VRN1-ratio test for polyploid wheat.

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conclusionThe duplications of the dominantVrn-A1alleles as well as theVRN-B1gene, revealed for the first time, are new sources of polymorphism in polyploid wheat at these agronomically valuable genomic locations. Flowering time is an important trait in wheat breeding. In spring wheat, this feature is mainly determined by the variants and number of the homoeologous dominant VRN1 alleles. Previously, multiplication of the recessive vrn-A1 allele was shown for winter hexaploid wheat (Würschum et al., BMC Genet 29:16-96, 2015). In the present study, VRN1 gene copy-number variation as well as the copy number of VRN-A1 with the alternative exon 4 haplotype were investigated in spring and winter accessions of different tetraploid and hexaploid wheat species. Two ratio tests were optimized based on end-point quantification of PCR fragments and results were verified by a qPCR assay. It was defined that since the genomic environment affects the accessibility of amplified VRN1 regions, the DNA template should be fragmented for proper quantification of VRN1 copy number during PCR-based assays. For the first time, it was shown that the dominant Vrn-A1 alleles are most often duplicated in hexaploid wheat. In tetraploid wheat, both the dominant and recessive alleles were represented as a single haploid copy, and in only two accessions of T. dicoccum, vrn-A1b.3 was duplicated. Multiplication of VRN-A1 was often associated with awnless spikes. Five haploid combinations of the recessive vrn-A1 copies with alternative exon 4 were identified in hexaploid wheat. Finally for the first time, duplication of VRN-B1 was found in hexaploid wheat of T. compactum and T. spelta. These results expand our knowledge of the genetic diversity of VRN1 genes in wheat and provide additional strategies for the manipulation of flowering time in this strategic crop.