微卫星三重PCR基因扫描技术在中国明对虾家系标识中的应用

Parentage identification of Chinese shrimp(Fenneropenaeus chinensis) using microsatellite-based triplex PCR Genescan technology

在开发中国明对虾(Fenneropenaeus chinensis)微卫星引物的基础上,利用3对微卫星引物,克隆编号分别为H081、RS0683和RS1101,构建中国明对虾的三重PCR稳定扩增体系。在此基础上,进一步利用荧光标记引物的基因扫描技术(Genescan)对利用精荚移植技术建立的中国明对虾7个半同胞家系和16个全同胞家系进行识别研究。结果表明,该项技术可以准确地把7个半同胞家系的子代个体间、16个全同胞家系的子代个体间以及总的31个全同胞家系的子代个体间区别开,且能准确把这些家系产生的任意子代个体定位到各个家系。该技术可以满足大批量家系材料样本的分析,具有较好的应用价值。[中国水产科学,2007,14(1):59-66]

In 2005 ,we developed many full- and half-sibs of Chinese shrimp（ Fenneropenaeus chinensis ）. In order to perform the genetic breeding program of Chinese shrimp, the offspring from deferent parentages were needed to cultivate together. Here, the problem is how to distinguish the offspring belonging to each parentage from each other. We attempted to use microsateUite molecular markers to resolve it. Through developing the microsatellite primers from the random genome sequences of F. chinensis, we obtained some microsateUite markers with rich genetic polymorphic information, in which three markers, RS1101,RS0683 and H081 were selected to establish a triplex PCR technique using a touch-down PCR method. The expected bands of three markers were clearly presented in the 8 % denature polyacrylamide gel by electrophoresing, and the optimal amplified PCR reaction volume was 15 μL containing 10 × Buffer 1.3 μL, MgCl2 （2.5 mmol/L） 1.0 μL, dNTP1.0 μL （ 2.5 mmol/L）, 0.5 μL of each forward or reverse primer（20 pmol/μL）, 1.5 μL template DNA（50ng/μL）,7μL ddH2O and 0.2μL Taq Polymerase（ 1 U）. Furthermore, the amplification products of trilex PCR were analyzed using ABI 3 700 Prism Genetic Analyzer and GeneScan 4.0 software（Perkin Elmer Applied Biosystems）, and Gene Maper3.0 software. In seven half-sibs and sixteen full-sibs,6,24 and 24 alleles were found on the RS1101 ,RS0683 and H081 loci,respectively. By the comparative analysis on genotypes between parents and offspring, the results showed that the offspring of seven half-sibs and sixteen full-sibs could be identified from each other. Additionally, by the help of Cervus bio-soft（Version 2.0, written by Triston）, any offspring cultivated together could be found which parentage they belong to. So, the triplex PCR is a useful tool for the parentage identification of Chinese shrimps.