摘 要 ：为了建立并优化大青叶SRAP-PCR扩增体系。以大青叶基因组DNA为模板，通过正交试验设计，从Mg2+、dNTP、引物、Taq DNA聚合酶和模板DNA 5种因素5个水平对大青叶SRAP-PCR反应体系进行优化。建立的大青叶SRAP-PCR最佳反应体系为25 μL反应体系中含1.5 mmol·L-1 Mg2+、0.15 mmol·L-1 dNTPs、0.60 μmol·L-1引物、2.0 U Taq DNA聚合酶和26 ng DNA模板。在这一体系下对不同大青叶材料的PCR扩增和在不同引物组合下扩增都证明该体系稳定可靠适合于大青叶SRAP分子标记。这一体系的建立为今后利用SRAP-PCR分子标记技术开展大青叶分子遗传学研究奠定了基础。
Abstract:We established and optimized SRAP-PCR amplification system for Isatis indigotica Fort by the orthogonal design. The order of factors affecting the result of SRAP-PCR were Mg2+, dNTPs, primers, template DNA and Taq DNA polymerase. A suitable SRAP-PCR system for Chinese bayberry was that total 25 μL reaction system containing 1.5 mmol·L-1 Mg2+, 0.15 mmol·L-1 dNTPs, 0.60 μmol·L-1 primers, 2.0 U Taq DNA polymerase and 26 ng template DNA could be able to amplified the most rich polymorphism and clear bands. This reaction system was experimentally validatied and and primers selected, and should be a suitable system for the genetic diversity analysis of I.indigotica Fort.