A chickpea genetic variation map based on the sequencing of 3,366 genomes.

Zero hunger and good health could be realized by 2030 through effective conservation, characterization and utilization of germplasm resources1. So far, few chickpea (Cicer arietinum) germplasm accessions have been characterized at the genome sequence level2. Here we present a detailed map of variation in 3,171 cultivated and 195 wild accessions to provide publicly available resources for chickpea genomics research and breeding. We constructed a chickpea pan-genome to describe genomic diversity across cultivated chickpea and its wild progenitor accessions. A divergence tree using genes present in around 80% of individuals in one species allowed us to estimate the divergence of Cicer over the last 21 million years. Our analysis found chromosomal segments and genes that show signatures of selection during domestication, migration and improvement. The chromosomal locations of deleterious mutations responsible for limited genetic diversity and decreased fitness were identified in elite germplasm. We identified superior haplotypes for improvement-related traits in landraces that can be introgressed into elite breeding lines through haplotype-based breeding, and found targets for purging deleterious alleles through genomics-assisted breeding and/or gene editing. Finally, we propose three crop breeding strategies based on genomic prediction to enhance crop productivity for 16 traits while avoiding the erosion of genetic diversity through optimal contribution selection (OCS)-based pre-breeding. The predicted performance for 100-seed weight, an important yield-related trait, increased by up to 23% and 12% with OCS- and haplotype-based genomic approaches, respectively.

Identification and characterization of NHX gene family for their role under salt stress in Vigna mungo.

In the current study, we have performed a comprehensive analysis of the Sodium Hydrogen Exchanger (NHX) gene family in Vigna mungo, and a total of 44 NHX genes were identified. A bimodal distribution based on domains, gene structure and phylogenetic analysis was evident. All intronpoor and intron-rich genes were clustered in clades I and II, respectively. Interestingly, all genes of subclade IIb were localized to vacuoles and possess only the NHX domain. The isoelectric point and trans-membrane domain analysis reflect the wide distribution of the NHX genes. Interestingly, Vm_NHX2 and Vm_NHX3 lacked trans-membrane domain but were found to interact with other NHX genes as well as vital salinity pathway genes, including calcium-mediated salt-responsive genes. The comparison of the mRNA sequences with that of V. marina, a halophytic species, reflects their independent evolution, majorly supporting the convergent evolution. The Ka/Ks ratio reflects the abundance of purifying selection supporting their conserved function during evolution. In our analysis, several abiotic stress and hormone-responsive elements and transcription factor binding sites were present in the promoter of the NHX genes. Further, the ion partitioning of a tolerant (K90) and a susceptible (K49) variety of V. mungo suggested that K90 managed the Na+/K+ ratio more affluently, which was also supported by profiling of superoxide radicals, hydrogen peroxide, phenol, peroxidase activity and superoxide dismutase activity. From the expression, we identified five candidate Vm_NHX genes, four of which, i.e. Vm_NHX16, Vm_NHX17, Vm_NHX29 and Vm_NHX33, were localized to the vacuolar and lysosomal membrane.

Comprehensive RNAseq analysis for identification of genes expressed under heat stress in lentil.

Lentils are highly sensitive to abrupt increases in temperature during the mid to late reproductive stages, leading to severe biomass and seed yield reduction. Therefore, we carried out an RNAseq analysis between IG4258 (heat tolerant) and IG3973 (heat sensitive) lentil genotypes at the reproductive stage under both normal and heat stress conditions in the field. It resulted in 209,549 assembled transcripts and among these 161,809 transcripts had coding regions, of which 94,437 transcripts were annotated. The differential gene expression analysis showed upregulation of 678 transcripts and downregulation of 680 transcripts between the tolerant and sensitive genotypes at the early reproductive stage. While 76 transcripts were upregulated and 47 transcripts were downregulated at the late reproductive stage under heat stress conditions. The validation of 12 up-or downregulated transcripts through RT-PCR corresponded well with the expression analysis data of RNAseq, with a correlation of R2  = 0.89. Among these transcripts, the DN364_c1_g1_i9 and DN2218_c0_g1_i5 transcripts encoded enzymes involved in the tryptophan pathway, indicating that tryptophan biosynthesis plays a role under heat stress in lentil. Moreover, KEGG pathways enrichment analysis identified transcripts associated with genes encoding proteins/regulating factors related to different metabolic pathways including signal transduction, fatty acid biosynthesis, rRNA processing, ribosome biogenesis, gibberellin (GA) biosynthesis, and riboflavin biosynthesis. This analysis also identified 6852 genic-SSRs leading to the development of 4968 SSR primers that are potential genomic resources for molecular mapping of heat-tolerant genes in lentil.

Genome-wide identification and functional prediction of salt- stress related long non-coding RNAs (lncRNAs) in chickpea (Cicer arietinum L.).

LncRNAs (long noncoding RNAs) are 200 bp length crucial RNA molecules, lacking coding potential and having important roles in regulating gene expression, particularly in response to abiotic stresses. In this study, we identified salt stress-induced lncRNAs in chickpea roots and predicted their intricate regulatory roles. A total of 3452 novel lncRNAs were identified to be distributed across all 08 chickpea chromosomes. On comparing salt-tolerant (ICCV 10, JG 11) and salt-sensitive cultivars (DCP 92-3, Pusa 256), 4446 differentially expressed lncRNAs were detected under various salt  treatments. We predicted 3373 lncRNAs to be regulating their target genes in cis regulating manner and 80 unique lncRNAs were observed as interacting with 136 different miRNAs, as eTMs (endogenous target mimic) targets of miRNAs and implicated them in the regulatory network of salt stress response. Functional analysis of these lncRNA revealed their association in targeting salt stress response-related genes like potassium transporter, transporter family genes, serine/threonine-protein kinase, aquaporins like TIP1-2, PIP2-5 and transcription factors like, AP2, NAC, bZIP, ERF, MYB and WRKY. Furthermore, about 614 lncRNA-SSRs (simple sequence repeats) were identified as a new generation of molecular markers with higher efficiency and specificity in chickpea. Overall, these findings will pave the understanding of comprehensive functional role of potential lncRNAs, which can help in providing insight into the molecular mechanism of salt tolerance in chickpea. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01093-0.

Genetic Dissection and Identification of Candidate Genes for Salinity Tolerance Using Axiom®CicerSNP Array in Chickpea.

Globally, chickpea production is severely affected by salinity stress. Understanding the genetic basis for salinity tolerance is important to develop salinity tolerant chickpeas. A recombinant inbred line (RIL) population developed using parental lines ICCV 10 (salt-tolerant) and DCP 92-3 (salt-sensitive) was screened under field conditions to collect information on agronomy, yield components, and stress tolerance indices. Genotyping data generated using Axiom®CicerSNP array was used to construct a linkage map comprising 1856 SNP markers spanning a distance of 1106.3 cM across eight chickpea chromosomes. Extensive analysis of the phenotyping and genotyping data identified 28 quantitative trait loci (QTLs) explaining up to 28.40% of the phenotypic variance in the population. We identified QTL clusters on CaLG03 and CaLG06, each harboring major QTLs for yield and yield component traits under salinity stress. The main-effect QTLs identified in these two clusters were associated with key genes such as calcium-dependent protein kinases, histidine kinases, cation proton antiporter, and WRKY and MYB transcription factors, which are known to impart salinity stress tolerance in crop plants. Molecular markers/genes associated with these major QTLs, after validation, will be useful to undertake marker-assisted breeding for developing better varieties with salinity tolerance.

Cloning and characterization of a gene encoding novel zinc finger protein transcription factor induced under water deficit stress from rice (Oryza sativa) cv. N-22.

A gene OsZnI encoding Cys3/His1-type zinc finger protein was isolated from the water stress-induced cDNA library of rice (Oryza sativa) cv. N-22, an early maturing, deep-rooted, drought-tolerant genotype adapted to upland conditions. The in-silico analysis revealed an insert of 800 bp with an ORF of 663 nucleotides, encoding 221 amino acids. OsZnI had three distinct features--nuclear localization signal (NLS) present in Arg152-Arg168, Zn finger domain between 185-193 amino acids and 12 amino acids conserved domain in 71-82 amino acids homologous to LEA motif, and belonged to C-type family of Zn finger protein. OsZnI showed induced expression under water deficit stress.

Construction of a high-density genetic map and QTL analysis for yield, yield components and agronomic traits in chickpea (Cicer arietinum L.).

Unravelling the genetic architecture underlying yield components and agronomic traits is important for enhancing crop productivity. Here, a recombinant inbred line (RIL) population, developed from ICC 4958 and DCP 92-3 cross, was used for constructing linkage map and QTL mapping analysis. The RIL population was genotyped using a high-throughput Axiom®CicerSNP array, which enabled the development of a high-density genetic map consisting of 3,818 SNP markers and spanning a distance of 1064.14 cM. Analysis of phenotyping data for yield, yield components and agronomic traits measured across three years together with genetic mapping data led to the identification of 10 major-effect QTLs and six minor-effect QTLs explaining up to 59.70% phenotypic variance. The major-effect QTLs identified for 100-seed weight, and plant height possessed key genes, such as C3HC4 RING finger protein, pentatricopeptide repeat (PPR) protein, sugar transporter, leucine zipper protein and NADH dehydrogenase, amongst others. The gene ontology studies highlighted the role of these genes in regulating seed weight and plant height in crop plants. The identified genomic regions for yield, yield components, and agronomic traits, and the closely linked markers will help advance genetics research and breeding programs in chickpea.

Global gene expression analysis of pigeonpea with male sterility conditioned by A2 cytoplasm.

Cytoplasmic male sterility(CMS), a maternally inherited trait, provides a promising means to harness yield gains associated with hybrid vigor. In pigeonpea [Cajanus cajan (L.) Huth], nine types of sterility-inducing cytoplasm have been reported, of which A2 and A4 have been successfully deployed in hybrid breeding. Unfortunately, molecular mechanism of the CMS trait is poorly understood because of limited research invested. More recently, an association between a mitochondrial gene (nad7) and A4 -CMS has been demonstrated in pigeonpea; however, the mechanism underlying A2 -CMS still remains obscure. The current investigation aimed to analyze the differences in A2 -CMS line (ICPL 88039A) and its isogenic maintainer line (ICPL 88039B) at transcriptome level using next-generation sequencing. Gene expression profiling uncovered a set of 505 genes that showed altered expression in response to CMS, of which, 412 genes were upregulated while 93 were downregulated in the fertile maintainer line vs. the CMS line. Further, gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) network analyses revealed association of CMS in pigeonpea with four major pathways: glucose and lipid metabolism, ATP production, pollen development and pollen tube growth, and reactive oxygen species (ROS) scavenging. Patterns of digital gene expression were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) of six candidate genes. This study elucidates candidate genes and metabolic pathways having potential associations with pollen development and male sterility in pigeonpea A2 -CMS. New insights on molecular mechanism of CMS trait in pigeonpea will be helpful to accelerate heterosis utilization for enhancing productivity gains in pigeonpea.

Translational Chickpea Genomics Consortium to Accelerate Genetic Gains in Chickpea (Cicer arietinum L.).

The Translational Chickpea Genomics Consortium (TCGC) was set up to increase the production and productivity of chickpea (Cicer arietinum L.). It represents research institutes from six major chickpea growing states (Madhya Pradesh, Maharashtra, Andhra Pradesh, Telangana, Karnataka and Uttar Pradesh) of India. The TCGC team has been engaged in deploying modern genomics approaches in breeding and popularizing improved varieties in farmers' fields across the states. Using marker-assisted backcrossing, introgression lines with enhanced drought tolerance and fusarium wilt resistance have been developed in the genetic background of 10 elite varieties of chickpea. Multi-location evaluation of 100 improved lines (70 desi and 30 kabuli) during 2016-2017 and 2018-2019 enabled the identification of top performing desi and kabuli lines. In total, 909 Farmer Participatory Varietal Selection trials were conducted in 158 villages in 16 districts of the five states, during 2017-2018, 2018-2019, and 2019-2020, involving 16 improved varieties. New molecular breeding lines developed in different genetic backgrounds are potential candidates for national trials under the ICAR-All India Coordinated Research Project on Chickpea. The comprehensive efforts of TCGC resulted in the development and adoption of high-yielding varieties that will increase chickpea productivity and the profitability of chickpea growing farmers.

Structural modelling and molecular dynamics of a multi-stress responsive WRKY TF-DNA complex towards elucidating its role in stress signalling mechanisms in chickpea.

Chickpea is a premier food legume crop with high nutritional quality and attains prime importance in the current era of 795 million people being undernourished worldwide. Chickpea production encounters setbacks due to various stresses and understanding the role of key transcription factors (TFs) involved in multiple stresses becomes inevitable. We have recently identified a multi-stress responsive WRKY TF in chickpea. The present study was conducted to predict the structure of WRKY TF to identify the DNA-interacting residues and decipher DNA-protein interactions. Comparative modelling approach produced 3D model of the WRKY TF with good stereochemistry, local/global quality and further revealed W19, R20, K21, and Y22 motifs within a vicinity of 5 Å to the DNA amongst R18, G23, Q24, K25, Y36, Y37, R38 and K47 and these positions were equivalent to the 2LEX WRKY domain of Arabidopsis. Molecular simulations analysis of reference protein -PDB ID 2LEX, along with Car-WRKY TF modelled structure with the DNA coordinates derived from PDB ID 2LEX and docked using HADDOCK were executed. Root Mean Square (RMS) Deviation and RMS Fluctuation values yielded consistently stable trajectories over 50 ns simulation. Strengthening the obtained results, neither radius of gyration, distance and total energy showed any signs of DNA-WRKY complex falling apart nor any significant dissociation event over 50 ns run. Therefore, the study provides first insights into the structural properties of multi-stress responsive WRKY TF-DNA complex in chickpea, enabling genome wide identification of TF binding sites and thereby deciphers their gene regulatory networks.