毛细管电泳四色荧光检测法分析茶树SSR标记

将毛细管电泳四色荧光栓测技术应用于茶树SSR标记分析.该方法采用三引物PCR扩增SSR位点,三引物即在5'端加有M13尾巴序列(5'-CACGACGTTGTAAAACGAC-3')的特异正向引物、特异反向引物及带有荧光标记的通用型M13引物:为了运用四色荧光检测系统使通过一次毛细管电泳能同时检测3个以上的SSR位点,采用蓝、绿、黑3种不同颜色的荧光染料分别对3个M13引物进行标记. 应用该方法对42个茶树品种(系)的16个SSR位点进行遗传分析的结果表明:此法具有简便、可靠、低成本及高通量的优点;且随着所分析SSR位点数的增加,降低成本的效果更加显著.采用建立的方法,还筛选获得了11个多态性丰富的可应用于茶树遗传研究的SSR标记.

Tea Plant Microsatellite Genotyping by Capillary Electrophoresis with Four-Color Fluorescent Detection Technique

The capillary electrophoresis with four-color fluorescent detection technique was used to analyze the microsatellites in tea plant. Three primers were used in one PCR reaction for amplification of a defined microsatellite locus by this method. The three primers were as follows: a sequence-specific forward primer tailed with M13-tail at its 5' end, a sequence-specific reverse primer and a universal fluorescent-labeled MI3 primer (5'-CACGACGTTGTAAAACGAC-3'). In order to detect three or more microsatellite loci simultaneously in each capillary electrophoresis through four-color fluorescent detection system, three universal M13 primers were labeled with three different fluorescence dyes (blue,green and black). With this method the genetic analysis of 16 microsatellite loci among 42 tea plants was carried out. The results showed that this method had merits of simple, reliable, cost-effective and high-throughput microsatellite genotyping, and the economy brought by it became greater with an increasing number of microsatellite markers. Also, 11 polymorphie microsatellite markers of tea plant were identified by this method, and these markers would be useful for investigating genetic questions of tea plant (Camellia sinensis ).