西伯利亚杏SRAP-PCR反应体系的优化研究

Optimization of SRAP-PCR system with orthogonal design in Prunus sibirica

以西伯利亚杏为试验材料,进行基因组DNA的提取、扩增及电泳试验。通过L16(45)正交试验,确定西伯利亚杏SRAP-PCR优化反应体系(反应体系总体积20μL):Mg2+2.0 mmol·L-1、dNTPs0.25 mmol·L-1、引物浓度1.2μmol·L-1、Taq聚合酶1.0 U、DNA模板20 ng。采用SRAP引物组合ME5/EM10,对优化的SRAP-PCR反应体系进行初步验证,24个西伯利亚杏无性系SRAP分子标记试验共扩增出谱带431条,引物扩增出23条谱带,其中338条为多态性谱带,多态百分率为78.42%。

With Prumts sibirica, genomic DNA extraction, amplification and electrophoresis tests were conducted. The optimal reac- tion system （20μL ） of SRAP markers were as follows： 2.0 mmol·L-1 of Mg2＋, 0.25 mmol· L-1 of dNTPs, 1.2μmol·L-1 of primer, 1.0U ofTaq DNApolymerase, 20.0 ng of DNA template. Using primer combination ME5/EM10, preliminary verification of the optimal SRAP-PCR system was carried, 24 Prunus sibirica clones from different provenances as samples, 431 loci were amplified, and a couple of primer amplified 23 loci. 338 loci were polymorphic, accounting for 78.42% of the total loci.