红树植物木榄SCoT-PCR体系优化及遗传多样性分析

System Optimization of SCoT-PCR and Genetic Diversity Analysis of a Mangrove Plant: Bruguiera gymnorrhiza

为优化建立木榄的SCo T-PCR体系,利用建立的SCo T方法进行分析木榄不同地理种源的遗传多样性,基于L25(56)正交设计,本研究在5个水平上优化试验了5个影响木榄SCo T-PCR的因素:模板DNA、Mg^(2+)、d NTPs、Taq酶和引物。同时,运用优化的SCo T体系对木榄8个不同地理种源进行遗传多样性分析。本研究结果建立了木榄SCo T-PCR最佳反应体系(20μL):模板DNA 20 ng,引物0.75μmol/L,d NTPs 0.5 mmol/L,Mg^(2+)2.5 mmol/L,Taq酶1.25 U。同时,筛选出了带谱清晰、多态性丰富的16条SCo T引物进行遗传多样性分析。共扩增出115个位点,其中73个为多态性位点,多态性比例为62.33%。从UPGMA聚类和PCo A分析结果可以看出8个地理种源间的遗传多样性及亲缘关系。研究表明SCo T分子标记可以用于木榄种群的遗传多样性。

To optimize SCoT-PCR system for the establishment of B. gymnorrhiza, we used the SCoT method to analyze genetic diversity of B. gymnorrhiza from different geographical provenances, and five factors （template DNA, Mg^2＋, dNTPs, Taq DNA polymerase and primer concentration） that effected the SCoT-PCR of B. gymnorrhiza were optimized based on the L25（5^6） orthogonal design at five concentration levels. Meanwhile, genetic diversity of 8 provenances of B. gymnorrhba was analyzed by the optimized SCoT system. The results of the study established optimal reaction system for B. gymnorrhiza （20 μL total volume）： template DNA 20 ng, Taq DNA polymerase 1.25 U, primer 0.75 μmol/L, Mg^2＋ 2.5 mmol/L, and dNTPs 0.5 mmol/L. At the same time, sixteen primers of SCoT with clearly separated bands, stable amplification and rich polymorphism were selected to study the genetic diversity. The results showed that 115 loci were generated, of which 73 loci were polymorphic, and the percent of polymorphic loci was 62.33%. The genetic diversity and relative relationships of 8 provenances of B. gymnorrhiza were showed based on the results of UPGMA cluster analysis and PCoA. This study indicated that SCoT molecular markers could be used in genetic diversity study of B. gymnorrhiza.