观赏海棠SSR-PCR体系优化及引物筛选应用

【目的】为探究适用于观赏海棠SSR反应分子标记研究的最佳PCR反应条件,采用正交设计法对观赏海棠SSR-PCR体系进行优化,并利用优化体系筛选适于观赏海棠的多态性SSR引物。【方法】以6个二倍体观赏海棠DNA模板为材料,采用L₁₆(4⁵)正交试验对DNA模板浓度、Taq酶浓度、引物浓度、Mg²⁺浓度和dNTP浓度进行优化实验,确立了观赏海棠SSR-PCR最佳反应体系。【结果】得出15 μL优化体系各成分为:DNA模板5 mg/L、Taq酶1.25 U、引物0.3 μmol/L、Mg²⁺ 2 mmol/L、dNTP 0.25 mmol/L、1×Buffer1.5 μL。利用优化体系在73对苹果属SSR引物中筛选出15对条带清晰、多态性丰富、重复性好的SSR 引物并确定各引物的退火温度,其多态信息含量均大于0.25。【结论】正交试验结果达到优化目的,利用优化体系筛选的15对多态性引物可直接应用于观赏海棠SSR分子标记实验中,为观赏海棠品种鉴定、分类以及遗传育种等研究提供了有效工具。

Optimization of SSR-PCR reaction system and primer sceening in Malus crabapple

Abstract: 【Objective】 To explore the optimal PCR conditions for SSR molecular markers of Malus crabapples, orthogonal design was used to optimize the SSR-PCR system, and SSR primers suitable for Malus crabapples were screened using the optimized system. 【Method】Six diploid Malus crabapple DNA templates were used as material. DNA template concentration, Taq enzyme concentration, primer concentration, Mg^{2 +} ion concentration, and dNTP concentration were optimized by the L₁₆(4⁵)orthogonal test and the optimum reaction system for SSR-PCR of crabapples was established. 【Results】 The results showed that 15 μL of optimized system was composed of DNA template 5 mg/L, Taq enzyme 1.25 U, primer 0.3 μmol/L, Mg²⁺ 2 mmol/L, dNTP 0.25 mmol/L and 1×Buffer 1.5 μL. Fifteen pairs of SSR primers from Malus with clear bands, abundant polymorphisms, and good repeatability were screened out using the optimization system. Its polymorphic information content was greater than 0.25. 【Conclusion】 Orthogonal test results achieved the purpose of optimization. The 15 polymorphic primers screened by the obtained system could be directly applied to the SSR molecular marker experiment for Malus crabapples, which provided an effective tool for identification, classification and genetic breeding of Malus crabapples.