阔叶十大功劳ISSR-PCR反应体系的建立与优化

采用单因子试验和正交设计方法,对影响阔叶十大功劳ISSR-PCR反应体系的引物、Taq DNA聚合酶、dNTPs、模板DNA及退火温度5个因素进行优化,为探讨阔叶十大功劳种质遗传多样性奠定基础.结果表明:阔叶十大功劳ISSR-PCR的最佳反应体系为:在20μL反应体系中,0.5μmol/L引物、0.5UTaq酶、150μmol/L dNTPs和20ng模板DNA.在最佳反应条件下,从80条引物中筛选出15条扩增稳定、多态性丰富的ISSR引物,并经过9份阔叶十大功劳种质检验,证明该体系具有扩增条带清晰、稳定、重复性好等优点.综上所述,本文所建立的ISSR-PCR反应体系可用于阔叶十大功劳的种质鉴定及遗传多样性分析.

Establishment and optimization of ISSR-PCR reaction system for Mahonia bealei

The single-factor test and orthogonal design were applied for optimizing five factors in the ISSR-PCR reaction system including primers,Taq DNA polymerase,dNTPs,template DNA and annealing temperature to establish and optimize ISSR-PCR reaction system for Mahonia bealei.The suitable PCR reaction system contained 0.5 μmol/L primer,0.5 U Taq DNA,150 μmol/L dNTPs,and 20 ng template DNA in total 20 μL reaction solution.15 primers were screened from 80 ISSR primers with stable amplification and rich polymorphism.The ISSR reaction system was proved to be stable and credible in the test of 9 Mahonia bealei populations.This optimized ISSR reaction system would provide the basis for the genetic analysis of Mahonia bealei.