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

item Gillman, Jason
item TETLOW, ASHLEY - University Of Missouri
item DEONG-LEE, JEONG - Kyungpook National University
item SHANNON, GROVER - University Of Missouri
item Bilyeu, Kristin

Submitted to: BMC Plant Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/9/2011
Publication Date: 11/9/2011
Citation: Gillman, J.D., Tetlow, A., Deong-Lee, J., Shannon, G.J., Bilyeu, K.D. 2011. Loss-of-function mutations affecting a specific Glycine max R2R3 MYB transcription factor result in brown hilum and brown seed coats. Biomed Central (BMC) Plant Biology. 11:155. Available:

Interpretive Summary: Seed producers have an obligation to maintain the purity of their seed stocks, especially when they contain novel technologies. The manipulation of seed color is a perfect way to mark a specific variety and one that allows the producer to quickly certify a seed lot as pure. Soybean seeds of modern varieties are almost exclusively yellow, with the only contrasting color present at the hilum, which is a small area of connection between the mother plant and the developing seed. Hilum coloration provides a ready means to detect contaminant seed in commercial seed lots. Over the last few decades, the genetics behind many of the unusual colors seen in soybean seed collections have been determined. One of the unusual seed colorations present in seed stocks is that of a brown seed coat and brown hilum. This combination has been determined to be the result of a single recessive locus but the specific molecular basis is of yet undetermined. We utilized resources derived from the recent sequencing of the soybean genome, along with classic genetic analysis methods, to identify mutations that disable a single gene regulator as being responsible for the brown hilum/seed coat coloration. We discovered multiple mutations exclusively in cultivated soybean lines (which are all yellow), but none were identified in wild soybeans. This suggests that ancient farmers/breeders may have selected this trait, perhaps for aesthetic reasons. This research furthers scientific understanding of the genetics behind soybean seed coloration, an important consideration for modern seed companies that rely on seed purity for their improved lines. In addition, because this trait is so widespread, it also provides a useful strategy for soybean breeders to follow and verify successful crossing attempts as part during soybean improvement breeding effort, making such efforts more efficient.

Technical Abstract: Although 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. In 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 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, but no upregulation of the UF3GT gene when a null allele of Glyma09g36990 was expressed. We 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. 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.