高粱抗丝黑穗病SSR-PCR反应体系的优化

Optimization of SSR-PCR System on Sorghum Resistance to Head Smut

目的:找到一种可用于高粱抗丝黑穗病SSR-PCR扩增最适宜的反应体系。方法:利用单因素体系优化的方法,对SSR-PCR反应体系中Taq酶、dNTPs、MgCl2、模板DNA以及引物用量进行了优化,并且用不同引物、不同模板DNA对该优化结果进行验证。结果:实验表明,Taq酶、dNTPs、MgCl2、模板DNA以及引物的不同用量均对PCR反应结果有显著的影响。最适宜高粱抗丝黑穗病20μl SSR-PCR的反应体系为1U/μl Taq酶2.5μl,10mmol/L dNTPs 1.0μl,25mmol/L MgCl21.5μl,模版DNA2.0μl(50ng),10μmol/L引物1.5μl,10×Buffer 2.0μl。验证结果表明,该体系扩增出的条带清晰且稳定。结论:该结果为今后利用SSR-PCR标记技术研究分析高粱抗丝黑穗病奠定了基础。

Objective： To find a conformable SSR- PCR reaction system of Sorghum Head Smut. Method： In this study, SSR- PCR reaction system was optimized on Sorghum resistance to Head Smut in Taq polymerase, dNTPs, MgCl2, genomic DNA templates and primers, respectively. Different random primers and template DNA were used to validate the result. Result： The results showed that the five main factors in different levels had the marked influence on the result of PCR. The optimal PCR reaction system for Sorghum resistance to Head Smut was 2.5μl 1U/t.d Taq polymerase, 1.0V.1 10mmol/L dNTPs, 1.5μl 25mmol/L MgCl2, 50ng genomic DNA templates, 1.5μl 10μmol/L primers and 10 × Buffer 2.0μl in a total volume of 20μl SSR - PCR reaction system. Validation result showed that the bands of this system were clear and steady. Conclusion： The reaction system was the most conformable one for SSR -PCR, and was established as the good foundation of SSR - PCR markering technique on Sorghum resistance to Head Smut.