Molecular analysis of glycinin genes in soybean mutants for development of gene-specific markers

Soybean mutant lines that differ in 11S glycinin and 7S β-conglycinin seed storage protein subunit compositions were developed. These proteins have significant influence on tofu quality. The molecular mechanisms underlying the mutant lines are unknown. In this study, gene-specific markers for five of the glycinin genes (Gy1 to Gy5) were developed using three 11S null lines, two A₄ null Japanese cultivars, Enrei and Raiden, and a control cultivar, Harovinton. Whereas gene-specific primers produced the appropriate products in the control cultivar for the Gy1, Gy2, Gy3 and Gy5 genes, they did not amplify in mutants missing the A_1aB₂, A₂B_1a, A_1b B_1b, and A₃B₄ subunits. However, ecotype targeting induced local lesions in genomes (EcoTILLING) and sequencing analysis revealed that the absence of the A₄ peptide in the mutants is due to the same point mutation as that in Enrei and Raiden. Selection efficiency of the gene-specific primer pairs was tested using a number of breeding lines segregating for the different subunits. Primer pairs specific to each of the Gy1, Gy2, Gy3, and Gy5 genes can be used to detect the presence or absence of amplification in normal or mutant lines. The Gy4 null allele can be selected for by temperature-switch PCR (TS-PCR) for identification of the A₄ (G4) null genotypes. In comparison to protein analysis by SDS-PAGE, gene-specific markers are easier, faster and more accurate for analysis, they do not have to use seed, and can be analyzed at any plant growth stage for marker-assisted selection.