本氏针茅SRAP PCR反应体系的建立及引物筛选

以本氏针茅（Stipa bungeana）幼嫩叶片为材料，建立适合本氏针茅基因组DNA提取的改良CTAB法，在此基础上采用正交试验设计和单因素分析相结合的方法，对本氏针茅SRAP PCR反应体系中的5个主要因素（DNA、Taq酶、dNTPs、Mg2+和引物）进行优化，旨在建立适合本氏针茅SRAP分析的反应体系。结果表明，在20 μL总的反应体系中各组分的加入量分别为：DNA（20 ng·μL-1）3 μL、Taq DNA酶(5 U·μL-1)0.2 μL、dNTPs（2.5 mmol·L-1)1.4 μL、引物（10 μmol·L-1）1.0 μL、Mg2+ (25 mmol·L-1)2.0 μL、10×Buffer 2.5 μL、ddH2O 8.9 μL。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，该体系的建立可为本氏针茅种质资源遗传多样性研究和黄土高原植被恢复及生态建设提供理论基础。

Establishment of the SRAP-PCR reaction system and selection of the primers in Stipa bungeana

An improved CTAB method,suitable for isolating genomic DNA of Stipa bungeana,using the young leaves of this grass speicies,was established in a previous study.Based on this result,an orthogonal design and a factor analysis method were combined to optimize the SRAP-PCR reaction system of S.bungeana,including five main factors: DNA,Taq Polymerase,dNTPs,Mg2+ and primers.The results indicated that the optimum concentrations of each component in the 20 μL reaction system were: DNA(20 ng·μL-1)3 μL,Taq Polymerase(5 U·μL-1) 0.2 μL,dNTPs(2.5 mmol·L-1) 1.4 μL,primer(10 μmol·L-1)1.0 μL,Mg2+(25 mmol·L-1) 2.0 μL,10×Buffer 2.5 μL,ddH2O 8.9 μL.This reaction system has been experimentally validatied and primers selected,and should be a suitable system for the genetic diversity analysis of S.bungeana.This SRAP-PCR reaction system could provide a basis for the analysis of genetic diversity within S.bungeana germplasm resources,with applications being vegetation restoration and ecological reconstruction in the loess plateau.