金花茶SCoT-PCR反应体系的正交优化及引物筛选

Orthogonal Optimization of SCoT-PCR Reaction System and Selection of Primers in Camellia petelotii

目的:建立适用于金花茶的SCoT-PCR反应体系,为金花茶组植物遗传多样性研究提供技术支持。方法:以金花茶基因组DNA为模板,采用SCoT分子标记方法,通过正交试验设计法对影响PCR反应体系的引物、DNA模板、2×Taq PCR Master Mix用量以及扩增程序进行筛选和优化。结果:最佳PCR反应体系为20μL:10 ng/μL的DNA模板1μL,10μmol/L的引物0.8μL,2×Taq PCR Master Mix 12μL,ddH_(2)O 6.2μL。PCR扩增程序为94℃预变性4 min;94℃变性35 s,退火60 s,72℃延伸80 s,30个循环,72℃延伸5 min,扩增产物4℃保存。结论:该研究所建立的SCoT-PCR反应体系经16份金花茶组样品的验证,反应体系稳定可靠,适于金花茶的SCoT分子标记研究。筛选出的10个引物均具有较高的多态性,为进一步深入开展金花茶遗传亲缘关系、品种鉴定、遗传育种等工作奠定了实验基础。

Objective:To establish a suitable SCoT-PCR reaction system of Camellia petelotii,so as to provide technical support for the study of plant genetic diversity of Camellia petelotii.Methods:Using Camellia petelotii genomic DNA as template,SCoT molecular marker method was used to screen and optimize primers,DNA templates,amount of 2×Taq PCR Master Mix and amplification procedures that affected the PCR reaction system by orthogonal experimental design.Results:The optimal PCR reaction system was 20μL:10 ng/μL DNA template 1μL,10μmol/L primer 0.8μL,2×Taq PCR Master Mix 12μL,ddH_(2)O 6.2μL.The best PCR procedure was 94℃predegeneration 4 min,then 30 circulations:94℃degeneration 35 seconds,annealing 60 seconds,72℃extend 80 seconds,72℃extend 5 min,at last 4℃keeping.Conclusion:The SCoT-PCR reaction system established in this study is verified by 16 samples of Camellia petelotii,and the reaction system is stable and reliable,suitable for the study of SCoT molecular labeling of Camellia petelotii.The 10 selected primers all show high polymorphism,which can lay the experimental foundation for further research on genetic relationship,variety identification and genetic breeding of Camellia petelotii.