Comparative genomics and association mapping approaches for opaque2 modifier genes in finger millet accessions using genic, genomic and candidate gene-based simple sequence repeat markers

Identification of alleles responsible for opaque2 modifiers (Opm) influencing tryptophan content in finger millet is a major aim for further improvement of the quality of the locally adapted finger millet germplasm. Since there is little genome sequence information available, comparative genomics plays a very important role in identification of genes/quantitative trait loci (QTLs) linked to the Opm genes using simple sequence repeat (SSR) markers. In the present study, a total of 74 genic SSRs were developed and then used for genetic diversity and population structure analysis of a global collection of 190 finger millet genotypes. The 74 SSRs yielded 133 scorable alleles and the polymorphism information content values varied from 0.186 to 0.707, with an average of 0.408. The gene diversity was in the range of 0.208–0.752, with an average of 0.501. The SSRs developed from the aspartate kinase2 gene of the lysine pathway showed more polymorphism than the other candidate genes. The 74 genic SSR loci grouped the 190 finger millet genotypes into three major clusters based on their tryptophan content, using both phylogenetic clustering and population structure analysis by STRUCTURE software. Association mapping for Opm was done using 120 (74 genic and 46 genomic) SSR loci for identification of QTLs linked to Opm influencing tryptophan content, and found two QTLs for tryptophan and one QTL for protein content. The QTLs for tryptophan content were associated with the genic marker OM5 at a P value of 0.009 and explained 11 % of phenotypic variance (R²). The OM5 marker was designed from the 27-kDa γ-zein gene of Opm, which influences the tryptophan content to a large extent, whereas the genomic marker FM8 was linked at a P value of 0.004 and explained 9 % of R². The QTLs for protein content were associated with the genic SSR marker FMO2EST1, which was designed from the RISBZ1 gene of rice and was linked at a P value of 0.002 and explained 9 % of R². The 220-bp allele of SSR locus OM5 was found to be present mostly in the high tryptophan-containing genotypes such as exotic genotypes, and among the Indian genotypes it was present in NW Himalayan genotypes. The markers linked to the QTLs for Opm found in the present study can be further used for cloning of the full-length gene, for fine mapping and in the marker-assisted breeding programmes for introgression of alleles into locally well-adapted germplasm.