Real-time PCR-mediated diagnosis of hemizygosity at the Tamyb10-D1 locus controlling grain color in wheat

Grain color of wheat is one of the most important economic traits, affecting both flour quality and grain dormancy. As grain color reflects the phenotype of the seed coat, which is a maternal tissue, genotyping of this trait in segregating F₂ plants requires an F₃ progeny test. Consequently, the development of co-dominant molecular markers for grain color enables genotyping on a single grain basis and would expedite wheat breeding. R-1 genes control grain color and have been identified as MYB-type transcription factors, denoted as Tamyb10, which reside on the 3A, 3B, and 3D chromosomes, respectively. In an earlier study we developed co-dominant markers for R-A1 and R-B1 on chromosomes 3A and 3B, but could only develop one dominant marker for R-D1 because the mutant allele deleted the entire Tamyb10-D1 fragment with an unknown deletion size. DNA of individuals of the F₂ population derived from crossing the white-grained line, 1–117, and the red-grained line, OW104, was isolated and used for real-time PCR. Relative amplification ratios of Tamyb10-D1 to Tamyb10-B1 in each of 96 individuals showed a bimodal distribution without overlapping, which allowed unequivocal classification of the F₂ plants into a 1.0 (dominant homozygous) or a 0.5 (heterozygous) class, in accordance with a 1:2 expected ratio. Genotypes with the R-D1 grain color (4 plants per each class) were tested by an F₃ progeny test. The results perfectly coincided with the genotypes deduced by the real-time PCR diagnosis. Consequently, the hemizygous status at the Tamyb10-D1 locus controlling grain color in wheat can now be diagnosed with the real-time PCR.