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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 optimize some factors that affect the amplification effect of medicinal chrysanthemum ISSR-PCR and lay the foundation for further study of its genetic diversity. Methods Based on analysis of variance (ANOVA), orthogonal design was used to optimize the reaction system of medicinal chrysanthemum from 4 levels and 3 levels of Taq enzyme, Mg2 +, dNTP and primers. Results The optimal reaction system of ISSR-PCR for medicinal chrysanthemum was: 1.00U Taq enzyme, 2.00mmol / L Mg2 +, 0.20mmol / L dNTP and 0.50μmol / L Taq enzyme in 20μL reaction system. Conclusion Taq enzyme, Mg2 +, dNTP and so on have a very significant impact on the results of ISSR reaction. The established ISSR system of medicinal chrysanthemum has the characteristics of clear marker site, stable reaction system, strong ability of detecting polymorphism, good repeatability and the like, and can be used for studying the genetic diversity of medicinal chrysanthemum.