国槐SSR-PCR反应体系的建立与优化

Establishment and Optimization of SSR-PCR Reaction System for Sophora japonica L.

利用单因素试验结合L_(16)(4^(5))正交试验设计对国槐SSR-PCR反应体系中的5个主要影响因素(Mg^(2+)、引物、dNTPs、ExTaq酶、模板DNA用量)进行优化,确定最佳反应体系为(10μL):25 mmol/L Mg^(2+)1.0μL,2.5 mmol/L dNTPs 0.2μL,10μmol/L上下游引物共0.5μL,5 U/μL ExTaq酶0.05μL,30 ng/μL模板DNA 1.0μL,10×PCR Buffer 1.0μL,ddH_(2)O 6.25μL。随机挑选4对SSR引物和8个国槐种质对试验确定的最佳反应体系进行验证,均能获得清晰、稳定的条带。该优化反应体系的建立为利用SSR标记对国槐种质资源进行深入研究和利用奠定了基础。

In order to establish optimum SSR-PCR reaction system for Sophora japonica L.,the L_(16)(4^(5))orthogonal design and single factor experiments were used to optimize 5 main factors affecting SSR-PCR system,which were ExTaq enzyme,Mg^(2+),dNTPs,template DNA and primer dosages.The results showed that the optimal SSR-PCR reaction system was 10μL and contained 25 mmol/L Mg^(2+)1.0μL,2.5 mmol/L dNTPs 0.2μL,10μmol/L primers 0.5μL,5 U/μL ExTaq enzyme 0.05μL,30 ng/μL template DNA 1.0μL,10×PCR Buffer 1.0μL,ddH_(2)O 6.25μL.Four pairs of primers and eight S.japonica germplasms were randomly selected to verify the optimized SSR-PCR system,and all could obtain clear and stable polymorphic bands.The establishment of this optimal SSR-PCR reaction system could lay foundations for further research and utilization of S.japonica L.by using SSR markers.