制干辣椒SCoT-PCR体系优化及引物筛选

Optimization of SCoT-PCR System and primers screening of dried-usage chili(Capsicum annuum L.)

【目的】优化制干辣椒SCo T-PCR反应体系,为新疆制干辣椒种质资源、分子遗传学研究奠定基础。【方法】以制干辣椒为材料,采用L25(55)正交设计方法对影响制干辣椒SCo T-PCR反应体系的Mg2+、模板DNA、引物、DNA聚合酶、d NTPs等因素进行优化筛选。【结果】从25个正交设计组合中筛选获得5个组合的扩增效果较好,扩增条带丰富且清晰度适中、分散性好;经引物SC-12、SC-13、SC-19筛选,获得适用于制干辣椒材料的SCo T-PCR最佳反应体系20.0μL,包含10×Buffer 2.0μL、30 ng模板DNA 0.8μL、2.0 mmol/L Mg2+1.8μL、1.0μmol/L引物1.0μL、1 U Taq DNA聚合酶0.4μL、0.25 mmol/L d NTPs 0.5μL、dd H2O 13.5μL。运用优化体系从80条引物中筛选出扩增条带清晰、多态性丰富的SCo T引物24条。【结论】优化的SCo T-PCR反应体系及筛选的引物可用于新疆制干辣椒种质鉴定、遗传图谱构建、遗传多样性的分析等。

[Objective]The present study was conducted to optimize ScoT-PCR system of dried-usage chili in order to lay the foundation for researching germplasm resources and molecular genetics of Xinjiang dried-usage chili（Capsicum annuum L.）. [Method]Dried-usage chili was used as tested materials, the L25 （5^5） orthogonal design method was applied to optimize SCoT-PCR amplification system of chili in five factors viz., Mg^2＋, template DNA, primer, Taq DNA polymerases and dNTPs at five levels. [Result]Among 25 designing combination, 5 combinations could be obtained better amplification results being polymorphic bands with suitable clearness. An optimal SCoT-PCR amplification system of dried-usage chili was established by screening of primers SC-12, SC-13 and SC-19, which 20.0 μL of reaction system contained 10× Buffer 2.0μ L, 0.8 μL template DNA（30 ng）, 1.8 μL Mg^2＋（2.0 mmol/L）, 1.0 μL primer （1.0 μmol/L）, 0.4 μL Taq DNA polymerase（1.0 U）, 0.5 μL dNTPs（0.25 mmol/L）, and 13.5 μL ddH2O. A total of 24 primer pairs with clear and polymorphic bands were chosen from 80 pairs by using optimized PCR system. [Conclusion ]The optimized SCoT-PCR reaction system and screening primers could be used for germplasm resources evaluation, genetic maps contruction and genetic diversity analysis of Xinjiang dried-usage chili.