基于方差分析优化菊花ISSR-PCR反应体系

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

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、Mg2+、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1.00U,Mg2+2.00mmol/L,dNTP0.20mmol/L,引物0.50μmol/L。结论Taq酶、Mg2+、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。

Objective To establish and optimize ISSR-PCR systems of Chrysanthemum morifolium 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. morifolium 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 20μL 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. morifolium.