棉花DUS测试标准品种的SSR指纹数据库构建

基于SSR核心引物,采用荧光毛细管电泳检测系统与多重PCR技术相结合,构建我国棉花DUS(Distinctness,Uniformity and Stability)测试标准品种的DNA指纹数据库并进行遗传多样性分析。依据多重PCR组合的基本原则与五色荧光检测系统的特点,采用40对核心引物构建了10个4重PCR组合,利用DNA遗传分析仪进行指纹检测与数据采集。40对荧光引物在30份DUS测试标准品种中共扩增产生146个等位变异,每对引物的等位变异数为2~7个不等,平均每对引物产生3.65个等位变异。海7124与陆地棉品种明显划分为2类,来源于新疆的新陆早1号区别于其他陆地棉品种,单独聚为1类。多色荧光检测系统相比常规聚丙烯酰胺凝胶电泳具有高精度、高通量、自动化程度高的优点,尤其适用于大规模指纹数据库的构建,提出了通过构建已知品种DNA指纹数据库,将分子标记技术应用于棉花DUS测试的初步设想。

Construction of SSR Fingerprinting Database of Standard Varieties on Cotton in DUS Testing

Core SSR primers were used for constructing DNA fingerprint database of cotton DUS testing standard varieties based on fluorescence detection system and multiplex PCR (polymerase chain reaction) technology, and the genetic diversity was analyzed. Ten sets of 4 multiplex PCR combinations were established according to fundamental principle of multiplex PCR and characteristic of fluorescent system. DNA genetic analyzer was used for construction of fingerprinting database. Among the 30 varieties, 40 primer pairs had 146 allelic variations, and 3.65 allelic variations were detected by each SSR primer pair on an average with the range from 2 to7. The number of homozygous loci and heterozygous loci is almost the same. Clustering analysis indicated that sea island cotton (Hai7124) and upland cotton varieties were obviously divided into two categories. Xinluzao1 from Xinjiang cotton region differed from other upland cotton varieties. Multiplex fluorescence detection system has many advantages such as high-precision, high-throughput, high degree of automation compared to conventional silver staining system, especially for construction of large-scale fingerprint database. A primary idea about the application of molecular marker technology in cotton DUS testing by the construction of fingerprinting database of known varieties was proposed.