Development of Genomic Microsatellite Multiplex PCR Using Dye-Labeled Universal Primer and Its Validation in Pedigree Analysis of Pacific Oyster(Crassostrea gigas)

作     者：LIU Ting LI Qi SONG Junlin YU Hong 

作者机构：Key Laboratory of Mariculture of Ministry of EducationOcean University of ChinaQingdao 266003P.R.China 

出 版 物：《Journal of Ocean University of China》 (中国海洋大学学报（英文版）)

年 卷 期：2017年第16卷第1期

页      面：151-160页

核心收录：

学科分类：0710[理学-生物学] 0908[农学-水产] 0707[理学-海洋科学] 09[农学] 0815[工学-水利工程] 0824[工学-船舶与海洋工程] 

基　　金：supported by the Shandong Seed Project the National Natural Science Foundation of China(No.31372524) Science and Technology Development Plan of Shandong Province,China(No.2014GHY 115002) 

主　　题：Crassostrea gigas traceability microsatellites universal primer multiplex PCR pedigree analysis 

摘      要：There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.