野杏SSR-PCR反应体系优化

为建立稳定可靠的野杏SSR-PCR反应体系,以3个野杏无性系170号、240号、263号和1个西伯利亚杏无性系508号为试验材料,采用L16(45)正交试验设计对影响野杏SSR-PCR反应的5个因素在4个水平上进行优化。结果表明:各因素对反应体系的影响由大到小依次为DNA模板浓度、引物、dNTP、Mg2+、Taq聚合酶。野杏SSR-PCR最佳反应体系(总体积20μL):dNTPs 0.45 mmol/L、Mg2+2 mmol/L、Taq酶1.125 U、引物0.125μmol/L、DNA模板20 ng。选用引物X128和60个野杏样木对该体系进行稳定性验证,扩增产物集中在100～200 bp,稳定度高,多态性良好,该体系可应用于野杏分子标记辅助育种等方面的研究中。

Optimization of SSR-PCR reaction system for Armeniaca vulgaris Lam. var. ansu

In order to establish a stable and reliable Armeniaca vulgaris var.ansu SSR-PCR reaction system,three A.vulgaris Lam.var.ansu clones No.170,No.240,No.263 and one A.sibiricat clone No.508 were used as experiment materials,and five factors affecting SSR-PCR reaction were optimized at four levels using the orthogonal design L16(45).The results showed that order of the factors was DNA template concentration,primer dosage,dNTP dosage,Mg^2+dosage,Taq DNA polymerase,based on effects on reaction system from high to low.The optimized reaction systems was:the total reaction system 20μL,dNTPs 0.45 mmol/L,Mg^2+2 mmol/L,Taq DNA polymerase 1.125 U,primer concentration 0.125μmol/L,and DNA template 20 ng.Stability of this reaction system was verified by using 60 samples from A.vulgaris Lam.var.ansu and primer X128,the amplified products ranged from 100 bp to 200 bp with high stability and good polymorphism,so the system could be used for molecular marker-assisted breeding of A.vulgaris var.ansu.