The Optimization of EST-SSR PCR Reaction System for Robinia pseudoacacia L.

doi:10.11924/j.issn.1000-6850.2014-0325

Abstract

Abstract: In order to establish EST-SSR Polymerase Chain Reaction (PCR) system of Robinia pseudoacacia L., L₁₆(4⁵) orthogonal design and single factor experiment was used to optimize 5 main factors affecting SSRPCR system: primer, template DNA, dNTPs, Mg²⁺ and Taq DNA polymerase. The results showed that the optimal reaction system was 20μL total reaction system containing 0.5μmol/L each primer, 30 ng temple DNA, 0.15 mmol/L dNTPS, 0.5 mmol/L Mg²⁺, 1.0 U Taq DNA polymerase. The author randomly selected 3 varieties of black locust clones and 7 EST-SSR primers to test this optimized EST-SSR system, stable and clear polymorphic bands were acquired. the establish of this optimal reaction system laid the foundation for the development of EST-SSR markers and using EST-SSR markers in-depth study and made use of the locust tree germplasm resources in China.

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