夏枯草ISSR分子标记技术的建立与体系优化

目的 建立并优化夏枯草ISSR-PCR反应体系和扩增程序.为探讨夏枯草种质问遗传多样性奠定基础.方法 采用单因子试验和正交设计方法,研究.Mg2+、dNTP、引物、Taq DNA聚合酶、模板DNA、退火温度及循环次数对PCR扩增的影响.结果 夏枯草ISSR-PCR的最佳反应体系为:在20 μL的反应体系中含模板DNA 30ng,Mg2+ 2.2 mmol/L、dNTP 175 μmol/L、引物0.75μmol/L、Taq DNA聚合酶1.0 U.在此基础上,从92条引物中筛选出18条扩增稳定、多态性丰富的ISSR引物.并通过梯度PCR试验.确定引物最佳退火温度.结论 采用单因子试验和正交设计方法可以快速建立ISSR-PCR反应体系,经过24份夏枯草种质检验,证明该体系稳定可靠,可用于夏枯草遗传分析.

Establishment of ISSR marker technology and optimization of its system in Prunella vulgaris

Objective To establish and optimize the ISSR-PCR reaction system for Prunella vulgaris and lay foundation for its genetic diversity research. Methods The single-factor and orthogonal design were applied for optimizing seven factors in the ISSR-PCR reaction system including Mg2+ , dNTP, prim-ers, Taq DNA polymerase, the template DNA, annealing temperature, and cycles. Results The suitable PCR reaction system contained 2. 2 mmol/L Mg2+ , 175 μmol/L dNTP, 0.75 μmol/L primer, 1. 0 U Taq DNA polymerase, and 30 ng template DNA in total 20 μL reaction solution. On this basis, 18 primers were screened with stable amplification and rich polymorphisrn from 92 ISSR primers. The optimal annea-ling temperature for ISSR-PCR reaction was proposed by gradient PCR. Conclusion It is a way to estab-lisb the ISSR-PCR system for orthogonal design combining with single-factor test. And it is proved to be stable and credible for the result of 24 P. Vulgaris populations. This optimized ISSR reactiofl system would provide the basis for the genetic analysis of P. Vulgaris.