本氏针茅ISSR-PCR反应体系的建立及引物筛选

Establishment and Optimization of the ISSR-PCR Reaction System in Stipa bungeana Trin.

以CTAB法提取的本氏针茅(Stipa bungeana Trin.)基因组DNA为模板,采用正交试验设计和单因素分析相结合的方法,对影响本氏针茅ISSR-PCR反应体系中的DNA、Taq酶、dNTPs、引物和Mg2+进行优化,旨在建立适合本氏针茅ISSR-PCR分析的最佳反应体系。结果表明,在20μL的反应体系中各组分的浓度分别为:DNA(20ng/μL)2.5μL、Taq DNA酶(5U/μL)0.1μL、dNTPs(2.5mmol/L)1.6μL、引物(10μmol/L)2.3μL、Mg2+(25mmol/L)1.4μL、10×Buffer 2.5μL、ddH2O 9.6μL。经过体系验证和引物筛选试验表明该体系适于本氏针茅遗传多样性分析,该体系的建立为本氏针茅种质资源遗传多样性研究提供了理论基础。

An improved CTAB method was used to extract the genomic DNA of Stipa bungeana, an orthogonal design was combined with a factor analysis method to optimize the ISSR-PCR reaction system of S. bungeana. Five main factors including DNA, Taq Polymerase, dNTPs, Mg2＋ and primers were opti- mized in order to establish a suitable PCR reaction system for Stipa bishbungeana. The results indicated that the concentration of each components in total 20/μ1 reaction system were： DNA（20ng/μL）2.5/μL, Taq polymerase （SU/μL）0.1/μL, dNTPs（2.5mmol/L） 1.6μL, primer（10/μmol/L）2.3μL, Mge＋ （25mmol/L） 1.4μL, 10XBuffer 2.5/μL, ddH20 9.6/μL, This reaction system is suitable for the genetic diversity analy- sis of Stipa bungeana. To establish the SRAP-PCR reaction system provides a theory basis for the genetic diversity analysis of S. bungeana germplasm resources.