Genome-wide profiling of histone (H3) lysine 4 (K4) tri-methylation (me3) under drought, heat, and combined stresses in switchgrass.

BACKGROUND: Switchgrass (Panicum virgatum L.) is a warm-season perennial (C4) grass identified as an important biofuel crop in the United States. It is well adapted to the marginal environment where heat and moisture stresses predominantly affect crop growth. However, the underlying molecular mechanisms associated with heat and drought stress tolerance still need to be fully understood in switchgrass. The methylation of H3K4 is often associated with transcriptional activation of genes, including stress-responsive. Therefore, this study aimed to analyze genome-wide histone H3K4-tri-methylation in switchgrass under heat, drought, and combined stress. RESULTS: In total, ~ 1.3 million H3K4me3 peaks were identified in this study using SICER. Among them, 7,342; 6,510; and 8,536 peaks responded under drought (DT), drought and heat (DTHT), and heat (HT) stresses, respectively. Most DT and DTHT peaks spanned 0 to + 2000 bases from the transcription start site [TSS]. By comparing differentially marked peaks with RNA-Seq data, we identified peaks associated with genes: 155 DT-responsive peaks with 118 DT-responsive genes, 121 DTHT-responsive peaks with 110 DTHT-responsive genes, and 175 HT-responsive peaks with 136 HT-responsive genes. We have identified various transcription factors involved in DT, DTHT, and HT stresses. Gene Ontology analysis using the AgriGO revealed that most genes belonged to biological processes. Most annotated peaks belonged to metabolite interconversion, RNA metabolism, transporter, protein modifying, defense/immunity, membrane traffic protein, transmembrane signal receptor, and transcriptional regulator protein families. Further, we identified significant peaks associated with TFs, hormones, signaling, fatty acid and carbohydrate metabolism, and secondary metabolites. qRT-PCR analysis revealed the relative expressions of six abiotic stress-responsive genes (transketolase, chromatin remodeling factor-CDH3, fatty-acid desaturase A, transmembrane protein 14C, beta-amylase 1, and integrase-type DNA binding protein genes) that were significantly (P < 0.05) marked during drought, heat, and combined stresses by comparing stress-induced against un-stressed and input controls. CONCLUSION: Our study provides a comprehensive and reproducible epigenomic analysis of drought, heat, and combined stress responses in switchgrass. Significant enrichment of H3K4me3 peaks downstream of the TSS of protein-coding genes was observed. In addition, the cost-effective experimental design, modified ChIP-Seq approach, and analyses presented here can serve as a prototype for other non-model plant species for conducting stress studies.

Aging and obesity prime the methylome and transcriptome of adipose stem cells for disease and dysfunction.

The epigenome of stem cells occupies a critical interface between genes and environment, serving to regulate expression through modification by intrinsic and extrinsic factors. We hypothesized that aging and obesity, which represent major risk factors for a variety of diseases, synergistically modify the epigenome of adult adipose stem cells (ASCs). Using integrated RNA- and targeted bisulfite-sequencing in murine ASCs from lean and obese mice at 5- and 12-months of age, we identified global DNA hypomethylation with either aging or obesity, and a synergistic effect of aging combined with obesity. The transcriptome of ASCs in lean mice was relatively stable to the effects of age, but this was not true in obese mice. Functional pathway analyses identified a subset of genes with critical roles in progenitors and in diseases of obesity and aging. Specifically, Mapt, Nr3c2, App, and Ctnnb1 emerged as potential hypomethylated upstream regulators in both aging and obesity (AL vs. YL and AO vs. YO), and App, Ctnnb1, Hipk2, Id2, and Tp53 exhibited additional effects of aging in obese animals. Furthermore, Foxo3 and Ccnd1 were potential hypermethylated upstream regulators of healthy aging (AL vs. YL), and of the effects of obesity in young animals (YO vs. YL), suggesting that these factors could play a role in accelerated aging with obesity. Finally, we identified candidate driver genes that appeared recurrently in all analyses and comparisons undertaken. Further mechanistic studies are needed to validate the roles of these genes capable of priming ASCs for dysfunction in aging- and obesity-associated pathologies.

Global analysis of switchgrass (Panicum virgatum L.) transcriptomes in response to interactive effects of drought and heat stresses.

BACKGROUND: Sustainable production of high-quality feedstock has been of great interest in bioenergy research. Despite the economic importance, high temperatures and water deficit are limiting factors for the successful cultivation of switchgrass in semi-arid areas. There are limited reports on the molecular basis of combined abiotic stress tolerance in switchgrass, particularly the combination of drought and heat stress. We used transcriptomic approaches to elucidate the changes in the response of switchgrass to drought and high temperature simultaneously. RESULTS: We conducted solely drought treatment in switchgrass plant Alamo AP13 by withholding water after 45 days of growing. For the combination of drought and heat effect, heat treatment (35 °C/25 °C day/night) was imposed after 72 h of the initiation of drought. Samples were collected at 0 h, 72 h, 96 h, 120 h, 144 h, and 168 h after treatment imposition, total RNA was extracted, and RNA-Seq conducted. Out of a total of 32,190 genes, we identified 3912, as drought (DT) responsive genes, 2339 and 4635 as, heat (HT) and drought and heat (DTHT) responsive genes, respectively. There were 209, 106, and 220 transcription factors (TFs) differentially expressed under DT, HT and DTHT respectively. Gene ontology annotation identified the metabolic process as the significant term enriched in DTHT genes. Other biological processes identified in DTHT responsive genes included: response to water, photosynthesis, oxidation-reduction processes, and response to stress. KEGG pathway enrichment analysis on DT and DTHT responsive genes revealed that TFs and genes controlling phenylpropanoid pathways were important for individual as well as combined stress response. For example, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) from the phenylpropanoid pathway was induced by single DT and combinations of DTHT stress. CONCLUSION: Through RNA-Seq analysis, we have identified unique and overlapping genes in response to DT and combined DTHT stress in switchgrass. The combination of DT and HT stress may affect the photosynthetic machinery and phenylpropanoid pathway of switchgrass which negatively impacts lignin synthesis and biomass production of switchgrass. The biological function of genes identified particularly in response to DTHT stress could further be confirmed by techniques such as single point mutation or RNAi.

Ten simple rules for providing effective bioinformatics research support.

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.

Comparative Hessian Fly Larval Transcriptomics Provides Novel Insight into Host and Nonhost Resistance.

The Hessian fly is a destructive pest of wheat. Employing additional molecular strategies can complement wheat's native insect resistance. However, this requires functional characterization of Hessian-fly-responsive genes, which is challenging because of wheat genome complexity. The diploid Brachypodium distachyon (Bd) exhibits nonhost resistance to Hessian fly and displays phenotypic/molecular responses intermediate between resistant and susceptible host wheat, offering a surrogate genome for gene characterization. Here, we compared the transcriptomes of Biotype L larvae residing on resistant/susceptible wheat, and nonhost Bd plants. Larvae from susceptible wheat and nonhost Bd plants revealed similar molecular responses that were distinct from avirulent larval responses on resistant wheat. Secreted salivary gland proteins were strongly up-regulated in all larvae. Genes from various biological pathways and molecular processes were up-regulated in larvae from both susceptible wheat and nonhost Bd plants. However, Bd larval expression levels were intermediate between larvae from susceptible and resistant wheat. Most genes were down-regulated or unchanged in avirulent larvae, correlating with their inability to establish feeding sites and dying within 4-5 days after egg-hatch. Decreased gene expression in Bd larvae, compared to ones on susceptible wheat, potentially led to developmentally delayed 2nd-instars, followed by eventually succumbing to nonhost resistance defense mechanisms.

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

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.

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

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.

Genome-wide identification of histone methylation (H3K9me2) and acetylation (H4K12ac) marks in two ecotypes of switchgrass (Panicum virgatum L.).

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- H3K9me2 and active- H4K12ac). 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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus).

Papaya, a fruit crop cultivated in tropical and subtropical regions, is known for its nutritional benefits and medicinal applications. Here we report a 3x draft genome sequence of 'SunUp' papaya, the first commercial virus-resistant transgenic fruit tree to be sequenced. The papaya genome is three times the size of the Arabidopsis genome, but contains fewer genes, including significantly fewer disease-resistance gene analogues. Comparison of the five sequenced genomes suggests a minimal angiosperm gene set of 13,311. A lack of recent genome duplication, atypical of other angiosperm genomes sequenced so far, may account for the smaller papaya gene number in most functional groups. Nonetheless, striking amplifications in gene number within particular functional groups suggest roles in the evolution of tree-like habit, deposition and remobilization of starch reserves, attraction of seed dispersal agents, and adaptation to tropical daylengths. Transgenesis at three locations is closely associated with chloroplast insertions into the nuclear genome, and with topoisomerase I recognition sites. Papaya offers numerous advantages as a system for fruit-tree functional genomics, and this draft genome sequence provides the foundation for revealing the basis of Carica's distinguishing morpho-physiological, medicinal and nutritional properties.