基于KASP技术的小麦条锈菌SNP分子标记开发与评价

为开发用于小麦条锈菌Puccinia striiformis f. sp. tritici群体遗传研究的竞争性等位基因特异性PCR-单核苷酸多态性(kompetitive allele specific PCR-single nucleotide polymorphism,KASPSNP)标记,以中国小麦条锈菌流行小种CYR32的基因组为参考,与美国小麦条锈菌流行小种PST78和印度小麦条锈菌流行小种38S102的基因组进行比对,根据比对到的SNP位点设计KASP-SNP引物,用64个中国小麦条锈菌标样对其进行筛选,同时用13对多态性引物组成的简单重复序列(sim‐ple sequence repeat,SSR)分子标记分析这64个标样,并利用Powermarker 3.25和Structure 2.3软件通过多态性指数和群体遗传结构分析来评价KASP-SNP和SSR两种分子标记。结果显示,共比对到29 929个SNP位点,设计出462对KASP-SNP引物,经64个中国小麦条锈菌标样筛选到43对多态性较好的引物,所开发的这43对KASP-SNP引物多态性信息含量指数平均为0.346,基因多样性指数平均为0.420,而SSR引物的2种指数分别为0.237和0.265,前者较后者分别高出46.0%和58.5%。2种标记结果的群体遗传结构分析可得到类似结果,最佳聚类数K值都为4,云南菌系是遗传结构相对最简单的菌系,湖北菌系是遗传结构相对最复杂的菌系,但个别菌株的遗传划分存在较大差异。表明本研究开发的KASP-SNP分子标记多态性较SSR分子标记更加丰富,具有较好的应用前景。

Development and evaluation of SNP molecular markers of wheat stripe rust based on KASP technology

To develop kompetitive allele specific PCR-single nucleotide polymorphism (KASP-SNP) primers for population genetic study of Puccinia striiformis f. sp. tritici (PST), the genome sequence of the epidemic race CYR32 in China was used as a reference in this study and compared with race PST78 in US and 38S102 in India. KASP-SNP primers were designed according to SNP sites, and polymorphism at each SNP locus was determined using 64 Chinese PST isolates. Meanwhile, 13 pairs of polymorphic simple sequence repeat (SSR) markers were also used to analyze these PST populations for comparison. Powermarker 3.25 and Structure 2.3 were used to evaluate these two molecular marker systems. The results showed that totally 29 929 SNP sites were found, and 462 pairs of KASP-SNP primers were designed, among which 43 pairs of polymorphic SNP primers were obtained. The average polymorphism information content (PIC) index of the SNP primers was 0.346 and the gene diversity index was 0.420, and both indexes were higher than those of the SSR primers, which were 0.237, 0.265, with the former 46.0% and 58.5% higher than the latter, respectively. The results of KASP-SNP markers were more credible when considering the polymorphism information content, and the optimum K values were both 4. Yunnan and Hubei PST populations had the simplest and the most complex genetic structure. The two marker systems supported similar patterns of population structure, while the genetic division of individual isolates was quite different. The KASP-SNP molecular marker was more polymorphic than SSR molecular markers, and therefore showed a good prospect for application.