Publications by authors named "Jyothi Thimmapuram"

47 Publications

RNA sequencing identifies transcriptional changes in the rabbit larynx in response to low humidity challenge.

BMC Genomics 2020 Dec 11;21(1):888. Epub 2020 Dec 11.

Department of Comparative Pathobiology, Purdue University, West Lafayette, IN, 47907, USA.

Background: Voice disorders are a worldwide problem impacting human health, particularly for occupational voice users. Avoidance of surface dehydration is commonly prescribed as a protective factor against the development of dysphonia. The available literature inconclusively supports this practice and a biological mechanism for how surface dehydration of the laryngeal tissue affects voice has not been described. In this study, we used an in vivo male New Zealand white rabbit model to elucidate biological changes based on gene expression within the vocal folds from surface dehydration. Surface dehydration was induced by exposure to low humidity air (18.6% + 4.3%) for 8 h. Exposure to moderate humidity (43.0% + 4.3%) served as the control condition. Ilumina-based RNA sequencing was performed and used for transcriptome analysis with validation by RT-qPCR.

Results: There were 103 statistically significant differentially expressed genes identified through Cuffdiff with 61 genes meeting significance by both false discovery rate and fold change. Functional annotation enrichment and predicted protein interaction mapping showed enrichment of various loci, including cellular stress and inflammatory response, ciliary function, and keratinocyte development. Eight genes were selected for RT-qPCR validation. Matrix metalloproteinase 12 (MMP12) and macrophage cationic peptide 1 (MCP1) were significantly upregulated and an epithelial chloride channel protein (ECCP) was significantly downregulated after surface dehydration by RNA-Seq and RT-qPCR. Suprabasin (SPBN) and zinc activated cationic channel (ZACN) were marginally, but non-significantly down- and upregulated as evidenced by RT-qPCR, respectively.

Conclusions: The data together support the notion that surface dehydration induces physiological changes in the vocal folds and justifies targeted analysis to further explore the underlying biology of compensatory fluid/ion flux and inflammatory mediators in response to airway surface dehydration.
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http://dx.doi.org/10.1186/s12864-020-07301-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733274PMC
December 2020

Changes in the core endophytic mycobiome of carrot taproots in response to crop management and genotype.

Sci Rep 2020 08 13;10(1):13685. Epub 2020 Aug 13.

Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN, USA.

Fungal endophytes can influence production and post-harvest challenges in carrot, though the identity of these microbes as well as factors affecting their composition have not yet been determined, which prevents growers from managing these organisms to improve crop performance. Consequently, we characterized the endophytic mycobiome in the taproots of three carrot genotypes that vary in resistance to two pathogens grown in a trial comparing organic and conventional crop management using Illumina sequencing of the internal transcribed spacer (ITS) gene. A total of 1,480 individual operational taxonomic units (OTUs) were identified. Most were consistent across samples, indicating that they are part of a core mycobiome, though crop management influenced richness and diversity, likely in response to differences in soil properties. There were also differences in individual OTUs among genotypes and the nematode resistant genotype was most responsive to management system indicating that it has greater control over its endophytic mycobiome, which could potentially play a role in resistance. Members of the Ascomycota were most dominant, though the exact function of most taxa remains unclear. Future studies aimed at overcoming difficulties associated with isolating fungal endophytes are needed to identify these microbes at the species level and elucidate their specific functional roles.
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http://dx.doi.org/10.1038/s41598-020-70683-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426841PMC
August 2020

Bioinformatics Core Survey Highlights the Challenges Facing Data Analysis Facilities.

J Biomol Tech 2020 07;31(2):66-73

Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Over the last decade, the cost of -omics data creation has decreased 10-fold, whereas the need for analytical support for those data has increased exponentially. Consequently, bioinformaticians face a second wave of challenges: novel applications of existing approaches (, single-cell RNA sequencing), integration of -omics data sets of differing size and scale (, spatial transcriptomics), as well as novel computational and statistical methods, all of which require more sophisticated pipelines and data management. Nonetheless, bioinformatics cores are often asked to operate under primarily a cost-recovery model, with limited institutional support. Seeing the need to assess bioinformatics core operations, the Association of Biomolecular Resource Facilities Genomics Bioinformatics Research Group conducted a survey to answer questions about staffing, services, financial models, and challenges to better understand the challenges bioinformatics core facilities are currently faced with and will need to address going forward. Of the respondent groups, we chose to focus on the survey data from smaller cores, which made up the majority. Although all cores indicated similar challenges in terms of changing technologies and analysis needs, small cores tended to have the added challenge of funding their operations largely through cost-recovery models with heavy administrative burdens.
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http://dx.doi.org/10.7171/jbt.20-3102-005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192196PMC
July 2020

Ten simple rules for providing effective bioinformatics research support.

PLoS Comput Biol 2020 03 26;16(3):e1007531. Epub 2020 Mar 26.

H3ABioNet, Centre for Proteomic and Genomic Research, Cape Town, South Africa.

Life scientists are increasingly turning to high-throughput sequencing technologies in their research programs, owing to the enormous potential of these methods. In a parallel manner, the number of core facilities that provide bioinformatics support are also increasing. Notably, the generation of complex large datasets has necessitated the development of bioinformatics support core facilities that aid laboratory scientists with cost-effective and efficient data management, analysis, and interpretation. In this article, we address the challenges-related to communication, good laboratory practice, and data handling-that may be encountered in core support facilities when providing bioinformatics support, drawing on our own experiences working as support bioinformaticians on multidisciplinary research projects. Most importantly, the article proposes a list of guidelines that outline how these challenges can be preemptively avoided and effectively managed to increase the value of outputs to the end user, covering the entire research project lifecycle, including experimental design, data analysis, and management (i.e., sharing and storage). In addition, we highlight the importance of clear and transparent communication, comprehensive preparation, appropriate handling of samples and data using monitoring systems, and the employment of appropriate tools and standard operating procedures to provide effective bioinformatics support.
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http://dx.doi.org/10.1371/journal.pcbi.1007531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098546PMC
March 2020

Transcriptomics in Erigeron canadensis reveals rapid photosynthetic and hormonal responses to auxin herbicide application.

J Exp Bot 2020 Jun;71(12):3701-3709

Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN, USA.

The perception pathway for endogenous auxin has been well described, yet the mode of action of synthetic auxin herbicides, used for >70 years, remains uncharacterized. We utilized transcriptomics and targeted physiological studies to investigate the unknown rapid response to synthetic auxin herbicides in the globally problematic weed species Erigeron canadensis. Synthetic auxin herbicide application consistently and rapidly down-regulated the photosynthetic machinery. At the same time, there was considerable perturbation to the expression of many genes related to phytohormone metabolism and perception. In particular, auxin herbicide application enhanced the expression of the key abscisic acid biosynthetic gene, 9-cis-epoxycarotenoid deoxygenase (NCED). The increase in NCED expression following auxin herbicide application led to a rapid biosynthesis of abscisic acid (ABA). This increase in ABA levels was independent of a loss of cell turgor or an increase in ethylene levels, both proposed triggers for rapid ABA biosynthesis. The levels of ABA in the leaf after auxin herbicide application continued to increase as plants approached death, up to >3-fold higher than in the leaves of plants that were drought stressed. We propose a new model in which synthetic auxin herbicides trigger plant death by the whole-scale, rapid, down-regulation of photosynthetic processes and an increase in ABA levels through up-regulation of NCED expression, independent of ethylene levels or a loss of cell turgor.
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http://dx.doi.org/10.1093/jxb/eraa124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307852PMC
June 2020

Multisite Evaluation of Next-Generation Methods for Small RNA Quantification.

J Biomol Tech 2020 07;31(2):47-56

MIT BioMicro Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Small RNAs (smRNAs) are important regulators of many biologic processes and are now most frequently characterized using Illumina sequencing. However, although standard RNA sequencing library preparation has become routine in most sequencing facilities, smRNA sequencing library preparation has historically been challenging because of high input requirements, laborious protocols involving gel purifications, inability to automate, and a lack of benchmarking standards. Additionally, studies have suggested that many of these methods are nonlinear and do not accurately reflect the amounts of smRNAs . Recently, a number of new kits have become available that permit lower input amounts and less laborious, gel-free protocol options. Several of these new kits claim to reduce RNA ligase-dependent sequence bias through novel adapter modifications and to lessen adapter-dimer contamination in the resulting libraries. With the increasing number of smRNA kits available, understanding the relative strengths of each method is crucial for appropriate experimental design. In this study, we systematically compared 9 commercially available smRNA library preparation kits as well as NanoString probe hybridization across multiple study sites. Although several of the new methodologies do reduce the amount of artificially over- and underrepresented microRNAs (miRNAs), we observed that none of the methods was able to remove all of the bias in the library preparation. Identical samples prepared with different methods show highly varied levels of different miRNAs. Even so, many methods excelled in ease of use, lower input requirement, fraction of usable reads, and reproducibility across sites. These differences may help users select the most appropriate methods for their specific question of interest.
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http://dx.doi.org/10.7171/jbt.20-3102-001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953595PMC
July 2020

Expression profiles of cell-wall related genes vary broadly between two common maize inbreds during stem development.

BMC Genomics 2019 Oct 29;20(1):785. Epub 2019 Oct 29.

Department of Botany & Plant Pathology, Purdue University, 915 West State Street, West Lafayette, IN, 47907, USA.

Background: The cellular machinery for cell wall synthesis and metabolism is encoded by members of large multi-gene families. Maize is both a genetic model for grass species and a potential source of lignocellulosic biomass from crop residues. Genetic improvement of maize for its utility as a bioenergy feedstock depends on identification of the specific gene family members expressed during secondary wall development in stems.

Results: High-throughput sequencing of transcripts expressed in developing rind tissues of stem internodes provided a comprehensive inventory of cell wall-related genes in maize (Zea mays, cultivar B73). Of 1239 of these genes, 854 were expressed among the internodes at ≥95 reads per 20 M, and 693 of them at ≥500 reads per 20 M. Grasses have cell wall compositions distinct from non-commelinid species; only one-quarter of maize cell wall-related genes expressed in stems were putatively orthologous with those of the eudicot Arabidopsis. Using a slope-metric algorithm, five distinct patterns for sub-sets of co-expressed genes were defined across a time course of stem development. For the subset of genes associated with secondary wall formation, fifteen sequence motifs were found in promoter regions. The same members of gene families were often expressed in two maize inbreds, B73 and Mo17, but levels of gene expression between them varied, with 30% of all genes exhibiting at least a 5-fold difference at any stage. Although presence-absence and copy-number variation might account for much of these differences, fold-changes of expression of a CADa and a FLA11 gene were attributed to polymorphisms in promoter response elements.

Conclusions: Large genetic variation in maize as a species precludes the extrapolation of cell wall-related gene expression networks even from one common inbred line to another. Elucidation of genotype-specific expression patterns and their regulatory controls will be needed for association panels of inbreds and landraces to fully exploit genetic variation in maize and other bioenergy grass species.
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http://dx.doi.org/10.1186/s12864-019-6117-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819468PMC
October 2019

Genome-wide identification of histone methylation (H3K9) and acetylation (H4K12) marks in two ecotypes of switchgrass (Panicum virgatum L.).

BMC Genomics 2019 Aug 22;20(1):667. Epub 2019 Aug 22.

Molecular Genetics and Epigenomics Laboratory, College of Agriculture and Related Sciences, Delaware State University, Dover, DE, USA.

Background: Histone modifications play a significant role in the regulation of transcription and various biological processes, such as development and regeneration. Though a few genomic (including DNA methylation patterns) and transcriptomic studies are currently available in switchgrass, the genome-wide distribution of histone modifications has not yet been studied to help elucidate gene regulation and its application to switchgrass improvement.

Results: This study provides a comprehensive epigenomic analyses of two contrasting switchgrass ecotypes, lowland (AP13) and upland (VS16), by employing chromatin immunoprecipitation sequencing (ChIP-Seq) with two histone marks (suppressive- H3K9 and active- H4K12). In this study, most of the histone binding was in non-genic regions, and the highest enrichment was seen between 0 and 2 kb regions from the transcriptional start site (TSS). Considering the economic importance and potential of switchgrass as a bioenergy crop, we focused on genes, transcription factors (TFs), and pathways that were associated with C4-photosynthesis, biomass, biofuel production, biotic stresses, and abiotic stresses. Using quantitative real-time PCR (qPCR) the relative expression of five genes selected from the phenylpropanoid-monolignol pathway showed preferential binding of acetylation marks in AP13 rather than in VS16.

Conclusions: The genome-wide histone modifications reported here can be utilized in understanding the regulation of genes important in the phenylpropanoid-monolignol biosynthesis pathway, which in turn, may help understand the recalcitrance associated with conversion of biomass to biofuel, a major roadblock in utilizing lignocellulosic feedstocks.
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http://dx.doi.org/10.1186/s12864-019-6038-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704705PMC
August 2019

The origin and biosynthesis of the naphthalenoid moiety of juglone in black walnut.

Hortic Res 2018 1;5:67. Epub 2018 Nov 1.

1Department of Horticulture and Landscape Architecture, Purdue University, 625 Agriculture Mall Drive, West Lafayette, IN 47907 USA.

Several members of the family produce juglone, a specialized 1,4-naphthoquinone (1,4-NQ) natural product that is responsible for the notorious allelopathic effects of black walnut (). Despite its documented ecological roles and potential for being developed as a novel natural product-based herbicide, none of the genes involved in synthesizing juglone have been identified. Based on classical labeling studies, we hypothesized that biosynthesis of juglone's naphthalenoid moiety is shared with biochemical steps of the phylloquinone pathway. Here, using comparative transcriptomics in combination with targeted metabolic profiling of 1,4-NQs in various black walnut organs, we provide evidence that phylloquinone pathway genes involved in 1,4-dihydroxynaphthoic acid (DHNA) formation are expressed in roots for synthesis of a compound other than phylloquinone. Feeding experiments using axenic black walnut root cultures revealed that stable isotopically labeled l-glutamate incorporates into juglone resulting in the same mass shift as that expected for labeling of the quinone ring in phylloquinone. Taken together, these results indicate that , an intermediate from the phylloquinone pathway provides the naphthalenoid moiety of juglone. Moreover, this work shows that juglone can be synthesized in roots without the contribution of immediate precursors translocated from aerial tissues. The present study illuminates all genes involved in synthesizing the juglone naphthoquinone ring and provides RNA-sequencing datasets that can be used with functional screening studies to elucidate the remaining juglone pathway genes. Translation of the generated knowledge is expected to inform future metabolic engineering strategies for harnessing juglone as a novel natural product-based herbicide.
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http://dx.doi.org/10.1038/s41438-018-0067-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210188PMC
November 2018

Meeting the challenge of tick-borne disease control: A proposal for 1000 Ixodes genomes.

Ticks Tick Borne Dis 2019 01 24;10(1):213-218. Epub 2018 Aug 24.

Department of Entomology, Purdue University, West Lafayette, IN, United States; Purdue Institute for Inflammation, Immunology and Infectious Diseases, Purdue University, West Lafayette, IN, United States. Electronic address:

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http://dx.doi.org/10.1016/j.ttbdis.2018.08.009DOI Listing
January 2019

Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction.

BMC Genomics 2018 Mar 15;19(1):199. Epub 2018 Mar 15.

MIT BioMicro Center, Massachusetts Institute of Technology, Cambridge, MA, USA.

Background: Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE and also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples.

Results: We find that all of the kits are capable of performing significant ribosomal depletion, though there are differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify ~ 14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes between kits suggests that transcript length may be a key factor in library production efficiency.

Conclusions: These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them.
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http://dx.doi.org/10.1186/s12864-018-4585-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389247PMC
March 2018

Analyses of methylomes of upland and lowland switchgrass (Panicum virgatum) ecotypes using MeDIP-seq and BS-seq.

BMC Genomics 2017 Nov 7;18(1):851. Epub 2017 Nov 7.

Molecular Genetics and Epigenomics Laboratory, Delaware State University, Dover, DE, 19901, USA.

Background: Switchgrass is a crop with many desirable traits for bioenergy production. Plant genomes have high DNA methylation levels throughout genes and transposable elements and DNA methylation is known to play a role in silencing transposable elements. Here we analyzed methylomes in two switchgrass genotypes AP13 and VS16. AP13 is derived from a lowland ecotype and VS16, typically considered drought-tolerant, is derived from an upland ecotype, both genotypes are tetraploid (2n = 4× = 36).

Results: Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and bisulfite-sequencing (BS-seq) were used to profile DNA methylation in genomic features of AP13 and VS16. The methylation patterns in genes and transposable elements were similar to other plants, however, overall CHH methylation levels were comparatively low. Differentially methylated regions (DMRs) were assessed and a total of 1777 CG-DMRs, 573 CHG-DMRs, and 3 CHH-DMRs were detected between the two genotypes. TEs and their flanking regions were higher than that of genic regions. Different types of TEs had different methylation patterns, but the two LTRs (Copia and Gypsy) were similarly methylated, while LINEs and DNA transposons typically had different methylation patterns. MeDIP-seq data was compared to BS-seq data and most of the peaks generated by MeDIP-seq were confirmed to be highly methylated by BS-seq.

Conclusions: DNA methylation in switchgrass genotypes obtained from the two ecotypes were found similar. Collinear gene pairs in two subgenomes (A and B) were not significantly differentially methylated. Both BS-seq and MeDIP-seq methodologies were found effective. Methylation levels were highest at CG and least in CHH. Increased DNA methylation was seen in TEs compared to genic regions. Exploitation of TE methylations can be a viable option in future crop improvement.
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http://dx.doi.org/10.1186/s12864-017-4218-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678558PMC
November 2017

ViewBS: a powerful toolkit for visualization of high-throughput bisulfite sequencing data.

Bioinformatics 2018 02;34(4):708-709

Bioinformatics Core, Purdue University, 155 South Grant Street, West Lafayette, Indiana 47907, USA.

Motivation: High throughput bisulfite sequencing (BS-seq) is an important technology to generate single-base DNA methylomes in both plants and animals. In order to accelerate the data analysis of BS-seq data, toolkits for visualization are required.

Results: ViewBS, an open-source toolkit, can extract and visualize the DNA methylome data easily and with flexibility. By using Tabix, ViewBS can visualize BS-seq for large datasets quickly. ViewBS can generate publication-quality figures, such as meta-plots, heat maps and violin-boxplots, which can help users to answer biological questions. We illustrate its application using BS-seq data from Arabidopsis thaliana.

Availability: ViewBS is freely available at: https://github.com/xie186/ViewBS.

Contact: xie186@purdue.edu.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btx633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5860610PMC
February 2018

Comparative analysis of the root transcriptomes of cultivated sweetpotato (Ipomoea batatas [L.] Lam) and its wild ancestor (Ipomoea trifida [Kunth] G. Don).

BMC Plant Biol 2017 01 13;17(1). Epub 2017 Jan 13.

Department of Agriculture, University of Arkansas at Pine Bluff, Pine Bluff, Arkansas, USA.

Background: The complex process of formation of storage roots (SRs) from adventitious roots affects sweetpotato yield. Identifying the genes that are uniquely expressed in the SR forming cultivated species, Ipomoea batatas (Ib), and its immediate ancestral species, Ipomoea trifida (It), which does not form SRs, may provide insights into the molecular mechanisms underlying SR formation in sweetpotato.

Results: Illumina paired-end sequencing generated ~208 and ~200 million reads for Ib and It, respectively. Trinity assembly of the reads resulted in 98,317 transcripts for Ib and 275,044 for It, after post-assembly removal of trans-chimeras. From these sequences, we identified 4,865 orthologous genes in both Ib and It, 60 paralogous genes in Ib and 2,286 paralogous genes in It. Among paralogous gene sets, transcripts encoding the transcription factor RKD, which may have a role in nitrogen regulation and starch formation, and rhamnogalacturonate lyase (RGL) family proteins, which produce the precursors of cell wall polysaccharides, were found only in Ib. In addition, transcripts encoding a K efflux antiporter (KEA5) and the ERECTA protein kinase, which function in phytohormonal regulation and root proliferation, respectively, were also found only in Ib. qRT-PCR indicated that starch and sucrose metabolism genes, such as those encoding ADP-glucose pyrophosphorylase and beta-amylase, showed lower expression in It than Ib, whereas lignin genes such as caffeoyl-CoA O-methyltransferase (CoMT) and cinnamyl alcohol dehydrogenase (CAD) showed higher expression in It than Ib. A total of 7,067 and 9,650 unique microsatellite markers, 1,037,396 and 495,931 single nucleotide polymorphisms (SNPs) and 103,439 and 69,194 InDels in Ib and It, respectively, were also identified from this study.

Conclusion: The detection of genes involved in the biosynthesis of RGL family proteins, the transcription factor RKD, and genes encoding a K efflux antiporter (KEA5) and the ERECTA protein kinase only in I. batatas indicate that these genes may have important functions in SR formation in sweetpotato. Potential molecular markers (SNPs, simple sequence repeats and InDels) and sequences identified in this study may represent a valuable resource for sweetpotato gene annotation and may serve as important tools for improving SR formation in sweetpotato through breeding.
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http://dx.doi.org/10.1186/s12870-016-0950-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5234144PMC
January 2017

Comparative transcriptome profiling of upland (VS16) and lowland (AP13) ecotypes of switchgrass.

Plant Cell Rep 2017 Jan 3;36(1):129-150. Epub 2016 Nov 3.

Molecular Genetics and Epigenomics Laboratory, College of Agriculture and Related Sciences, Delaware State University, Dover, DE, USA.

Key Message: Transcriptomes of two switchgrass genotypes representing the upland and lowland ecotypes will be key tools in switchgrass genome annotation and biotic and abiotic stress functional genomics. Switchgrass (Panicum virgatum L.) is an important bioenergy feedstock for cellulosic ethanol production. We report genome-wide transcriptome profiling of two contrasting tetraploid switchgrass genotypes, VS16 and AP13, representing the upland and lowland ecotypes, respectively. A total of 268 million Illumina short reads (50 nt) were generated, of which, 133 million were obtained in AP13 and the rest 135 million in VS16. More than 90% of these reads were mapped to the switchgrass reference genome (V1.1). We identified 6619 and 5369 differentially expressed genes in VS16 and AP13, respectively. Gene ontology and KEGG pathway analysis identified key genes that regulate important pathways including C4 photosynthesis, photorespiration and phenylpropanoid metabolism. A series of genes (33) involved in photosynthetic pathway were up-regulated in AP13 but only two genes showed higher expression in VS16. We identified three dicarboxylate transporter homologs that were highly expressed in AP13. Additionally, genes that mediate drought, heat, and salinity tolerance were also identified. Vesicular transport proteins, syntaxin and signal recognition particles were seen to be up-regulated in VS16. Analyses of selected genes involved in biosynthesis of secondary metabolites, plant-pathogen interaction, membrane transporters, heat, drought and salinity stress responses confirmed significant variation in the relative expression reflected in RNA-Seq data between VS16 and AP13 genotypes. The phenylpropanoid pathway genes identified here are potential targets for biofuel conversion.
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http://dx.doi.org/10.1007/s00299-016-2065-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206262PMC
January 2017

The impact of orally administered phages on host immune response and surrounding microbial communities.

Bacteriophage 2016 Jul-Sep;6(3):e1211066. Epub 2016 Jul 13.

Department of Animal Sciences, Purdue University , West Lafayette, IN, USA.

Numerous studies have shown the efficacy of phage therapy in reducing foodborne pathogen carriage in food animals. Fewer studies have focused on host reactions, especially in terms of phage-mediated acute immune responses and effects on the gut microbiome. Here we administered O157:H7 phages in low (single dose of 10 PFU) or high (single dose of 10 PFU) quantities to mice. While there were time points at which cytokine levels in different treatment groups differed from one another, all cytokine levels remained within normal ranges for mice regardless of treatment. Similarly, the patterns of these differences were not dose related, indicating that phage treatment did not result in a strong acute immune response as measured here. In separate experiments, 3-week-old pigs received a diet containing an in-feed antibiotic or daily phage treatment. After two weeks, microbial DNA of ileal, cecal, and fecal contents was characterized using 16S rRNA sequencing. There were no statistical differences in performance among the different groups. Compared to control pigs (no antibiotic, no phage), antibiotic treatment significantly altered ileal microbiome composition (P < 0.05), with being most affected (antibiotic treated: 22%; control: 76%; FDR = 0.0572). No significant differences were observed in cecal and fecal microbiome composition between antibiotic-treated and control pigs, and there were no differences in gut microbiome composition between phage treated and control pigs in any intestinal compartment. Significant abundance differences were observed at the OTU level, with OTUs belonging to genera such as and being over- or under-represented in either antibiotic or phage treated groups compared to control pigs. Determining whether these changes are deleterious to host, however, requires further study.
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http://dx.doi.org/10.1080/21597081.2016.1211066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056770PMC
July 2016

Genomic insights into the Ixodes scapularis tick vector of Lyme disease.

Authors:
Monika Gulia-Nuss Andrew B Nuss Jason M Meyer Daniel E Sonenshine R Michael Roe Robert M Waterhouse David B Sattelle José de la Fuente Jose M Ribeiro Karine Megy Jyothi Thimmapuram Jason R Miller Brian P Walenz Sergey Koren Jessica B Hostetler Mathangi Thiagarajan Vinita S Joardar Linda I Hannick Shelby Bidwell Martin P Hammond Sarah Young Qiandong Zeng Jenica L Abrudan Francisca C Almeida Nieves Ayllón Ketaki Bhide Brooke W Bissinger Elena Bonzon-Kulichenko Steven D Buckingham Daniel R Caffrey Melissa J Caimano Vincent Croset Timothy Driscoll Don Gilbert Joseph J Gillespie Gloria I Giraldo-Calderón Jeffrey M Grabowski David Jiang Sayed M S Khalil Donghun Kim Katherine M Kocan Juraj Koči Richard J Kuhn Timothy J Kurtti Kristin Lees Emma G Lang Ryan C Kennedy Hyeogsun Kwon Rushika Perera Yumin Qi Justin D Radolf Joyce M Sakamoto Alejandro Sánchez-Gracia Maiara S Severo Neal Silverman Ladislav Šimo Marta Tojo Cristian Tornador Janice P Van Zee Jesús Vázquez Filipe G Vieira Margarita Villar Adam R Wespiser Yunlong Yang Jiwei Zhu Peter Arensburger Patricia V Pietrantonio Stephen C Barker Renfu Shao Evgeny M Zdobnov Frank Hauser Cornelis J P Grimmelikhuijzen Yoonseong Park Julio Rozas Richard Benton Joao H F Pedra David R Nelson Maria F Unger Jose M C Tubio Zhijian Tu Hugh M Robertson Martin Shumway Granger Sutton Jennifer R Wortman Daniel Lawson Stephen K Wikel Vishvanath M Nene Claire M Fraser Frank H Collins Bruce Birren Karen E Nelson Elisabet Caler Catherine A Hill

Nat Commun 2016 Feb 9;7:10507. Epub 2016 Feb 9.

Department of Entomology, Purdue University, West Lafayette, Indiana 47907, USA.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.
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http://dx.doi.org/10.1038/ncomms10507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4748124PMC
February 2016

A Study of Alterations in DNA Epigenetic Modifications (5mC and 5hmC) and Gene Expression Influenced by Simulated Microgravity in Human Lymphoblastoid Cells.

PLoS One 2016 28;11(1):e0147514. Epub 2016 Jan 28.

Bindley Biosciences Center, Discovery Park, Purdue University, West Lafayette IN, 47907, United States of America.

Cells alter their gene expression in response to exposure to various environmental changes. Epigenetic mechanisms such as DNA methylation are believed to regulate the alterations in gene expression patterns. In vitro and in vivo studies have documented changes in cellular proliferation, cytoskeletal remodeling, signal transduction, bone mineralization and immune deficiency under the influence of microgravity conditions experienced in space. However microgravity induced changes in the epigenome have not been well characterized. In this study we have used Next-generation Sequencing (NGS) to profile ground-based "simulated" microgravity induced changes on DNA methylation (5-methylcytosine or 5mC), hydroxymethylation (5-hydroxymethylcytosine or 5hmC), and simultaneous gene expression in cultured human lymphoblastoid cells. Our results indicate that simulated microgravity induced alterations in the methylome (~60% of the differentially methylated regions or DMRs are hypomethylated and ~92% of the differentially hydroxymethylated regions or DHMRs are hyperhydroxymethylated). Simulated microgravity also induced differential expression in 370 transcripts that were associated with crucial biological processes such as oxidative stress response, carbohydrate metabolism and regulation of transcription. While we were not able to obtain any global trend correlating the changes of methylation/ hydroxylation with gene expression, we have been able to profile the simulated microgravity induced changes of 5mC over some of the differentially expressed genes that includes five genes undergoing differential methylation over their promoters and twenty five genes undergoing differential methylation over their gene-bodies. To the best of our knowledge, this is the first NGS-based study to profile epigenomic patterns induced by short time exposure of simulated microgravity and we believe that our findings can be a valuable resource for future explorations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147514PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731572PMC
July 2016

Identification of a plastidial phenylalanine exporter that influences flux distribution through the phenylalanine biosynthetic network.

Nat Commun 2015 Sep 10;6:8142. Epub 2015 Sep 10.

Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, Indiana 47907-2063, USA.

In addition to proteins, L-phenylalanine is a versatile precursor for thousands of plant metabolites. Production of phenylalanine-derived compounds is a complex multi-compartmental process using phenylalanine synthesized predominantly in plastids as precursor. The transporter(s) exporting phenylalanine from plastids, however, remains unknown. Here, a gene encoding a Petunia hybrida plastidial cationic amino-acid transporter (PhpCAT) functioning in plastidial phenylalanine export is identified based on homology to an Escherichia coli phenylalanine transporter and co-expression with phenylalanine metabolic genes. Radiolabel transport assays show that PhpCAT exports all three aromatic amino acids. PhpCAT downregulation and overexpression result in decreased and increased levels, respectively, of phenylalanine-derived volatiles, as well as phenylalanine, tyrosine and their biosynthetic intermediates. Metabolic flux analysis reveals that flux through the plastidial phenylalanine biosynthetic pathway is reduced in PhpCAT RNAi lines, suggesting that the rate of phenylalanine export from plastids contributes to regulating flux through the aromatic amino-acid network.
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http://dx.doi.org/10.1038/ncomms9142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4647861PMC
September 2015

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

PLoS One 2015 13;10(7):e0132176. Epub 2015 Jul 13.

Sequencing and Genotyping Center, Delaware Biotechnology Institute, Newark, Delaware, United States of America.

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132176PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4500563PMC
April 2016

Metatranscriptomic profiles of Eastern subterranean termites, Reticulitermes flavipes (Kollar) fed on second generation feedstocks.

BMC Genomics 2015 Apr 22;16:332. Epub 2015 Apr 22.

Department of Entomology, Purdue University, West Lafayette, 47907-2089, Indiana.

Background: Second generation lignocellulosic feedstocks are being considered as an alternative to first generation biofuels that are derived from grain starches and sugars. However, the current pre-treatment methods for second generation biofuel production are inefficient and expensive due to the recalcitrant nature of lignocellulose. In this study, we used the lower termite Reticulitermes flavipes (Kollar), as a model to identify potential pretreatment genes/enzymes specifically adapted for use against agricultural feedstocks.

Results: Metatranscriptomic profiling was performed on worker termite guts after feeding on corn stover (CS), soybean residue (SR), or 98% pure cellulose (paper) to identify (i) microbial community, (ii) pathway level and (iii) gene-level responses. Microbial community profiles after CS and SR feeding were different from the paper feeding profile, and protist symbiont abundance decreased significantly in termites feeding on SR and CS relative to paper. Functional profiles after CS feeding were similar to paper and SR; whereas paper and SR showed different profiles. Amino acid and carbohydrate metabolism pathways were downregulated in termites feeding on SR relative to paper and CS. Gene expression analyses showed more significant down regulation of genes after SR feeding relative to paper and CS. Stereotypical lignocellulase genes/enzymes were not differentially expressed, but rather were among the most abundant/constitutively-expressed genes.

Conclusions: These results suggest that the effect of CS and SR feeding on termite gut lignocellulase composition is minimal and thus, the most abundantly expressed enzymes appear to encode the best candidate catalysts for use in saccharification of these and related second-generation feedstocks. Further, based on these findings we hypothesize that the most abundantly expressed lignocellulases, rather than those that are differentially expressed have the best potential as pretreatment enzymes for CS and SR feedstocks.
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http://dx.doi.org/10.1186/s12864-015-1502-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4411656PMC
April 2015

Coordination of MicroRNAs, PhasiRNAs, and NB-LRR Genes in Response to a Plant Pathogen: Insights from Analyses of a Set of Soybean Rps Gene Near-Isogenic Lines.

Plant Genome 2015 Mar;8(1):eplantgenome2014.09.0044

Dep. of Agronomy, Purdue Univ., West Lafayette, IN, 47907.

Disease-related genes, particularly the nucleotide binding site (NB)-leucine-rich repeat (LRR) class of R plant genes can be triggered by microRNAs (miRNAs) to generate phased small interfering RNAs (phasiRNAs), which could reduce the transcript levels of their targets. However, how global changes in NB-LRR transcript levels coordinate with changes in miRNA and phasiRNA levels in defense responses remains largely unknown. Here, we investigated changes in the relative abundance of small RNAs (sRNAs), with a focus on miRNAs and phasiRNAs and their potential targets in response to the pathogen Phytophthora sojae in the susceptible soybean [Glycine max (L.) Merr.] 'Williams' and nine resistant near-isogenic lines (NILs), each carrying a unique resistance to P. sojae (Rps) gene. In total, 369 distinct miRNAs, including 78 new ones, were identified in the 10 soybean lines. The majority of miRNAs were downregulated by the pathogen. Of the 525 NB-LRR genes found in the soybean reference genome, 257 were predicted to be the targets of eight abundant miRNA families and 126 (dubbed phasi-NB-LRRs or pNLs) were predicted to have produced phasiRNAs. Upregulation of 15 phasi-NB-LRRs was associated with downregulation of their corresponding phasiRNAs in the NILs; these phasiRNAs were predicted to regulate 75 additional NB-LRRs in trans. In addition, we identified putative 24-nucleotide (nt) phasiRNAs from transposons, possibly representing a novel general epigenetic mechanism for regulation of transposon activity under biotic stresses. Together, these observations suggest that miRNAs and phasiRNAs play an important role in response to plant pathogens through complex, multiple layers of post-transcriptional regulation.
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http://dx.doi.org/10.3835/plantgenome2014.09.0044DOI Listing
March 2015

Transcriptomic and proteomic dynamics in the metabolism of a diazotrophic cyanobacterium, Cyanothece sp. PCC 7822 during a diurnal light-dark cycle.

BMC Genomics 2014 Dec 29;15:1185. Epub 2014 Dec 29.

Department of Biological Sciences, Purdue University, West Lafayette, IN, USA.

Background: Cyanothece sp. PCC 7822 is an excellent cyanobacterial model organism with great potential to be applied as a biocatalyst for the production of high value compounds. Like other unicellular diazotrophic cyanobacterial species, it has a tightly regulated metabolism synchronized to the light-dark cycle. Utilizing transcriptomic and proteomic methods, we quantified the relationships between transcription and translation underlying central and secondary metabolism in response to nitrogen free, 12 hour light and 12 hour dark conditions.

Results: By combining mass-spectrometry based proteomics and RNA-sequencing transcriptomics, we quantitatively measured a total of 6766 mRNAs and 1322 proteins at four time points across a 24 hour light-dark cycle. Photosynthesis, nitrogen fixation, and carbon storage relevant genes were expressed during the preceding light or dark period, concurrent with measured nitrogenase activity in the late light period. We describe many instances of disparity in peak mRNA and protein abundances, and strong correlation of light dependent expression of both antisense and CRISPR-related gene expression. The proteins for nitrogenase and the pentose phosphate pathway were highest in the dark, whereas those for glycolysis and the TCA cycle were more prominent in the light. Interestingly, one copy of the psbA gene encoding the photosystem II (PSII) reaction center protein D1 (psbA4) was highly upregulated only in the dark. This protein likely cannot catalyze O2 evolution and so may be used by the cell to keep PSII intact during N2 fixation. The CRISPR elements were found exclusively at the ends of the large plasmid and we speculate that their presence is crucial to the maintenance of this plasmid.

Conclusions: This investigation of parallel transcriptional and translational activity within Cyanothece sp. PCC 7822 provided quantitative information on expression levels of metabolic pathways relevant to engineering efforts. The identification of expression patterns for both mRNA and protein affords a basis for improving biofuel production in this strain and for further genetic manipulations. Expression analysis of the genes encoded on the 6 plasmids provided insight into the possible acquisition and maintenance of some of these extra-chromosomal elements.
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http://dx.doi.org/10.1186/1471-2164-15-1185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320622PMC
December 2014

CYCLIN-DEPENDENT KINASE8 differentially regulates plant immunity to fungal pathogens through kinase-dependent and -independent functions in Arabidopsis.

Plant Cell 2014 Oct 3;26(10):4149-70. Epub 2014 Oct 3.

Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907

CYCLIN-DEPENDENT KINASE8 (CDK8) is a widely studied component of eukaryotic Mediator complexes. However, the biological and molecular functions of plant CDK8 are not well understood. Here, we provide evidence for regulatory functions of Arabidopsis thaliana CDK8 in defense and demonstrate its functional and molecular interactions with other Mediator and non-Mediator subunits. The cdk8 mutant exhibits enhanced resistance to Botrytis cinerea but susceptibility to Alternaria brassicicola. The contributions of CDK8 to the transcriptional activation of defensin gene PDF1.2 and its interaction with MEDIATOR COMPLEX SUBUNIT25 (MED25) implicate CDK8 in jasmonate-mediated defense. Moreover, CDK8 associates with the promoter of AGMATINE COUMAROYLTRANSFERASE to promote its transcription and regulate the biosynthesis of the defense-active secondary metabolites hydroxycinnamic acid amides. CDK8 also interacts with the transcription factor WAX INDUCER1, implying its additional role in cuticle development. In addition, overlapping functions of CDK8 with MED12 and MED13 and interactions between CDK8 and C-type cyclins suggest the conserved configuration of the plant Mediator kinase module. In summary, while CDK8's positive transcriptional regulation of target genes and its phosphorylation activities underpin its defense functions, the impaired defense responses in the mutant are masked by its altered cuticle, resulting in specific resistance to B. cinerea.
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http://dx.doi.org/10.1105/tpc.114.128611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4247566PMC
October 2014

Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population.

Plant Physiol 2014 Aug 27;165(4):1475-1487. Epub 2014 Jun 27.

Departments of Biological Sciences (B.W.P., M.C.M., N.C.C.), Botany and Plant Pathology (C.K.D., M.A.H., J.F.K., N.C.C.), and Agronomy (N.C.B., C.F.W.), Laboratory of Renewable Resources Engineering and Agricultural and Biological Engineering (N.S.M.), and Bioinformatics Core (J.T.S., J.T.), Purdue University, West Lafayette, Indiana 47907;National Bioenergy Center, National Renewable Energy Laboratory, Golden, Colorado 80401 (R.W.S., M.F., A.Z., M.F.D., S.R.D., G.B.T.); andDepartment of Plant Biology, University of Minnesota, St. Paul, Minnesota 55108 (N.M.S.)

Biotechnological approaches to reduce or modify lignin in biomass crops are predicated on the assumption that it is the principal determinant of the recalcitrance of biomass to enzymatic digestion for biofuels production. We defined quantitative trait loci (QTL) in the Intermated B73 × Mo17 recombinant inbred maize (Zea mays) population using pyrolysis molecular-beam mass spectrometry to establish stem lignin content and an enzymatic hydrolysis assay to measure glucose and xylose yield. Among five multiyear QTL for lignin abundance, two for 4-vinylphenol abundance, and four for glucose and/or xylose yield, not a single QTL for aromatic abundance and sugar yield was shared. A genome-wide association study for lignin abundance and sugar yield of the 282-member maize association panel provided candidate genes in the 11 QTL of the B73 and Mo17 parents but showed that many other alleles impacting these traits exist among this broader pool of maize genetic diversity. B73 and Mo17 genotypes exhibited large differences in gene expression in developing stem tissues independent of allelic variation. Combining these complementary genetic approaches provides a narrowed list of candidate genes. A cluster of SCARECROW-LIKE9 and SCARECROW-LIKE14 transcription factor genes provides exceptionally strong candidate genes emerging from the genome-wide association study. In addition to these and genes associated with cell wall metabolism, candidates include several other transcription factors associated with vascularization and fiber formation and components of cellular signaling pathways. These results provide new insights and strategies beyond the modification of lignin to enhance yields of biofuels from genetically modified biomass.
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http://dx.doi.org/10.1104/pp.114.242446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4119032PMC
August 2014

Draft Genome Sequence of Acetobacter aceti Strain 1023, a Vinegar Factory Isolate.

Genome Announc 2014 Jun 5;2(3). Epub 2014 Jun 5.

Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA

The genome sequence of Acetobacter aceti 1023, an acetic acid bacterium adapted to traditional vinegar fermentation, comprises 3.0 Mb (chromosome plus plasmids). A. aceti 1023 is closely related to the cocoa fermenter Acetobacter pasteurianus 386B but possesses many additional insertion sequence elements.
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http://dx.doi.org/10.1128/genomeA.00550-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047455PMC
June 2014

MED18 interaction with distinct transcription factors regulates multiple plant functions.

Nat Commun 2014 ;5:3064

Department of Botany and Plant Pathology, Purdue University, 915 W State Street, West Lafayette, Indiana 47907, USA.

Mediator is an evolutionarily conserved transcriptional regulatory complex. Mechanisms of Mediator function are poorly understood. Here we show that Arabidopsis MED18 is a multifunctional protein regulating plant immunity, flowering time and responses to hormones through interactions with distinct transcription factors. MED18 interacts with YIN YANG1 to suppress disease susceptibility genes glutaredoxins GRX480, GRXS13 and thioredoxin TRX-h5. Consequently, yy1 and med18 mutants exhibit deregulated expression of these genes and enhanced susceptibility to fungal infection. In addition, MED18 interacts with ABA INSENSITIVE 4 and SUPPRESSOR OF FRIGIDA4 to regulate abscisic acid responses and flowering time, respectively. MED18 associates with the promoter, coding and terminator regions of target genes suggesting its function in transcription initiation, elongation and termination. Notably, RNA polymerase II occupancy and histone H3 lysine tri-methylation of target genes are affected in the med18 mutant, reinforcing MED18 function in different mechanisms of transcriptional control. Overall, MED18 conveys distinct cues to engender transcription underpinning plant responses.
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http://dx.doi.org/10.1038/ncomms4064DOI Listing
November 2015

The bench scientist's guide to statistical analysis of RNA-Seq data.

BMC Res Notes 2012 Sep 14;5:506. Epub 2012 Sep 14.

USDA ARS Global Change and Photosynthesis Research Unit, 1201 W. Gregory Drive, Urbana, IL 61801, USA.

Background: RNA sequencing (RNA-Seq) is emerging as a highly accurate method to quantify transcript abundance. However, analyses of the large data sets obtained by sequencing the entire transcriptome of organisms have generally been performed by bioinformatics specialists. Here we provide a step-by-step guide and outline a strategy using currently available statistical tools that results in a conservative list of differentially expressed genes. We also discuss potential sources of error in RNA-Seq analysis that could alter interpretation of global changes in gene expression.

Findings: When comparing statistical tools, the negative binomial distribution-based methods, edgeR and DESeq, respectively identified 11,995 and 11,317 differentially expressed genes from an RNA-seq dataset generated from soybean leaf tissue grown in elevated O3. However, the number of genes in common between these two methods was only 10,535, resulting in 2,242 genes determined to be differentially expressed by only one method. Upon analysis of the non-significant genes, several limitations of these analytic tools were revealed, including evidence for overly stringent parameters for determining statistical significance of differentially expressed genes as well as increased type II error for high abundance transcripts.

Conclusions: Because of the high variability between methods for determining differential expression of RNA-Seq data, we suggest using several bioinformatics tools, as outlined here, to ensure that a conservative list of differentially expressed genes is obtained. We also conclude that despite these analytical limitations, RNA-Seq provides highly accurate transcript abundance quantification that is comparable to qRT-PCR.
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http://dx.doi.org/10.1186/1756-0500-5-506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522531PMC
September 2012

Auxin and ABA act as central regulators of developmental networks associated with paradormancy in Canada thistle (Cirsium arvense).

Funct Integr Genomics 2012 Aug 13;12(3):515-31. Epub 2012 May 13.

USDA-Agricultural Research Service, Biosciences Research Laboratory, U.S. Department of Agriculture, 1605 Albrecht Blvd., Fargo, ND 58102-2765, USA.

Dormancy in underground vegetative buds of Canada thistle, an herbaceous perennial weed, allows escape from current control methods and contributes to its invasive nature. In this study, ~65 % of root sections obtained from greenhouse propagated Canada thistle produced new vegetative shoots by 14 days post-sectioning. RNA samples obtained from sectioned roots incubated 0, 24, 48, and 72 h at 25°C under 16:8 h light-dark conditions were used to construct four MID-tagged cDNA libraries. Analysis of in silico data obtained using Roche 454 GS-FLX pyrosequencing technologies identified molecular networks associated with paradormancy release in underground vegetative buds of Canada thistle. Sequencing of two replicate plates produced ~2.5 million ESTs with an average read length of 362 bases. These ESTs assembled into 67358 unique sequences (21777 contigs and 45581 singlets) and annotation against the Arabidopsis database identified 15232 unigenes. Among the 15232 unigenes, we identified processes enriched with transcripts involved in plant hormone signaling networks. To follow-up on these results, we examined hormone profiles in roots, which identified changes in abscisic acid (ABA) and ABA metabolites, auxins, and cytokinins post-sectioning. Transcriptome and hormone profiling data suggest that interaction between auxin- and ABA-signaling regulate paradormancy maintenance and release in underground adventitious buds of Canada thistle. Our proposed model shows that sectioning-induced changes in polar auxin transport alters ABA metabolism and signaling, which further impacts gibberellic acid signaling involving interactions between ABA and FUSCA3. Here we report that reduced auxin and ABA-signaling, in conjunction with increased cytokinin biosynthesis post-sectioning supports a model where interactions among hormones drives molecular networks leading to cell division, differentiation, and vegetative outgrowth.
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http://dx.doi.org/10.1007/s10142-012-0280-5DOI Listing
August 2012

Whole-genome resequencing of two elite sires for the detection of haplotypes under selection in dairy cattle.

Proc Natl Acad Sci U S A 2012 May 23;109(20):7693-8. Epub 2012 Apr 23.

Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

Using a combination of whole-genome resequencing and high-density genotyping arrays, genome-wide haplotypes were reconstructed for two of the most important bulls in the history of the dairy cattle industry, Pawnee Farm Arlinda Chief ("Chief") and his son Walkway Chief Mark ("Mark"), each accounting for ∼7% of all current genomes. We aligned 20.5 Gbp (∼7.3× coverage) and 37.9 Gbp (∼13.5× coverage) of the Chief and Mark genomic sequences, respectively. More than 1.3 million high-quality SNPs were detected in Chief and Mark sequences. The genome-wide haplotypes inherited by Mark from Chief were reconstructed using ∼1 million informative SNPs. Comparison of a set of 15,826 SNPs that overlapped in the sequence-based and BovineSNP50 SNPs showed the accuracy of the sequence-based haplotype reconstruction to be as high as 97%. By using the BovineSNP50 genotypes, the frequencies of Chief alleles on his two haplotypes then were determined in 1,149 of his descendants, and the distribution was compared with the frequencies that would be expected assuming no selection. We identified 49 chromosomal segments in which Chief alleles showed strong evidence of selection. Candidate polymorphisms for traits that have been under selection in the dairy cattle population then were identified by referencing Chief's DNA sequence within these selected chromosome blocks. Eleven candidate genes were identified with functions related to milk-production, fertility, and disease-resistance traits. These data demonstrate that haplotype reconstruction of an ancestral proband by whole-genome resequencing in combination with high-density SNP genotyping of descendants can be used for rapid, genome-wide identification of the ancestor's alleles that have been subjected to artificial selection.
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http://dx.doi.org/10.1073/pnas.1114546109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3356612PMC
May 2012