SNPs genotyping of hexaploid wheat by the 'Allele-Specific Pyrosequencing'.

摘要

Single Nucleotide Polymorphisms (SNPs) are the most abundant variations in genome and have been quite useful markers for genotyping and mapping. In common wheat, because of its polyploid nature, it is difficult to identify the SNPs for genotyping of strains by the ordinary methods. To discover the locus specific SNPs among homoeologous genes of three genomes, we have developed wheat ESTs clustering pipeline (WECP) based on PHRAP and BLAST programs. The 116232 ESTs of common wheat (Triticum aestivum cv. Chinese Spring) were classified into 14906 gene groups. Then, we found haplotypes on each homoeologous sequence showing the locus specific polymorphism. These haplotypes can become the candidates for anchors of locus specific SNPs genotyping. In orderto detect SNPs of each homoeologous gene among wheat strains, Pyrosequencing~(TM) technology was adopted. We developed Allele-Specific Pyrosequencing (AS-Pyrosequencing) method in the hexaploid background by specific sequence extension using locus specific polymorphism. This method was possible to identify the SNPs as co-dominant pairs of peaks between wheat strains. For example, AS-Pyrosequencing allowed genotyping of the SNPs for purothionin gene among wheat strains. We are trying to map those SNPs on each of the homoeologous chromosomes in hexaploid wheat.