山东鹅观草SSR-PCR反应体系的优化和验证

山东鹅观草是一重要的小麦野生近缘种质资源,具有对黄矮病的高抗性和易于小麦杂交的高亲和性等优良性状.以CTAB法提取山东鹅观草叶片DNA为模板,进行反应PCR体系优化.选用20 μL的PCR 反应,对Mg2+浓度、dNTP浓度、引物、DNA模板和Taq酶5个因素设计5个水平进行体系优化和验证试验.先以1个SSR标记(Xgwm43-7B )对5个因素进行筛选.为了验证所选最优体系的稳定性,再选用9个SSR标记(Xgwm18-1B、Xgwm32-3A、Xgwm6-4B、Xgwm60-7A、Xgwm67-5B、Xgwm77-3B、Xgwm88-6B、Xgwm95-2A和Xgwm99-1A)进行验证性试验.单因素试验结果表明PCR反应最佳体系为:20 μL 反应体系,2μL的 10×PCR Buffer,2.875mmol/L 的Mg2+,200μmol/L 的dNTP,3 pmol的引物,45 ng的模板DNA,1.5 U的 Taq 聚合酶,双蒸水以补足20 μL反应体系.验证性试验结果表明该反应体系有一定通用性,但扩增效果及PCR产量有差异.

Optimization and testing for SSR-PCR system of Roegneria shandongensis

The tribe triticeae represents a potential gene pool for improvement of crops such as wheat,rye and barley.Roegneria shandongensis is a tetraploid species widely distributing in the Eastern part of China.The species contains resistance to wheat yellow dwarf disease.However,the molecular markers used to investigate the genetic diversity of R.shandongensis were poorly studied.In this paper,a SSR-PCR system of R.shandongensis was optimized and tested.PCR system was optimized in five factors(Mg2+,dNTP,primer,DNA template and Taq DNA polymerase) at five levels respectively.To discover the economic,rapid,and stable PCR system to screen SSR primers of R.shandongensis and detect the generality of the established system,one SSR primer Xgwm43-7B was used to screen the best PCR reaction system,and nine pairs of SSR primers(Xgwm18-1B,Xgwm32-3A,Xgwm6-4B,Xgwm60-7A,Xgwm67-5B,Xgwm77-3B,Xgwm88-6B,Xgwm95-2A and Xgwm99-1A) were used to test the generality.As a result,a satisfactory SSR-PCR reaction system for R.shandongensis with desirable repeatability and polymorphic bands was established.20μL SSR-PCR system contained 1×buffer,2.875mmol/L Mg2+,200μmol/L dNTP,3 pmol primer,45 ng template DNA,1.5 U Taq polymerase,and ddH2O then added up to terminal volume of 20 μL.The generality test of PCR optimized system of R.shandongensis was carried out.The test result indicated that this system is also suitable for the amplification done by other SSR primers.