Detection of SNPs in the VRN-A1 gene of common wheat (Triticum aestivum L.) by a modified Ecotilling method using agarose gel electrophoresis

Vernalization is one of the most important traits for the adaptability of wheat; detecting variation in genes regulating the response of wheat to vernalization is of importance for classifying winter phenotypes. Ecotilling is a high throughput reverse genetics approach for detecting single nucleotide polymorphisms (SNPs) in the genes of a natural population. To simplify methodology and decrease costs, a modified Ecotilling method based on agarose gel electrophoresis was used to discover the occurrence of SNPs in the wheat vernalization gene VRN-A1 in a natural population of 106 accessions of hexaploid wheat varieties and their association with the winter/spring phenotype. Based on the SNPs detected, these accessions fell into three haplotypes, namely, 'A', 'B' and 'C', each with five, six, and three variations, respectively. The winter phenotype was mainly determined by haplotype 'A' and 'B', while the spring phenotype was mainly determined by haplotype 'C'. After sequence confirmation, three SNPs ('A/-', in the fourth intron; '-/A', in the sixth intron; 'T/C', in the seventh exon) were associated with wheat vernalization and another three mutations ('G/T' in the second intron; 'T/C' in the fourth exon; 'CA/--', in the fourth intron) had no identified effect. Therefore, Ecotilling with detection of DNA fragments by agarose gel electrophoresis can be used as an efficient method for detecting SNPs in genes of polyploidy materials, such as wheat.