Genome-wide mapping of DNase I hypersensitive sites revealed differential chromatin accessibility and regulatory DNA elements under drought stress in rice cultivars.

Drought stress (DS) is one of the major constraints limiting yield in crop plants including rice. Gene regulation under DS is largely governed by accessibility of the transcription factors (TFs) to their cognate cis-regulatory elements (CREs). In this study, we used DNase I hypersensitive assays followed by sequencing to identify the accessible chromatin regions under DS in a drought-sensitive (IR64) and a drought-tolerant (N22) rice cultivar. Our results indicated that DNase I hypersensitive sites (DHSs) were highly enriched at transcription start sites (TSSs) and numerous DHSs were detected in the promoter regions. DHSs were concurrent with epigenetic marks and the genes harboring DHSs in their TSS and promoter regions were highly expressed. In addition, DS induced changes in DHSs (∆DHSs) in TSS and promoter regions were positively correlated with upregulation of several genes involved in drought/abiotic stress response, those encoding TFs and located within drought-associated quantitative trait loci, much preferentially in the drought-tolerant cultivar. The CREs representing the binding sites of TFs involved in DS response were detected within the ∆DHSs, suggesting differential accessibility of TFs to their cognate sites under DS in different rice cultivars, which may be further deployed for enhancing drought tolerance in rice.

Unravelling the molecular mechanism underlying drought stress response in chickpea via integrated multi-omics analysis.

Drought stress affects growth and productivity significantly in chickpea. An integrated multi-omics analysis can provide a better molecular-level understanding of drought stress tolerance. In the present study, comparative transcriptome, proteome and metabolome analyses of two chickpea genotypes with contrasting responses to drought stress, ICC 4958 (drought-tolerant, DT) and ICC 1882 (drought-sensitive, DS), was performed to gain insights into the molecular mechanisms underlying drought stress response/tolerance. Pathway enrichment analysis of differentially abundant transcripts and proteins suggested the involvement of glycolysis/gluconeogenesis, galactose metabolism, and starch and sucrose metabolism in the DT genotype. An integrated multi-omics analysis of transcriptome, proteome and metabolome data revealed co-expressed genes, proteins and metabolites involved in phosphatidylinositol signaling, glutathione metabolism and glycolysis/gluconeogenesis pathways, specifically in the DT genotype under drought. These stress-responsive pathways were coordinately regulated by the differentially abundant transcripts, proteins and metabolites to circumvent the drought stress response/tolerance in the DT genotype. The QTL-hotspot associated genes, proteins and transcription factors may further contribute to improved drought tolerance in the DT genotype. Altogether, the multi-omics approach provided an in-depth understanding of stress-responsive pathways and candidate genes involved in drought tolerance in chickpea.

Unravelling Differential DNA Methylation Patterns in Genotype Dependent Manner under Salinity Stress Response in Chickpea.

DNA methylation is one of the epigenetic mechanisms that govern gene regulation in response to abiotic stress in plants. Here, we analyzed the role of epigenetic variations by exploring global DNA methylation and integrating it with differential gene expression in response to salinity stress in tolerant and sensitive chickpea genotypes. Genome-wide DNA methylation profiles showed higher CG methylation in the gene body regions and higher CHH methylation in the TE body regions. The analysis of differentially methylated regions (DMRs) suggested more hyper-methylation in response to stress in the tolerant genotype compared to the sensitive genotype. We observed higher enrichment of CG DMRs in genes and CHH DMRs in transposable elements (TEs). A positive correlation of gene expression with CG gene body methylation was observed. The enrichment analysis of DMR-associated differentially expressed genes revealed they are involved in biological processes, such as lateral root development, transmembrane transporter activity, GTPase activity, and regulation of gene expression. Further, a high correlation of CG methylation with CHG and CHH methylation under salinity stress was revealed, suggesting crosstalk among the methylation contexts. Further, we observed small RNA-mediated CHH hypermethylation in TEs. Overall, the interplay between DNA methylation, small RNAs, and gene expression provides new insights into the regulatory mechanism underlying salinity stress response in chickpeas.

An integrated transcriptome mapping the regulatory network of coding and long non-coding RNAs provides a genomics resource in chickpea.

Large-scale transcriptome analysis can provide a systems-level understanding of biological processes. To accelerate functional genomic studies in chickpea, we perform a comprehensive transcriptome analysis to generate full-length transcriptome and expression atlas of protein-coding genes (PCGs) and long non-coding RNAs (lncRNAs) from 32 different tissues/organs via deep sequencing. The high-depth RNA-seq dataset reveal expression dynamics and tissue-specificity along with associated biological functions of PCGs and lncRNAs during development. The coexpression network analysis reveal modules associated with a particular tissue or a set of related tissues. The components of transcriptional regulatory networks (TRNs), including transcription factors, their cognate cis-regulatory motifs, and target PCGs/lncRNAs that determine developmental programs of different tissues/organs, are identified. Several candidate tissue-specific and abiotic stress-responsive transcripts associated with quantitative trait loci that determine important agronomic traits are also identified. These results provide an important resource to advance functional/translational genomic and genetic studies during chickpea development and environmental conditions.

Genome-wide analysis suggests the potential role of lncRNAs during seed development and seed size/weight determination in chickpea.

MAIN CONCLUSION: The integrated transcriptome data analyses suggested the plausible roles of lncRNAs during seed development in chickpea. The candidate lncRNAs associated with QTLs and those involved in miRNA-mediated seed size/weight determination in chickpea have been identified. Long non-coding RNAs (lncRNAs) are important regulators of various biological processes. Here, we identified lncRNAs at seven successive stages of seed development in small-seeded and large-seeded chickpea cultivars. In total, 4751 lncRNAs implicated in diverse biological processes were identified. Most of lncRNAs were conserved between the two cultivars, whereas only a few of them were conserved in other plants, suggesting their species-specificity. A large number of lncRNAs differentially expressed between the two chickpea cultivars associated with seed development-related processes were identified. The lncRNAs acting as precursors of miRNAs and those mimicking target protein-coding genes of miRNAs involved in seed size/weight determination, including HAIKU1, BIG SEEDS1, and SHB1, were also revealed. Further, lncRNAs located within seed size/weight associated quantitative trait loci were also detected. Overall, we present a comprehensive resource and identified candidate lncRNAs that may play important roles during seed development and seed size/weight determination in chickpea.

Genome-wide discovery of DNA polymorphisms via resequencing of chickpea cultivars with contrasting response to drought stress.

Drought stress limits plant growth, resulting in a significant yield loss in chickpea. The diversification in genome sequence and selective sweep of allele(s) in different genotypes of a crop plant may play an important role in the determination of agronomic traits, including drought stress response. We investigated, via whole genome resequencing, the DNA polymorphisms between two sets of chickpea genotypes with contrasting drought stress responses (3 drought-sensitive vs. 6 drought-tolerant). In total, 36,406 single nucleotide polymorphisms (SNPs) and 3407 insertions or deletions (InDels) differentiating drought-sensitive and drought-tolerant chickpea genotypes were identified. Interestingly, most (91%) of these DNA polymorphisms were located in chromosomes 1 and 4. The genes harboring DNA polymorphisms in their promoter and/or coding regions and exhibiting differential expression under control and/or drought stress conditions between/within the drought-sensitive and tolerant genotypes were found implicated in the stress response. Furthermore, we identified DNA polymorphisms within the cis-regulatory motifs in the promoter region of abiotic stress-related and QTL-associated genes, which might contribute to the differential expression of the candidate drought-responsive genes. In addition, we revealed the effect of nonsynonymous SNPs on mutational sensitivity and stability of the encoded proteins. Taken together, we identified DNA polymorphisms having relevance in drought stress response and revealed candidate genes to engineer drought tolerance in chickpea.

Discovery of DNA polymorphisms via whole genome resequencing and their functional relevance in salinity stress response in chickpea.

Salinity stress is one of the major constraints for plant growth and yield. The salinity stress response of different genotypes of crop plants may largely be governed by DNA polymorphisms. To determine the molecular genetic factors involved in salinity stress tolerance in chickpea, we performed a whole genome resequencing data analysis of three each of salinity-sensitive and salinity-tolerant genotypes. A total of 6173 single nucleotide polymorphisms and 920 insertions and deletions differentiating the chickpea genotypes with contrasting salinity stress responses were identified. Gene ontology analysis revealed the enrichment of functional terms related to stress response and development among the genes harboring DNA polymorphisms in their promoter and/or coding regions. DNA polymorphisms located within the cis-regulatory motifs of the quantitative trait loci (QTL)-associated and abiotic stress related genes were identified, which may influence salinity stress response via modulating binding affinity of the transcription factors. Several genes including QTL-associated and abiotic stress response related genes harboring DNA polymorphisms exhibited differential expression in response to salinity stress especially at the reproductive stage of development in the salinity-tolerant genotype. Furthermore, effects of non-synonymous DNA polymorphisms on mutational sensitivity and structural integrity of the encoded proteins by the candidate QTL-associated and abiotic stress response related genes were revealed. The results suggest that DNA polymorphisms may determine salinity stress response via influencing differential gene expression in genotype and/or stage-dependent manner. Altogether, we provide a high-quality set of DNA polymorphisms and candidate genes that may govern salinity stress tolerance in chickpea.

Bacillus cereus: Beyond Gastroenteritis.

INTRODUCTION: Bacillus cereus (B cereus) has been found within the gastrointestinal flora. Due to its ubiquity, B cereus is usually considered a contaminant. However, it can cause serious infections in certain populations. CASE PRESENTATION: A 39-year-old woman with refractory gastroparesis requiring gastric pacemaker with a jejunostomy tube and cervical cancer status post chemotherapy presented with fever and fatigue. Initial and repeat blood cultures (from peripheral and port-a-cath access) grew B cereus and the port-a-cath was removed. She was treated with appropriate antibiotics and bacteremia resolved. DISCUSSION: B cereus is often associated with toxin-mediated emetic or diarrheal gastroenteritis. However, in patients with prosthetic devices or intravenous (IV) drug users, B cereus can cause serious infection. Biofilms produced by B cereus attach to indwelling catheters, allowing persistent infection until catheter removal. CONCLUSION: In patients with prosthetic devices or IV drug use, B cereus should be treated with appropriated antibiotics and any indwelling catheters should be removed.

Enhancers as potential targets for engineering salinity stress tolerance in crop plants.

Enhancers represent noncoding regulatory regions of the genome located distantly from their target genes. They regulate gene expression programs in a context-specific manner via interacting with promoters of one or more target genes and are generally associated with transcription factor binding sites and epi(genomic)/chromatin features, such as regions of chromatin accessibility and histone modifications. The enhancers are difficult to identify due to the modularity of their associated features. Although enhancers have been studied extensively in human and animals, only a handful of them has been identified in few plant species till date due to nonavailability of plant-specific experimental and computational approaches for their discovery. Being an important regulatory component of the genome, enhancers represent potential targets for engineering agronomic traits, including salinity stress tolerance in plants. Here, we provide a review of the available experimental and computational approaches along with the associated sequence and chromatin/epigenetic features for the discovery of enhancers in plants. In addition, we provide insights into the challenges and future prospects of enhancer research in plant biology with emphasis on potential applications in engineering salinity stress tolerance in crop plants.

Genome resequencing reveals DNA polymorphisms associated with seed size/weight determination in chickpea.

Crop productivity in legumes is determined by number and size/weight of seeds. To understand the genetic basis of seed size/weight in chickpea, we performed genome resequencing of 13 small- and 5 large-seeded genotypes using Illumina platform. Single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) differentiating small- and large-seeded genotypes were identified. A total of 17,902 SNPs and 2594 InDels located in promoter and/or coding regions that may contribute to seed size/weight were detected. Of these, 266 SNPs showed significant association with seed size/weight trait. Twenty-three genes including those involved in cell growth/division, encoding transcription factors and located within QTLs associated with seed size/weight harbored SNPs within transcription factor binding motif(s) and/or coding region. The non-synonymous SNPs were found to affect the mutational sensitivity and stability of the encoded proteins. Overall, we provided a high-quality SNP map for large-scale genotyping applications and identified candidate genes that determine seed size/weight in chickpea.

Genome-wide profiling of miRNAs during seed development reveals their functional relevance in seed size/weight determination in chickpea.

MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at transcriptional and post-transcriptional levels. The role of miRNAs in seed development and seed size/weight determination is poorly understood in legumes. In this study, we profiled miRNAs at seven successive stages of seed development in a small-seeded and a large-seeded chickpea cultivar via small RNA sequencing. In total, 113 known and 243 novel miRNAs were identified. Gene ontology analysis revealed the enrichment of seed/reproductive/post-embryonic development and signaling pathways processes among the miRNA target genes. A large fraction of the target genes exhibited antagonistic correlation with miRNA expression. The sets of co-expressed miRNAs showing differential expression between the two cultivars were recognized. Known transcription factor (TF) encoding genes involved in seed size/weight determination, including SPL, GRF, MYB, ARF, HAIKU1, SHB1, KLUH/CYP78A5, and E2Fb along with novel genes were found to be targeted by the predicted miRNAs. Differential expression analysis revealed higher transcript levels of members of SPL and REVOLUTA TF families and lower expression of their corresponding miRNAs in the large-seeded cultivar. At least 19 miRNAs known to be involved in seed development or differentially expressed between small-seeded and large-seeded cultivars at late-embryogenesis and/or mid-maturation stages were located within known quantitative trait loci (QTLs) associated with seed size/weight determination. Moreover, 41 target genes of these miRNAs were also located within these QTLs. Altogether, we revealed important roles of miRNAs in seed development and identified candidate miRNAs and their target genes that have functional relevance in determining seed size/weight in chickpea.

Draft genome and transcriptome analyses of halophyte rice Oryza coarctata provide resources for salinity and submergence stress response factors.

Oryza coarctata is a wild relative of rice that has adapted to diverse ecological environments, including high salinity and submergence. Thus, it can provide an important resource for discovering candidate genes/factors involved in tolerance to these stresses. Here, we report a draft genome assembly of 573 Mb comprised of 8877 scaffolds with N50 length of 205 kb. We predicted a total of 50,562 protein-coding genes, of which a significant fraction was found to be involved in secondary metabolite biosynthesis and hormone signal transduction pathways. Several salinity and submergence stress-responsive protein-coding and long noncoding RNAs involved in diverse biological processes were identified using RNA-sequencing data. Based on small RNA sequencing, we identified 168 unique miRNAs and 3219 target transcripts (coding and noncoding) involved in several biological processes, including abiotic stress responses. Further, whole genome bisulphite sequencing data analysis revealed at least 19%-48% methylcytosines in different sequence contexts and the influence of methylation status on gene expression. The genome assembly along with other datasets have been made publicly available at http://ccbb.jnu.ac.in/ory-coar. Altogether, we provide a comprehensive genomic resource for understanding the regulation of salinity and submergence stress responses and identification of candidate genes/factors involved for functional genomics studies.

DNA methylation reprogramming during seed development and its functional relevance in seed size/weight determination in chickpea.

Seed development is orchestrated via complex gene regulatory networks and pathways. Epigenetic factors may also govern seed development and seed size/weight. Here, we analyzed DNA methylation in a large-seeded chickpea cultivar (JGK 3) during seed development stages. Progressive gain of CHH context DNA methylation in transposable elements (TEs) and higher frequency of small RNAs in hypermethylated TEs during seed development suggested a role of the RNA-dependent DNA methylation pathway. Frequency of intragenic TEs was higher in CHH context differentially methylated region (DMR) associated differentially expressed genes (DEGs). CG context hyper/hypomethylation within the gene body was observed for most of DMR-associated DEGs in JGK 3 as compared to small-seeded chickpea cultivar (Himchana 1). We identified candidate genes involved in seed size/weight determination exhibiting CG context hypermethylation within the gene body and higher expression in JGK 3. This study provides insights into the role of DNA methylation in seed development and seed size/weight determination in chickpea.

Bisulphite sequencing reveals dynamic DNA methylation under desiccation and salinity stresses in rice cultivars.

DNA methylation governs gene regulation in plants in response to environmental conditions. Here, we analyzed role of DNA methylation under desiccation and salinity stresses in three (IR64, stress-sensitive; Nagina 22, drought-tolerant and Pokkali, salinity-tolerant) rice cultivars via bisulphite sequencing. Methylation in CG context within gene body and methylation in CHH context in distal promoter regions were positively correlated with gene expression. Hypomethylation in Nagina 22 and hypermethylation in Pokkali in response to desiccation and salinity stresses, respectively, were correlated with higher expression of few abiotic stress response related genes. Most of the differentially methylated and differentially expressed genes (DMR-DEGs) were cultivar-specific, suggesting an important role of DNA methylation in abiotic stress responses in rice in cultivar-specific manner. DMR-DEGs harboring differentially methylated cytosines due to DNA polymorphisms between the sensitive and tolerant cultivars in their promoter regions and/or coding regions were identified, suggesting the role of epialleles in abiotic stress responses.

Genome-wide analysis of glutathione S-transferase gene family in chickpea suggests its role during seed development and abiotic stress.

Glutathione S-transferases (GSTs) are multifunctional proteins that help in oxidative stress metabolism and detoxification of xenobiotic compounds. Studies pertaining to GST gene family have been undertaken in various plant species, however no information is available with respect to GST genes in chickpea. In the current study, we identified a total of 51 GST encoding genes in chickpea (CaGST) genome. Phylogenetic analysis revealed that GST gene family can be divided into eleven distinct classes. Tau and phi were the major classes in chickpea and one third of the CaGST genes represented segmental duplication and purifying selection was common among these genes. Expression of many CaGST genes, in particular, members of tau class were found to be upregulated under abiotic stress conditions. In addition, CaGST genes displayed differential expression patterns across diverse organs/tissues, suggesting their roles in developmental processes. Many CaGST genes showed opposite expression pattern in small- and large-seeded chickpea cultivars during seed development. Higher expression of CaGST genes in small-seeded cultivar at maturation stages of seed development suggested their important role in seed development and seed size/weight determination in chickpea. Overall, these results provide a comprehensive information on GST gene family members in chickpea and is expected to provide a rational platform to explore versatile role of these genes in semi-arid legume crops.

Updates on Genomic Resources in Chickpea for Crop Improvement.

In recent years, rapid advancement has been done in generation of genomic resources for the important legume crop chickpea. Here, we provide an update on important advancements made on availability of genomic resources for this crop. The availability of reference genome and transcriptome sequences, and resequencing of several accessions have enabled the discovery of gene space and molecular markers in chickpea. These resources have helped in elucidating evolutionary relationship and identification of quantitative trait loci for important agronomic traits. Gene expression in different tissues/organs during development and under abiotic/biotic stresses has been interrogated. In addition, single-base resolution DNA methylation patterns in different organs have been analyzed to understand gene regulation. Overall, we provide a consolidated overview of available genomic resources of chickpea that may help in fulfilling the promises for improvement of this important crop.

Method for Bisulfite Sequencing Data Analysis for Whole-Genome Level DNA Methylation Detection in Legumes.

Methylation of cytosines in DNA is the most stable type of epigenetic modification that is established and maintained by different enzymes. In plants, DNA methylation is inherited from one generation to another leaving an epigenetic mark as a memory of previous state, which may include encounter with stress or pathogen. Advancement in the next generation sequencing technologies has enabled the profiling of methylation marks. Whole-genome bisulfite sequencing (WGBS) has the potential to unravel the patterns of DNA methylation at single-base resolution. Though the sequencing technologies have evolved drastically, analysis of WGBS data still remains challenging. Here, we provide a methodology for performing WGBS data analysis along with critical steps for identification of methylation marks in plant genomes including legumes.

Methods for Identification and Validation of G-Quadruplex Sequences in Legumes.

Among the different secondary structures that DNA can adopt, G-quadruplex is a noncanonical form that has recently started to garner attention about the possible layers of regulation they could introduce in cellular processes. Here, we outline how the presence of G-quadruplexes can be probed in legumes and other plant genomes. This chapter describes various in silico approaches that can be utilized to identify putative G-quadruplex forming sequences (GQSes) and validate their formation through in vitro experimental approaches.