Detection of Snps in the VRN-A1 Gene of Common Wheat ('Triticum aestivum' L.) by a Modified Ecotilling Method Using Agarose Gel Electrophoresis
Chen, Liang; Wang, Shi-Qiang; Hu, Yin-Gang
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.
Keywords: Agarose gel electrophoresis; Ecotilling; SNP detection;
Vernalization; VRN-A1 gene; Wheat.
Abbreviations: CELI- a single-strand specific endonuclease
extracted from celery; EB- Ethidium bromide; Ecotilling- A high
throughput reverse genetics approach for detecting single nucleotide
polymorphisms in the genes of a natural population; N5AT5D- Chinese
Spring nulli-tetrasomic line without the 5A chromosome but with two
pairs of the 5D chromosomes; N5BT5DChinese Spring nulli-tetrasomic line
without the 5B chromosome but with two pairs of 5D chromosomes; N5DT5B-
Chinese Spring nulli-tetrasomic line without the 5D chromosome but with
two pairs of 5B chromosomes; SNPs- Single Nucleotide Polymorphisms; TAE-
Tris, Acetic acid and EDTA buffer for agarose gel electrophoresis;
Tilling- Targeting induced local lesions in genomes; VRN-A1- The
vernalization gene VRN1 in the 5A chromosome of hexaploid wheat.
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