Molecular analysis of two novel missense mutations in the sg-1 gene associated with group a saponin biosynthesis in soybean

Soybean [Glycine max (L.) Merr.] accumulates several different types of saponins, including the Aa and Ab series of saponins, which contribute to the bitter and astringent aftertaste in soy food products. Modification of the soybean genome is being applied to develop bitter-free soybean cultivars. In this study, two new ethyl-meth-anesulfonate (EMS)-induced mutants, PE937 and PE1015, were characterized for their ability to accumulate the bitter-free A0-αg saponin and low level of the bitter Ab-series saponins compared with the wild cultivar. These pheno-types resemble that of previously reported sg-1° mutants that accumulate A0-αg and no Ab-series saponins, which are controlled by the Sg-1^b (Glyma.07G254600) gene. Sequence analysis of Sg-1^b in PE937 and PE1015 revealed that each mutant has a single-nucleotide polymorphism that causes an amino acid change from Gly₃₄₉ to Asp₃₄₉ and from Gly₂₄₁ to Asp₂₄₁ in the Sg-1^b proteins. We therefore designated these mutations as alleles sg-1^b1 and sg-1^b2, respectively. Multiple alignment analysis showed that the Gly₃₄₉ and Gly₂₄₁ are highly conserved among the UDP-dependent glycosyltransferase (UGT) proteins. In light of the above results, the fact that there are low levels of Ab-series saponins in both of the mutants indicates that these mutations may reduce the UGT activity of Sg-1^b proteins in the biosynthesis of Ab-series saponins. Cosegregation analysis revealed that the novel sg-1^b1 and sg-1^b2 variants are recessive to Sg-1^b and are tightly linked to A0-αg saponin accumulation.