基于方差分析优化菊花ISSR-PCR反应体系

目的 对影响药用菊花ISSR-PCR扩增效果的一些因素进行优化,为进一步研究其遗传多样性奠定基础.方法 基于方差分析方法,利用正交试验从Taq酶、Mg~(2+)、dNTP和引物等4因素3水平对药用菊花反应体系进行优化.结果 药用菊花ISSR-PCR的最佳反应体系:在20μL的反应体系中,Taq酶1.00 U,Mg~(2+)2.00 mmol/L,dNTP 0.20 mmol/L,引物0.50μmol/L.结论 Taq酶、Mg~(2+)、dNTP等对ISSR反应结果有极显著影响.所建立的药用菊花ISSR反应体系具有标记位点清晰、反应系统稳定、检测多态性能力较强、重复性好等特点,可用于药用菊花的遗传多样性研究.

Optimization of ISSR-PCR reaction system for Chrysanthemum morifolium based on analysis of variance

Objective To establish and optimize ISSR-PCR systems of Chrysanthemum mori folium and lay the foundation for its genetic diversity research. Methods Based on the analysis of variance, an orthogonal design was used to optimize the ISSR-PCR amplification system on C. mori folium by four factors (Taq polymerase, Mg~(2+) , dNTP, and primer) at three concentration levels, respectively. Results A suitable ISSR reaction system was constructed with the 20 μL reaction system containing 1. 00 U Taq polymerase, 2. 00 mmol/L Mg~(2+) , 0. 20 mmol/L dNTP, and 0. 50 μmol/L primer. Conclusion ISSR-PCR is significantly influenced by the concentration of Taq polymerase, Mg~(2+) , and dNTP. This ISSR-PCR system could provide clear bands, reliable reaction system, and abundant polymorphisms . It is proved to be suitable for the study of the genetic diversity of C. mori folium.