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Dihydroflavonol-4-reductase (DFR) plays a crucial role in anthocyanin biosynthesis. In cultivated soybean [Glycine max (L.) Merr.], the W3 and W4 loci encode DFR1 and DFR2, respectively and are epistatic to each other. In this study, we discovered a new flower color variant of Glycine soja (L.) Merr., CW13133, which has pinkish-white flowers and investigated the genetic and molecular basis of the variation in flower color. We found that the W4 locus encoding DFR2 was responsible for the variation in flower color. Sequence analysis of the DFR2 gene of CW13133 revealed three single-nucleotide polymorphisms (SNPs). Among these, an SNP (T to C) at nucleotide position 2856 was noteworthy, since it led to a change in an amino acid residue (leucine to proline) at position 326 of DFR2. This amino acid substitution was located in a region that is highly conserved among different plant species. The new allele was designated w4-s1. A cleaved amplified polymorphic sequence analysis showed cosegregation of the w4-s1 allele with pinkish-white flowers. Interestingly, DFR1 was not expressed in the w4-s1 mutant, indicating that the W3 locus has no association with flower color variation. Based on a randomized comparative analysis using 10 different purple-flowered accessions of G. soja, we provide evidence for the nonassociation of DFR1 expression with total anthocyanin contents and flower color determination in G. soja. In conclusion, we found that the W3 locus in G. soja does not contribute to flower color determination and consequently there is no epistatic interaction between the W3 and W4 loci, as shown in G. max. [ABSTRACT FROM AUTHOR]

An ethyl methanesulfonate-induced mutant (PE1033) with near-white flowers was isolated from the soybean [Glycine max (L.) Merr.] cultivar Pungsannamul with purple flowers that had the W1w3W4 genotype. In the present study, we aimed to investigate the genetic and molecular bases of the flower color variation displayed by the PE1033 mutant. Analyses of genomic sequences and reverse transcription-polymerase chain reaction (RT-PCR) revealed that the color variation of PE1033 flowers originated from a recessive mutation in the W4 locus that encodes dihydroflavonol 4-reductase (DFR) 2. The new mutant allele, named w4-nw, harbored a single-nucleotide substitution (G to A) at the 5' spliceosome recognition site in the first intron of DFR2, resulting in the failure of splicing and producing two different abnormal transcripts: w4-nw-a and w4-nw-b. The former encoded a truncated DFR2 protein because of the occurrence of a premature stop codon in the first intron, which was retained in the transcript; the latter resulted from alternative splicing of the first intron and encoded a DFR2 protein that lacked 11 amino acid residues in the N-terminus. Derived cleaved amplified polymorphism sequence (dCAPS) analysis revealed a tight cosegregation pattern between the w4-nw mutant allele and near-white color phenotype. In conclusion, the novel mutation in the W4 locus, w4-nw, caused the production of near-white flowers under the w3 allelic background of PE1033. [ABSTRACT FROM AUTHOR]

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 the w1 recessive allele of the W1 gene 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 the W1 locus. We not only found that the w1 alleles of the 99 white-flowered landraces were identical to those of the white-flowered Williams 82, but also found that these w1 alleles displayed no polymorphism at all. By carrying out a phylogenetic analysis, we were able to identify a group with W1 alleles from which the w1 allele might have diverged. [ABSTRACT FROM AUTHOR]

The determination of flower color mainly depends on the anthocyanin biosynthesis pathway and vacuolar pH; however, unlike the former, the mechanism of vacuolar acidification in soybean remains uncharacterized at the molecular level. To investigate this mechanism, we isolated four recessive purple-blue EMS-induced flower mutants from the purple flower soybean cultivar, Pungsannamul. The petals of all the mutants had increased pH compared with those of wild Pungsannamul. One of the mutants had a single nucleotide substitution in GmPH4 , a regulator gene encoding an MYB transcription factor, and the substitution resulted in a premature stop codon in its first exon. The other three mutants had nucleotide substitutions in GmPH5 , a single new gene that we identified by physical mapping. It corresponds to Glyma.03G262600 in chromosome 3 and encodes a proton pump that belongs to the P 3A -ATPase family. The substitutions resulted in a premature stop codon, which may be a defect in the ATP-binding capacity of GmPH5 and possibly a catalytic inefficiency of GmPH5. The result is consistent with their genetic recessiveness as well as the high pH of mutant petals, suggesting that GmPH5 is directly involved in vacuolar acidification. We also found that the expression of GmPH5 and several putative "acidifying" genes in the gmph4 mutant was remarkably reduced, indicating that GmPH4 may regulate the genes involved in determining the vacuolar pH of soybean petals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Sundaramoorthy, Park, Lee, Kim, Seo and Song.)