Browsing by Author "Verma, Subodh"

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Analysis of genes encoding seed storage proteins (SSPs) in chickpea (Cicer arietinum L.) reveals co-expressing transcription factors and a seed-specific promoter
(Springer Nature, 2019) Verma, Subodh; Bhatia, Sabhyata
Improvement of the quality and quantity of chickpea seed protein can be greatly facilitated by an understanding of the genic organization and the genetic architecture of the genes encoding seed storage proteins (SSPs). The aim of this study was to provide a comprehensive analysis of the chickpea SSP genes, putative co-expressing transcription factors (TFs), and to identify a seedspecific SSP gene promoter. A genome-wide identification of SSP genes in chickpea led to the identification of 21 non-redundant SSP encoding genes located on 6 chromosomes. Phylogenetic analysis grouped SSP genes into 3 subgroups where members within the same clade demonstrated similar motif composition and intron-exon organization. Tandem duplications were identified to be the major contributors to the expansion of the SSP gene family in chickpea. Co-expression analysis revealed 14 TFs having expression profiles similar to the SSP genes that included members of important TF families that are known to regulate seed development. Expression analysis of SSP genes and TFs revealed significantly higher expression in late stages of seed development as well as in high seed protein content (HPC) genotypes. In silico analysis of the promoter regions of the SSP encoding genes revealed several seed-specific cis-regulatory elements such as RY repeats, ACGT motifs, CAANTG, and GCN4. A candidate promoter was analyzed for seed specificity by generating stable transgenics in Arabidopsis. Overall, this study provides a useful resource to explore the regulatory networks involved in SSP synthesis and/or accumulation for utilization in developing nutritionally improved chickpea genotypes.
A comprehensive analysis of the B3 superfamily identifies tissue-specific and stress-responsive genes in chickpea (Cicer arietinum L.)
(Springer Nature Publishing AG, 2019) Verma, Subodh; Bhatia, Sabhyata
The aim of this study was to provide a comprehensive analysis of the plant-specifc B3 domain-containing transcription factors (TFs) in chickpea. Scanning of the chickpea genome resulted in the identifcation of 51 B3 domain-containing TFs that were located on seven out of eight chickpea chromosomes. Based on the presence of additional domains other than the B3 domain, the candidates were classifed into four subfamilies, i.e., ARF (24), REM (19), LAV (6) and RAV (2). Phylogenetic analysis classifed them into four groups in which members of the same group had similar intron–exon organization and motif composition. Genome duplication analysis of the candidate B3 genes revealed an event of segmental duplication that was instrumental in the expansion of the B3 gene family. Ka/Ks analysis showed that the B3 gene family was under purifying selection. Further, chickpea B3 genes showed maximum orthology with Medicago followed by soybean and Arabidopsis. Promoter analyses of the B3 genes led to the identifcation of several tissue-specifc and stress-responsive cis-regulatory elements. Expression profling of the candidate B3 genes using publicly available RNA-seq data of several chickpea tissues indicated their putative role in plant development and abiotic stress response. These fndings were further validated by realtime expression analysis. Overall, this study provides a comprehensive analysis of the B3 domain-containing proteins in chickpea that would aid in devising strategies for crop manipulation in chickpea.
Development of gene-based markers for use in construction of the chickpea (Cicer arietinum L.) genetic linkage map and identification of QTLs associated with seed weight and plant height
(Springer, 2015) Gupta, Shefali; Kumar, Tapan; Verma, Subodh; Bharadwaj, Chellapilla; Bhatia, Sabhyata
Seed weight and plant height are important agronomic traits and contribute to seed yield. The objective of this study was to identify QTLs underlying these traits using an intra-specific mapping population of chickpea. A F11 population of 177 recombinant inbred lines derived from a cross between SBD377 (100-seed weight- 48 g and plant height- 53 cm) and BGD112 (100-seed weight- 15 g and plant height- 65 cm) was used. A total of 367 novel EST-derived functional markers were developed which included 187 EST-SSRs, 130 potential intron polymorphisms (PIPs) and 50 expressed sequence tag polymorphisms (ESTPs). Along with these, 590 previously published markers including 385 EST-based markers and 205 genomic SSRs were utilized. Of the 957 markers tested for analysis of parental polymorphism between the two parents of the mapping population, 135 (14.64 %) were found to be polymorphic. Of these, 131 polymorphic markers could be mapped to the 8 linkage groups. The linkage map had a total length of 1140.54 cM with an average marker density of 8.7 cM. The map was further used for QTL identification using composite interval mapping method (CIM). Two QTLs each for seed weight, qSW-1 and qSW-2 (explaining 11.54 and 19.24 % of phenotypic variance, respectively) and plant height, qPH-1 and qPH-2 (explaining 13.98 and 12.17 % of phenotypic variance, respectively) were detected. The novel set of genic markers, the intra-specific linkage map and the QTLs identified in the present study will serve as valuable genomic resources in improving the chickpea seed yield using marker-assisted selection (MAS) strategies.
Genome-wide analysis of the CCCH zinc finger family identifies tissue specific and stress responsive candidates in chickpea (Cicer arietinum L.)
(PLOS, 2017) Pradhan, Seema; Kant, Chandra; Verma, Subodh; Bhatia, Sabhyata
The CCCH zinc finger is a group of proteins characterised by a typical motif consisting of three cysteine residues and one histidine residue. These proteins have been reported to play important roles in regulation of plant growth, developmental processes and environmental responses. In the present study, genome wide analysis of the CCCH zinc finger gene family was carried out in the available chickpea genome. Various bioinformatics tools were employed to predict 58 CCCH zinc finger genes in chickpea (designated CarC3H1-58), which were analysed for their physio-chemical properties. Phylogenetic analysis classified the proteins into 12 groups in which members of a particular group had similar structural organization. Further, the numbers as well as the types of CCCH motifs present in the CarC3H proteins were compared with those from Arabidopsis and Medicago truncatula. Synteny analysis revealed valuable information regarding the evolution of this gene family. Tandem and segmental duplication events were identified and their Ka/Ks values revealed that the CarC3H gene family in chickpea had undergone purifying selection. Digital, as well as real time qRT-PCR expression analysis was performed which helped in identification of several CarC3H members that expressed preferentially in specific chickpea tissues as well as during abiotic stresses (desiccation, cold, salinity). Moreover, molecular characterization of an important member CarC3H45 was carried out. This study provides comprehensive genomic information about the important CCCH zinc finger gene family in chickpea. The identified tissue specific and abiotic stress specific CCCH genes could be potential candidates for further characterization to delineate their functional roles in development and stress.
High-density linkage map construction and mapping of seed trait QTLs in chickpea (Cicer arietinum L.) using genotyping-by-sequencing (GBS)
(Nature Publishing Group, 2015) Verma, Subodh; Gupta, Shefali; Bandhiwal, Nitesh; Kumar, Tapan; Bharadwaj, Chellapilla; Bhatia, Sabhyata
This study reports the use of Genotyping-by-Sequencing (GBS) for large-scale SNP discovery and simultaneous genotyping of recombinant inbred lines (RILs) of an intra-specific mapping population of chickpea contrasting for seed traits. A total of 119,672 raw SNPs were discovered, which after stringent filtering revealed 3,977 high quality SNPs of which 39.5% were present in genic regions. Comparative analysis using physically mapped marker loci revealed a higher degree of synteny with Medicago in comparison to soybean. The SNP genotyping data was utilized to construct one of the most saturated intra-specific genetic linkage maps of chickpea having 3,363 mapped positions including 3,228 SNPs on 8 linkage groups spanning 1006.98 cM at an average inter marker distance of 0.33 cM. The map was utilized to identify 20 quantitative trait loci (QTLs) associated with seed traits accounting for phenotypic variations ranging from 9.97% to 29.71%. Analysis of the genomic sequence corresponding to five robust QTLs led to the identification of 684 putative candidate genes whose expression profiling revealed that 101 genes exhibited seed specific expression. The integrated approach utilizing the identified QTLs along with the available genome and transcriptome could serve as a platform for candidate gene identification for molecular breeding of chickpea.
A high-density SNP-based linkage map using genotyping-by-sequencing and its utilization for improved genome assembly of chickpea (Cicer arietinum L.)
(Springer Nature Publishing AG, 2020) Gaur, Rashmi; Verma, Subodh; Pradhan, Seema; Ambreen, Heena; Bhatia, Sabhyata
Genotyping-by-sequencing (GBS) allows rapid identification of markers for use in development of linkage maps, which expedite efficient breeding programs. In the present study, we have utilized GBS approach to identify and genotype single-nucleotide polymorphism (SNP) markers in an inter-specific RIL population of Cicer arietinum L. X C. reticulatum. A total of 141,639 raw SNPs were identified using the TASSEL-GBS pipeline. After stringent filtering, 8208 candidate SNPs were identified of which ~ 37% were localized in the intragenic regions followed by genic regions (~ 30%) and intergenic regions (~ 27%). We then utilized 6920 stringent selected SNPs from present study and 6714 SNPs and microsatellite markers available from previous studies for construction of linkage map. The resulting high-density linkage map comprising of eight linkage groups contained 13,590 markers which spanned 1299.14 cM of map length with an average marker density of 0.095 cM. Further, the derived linkage map was used to improve the available assembly of desi chickpea genome by anchoring 443 previously unplaced scaffolds onto eight linkage groups. The present efforts have refined anchoring of the desi chickpea genome assembly to 55.57% of the ~ 520 Mb of assembled desi genome. To the best of our knowledge, the linkage map generated in the present study represents one of the most dense linkage map developed for the crop till date. It will serve as a valuable resource for fine mapping and positional cloning of important quantitative trait loci (QTLs) associated with agronomical traits and also for anchoring and ordering of future genome sequence assemblies.
Identification and molecular characterization of miRNAs and their target genes associated with seed development through small RNA sequencing in chickpea
(Springer Nature Publishing AG, 2021) Pradhan, Seema; Verma, Subodh; Chakraborty, Anirban; Bhatia, Sabhyata
Multiple studies have attempted to dissect the molecular mechanism underlying seed development in chickpea (Cicer arietinum L.). These studies highlight the need to focus on the role of miRNAs in regulating storage protein accumulation in seeds. Therefore, a total of 8,856,691 short-read sequences were generated from a small RNA library of developing chickpea seeds and were analyzed using miRDeep-P to identify 74 known and 26 novel miRNA sequences. Known miRNAs were classified into 22 miRNA families with miRNA156 family being most abundant. Of the 26 putative novel miRNAs identified, only 22 could be experimentally validated using stem loop end point PCR. Differential expression analyses led to the identification of known as well as novel miRNAs that could regulate various stages of chickpea seed development. In silico target prediction revealed several important target genes and transcription factors like SPL, mediator of RNA Polymerase II transcription subunit 12, aspartic proteinase and NACs, which were further validated by real-time PCR analysis. A comparative expression analysis in chickpea genotypes with contrasting seed protein content revealed one known (Car-miR156h) and two novel miRNA (CarnovmiR7 and Car-novmiR23) candidates to be highly expressed in the LPC (low protein content) chickpea genotypes, targets of which are known to regulate seed storage protein accumulation. Therefore, this study provides a useful resource in the form of miRNA and their targets which can be further utilized to understand and manipulate various regulatory mechanisms involved in seed development with the overall aim of improving yield and nutrition attributes in chickpea.