Publications of NIPGR Scientists
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Item Genome-wide discovery of DNA polymorphisms among chickpea cultivars with contrasting seed size/weight and their functional relevance(Springer Nature, 2018) Rajkumar, Mohan Singh; Garg, Rohini; Jain, MukeshSeed size/weight is a major agronomic trait which determine crop productivity in legumes. To understand the genetic basis of seed size determination, we sought to identify DNA polymorphisms between two small (Himchana 1 and Pusa 362) and two large-seeded (JGK 3 and PG 0515) chickpea cultivars via whole genome resequencing. We identified a total of 75535 single nucleotide polymorphisms (SNPs), 6486 insertions and deletions (InDels), 1938 multi-nucleotide polymorphisms (MNPs) and 5025 complex variants between the two small and two large-seeded chickpea cultivars. Our analysis revealed 814, 244 and 72 seed-specific genes harboring DNA polymorphisms in promoter or non-synonymous and large-effect DNA polymorphisms, respectively. Gene ontology analysis revealed enrichment of cell growth and division related terms in these genes. Among them, at least 22 genes associated with quantitative trait loci, and those involved in cell growth and division and encoding transcription factors harbored promoter and/or large-effect/non-synonymous DNA polymorphisms. These also showed higher expression at late-embryogenesis and/or mid-maturation stages of seed development in the large-seeded cultivar, suggesting their role in seed size/weight determination in chickpea. Altogether, this study provided a valuable resource for large-scale genotyping applications and a few putative candidate genes that might play crucial role in governing seed size/weight in chickpea.Item Genome-wide bisulphite-sequencing reveals organ-specific methylation patterns in chickpea(Springer Nature, 2018) Bhatia, Himanshi; Khemka, Niraj; Jain, Mukesh; Garg, RohiniDNA methylation is widely known to regulate gene expression in eukaryotes. Here, we unraveled DNA methylation patterns in cultivated chickpea to understand the regulation of gene expression in different organs. We analyzed the methylation pattern in leaf tissue of wild chickpea too, and compared it with cultivated chickpea. Our analysis indicated abundant CG methylation within gene-body and CHH methylation in intergenic regions of the chickpea genome in all the organs examined. Analysis of differentially methylated regions (DMRs) demonstrated a higher number of CG context DMRs in wild chickpea and CHH context DMRs in cultivated chickpea. We observed increased preponderance of hypermethylated DMRs in the promoter regions and hypomethylated DMRs in the genic regions in cultivated chickpea. Genomic location and context of the DMRs correlated well with expression of proximal genes. Our results put forth a positive correlation of promoter hypermethylation with increased transcript abundance via identification of DMR-associated genes involved in flower development in cultivated chickpea. The atypical correlation observed between promoter hypermethylation and increased transcript abundance might be dependent on 24-nt small RNAs and transcription factors binding to the promoter region. This study provides novel insights into DNA methylation patterns in chickpea and their role in regulation of gene expression.Item Genome-wide identification and co-expression network analysis provide insights into the roles of auxin response factor gene family in chickpea(Nature Publishing Group, 2017) Singh, Vikash K.; Rajkumar, Mohan Singh; Garg, Rohini; Jain, MukeshAuxin response factors (ARFs) are the transcription factors that regulate auxin responses in various aspects of plant growth and development. Although genome-wide analysis of ARF gene family has been done in some species, no information is available regarding ARF genes in chickpea. In this study, we identified 28 ARF genes (CaARF) in the chickpea genome. Phylogenetic analysis revealed that CaARFs can be divided into four different groups. Duplication analysis revealed that 50% of CaARF genes arose from duplication events. We analyzed expression pattern of CaARFs in various developmental stages. CaARF16.3, CaARF17.1 and CaARF17.2 showed highest expression at initial stages of flower bud development, while CaARF6.2 had higher expression at later stages of flower development. Further, CaARF4.2, CaARF9.2, CaARF16.2 and CaARF7.1 exhibited differential expression under different abiotic stress conditions, suggesting their role in abiotic stress responses. Co-expression network analysis among CaARF, CaIAA and CaGH3 genes enabled us to recognize components involved in the regulatory network associated with CaARFs. Further, we identified microRNAs that target CaARFs and TAS3 locus that trigger production of trans-acting siRNAs targeting CaARFs. The analyses presented here provide comprehensive information on ARF family members and will help in elucidating their exact function in chickpea.Item Global transcriptome and co-expression network analyses reveal cultivar-specific molecular signatures associated with seed development and seed size/weight determination in chickpea(John Wiley & Sons, 2017) Garg, Rohini; Singh, Vikash K.; Rajkumar, Mohan Singh; Kumar, Vinay; Jain, MukeshSeed 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. About one-fourth of total genes exhibited significant differential expression in JGK 3 as compared to Himchana 1. We found that different seed development stages can be delineated by modules of co-expressed 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.Item Genome-wide analysis of long intergenic non-coding RNAs in chickpea and their potential role in flower development(Nature Publishing Group, 2016) Khemka, Niraj; Singh, Vikash Kumar; Garg, Rohini; Jain, MukeshNon-coding RNAs constitute a major portion of the transcriptome in most of eukaryotes. Long non-coding transcripts originating from the DNA segment present between the protein coding genes are termed as long intergenic non-coding RNAs (lincRNAs). Several evidences suggest the role of lincRNAs in regulation of various biological processes. In this study, we identified a total of 2248 lincRNAs in chickpea using RNA-seq data from eight successive stages of flower development and three vegetative tissues via an optimized pipeline. Different characteristic features of lincRNAs were studied and compared with those of predicted mRNAs in chickpea. Further, we utilized a method using network propagation algorithm to reveal the putative function of lincRNAs in plants. In total, at least 79% of the identified chickpea lincRNAs were assigned with a putative function. A comprehensive expression profiling revealed differential expression patterns and tissue specificity of lincRNAs in different stages of flower development in chickpea. In addition, potential lincRNAs-miRNA interactions were explored for the predicted lincRNAs in chickpea. These findings will pave the way for understanding the role of lincRNAs in the regulatory mechanism underlying flower development in chickpea and other legumes.Item Genome-wide discovery of G-quadruplex forming sequences and their functional relevance in plants(Nature Publishing Group, 2016) Garg, Rohini; Aggarwal, Jyoti; Thakkar, BijalDNA, in addition to the canonical B-form, can acquire a variety of alternate structures, such as G-quadruplexes. These structures have been implicated in several cellular processes in animals. In this study, we identified different types of G-quadruplex forming sequences (GQSes) in 15 sequenced plants and analyzed their distribution in various genomic features, including gene body, coding, intergenic and promoter regions. G2-type GQSes were most abundant in all the plant species analyzed. A strong association of G3-type GQSes with intergenic, promoter and intronic regions was found. However, G2-type GQSes were enriched in genic, CDS, exonic and untranslated regions. Further, we identified GQSes present in the conserved genes among monocots and dicots. The genes involved in development, cell growth and size, transmembrane transporter, and regulation of gene expression were found to be significantly enriched. In the promoter region, we detected strong co-occurrence of Telobox, ERF, MYB, RAV1B and E2F motifs with GQSes. Further, we validated the structure formation of several plant GQSes, demonstrated their effect on stalling in-vitro replication and revealed their interaction with plant nuclear proteins. Our data provide insights into the prevalence of GQSes in plants, establish their association with different genomic features and functional relevance.Item De novo transcriptome assembly and comprehensive expression profiling in Crocus sativus to gain insights into apocarotenoid biosynthesis(Nature Publishing Group, 2016) Jain, Mukesh; Srivastava, Prabhakar Lal; Verma, Mohit; Ghangal, Rajesh; Garg, RohiniSaffron (Crocus sativus L.) is commonly known as world’s most expensive spice with rich source of apocarotenoids and possesses magnificent medicinal properties. To understand the molecular basis of apocarotenoid biosynthesis/accumulation, we performed transcriptome sequencing from five different tissues/organs of C. sativus using Illumina platform. After comprehensive optimization of de novo transcriptome assembly, a total of 105, 269 unique transcripts (average length of 1047bp and N50 length of 1404bp) were obtained from 206 million high-quality paired-end reads. Functional annotation led to the identification of many genes involved in various biological processes and molecular functions. In total, 54% of C. sativus transcripts could be functionally annotated using public databases. Transcriptome analysis of C. sativus revealed the presence of 16721 SSRs and 3819 transcription factor encoding transcripts. Differential expression analysis revealed preferential/specific expression of many transcripts involved in apocarotenoid biosynthesis in stigma. We have revealed the differential expression of transcripts encoding for transcription factors (MYB, MYB related, WRKY, C2C2-YABBY and bHLH) involved in secondary metabolism. Overall, these results will pave the way for understanding the molecular basis of apocarotenoid biosynthesis and other aspects of stigma development in C. sativus.Item Transcriptome analyses reveal genotype- and developmental stage-specific molecular responses to drought and salinity stresses in chickpea(Nature Publishing Group, 2016) Garg, Rohini; Shankar, Rama; Thakkar, Bijal; Kudapa, Himabindu; Krishnamurthy, Lakshmanan; Mantri, Nitin; Varshney, Rajeev K.; Bhatia, Sabhyata; Jain, MukeshDrought and salinity are the major factors that limit chickpea production worldwide. We performed whole transcriptome analyses of chickpea genotypes to investigate the molecular basis of drought and salinity stress response/adaptation. Phenotypic analyses confirmed the contrasting responses of the chickpea genotypes to drought or salinity stress. RNA-seq of the roots of drought and salinity related genotypes was carried out under control and stress conditions at vegetative and/or reproductive stages. Comparative analysis of the transcriptomes revealed divergent gene expression in the chickpea genotypes at different developmental stages. We identified a total of 4954 and 5545 genes exclusively regulated in drought-tolerant and salinity-tolerant genotypes, respectively. A significant fraction (~47%) of the transcription factor encoding genes showed differential expression under stress. The key enzymes involved in metabolic pathways, such as carbohydrate metabolism, photosynthesis, lipid metabolism, generation of precursor metabolites/energy, protein modification, redox homeostasis and cell wall component biogenesis, were affected by drought and/or salinity stresses. Interestingly, transcript isoforms showed expression specificity across the chickpea genotypes and/or developmental stages as illustrated by the AP2-EREBP family members. Our findings provide insights into the transcriptome dynamics and components of regulatory network associated with drought and salinity stress responses in chickpea.Item CTDB: an integrated chickpea transcriptome database for functional and applied genomics(PLOS, 2015) Verma, Mohit; Kumar, Vinay; Patel, Ravi K.; Garg, Rohini; Jain, MukeshChickpea is an important grain legume used as a rich source of protein in human diet. The narrow genetic diversity and limited availability of genomic resources are the major constraints in implementing breeding strategies and biotechnological interventions for genetic enhancement of chickpea. We developed an integrated Chickpea Transcriptome Database (CTDB), which provides the comprehensive web interface for visualization and easy retrieval of transcriptome data in chickpea. The database features many tools for similarity search, functional annotation (putative function, PFAM domain and gene ontology) search and comparative gene expression analysis. The current release of CTDB (v2.0) hosts transcriptome datasets with high quality functional annotation from cultivated (desi and kabuli types) and wild chickpea. A catalog of transcription factor families and their expression profiles in chickpea are available in the database. The gene expression data have been integrated to study the expression profiles of chickpea transcripts in major tissues/organs and various stages of flower development. The utilities, such as similarity search, ortholog identification and comparative gene expression have also been implemented in the database to facilitate comparative genomic studies among different legumes and Arabidopsis. Furthermore, the CTDB represents a resource for the discovery of functional molecular markers (microsatellites and single nucleotide polymorphisms) between different chickpea types. We anticipate that integrated information content of this database will accelerate the functional and applied genomic research for improvement of chickpea. The CTDB web service is freely available at http://nipgr.res.in/ctdb.html.Item Genome-wide analysis and expression profiling suggest diverse roles of GH3 genes during development and abiotic stress responses in legumes(Frontiers Media S.A., 2015) Singh, Vikash K.; Jain, Mukesh; Garg, RohiniGrowth hormone auxin regulates various cellular processes by altering the expression of diverse genes in plants. Among various auxin-responsive genes, GH3 genes maintain endogenous auxin homeostasis by conjugating excess of auxin with amino acids. GH3 genes have been characterized in many plant species, but not in legumes. In the present work, we identified members of GH3 gene family and analyzed their chromosomal distribution, gene structure, gene duplication and phylogenetic analysis in different legumes, including chickpea, soybean, Medicago, and Lotus. A comprehensive expression analysis in different vegetative and reproductive tissues/stages revealed that many of GH3 genes were expressed in a tissue-specific manner. Notably, chickpea CaGH3-3, soybean GmGH3-8 and -25, and Lotus LjGH3-4, -5, -9 and -18 genes were up-regulated in root, indicating their putative role in root development. In addition, chickpea CaGH3-1 and -7, and Medicago MtGH3-7, -8, and -9 were found to be highly induced under drought and/or salt stresses, suggesting their role in abiotic stress responses. We also observed the examples of differential expression pattern of duplicated GH3 genes in soybean, indicating their functional diversification. Furthermore, analyses of three-dimensional structures, active site residues and ligand preferences provided molecular insights into function of GH3 genes in legumes. The analysis presented here would help in investigation of precise function of GH3 genes in legumes during development and stress conditions.