药用菊花SCoT-PCR反应体系的正交优化

Optimization of Start Codon Targeted Polymorphism System in Medicinal Chrysanthemum Morifolium Based on Orthogonal Design

为进一步开发利用药用菊花种质资源,为药用菊花的遗传多样性及分子鉴定研究提供技术支持,建立并优化药用菊花的目标起始密码子多态性-聚合酶链式反应(SCoT-PCR)体系,在方差分析基础上,运用L25(56)正交设计在5个水平上对影响药用菊花SCoT-PCR反应的模板DNA、Mg2+、dNTPs、Taq酶、引物5个因素进行优化试验,并对PCR结果进行分析。建立了药用菊花SCoT-PCR最佳反应体系(20μL):模板DNA 10 ng,引物0.8μmol·L-1,dNTPs 0.2 mmol·L-1,Mg2+2.0 mmol·L-1,Taq酶1.5 U,SCoT反应的影响因素依次为:Taq酶>引物>dNTPs>Mg2+>模板DNA。优化的SCoT-PCR反应体系在多个药用菊花品种遗传多样性研究中得到了验证,结果表现出良好的稳定性、重复性和多态性丰富等特点,该体系的建立为药用菊花品种遗传多样性分析、系统发育研究、遗传图谱构建、基因定位和分子标记辅助育种研究奠定了基础。

In order to develop and utilize of medicinal Chrysanthemum morifoliumthe germplasm resources, as well as provide technical support for genetic diversity and molecular identification studies of medicinal Chrysanthemum morifolium, the SCoT （start codon targeted polymorphism） -PCR （polymerase chain reaction） system was established and optimized. In this research, the L25 （56） orthogonal design was applied to optimize the five factors （ Taq DNA polymerase, Mg2＋, template DNA, dNTPs and primer concentrations） of SCoT-PCR amplification system at five concentration ration levels. The results of optimized system by both intuitive analysis and variance analysis were as follows ： a total volume of 20 μL system including 10 ng template DNA, 1.5 U Taq DNA polymerase, 0.8 μmol·L-1 primer, 2.0 mmol·L-1 Mg2＋ , 0.2 mmol·L-1 dNTPs. The effective degree of the factors on the SCoT reaction was in the order： Taq DNA polymerase 〉 primer 〉 dNTPs 〉 Mg2＋ 〉 template DNA. The key factor affecting the SCoT-PCR system was the concentration of Taq DNA polymerase. Furthermore, the optimal reaction system was further used to verify with multiple medicinal Chrysanthemum morifolium varieties, which showed good stability and repeatability. Our results indicate the SCoT-PCR system is informative and would facilitate further lay the solid foundation for genetic diversity, genetic maps, gene localizations and molecular marker assisted breeding studies in medicinal Chrysanthemum morifolium.