小麦基因组SSR体系及反应条件优化研究

Study on optimization of wheat SSR reaction system and condition

以小麦叶锈病抗病亲本TcLr45和感病亲本Thatcher为材料,探索影响SSR扩增结果的各因素(模板DNA、dNTP、引物、Taq酶)的最佳用量及不同引物的退火温度.结果表明:dNTP对扩增影响较大,Taq酶浓度在聚丙烯酰胺凝胶电泳检测时表现对扩增有一定影响,每对引物都有其扩增适合的退火温度.确立了适合小麦SSR分子标记研究的优化体系.最终确定总反应体系为25 μL,其中模板DNA 20～50 ng、dNTP 240 μmol/L、引物各0.2 μmol/L、Taq酶1.5 U.

Resistance parent TcLr45 and susceptible parent Thatcher against wheat leaf rust were used in this study to optimize the several factors applied in SSR technique system, including the concentration of template, dNTP, primer and Taq polymerase, dNTP was found to have distinct effect to amplification, concentration of 7aq polymerase have some effect to amplification when checked on polyacrilamide gel electrophsis, annealing temperature specific to primer pair was also checked. Optimized SSR system for wheat was established： 20～50 ng of template DNA, 240 μmol/L dNTP, 0.2 μmol/L of each primer and 1.5 U Taq polymerase were added to the 25 μL PCR mixture.