草莓SRAP反应体系优化及引物筛选

Optimization of SRAP Reaction System and Selection of Primers for Strawberry

为建立草莓SRAP-PCR适宜的反应体系,以草莓品种‘丰香’为实验材料,采用单因素实验设计,对Mg2+、dNTPs、Taq DNA聚合酶及引物浓度4个因素4水平进行优化,并在此基础上对模板DNA的浓度和退火温度进行优化。结果表明,草莓SRAP-PCR最佳反应体系为:20μL的反应体系中含10×PCR buffer 2μL,Mg2+2.0 mmol/L,dNTPs 0.3 mmol/L,正反向引物各为0.6μmol/L,Taq DNA聚合酶1.0 U,模板DNA为100 ng。扩增程序为:94℃预变性5 min;94℃变性1 min,35℃退火1 min,72℃延伸1 min,共5个循环;94℃变性1 min,54℃退火1 min,72℃延伸1 min,共35个循环;72℃延伸5 min;4℃保存。利用该优化体系筛选引物,从110对SRAP引物组合中筛选出29对条带清晰丰富、多态性好的引物,证明了此优化体系稳定可靠,能够用于草莓种质资源的鉴定、分子标记辅助育种等研究。

The study took strawberry variety ＇ Fengxiang＇ as tested material and used one-factor experimental design to establish the SRAP-PCR reaction system of strawberry, which including four different gradients of Mg2＋, dNTPs, TaqDNA polymerase, primer concentration, and based on this, optimized the template DNA concentration and annealing temperature. The results showed that the optimum SRAP-PCR system of strawberry was as follows： 20 μL capacity of reaction system contained 10×PCR buffer 2 μL, Mg2＋ 2.0 mmol/L, dNTPs 0.3 mmol/L, forward primer and reverse primer 0.6 μmol/L, Taq DNA polymerase 1.0 U, template DNA 100 ng. The SRAP-PCR amplification procedure was as follows： pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 1 min, anneal at 35℃ for 1 min, extension at 72℃ for 1 min and in total 5 cycles; denaturation at 94℃ for 1 min, anneal at 54℃ for 1 min, extension at 72℃ for 1 min and in total 35 cycles; extension at 72℃ for 5 min; preservation at 4℃. Twenty-nine primer pairs were screened out from 110 SRAP primer pair combinations based on their clear and rich amplification bands and good polymorphism by utilizing the optimal SRAP reaction system. So the optimal system was reliable which could be applied in the germplasm identification and molecular marker assisted breeding of strawberry.