The wide range of flower colors in soybean is controlled by six independent loci (W1, W2, W3, W4, Wm, and Wp). Among these loci, mutations in the W3 locus under the w4 allelic background (i.e., w3w4) produce near-white flowers, while the W3w4 genotype produces purple throat flowers. Although a gene encoding dihydroflavonol 4-reductase, DFR1, has been known to be closely associated with the W3 locus, its molecular identity has not yet been characterized. In the present study, we aimed to determine whether DFR1 is responsible for allelic variations in the W3 locus. On the basis of the sequence of a DFR probe, Glyma.14G072700 was identified as a candidate gene for DFR1, and nucleotide sequences of Glyma.14G072700 from cultivars with previously validated genotypes for the W3 locus were determined. As a result, a number of nucleotide polymorphisms, mainly single-base substitutions, between both coding and 5′-upstream region sequences of the W3 and w3 alleles were identified. Among them, an indel of 311-bp in the 5′-upstream region was noteworthy, since the Glyma.14G072700 in all the w3 alleles examined contained the indel, whereas that in all the W3 alleles did not; the former was barely expressed, but the latter was well expressed. These results suggest that Glyma.14G072700 is likely to correspond to DFR1 for the W3 locus and that its expression patterns may lead to allelic color phenotypes of W3 and w3 alleles under the w4 allelic background.

Cultivated soybeans [Glycinemax(L.) Merr.] have various flower colours such as dark purple, purple, light purple, pink, magenta, near white and white. About one-third of the soybean accessions in the United States Department of Agriculture – Germplasm Resource Information Network (USDA-GRIN) Soybean Germplasm Collections have white flowers and are the second dominant accessions after the purple-flowered accessions. Earlier studies have shown that thew1recessive allele of theW1gene encoding flavonoid 3′,5′-hydroxylase produces white flowers. In the present study, we aimed to understand why the white-flowered accessions have become abundant among the cultivated soybeans and what their genetic and regional origin is. For this purpose, 99 landraces with white flowers and 39 landraces with purple flowers from eight Asian countries and Russia were analysed with regard to the nucleotide sequences of theW1locus. We not only found that thew1alleles of the 99 white-flowered landraces were identical to those of the white-flowered Williams 82, but also found that thesew1alleles displayed no polymorphism at all. By carrying out a phylogenetic analysis, we were able to identify a group withW1alleles from which thew1allele might have diverged.

In soybean, triterpenoid saponin is one of the major secondary metabolites and is further classified into group A and DDMP saponins. Although they have known health benefits for humans and animals, acetylation of group A saponins causes bitterness and gives an astringent taste to soy products. Therefore, several studies are being conducted to eliminate acetylated group A saponins. Previous studies have isolated and characterized the Sg-5 (Glyma.15g243300) gene, which encodes the cytochrome P450 72A69 enzyme and is responsible for soyasapogenol A biosynthesis. In this study, we elucidated the molecular identity of a novel mutant of Glycine soja, ′CWS5095′. Phenotypic analysis using TLC and LC-PDA/MS/MS showed that the mutant ′CWS5095′ did not produce any group A saponins. Segregation analysis showed that the absence of group A saponins is controlled by a single recessive allele. The locus was mapped on chromosome 15 (4.3 Mb) between Affx-89193969 and Affx-89134397 where the previously identified Glyma.15g243300 gene is positioned. Sequence analysis of the coding region for the Glyma.15g243300 gene revealed the presence of four SNPs in ′CWS5095′ compared to the control lines. One of these four SNPs (G1127A) leads to the amino acid change Arg376Lys in the EXXR motif, which is invariably conserved among the CYP450 superfamily proteins. Co-segregation analysis showed that the missense mutation (Arg376Lys) was tightly linked with the absence of group A saponins in ′CWS5095′. Even though Arg and Lys have similar chemical features, the 3D modelled protein structure indicates that the replacement of Arg with Lys may cause a loss-of-function of the Sg-5 protein by inhibiting the stable binding of a heme cofactor to the CYP72A69 apoenzyme.