Functional allele of a MATE gene selected during domestication modulates seed color in chickpea.

Seed color is one of the key target traits of domestication and artificial selection in chickpeas due to its implications on consumer preference and market value. The complex seed color trait has been well dissected in several crop species; however, the genetic mechanism underlying seed color variation in chickpea remains poorly understood. Here, we employed an integrated genomics strategy involving QTL mapping, high-density mapping, map-based cloning, association analysis, and molecular haplotyping in an inter-specific RIL mapping population, association panel, wild accessions, and introgression lines (ILs) of Cicer gene pool. This delineated a MATE gene, CaMATE23, encoding a Transparent Testa (TT) and its natural allele (8-bp insertion) and haplotype underlying a major QTL governing seed color on chickpea chromosome 4. Signatures of selective sweep and a strong purifying selection reflected that CaMATE23, especially its 8-bp insertion natural allelic variant, underwent selection during chickpea domestication. Functional investigations revealed that the 8-bp insertion containing the third cis-regulatory RY-motif element in the CaMATE23 promoter is critical for enhanced binding of CaFUSCA3 transcription factor, a key regulator of seed development and flavonoid biosynthesis, thereby affecting CaMATE23 expression and proanthocyanidin (PA) accumulation in the seed coat to impart varied seed color in chickpea. Consequently, overexpression of CaMATE23 in Arabidopsis tt12 mutant partially restored the seed color phenotype to brown pigmentation, ascertaining its functional role in PA accumulation in the seed coat. These findings shed new light on the seed color regulation and evolutionary history, and highlight the transcriptional regulation of CaMATE23 by CaFUSCA3 in modulating seed color in chickpea. The functionally relevant InDel variation, natural allele, and haplotype from CaMATE23 are vital for translational genomic research, including marker-assisted breeding, for developing chickpea cultivars with desirable seed color that appeal to consumers and meet global market demand.

A superior gene allele involved in abscisic acid signaling enhances drought tolerance and yield in chickpea.

Identifying potential molecular tags for drought tolerance is essential for achieving higher crop productivity under drought stress. We employed an integrated genomics-assisted breeding and functional genomics strategy involving association mapping, fine mapping, map-based cloning, molecular haplotyping and transcript profiling in the introgression lines (ILs)- and near isogenic lines (NILs)-based association panel and mapping population of chickpea (Cicer arietinum). This combinatorial approach delineated a bHLH (basic helix-loop-helix) transcription factor, CabHLH10 (Cicer arietinum bHLH10) underlying a major QTL, along with its derived natural alleles/haplotypes governing yield traits under drought stress in chickpea. CabHLH10 binds to a cis-regulatory G-box promoter element to modulate the expression of RD22 (responsive to desiccation 22), a drought/abscisic acid (ABA)-responsive gene (via a trans-expression QTL), and two strong yield-enhancement photosynthetic efficiency (PE) genes. This, in turn, upregulates other downstream drought-responsive and ABA signaling genes, as well as yield-enhancing PE genes, thus increasing plant adaptation to drought with reduced yield penalty. We showed that a superior allele of CabHLH10 introgressed into the NILs improved root and shoot biomass and PE, thereby enhancing yield and productivity during drought without compromising agronomic performance. Furthermore, overexpression of CabHLH10 in chickpea and Arabidopsis (Arabidopsis thaliana) conferred enhanced drought tolerance by improving root and shoot agro-morphological traits. These findings facilitate translational genomics for crop improvement and the development of genetically tailored, climate-resilient, high-yielding chickpea cultivars.

Transcriptional signatures modulating shoot apical meristem morphometric and plant architectural traits enhance yield and productivity in chickpea.

Plant height (PH) and plant width (PW), two of the major plant architectural traits determining the yield and productivity of a crop, are defined by diverse morphometric characteristics of the shoot apical meristem (SAM). The identification of potential molecular tags from a single gene that simultaneously modulates these plant/SAM architectural traits is therefore prerequisite to achieve enhanced yield and productivity in crop plants, including chickpea. Large-scale multienvironment phenotyping of the association panel and mapping population have ascertained the efficacy of three vital SAM morphometric trait parameters, SAM width, SAM height and SAM area, as key indicators to unravel the genetic basis of the wide PW and PH trait variations observed in desi chickpea. This study integrated a genome-wide association study (GWAS); quantitative trait locus (QTL)/fine-mapping and map-based cloning with molecular haplotyping; transcript profiling; and protein-DNA interaction assays for the dissection of plant architectural traits in chickpea. These exertions delineated natural alleles and superior haplotypes from a CabHLH121 transcription factor (TF) gene within the major QTL governing PW, PH and SAM morphometric traits. A genome-wide protein-DNA interaction assay assured the direct binding of a known stem cell master regulator, CaWUS, to the WOX-homeodomain TF binding sites of a CabHLH121 gene and its constituted haplotypes. The differential expression of CaWUS and transcriptional regulation of its target CabHLH121 gene/haplotypes were apparent, suggesting their collective role in altering SAM morphometric characteristics and plant architectural traits in the contrasting near isogenic lines (NILs). The NILs introgressed with a superior haplotype of a CabHLH121 exhibited optimal PW and desirable PH as well as enhanced yield and productivity without compromising any component of agronomic performance. These molecular signatures of the CabHLH121 TF gene have the potential to regulate both PW and PH traits through the modulation of proliferation, differentiation and maintenance of the meristematic stem cell population in the SAM; therefore, these signatures will be useful in the translational genomic study of chickpea genetic enhancement. The restructured cultivars with desirable PH (semidwarf) and PW will ensure maximal planting density in a specified cultivable field area, thereby enhancing the overall yield and productivity of chickpea. This can essentially facilitate the achievement of better remunerative outputs by farmers with rational land use, therefore ensuring global food security in the present scenario of an increasing population density and shrinking per capita land area.

ABC Transporter-Mediated Transport of Glutathione Conjugates Enhances Seed Yield and Quality in Chickpea.

The identification of functionally relevant molecular tags is vital for genomics-assisted crop improvement and enhancement of seed yield, quality, and productivity in chickpea (Cicer arietinum). The simultaneous improvement of yield/productivity as well as quality traits often requires pyramiding of multiple genes, which remains a major hurdle given various associated epistatic and pleotropic effects. Unfortunately, no single gene that can improve yield/productivity along with quality and other desirable agromorphological traits is known, hampering the genetic enhancement of chickpea. Using a combinatorial genomics-assisted breeding and functional genomics strategy, this study identified natural alleles and haplotypes of an ABCC3-type transporter gene that regulates seed weight, an important domestication trait, by transcriptional regulation and modulation of the transport of glutathione conjugates in seeds of desi and kabuli chickpea. The superior allele/haplotype of this gene introgressed in desi and kabuli near-isogenic lines enhances the seed weight, yield, productivity, and multiple desirable plant architecture and seed-quality traits without compromising agronomic performance. These salient findings can expedite crop improvement endeavors and the development of nutritionally enriched high-yielding cultivars in chickpea.

Genome-wide cis-regulatory signatures for modulation of agronomic traits as exemplified by drought yield index (DYI) in chickpea.

Developing functional molecular tags from the cis-regulatory sequence components of genes is vital for their deployment in efficient genetic dissection of complex quantitative traits in crop plants including chickpea. The current study identified 431,194 conserved non-coding SNP (CNSNP) from the cis-regulatory element regions of genes which were annotated on a chickpea genome. These genome-wide CNSNP marker resources are made publicly accessible through a user-friendly web-database ( http://www.cnsnpcicarbase.com ). The CNSNP-based quantitative trait loci (QTL) and expression QTL (eQTL) mapping and genome-wide association study (GWAS) were further integrated with global gene expression landscapes, molecular haplotyping, and DNA-protein interaction study in the association panel and recombinant inbred lines (RIL) mapping population to decode complex genetic architecture of one of the vital seed yield trait under drought stress, drought yield index (DYI), in chickpea. This delineated two constituted natural haplotypes and alleles from a histone H3 protein-coding gene and its transcriptional regulator NAC transcription factor (TF) harboring the major QTLs and trans-acting eQTL governing DYI in chickpea. The effect of CNSNPs in TF-binding cis-element of a histone H3 gene in altering the binding affinity and transcriptional activity of NAC TF based on chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assay was evident. The CNSNP-led promising molecular tags scanned will essentially have functional significance to decode transcriptional gene regulatory function and thus can drive translational genomic analysis in chickpea.

Genetic dissection of photosynthetic efficiency traits for enhancing seed yield in chickpea.

Understanding the genetic basis of photosynthetic efficiency (PE) contributing to enhanced seed yield per plant (SYP) is vital for genomics-assisted crop improvement of chickpea. The current study employed an integrated genomic strategy involving photosynthesis pathway gene-based association mapping, genome-wide association study, quantitative trait loci (QTL) mapping, and expression profiling. This identified 16 potential single nucleotide polymorphism loci linked to major QTLs underlying 16 candidate genes significantly associated with PE and SYP traits in chickpea. The allelic variants were tightly linked to positively interacting QTLs regulating both enhanced PE and SYP traits as exemplified by a chlorophyll A-B binding protein-coding gene. The leaf tissue-specific pronounced up-regulated expression of 16 associated genes in germplasm accessions and homozygous individuals of mapping population was evident. Such combinatorial genomic strategy coupled with gene haplotype-specific association and in silico protein-protein interaction study delineated natural alleles and superior haplotypes from a chlorophyll A-B binding (CAB) protein-coding gene and its interacting gene, Timing of CAB Expression 1 (TOC1), which appear to be most promising candidates in modulating chickpea PE and SYP traits. These functionally pertinent molecular signatures identified have efficacy to drive marker-assisted selection for developing PE-enriched cultivars with high seed yield in chickpea.

CLAVATA signaling pathway genes modulating flowering time and flower number in chickpea.

KEY MESSAGE: A combinatorial genomic strategy delineated functionally relevant natural allele of a CLAVATA gene and its marker (haplotype)-assisted introgression led to development of the early-flowering chickpea cultivars with high flower number and enhanced yield/productivity. Unraveling the genetic components involved in CLAVATA (CLV) signaling is crucial for modulating important shoot apical meristem (SAM) characteristics and ultimately regulating diverse SAM-regulated agromorphological traits in crop plants. A genome-wide scan identified 142 CLV1-, 28 CLV2- and 6 CLV3-like genes, and their comprehensive genomic constitution and phylogenetic relationships were deciphered in chickpea. The QTL/fine mapping and map-based cloning integrated with high-resolution association analysis identified SNP loci from CaCLV3_01 gene within a major CaqDTF1.1/CaqFN1.1 QTL associated with DTF (days to 50% flowering) and FN (flower number) traits in chickpea, which was further ascertained by quantitative expression profiling. Molecular haplotyping of CaCLV3_01 gene, expressed specifically in SAM, constituted two major haplotypes that differentiated the early-DTF and high-FN chickpea accessions from late-DTF and low-FN. Enhanced accumulation of transcripts of superior CaCLV3_01 gene haplotype and known flowering promoting genes was observed in the corresponding haplotype-introgressed early-DTF and high-FN near-isogenic lines (NILs) with narrow SAM width. The superior haplotype-introgressed NILs exhibited early-flowering, high-FN and enhanced seed yield/productivity without compromising agronomic performance. These delineated molecular signatures can regulate DTF and FN traits through SAM proliferation and differentiation and thereby will be useful for translational genomic study to develop early-flowering cultivars with enhanced yield/productivity.

Uncovering DNA methylation landscapes to decipher evolutionary footprints of phenotypic diversity in chickpea.

Genetic diversity and environmental factors are long believed to be the dominant contributors to phenotypic diversity in crop plants. However, it has been recently established that, besides genetic variation, epigenetic variation, especially variation in DNA methylation, plays a significant role in determining phenotypic diversity in crop plants. Therefore, assessing DNA methylation diversity in crop plants becomes vital, especially in the case of crops like chickpea, which has a narrow genetic base. Thus, in the present study, we employed whole-genome bisulfite sequencing to assess DNA methylation diversity in wild and cultivated (desi and kabuli) chickpea. This revealed extensive DNA methylation diversity in both wild and cultivated chickpea. Interestingly, the methylation diversity was found to be significantly higher than genetic diversity, suggesting its potential role in providing vital phenotypic diversity for the evolution and domestication of the Cicer gene pool. The phylogeny based on DNA methylation variation also indicates a potential complementary role of DNA methylation variation in addition to DNA sequence variation in shaping chickpea evolution. Besides, the study also identified diverse epi-alleles of many previously known genes of agronomic importance. The Cicer MethVarMap database developed in this study enables researchers to readily visualize methylation variation within the genes and genomic regions of their interest (http://223.31.159.7/cicer/public/). Therefore, epigenetic variation like DNA methylation variation can potentially explain the paradox of high phenotypic diversity despite the narrow genetic base in chickpea and can potentially be employed for crop improvement.

Genome-Wide Scans for Delineation of Candidate Genes Regulating Seed-Protein Content in Chickpea.

Identification of potential genes/alleles governing complex seed-protein content (SPC) is essential in marker-assisted breeding for quality trait improvement of chickpea. Henceforth, the present study utilized an integrated genomics-assisted breeding strategy encompassing trait association analysis, selective genotyping in traditional bi-parental mapping population and differential expression profiling for the first-time to understand the complex genetic architecture of quantitative SPC trait in chickpea. For GWAS (genome-wide association study), high-throughput genotyping information of 16376 genome-based SNPs (single nucleotide polymorphism) discovered from a structured population of 336 sequenced desi and kabuli accessions [with 150-200 kb LD (linkage disequilibrium) decay] was utilized. This led to identification of seven most effective genomic loci (genes) associated [10-20% with 41% combined PVE (phenotypic variation explained)] with SPC trait in chickpea. Regardless of the diverse desi and kabuli genetic backgrounds, a comparable level of association potential of the identified seven genomic loci with SPC trait was observed. Five SPC-associated genes were validated successfully in parental accessions and homozygous individuals of an intra-specific desi RIL (recombinant inbred line) mapping population (ICC 12299 × ICC 4958) by selective genotyping. The seed-specific expression, including differential up-regulation (>four fold) of six SPC-associated genes particularly in accessions, parents and homozygous individuals of the aforementioned mapping population with a high level of contrasting SPC (21-22%) was evident. Collectively, the integrated genomic approach delineated diverse naturally occurring novel functional SNP allelic variants in six potential candidate genes regulating SPC trait in chickpea. Of these, a non-synonymous SNP allele-carrying zinc finger transcription factor gene exhibiting strong association with SPC trait was found to be the most promising in chickpea. The informative functionally relevant molecular tags scaled-down essentially have potential to accelerate marker-assisted genetic improvement by developing nutritionally rich chickpea cultivars with enhanced SPC.

JAZ Repressors: Potential Involvement in Nutrients Deficiency Response in Rice and Chickpea.

Jasmonates (JA) are well-known phytohormones which play important roles in plant development and defense against pathogens. Jasmonate ZIM domain (JAZ) proteins are plant-specific proteins and act as transcriptional repressors of JA-responsive genes. JA regulates both biotic and abiotic stress responses in plants; however, its role in nutrient deficiency responses is very elusive. Although, JA is well-known for root growth inhibition, little is known about behavior of JAZ genes in response to nutrient deficiencies, under which root architectural alteration is an important adaptation. Using protein sequence homology and a conserved-domains approach, here we identify 10 novel JAZ genes from the recently sequenced Chickpea genome, which is one of the most nutrient efficient crops. Both rice and chickpea JAZ genes express in tissue- and stimuli-specific manners. Many of which are preferentially expressed in root. Our analysis further showed differential expression of JAZ genes under macro (NPK) and micronutrients (Zn, Fe) deficiency in rice and chickpea roots. While both rice and chickpea JAZ genes showed a certain level of specificity toward type of nutrient deficiency, generally majority of them showed induction under K deficiency. Generally, JAZ genes showed an induction at early stages of stress and expression declined at later stages of macro-nutrient deficiency. Our results suggest that JAZ genes might play a role in early nutrient deficiency response both in monocot and dicot roots, and information generated here can be further used for understanding the possible roles of JA in root architectural alterations for nutrient deficiency adaptations.