Loss-of-function mutations affecting a specific Glycine max R2R3 MYB transcription factor result in brown hilum and brown seed coats

Abstract

BackgroundAlthough modern soybean cultivars feature yellow seed coats, with the only color variation found at the hila, the ancestral condition is black seed coats. Both seed coat and hila coloration are due to the presence of phenylpropanoid pathway derivatives, principally anthocyanins. The genetics of soybean seed coat and hilum coloration were first investigated during the resurgence of genetics during the 1920s, following the rediscovery of Mendel's work. Despite the inclusion of this phenotypic marker into the extensive genetic maps developed for soybean over the last twenty years, the genetic basis behind the phenomenon of brown seed coats (the R locus) has remained undetermined until now.ResultsIn order to identify the gene responsible for the r gene effect (brown hilum or seed coat color), we utilized bulk segregant analysis and identified recombinant lines derived from a population segregating for two phenotypically distinct alleles of the R locus. Fine mapping was accelerated through use of a novel, bioinformatically determined set of Simple Sequence Repeat (SSR) markers which allowed us to delimit the genomic region containing the r gene to less than 200 kbp, despite the use of a mapping population of only 100 F6 lines. Candidate gene analysis identified a loss of function mutation affecting a seed coat-specific expressed R2R3 MYB transcription factor gene (Glyma09g36990) as a strong candidate for the brown hilum phenotype. We observed a near perfect correlation between the mRNA expression levels of the functional R gene candidate and an UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) gene, which is responsible for the final step in anthocyanin biosynthesis. In contrast, when a null allele of Glyma09g36990 is expressed no upregulation of the UF3GT gene was found.ConclusionsWe discovered an allelic series of four loss of function mutations affecting our R locus gene candidate. The presence of any one of these mutations was perfectly correlated with the brown seed coat/hilum phenotype in a broadly distributed survey of soybean cultivars, barring the presence of the epistatic dominant I allele or gray pubescence, both of which can mask the effect of the r allele, resulting in yellow or buff hila. These findings strongly suggest that loss of function for one particular seed coat-expressed R2R3 MYB gene is responsible for the brown seed coat/hilum phenotype in soybean.

Methylation Affects Transposition and Splicing of a Large CACTA Transposon from a MYB Transcription Factor Regulating Anthocyanin Synthase Genes in Soybean Seed Coats

“…Based on a combined analysis of transcriptome and metabolome data, another recent study identified 20 anthocyanin, flavonoid and phenylpropanoid isogenes that were differentially expressed between black ( iRT ) and brown ( irT ) seeded soybean isolines [5]. This same study suggested that R locus candidate might be a transcription factor at the distal end of chromosome Gm09 at or near Glyma09g36840, 120 kb from the Glyma09g36983 MYB factor mapped by Gillman et al [4].…”

Overexpression of soybean R2R3-MYB transcription factor, GmMYB12B2, and tolerance to UV radiation and salt stress in transgenic Arabidopsis

“…An R1 MYB transcription factor, GmMYB176, regulates CHS8 expression and isoflavonoid synthesis in soybean (Yi et al, 2010). A loss-of-function mutation in a specific R2R3 MYB transcription factor gene (Glyma09g36990) in soybean resulted in a brown hilum and brown seed coat (Gillman et al, 2011). GmMYB-G20-1 is a candidate gene for the W2 locus, which generates a purple-blue color and a high vacuolar pH in soybean flower petals (Takahashi et al, 2011;Takahashi et al, 2013).…”

“…There are 244 typical R2R3-MYB proteins in a primary soybean genome data set (Du et al, 2012). However, only 12 members of the MYB family have been functionally characterized, thus far, in soybean (Miyake et al, 2003;Yang, 2007;Liao et al, 2008;Du et al, 2008;Libault et al, 2009;Yi et al, 2010;Gillman et al, 2011;Takahashi et al, 2011). The functions of most plant MYB genes are unknown.…”

Inheritance of R Locus Expressing Brown Hilum on Black Seed Coat in Soybean

“…One recessive gene conditioning the phenotype spread hilum in the T48 genotype was found. The expression of the spread hilum is dependent of locus T_, which controls pubescence color; therefore it occurs only in genotypes with brown pubescence (T_), which characterizes pleiotropic effect of this locus on the trait spread hilum [10].…”

“…More recently Gillman et al [10] identified the specific gene and causative basis behind the phenomenon of brown hilum/seed coat coloration in soybean (R locus), and discovered an allelic series of four loss of function mutations affecting our R locus gene candidate (R 2 R 3 MYB gene). The presence of each one of these mutations was perfectly correlated with the brown seed coat/hilum phenotype in a broadly distributed survey of soybean cultivars.…”