烟草疫霉SSR分子标记的开发与初步应用

Development of SSR markers for Phytophthora nicotianae and its preliminary application

为开发烟草疫霉的SSR分子标记,利用MISA软件搜索烟草疫霉基因组序列中的SSR位点,共发现1311个SSR位点,优势SSR位点为二核苷酸和三核苷酸,分别占总SSR位点的56.45%和39.36%;根据分析到的SSR位点使用Primer 5.0软件设计48对SSR引物,以7株烟草疫霉的DNA为模板对这些引物进行筛选,共获得扩增条带清晰且具有多态性的SSR引物20对,然后使用其中的6对SSR对32株烟草疫霉进行UPGMA聚类分析,遗传相似系数在0.60~1.00之间,在相似系数0.70水平上,可将其划分为3个类群,类群Ⅰ包含29个菌株,类群Ⅱ包含1个菌株,类群Ⅲ包含2个菌株,显示出较低的遗传分化水平。这些SSR引物的开发将为研究我国烟草疫霉的遗传多样性、群体遗传结构以及遗传图谱构建等奠定基础。

The objective of this research is to develop SSR markers in Phytophthora nicotianae.A total of 1311 SSR loci were identified from the P.nicotianae genome using MISA software.Trinucleotide and tetranucleotide were dominant motifs which accounted for 56.45% and 39.36%,respectively.Based on identified SSR loci,48 pairs of primers were designed using Primer 5.0 software.DNAs of seven P.nicotianae isolates were used as template in order to screen these primers.Twenty pairs of primers of which amplified bands were clear and polymorphic were identified.Based on amplification results of six pairs of primers,the UPGMA dendrogram of 32 isolates illustrated that they were separated clearly into three groups at 0.70 genetic similarity.Group Ⅰ included 29 isolates.Group Ⅱ included one isolate and Group Ⅲ included two isolates.The genetic similarity coefficient of 32 isolates varied from 0.60 to 1.00.Genetic differentiation among isolates was low.Development of these SSR markers laid foundation for studying genetic diversity,population genetic structure,and linkage map construction of P.nicotianae.