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 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.

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.

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.

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.

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.

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.

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.

The H1047R PIK3CA oncogene induces a senescence-like state, pleiotropy and acute HSP90 dependency in HER2+ mammary epithelial cells.

In pre-clinical models, co-existence of Human Epidermal Growth Factor Receptor-2 (HER2)-amplification and PI3K catalytic subunit (PIK3CA) mutations results in aggressive, anti-HER2 therapy-resistant breast tumors. This is not always reflected in clinical setting. We speculated that the complex interaction between the HER2 and PIK3CA oncogenes is responsible for such inconsistency. We performed series of biochemical, molecular and cellular assays on genetically engineered isogenic mammary epithelial cell lines and breast cancer cells expressing both oncogenes. In vitro observations were validated in xenografts models. We showed that H1047R, one of the most common PIK3CA mutations, is responsible for endowing a senescence-like state in mammary epithelial cells overexpressing HER2. Instead of imposing a permanent growth arrest characteristic of oncogene-induced senescence, the proteome secreted by the mutant cells promotes stem cell enrichment, angiogenesis, epithelial-to-mesenchymal transition, altered immune surveillance and acute vulnerability toward HSP90 inhibition. We inferred that the pleiotropism, as observed here, conferred by the mutated oncogene, depending on the host microenvironment, contributes to conflicting pre-clinical and clinical characteristics of HER2+, mutated PIK3CA-bearing tumor cells. We also came up with a plausible model for evolution of breast tumors from mammary epithelial cells harboring these two molecular lesions.

PLncPRO for prediction of long non-coding RNAs (lncRNAs) in plants and its application for discovery of abiotic stress-responsive lncRNAs in rice and chickpea.

Long non-coding RNAs (lncRNAs) make up a significant portion of non-coding RNAs and are involved in a variety of biological processes. Accurate identification/annotation of lncRNAs is the primary step for gaining deeper insights into their functions. In this study, we report a novel tool, PLncPRO, for prediction of lncRNAs in plants using transcriptome data. PLncPRO is based on machine learning and uses random forest algorithm to classify coding and long non-coding transcripts. PLncPRO has better prediction accuracy as compared to other existing tools and is particularly well-suited for plants. We developed consensus models for dicots and monocots to facilitate prediction of lncRNAs in non-model/orphan plants. The performance of PLncPRO was quite better with vertebrate transcriptome data as well. Using PLncPRO, we discovered 3714 and 3457 high-confidence lncRNAs in rice and chickpea, respectively, under drought or salinity stress conditions. We investigated different characteristics and differential expression under drought/salinity stress conditions, and validated lncRNAs via RT-qPCR. Overall, we developed a new tool for the prediction of lncRNAs in plants and showed its utility via identification of lncRNAs in rice and chickpea.

Global transcriptome and coexpression network analyses reveal cultivar-specific molecular signatures associated with seed development and seed size/weight determination in chickpea.

Seed development is an intricate process regulated via a complex transcriptional regulatory network. To understand the molecular mechanisms governing seed development and seed size/weight in chickpea, we performed a comprehensive analysis of transcriptome dynamics during seed development in two cultivars with contrasting seed size/weight (small-seeded, Himchana 1 and large-seeded, JGK 3). Our analysis identified stage-specific expression for a significant proportion (>13%) of the genes in each cultivar. About one half of the total genes exhibited significant differential expression in JGK 3 as compared with Himchana 1. We found that different seed development stages can be delineated by modules of coexpressed genes. A comparative analysis revealed differential developmental stage specificity of some modules between the two cultivars. Furthermore, we constructed transcriptional regulatory networks and identified key components determining seed size/weight. The results suggested that extended period of cell division during embryogenesis and higher level of endoreduplication along with more accumulation of storage compounds during maturation determine large seed size/weight. Further, we identified quantitative trait loci-associated candidate genes harboring single nucleotide polymorphisms in the promoter sequences that differentiate small- and large-seeded chickpea cultivars. The results provide a valuable resource to dissect the role of candidate genes governing seed development and seed size/weight in chickpea.

A draft genome sequence of the pulse crop chickpea (Cicer arietinum L.).

Cicer arietinum L. (chickpea) is the third most important food legume crop. We have generated the draft sequence of a desi-type chickpea genome using next-generation sequencing platforms, bacterial artificial chromosome end sequences and a genetic map. The 520-Mb assembly covers 70% of the predicted 740-Mb genome length, and more than 80% of the gene space. Genome analysis predicts the presence of 27,571 genes and 210 Mb as repeat elements. The gene expression analysis performed using 274 million RNA-Seq reads identified several tissue-specific and stress-responsive genes. Although segmental duplicated blocks are observed, the chickpea genome does not exhibit any indication of recent whole-genome duplication. Nucleotide diversity analysis provides an assessment of a narrow genetic base within the chickpea cultivars. We have developed a resource for genetic markers by comparing the genome sequences of one wild and three cultivated chickpea genotypes. The draft genome sequence is expected to facilitate genetic enhancement and breeding to develop improved chickpea varieties.

Genomewide discovery of DNA polymorphisms in rice cultivars with contrasting drought and salinity stress response and their functional relevance.

Next-generation sequencing technologies provide opportunities to understand the genetic basis of phenotypic differences, such as abiotic stress response, even in the closely related cultivars via identification of large number of DNA polymorphisms. We performed whole-genome resequencing of three rice cultivars with contrasting responses to drought and salinity stress (sensitive IR64, drought-tolerant Nagina 22 and salinity-tolerant Pokkali). More than 356 million 90-bp paired-end reads were generated, which provided about 85% coverage of the rice genome. Applying stringent parameters, we identified a total of 1 784 583 nonredundant single-nucleotide polymorphisms (SNPs) and 154 275 InDels between reference (Nipponbare) and the three resequenced cultivars. We detected 401 683 and 662 509 SNPs between IR64 and Pokkali, and IR64 and N22 cultivars, respectively. The distribution of DNA polymorphisms was found to be uneven across and within the rice chromosomes. One-fourth of the SNPs and InDels were detected in genic regions, and about 3.5% of the total SNPs resulted in nonsynonymous changes. Large-effect SNPs and InDels, which affect the integrity of the encoded protein, were also identified. Further, we identified DNA polymorphisms present in the differentially expressed genes within the known quantitative trait loci. Among these, a total of 548 SNPs in 232 genes, located in the conserved functional domains, were identified. The data presented in this study provide functional markers and promising target genes for salinity and drought tolerance and present a valuable resource for high-throughput genotyping and molecular breeding for abiotic stress traits in rice.

Genome-wide discovery and differential regulation of conserved and novel microRNAs in chickpea via deep sequencing.

MicroRNAs (miRNAs) are essential components of complex gene regulatory networks that orchestrate plant development. Although several genomic resources have been developed for the legume crop chickpea, miRNAs have not been discovered until now. For genome-wide discovery of miRNAs in chickpea (Cicer arietinum), we sequenced the small RNA content from seven major tissues/organs employing Illumina technology. About 154 million reads were generated, which represented more than 20 million distinct small RNA sequences. We identified a total of 440 conserved miRNAs in chickpea based on sequence similarity with known miRNAs in other plants. In addition, 178 novel miRNAs were identified using a miRDeep pipeline with plant-specific scoring. Some of the conserved and novel miRNAs with significant sequence similarity were grouped into families. The chickpea miRNAs targeted a wide range of mRNAs involved in diverse cellular processes, including transcriptional regulation (transcription factors), protein modification and turnover, signal transduction, and metabolism. Our analysis revealed several miRNAs with differential spatial expression. Many of the chickpea miRNAs were expressed in a tissue-specific manner. The conserved and differential expression of members of the same miRNA family in different tissues was also observed. Some of the same family members were predicted to target different chickpea mRNAs, which suggested the specificity and complexity of miRNA-mediated developmental regulation. This study, for the first time, reveals a comprehensive set of conserved and novel miRNAs along with their expression patterns and putative targets in chickpea, and provides a framework for understanding regulation of developmental processes in legumes.

A global view of transcriptome dynamics during flower development in chickpea by deep sequencing.

Measurement of gene expression can provide important clues about gene function and molecular basis of developmental processes. Here, we have analysed the chickpea transcriptome in vegetative and flower tissues by exploiting the potential of high-throughput sequencing to measure gene expression. We mapped more than 295 million reads to quantify the transcript abundance during flower development. We detected the expression of more than 90% genes in at least one tissue analysed. We found quite a large number of genes were differentially expressed during flower development as compared to vegetative tissues. Further, we identified several genes expressed in a stage-specific manner. Various transcription factor families and metabolic pathways involved in flower development were elucidated. The members of MADS-box family were most represented among the transcription factor genes up-regulated during various stages of flower development. The abundant expression of several well-known genes implicated in flower development in chickpea flower development stages confirmed our results. In addition, we detected the expression specificities of lineage-specific genes during flower development. The expression data presented in this study is the most comprehensive dataset available for chickpea as of now and will serve as resource for unraveling the functions of many specific genes involved in flower development in chickpea and other legumes.

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.

Transcriptome sequencing of wild chickpea as a rich resource for marker development.

The transcriptome of cultivated chickpea (Cicer arietinum L.), an important crop legume, has recently been sequenced. Here, we report sequencing of the transcriptome of wild chickpea, C. reticulatum (PI489777), the progenitor of cultivated chickpea, by GS-FLX 454 technology. The optimized assembly of C. reticulatum transcriptome generated 37 265 transcripts in total with an average length of 946 bp. A total of 4072 simple sequence repeats (SSRs) could be identified in these transcript sequences, of which at least 561 SSRs were polymorphic between C. arietinum and C. reticulatum. In addition, a total of 36 446 single-nucleotide polymorphisms (SNPs) were identified after optimization of probability score, quality score, read depth and consensus base ratio. Several of these SSRs and SNPs could be associated with tissue-specific and transcription factor encoding transcripts. A high proportion (92-94%) of polymorphic SSRs and SNPs identified between the two chickpea species were validated successfully. Further, the estimation of synonymous substitution rates of orthologous transcript pairs suggested that the speciation event for divergence of C. arietinum and C. reticulatum may have happened approximately 0.53 million years ago. The results of our study provide a rich resource for exploiting genetic variations in chickpea for breeding programmes.