An integrated genomic approach for rapid delineation of candidate genes regulating agro-morphological traits in chickpea

Abstract

The identification and fine mapping of robust quantitative trait loci (QTLs)/genes governing important agro-morphological traits in chickpea still lacks systematic efforts at a genome-wide scale involving wild Cicer accessions. In this context, an 834 simple sequence repeat and single-nucleotide polymorphism marker-based high-density genetic linkage map between cultivated and wild parental accessions (Cicer arietinum desi cv. ICC 4958 and Cicer reticulatum wild cv. ICC 17160) was constructed. This inter-specific genetic map comprising eight linkage groups spanned a map length of 949.4 cM with an average inter-marker distance of 1.14 cM. Eleven novel major genomic regions harbouring 15 robust QTLs (15.6-39.8% R(2) at 4.2-15.7 logarithm of odds) associated with four agro-morphological traits (100-seed weight, pod and branch number/plant and plant hairiness) were identified and mapped on chickpea chromosomes. Most of these QTLs showed positive additive gene effects with effective allelic contribution from ICC 4958, particularly for increasing seed weight (SW) and pod and branch number. One robust SW-influencing major QTL region (qSW4.2) has been narrowed down by combining QTL mapping with high-resolution QTL region-specific association analysis, differential expression profiling and gene haplotype-based association/LD mapping. This enabled to delineate a strong SW-regulating ABI3VP1 transcription factor (TF) gene at trait-specific QTL interval and consequently identified favourable natural allelic variants and superior high seed weight-specific haplotypes in the upstream regulatory region of this gene showing increased transcript expression during seed development. The genes (TFs) harbouring diverse trait-regulating QTLs, once validated and fine-mapped by our developed rapid integrated genomic approach and through gene/QTL map-based cloning, can be utilized as potential candidates for marker-assisted genetic enhancement of chickpea.

Keywords: QTLs; SNP; SSR; chickpea; transcription factor; wild.

Figure 1.

Nineteen genomic regions harbouring 27 significant QTLs (15.6–39.8% R²) associated with SW, NP, NB and PH were identified and mapped on eight LGs (LOD > 4.0 at P < 0.05) using a 229 RIL mapping population (ICC 4958 × ICC 17160) of chickpea. The genetic distance (cM) and identity of the marker loci integrated on the chromosomes are indicated on the left and right sides of the LGs, respectively. The 15 major and robust QTLs are marked with yellow boxes. The marker pairs flanking the QTLs are coloured with red and blue. Blue, violet, green and brown lines indicate the QTLs regulating SW, NB, NP and PH mapped on eight LGs. The direction of QTLs (additive effects) are designated with empty (ICC 17160-specific alleles) vs. filled (ICC 4958-specific alleles) boxes. *qSW2.1, qSW4.1, qNP8.1 and qNB1.2 correspond to known QTLs from previous studies by Cobos et al.,^, Hossain et al., Varshney et al. and Gowda et al. This figure appears in colour in the online version of DNA Research.

Figure 2.

Integration of genetic (A) and physical (B) map of target genomic region harbouring one robust SW-regulating major QTL (qSW4.2) identified and mapped QTL on 239.1 kb sequence interval (indicated by red-coloured flanking marker pairs) of chickpea chromosome 4. This QTL was further delimited to an ∼62.7 kb sequenced region on chromosome 4 by integrating traditional QTL mapping with QTL region-specific association analysis (B). Five protein-coding candidate genes annotated within 62.7 kb sequenced interval between markers CaSNP121 and CaSNP194 (marked with blue colour), of which ABI3VP1 TF gene showed strong association with SW in chickpea (C). The genetic (cM)/physical (bp) distance and identity of the markers mapped on the chromosomes are indicated on the left and right sides of the chromosomes, respectively. The green-coloured markers are derived from the chickpea genes annotated at 62.7 kb QTL (qSW4.2) interval. The markers genetically and physically mapped on chickpea chromosome 4 are coloured with black. This figure appears in colour in the online version of DNA Research.

Figure 3.

The molecular haplotyping, LD mapping and gene haplotype-specific association analysis in an ABI3VP1 TF gene validating its strong association potential for SW in chickpea. The genotyping of 16 SNPs including one non-synonymous SNP (T/C) [encoding valine (GTC) to alanine (GCC)] in the B3 functional domain and five regulatory SNPs in the URR of this gene (A) among 244 cultivated and 81 wild chickpea accessions constituted seven haplotypes (B). Thirty-seven low seed weight (1.2–3.7 g) accessions represented by single haplotype group 7 (AGAG) and two other haplotypes (GAGC) consisting 24 accessions of high seed weight (13–57.6 g) in the TF gene showed strong association potential for high and low SW differentiation. The seven SNP haplotype-based genotyping information produced higher LD estimates (r² > 0.60 and P < 0.0001) covering the entire 4,086 bp sequenced region of gene (C). The high (GAGC) and low (AGAG) seed weight-specific haplotypes constituted by four SNPs (shaded with yellow colour in B) in URR of TF gene are depicted (B). (D) The differential expression profiling of ABI3VP1 TF gene in high and low seed weight accessions during seed development compared with the leaf. A superior favourable high seed weight-regulating haplotype (GAGC) with increased transcript expression was identified in the URR of TF gene (D). Each bars represent the mean (±standard error) of three independent biological replicates with two technical replicates for each sample used in quantitative RT–PCR assay. *Significant differences in expression of gene haplotypes at two seed developmental stages of low and high seed weight accessions compared with leaf (LSD-ANOVA significance test at P < 0.01). This figure appears in colour in the online version of DNA Research.