Figure 4.

The gene haplotype-based LD/association mapping, expression profiling and PA content estimation in a strong seed coat color-associated MATE secondary transporter kabuli gene (Ca18123), validating its potential for seed coat color regulation in chickpea. The genotyping of four SNP (one non-synonymous SNPs) and one SSR markers in different coding and non-coding sequence components of gene (A) among 172 cultivated and wild chickpea accessions constituted four haplotypes (B). The haplotypes I: [C-(GTTG)₃-T-T-A] and II: [C-(GTTG)₃-G-T-A] discriminated by one non-synonymous SNP (T/G at 1708 bp, yellow shaded) in the CDS of gene showed strong association potential for BE and LB/YB seed coat color differentiation, respectively. The four haplotype marker-based genotyping information imparted higher LD estimate covering the entire 6843 bp sequenced region of the gene (C). The columns below the diagonal denote the correlation frequency (r²) among a pair of four different haplotypes constituted in a gene, whereas columns above the diagonal indicate the P-value significance (P < 0.01) of LD estimates (r²) for these haplotype combinations at 1000 permutation. (D) The differential expression of BE and YB/LB seed color-associated MATE gene haplotypes (I and II) in the mature seed coats of accessions, mapping parents and homozygous RIL individuals representing BE and LB/YB seed colored haplotype groups. The elongation factor-1 alpha gene was utilized as an internal control in the RT-PCR assay. (E) The soluble and insoluble PA contents (nmol/mg seeds) estimated in the mature seed coats of desi and kabuli chickpea accessions, mapping parents and homozygous RIL individuals representing BE and LB/YB seed colored haplotypes. Each bars represent the mean (± standard error) of three independent biological replicates along with two technical replicates for each sample used in RT-PCR and PA estimation assays. ^*Significant differences in expression of gene haplotypes in mature seeds of LB/YB and BE seed colored accessions as compared to their leaf and seed (without seed coats) tissues at p < 0.001 (LSD-ANOVA significance). ^*Significant variation in PA contents in the matured seeds of LB/YB seed colored accessions as compared to BE seed coat colored accessions at p < 0.001 (ANOVA significance). YBH (yellow brown homozygous) and BEH (beige homozygous) RIL mapping individuals.