Genome-wide conserved non-coding microsatellite (CNMS) marker-based integrative genetical genomics for quantitative dissection of seed weight in chickpea

Abstract

Phylogenetic footprinting identified 666 genome-wide paralogous and orthologous CNMS (conserved non-coding microsatellite) markers from 5'-untranslated and regulatory regions (URRs) of 603 protein-coding chickpea genes. The (CT)n and (GA)n CNMS carrying CTRMCAMV35S and GAGA8BKN3 regulatory elements, respectively, are abundant in the chickpea genome. The mapped genic CNMS markers with robust amplification efficiencies (94.7%) detected higher intraspecific polymorphic potential (37.6%) among genotypes, implying their immense utility in chickpea breeding and genetic analyses. Seventeen differentially expressed CNMS marker-associated genes showing strong preferential and seed tissue/developmental stage-specific expression in contrasting genotypes were selected to narrow down the gene targets underlying seed weight quantitative trait loci (QTLs)/eQTLs (expression QTLs) through integrative genetical genomics. The integration of transcript profiling with seed weight QTL/eQTL mapping, molecular haplotyping, and association analyses identified potential molecular tags (GAGA8BKN3 and RAV1AAT regulatory elements and alleles/haplotypes) in the LOB-domain-containing protein- and KANADI protein-encoding transcription factor genes controlling the cis-regulated expression for seed weight in the chickpea. This emphasizes the potential of CNMS marker-based integrative genetical genomics for the quantitative genetic dissection of complex seed weight in chickpea.

Keywords: CNMS; Chickpea; Cicer arietinum; SNPs; eQTLs; microsatellites; seed weight; transcription factor..

Fig. 1.

Development of CNMS markers from the known regulatory elements/TFBS present in the sequences upstream of the translation initiation codons (ATG) of protein-coding chickpea genes. The forward and reverse primers designed from the sequences flanking the (GA)_n and (CT)_n CNMS repeat motifs carrying known GAGA8HVBKN3 and CTRMCAMV35S regulatory elements/TFBS of LOB- (A) and homeobox (B) domain protein-encoding TF genes, respectively, were used to develop CNMS markers in chickpea. (This figure is available in colour at JXB online.)

Fig. 2.

(A) Distribution frequency of CNMS repeat motifs containing gene regulatory elements in the chickpea genome. (B) Proportionate distribution of different CNMS repeat motif classes on the eight chickpea chromosomes. Dinucleotide CNMS repeats (45.6%) were the abundant microsatellite classes, followed by trinucleotide CNMS (30%) in the chickpea genome. (C) Positional distribution of CNMS repeat motifs carrying regulatory elements in the non-coding sequences upstream of the translation initiation codons (ATG) of chickpea genes. (D) Proportionate distribution of different CNMS repeat motifs containing gene regulatory elements on the eight chickpea chromosomes. The detailed characteristics of these regulatory elements and CNMS are described in Supplementary Table S2 at JXB online. (This figure is available in colour at JXB online.)

Fig. 3.

Allelic variation detected among a representative set of 20 desi and kabuli genotypes using the normal unlabelled and fluorescent dye-labelled CNMS marker designed from the flanking sequences of the (GAA)₁₈ repeat motif present in the signal sequence-binding site (CTGAAGAAGAA) of the TL1ATSAR regulatory element containing the bZIP (basic leucine zipper) TF gene by gel-based assay (A) and automated fragment analyser (B), respectively. A maximum of four polymorphic alleles were amplified by the genic CNMS marker among chickpea genotypes using the gel-based assay (A) and automated fragment analyser (B), respectively. The fragment sizes (bp) of all amplified polymorphic alleles are indicated. The identities of genotypes correspond to serial numbers that are provided in Supplementary Table S1 at JXB online. (This figure is available in colour at JXB online.)

Fig. 4.

Multiple sequence alignments of cDNA and genomic DNA amplicons amplified from 10 low and high/very high seed weight homozygous RIL mapping individuals, parental genotypes (ICC 4958 and ICC 17163), and 12 contrasting germplasm lines using the two seed weight-regulating differentially expressed CNMS marker-associated LOB-domain proten- (A) and KANADI protein-encoding (B) genes validated the presence of microsatellite repeat motifs. The presence of variable CNMS repeat units, such as (GA)_n and (CAA)_n, in the signal sequence-binding sites of the GAGA8HVBKN3 and RAV1AAT regulatory elements of these two genes, respectively, which differentiated all of the low seed weight germplasm lines, parents, and homozygous mapping individuals (amplifying 158bp and 150bp alleles) from the high/very high seed weight germplasm lines, parents, and homozygous RILs (160/162bp and 156bp alleles) was evident. HSL, homozygous lines. (This figure is available in colour at JXB online.)

Fig. 5.

Hierarchical cluster display of the expression profile for 17 CNMS marker-associated genes showing high levels of seed-specific expression at two seed developmental stages/tissues compared with vegetative leaf tissues of three contrasting low and high/very high seed weight chickpea genotypes (ICCX-810800, ICC 4958, and ICC 20268). The scale at the top represents the average log of the signal expression values (expression levels) of the genes in the various tissues and developmental stages. The tissues/seed developmental stages of the contrasting genotypes and CNMS marker-associated genes that were used for the expression profiling analysis are indicated on the right side and top of the expression map, respectively. Details of CNMS marker-associated genes are given in Table 1. Seven CNMS marker-associated genes harbouring the seed weight QTLs/eQTLs are marked with arrows. S1, seed development stage 1 (10–20 DAP); S2, seed development stage 2 (21–30 DAP). The expression values across different tissues/developmental stages of genotypes were normalized against the endogenous control elongation factor-1 alpha in the quantitative RT-PCR assay. The gene expression in leaf tissues of all the genotypes was considered as the reference calibrator and assigned as 1. (This figure is available in colour at JXB online.)

Fig. 6.

Integration of the genetic and physical maps of the target genomic regions underlying seed weight QTLs and eQTLs identified on chickpea chromosomes 2 and 7. The target seed weight cis- and trans-eQTLs (LOD >5.0) for three CNMS marker-associated genes identified on chromosomes 2 and 7 are indicated. The genetic (cM) and physical (bp) distances and identities of the CNMS marker loci integrated on the chromosomes are indicated on the left and right sides of the chromosomes, respectively. (This figure is available in colour at JXB online.)

Fig. 7.

Differential expression profiling of seed weight-regulating CNMS marker-associated LOB-domain protein- (A) and KANADI (B) protein-encoding genes at two seed developmental stages of 10 low and high/very high seed weight homozygous RIL mapping individuals, parental genotypes (ICC 4958 and ICC 17163), and 12 contrasting germplasm lines compared with their vegetative leaf tissues using quantitative RT-PCR assay. HL, homozygous lines; S1, seed development stage 1 (10–20 DAP); S2, seed development stage 2 (21–30 DAP). The expression values across different tissues/developmental stages of germplasm lines, parents, and mapping individuals were normalized against the endogenous control elongation factor-1 alpha in the quantitative RT-PCR assay. The gene expression in leaf tissues (indicated as All-Leaf) of all the germplasm lines and mapping individuals was considered as the reference calibrator and assigned as 1. Each bar represents the mean (± SE) of three independent biological replicates with two technical replicates for each sample used in the quantitative RT-PCR assay. *Significant differences in expression of CNMS marker-associated genes at two seed developmental stages of low and high seed weight germplasm lines, parents, and mapping individuals as compared with leaf at P<0.01 (LSD-ANOVA significance test). (This figure is available in colour at JXB online.)

Fig. 8.

The seed weight-specific molecular haplotyping, LD mapping, genetic association analysis, and gene haplotype-specific expression profiling of a CNMS marker-associated LOB-domain protein-encoding TF gene, validating its potential for seed weight regulation in chickpea. The genotyping of one CNMS marker in the regulatory element/TFBS and one synonymous SNP (A/G) identified in the coding sequence component of this TF gene (A) among 96 contrasting low and high seed weight germplasm lines (association panel) constituted six haplotypes (B). Three specific haplotype (B) groups [(GA)₁₂-A/G], [(GA)₁₃-A/G], and [(GA)₁₄-A/G], which were represented by 42, 23, and 31 germplasm lines showed significant association potentials for low (100 seed weight varied from 5g to 13g) and high (31–41g)/very high (47–55g) seed weight differentiation (C), respectively. (D) The LD mapping with genotyping data of six haplotypes produced higher LD estimates (r ²>0.20 and P<0.0001) across the entire 3400bp sequenced region of the TF gene. The columns below the diagonal indicate the correlation frequency (r ²) among a pair of six different haplotypes constituted in a gene, whereas columns above the diagonal specify the P-value (P<0.01) of LD estimates (r ²) for these haplotype combinations at 1000 permutations. (E) The differential up-regulated expression of the haplotypes of this gene in seed developmental stages of low and high/very high seed weight germplasm lines, parents, and homozygous RIL mapping individuals representing haplotype groups compared with the leaf was also observed. Each bar represents the mean (± SE) of three independent biological replicates with two technical replicates for each sample used in the quantitative RT-PCR assay. *Significant differences in expression of gene haplotypes at two seed developmental stages of low and high seed weight germplasm lines, parents, and mapping individuals as compared with leaf at P<0.01 (LSD-ANOVA significance test). (This figure is available in colour at JXB online.)