the combined stressor treatments in sandy sediments. Our results indicated that marine benthic microorganisms, especially in muddy sediments, are susceptible to changes in ocean carbonate chemistry and seawater temperature, which ultimately may have an impact upon key benthic biogeochemical cycles.

Molecular Dissection of a Major Gene Effect on a Quantitative Trait: The Level of Alcohol Dehydrogenase Expression in Drosophila Melanogaster

PubMed Central

Stam, L. F.; Laurie, C. C.

1996-01-01

A molecular mapping experiment shows that a major gene effect on a quantitative trait, the level of alcohol dehydrogenase expression in Drosophila melanogaster, is due to multiple polymorphisms within the Adh gene. These polymorphisms are located in an intron, the coding sequence, and the 3' untranslated region. Because of nonrandom associations among polymorphisms at different sites, the individual effects combine (in some cases epistatically) to produce ``superalleles'' with large effect. These results have implications for the interpretation of major gene effects detected by quantitative trait locus mapping methods. They show that large effects due to a single locus may be due to multiple associated polymorphisms (or sequential fixations in isolated populations) rather than individual mutations of large effect. PMID:8978044

Selection of Reference Genes for Normalization of MicroRNA Expression by RT-qPCR in Sugarcane Buds under Cold Stress

PubMed Central

Yang, Yuting; Zhang, Xu; Chen, Yun; Guo, Jinlong; Ling, Hui; Gao, Shiwu; Su, Yachun; Que, Youxiong; Xu, Liping

2016-01-01

Sugarcane, accounting for 80% of world's sugar, originates in the tropics but is cultivated mainly in the subtropics. Therefore, chilling injury frequently occurs and results in serious losses. Recent studies in various plant species have established microRNAs as key elements in the post-transcriptional regulation of response to biotic and abiotic stresses including cold stress. Though, its accuracy is largely influenced by the use of reference gene for normalization, quantitative PCR is undoubtedly a popular method used for identification of microRNAs. For identifying the most suitable reference genes for normalizing miRNAs expression in sugarcane under cold stress, 13 candidates among 17 were investigated using four algorithms: geNorm, NormFinder, deltaCt, and Bestkeeper, and four candidates were excluded because of unsatisfactory efficiency and specificity. Verification was carried out using cold-related genes miR319 and miR393 in cold-tolerant and sensitive cultivars. The results suggested that miR171/18S rRNA and miR171/miR5059 were the best reference gene sets for normalization for miRNA RT-qPCR, followed by the single miR171 and 18S rRNA. These results can aid research on miRNA responses during sugarcane stress, and the development of sugarcane tolerant to cold stress. This study is the first report concerning the reference gene selection of miRNA RT-qPCR in sugarcane. PMID:26904058

Characterization of barley Prp1 gene and its expression during seed development and under abiotic stress.

PubMed

Jiang, Qian-Tao; Liu, Tao; Ma, Jian; Wei, Yu-Ming; Lu, Zhen-Xiang; Lan, Xiu-Jin; Dai, Shou-Fen; Zheng, You-Liang

2011-10-01

The pre-mRNA processing (Prp1) gene encodes a spliceosomal protein. It was firstly identified in fission yeast and plays a regular role during spliceosome activation and cell cycle. Plant Prp1 genes have only been identified from rice, Sorghum and Arabidopsis thaliana. In this study, we reported the identification and isolation of a novel Prp1 gene from barley, and further explored its expressional pattern by using real-time quantitative RTPCR, promoter prediction and analysis of microarray data. The putative barley Prp1 protein has a similar primary structure features to those of other known Prp1 protein in this family. The results of amino acid comparison indicated that Prp1 protein of barley and other plant species has a highly conserved 30 termnal region while their 50 sequences greatly varied. The results of expressional analysis revealed that the expression level of barley Prp1 gene is always stable in different vegetative tissues, except it is up-regulated at the mid- and late stages of seed development or under the condition of cold stress. This kind of expressional pattern for barley Prp1 is also supported by our results of comparison of microarray data from barley, rice and Arabidopsis. For the molecular mechanism of its expressional pattern, we conclude that the expression of Prp1 gene may be up-regulated by the increase of pre-mRNAs and not be constitutive or ubiquitous.

An R2R3-type MYB transcription factor, GmMYB29, regulates isoflavone biosynthesis in soybean

PubMed Central

Liu, Shulin; Zhou, Xiaoqiong; Zhang, Huairen; Wang, Chun-e; Yang, Wenming; Tian, Zhixi; Cheng, Hao; Yu, Deyue

2017-01-01

Isoflavones comprise a group of secondary metabolites produced almost exclusively by plants in the legume family, including soybean [Glycine max (L.) Merr.]. They play vital roles in plant defense and have many beneficial effects on human health. Isoflavone content is a complex quantitative trait controlled by multiple genes, and the genetic mechanisms underlying isoflavone biosynthesis remain largely unknown. Via a genome-wide association study (GWAS), we identified 28 single nucleotide polymorphisms (SNPs) that are significantly associated with isoflavone concentrations in soybean. One of these 28 SNPs was located in the 5’-untranslated region (5’-UTR) of an R2R3-type MYB transcription factor, GmMYB29, and this gene was thus selected as a candidate gene for further analyses. A subcellular localization study confirmed that GmMYB29 was located in the nucleus. Transient reporter gene assays demonstrated that GmMYB29 activated the IFS2 (isoflavone synthase 2) and CHS8 (chalcone synthase 8) gene promoters. Overexpression and RNAi-mediated silencing of GmMYB29 in soybean hairy roots resulted in increased and decreased isoflavone content, respectively. Moreover, a candidate-gene association analysis revealed that 11 natural GmMYB29 polymorphisms were significantly associated with isoflavone contents, and regulation of GmMYB29 expression could partially contribute to the observed phenotypic variation. Taken together, these results provide important genetic insights into the molecular mechanisms underlying isoflavone biosynthesis in soybean. PMID:28489859

Behavior-specific changes in transcriptional modules lead to distinct and predictable neurogenomic states

PubMed Central

Chandrasekaran, Sriram; Ament, Seth A.; Eddy, James A.; Rodriguez-Zas, Sandra L.; Schatz, Bruce R.; Price, Nathan D.; Robinson, Gene E.

2011-01-01

Using brain transcriptomic profiles from 853 individual honey bees exhibiting 48 distinct behavioral phenotypes in naturalistic contexts, we report that behavior-specific neurogenomic states can be inferred from the coordinated action of transcription factors (TFs) and their predicted target genes. Unsupervised hierarchical clustering of these transcriptomic profiles showed three clusters that correspond to three ecologically important behavioral categories: aggression, maturation, and foraging. To explore the genetic influences potentially regulating these behavior-specific neurogenomic states, we reconstructed a brain transcriptional regulatory network (TRN) model. This brain TRN quantitatively predicts with high accuracy gene expression changes of more than 2,000 genes involved in behavior, even for behavioral phenotypes on which it was not trained, suggesting that there is a core set of TFs that regulates behavior-specific gene expression in the bee brain, and other TFs more specific to particular categories. TFs playing key roles in the TRN include well-known regulators of neural and behavioral plasticity, e.g., Creb, as well as TFs better known in other biological contexts, e.g., NF-κB (immunity). Our results reveal three insights concerning the relationship between genes and behavior. First, distinct behaviors are subserved by distinct neurogenomic states in the brain. Second, the neurogenomic states underlying different behaviors rely upon both shared and distinct transcriptional modules. Third, despite the complexity of the brain, simple linear relationships between TFs and their putative target genes are a surprisingly prominent feature of the networks underlying behavior. PMID:21960440

How powerful are summary-based methods for identifying expression-trait associations under different genetic architectures?

PubMed

Veturi, Yogasudha; Ritchie, Marylyn D

2018-01-01

Transcriptome-wide association studies (TWAS) have recently been employed as an approach that can draw upon the advantages of genome-wide association studies (GWAS) and gene expression studies to identify genes associated with complex traits. Unlike standard GWAS, summary level data suffices for TWAS and offers improved statistical power. Two popular TWAS methods include either (a) imputing the cis genetic component of gene expression from smaller sized studies (using multi-SNP prediction or MP) into much larger effective sample sizes afforded by GWAS - TWAS-MP or (b) using summary-based Mendelian randomization - TWAS-SMR. Although these methods have been effective at detecting functional variants, it remains unclear how extensive variability in the genetic architecture of complex traits and diseases impacts TWAS results. Our goal was to investigate the different scenarios under which these methods yielded enough power to detect significant expression-trait associations. In this study, we conducted extensive simulations based on 6000 randomly chosen, unrelated Caucasian males from Geisinger's MyCode population to compare the power to detect cis expression-trait associations (within 500 kb of a gene) using the above-described approaches. To test TWAS across varying genetic backgrounds we simulated gene expression and phenotype using different quantitative trait loci per gene and cis-expression /trait heritability under genetic models that differentiate the effect of causality from that of pleiotropy. For each gene, on a training set ranging from 100 to 1000 individuals, we either (a) estimated regression coefficients with gene expression as the response using five different methods: LASSO, elastic net, Bayesian LASSO, Bayesian spike-slab, and Bayesian ridge regression or (b) performed eQTL analysis. We then sampled with replacement 50,000, 150,000, and 300,000 individuals respectively from the testing set of the remaining 5000 individuals and conducted GWAS on each set. Subsequently, we integrated the GWAS summary statistics derived from the testing set with the weights (or eQTLs) derived from the training set to identify expression-trait associations using (a) TWAS-MP (b) TWAS-SMR (c) eQTL-based GWAS, or (d) standalone GWAS. Finally, we examined the power to detect functionally relevant genes using the different approaches under the considered simulation scenarios. In general, we observed great similarities among TWAS-MP methods although the Bayesian methods resulted in improved power in comparison to LASSO and elastic net as the trait architecture grew more complex while training sample sizes and expression heritability remained small. Finally, we observed high power under causality but very low to moderate power under pleiotropy.

Multi-Platform Next-Generation Sequencing of the Domestic Turkey (Meleagris gallopavo): Genome Assembly and Analysis

PubMed Central

Aslam, Luqman; Beal, Kathryn; Ann Blomberg, Le; Bouffard, Pascal; Burt, David W.; Crasta, Oswald; Crooijmans, Richard P. M. A.; Cooper, Kristal; Coulombe, Roger A.; De, Supriyo; Delany, Mary E.; Dodgson, Jerry B.; Dong, Jennifer J.; Evans, Clive; Frederickson, Karin M.; Flicek, Paul; Florea, Liliana; Folkerts, Otto; Groenen, Martien A. M.; Harkins, Tim T.; Herrero, Javier; Hoffmann, Steve; Megens, Hendrik-Jan; Jiang, Andrew; de Jong, Pieter; Kaiser, Pete; Kim, Heebal; Kim, Kyu-Won; Kim, Sungwon; Langenberger, David; Lee, Mi-Kyung; Lee, Taeheon; Mane, Shrinivasrao; Marcais, Guillaume; Marz, Manja; McElroy, Audrey P.; Modise, Thero; Nefedov, Mikhail; Notredame, Cédric; Paton, Ian R.; Payne, William S.; Pertea, Geo; Prickett, Dennis; Puiu, Daniela; Qioa, Dan; Raineri, Emanuele; Ruffier, Magali; Salzberg, Steven L.; Schatz, Michael C.; Scheuring, Chantel; Schmidt, Carl J.; Schroeder, Steven; Searle, Stephen M. J.; Smith, Edward J.; Smith, Jacqueline; Sonstegard, Tad S.; Stadler, Peter F.; Tafer, Hakim; Tu, Zhijian (Jake); Van Tassell, Curtis P.; Vilella, Albert J.; Williams, Kelly P.; Yorke, James A.; Zhang, Liqing; Zhang, Hong-Bin; Zhang, Xiaojun; Zhang, Yang; Reed, Kent M.

2010-01-01

A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest. PMID:20838655

Simulated microgravity alters the expression of cytoskeleton- and ATP-binding-related genes in MLO-Y4 osteocytes

NASA Astrophysics Data System (ADS)

Chen, Zhihao; Zhao, Fan; Qi, Yiduo; Hu, Lifang; Li, Dijie; Yin, Chong; Su, Peihong; Zhang, Yan; Ma, Jianhua; Qian, Jing; Zhou, Hongpo; Zou, Yiwei; Qian, Airong

2016-12-01

Bone undergoes dynamic modelling and remodelling processes, and it requires gravity-mediated mechanical stimulation for the maintenance of mineral content and structure. Osteocytes are the most commonly found cells in the mature bone, and they are sensitive to mechanical changes. The purpose of this study was to investigate the effects of microgravity simulated with a random position machine (RPM) on the gene expression profile of osteocytes. Genes sensitive to RPM treatment were sorted on the basis of biological processes, interactions and signalling pathways. Overall, 504 differentially expressed genes (DEGs) in osteocytes cultured under RPM conditions were found. The DEGs were further analysed using bioinformatics tools such as DAVID and iReport. A total of 15 ATP-binding and cytoskeleton-related genes were further confirmed by quantitative real-time PCR (qRT-PCR). Our findings demonstrate that the RPM affected the expression of genes involved in cytoskeleton remodelling and the energy-transfer process in osteocytes. The identification of mechanosensitive genes may enhance our understanding of the roles of osteocytes in mechanosensation and may provide some potential targets for preventing and treating bone-related diseases.

Identification of expression quantitative trait loci by the interaction analysis using genetic algorithm.

PubMed

Namkung, Junghyun; Nam, Jin-Wu; Park, Taesung

2007-01-01

Many genes with major effects on quantitative traits have been reported to interact with other genes. However, finding a group of interacting genes from thousands of SNPs is challenging. Hence, an efficient and robust algorithm is needed. The genetic algorithm (GA) is useful in searching for the optimal solution from a very large searchable space. In this study, we show that genome-wide interaction analysis using GA and a statistical interaction model can provide a practical method to detect biologically interacting loci. We focus our search on transcriptional regulators by analyzing gene x gene interactions for cancer-related genes. The expression values of three cancer-related genes were selected from the expression data of the Genetic Analysis Workshop 15 Problem 1 data set. We implemented a GA to identify the expression quantitative trait loci that are significantly associated with expression levels of the cancer-related genes. The time complexity of the GA was compared with that of an exhaustive search algorithm. As a result, our GA, which included heuristic methods, such as archive, elitism, and local search, has greatly reduced computational time in a genome-wide search for gene x gene interactions. In general, the GA took one-fifth the computation time of an exhaustive search for the most significant pair of single-nucleotide polymorphisms.

Identification of expression quantitative trait loci by the interaction analysis using genetic algorithm

PubMed Central

Namkung, Junghyun; Nam, Jin-Wu; Park, Taesung

2007-01-01

Many genes with major effects on quantitative traits have been reported to interact with other genes. However, finding a group of interacting genes from thousands of SNPs is challenging. Hence, an efficient and robust algorithm is needed. The genetic algorithm (GA) is useful in searching for the optimal solution from a very large searchable space. In this study, we show that genome-wide interaction analysis using GA and a statistical interaction model can provide a practical method to detect biologically interacting loci. We focus our search on transcriptional regulators by analyzing gene × gene interactions for cancer-related genes. The expression values of three cancer-related genes were selected from the expression data of the Genetic Analysis Workshop 15 Problem 1 data set. We implemented a GA to identify the expression quantitative trait loci that are significantly associated with expression levels of the cancer-related genes. The time complexity of the GA was compared with that of an exhaustive search algorithm. As a result, our GA, which included heuristic methods, such as archive, elitism, and local search, has greatly reduced computational time in a genome-wide search for gene × gene interactions. In general, the GA took one-fifth the computation time of an exhaustive search for the most significant pair of single-nucleotide polymorphisms. PMID:18466570

Integrating Genomic Analysis with the Genetic Basis of Gene Expression: Preliminary Evidence of the Identification of Causal Genes for Cardiovascular and Metabolic Traits Related to Nutrition in Mexicans123

PubMed Central

Bastarrachea, Raúl A.; Gallegos-Cabriales, Esther C.; Nava-González, Edna J.; Haack, Karin; Voruganti, V. Saroja; Charlesworth, Jac; Laviada-Molina, Hugo A.; Veloz-Garza, Rosa A.; Cardenas-Villarreal, Velia Margarita; Valdovinos-Chavez, Salvador B.; Gomez-Aguilar, Patricia; Meléndez, Guillermo; López-Alvarenga, Juan Carlos; Göring, Harald H. H.; Cole, Shelley A.; Blangero, John; Comuzzie, Anthony G.; Kent, Jack W.

2012-01-01

Whole-transcriptome expression profiling provides novel phenotypes for analysis of complex traits. Gene expression measurements reflect quantitative variation in transcript-specific messenger RNA levels and represent phenotypes lying close to the action of genes. Understanding the genetic basis of gene expression will provide insight into the processes that connect genotype to clinically significant traits representing a central tenet of system biology. Synchronous in vivo expression profiles of lymphocytes, muscle, and subcutaneous fat were obtained from healthy Mexican men. Most genes were expressed at detectable levels in multiple tissues, and RNA levels were correlated between tissue types. A subset of transcripts with high reliability of expression across tissues (estimated by intraclass correlation coefficients) was enriched for cis-regulated genes, suggesting that proximal sequence variants may influence expression similarly in different cellular environments. This integrative global gene expression profiling approach is proving extremely useful for identifying genes and pathways that contribute to complex clinical traits. Clearly, the coincidence of clinical trait quantitative trait loci and expression quantitative trait loci can help in the prioritization of positional candidate genes. Such data will be crucial for the formal integration of positional and transcriptomic information characterized as genetical genomics. PMID:22797999

Genetic dissection of a major anthocyanin QTL contributing to pollinator-mediated reproductive isolation between sister species of Mimulus.

PubMed

Yuan, Yao-Wu; Sagawa, Janelle M; Young, Riane C; Christensen, Brian J; Bradshaw, Harvey D

2013-05-01

Prezygotic barriers play a major role in the evolution of reproductive isolation, which is a prerequisite for speciation. However, despite considerable progress in identifying genes and mutations responsible for postzygotic isolation, little is known about the genetic and molecular basis underlying prezygotic barriers. The bumblebee-pollinated Mimulus lewisii and the hummingbird-pollinated M. cardinalis represent a classic example of pollinator-mediated prezygotic isolation between two sister species in sympatry. Flower color differences resulting from both carotenoid and anthocyanin pigments contribute to pollinator discrimination between the two species in nature. Through fine-scale genetic mapping, site-directed mutagenesis, and transgenic experiments, we demonstrate that a single-repeat R3 MYB repressor, ROSE INTENSITY1 (ROI1), is the causal gene underlying a major quantitative trait locus (QTL) with the largest effect on anthocyanin concentration and that cis-regulatory change rather than coding DNA mutations cause the allelic difference between M. lewisii and M. cardinalis. Together with the genomic resources and stable transgenic tools developed here, these results suggest that Mimulus is an excellent platform for studying the genetics of pollinator-mediated reproductive isolation and the molecular basis of morphological evolution at the most fundamental level-gene by gene, mutation by mutation.

Genome-Wide Association Study in Arabidopsis thaliana of Natural Variation in Seed Oil Melting Point: A Widespread Adaptive Trait in Plants.

PubMed

Branham, Sandra E; Wright, Sara J; Reba, Aaron; Morrison, Ginnie D; Linder, C Randal

2016-05-01

Seed oil melting point is an adaptive, quantitative trait determined by the relative proportions of the fatty acids that compose the oil. Micro- and macro-evolutionary evidence suggests selection has changed the melting point of seed oils to covary with germination temperatures because of a trade-off between total energy stores and the rate of energy acquisition during germination under competition. The seed oil compositions of 391 natural accessions of Arabidopsis thaliana, grown under common-garden conditions, were used to assess whether seed oil melting point within a species varied with germination temperature. In support of the adaptive explanation, long-term monthly spring and fall field temperatures of the accession collection sites significantly predicted their seed oil melting points. In addition, a genome-wide association study (GWAS) was performed to determine which genes were most likely responsible for the natural variation in seed oil melting point. The GWAS found a single highly significant association within the coding region of FAD2, which encodes a fatty acid desaturase central to the oil biosynthesis pathway. In a separate analysis of 15 a priori oil synthesis candidate genes, 2 (FAD2 and FATB) were located near significant SNPs associated with seed oil melting point. These results comport with others' molecular work showing that lines with alterations in these genes affect seed oil melting point as expected. Our results suggest natural selection has acted on a small number of loci to alter a quantitative trait in response to local environmental conditions. © The American Genetic Association. 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A transposon-directed epigenetic change in ZmCCT underlies quantitative resistance to Gibberella stalk rot in maize.

PubMed

Wang, Chao; Yang, Qin; Wang, Weixiang; Li, Yipu; Guo, Yanling; Zhang, Dongfeng; Ma, Xuena; Song, Wei; Zhao, Jiuran; Xu, Mingliang

2017-09-01

A major resistance quantitative trait locus, qRfg1, significantly enhances maize resistance to Gibberella stalk rot, a devastating disease caused by Fusarium graminearum. However, the underlying molecular mechanism remains unknown. We adopted a map-based cloning approach to identify the resistance gene at qRfg1 and examined the dynamic epigenetic changes during qRfg1-mediated maize resistance to the disease. A CCT domain-containing gene, ZmCCT, is the causal gene at the qRfg1 locus and a polymorphic CACTA-like transposable element (TE1) c. 2.4 kb upstream of ZmCCT is the genetic determinant of allelic variation. The non-TE1 ZmCCT allele is in a poised state, with predictive bivalent chromatin enriched for both repressive (H3K27me3/H3K9me3) and active (H3K4me3) histone marks. Upon pathogen challenge, this non-TE1 ZmCCT allele was promptly induced by a rapid yet transient reduction in H3K27me3/H3K9me3 and a progressive decrease in H3K4me3, leading to disease resistance. However, TE1 insertion in ZmCCT caused selective depletion of H3K4me3 and enrichment of methylated GC to suppress the pathogen-induced ZmCCT expression, resulting in disease susceptibility. Moreover, ZmCCT-mediated resistance to Gibberella stalk rot is not affected by photoperiod sensitivity. This chromatin-based regulatory mechanism enables ZmCCT to be more precise and timely in defense against F. graminearum infection. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Dehydrins from wheat x Thinopyrum ponticum amphiploid increase salinity and drought tolerance under their own inducible promoters without growth retardation.

PubMed

Qin, Yu-Xiang; Qin, Fangyuan

2016-02-01

Dehydrins confer abiotic stress tolerance in seedlings, but few dehydrins have been studied by transgenic analysis under their own promoters in relation to abiotic stress tolerance. Also the inducible promoters for transgenic engineering are limited. In this study, we isolated from wheat three salt-induced YSK2 dehydrin genes and their promoters. The cDNA sequences were 711, 785, and 932 bp in length, encoding proteins containing 133, 166 and 231 amino acids, respectively, and were named TaDHN1, TaDHN2, and TaDHN3. TaDHN2 doesn't contain introns, while the other two genes each contain one. Semi-quantitative reverse transcription PCR analysis revealed all three dehydrin genes are substantially induced by ABA and NaCl, but only TaDHN2 is induced in seedlings by PEG and by cold (4 °C). Regulatory sequences upstream of the first translation codon (775, 1615 and 889 bp) of the three dehydrin genes were also cloned. Cis-element prediction indicated the presence of ABRE and other abiotic-stress-related elements. Histochemical analysis using GUS expression demonstrated that all three promoters were induced by ABA, cold or NaCl. Ectopic over-expression of TaDHN1 or TaDHN3 in Arabidopsis under their own inducible promoters enhanced NaCl- and drought-stress tolerance without growth retardation. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Evaluation of Reference Genes for RT-qPCR Studies in the Seagrass Zostera muelleri Exposed to Light Limitation

PubMed Central

Schliep, M.; Pernice, M.; Sinutok, S.; Bryant, C. V.; York, P. H.; Rasheed, M. A.; Ralph, P. J.

2015-01-01

Seagrass meadows are threatened by coastal development and global change. In the face of these pressures, molecular techniques such as reverse transcription quantitative real-time PCR (RT-qPCR) have great potential to improve management of these ecosystems by allowing early detection of chronic stress. In RT-qPCR, the expression levels of target genes are estimated on the basis of reference genes, in order to control for RNA variations. Although determination of suitable reference genes is critical for RT-qPCR studies, reports on the evaluation of reference genes are still absent for the major Australian species Zostera muelleri subsp. capricorni (Z. muelleri). Here, we used three different software (geNorm, NormFinder and Bestkeeper) to evaluate ten widely used reference genes according to their expression stability in Z. muelleri exposed to light limitation. We then combined results from different software and used a consensus rank of four best reference genes to validate regulation in Photosystem I reaction center subunit IV B and Heat Stress Transcription factor A- gene expression in Z. muelleri under light limitation. This study provides the first comprehensive list of reference genes in Z. muelleri and demonstrates RT-qPCR as an effective tool to identify early responses to light limitation in seagrass. PMID:26592440

Correlation Analysis Between Expression Levels of Hepatic Growth Hormone Receptor, Janus Kinase 2, Insulin-Like Growth Factor-I Genes and Dwarfism Phenotype in Bama Minipig.

PubMed

Yang, Haowen; Jiang, Qinyang; Wu, Dan; Lan, Ganqiu; Fan, Jing; Guo, Yafen; Chen, Baojian; Yang, Xiurong; Jiang, Hesheng

2015-02-01

Animal growth and development are complex and sophisticated biological metabolic processes, in which genes plays an important role. In this paper, we employed real-time quantitative PCR (RT-qPCR) to analyze the expression levels of hepatic GHR, JAK2 and IGF-I genes in 1, 30, 180 day of Bama minipig and Landrace with attempt to verify the correlation between the expression of these growth-associated genes and the dwarfism phenotype of Bama minipig. The results showed that the expression levels of these 3 genes in Bama minipigs were down-regulated expressed from 1 day to 30 day, and which was up-regulated expressed in Landrace. The expression levels of the 3 genes on 1, 30, 180 day were prominently higher in Landrace than in Bama minipigs. The significant differences of the 3 genes expression levels on 1 day between this two breeds indicate that different expressions of these genes might occur before birth. It is speculated that the down-regulated expression of the 3 genes may have a close correlation with the dwarfism phenotype of Bama minipig. More investigations in depth of this study is under progress with the help of biochip nanotechnology.

Gene expression in gut symbiotic organ of stinkbug affected by extracellular bacterial symbiont.

PubMed

Futahashi, Ryo; Tanaka, Kohjiro; Tanahashi, Masahiko; Nikoh, Naruo; Kikuchi, Yoshitomo; Lee, Bok Luel; Fukatsu, Takema

2013-01-01

The bean bug Riptortus pedestris possesses a specialized symbiotic organ in a posterior region of the midgut, where numerous crypts harbor extracellular betaproteobacterial symbionts of the genus Burkholderia. Second instar nymphs orally acquire the symbiont from the environment, and the symbiont infection benefits the host by facilitating growth and by occasionally conferring insecticide resistance. Here we performed comparative transcriptomic analyses of insect genes expressed in symbiotic and non-symbiotic regions of the midgut dissected from Burkholderia-infected and uninfected R. pedestris. Expression sequence tag analysis of cDNA libraries and quantitative reverse transcription PCR identified a number of insect genes expressed in symbiosis- or aposymbiosis-associated patterns. For example, genes up-regulated in symbiotic relative to aposymbiotic individuals, including many cysteine-rich secreted protein genes and many cathepsin protease genes, are likely to play a role in regulating the symbiosis. Conversely, genes up-regulated in aposymbiotic relative to symbiotic individuals, including a chicken-type lysozyme gene and a defensin-like protein gene, are possibly involved in regulation of non-symbiotic bacterial infections. Our study presents the first transcriptomic data on gut symbiotic organ of a stinkbug, which provides initial clues to understanding of molecular mechanisms underlying the insect-bacterium gut symbiosis and sheds light on several intriguing commonalities between endocellular and extracellular symbiotic associations.

Gene Expression in Gut Symbiotic Organ of Stinkbug Affected by Extracellular Bacterial Symbiont

PubMed Central

Futahashi, Ryo; Tanaka, Kohjiro; Tanahashi, Masahiko; Nikoh, Naruo; Kikuchi, Yoshitomo; Lee, Bok Luel; Fukatsu, Takema

2013-01-01

The bean bug Riptortus pedestris possesses a specialized symbiotic organ in a posterior region of the midgut, where numerous crypts harbor extracellular betaproteobacterial symbionts of the genus Burkholderia. Second instar nymphs orally acquire the symbiont from the environment, and the symbiont infection benefits the host by facilitating growth and by occasionally conferring insecticide resistance. Here we performed comparative transcriptomic analyses of insect genes expressed in symbiotic and non-symbiotic regions of the midgut dissected from Burkholderia-infected and uninfected R. pedestris. Expression sequence tag analysis of cDNA libraries and quantitative reverse transcription PCR identified a number of insect genes expressed in symbiosis- or aposymbiosis-associated patterns. For example, genes up-regulated in symbiotic relative to aposymbiotic individuals, including many cysteine-rich secreted protein genes and many cathepsin protease genes, are likely to play a role in regulating the symbiosis. Conversely, genes up-regulated in aposymbiotic relative to symbiotic individuals, including a chicken-type lysozyme gene and a defensin-like protein gene, are possibly involved in regulation of non-symbiotic bacterial infections. Our study presents the first transcriptomic data on gut symbiotic organ of a stinkbug, which provides initial clues to understanding of molecular mechanisms underlying the insect-bacterium gut symbiosis and sheds light on several intriguing commonalities between endocellular and extracellular symbiotic associations. PMID:23691247

A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep.

PubMed

Clop, Alex; Marcq, Fabienne; Takeda, Haruko; Pirottin, Dimitri; Tordoir, Xavier; Bibé, Bernard; Bouix, Jacques; Caiment, Florian; Elsen, Jean-Michel; Eychenne, Francis; Larzul, Catherine; Laville, Elisabeth; Meish, Françoise; Milenkovic, Dragan; Tobin, James; Charlier, Carole; Georges, Michel

2006-07-01

Texel sheep are renowned for their exceptional meatiness. To identify the genes underlying this economically important feature, we performed a whole-genome scan in a Romanov x Texel F2 population. We mapped a quantitative trait locus with a major effect on muscle mass to chromosome 2 and subsequently fine-mapped it to a chromosome interval encompassing the myostatin (GDF8) gene. We herein demonstrate that the GDF8 allele of Texel sheep is characterized by a G to A transition in the 3' UTR that creates a target site for mir1 and mir206, microRNAs (miRNAs) that are highly expressed in skeletal muscle. This causes translational inhibition of the myostatin gene and hence contributes to the muscular hypertrophy of Texel sheep. Analysis of SNP databases for humans and mice demonstrates that mutations creating or destroying putative miRNA target sites are abundant and might be important effectors of phenotypic variation.

Gene Expression Profiling in Fish Toxicology: A Review.

PubMed

Kumar, Girish; Denslow, Nancy D

In this review, we present an overview of transcriptomic responses to chemical exposures in a variety of fish species. We have discussed the use of several molecular approaches such as northern blotting, differential display reverse transcription-polymerase chain reaction (DDRT-PCR), suppression subtractive hybridization (SSH), real time quantitative PCR (RT-qPCR), microarrays, and next-generation sequencing (NGS) for measuring gene expression. These techniques have been mainly used to measure the toxic effects of single compounds or simple mixtures in laboratory conditions. In addition, only few studies have been conducted to examine the biological significance of differentially expressed gene sets following chemical exposure. Therefore, future studies should focus more under field conditions using a multidisciplinary approach (genomics, proteomics and metabolomics) to understand the synergetic effects of multiple environmental stressors and to determine the functional significance of differentially expressed genes. Nevertheless, recent developments in NGS technologies and decreasing costs of sequencing holds the promise to uncover the complexity of anthropogenic impacts and biological effects in wild fish populations.

Global gene expression analysis by combinatorial optimization.

PubMed

Ameur, Adam; Aurell, Erik; Carlsson, Mats; Westholm, Jakub Orzechowski

2004-01-01

Generally, there is a trade-off between methods of gene expression analysis that are precise but labor-intensive, e.g. RT-PCR, and methods that scale up to global coverage but are not quite as quantitative, e.g. microarrays. In the present paper, we show how how a known method of gene expression profiling (K. Kato, Nucleic Acids Res. 23, 3685-3690 (1995)), which relies on a fairly small number of steps, can be turned into a global gene expression measurement by advanced data post-processing, with potentially little loss of accuracy. Post-processing here entails solving an ancillary combinatorial optimization problem. Validation is performed on in silico experiments generated from the FANTOM data base of full-length mouse cDNA. We present two variants of the method. One uses state-of-the-art commercial software for solving problems of this kind, the other a code developed by us specifically for this purpose, released in the public domain under GPL license.

Reference genes for measuring mRNA expression.

PubMed

Dundas, Jitesh; Ling, Maurice

2012-12-01

The aim of this review is to find answers to some of the questions surrounding reference genes and their reliability for quantitative experiments. Reference genes are assumed to be at a constant expression level, over a range of conditions such as temperature. These genes, such as GADPH and beta-actin, are used extensively for gene expression studies using techniques like quantitative PCR. There have been several studies carried out on identifying reference genes. However, a lot of evidence indicates issues to the general suitability of these genes. Recent studies had shown that different factors, including the environment and methods, play an important role in changing the expression levels of the reference genes. Thus, we conclude that there is no reference gene that can deemed suitable for all the experimental conditions. In addition, we believe that every experiment will require the scientific evaluation and selection of the best candidate gene for use as a reference gene to obtain reliable scientific results.

Functional characterization of MAT1-1-specific mating-type genes in the homothallic ascomycete Sordaria macrospora provides new insights into essential and nonessential sexual regulators.

PubMed

Klix, V; Nowrousian, M; Ringelberg, C; Loros, J J; Dunlap, J C; Pöggeler, S

2010-06-01

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA-2 is essential for sexual reproduction, whereas the alpha domain protein-encoding genes SmtA-1 and SmtA-3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA-1 and SmtA-2. Both genes are involved in the regulation of gene expression, including that of pheromone genes.

Functional Characterization of MAT1-1-Specific Mating-Type Genes in the Homothallic Ascomycete Sordaria macrospora Provides New Insights into Essential and Nonessential Sexual Regulators▿†

PubMed Central

Klix, V.; Nowrousian, M.; Ringelberg, C.; Loros, J. J.; Dunlap, J. C.; Pöggeler, S.

2010-01-01

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA-2 is essential for sexual reproduction, whereas the α domain protein-encoding genes SmtA-1 and SmtA-3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA-1 and SmtA-2. Both genes are involved in the regulation of gene expression, including that of pheromone genes. PMID:20435701

Validation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in strawberry fruits using different cultivars and osmotic stresses.

PubMed

Galli, Vanessa; Borowski, Joyce Moura; Perin, Ellen Cristina; Messias, Rafael da Silva; Labonde, Julia; Pereira, Ivan dos Santos; Silva, Sérgio Delmar Dos Anjos; Rombaldi, Cesar Valmor

2015-01-10

The increasing demand of strawberry (Fragaria×ananassa Duch) fruits is associated mainly with their sensorial characteristics and the content of antioxidant compounds. Nevertheless, the strawberry production has been hampered due to its sensitivity to abiotic stresses. Therefore, to understand the molecular mechanisms highlighting stress response is of great importance to enable genetic engineering approaches aiming to improve strawberry tolerance. However, the study of expression of genes in strawberry requires the use of suitable reference genes. In the present study, seven traditional and novel candidate reference genes were evaluated for transcript normalization in fruits of ten strawberry cultivars and two abiotic stresses, using RefFinder, which integrates the four major currently available software programs: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. The results indicate that the expression stability is dependent on the experimental conditions. The candidate reference gene DBP (DNA binding protein) was considered the most suitable to normalize expression data in samples of strawberry cultivars and under drought stress condition, and the candidate reference gene HISTH4 (histone H4) was the most stable under osmotic stresses and salt stress. The traditional genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 18S (18S ribosomal RNA) were considered the most unstable genes in all conditions. The expression of phenylalanine ammonia lyase (PAL) and 9-cis epoxycarotenoid dioxygenase (NCED1) genes were used to further confirm the validated candidate reference genes, showing that the use of an inappropriate reference gene may induce erroneous results. This study is the first survey on the stability of reference genes in strawberry cultivars and osmotic stresses and provides guidelines to obtain more accurate RT-qPCR results for future breeding efforts. Copyright © 2014 Elsevier B.V. All rights reserved.

Genetic regulation of gene expression in the lung identifies CST3 and CD22 as potential causal genes for airflow obstruction.

PubMed

Lamontagne, Maxime; Timens, Wim; Hao, Ke; Bossé, Yohan; Laviolette, Michel; Steiling, Katrina; Campbell, Joshua D; Couture, Christian; Conti, Massimo; Sherwood, Karen; Hogg, James C; Brandsma, Corry-Anke; van den Berge, Maarten; Sandford, Andrew; Lam, Stephen; Lenburg, Marc E; Spira, Avrum; Paré, Peter D; Nickle, David; Sin, Don D; Postma, Dirkje S

2014-11-01

COPD is a complex chronic disease with poorly understood pathogenesis. Integrative genomic approaches have the potential to elucidate the biological networks underlying COPD and lung function. We recently combined genome-wide genotyping and gene expression in 1111 human lung specimens to map expression quantitative trait loci (eQTL). To determine causal associations between COPD and lung function-associated single nucleotide polymorphisms (SNPs) and lung tissue gene expression changes in our lung eQTL dataset. We evaluated causality between SNPs and gene expression for three COPD phenotypes: FEV(1)% predicted, FEV(1)/FVC and COPD as a categorical variable. Different models were assessed in the three cohorts independently and in a meta-analysis. SNPs associated with a COPD phenotype and gene expression were subjected to causal pathway modelling and manual curation. In silico analyses evaluated functional enrichment of biological pathways among newly identified causal genes. Biologically relevant causal genes were validated in two separate gene expression datasets of lung tissues and bronchial airway brushings. High reliability causal relations were found in SNP-mRNA-phenotype triplets for FEV(1)% predicted (n=169) and FEV(1)/FVC (n=80). Several genes of potential biological relevance for COPD were revealed. eQTL-SNPs upregulating cystatin C (CST3) and CD22 were associated with worse lung function. Signalling pathways enriched with causal genes included xenobiotic metabolism, apoptosis, protease-antiprotease and oxidant-antioxidant balance. By using integrative genomics and analysing the relationships of COPD phenotypes with SNPs and gene expression in lung tissue, we identified CST3 and CD22 as potential causal genes for airflow obstruction. This study also augmented the understanding of previously described COPD pathways. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Isolation, characterization, and evaluation of three Citrus sinensis-derived constitutive gene promoters.

PubMed

Erpen, L; Tavano, E C R; Harakava, R; Dutt, M; Grosser, J W; Piedade, S M S; Mendes, B M J; Mourão Filho, F A A

2018-05-23

Regulatory sequences from the citrus constitutive genes cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1) were isolated, fused to the uidA gene, and qualitatively and quantitatively evaluated in transgenic sweet orange plants. The 5' upstream region of a gene (the promoter) is the most important component for the initiation and regulation of gene transcription of both native genes and transgenes in plants. The isolation and characterization of gene regulatory sequences are essential to the development of intragenic or cisgenic genetic manipulation strategies, which imply the use of genetic material from the same species or from closely related species. We describe herein the isolation and evaluation of the promoter sequence from three constitutively expressed citrus genes: cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1). The functionality of the promoters was confirmed by a histochemical GUS assay in leaves, stems, and roots of stably transformed citrus plants expressing the promoter-uidA construct. Lower uidA mRNA levels were detected when the transgene was under the control of citrus promoters as compared to the expression under the control of the CaMV35S promoter. The association of the uidA gene with the citrus-derived promoters resulted in mRNA levels of up to 60-41.8% of the value obtained with the construct containing CaMV35S driving the uidA gene. Moreover, a lower inter-individual variability in transgene expression was observed amongst the different transgenic lines, where gene constructs containing citrus-derived promoters were used. In silico analysis of the citrus-derived promoter sequences revealed that their activity may be controlled by several putative cis-regulatory elements. These citrus promoters will expand the availability of regulatory sequences for driving gene expression in citrus gene-modification programs.

Gene profiling reveals a role for stress hormones in the molecular and behavioral response to food restriction

PubMed Central

Guarnieri, Douglas J.; Brayton, Catherine E.; Richards, Sarah M.; Maldonado-Aviles, Jaime; Trinko, Joseph R.; Nelson, Jessica; Taylor, Jane R.; Gourley, Shannon L.; DiLeone, Ralph J.

2011-01-01

Background Food restriction is known to enhance learning and motivation. The neural mechanisms underlying these responses likely involve alterations in gene expression in brain regions mediating the motivation to feed. Methods Analysis of gene expression profiles in male C57BL6/J mice using whole-genome microarrays was completed in the medial prefrontal cortex, nucleus accumbens, ventral tegmental area, and the hypothalamus following a five day food restriction. Quantitative PCR was used to validate these findings and determine the time-course of expression changes. Plasma levels of the stress hormone corticosterone (CORT) were measured by ELISA. Expression changes were measured in adrenalectomized animals that underwent food restriction, as well as in animals receiving daily injections of CORT. Progressive ratio responding for food, a measure of motivated behavior, was assessed after CORT treatment in restricted and fed animals. Results Brief food restriction results in an upregulation of peripheral stress responsive genes in the mammalian brain. Time-course analysis demonstrated rapid and persistent expression changes in all four brain regions under study. Administration of CORT to non-restricted animals was sufficient to induce a subset of the genes, and alterations in gene expression after food restriction were dependent on intact adrenal glands. CORT can increase the motivation to work for food only in the restricted state. Conclusions These data demonstrate a central role for CORT in mediating both molecular and behavioral responses to food restriction. The stress hormone-induced alterations in gene expression described here may be relevant for both adaptive and pathological responses to stress. PMID:21855858

Transcriptome Sequencing Identified Genes and Gene Ontologies Associated with Early Freezing Tolerance in Maize

PubMed Central

Li, Zhao; Hu, Guanghui; Liu, Xiangfeng; Zhou, Yao; Li, Yu; Zhang, Xu; Yuan, Xiaohui; Zhang, Qian; Yang, Deguang; Wang, Tianyu; Zhang, Zhiwu

2016-01-01

Originating in a tropical climate, maize has faced great challenges as cultivation has expanded to the majority of the world's temperate zones. In these zones, frost and cold temperatures are major factors that prevent maize from reaching its full yield potential. Among 30 elite maize inbred lines adapted to northern China, we identified two lines of extreme, but opposite, freezing tolerance levels—highly tolerant and highly sensitive. During the seedling stage of these two lines, we used RNA-seq to measure changes in maize whole genome transcriptome before and after freezing treatment. In total, 19,794 genes were expressed, of which 4550 exhibited differential expression due to either treatment (before or after freezing) or line type (tolerant or sensitive). Of the 4550 differently expressed genes, 948 exhibited differential expression due to treatment within line or lines under freezing condition. Analysis of gene ontology found that these 948 genes were significantly enriched for binding functions (DNA binding, ATP binding, and metal ion binding), protein kinase activity, and peptidase activity. Based on their enrichment, literature support, and significant levels of differential expression, 30 of these 948 genes were selected for quantitative real-time PCR (qRT-PCR) validation. The validation confirmed our RNA-Seq-based findings, with squared correlation coefficients of 80% and 50% in the tolerance and sensitive lines, respectively. This study provided valuable resources for further studies to enhance understanding of the molecular mechanisms underlying maize early freezing response and enable targeted breeding strategies for developing varieties with superior frost resistance to achieve yield potential. PMID:27774095

Differential gene expression underlying ovarian phenotype determination in honey bee, Apis mellifera L., caste development.

PubMed

Lago, Denyse Cavalcante; Humann, Fernanda Carvalho; Barchuk, Angel Roberto; Abraham, Kuruvilla Joseph; Hartfelder, Klaus

2016-12-01

Adult honey bee queens and workers drastically differ in ovary size. This adult ovary phenotype difference becomes established during the final larval instar, when massive programmed cell death leads to the degeneration of 95-99% of the ovariole anlagen in workers. The higher juvenile hormone (JH) levels in queen larvae protect the ovaries against such degeneration. To gain insights into the molecular architecture underlying this divergence critical for adult caste fate and worker sterility, we performed a microarray analysis on fourth and early fifth instar queen and worker ovaries. For the fourth instar we found nine differentially expressed genes (DEGs) with log 2 FC > 1.0, but this number increased to 56 in early fifth-instar ovaries. We selected 15 DEGs for quantitative PCR (RT-qPCR) analysis. Nine differed significantly by the variables caste and/or development. Interestingly, genes with enzyme functions were higher expressed in workers, while those related to transcription and signaling had higher transcript levels in queens. For the RT-qPCR confirmed genes we analyzed their response to JH. This revealed a significant up-regulation for two genes, a short chain dehydrogenase reductase (sdr) and a heat shock protein 90 (hsp90). Five other genes, including hsp60 and hexamerin 70b (hex70b), were significantly down-regulated by JH. The sdr gene had previously come up as differentially expressed in other transcriptome analyses on honey bee larvae and heat shock proteins are frequently involved in insect hormone responses, this making them interesting candidates for further functional assays. Copyright © 2016. Published by Elsevier Ltd.

Differential expression of type X collagen in a mechanically active 3-D chondrocyte culture system: a quantitative study

PubMed Central

Yang, Xu; Vezeridis, Peter S; Nicholas, Brian; Crisco, Joseph J; Moore, Douglas C; Chen, Qian

2006-01-01

Objective Mechanical loading of cartilage influences chondrocyte metabolism and gene expression. The gene encoding type X collagen is expressed specifically by hypertrophic chondrocytes and up regulated during osteoarthritis. In this study we tested the hypothesis that the mechanical microenvironment resulting from higher levels of local strain in a three dimensional cell culture construct would lead to an increase in the expression of type X collagen mRNA by chondrocytes in those areas. Methods Hypertrophic chondrocytes were isolated from embryonic chick sterna and seeded onto rectangular Gelfoam sponges. Seeded sponges were subjected to various levels of cyclic uniaxial tensile strains at 1 Hz with the computer-controlled Bio-Stretch system. Strain distribution across the sponge was quantified by digital image analysis. After mechanical loading, sponges were cut and the end and center regions were separated according to construct strain distribution. Total RNA was extracted from the cells harvested from these regions, and real-time quantitative RT-PCR was performed to quantify mRNA levels for type X collagen and a housing-keeping gene 18S RNA. Results Chondrocytes distributed in high (9%) local strain areas produced more than two times type X collagen mRNA compared to the those under no load conditions, while chondrocytes located in low (2.5%) local strain areas had no appreciable difference in type X collagen mRNA production in comparison to non-loaded samples. Increasing local strains above 2.5%, either in the center or end regions of the sponge, resulted in increased expression of Col X mRNA by chondrocytes in that region. Conclusion These findings suggest that the threshold of chondrocyte sensitivity to inducing type X collagen mRNA production is more than 2.5% local strain, and that increased local strains above the threshold results in an increase of Col X mRNA expression. Such quantitative analysis has important implications for our understanding of mechanosensitivity of cartilage and mechanical regulation of chondrocyte gene expression. PMID:17150098

Identification and Evaluation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis in Tea Plant (Camellia sinensis (L.) O. Kuntze)

PubMed Central

Hao, Xinyuan; Horvath, David P.; Chao, Wun S.; Yang, Yajun; Wang, Xinchao; Xiao, Bin

2014-01-01

Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ∆CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions. PMID:25474086

Transcriptome sequencing and annotation of the halophytic microalga Dunaliella salina * #

PubMed Central

Hong, Ling; Liu, Jun-li; Midoun, Samira Z.; Miller, Philip C.

2017-01-01

The unicellular green alga Dunaliella salina is well adapted to salt stress and contains compounds (including β-carotene and vitamins) with potential commercial value. A large transcriptome database of D. salina during the adjustment, exponential and stationary growth phases was generated using a high throughput sequencing platform. We characterized the metabolic processes in D. salina with a focus on valuable metabolites, with the aim of manipulating D. salina to achieve greater economic value in large-scale production through a bioengineering strategy. Gene expression profiles under salt stress verified using quantitative polymerase chain reaction (qPCR) implied that salt can regulate the expression of key genes. This study generated a substantial fraction of D. salina transcriptional sequences for the entire growth cycle, providing a basis for the discovery of novel genes. This first full-scale transcriptome study of D. salina establishes a foundation for further comparative genomic studies. PMID:28990374

Evolutionary Inference across Eukaryotes Identifies Specific Pressures Favoring Mitochondrial Gene Retention.

PubMed

Johnston, Iain G; Williams, Ben P

2016-02-24

Since their endosymbiotic origin, mitochondria have lost most of their genes. Although many selective mechanisms underlying the evolution of mitochondrial genomes have been proposed, a data-driven exploration of these hypotheses is lacking, and a quantitatively supported consensus remains absent. We developed HyperTraPS, a methodology coupling stochastic modeling with Bayesian inference, to identify the ordering of evolutionary events and suggest their causes. Using 2015 complete mitochondrial genomes, we inferred evolutionary trajectories of mtDNA gene loss across the eukaryotic tree of life. We find that proteins comprising the structural cores of the electron transport chain are preferentially encoded within mitochondrial genomes across eukaryotes. A combination of high GC content and high protein hydrophobicity is required to explain patterns of mtDNA gene retention; a model that accounts for these selective pressures can also predict the success of artificial gene transfer experiments in vivo. This work provides a general method for data-driven inference of the ordering of evolutionary and progressive events, here identifying the distinct features shaping mitochondrial genomes of present-day species. Copyright © 2016 Elsevier Inc. All rights reserved.

Mapping eQTL Networks with Mixed Graphical Markov Models

PubMed Central

Tur, Inma; Roverato, Alberto; Castelo, Robert

2014-01-01

Expression quantitative trait loci (eQTL) mapping constitutes a challenging problem due to, among other reasons, the high-dimensional multivariate nature of gene-expression traits. Next to the expression heterogeneity produced by confounding factors and other sources of unwanted variation, indirect effects spread throughout genes as a result of genetic, molecular, and environmental perturbations. From a multivariate perspective one would like to adjust for the effect of all of these factors to end up with a network of direct associations connecting the path from genotype to phenotype. In this article we approach this challenge with mixed graphical Markov models, higher-order conditional independences, and q-order correlation graphs. These models show that additive genetic effects propagate through the network as function of gene–gene correlations. Our estimation of the eQTL network underlying a well-studied yeast data set leads to a sparse structure with more direct genetic and regulatory associations that enable a straightforward comparison of the genetic control of gene expression across chromosomes. Interestingly, it also reveals that eQTLs explain most of the expression variability of network hub genes. PMID:25271303

Simplification of genotyping techniques of the ABO blood type experiment and exploration of population genetics.

PubMed

Hu, Jian; Zhou, Yi-ren; Ding, Jia-lin; Wang, Zhi-yuan; Liu, Ling; Wang, Ye-kai; Lou, Hui-ling; Qiao, Shou-yi; Wu, Yan-hua

2017-05-20

The ABO blood type is one of the most common and widely used genetic traits in humans. Three glycosyltransferase-encoding gene alleles, I A , I B and i, produce three red blood cell surface antigens, by which the ABO blood type is classified. By using the ABO blood type experiment as an ideal case for genetics teaching, we can easily introduce to the students several genetic concepts, including multiple alleles, gene interaction, single nucleotide polymorphism (SNP) and gene evolution. Herein we have innovated and integrated our ABO blood type genetics experiments. First, in the section of Molecular Genetics, a new method of ABO blood genotyping was established: specific primers based on SNP sites were designed to distinguish three alleles through quantitative real-time PCR. Next, the experimental teaching method of Gene Evolution was innovated in the Population Genetics section: a gene-evolution software was developed to simulate the evolutionary tendency of the ABO genotype encoding alleles under diverse conditions. Our reform aims to extend the contents of genetics experiments, to provide additional teaching approaches, and to improve the learning efficiency of our students eventually.

Validating internal controls for quantitative plant gene expression studies

PubMed Central

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-01-01

Background Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Results Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Conclusion Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments. PMID:15317655

Identification of Causal Genes, Networks, and Transcriptional Regulators of REM Sleep and Wake

PubMed Central

Millstein, Joshua; Winrow, Christopher J.; Kasarskis, Andrew; Owens, Joseph R.; Zhou, Lili; Summa, Keith C.; Fitzpatrick, Karrie; Zhang, Bin; Vitaterna, Martha H.; Schadt, Eric E.; Renger, John J.; Turek, Fred W.

2011-01-01

Study Objective: Sleep-wake traits are well-known to be under substantial genetic control, but the specific genes and gene networks underlying primary sleep-wake traits have largely eluded identification using conventional approaches, especially in mammals. Thus, the aim of this study was to use systems genetics and statistical approaches to uncover the genetic networks underlying 2 primary sleep traits in the mouse: 24-h duration of REM sleep and wake. Design: Genome-wide RNA expression data from 3 tissues (anterior cortex, hypothalamus, thalamus/midbrain) were used in conjunction with high-density genotyping to identify candidate causal genes and networks mediating the effects of 2 QTL regulating the 24-h duration of REM sleep and one regulating the 24-h duration of wake. Setting: Basic sleep research laboratory. Patients or Participants: Male [C57BL/6J × (BALB/cByJ × C57BL/6J*) F1] N2 mice (n = 283). Interventions: None. Measurements and Results: The genetic variation of a mouse N2 mapping cross was leveraged against sleep-state phenotypic variation as well as quantitative gene expression measurement in key brain regions using integrative genomics approaches to uncover multiple causal sleep-state regulatory genes, including several surprising novel candidates, which interact as components of networks that modulate REM sleep and wake. In particular, it was discovered that a core network module, consisting of 20 genes, involved in the regulation of REM sleep duration is conserved across the cortex, hypothalamus, and thalamus. A novel application of a formal causal inference test was also used to identify those genes directly regulating sleep via control of expression. Conclusion: Systems genetics approaches reveal novel candidate genes, complex networks and specific transcriptional regulators of REM sleep and wake duration in mammals. Citation: Millstein J; Winrow CJ; Kasarskis A; Owens JR; Zhou L; Summa KC; Fitzpatrick K; Zhang B; Vitaterna MH; Schadt EE; Renger JJ; Turek FW. Identification of causal genes, networks, and transcriptional regulators of REM sleep and wake. SLEEP 2011;34(11):1469-1477. PMID:22043117

The detection of Yersinia enterocolitica in surface water by quantitative PCR amplification of the ail and yadA genes.

PubMed

Cheyne, Bo M; Van Dyke, Michele I; Anderson, William B; Huck, Peter M

2010-09-01

Yersinia enterocolitica has been detected in surface water, and drinking untreated water is a risk factor for infection. PCR-based methods have been used to detect Y. enterocolitica in various sample types, but quantitative studies have not been conducted in water. In this study, quantitative PCR (qPCR)-based methods targeting the Yersinia virulence genes ail and yadA were used to survey the Grand River watershed in southern Ontario, Canada. Initial testing of reference strains showed that ail and yadA PCR assays were specific for pathogenic biotypes of Y. enterocolitica; however the genes were also detected in one clinical Yersinia intermedia isolate. A survey of surface water from the Grand River watershed showed that both genes were detected at five sampling locations, with the ail and yadA genes detected in 38 and 21% of samples, respectively. Both genes were detected more frequently at colder water temperatures. A screening of Yersinia strains isolated from the watershed showed that the ail gene was detected in three Y. enterocolitica 1A/O:5 isolates. Results of this study show that Yersinia virulence genes were commonly detected in a watershed used as a source of drinking water, and that the occurrence of these genes was seasonal.

Transcriptome and key genes expression related to carbon fixation pathways in Chlorella PY-ZU1 cells and their growth under high concentrations of CO2.

PubMed

Huang, Yun; Cheng, Jun; Lu, Hongxiang; He, Yong; Zhou, Junhu; Cen, Kefa

2017-01-01

The biomass yield of Chlorella PY-ZU1 drastically increased when cultivated under high CO 2 condition compared with that cultivated under air condition. However, less attention has been given to the microalgae photosynthetic mechanisms response to different CO 2 concentrations. The genetic reasons for the higher growth rate, CO 2 fixation rate, and photosynthetic efficiency of microalgal cells under higher CO 2 concentration have not been clearly defined yet. In this study, the Illumina sequencing and de novo transcriptome assembly of Chlorella PY-ZU1 cells cultivated under 15% CO 2 were performed and compared with those of cells grown under air. It was found that carbonic anhydrase (CAs, enzyme for interconversion of bicarbonate to CO 2 ) dramatically decreased to near 0 in 15% CO 2 -grown cells, which indicated that CO 2 molecules directly permeated into cells under high CO 2 stress without CO 2 -concentrating mechanism. Extrapolating from the growth conditions and quantitative Real-Time PCR of CCM-related genes, the K m (CO 2 ) (the minimum intracellular CO 2 concentration that rubisco required) of Chlorella PY-ZU1 might be in the range of 80-192 μM. More adenosine triphosphates was saved for carbon fixation-related pathways. The transcript abundance of rubisco (the most important enzyme of CO 2 fixation reaction) was 16.3 times higher in 15% CO 2 -grown cells than that under air. Besides, the transcript abundances of most key genes involved in carbon fixation pathways were also enhanced in 15% CO 2 -grown cells. Carbon fixation and nitrogen metabolism are the two most important metabolisms in the photosynthetic cells. These genes related to the two most metabolisms with significantly differential expressions were beneficial for microalgal growth (2.85 g L -1 ) under 15% CO 2 concentration. Considering the micro and macro growth phenomena of Chlorella PY-ZU1 under different concentrations of CO 2 (0.04-60%), CO 2 transport pathways responses to different CO 2 (0.04-60%) concentrations was reconstructed.

Advances in cereal genomics and applications in crop breeding.

PubMed

Varshney, Rajeev K; Hoisington, David A; Tyagi, Akhilesh K

2006-11-01

Recent advances in cereal genomics have made it possible to analyse the architecture of cereal genomes and their expressed components, leading to an increase in our knowledge of the genes that are linked to key agronomically important traits. These studies have used molecular genetic mapping of quantitative trait loci (QTL) of several complex traits that are important in breeding. The identification and molecular cloning of genes underlying QTLs offers the possibility to examine the naturally occurring allelic variation for respective complex traits. Novel alleles, identified by functional genomics or haplotype analysis, can enrich the genetic basis of cultivated crops to improve productivity. Advances made in cereal genomics research in recent years thus offer the opportunities to enhance the prediction of phenotypes from genotypes for cereal breeding.

Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L.).

PubMed

Taylor, Candy M; Jost, Ricarda; Erskine, William; Nelson, Matthew N

2016-01-01

Quantitative Reverse Transcription PCR (qRT-PCR) is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop) using a selection of seven genes previously trialed as reference genes for the model legume, Medicago truncatula. In a preliminary evaluation, the seven candidate reference genes were assessed on the basis of primer specificity for their respective targeted region, PCR amplification efficiency, and ability to discriminate between cDNA and gDNA. Following this assessment, expression of the three most promising candidates [Ubiquitin C (UBC), Helicase (HEL), and Polypyrimidine tract-binding protein (PTB)] was evaluated using the NormFinder and RefFinder statistical algorithms in two narrow-leafed lupin lines, both with and without vernalisation treatment, and across seven organ types (cotyledons, stem, leaves, shoot apical meristem, flowers, pods and roots) encompassing three developmental stages. UBC was consistently identified as the most stable candidate and has sufficiently uniform expression that it may be used as a sole reference gene under the experimental conditions tested here. However, as organ type and developmental stage were associated with greater variability in relative expression, it is recommended using UBC and HEL as a pair to achieve optimal normalisation. These results highlight the importance of rigorously assessing candidate reference genes for each species across a diverse range of organs and developmental stages. With emerging technologies, such as RNAseq, and the completion of valuable transcriptome data sets, it is possible that other potentially more suitable reference genes will be identified for this species in future.

Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L.)

PubMed Central

Erskine, William; Nelson, Matthew N.

2016-01-01

Quantitative Reverse Transcription PCR (qRT-PCR) is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop) using a selection of seven genes previously trialed as reference genes for the model legume, Medicago truncatula. In a preliminary evaluation, the seven candidate reference genes were assessed on the basis of primer specificity for their respective targeted region, PCR amplification efficiency, and ability to discriminate between cDNA and gDNA. Following this assessment, expression of the three most promising candidates [Ubiquitin C (UBC), Helicase (HEL), and Polypyrimidine tract-binding protein (PTB)] was evaluated using the NormFinder and RefFinder statistical algorithms in two narrow-leafed lupin lines, both with and without vernalisation treatment, and across seven organ types (cotyledons, stem, leaves, shoot apical meristem, flowers, pods and roots) encompassing three developmental stages. UBC was consistently identified as the most stable candidate and has sufficiently uniform expression that it may be used as a sole reference gene under the experimental conditions tested here. However, as organ type and developmental stage were associated with greater variability in relative expression, it is recommended using UBC and HEL as a pair to achieve optimal normalisation. These results highlight the importance of rigorously assessing candidate reference genes for each species across a diverse range of organs and developmental stages. With emerging technologies, such as RNAseq, and the completion of valuable transcriptome data sets, it is possible that other potentially more suitable reference genes will be identified for this species in future. PMID:26872362

Molecular characterization of BrMYB28 and BrMYB29 paralogous transcription factors involved in the regulation of aliphatic glucosinolate profiles in Brassica rapa ssp. pekinensis.

PubMed

Baskar, Venkidasamy; Park, Se Won

2015-07-01

Glucosinolates (GSL) are one of the major secondary metabolites of the Brassicaceae family. In the present study, we aim at characterizing the multiple paralogs of aliphatic GSL regulators, such as BrMYB28 and BrMYB29 genes in Brassica rapa ssp. pekinensis, by quantitative real-time PCR (qRT-PCR) analysis in different tissues and at various developmental stages. An overlapping gene expression pattern between the BrMYBs as well as their downstream genes (DSGs) was found at different developmental stages. Among the BrMYB28 and BrMYB29 paralogous genes, the BrMYB28.3 and BrMYB29.1 genes were dominantly expressed in most of the developmental stages, compared to the other paralogs of the BrMYB genes. Furthermore, the differential expression pattern of the BrMYBs was observed under various stress treatments. Interestingly, BrMYB28.2 showed the least expression in most developmental stages, while its expression was remarkably high in different stress conditions. More specifically, the BrMYB28.2, BrMYB28.3, and BrMYB29.1 genes were highly responsive to various abiotic and biotic stresses, further indicating their possible role in stress tolerance. Moreover, the in silico cis motif analysis in the upstream regulatory regions of BrMYBs showed the presence of various putative stress-specific motifs, which further indicated their responsiveness to biotic and abiotic stresses. These observations suggest that the dominantly expressed BrMYBs, both in different developmental stages and under various stress treatments (BrMYB28.3 and BrMYB29.1), may be potential candidate genes for altering the GSL level through genetic modification studies in B. rapa ssp. pekinensis. Copyright © 2015. Published by Elsevier SAS.

Overexpression of GmHsp90s, a Heat Shock Protein 90 (Hsp90) Gene Family Cloning from Soybean, Decrease Damage of Abiotic Stresses in Arabidopsis thaliana

PubMed Central

Xue, Dong; Zhao, Jinming; Gai, Junyi; Guo, Na; Xing, Han

2013-01-01

Hsp90 is one of the most conserved and abundant molecular chaperones and is an essential component of the protective stress response; however, its roles in abiotic stress responses in soybean (Glycine max) remain obscure. Here, 12 GmHsp90 genes from soybean were identified and found to be expressed and to function differentially under abiotic stresses. The 12 GmHsp90 genes were isolated and named GmHsp90A1–GmHsp90A6, GmHsp90B1, GmHsp90B2, GmHsp90C1.1, GmHsp90C1.2, GmHsp90C2.1 and GmHsp90C2.2 based on their characteristics and high homology to other Hsp90s according to a new nomenclature system. Quantitative real-time PCR expression data revealed that all the genes exhibited higher transcript levels in leaves and could be strongly induced under heat, osmotic and salt stress but not cold stress. Overexpression of five typical genes (GmHsp90A2, GmHsp90A4, GmHsp90B1, GmHsp90C1.1 and GmHsp90C2.1) in Arabidopsis thaliana provided useful evidences that GmHsp90 genes can decrease damage of abiotic stresses. In addition, an abnormal accumulation of proline was detected in some transgenic Arabidopsis plants suggested overexpressing GmHsp90s may affect the synthesis and response system of proline. Our work represents a systematic determination of soybean genes encoding Hsp90s, and provides useful evidence that GmHsp90 genes function differently in response to abiotic stresses and may affect the synthesis and response system of proline. PMID:23936107

Comprehensive Genomic Analysis and Expression Profiling of the NOX Gene Families under Abiotic Stresses and Hormones in Plants.

PubMed

Chang, Yan-Li; Li, Wen-Yan; Miao, Hai; Yang, Shuai-Qi; Li, Ri; Wang, Xiang; Li, Wen-Qiang; Chen, Kun-Ming

2016-02-23

Plasma membrane NADPH oxidases (NOXs) are key producers of reactive oxygen species under both normal and stress conditions in plants and they form functional subfamilies. Studies of these subfamilies indicated that they show considerable evolutionary selection. We performed a comparative genomic analysis that identified 50 ferric reduction oxidases (FRO) and 77 NOX gene homologs from 20 species representing the eight major plant lineages within the supergroup Plantae: glaucophytes, rhodophytes, chlorophytes, bryophytes, lycophytes, gymnosperms, monocots, and eudicots. Phylogenetic and structural analysis classified these FRO and NOX genes into four well-conserved groups represented as NOX, FRO I, FRO II, and FRO III. Further analysis of NOXs of phylogenetic and exon/intron structures showed that single intron loss and gain had occurred, yielding the diversified gene structures during the evolution of NOXs family genes and which were classified into four conserved subfamilies which are represented as Sub.I, Sub.II, Sub.III, and Sub.IV. Additionally, both available global microarray data analysis and quantitative real-time PCR experiments revealed that the NOX genes in Arabidopsis and rice (Oryza sativa) have different expression patterns in different developmental stages, various abiotic stresses and hormone treatments. Finally, coexpression network analysis of NOX genes in Arabidopsis and rice revealed that NOXs have significantly correlated expression profiles with genes which are involved in plants metabolic and resistance progresses. All these results suggest that NOX family underscores the functional diversity and divergence in plants. This finding will facilitate further studies of the NOX family and provide valuable information for functional validation of this family in plants. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

A gene regulatory network controlled by the NAC transcription factor ANAC092/AtNAC2/ORE1 during salt-promoted senescence.

PubMed

Balazadeh, Salma; Siddiqui, Hamad; Allu, Annapurna D; Matallana-Ramirez, Lilian P; Caldana, Camila; Mehrnia, Mohammad; Zanor, Maria-Inés; Köhler, Barbara; Mueller-Roeber, Bernd

2010-04-01

The onset and progression of senescence are under genetic and environmental control. The Arabidopsis thaliana NAC transcription factor ANAC092 (also called AtNAC2 and ORE1) has recently been shown to control age-dependent senescence, but its mode of action has not been analysed yet. To explore the regulatory network administered by ANAC092 we performed microarray-based expression profiling using estradiol-inducible ANAC092 overexpression lines. Approximately 46% of the 170 genes up-regulated upon ANAC092 induction are known senescence-associated genes, suggesting that the NAC factor exerts its role in senescence through a regulatory network that includes many of the genes previously reported to be senescence regulated. We selected 39 candidate genes and confirmed their time-dependent response to enhanced ANAC092 expression by quantitative RT-PCR. We also found that the majority of them (24 genes) are up-regulated by salt stress, a major promoter of plant senescence, in a manner similar to that of ANAC092, which itself is salt responsive. Furthermore, 24 genes like ANAC092 turned out to be stage-dependently expressed during seed growth with low expression at early and elevated expression at late stages of seed development. Disruption of ANAC092 increased the rate of seed germination under saline conditions, whereas the opposite occurred in respective overexpression plants. We also detected a delay of salinity-induced chlorophyll loss in detached anac092-1 mutant leaves. Promoter-reporter (GUS) studies revealed transcriptional control of ANAC092 expression during leaf and flower ageing and in response to salt stress. We conclude that ANAC092 exerts its functions during senescence and seed germination through partly overlapping target gene sets.

Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae

PubMed Central

Teste, Marie-Ange; Duquenne, Manon; François, Jean M; Parrou, Jean-Luc

2009-01-01

Background Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae. Results From public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, ALG9, TAF10, TFC1 and UBC6 turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with ACT1. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for GPH1 and 20-fold for GSY2 between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, SGA1 expression was independent of the carbon source and increased by 3-fold in stationary phase. Conclusion In this work, we provided a set of genes that are suitable reference genes for quantitative gene expression analysis by real-time RT-PCR in yeast biological samples covering a large panel of physiological states. In contrast, we invalidated and discourage the use of ACT1 as well as other commonly used reference genes (PDA1, TDH3, RDN18, etc) as internal controls for quantitative gene expression analysis in yeast. PMID:19874630

Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae.

PubMed

Teste, Marie-Ange; Duquenne, Manon; François, Jean M; Parrou, Jean-Luc

2009-10-30

Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae. From public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, ALG9, TAF10, TFC1 and UBC6 turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with ACT1. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for GPH1 and 20-fold for GSY2 between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, SGA1 expression was independent of the carbon source and increased by 3-fold in stationary phase. In this work, we provided a set of genes that are suitable reference genes for quantitative gene expression analysis by real-time RT-PCR in yeast biological samples covering a large panel of physiological states. In contrast, we invalidated and discourage the use of ACT1 as well as other commonly used reference genes (PDA1, TDH3, RDN18, etc) as internal controls for quantitative gene expression analysis in yeast.

Inference of quantitative models of bacterial promoters from time-series reporter gene data.

PubMed

Stefan, Diana; Pinel, Corinne; Pinhal, Stéphane; Cinquemani, Eugenio; Geiselmann, Johannes; de Jong, Hidde

2015-01-01

The inference of regulatory interactions and quantitative models of gene regulation from time-series transcriptomics data has been extensively studied and applied to a range of problems in drug discovery, cancer research, and biotechnology. The application of existing methods is commonly based on implicit assumptions on the biological processes under study. First, the measurements of mRNA abundance obtained in transcriptomics experiments are taken to be representative of protein concentrations. Second, the observed changes in gene expression are assumed to be solely due to transcription factors and other specific regulators, while changes in the activity of the gene expression machinery and other global physiological effects are neglected. While convenient in practice, these assumptions are often not valid and bias the reverse engineering process. Here we systematically investigate, using a combination of models and experiments, the importance of this bias and possible corrections. We measure in real time and in vivo the activity of genes involved in the FliA-FlgM module of the E. coli motility network. From these data, we estimate protein concentrations and global physiological effects by means of kinetic models of gene expression. Our results indicate that correcting for the bias of commonly-made assumptions improves the quality of the models inferred from the data. Moreover, we show by simulation that these improvements are expected to be even stronger for systems in which protein concentrations have longer half-lives and the activity of the gene expression machinery varies more strongly across conditions than in the FliA-FlgM module. The approach proposed in this study is broadly applicable when using time-series transcriptome data to learn about the structure and dynamics of regulatory networks. In the case of the FliA-FlgM module, our results demonstrate the importance of global physiological effects and the active regulation of FliA and FlgM half-lives for the dynamics of FliA-dependent promoters.

Association between VDR and ESR1 gene polymorphisms with bone and obesity phenotypes in Chinese male nuclear families.

PubMed

Gu, Jie-mei; Xiao, Wen-jin; He, Jin-wei; Zhang, Hao; Hu, Wei-wei; Hu, Yun-qiu; Li, Miao; Liu, Yu-juan; Fu, Wen-zhen; Yu, Jin-bo; Gao, Gao; Yue, Hua; Ke, Yao-hua; Zhang, Zhen-lin

2009-12-01

The goal of this study was to determine whether polymorphisms in the vitamin D receptor (VDR) and estrogen receptor alpha (ESR1) genes are associated with variations of peak bone mineral density (BMD) and obesity phenotypes in young Chinese men. A total of 1215 subjects from 400 Chinese nuclear families were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allele-specific multiple PCR (ASM-PCR) analysis at the ApaI, FokI, and CDX2 sites in the VDR gene and the PvuII and XbaI sites in the ESR1 gene. BMD at the lumbar spine and hip, total fat mass, and total lean mass were measured using dual energy X-ray absorptiometry. The associations between VDR and ESR1 gene polymorphisms with peak BMD, body mass index (BMI), total fat mass, total lean mass, and percentage fat mass (PFM) were determined using quantitative transmission disequilibrium tests (QTDTs). Using QTDTs, no significant within-family associations were obtained between genotypes or haplotypes of the VDR and ESR1 genes and peak BMD. For the obesity phenotypes, the within-family associations were significant between CDX2 genotypes and BMI (P=0.046), fat mass (P=0.004), and PFM (P=0.020). Further, PvuII was significantly associated with the variation of fat mass and PFM (P=0.002 and P=0.039, respectively). A subsequent 1000 permutations were in agreement with these within-family association results. Our findings showed that VDR and ESR1 polymorphisms were associated with total fat mass in young Chinese men, but we failed to find a significant association between VDR and ESR1 genotypes and peak BMD. These findings suggested that the VDR and ESR1 genes are quantitative trait loci (QTL) underlying fat mass variation in young Chinese men.

Altered Expression of Diabetes-Related Genes in Alzheimer's Disease Brains: The Hisayama Study

PubMed Central

Hokama, Masaaki; Oka, Sugako; Leon, Julio; Ninomiya, Toshiharu; Honda, Hiroyuki; Sasaki, Kensuke; Iwaki, Toru; Ohara, Tomoyuki; Sasaki, Tomio; LaFerla, Frank M.; Kiyohara, Yutaka; Nakabeppu, Yusaku

2014-01-01

Diabetes mellitus (DM) is considered to be a risk factor for dementia including Alzheimer's disease (AD). However, the molecular mechanism underlying this risk is not well understood. We examined gene expression profiles in postmortem human brains donated for the Hisayama study. Three-way analysis of variance of microarray data from frontal cortex, temporal cortex, and hippocampus was performed with the presence/absence of AD and vascular dementia, and sex, as factors. Comparative analyses of expression changes in the brains of AD patients and a mouse model of AD were also performed. Relevant changes in gene expression identified by microarray analysis were validated by quantitative real-time reverse-transcription polymerase chain reaction and western blotting. The hippocampi of AD brains showed the most significant alteration in gene expression profile. Genes involved in noninsulin-dependent DM and obesity were significantly altered in both AD brains and the AD mouse model, as were genes related to psychiatric disorders and AD. The alterations in the expression profiles of DM-related genes in AD brains were independent of peripheral DM-related abnormalities. These results indicate that altered expression of genes related to DM in AD brains is a result of AD pathology, which may thereby be exacerbated by peripheral insulin resistance or DM. PMID:23595620

Integration of QTL and bioinformatic tools to identify candidate genes for triglycerides in mice[S

PubMed Central

Leduc, Magalie S.; Hageman, Rachael S.; Verdugo, Ricardo A.; Tsaih, Shirng-Wern; Walsh, Kenneth; Churchill, Gary A.; Paigen, Beverly

2011-01-01

To identify genetic loci influencing lipid levels, we performed quantitative trait loci (QTL) analysis between inbred mouse strains MRL/MpJ and SM/J, measuring triglyceride levels at 8 weeks of age in F2 mice fed a chow diet. We identified one significant QTL on chromosome (Chr) 15 and three suggestive QTL on Chrs 2, 7, and 17. We also carried out microarray analysis on the livers of parental strains of 282 F2 mice and used these data to find cis-regulated expression QTL. We then narrowed the list of candidate genes under significant QTL using a “toolbox” of bioinformatic resources, including haplotype analysis; parental strain comparison for gene expression differences and nonsynonymous coding single nucleotide polymorphisms (SNP); cis-regulated eQTL in livers of F2 mice; correlation between gene expression and phenotype; and conditioning of expression on the phenotype. We suggest Slc25a7 as a candidate gene for the Chr 7 QTL and, based on expression differences, five genes (Polr3 h, Cyp2d22, Cyp2d26, Tspo, and Ttll12) as candidate genes for Chr 15 QTL. This study shows how bioinformatics can be used effectively to reduce candidate gene lists for QTL related to complex traits. PMID:21622629

Long-distance gene flow and adaptation of forest trees to rapid climate change

PubMed Central

Kremer, Antoine; Ronce, Ophélie; Robledo-Arnuncio, Juan J; Guillaume, Frédéric; Bohrer, Gil; Nathan, Ran; Bridle, Jon R; Gomulkiewicz, Richard; Klein, Etienne K; Ritland, Kermit; Kuparinen, Anna; Gerber, Sophie; Schueler, Silvio

2012-01-01

Forest trees are the dominant species in many parts of the world and predicting how they might respond to climate change is a vital global concern. Trees are capable of long-distance gene flow, which can promote adaptive evolution in novel environments by increasing genetic variation for fitness. It is unclear, however, if this can compensate for maladaptive effects of gene flow and for the long-generation times of trees. We critically review data on the extent of long-distance gene flow and summarise theory that allows us to predict evolutionary responses of trees to climate change. Estimates of long-distance gene flow based both on direct observations and on genetic methods provide evidence that genes can move over spatial scales larger than habitat shifts predicted under climate change within one generation. Both theoretical and empirical data suggest that the positive effects of gene flow on adaptation may dominate in many instances. The balance of positive to negative consequences of gene flow may, however, differ for leading edge, core and rear sections of forest distributions. We propose future experimental and theoretical research that would better integrate dispersal biology with evolutionary quantitative genetics and improve predictions of tree responses to climate change. PMID:22372546

Long-distance gene flow and adaptation of forest trees to rapid climate change.

PubMed

Kremer, Antoine; Ronce, Ophélie; Robledo-Arnuncio, Juan J; Guillaume, Frédéric; Bohrer, Gil; Nathan, Ran; Bridle, Jon R; Gomulkiewicz, Richard; Klein, Etienne K; Ritland, Kermit; Kuparinen, Anna; Gerber, Sophie; Schueler, Silvio

2012-04-01

Forest trees are the dominant species in many parts of the world and predicting how they might respond to climate change is a vital global concern. Trees are capable of long-distance gene flow, which can promote adaptive evolution in novel environments by increasing genetic variation for fitness. It is unclear, however, if this can compensate for maladaptive effects of gene flow and for the long-generation times of trees. We critically review data on the extent of long-distance gene flow and summarise theory that allows us to predict evolutionary responses of trees to climate change. Estimates of long-distance gene flow based both on direct observations and on genetic methods provide evidence that genes can move over spatial scales larger than habitat shifts predicted under climate change within one generation. Both theoretical and empirical data suggest that the positive effects of gene flow on adaptation may dominate in many instances. The balance of positive to negative consequences of gene flow may, however, differ for leading edge, core and rear sections of forest distributions. We propose future experimental and theoretical research that would better integrate dispersal biology with evolutionary quantitative genetics and improve predictions of tree responses to climate change. © 2012 Blackwell Publishing Ltd/CNRS.

[DNA microarray reveals changes in gene expression of endothelial cells under shear stress].

PubMed

Cheng, Min; Zhang, Wensheng; Chen, Huaiqing; Wu, Wenchao; Huang, Hua

2004-04-01

cDNA microarray technology is used as a powerful tool for rapid, comprehensive, and quantitative analysis of gene profiles of cultured human umbilical vein endothelial cells(HUVECs) in the normal static group and the shear stressed (4.20 dyne/cm2, 2 h) group. The total RNA from normal static cultured HUVECs was labeled by Cy3-dCTP, and total RNA of HUVECs from the paired shear stressed experiment was labeled by Cy5-dCTP. The expression ratios reported are the average from the two separate experiments. After bioinformatics analysis, we identified a total of 108 genes (approximately 0.026%) revealing differential expression. Of these 53 genes expressions were up-regulated, the most enhanced ones being human homolog of yeast IPP isomerase, human low density lipoprotein receptor gene, Squalene epoxidase gene, 7-dehydrocholesterol reductase, and 55 were down-regulated, the most decreased ones being heat shock 70 kD protein 1, TCB gene encoding cytosolic thyroid hormone-binding protein in HUVECs exposed to low shear stress. These results indicate that the cDNA microarray technique is effective in screening the differentially expressed genes in endothelial cells induced by various experimental conditions and the data may serve as stimuli to further researches.

Novel Combination of Sorafenib and Celecoxib Provides Synergistic Anti-Proliferative and Pro-Apoptotic Effects in Human Liver Cancer Cells

PubMed Central

Lampiasi, Nadia; Cusimano, Antonella; Azzolina, Antonina; McCubrey, James A.; Montalto, Giuseppe

2013-01-01

Molecular targeted therapy has shown promise as a treatment for advanced hepatocellular carcinoma (HCC). Sorafenib, a multikinase inhibitor, recently received FDA approval for the treatment of advanced HCC. However, although sorafenib is well tolerated, concern for its safety has been expressed. Celecoxib (Celebrex®) is a selective cyclooxygenase-2 (COX-2) inhibitor which exhibits antitumor effects in human HCC cells. The present study examined the interaction between celecoxib and sorafenib in two human liver tumor cell lines HepG2 and Huh7. Our data showed that each inhibitor alone reduced cell growth and the combination of celecoxib with sorafenib synergistically inhibited cell growth and increased apoptosis. To better understand the molecular mechanisms underlying the synergistic antitumor activity of the combination, we investigated the expression profile of the combination-treated liver cancer cell lines using microarray analysis. Combination treatment significantly altered expression levels of 1,986 and 2,483 transcripts in HepG2 and Huh7 cells, respectively. Genes functionally involved in cell death, signal transduction and regulation of transcription were predominantly up-regulated, while genes implicated in metabolism, cell-cycle control and DNA replication and repair were mainly down-regulated upon treatment. However, combination-treated HCC cell lines displayed specificity in the expression and activity of crucial factors involved in hepatocarcinogenesis. The altered expression of some of these genes was confirmed by semi-quantitative and quantitative RT-PCR and by Western blotting. Many novel genes emerged from our transcriptomic analyses, and further functional analyses may determine whether these genes can serve as potential molecular targets for more effective anti-HCC strategies. PMID:23776502

Genome investigation suggests MdSHN3, an APETALA2-domain transcription factor gene, to be a positive regulator of apple fruit cuticle formation and an inhibitor of russet development

PubMed Central

Lashbrooke, Justin; Aharoni, Asaph; Costa, Fabrizio

2015-01-01

The outer epidermal layer of apple fruit is covered by a protective cuticle. Composed of a polymerized cutin matrix embedded with waxes, the cuticle is a natural waterproof barrier and protects against several abiotic and biotic stresses. In terms of apple production, the cuticle is essential to maintain long post-harvest storage, while severe failure of the cuticle can result in the formation of a disorder known as russet. Apple russet results from micro-cracking of the cuticle and the formation of a corky suberized layer. This is typically an undesirable consumer trait, and negatively impacts the post-harvest storage of apples. In order to identify genetic factors controlling cuticle biosynthesis (and thus preventing russet) in apple, a quantitative trait locus (QTL) mapping survey was performed on a full-sib population. Two genomic regions located on chromosomes 2 and 15 that could be associated with russeting were identified. Apples with compromised cuticles were identified through a novel and high-throughput tensile analysis of the skin, while histological analysis confirmed cuticle failure in a subset of the progeny. Additional genomic investigation of the determined QTL regions identified a set of underlying genes involved in cuticle biosynthesis. Candidate gene expression profiling by quantitative real-time PCR on a subset of the progeny highlighted the specific expression pattern of a SHN1/WIN1 transcription factor gene (termed MdSHN3) on chromosome 15. Orthologues of SHN1/WIN1 have been previously shown to regulate cuticle formation in Arabidopsis, tomato, and barley. The MdSHN3 transcription factor gene displayed extremely low expression in lines with improper cuticle formation, suggesting it to be a fundamental regulator of cuticle biosynthesis in apple fruit. PMID:26220084

Genetic Architecture of Ear Fasciation in Maize (Zea mays) under QTL Scrutiny

PubMed Central

Mendes-Moreira, Pedro; Alves, Mara L.; Satovic, Zlatko; dos Santos, João Pacheco; Santos, João Nina; Souza, João Cândido; Pêgo, Silas E.; Hallauer, Arnel R.; Vaz Patto, Maria Carlota

2015-01-01

Maize ear fasciation Knowledge of the genes affecting maize ear inflorescence may lead to better grain yield modeling. Maize ear fasciation, defined as abnormal flattened ears with high kernel row number, is a quantitative trait widely present in Portuguese maize landraces. Material and Methods Using a segregating population derived from an ear fasciation contrasting cross (consisting of 149 F2:3 families) we established a two location field trial using a complete randomized block design. Correlations and heritabilities for several ear fasciation-related traits and yield were determined. Quantitative Trait Loci (QTL) involved in the inheritance of those traits were identified and candidate genes for these QTL proposed. Results and Discussion Ear fasciation broad-sense heritability was 0.73. Highly significant correlations were found between ear fasciation and some ear and cob diameters and row number traits. For the 23 yield and ear fasciation-related traits, 65 QTL were identified, out of which 11 were detected in both environments, while for the three principal components, five to six QTL were detected per environment. Detected QTL were distributed across 17 genomic regions and explained individually, 8.7% to 22.4% of the individual traits or principal components phenotypic variance. Several candidate genes for these QTL regions were proposed, such as bearded-ear1, branched silkless1, compact plant1, ramosa2, ramosa3, tasselseed4 and terminal ear1. However, many QTL mapped to regions without known candidate genes, indicating potential chromosomal regions not yet targeted for maize ear traits selection. Conclusions Portuguese maize germplasm represents a valuable source of genes or allelic variants for yield improvement and elucidation of the genetic basis of ear fasciation traits. Future studies should focus on fine mapping of the identified genomic regions with the aim of map-based cloning. PMID:25923975

Genetic Architecture of Ear Fasciation in Maize (Zea mays) under QTL Scrutiny.

PubMed

Mendes-Moreira, Pedro; Alves, Mara L; Satovic, Zlatko; Dos Santos, João Pacheco; Santos, João Nina; Souza, João Cândido; Pêgo, Silas E; Hallauer, Arnel R; Vaz Patto, Maria Carlota

2015-01-01

Knowledge of the genes affecting maize ear inflorescence may lead to better grain yield modeling. Maize ear fasciation, defined as abnormal flattened ears with high kernel row number, is a quantitative trait widely present in Portuguese maize landraces. Using a segregating population derived from an ear fasciation contrasting cross (consisting of 149 F2:3 families) we established a two location field trial using a complete randomized block design. Correlations and heritabilities for several ear fasciation-related traits and yield were determined. Quantitative Trait Loci (QTL) involved in the inheritance of those traits were identified and candidate genes for these QTL proposed. Ear fasciation broad-sense heritability was 0.73. Highly significant correlations were found between ear fasciation and some ear and cob diameters and row number traits. For the 23 yield and ear fasciation-related traits, 65 QTL were identified, out of which 11 were detected in both environments, while for the three principal components, five to six QTL were detected per environment. Detected QTL were distributed across 17 genomic regions and explained individually, 8.7% to 22.4% of the individual traits or principal components phenotypic variance. Several candidate genes for these QTL regions were proposed, such as bearded-ear1, branched silkless1, compact plant1, ramosa2, ramosa3, tasselseed4 and terminal ear1. However, many QTL mapped to regions without known candidate genes, indicating potential chromosomal regions not yet targeted for maize ear traits selection. Portuguese maize germplasm represents a valuable source of genes or allelic variants for yield improvement and elucidation of the genetic basis of ear fasciation traits. Future studies should focus on fine mapping of the identified genomic regions with the aim of map-based cloning.

Quantitative system drift compensates for altered maternal inputs to the gap gene network of the scuttle fly Megaselia abdita

PubMed Central

Wotton, Karl R; Jiménez-Guri, Eva; Crombach, Anton; Janssens, Hilde; Alcaine-Colet, Anna; Lemke, Steffen; Schmidt-Ott, Urs; Jaeger, Johannes

2015-01-01

The segmentation gene network in insects can produce equivalent phenotypic outputs despite differences in upstream regulatory inputs between species. We investigate the mechanistic basis of this phenomenon through a systems-level analysis of the gap gene network in the scuttle fly Megaselia abdita (Phoridae). It combines quantification of gene expression at high spatio-temporal resolution with systematic knock-downs by RNA interference (RNAi). Initiation and dynamics of gap gene expression differ markedly between M. abdita and Drosophila melanogaster, while the output of the system converges to equivalent patterns at the end of the blastoderm stage. Although the qualitative structure of the gap gene network is conserved, there are differences in the strength of regulatory interactions between species. We term such network rewiring ‘quantitative system drift’. It provides a mechanistic explanation for the developmental hourglass model in the dipteran lineage. Quantitative system drift is likely to be a widespread mechanism for developmental evolution. DOI: http://dx.doi.org/10.7554/eLife.04785.001 PMID:25560971

Targeting neuroendocrine differentiation for prostate cancer radiosensitization

DTIC Science & Technology

2017-12-01

Secondary HRP-conjugated antibodies were purchased fromGEHealthcare UK Ltd. (Buckinghamshire, UK). 2.5. RNA isolation and quantitative real- time PCR (qRT...gene expression data using real- time quantitative PCR and the 2(-Delta Delta C(T))Method,Methods 25 (2001) 402–408. [47] T.K. Kelly, T.B. Miranda, G...relative gene expression data using real- time quantitative PCR and the 2(−Delta Delta C(T)) method, Methods 25 (2001) 402–408. [37] J. Ren, L. Wen, X

Genome-wide transcriptional analysis of two soybean genotypes under dehydration and rehydration conditions

PubMed Central

2013-01-01

Background Soybean is an important crop that provides valuable proteins and oils for human use. Because soybean growth and development is extremely sensitive to water deficit, quality and crop yields are severely impacted by drought stress. In the face of limited water resources, drought-responsive genes are therefore of interest. Identification and analysis of dehydration- and rehydration-inducible differentially expressed genes (DEGs) would not only aid elucidation of molecular mechanisms of stress response, but also enable improvement of crop stress tolerance via gene transfer. Using Digital Gene Expression Tag profiling (DGE), a new technique based on Illumina sequencing, we analyzed expression profiles between two soybean genotypes to identify drought-responsive genes. Results Two soybean genotypes—drought-tolerant Jindou21 and drought-sensitive Zhongdou33—were subjected to dehydration and rehydration conditions. For analysis of DEGs under dehydration conditions, 20 cDNA libraries were generated from roots and leaves at two different time points under well-watered and dehydration conditions. We also generated eight libraries for analysis under rehydration conditions. Sequencing of the 28 libraries produced 25,000–33,000 unambiguous tags, which were mapped to reference sequences for annotation of expressed genes. Many genes exhibited significant expression differences among the libraries. DEGs in the drought-tolerant genotype were identified by comparison of DEGs among treatments and genotypes. In Jindou21, 518 and 614 genes were differentially expressed under dehydration in leaves and roots, respectively, with 24 identified both in leaves and roots. The main functional categories enriched in these DEGs were metabolic process, response to stresses, plant hormone signal transduction, protein processing, and plant-pathogen interaction pathway; the associated genes primarily encoded transcription factors, protein kinases, and other regulatory proteins. The seven most significantly expressed (|log2 ratio| ≥ 8) genes— Glyma15g03920, Glyma05g02470, Glyma15g15010, Glyma05g09070, Glyma06g35630, Glyma08g12590, and Glyma11g16000—are more likely to determine drought stress tolerance. The expression patterns of eight randomly-selected genes were confirmed by quantitative RT-PCR; the results of QRT-PCR analysis agreed with transcriptional profile data for 96 out of 128 (75%) data points. Conclusions Many soybean genes were differentially expressed between drought-tolerant and drought-sensitive genotypes. Based on GO functional annotation and pathway enrichment analysis, some of these genes encoded transcription factors, protein kinases, and other regulatory proteins. The seven most significant DEGs are candidates for improving soybean drought tolerance. These findings will be helpful for analysis and elucidation of molecular mechanisms of drought tolerance; they also provide a basis for cultivating new varieties of drought-tolerant soybean. PMID:24093224

Building quantitative, three-dimensional atlases of gene expression and morphology at cellular resolution.

PubMed

Knowles, David W; Biggin, Mark D

2013-01-01

Animals comprise dynamic three-dimensional arrays of cells that express gene products in intricate spatial and temporal patterns that determine cellular differentiation and morphogenesis. A rigorous understanding of these developmental processes requires automated methods that quantitatively record and analyze complex morphologies and their associated patterns of gene expression at cellular resolution. Here we summarize light microscopy-based approaches to establish permanent, quantitative datasets-atlases-that record this information. We focus on experiments that capture data for whole embryos or large areas of tissue in three dimensions, often at multiple time points. We compare and contrast the advantages and limitations of different methods and highlight some of the discoveries made. We emphasize the need for interdisciplinary collaborations and integrated experimental pipelines that link sample preparation, image acquisition, image analysis, database design, visualization, and quantitative analysis. Copyright © 2013 Wiley Periodicals, Inc.

Effects of co-overexpression of the genes of Rubisco and transketolase on photosynthesis in rice.

PubMed

Suzuki, Yuji; Kondo, Eri; Makino, Amane

2017-03-01

Metabolome analyses have indicated an accumulation of sedoheptulose 7-phosphate in transgenic rice plants with overproduction of Rubisco (Suzuki et al. in Plant Cell Environ 35:1369-1379, 2012. doi: 10.1111/j.1365-3040.2012.02494.x ). Since Rubisco overproduction did not quantitatively enhance photosynthesis even under CO 2 -limited conditions, it is suspected that such an accumulation of sedoheptulose 7-phosphate hampers the improvement of photosynthetic capacity. In the present study, the gene of transketolase, which is involved in the metabolism of sedoheptulose 7-phosphate, was co-overexpressed with the Rubisco small subunit gene in rice. Rubisco and transketolase were successfully overproduced in comparison with those in wild-type plants by 35-53 and 39-84 %, respectively. These changes in the amounts of the proteins were associated with those of the mRNA levels. However, the rate of CO 2 assimilation under high irradiance and different [CO 2 ] did not differ between co-overexpressed plants and wild-type plants. Thus, co-overproduction of Rubisco and transketolase did not improve photosynthesis in rice. Transketolase was probably not a limiting factor of photosynthesis as overproduction of transketolase alone by 80-94 % did not affect photosynthesis.

Identification of QTL controlling domestication-related traits in cowpea (Vigna unguiculata L. Walp).

PubMed

Lo, Sassoum; Muñoz-Amatriaín, María; Boukar, Ousmane; Herniter, Ira; Cisse, Ndiaga; Guo, Yi-Ning; Roberts, Philip A; Xu, Shizhong; Fatokun, Christian; Close, Timothy J

2018-04-19

Cowpea (Vigna unguiculata L. Walp) is a warm-season legume with a genetically diverse gene-pool composed of wild and cultivated forms. Cowpea domestication involved considerable phenotypic changes from the wild progenitor, including reduction of pod shattering, increased organ size, and changes in flowering time. Little is known about the genetic basis underlying these changes. In this study, 215 recombinant inbred lines derived from a cross between a cultivated and a wild cowpea accession were used to evaluate nine domestication-related traits (pod shattering, peduncle length, flower color, days to flowering, 100-seed weight, pod length, leaf length, leaf width and seed number per pod). A high-density genetic map containing 17,739 single nucleotide polymorphisms was constructed and used to identify 16 quantitative trait loci (QTL) for these nine traits. Based on annotations of the cowpea reference genome, genes within these regions are reported. Four regions with clusters of QTL were identified, including one on chromosome 8 related to increased organ size. This study provides new knowledge of the genomic regions controlling domestication-related traits in cowpea as well as candidate genes underlying those QTL. This information can help to exploit wild relatives in cowpea breeding programs.

Molecular characterization of a microbial consortium involved in methane oxidation coupled to denitrification under micro-aerobic conditions

PubMed Central

Liu, Jingjing; Sun, Faqian; Wang, Liang; Ju, Xi; Wu, Weixiang; Chen, Yingxu

2014-01-01

Methane can be used as an alternative carbon source in biological denitrification because it is nontoxic, widely available and relatively inexpensive. A microbial consortium involved in methane oxidation coupled to denitrification (MOD) was enriched with nitrite and nitrate as electron acceptors under micro-aerobic conditions. The 16S rRNA gene combined with pmoA phylogeny of methanotrophs and nirK phylogeny of denitrifiers were analysed to reveal the dominant microbial populations and functional microorganisms. Real-time quantitative polymerase chain reaction results showed high numbers of methanotrophs and denitrifiers in the enriched consortium. The 16S rRNA gene clone library revealed that Methylococcaceae and Methylophilaceae were the dominant populations in the MOD ecosystem. Phylogenetic analyses of pmoA gene clone libraries indicated that all methanotrophs belonged to Methylococcaceae, a type I methanotroph employing the ribulose monophosphate pathway for methane oxidation. Methylotrophic denitrifiers of the Methylophilaceae that can utilize organic intermediates (i.e. formaldehyde, citrate and acetate) released from the methanotrophs played a vital role in aerobic denitrification. This study is the first report to confirm micro-aerobic denitrification and to make phylogenetic and functional assignments for some members of the microbial assemblages involved in MOD. PMID:24245852

A GA-insensitive dwarf mutant of Brassica napus L. correlated with mutation in pyrimidine box in the promoter of GID1.

PubMed

Li, Huapeng; Wang, Yun; Li, Xiaocheng; Gao, Yong; Wang, Zhijun; Zhao, Yun; Wang, Maolin

2011-01-01

A dwarf mutant from Brassica napus, namely NDF-1, which was derived from a high doubled haploid (DH) line '3529'(Brassica napus L.) of which seeds were jointly treated with chemical inducers and fast neutron bombardment, was revealed that dwarfism is under the control of a major gene(designated as ndf1) with a mainly additive effect and non-significant dominance effect. The germination and hypocotyls elongation response of dwarf mutants after exogenous GA and uniconazol application showed NDF-1 was a gibberellin insensitive dwarf. We cloned the Brassica napus GID1 gene, named BnGID1, and found it was the ortholog of AtGID1a. The sequence blasting of the BnGID1 genes from NDF-1 and wild type showed there was no mutant in the gene. But the quantitative RT-PCR analysis of GID1 EST pointed out the mutation was caused by the low-level expression of BnGID1 gene. After sequenced the BnGID1 gene's upstream, we found three bases mutated in the pyrimidine box (P-box) of the BnGID1 promoter, which is linkage with the dwarf mutant.

The rec A operon: a novel stress response gene cluster in Bacteroides fragilis

PubMed Central

Nicholson, Samantha A; Smalley, Darren; Smith, C. Jeffrey; Abratt, Valerie R

2014-01-01

Bacteroides fragilis, an opportunistic pathogen of humans, is a leading cause of bacteraemias and anaerobic abscesses which are often treated with metronidazole, a drug which damages DNA. This study investigated the responses of the B. fragilis recA three gene operon to the stress experienced during metronidazole treatment and exposure to reactive oxygen species simulating those generated by the host immune system during infection. A transcriptionally regulated response was observed using quantitative RT-PCR after metronidazole and hydrogen peroxide treatment, with all three genes being upregulated under stress conditions. In vivo and in vitro analysis of the functional role of the second gene of the operon was done using heterologous complementation and protein expression (in Escherichia coli), with subsequent biochemical assay. This gene encoded a functional bacterioferritin co-migratory protein (BCP) which was thiol-specific and had antioxidant properties, including protection of the glutamine synthetase III enzyme. This in vitro data supports the hypothesis that the genes of the operon may be involved in protection of the bacteria from the oxidative burst during tissue invasion and may play a significant role in bacterial survival and metronidazole resistance during treatment of B. fragilis infections. PMID:24703997

Growth arrest specific gene 2 in tilapia (Oreochromis niloticus): molecular characterization and functional analysis under low-temperature stress.

PubMed

Yang, ChangGeng; Wu, Fan; Lu, Xing; Jiang, Ming; Liu, Wei; Yu, Lijuan; Tian, Juan; Wen, Hua

2017-07-17

Growth arrest specific 2 (gas2) gene is a component of the microfilament system that plays a major role in the cell cycle, regulation of microfilaments, and cell morphology during apoptotic processes. However, little information is available on fish gas2. In this study, the tilapia (Oreochromis niloticus) gas2 gene was cloned and characterized for the first time. The open reading frame was 1020 bp, encoding 340 amino acids; the 5'-untranslated region (UTR) was 140 bp and the 3'-UTR was 70 bp, with a poly (A) tail. The highest promoter activity occurred in the regulatory region (-3000 to -2400 bp). The Gas2-GFP fusion protein was distributed within the cytoplasm. Quantitative reverse transcription-polymerase chain reaction and western blot analyses revealed that gas2 gene expression levels in the liver, muscle, and brain were clearly affected by low temperature stress. The results of gas2 RNAi showed decreased expression of the gas2 and P53 genes. These results suggest that the tilapia gas2 gene may be involved in low temperature stress-induced apoptosis.

Genomic linkage of male song and female acoustic preference QTL underlying a rapid species radiation

PubMed Central

Shaw, Kerry L.; Lesnick, Sky C.

2009-01-01

The genetic coupling hypothesis of signal-preference evolution, whereby the same genes control male signal and female preference for that signal, was first inspired by the evolution of cricket acoustic communication nearly 50 years ago. To examine this hypothesis, we compared the genomic location of quantitative trait loci (QTL) underlying male song and female acoustic preference variation in the Hawaiian cricket genus Laupala. We document a QTL underlying female acoustic preference variation between 2 closely related species (Laupala kohalensis and Laupala paranigra). This preference QTL colocalizes with a song QTL identified previously, providing compelling evidence for a genomic linkage of the genes underlying these traits. We show that both song and preference QTL make small to moderate contributions to the behavioral difference between species, suggesting that divergence in mating behavior among Laupala species is due to the fixation of many genes of minor effect. The diversity of acoustic signaling systems in crickets exemplifies the evolution of elaborate male displays by sexual selection through female choice. Our data reveal genetic conditions that would enable functional coordination between song and acoustic preference divergence during speciation, resulting in a behaviorally coupled mode of signal-preference evolution. Interestingly, Laupala exhibits one of the fastest rates of speciation in animals, concomitant with equally rapid evolution in sexual signaling behaviors. Genomic linkage may facilitate rapid speciation by contributing to genetic correlations between sexual signaling behaviors that eventually cause sexual isolation between diverging populations. PMID:19487670

Differential Expression Analysis of a Subset of Drought-Responsive GmNAC Genes in Two Soybean Cultivars Differing in Drought Tolerance

PubMed Central

Thao, Nguyen Phuong; Thu, Nguyen Binh Anh; Hoang, Xuan Lan Thi; Van Ha, Chien; Tran, Lam-Son Phan

2013-01-01

The plant-specific NAC transcription factors play important roles in plant response to drought stress. Here, we have compared the expression levels of a subset of GmNAC genes in drought-tolerant DT51 and drought-sensitive MTD720 under both normal and drought stress conditions aimed at identifying correlation between GmNAC expression levels and drought tolerance degree, as well as potential GmNAC candidates for genetic engineering. The expression of 23 selected dehydration-responsive GmNACs was assessed in both stressed and unstressed root tissues of DT51 and MTD720 using real-time quantitative PCR. The results indicated that expression of GmNACs was genotype-dependent. Seven and 13 of 23 tested GmNACs showed higher expression levels in roots of DT51 in comparison with MTD720 under normal and drought stress conditions, respectively, whereas none of them displayed lower transcript levels under any conditions. This finding suggests that the higher drought tolerance of DT51 might be positively correlated with the higher induction of the GmNAC genes during water deficit. The drought-inducible GmNAC011 needs to be mentioned as its transcript accumulation was more than 76-fold higher in drought-stressed DT51 roots relative to MTD720 roots. Additionally, among the GmNAC genes examined, GmNAC085, 092, 095, 101 and 109 were not only drought-inducible but also more highly up-regulated in DT51 roots than in that of MTD720 under both treatment conditions. These data together suggest that GmNAC011, 085, 092, 095, 101 and 109 might be promising candidates for improvement of drought tolerance in soybean by biotechnological approaches. PMID:24322442

Genome-Wide Identification of the PHD-Finger Family Genes and Their Responses to Environmental Stresses in Oryza sativa L.

PubMed Central

Sun, Mingzhe; Yang, Junkai; Cui, Na; Zhu, Yanming

2017-01-01

The PHD-finger family has been demonstrated to be involved in regulating plant growth and development. However, little information is given for its role in environmental stress responses. Here, we identified a total of 59 PHD family genes in the rice genome. These OsPHDs genes were located on eleven chromosomes and synteny analysis only revealed nine duplicated pairs within the rice PHD family. Phylogenetic analysis of all OsPHDs and PHDs from other species revealed that they could be grouped into two major clusters. Furthermore, OsPHDs were clustered into eight groups and members from different groups displayed a great divergence in terms of gene structure, functional domains and conserved motifs. We also found that with the exception of OsPHD6, all OsPHDs were expressed in at least one of the ten tested tissues and OsPHDs from certain groups were expressed in specific tissues. Moreover, our results also uncovered differential responses of OsPHDs expression to environmental stresses, including ABA (abscisic acid), water deficit, cold and high Cd. By using quantitative real-time PCR, we further confirmed the differential expression of OsPHDs under these stresses. OsPHD1/7/8/13/33 were differentially expressed under water deficit and Cd stresses, while OsPHD5/17 showed altered expression under water deficit and cold stresses. Moreover, OsPHD3/44/28 displayed differential expression under ABA and Cd stresses. In conclusion, our results provide valuable information on the rice PHD family in plant responses to environmental stress, which will be helpful for further characterizing their biological roles in responding to environmental stresses.

The integration of quantitative genetics, paleontology, and neontology reveals genetic underpinnings of primate dental evolution.

PubMed

Hlusko, Leslea J; Schmitt, Christopher A; Monson, Tesla A; Brasil, Marianne F; Mahaney, Michael C

2016-08-16

Developmental genetics research on mice provides a relatively sound understanding of the genes necessary and sufficient to make mammalian teeth. However, mouse dentitions are highly derived compared with human dentitions, complicating the application of these insights to human biology. We used quantitative genetic analyses of data from living nonhuman primates and extensive osteological and paleontological collections to refine our assessment of dental phenotypes so that they better represent how the underlying genetic mechanisms actually influence anatomical variation. We identify ratios that better characterize the output of two dental genetic patterning mechanisms for primate dentitions. These two newly defined phenotypes are heritable with no measurable pleiotropic effects. When we consider how these two phenotypes vary across neontological and paleontological datasets, we find that the major Middle Miocene taxonomic shift in primate diversity is characterized by a shift in these two genetic outputs. Our results build on the mouse model by combining quantitative genetics and paleontology, and thereby elucidate how genetic mechanisms likely underlie major events in primate evolution.

Genetic and genomic analysis of hyperlipidemia, obesity and diabetes using (C57BL/6J × TALLYHO/JngJ) F2 mice.

PubMed

Stewart, Taryn P; Kim, Hyoung Yon; Saxton, Arnold M; Kim, Jung Han

2010-12-19

Type 2 diabetes (T2D) is the most common form of diabetes in humans and is closely associated with dyslipidemia and obesity that magnifies the mortality and morbidity related to T2D. The genetic contribution to human T2D and related metabolic disorders is evident, and mostly follows polygenic inheritance. The TALLYHO/JngJ (TH) mice are a polygenic model for T2D characterized by obesity, hyperinsulinemia, impaired glucose uptake and tolerance, hyperlipidemia, and hyperglycemia. In order to determine the genetic factors that contribute to these T2D related characteristics in TH mice, we interbred TH mice with C57BL/6J (B6) mice. The parental, F1, and F2 mice were phenotyped at 8, 12, 16, 20, and 24 weeks of age for 4-hour fasting plasma triglyceride, cholesterol, insulin, and glucose levels and body, fat pad and carcass weights. The F2 mice were genotyped genome-wide and used for quantitative trait locus (QTL) mapping. We also applied a genetical genomic approach using a subset of the F2 mice to seek candidate genes underlying the QTLs. Major QTLs were detected on chromosomes (Chrs) 1, 11, 4, and 8 for hypertriglyceridemia, 1 and 3 for hypercholesterolemia, 4 for hyperglycemia, 11 and 1 for body weight, 1 for fat pad weight, and 11 and 14 for carcass weight. Most alleles, except for Chr 3 and 14 QTLs, increased phenotypic values when contributed by the TH strain. Fourteen pairs of interacting loci were detected, none of which overlapped the major QTLs. The QTL interval linked to hypercholesterolemia and hypertriglyceridemia on distal Chr 1 contains Apoa2 gene. Sequencing analysis revealed polymorphisms of Apoa2 in TH mice, suggesting Apoa2 as the candidate gene for the hyperlipidemia QTL. Gene expression analysis added novel information and aided in selection of candidates underlying the QTLs. We identified several genetic loci that affect the quantitative variations of plasma lipid and glucose levels and obesity traits in a TH × B6 intercross. Polymorphisms in Apoa2 gene are suggested to be responsible for the Chr 1 QTL linked to hypercholesterolemia and hypertriglyceridemia. Further, genetical genomic analysis led to potential candidate genes for the QTLs.

Genetic and genomic analysis of hyperlipidemia, obesity and diabetes using (C57BL/6J × TALLYHO/JngJ) F2 mice

PubMed Central

2010-01-01

Background Type 2 diabetes (T2D) is the most common form of diabetes in humans and is closely associated with dyslipidemia and obesity that magnifies the mortality and morbidity related to T2D. The genetic contribution to human T2D and related metabolic disorders is evident, and mostly follows polygenic inheritance. The TALLYHO/JngJ (TH) mice are a polygenic model for T2D characterized by obesity, hyperinsulinemia, impaired glucose uptake and tolerance, hyperlipidemia, and hyperglycemia. Results In order to determine the genetic factors that contribute to these T2D related characteristics in TH mice, we interbred TH mice with C57BL/6J (B6) mice. The parental, F1, and F2 mice were phenotyped at 8, 12, 16, 20, and 24 weeks of age for 4-hour fasting plasma triglyceride, cholesterol, insulin, and glucose levels and body, fat pad and carcass weights. The F2 mice were genotyped genome-wide and used for quantitative trait locus (QTL) mapping. We also applied a genetical genomic approach using a subset of the F2 mice to seek candidate genes underlying the QTLs. Major QTLs were detected on chromosomes (Chrs) 1, 11, 4, and 8 for hypertriglyceridemia, 1 and 3 for hypercholesterolemia, 4 for hyperglycemia, 11 and 1 for body weight, 1 for fat pad weight, and 11 and 14 for carcass weight. Most alleles, except for Chr 3 and 14 QTLs, increased phenotypic values when contributed by the TH strain. Fourteen pairs of interacting loci were detected, none of which overlapped the major QTLs. The QTL interval linked to hypercholesterolemia and hypertriglyceridemia on distal Chr 1 contains Apoa2 gene. Sequencing analysis revealed polymorphisms of Apoa2 in TH mice, suggesting Apoa2 as the candidate gene for the hyperlipidemia QTL. Gene expression analysis added novel information and aided in selection of candidates underlying the QTLs. Conclusions We identified several genetic loci that affect the quantitative variations of plasma lipid and glucose levels and obesity traits in a TH × B6 intercross. Polymorphisms in Apoa2 gene are suggested to be responsible for the Chr 1 QTL linked to hypercholesterolemia and hypertriglyceridemia. Further, genetical genomic analysis led to potential candidate genes for the QTLs. PMID:21167066

A myo-inositol-1-phosphate synthase gene, IbMIPS1, enhances salt and drought tolerance and stem nematode resistance in transgenic sweet potato.

PubMed

Zhai, Hong; Wang, Feibing; Si, Zengzhi; Huo, Jinxi; Xing, Lei; An, Yanyan; He, Shaozhen; Liu, Qingchang

2016-02-01

Myo-inositol-1-phosphate synthase (MIPS) is a key rate limiting enzyme in myo-inositol biosynthesis. The MIPS gene has been shown to improve tolerance to abiotic stresses in several plant species. However, its role in resistance to biotic stresses has not been reported. In this study, we found that expression of the sweet potato IbMIPS1 gene was induced by NaCl, polyethylene glycol (PEG), abscisic acid (ABA) and stem nematodes. Its overexpression significantly enhanced stem nematode resistance as well as salt and drought tolerance in transgenic sweet potato under field conditions. Transcriptome and real-time quantitative PCR analyses showed that overexpression of IbMIPS1 up-regulated the genes involved in inositol biosynthesis, phosphatidylinositol (PI) and ABA signalling pathways, stress responses, photosynthesis and ROS-scavenging system under salt, drought and stem nematode stresses. Inositol, inositol-1,4,5-trisphosphate (IP3 ), phosphatidic acid (PA), Ca(2+) , ABA, K(+) , proline and trehalose content was significantly increased, whereas malonaldehyde (MDA), Na(+) and H2 O2 content was significantly decreased in the transgenic plants under salt and drought stresses. After stem nematode infection, the significant increase of inositol, IP3 , PA, Ca(2+) , ABA, callose and lignin content and significant reduction of MDA content were found, and a rapid increase of H2 O2 levels was observed, peaked at 1 to 2 days and thereafter declined in the transgenic plants. This study indicates that the IbMIPS1 gene has the potential to be used to improve the resistance to biotic and abiotic stresses in plants. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Making lineage decisions with biological noise: Lessons from the early mouse embryo.

PubMed

Simon, Claire S; Hadjantonakis, Anna-Katerina; Schröter, Christian

2018-04-30

Understanding how individual cells make fate decisions that lead to the faithful formation and homeostatic maintenance of tissues is a fundamental goal of contemporary developmental and stem cell biology. Seemingly uniform populations of stem cells and multipotent progenitors display a surprising degree of heterogeneity, primarily originating from the inherent stochastic nature of molecular processes underlying gene expression. Despite this heterogeneity, lineage decisions result in tissues of a defined size and with consistent proportions of differentiated cell types. Using the early mouse embryo as a model we review recent developments that have allowed the quantification of molecular intercellular heterogeneity during cell differentiation. We first discuss the relationship between these heterogeneities and developmental cellular potential. We then review recent theoretical approaches that formalize the mechanisms underlying fate decisions in the inner cell mass of the blastocyst stage embryo. These models build on our extensive knowledge of the genetic control of fate decisions in this system and will become essential tools for a rigorous understanding of the connection between noisy molecular processes and reproducible outcomes at the multicellular level. We conclude by suggesting that cell-to-cell communication provides a mechanism to exploit and buffer intercellular variability in a self-organized process that culminates in the reproducible formation of the mature mammalian blastocyst stage embryo that is ready for implantation into the maternal uterus. This article is categorized under: Gene Expression and Transcriptional Hierarchies > Cellular Differentiation Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Gene Expression and Transcriptional Hierarchies > Quantitative Methods and Models. © 2018 Wiley Periodicals, Inc.

Primers to block the amplification of symbiotic apostome ciliate 18S rRNA gene in a PCR-based copepod diet study

NASA Astrophysics Data System (ADS)

Yi, Xiaoyan; Zhang, Huan; Liu, Guangxing

2014-05-01

Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene (18S rDNA) libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18s rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.

CsGOGAT Is Important in Dynamic Changes of Theanine Content in Postharvest Tea Plant Leaves under Different Temperature and Shading Spreadings.

PubMed

Liu, Zhi-Wei; Li, Hui; Wang, Wen-Li; Wu, Zhi-Jun; Cui, Xin; Zhuang, Jing

2017-11-08

We analyzed the changes of theanine content in postharvest tea leaves under high temperature (38 °C), low temperature (4 °C), and shading spreadings by using ultrahigh-performance liquid chromatography. The differentially expressed proteins (DEPs), CsFd-GOGAT and CsNADH-GOGAT, which are involved in theanine biosynthesis pathway, were identified from the corresponding proteome data. The protein-protein interactions of CsFd-GOGAT and CsNADH-GOGAT, CsTS1, or CsNiR were verified by yeast two-hybrid technology. The expression profiles of 17 genes in theanine metabolism, including CsFd-GOGAT and CsNADH-GOGAT, were analyzed by quantitative real-time polymerase chain reaction. The correlations between the dynamic changes of theanine content and expression profiles of related genes and DEPs were analyzed. This study preliminarily proved the importance of CsGOGAT in dynamic changes of theanine content in postharvest tea leaves during spreading.

Six quantitative trait loci influence task thresholds for hygienic behaviour in honeybees (Apis mellifera).

PubMed

Oxley, Peter R; Spivak, Marla; Oldroyd, Benjamin P

2010-04-01

Honeybee hygienic behaviour provides colonies with protection from many pathogens and is an important model system of the genetics of a complex behaviour. It is a textbook example of complex behaviour under simple genetic control: hygienic behaviour consists of two components--uncapping a diseased brood cell, followed by removal of the contents--each of which are thought to be modulated independently by a few loci of medium to large effect. A worker's genetic propensity to engage in hygienic tasks affects the intensity of the stimulus required before she initiates the behaviour. Genetic diversity within colonies leads to task specialization among workers, with a minority of workers performing the majority of nest-cleaning tasks. We identify three quantitative trait loci that influence the likelihood that workers will engage in hygienic behaviour and account for up to 30% of the phenotypic variability in hygienic behaviour in our population. Furthermore, we identify two loci that influence the likelihood that a worker will perform uncapping behaviour only, and one locus that influences removal behaviour. We report the first candidate genes associated with engaging in hygienic behaviour, including four genes involved in olfaction, learning and social behaviour, and one gene involved in circadian locomotion. These candidates will allow molecular characterization of this distinctive behavioural mode of disease resistance, as well as providing the opportunity for marker-assisted selection for this commercially significant trait.

Finding the joker among the maize endogenous reference genes for genetically modified organism (GMO) detection.

PubMed

Paternò, Annalisa; Marchesi, Ugo; Gatto, Francesco; Verginelli, Daniela; Quarchioni, Cinzia; Fusco, Cristiana; Zepparoni, Alessia; Amaddeo, Demetrio; Ciabatti, Ilaria

2009-12-09

The comparison of five real-time polymerase chain reaction (PCR) methods targeted at maize ( Zea mays ) endogenous sequences is reported. PCR targets were the alcohol dehydrogenase (adh) gene for three methods and high-mobility group (hmg) gene for the other two. The five real-time PCR methods have been checked under repeatability conditions at several dilution levels on both pooled DNA template from several genetically modified (GM) maize certified reference materials (CRMs) and single CRM DNA extracts. Slopes and R(2) coefficients of all of the curves obtained from the adopted regression model were compared within the same method and among all of the five methods, and the limit of detection and limit of quantitation were analyzed for each PCR system. Furthermore, method equivalency was evaluated on the basis of the ability to estimate the target haploid genome copy number at each concentration level. Results indicated that, among the five methods tested, one of the hmg-targeted PCR systems can be considered equivalent to the others but shows the best regression parameters and a higher repeteability along the dilution range. Thereby, it is proposed as a valid module to be coupled to different event-specific real-time PCR for maize genetically modified organism (GMO) quantitation. The resulting practicability improvement on the analytical control of GMOs is discussed.

Analysis of Sequence Data Under Multivariate Trait-Dependent Sampling.

PubMed

Tao, Ran; Zeng, Donglin; Franceschini, Nora; North, Kari E; Boerwinkle, Eric; Lin, Dan-Yu

2015-06-01

High-throughput DNA sequencing allows for the genotyping of common and rare variants for genetic association studies. At the present time and for the foreseeable future, it is not economically feasible to sequence all individuals in a large cohort. A cost-effective strategy is to sequence those individuals with extreme values of a quantitative trait. We consider the design under which the sampling depends on multiple quantitative traits. Under such trait-dependent sampling, standard linear regression analysis can result in bias of parameter estimation, inflation of type I error, and loss of power. We construct a likelihood function that properly reflects the sampling mechanism and utilizes all available data. We implement a computationally efficient EM algorithm and establish the theoretical properties of the resulting maximum likelihood estimators. Our methods can be used to perform separate inference on each trait or simultaneous inference on multiple traits. We pay special attention to gene-level association tests for rare variants. We demonstrate the superiority of the proposed methods over standard linear regression through extensive simulation studies. We provide applications to the Cohorts for Heart and Aging Research in Genomic Epidemiology Targeted Sequencing Study and the National Heart, Lung, and Blood Institute Exome Sequencing Project.

Gene expression patterns during the larval development of European sea bass (dicentrarchus labrax) by microarray analysis.

PubMed

Darias, M J; Zambonino-Infante, J L; Hugot, K; Cahu, C L; Mazurais, D

2008-01-01

During the larval period, marine teleosts undergo very fast growth and dramatic changes in morphology, metabolism, and behavior to accomplish their metamorphosis into juvenile fish. Regulation of gene expression is widely thought to be a key mechanism underlying the management of the biological processes required for harmonious development over this phase of life. To provide an overall analysis of gene expression in the whole body during sea bass larval development, we monitored the expression of 6,626 distinct genes at 10 different points in time between 7 and 43 days post-hatching (dph) by using heterologous hybridization of a rainbow trout cDNA microarray. The differentially expressed genes (n = 485) could be grouped into two categories: genes that were generally up-expressed early, between 7 and 23 dph, and genes up-expressed between 25 and 43 dph. Interestingly, among the genes regulated during the larval period, those related to organogenesis, energy pathways, biosynthesis, and digestion were over-represented compared with total set of analyzed genes. We discuss the quantitative regulation of whole-body contents of these specific transcripts with regard to the ontogenesis and maturation of essential functions that take place over larval development. Our study is the first utilization of a transcriptomic approach in sea bass and reveals dynamic changes in gene expression patterns in relation to marine finfish larval development.

Targeted and Untargeted Approaches Unravel Novel Candidate Genes and Diagnostic SNPs for Quantitative Resistance of the Potato (Solanum tuberosum L.) to Phytophthora infestans Causing the Late Blight Disease.

PubMed

Mosquera, Teresa; Alvarez, Maria Fernanda; Jiménez-Gómez, José M; Muktar, Meki Shehabu; Paulo, Maria João; Steinemann, Sebastian; Li, Jinquan; Draffehn, Astrid; Hofmann, Andrea; Lübeck, Jens; Strahwald, Josef; Tacke, Eckhard; Hofferbert, Hans-Reinhardt; Walkemeier, Birgit; Gebhardt, Christiane

2016-01-01

The oomycete Phytophthora infestans causes late blight of potato, which can completely destroy the crop. Therefore, for the past 160 years, late blight has been the most important potato disease worldwide. The identification of cultivars with high and durable field resistance to P. infestans is an objective of most potato breeding programs. This type of resistance is polygenic and therefore quantitative. Its evaluation requires multi-year and location trials. Furthermore, quantitative resistance to late blight correlates with late plant maturity, a negative agricultural trait. Knowledge of the molecular genetic basis of quantitative resistance to late blight not compromised by late maturity is very limited. It is however essential for developing diagnostic DNA markers that facilitate the efficient combination of superior resistance alleles in improved cultivars. We used association genetics in a population of 184 tetraploid potato cultivars in order to identify single nucleotide polymorphisms (SNPs) that are associated with maturity corrected resistance (MCR) to late blight. The population was genotyped for almost 9000 SNPs from three different sources. The first source was candidate genes specifically selected for their function in the jasmonate pathway. The second source was novel candidate genes selected based on comparative transcript profiling (RNA-Seq) of groups of genotypes with contrasting levels of quantitative resistance to P. infestans. The third source was the first generation 8.3k SolCAP SNP genotyping array available in potato for genome wide association studies (GWAS). Twenty seven SNPs from all three sources showed robust association with MCR. Some of those were located in genes that are strong candidates for directly controlling quantitative resistance, based on functional annotation. Most important were: a lipoxygenase (jasmonate pathway), a 3-hydroxy-3-methylglutaryl coenzyme A reductase (mevalonate pathway), a P450 protein (terpene biosynthesis), a transcription factor and a homolog of a major gene for resistance to P. infestans from the wild potato species Solanum venturii. The candidate gene approach and GWAS complemented each other as they identified different genes. The results of this study provide new insight in the molecular genetic basis of quantitative resistance in potato and a toolbox of diagnostic SNP markers for breeding applications.

Targeted and Untargeted Approaches Unravel Novel Candidate Genes and Diagnostic SNPs for Quantitative Resistance of the Potato (Solanum tuberosum L.) to Phytophthora infestans Causing the Late Blight Disease

PubMed Central

Jiménez-Gómez, José M.; Muktar, Meki Shehabu; Paulo, Maria João; Steinemann, Sebastian; Li, Jinquan; Draffehn, Astrid; Hofmann, Andrea; Lübeck, Jens; Strahwald, Josef; Tacke, Eckhard; Hofferbert, Hans-Reinhardt; Walkemeier, Birgit; Gebhardt, Christiane

2016-01-01

The oomycete Phytophthora infestans causes late blight of potato, which can completely destroy the crop. Therefore, for the past 160 years, late blight has been the most important potato disease worldwide. The identification of cultivars with high and durable field resistance to P. infestans is an objective of most potato breeding programs. This type of resistance is polygenic and therefore quantitative. Its evaluation requires multi-year and location trials. Furthermore, quantitative resistance to late blight correlates with late plant maturity, a negative agricultural trait. Knowledge of the molecular genetic basis of quantitative resistance to late blight not compromised by late maturity is very limited. It is however essential for developing diagnostic DNA markers that facilitate the efficient combination of superior resistance alleles in improved cultivars. We used association genetics in a population of 184 tetraploid potato cultivars in order to identify single nucleotide polymorphisms (SNPs) that are associated with maturity corrected resistance (MCR) to late blight. The population was genotyped for almost 9000 SNPs from three different sources. The first source was candidate genes specifically selected for their function in the jasmonate pathway. The second source was novel candidate genes selected based on comparative transcript profiling (RNA-Seq) of groups of genotypes with contrasting levels of quantitative resistance to P. infestans. The third source was the first generation 8.3k SolCAP SNP genotyping array available in potato for genome wide association studies (GWAS). Twenty seven SNPs from all three sources showed robust association with MCR. Some of those were located in genes that are strong candidates for directly controlling quantitative resistance, based on functional annotation. Most important were: a lipoxygenase (jasmonate pathway), a 3-hydroxy-3-methylglutaryl coenzyme A reductase (mevalonate pathway), a P450 protein (terpene biosynthesis), a transcription factor and a homolog of a major gene for resistance to P. infestans from the wild potato species Solanum venturii. The candidate gene approach and GWAS complemented each other as they identified different genes. The results of this study provide new insight in the molecular genetic basis of quantitative resistance in potato and a toolbox of diagnostic SNP markers for breeding applications. PMID:27281327

Quantitative developmental transcriptomes of the Mediterranean sea urchin Paracentrotus lividus.

PubMed

Gildor, Tsvia; Malik, Assaf; Sher, Noa; Avraham, Linor; Ben-Tabou de-Leon, Smadar

2016-02-01

Embryonic development progresses through the timely activation of thousands of differentially activated genes. Quantitative developmental transcriptomes provide the means to relate global patterns of differentially expressed genes to the emerging body plans they generate. The sea urchin is one of the classic model systems for embryogenesis and the models of its developmental gene regulatory networks are of the most comprehensive of their kind. Thus, the sea urchin embryo is an excellent system for studies of its global developmental transcriptional profiles. Here we produced quantitative developmental transcriptomes of the sea urchin Paracentrotus lividus (P. lividus) at seven developmental stages from the fertilized egg to prism stage. We generated de-novo reference transcriptome and identified 29,817 genes that are expressed at this time period. We annotated and quantified gene expression at the different developmental stages and confirmed the reliability of the expression profiles by QPCR measurement of a subset of genes. The progression of embryo development is reflected in the observed global expression patterns and in our principle component analysis. Our study illuminates the rich patterns of gene expression that participate in sea urchin embryogenesis and provide an essential resource for further studies of the dynamic expression of P. lividus genes. Copyright © 2015 Elsevier B.V. All rights reserved.

Generation of mammalian cells stably expressing multiple genes at predetermined levels.

PubMed

Liu, X; Constantinescu, S N; Sun, Y; Bogan, J S; Hirsch, D; Weinberg, R A; Lodish, H F

2000-04-10

Expression of cloned genes at desired levels in cultured mammalian cells is essential for studying protein function. Controlled levels of expression have been difficult to achieve, especially for cell lines with low transfection efficiency or when expression of multiple genes is required. An internal ribosomal entry site (IRES) has been incorporated into many types of expression vectors to allow simultaneous expression of two genes. However, there has been no systematic quantitative analysis of expression levels in individual cells of genes linked by an IRES, and thus the broad use of these vectors in functional analysis has been limited. We constructed a set of retroviral expression vectors containing an IRES followed by a quantitative selectable marker such as green fluorescent protein (GFP) or truncated cell surface proteins CD2 or CD4. The gene of interest is placed in a multiple cloning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the approximately 100-fold differences in levels of expression of a retrovirus vector depending on its site of insertion in the host chromosome. We show that the level of expression of the gene downstream of the IRES and the expression level and functional activity of the gene cloned upstream of the IRES are highly correlated in stably infected target cells. This feature makes our vectors extremely useful for the rapid generation of stably transfected cell populations or clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of expression of the gene downstream of the IRES. We show how these vectors can be used to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressing two different cloned proteins, Ski and Smad4. Correlation of a biologic effect with the level of expression of the protein downstream of the IRES provides strong evidence for the function of the protein placed upstream of the IRES.

Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis.

PubMed

dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

2015-01-01

Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis.