A new multiplex PCR test for the determination of Vrn-B1 alleles in bread wheat (Triticum aestivum L.)

Winter wheat requires vernalization, a long exposure to low but non-freezing temperatures, to promote reproductive development. The vernalization requirement in bread wheat (Triticum aestivum L.) is mainly controlled by the Vrn-1 genes that are located on chromosomes 5A, 5B and 5D. Dominant alleles confer spring habit and are epistatic to the recessive winter alleles which means that spring varieties carry at least one dominant allele. To date, two dominant and one recessive Vrn-B1 alleles have been described. Vrn-B1a (formerly designated as Vrn-B1) differs from the winter vrn-B1 allele by a large deletion in intron 1. Vrn-B1b has an additional small deletion and is probably derived from Vrn-B1a. The novel allele described here and designated as Vrn-B1c also has a large deletion within intron 1 but with different breakpoints from Vrn-B1a or b, and sequence duplication, showing that this is an independently derived spring allele. By combining an exon 1 primer with previously published PCR primers it was possible to develop a multiplex PCR that distinguished all four alleles simultaneously. The multiplex PCR was validated by testing 320 winter wheat and 137 spring wheat varieties. This demonstrated that the novel Vrn-B1c allele was present in 25 spring varieties of diverse origin, showing this allele to be widely distributed.