苹果SSR-PCR反应体系的建立与优化

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-P...

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

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 25 μL for SSR-PCR was finally determined to be as follows: 30-60 ng DNA template, 1.2 μL forward and reverse primer (5 μmol/L), 0.5 μL dNTPs (2.5 mmol/L), 0.5 μL Top Taq enzyme (2.5 U/μL) and 2.5 μL 10×Top Taq buffer (containing Mg²⁺). 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.