Genome-wide discovery of DNA polymorphisms among chickpea cultivars with contrasting seed size/weight and their functional relevance.
ABSTRACT: Seed 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.
Project description:Chickpea (Cicer arietinum L.) is the third most important food legume crop. Seed size is the most economically important trait for chickpea. To understand the genetic regulation of seed size in chickpea, the present study established a three-way association of CT repeat length variation of a simple sequence repeat (SSR) in myo-inositol monophosphatase gene (CaIMP) with seed weight and phytic acid content by large scale validation and genotyping in a set of genetically diverse germplasm accessions and two reciprocal intra-specific mapping populations. Germplasms and mapping individuals with CT repeat-length expansion in the 5' untranslated region of CaIMP exhibited a pronounced increase in CaIMP protein level, enzymatic activity, seed-phytate content and seed weight. A chickpea transient expression system demonstrated this repeat-length variation influenced the translation of CaIMP mRNA, apparently by facilitating translation initiation. Our analyses proposed that the SSR marker derived from 5' UTR of a CaIMP gene is a promising candidate for selection of seed size/weight for agronomic trait improvement of chickpea.
Project description:The SNP-based high-resolution QTL mapping mapped eight major genomic regions harbouring robust QTLs governing seed-Fe and Zn concentrations (39.4% combined phenotypic variation explained/PVE) on six chromosomes of an intra-specific high-density genetic linkage map (1.56 cM map-density). 24620 SNPs discovered from genome-wide GBS (genotyping-by-sequencing) and 13 known cloned Fe and Zn contents-related chickpea gene-orthologs were genotyped in a structured population of 92 sequenced desi and kabuli accessions. The large-scale 16591 SNP genotyping- and phenotyping-based GWAS (genome-wide association study) identified 16 genomic loci/genes associated (29% combined PVE) with seed-Fe and Zn concentrations. Of these, 11 trait-associated SNPs in the genes linked tightly with eight QTLs were validated by QTL mapping. The seed-specific expression, including pronounced differential-regulation of 16 trait-associated genes particularly in accessions/mapping individuals with contrasting level of seed-Fe and Zn contents was apparent. Collectively, the aforementioned rapid integrated genomic strategy led to delineate novel functional non-synonymous and regulatory SNP allelic-variants from 16 known/candidate genes, including three strong trait-associated genes (encoding late embryogenesis abundant and yellow stripe-like 1 protein, and vacuolar protein sorting-associated protein) and eight major QTLs regulating seed-Fe and Zn concentrations in chickpea. These essential inputs thus have potential to be deployed in marker-assisted genetic enhancement for developing nutritionally-rich iron/zinc-biofortified chickpea cultivars.
Project description:Plant carotenoids have a key role in preventing various diseases in human because of their antioxidant and provitamin A properties. Chickpea is a good source of carotenoid among legumes and its diverse germplasm and genome accessibility makes it a good model for carotenogenesis studies. The structure, location, and copy numbers of genes involved in carotenoid biosynthesis were retrieved from the chickpea genome. The majority of the single nucleotide polymorphism (SNPs) within these genes across five diverse chickpea cultivars was synonymous mutation. We examined the expression of the carotenogenesis genes and their association with carotenoid concentration at different seed development stages of five chickpea cultivars. Total carotenoid concentration ranged from 22 ?g g-1 in yellow cotyledon kabuli to 44 ?g g-1 in green cotyledon desi at 32 days post anthesis (DPA). The majority of carotenoids in chickpea seeds consists of lutein and zeaxanthin. The expression of the selected 19 genes involved in carotenoid biosynthesis pathway showed common pattern across five cultivars with higher expression at 8 and/or 16 DPA then dropped considerably at 24 and 32 DPA. Almost all genes were up-regulated in CDC Jade cultivar. Correlation analysis between gene expression and carotenoid concentration showed that the genes involved in the primary step of carotenoid biosynthesis pathway including carotenoid desaturase and isomerase positively correlated with various carotenoid components in chickpea seeds. A negative correlation was found between hydroxylation activity and provitamin A concentration in the seeds. The highest provitamin A concentration including ?-carotene and ?-cryptoxanthin were found in green cotyledon chickpea cultivars.
Project description:The study identified 9045 high-quality SNPs employing both genome-wide GBS- and candidate gene-based SNP genotyping assays in 172, including 93 cultivated (desi and kabuli) and 79 wild chickpea accessions. The GWAS in a structured population of 93 sequenced accessions detected 15 major genomic loci exhibiting significant association with seed coat color. Five seed color-associated major genomic loci underlying robust QTLs mapped on a high-density intra-specific genetic linkage map were validated by QTL mapping. The integration of association and QTL mapping with gene haplotype-specific LD mapping and transcript profiling identified novel allelic variants (non-synonymous SNPs) and haplotypes in a MATE secondary transporter gene regulating light/yellow brown and beige seed coat color differentiation in chickpea. The down-regulation and decreased transcript expression of beige seed coat color-associated MATE gene haplotype was correlated with reduced proanthocyanidins accumulation in the mature seed coats of beige than light/yellow brown seed colored desi and kabuli accessions for their coloration/pigmentation. This seed color-regulating MATE gene revealed strong purifying selection pressure primarily in LB/YB seed colored desi and wild Cicer reticulatum accessions compared with the BE seed colored kabuli accessions. The functionally relevant molecular tags identified have potential to decipher the complex transcriptional regulatory gene function of seed coat coloration and for understanding the selective sweep-based seed color trait evolutionary pattern in cultivated and wild accessions during chickpea domestication. The genome-wide integrated approach employed will expedite marker-assisted genetic enhancement for developing cultivars with desirable seed coat color types in chickpea.
Project description:Single seed weight (SSW), or seed size, is a seed yield components (SYC) in soybean, and it is suggested that the genetic factors regulating SSW are involved in the control of other SYCs. The quantitative trait loci (QTLs) for SSW and their effects on the other SYCs were investigated using a recombinant inbred line population derived from typical small- and large-seeded cultivars that were cultivated in two different environments. QTL analysis detected four environmentally stable QTLs for SSW, two of which coincided with the defined loci, qSw17-1 and Ln. The effects of the other loci, qSw12-1 and qSw13-1, were confirmed by analyzing residual heterozygous line progenies derived from the recombinant population. These four QTL regions were also involved in the control of an additional SYC, namely the large-seeded allele at each locus that reduced either the number of pods per plant or the number of ovules per pod. These results suggest the presence of at least two different regulatory mechanisms for SSW. Isolation of genes responsible for these QTLs provides an important tool in the understanding and utilization of SSW diversity for soybean breeding.
Project description:We discovered 2150 desi and 2199 kabuli accessions-derived SNPs by cultivar-wise individual assembling of sequence-reads generated through genotyping-by-sequencing of 92 chickpea accessions. Subsequent large-scale validation and genotyping of these SNPs discovered 619 desi accessions-derived (DAD) SNPs, 531 kabuli accessions-derived (KAD) SNPs, 884 multiple accessions-derived (MAD) SNPs and 1083 two accessions (desi ICC 4958 and kabuli CDC Frontier)-derived (TAD) SNPs that were mapped on eight chromosomes. These informative SNPs were annotated in coding/non-coding regulatory sequence components of genes. The MAD-SNPs were efficient to detect high intra-specific polymorphic potential and wide natural allelic diversity level including high-resolution admixed-population genetic structure and precise phylogenetic relationship among 291 desi and kabuli accessions. This signifies their effectiveness in introgression breeding and varietal improvement studies targeting useful agronomic traits of chickpea. Six trait-associated genes with SNPs including quantitative trait nucleotides (QTNs) in combination explained 27.5% phenotypic variation for seed yield per plant (SYP). A pentatricopeptide repeat (PPR) gene with a synonymous-coding SNP/QTN significantly associated with SYP trait was found most-promising in chickpea. The essential information delineated can be of immense utility in genomics-assisted breeding applications to develop high-yielding chickpea cultivars.
Project description:BACKGROUND:The homeobox transcription factor has a diversity of functions during plant growth and development process. Previous transcriptome analyses of seed development in grape hybrids suggested that specific homeodomain transcription factors are involved in seed development in seedless cultivars. However, the molecular mechanism of homeobox gene regulating seed development in grape is rarely reported. RESULTS:Here, we report that the grapevine VvHB58 gene, encoding a homeodomain-leucine zipper (HD-Zip I) transcription factor, participates in regulating fruit size and seed number. The VvHB58 gene was differentially expressed during seed development between seedless and seeded cultivars. Subcellular localization assays revealed that the VvHB58 protein was located in the nucleus. Transgenic expression of VvHB58 in tomato led to loss of apical dominance, a reduction in fruit pericarp expansion, reduced fruit size and seed number, and larger endosperm cells. Analysis of the cytosine methylation levels within the VvHB58 promoter indicated that the differential expression during seed development between seedless and seeded grapes may be caused by different transcriptional regulatory mechanisms rather than promoter DNA methylation. Measurements of five classic endogenous hormones and expression analysis of hormone-related genes between VvHB58 transgenic and nontransgenic control plants showed that expression of VvHB58 resulted in significant changes in auxin, gibberellin and ethylene signaling pathways. Additionally, several DNA methylation-related genes were expressed differentially during seed development stages in seedless and seeded grapes, suggesting changes in methylation levels during seed development may be associated with seed abortion. CONCLUSION:VvHB58 has a potential function in regulating fruit and seed development by impacting multiple hormonal pathways. These results expand understanding of homeodomain transcription factors and potential regulatory mechanism of seed development in grapevine, and provided insights into molecular breeding for grapes.
Project description:The large-scale mining and high-throughput genotyping of novel gene-based allelic variants in natural mapping population are essential for association mapping to identify functionally relevant molecular tags governing useful agronomic traits in chickpea. The present study employs an alternative time-saving, non-laborious and economical pool-based EcoTILLING approach coupled with agarose gel detection assay to discover 1133 novel SNP allelic variants from diverse coding and regulatory sequence components of 1133 transcription factor (TF) genes by genotyping in 192 diverse desi and kabuli chickpea accessions constituting a seed weight association panel. Integrating these SNP genotyping data with seed weight field phenotypic information of 192 structured association panel identified eight SNP alleles in the eight TF genes regulating seed weight of chickpea. The associated individual and combination of all SNPs explained 10-15 and 31% phenotypic variation for seed weight, respectively. The EcoTILLING-based large-scale allele mining and genotyping strategy implemented for association mapping is found much effective for a diploid genome crop species like chickpea with narrow genetic base and low genetic polymorphism. This optimized approach thus can be deployed for various genomics-assisted breeding applications with optimal expense of resources in domesticated chickpea. The seed weight-associated natural allelic variants and candidate TF genes delineated have potential to accelerate marker-assisted genetic improvement of chickpea.
Project description:The breeding of new soft-seeded pomegranate cultivars provides new products for the market and increases farmers' incomes, yet the genetic architecture mediating seed hardness is largely unknown. Here, the seed hardness and hundred-seed weights of 26 cultivars were determined in 2 successive years. We conducted miRNA and mRNA sequencing to analyse the seeds of two varieties of Punica granatum: soft-seeded Tunisia and hard-seeded Sanbai, at 60 and 120 d after flowering. Seed hardness was strongly positively correlated with hundred-seed weight. We detected 25 and 12 differentially expressed miRNA-mRNA pairs with negative regulatory relationships between the two genotypes at 60 and 120 d after flowering, respectively. These miRNA-mRNA pairs mainly regulated seed hardness by altering cell wall structure. Transcription factors including NAC1, WRKY and MYC, which are involved in seed hardness, were targeted by differentially expressed mdm-miR164e and mdm-miR172b. Thus, seed hardness is the result of a complex biological process regulated by a miRNA-mRNA network in pomegranate. These results will help us understand the complexity of seed hardness and help to elucidate the miRNA-mediated molecular mechanisms that contribute to seed hardness in pomegranate.