长柄石杉ISSR-PCR反应体系的建立与正交优化

长柄石杉ISSR反应体系的建立能为利用ISSR标记技术进行长柄石杉品种鉴别、分类、种质资源遗传多样性分析奠定良好基础。利用正交试验设计的方法，从dNTP浓度、Taq DNA聚合酶浓度、引物浓度和Mg2+浓度4种因素，对长柄石杉ISSR-PCR反应体系进行优化分析，并在此基础上对模板DNA浓度进行梯度检测。结果表明，20 μL ISSR-PCR反应体系中各因素的最佳浓度为1×PCR buffer、300 μmol/L dNTP、0.2 μmol/L引物、2.0 mmol/L Mg2+、1.75 U Taq DNA聚合酶、40 ng模板DNA。该优化体系的建立为下一步进行长柄石杉ISSR分子标记奠定了基础。

Establishment and Orthogonal Optimization of ISSR-PCR Amplification System for Huperzia javanica

The establishment of the ISSR-PCR amplification system could lay a good foundation for identification and classification and analysis on the genetic diversity of germplasm resources of Huperzia javanica using ISSR molecular marker techniques.In this paper,the orthogonal design was used to optimize the ISSR-PCR amplification system of Huperzia javanica in four factors （the concentration of dNTP,Taq DNA polymerase,primers and Mg 2 ＋） respectively.Then,based on the optimal ISSR-PCR amplification system,the concentration of template DNA was proposed by gradient PCR.The results showed that a suitable ISSR-PCR amplification system of Huperzia javanica was established.In a total volume of 20 μL ISSR-PCR amplification system,it contained 1×PCR buffer,300 μmol/L dNTP,0.2 μmol/L primer,2.0 mmol/L Mg 2＋,1.75 U Taq DNA polymerase and 40 ng template DNA.The optimized system laid the groundwork for ISSR molecular analysis of Huperzia javanica.