苹果SSR-PCR反应体系的建立与优化

Establishment and Optimization of SSR-PCR Reaction System of Apple Plants

为建立苹果属植物SSR-PCR反应优化体系,采用L16(45)正交实验和退火温度梯度实验,分析反应体系中使用的模板、引物、Mg2+、dNTPs以及Top Taq酶浓度对扩增产物的影响,并对这5个因素进行4个水平不同浓度梯度的筛选和优化。结果表明,引物浓度对SSR-PCR反应体系的影响最大,通过综合分析最终确定SSR-PCR的25μL最佳反应体系为:30~60 ng DNA模板,1.2μL正反向引物(5μmol/L),0.5μL dNTPs(2.5 mmol/L),0.5μL Top Taq酶(2.5 U/μL)以及2.5μL 10×Top Taq buffer(含Mg^(2+))。利用优化后的反应体系对蔷薇科苹果属的21种植物材料进行检测,实验结果表明扩增产物均在100~200 bp左右,可扩增出2~3个有效等位基因。通过优化反应体系中的各影响因素,建立了最佳的SSR-PCR反应体系,可为苹果属植物不同种群的遗传变异研究及亲缘关系的分子鉴定提供前期研究基础。

To establish an optimal SSR-PCR system for apple plants,the L16(45)orthogonal test and the annealing temperature gradient experiment were used to analyze the effect of template concentration,primer concentration,Mg2+concentration,dNTPs concentration and Top Taq enzyme concentration in the SSR reaction system on the amplification products.Additionally,the above five factors were screened and optimized at 4 different levels.The results showed that the primer concentration factor had the greatest impact on the SSR-PCR reaction system.Though analysis,the optimal reaction system of 25μL for SSRPCR was finally determined to be as follows:30-60 ng DNA template,1.2μL forward and reverse primer(5μmol/L),0.5μL dNTPs(2.5 mmol/L),0.5μL Top Taq enzyme(2.5 U/μL)and 2.5μL 10×Top Taq buffer(containing Mg^(2+)).Finally,optimized reaction system was used for PCR amplification using 21 different apple plants,and the amplification products were about 100-200 bp displaying 2-3 alleles.The optimal SSR-PCR reaction system can provide a preliminary basis for the genetic variation research and molecular identification of genetic relationships in different populations of apple plants.