Establishment and Optimization on the SSR-PCR Reaction System of Psammosilene tunicoides

Purpose Establish and optimize the SSR-PCR reaction system of Psammosilene tunicoides.

Method The leaves of P. tunicoides were used as material to extract DNA by three method of Ezup column plant genomic DNA extraction kit, SDS and CTAB, and the extraction results were compared. Twenty pairs of EST-SSR primers screened and synthesized were used to establish a stable SSR-PCR system of P. tunicoides with L₁₆(4⁴) orthogonal experiments of four factors on the dosage of template DNA, primers, Taq DNA polymerase and dNTP.

Result The quality of P. tunicoides DNA extracted by Ezup column plant genomic DNA extraction kit was the best and suitable for SSR molecular marker test. Among twenty pairs of P. tunicoides EST-SSR primers, two pairs could be used for SSR polymorphism analysis. The influence degree of each factor on the amplification effect of PCR was as follows: primer > Taq DNA polymerase > template DNA > dNTP. The optimal SSR-PCR reaction system (20 μL) was established as follows: 1.0 μL template DNA (40 ng/μL), 1.5 μL primer (10mmol/L), 0.3 μL dNTP (10 mmol/L), 0.3 μL Taq DNA polymerase (5 U/μL) and 2.0 μL 10×PCR Buffer (containing Mg²⁺ 15 mmol/L), supplemented with ddH₂O to 20 μL.

ConclusionA research basis on genetic variation of different populations and molecular identification of genetic relationship in crossbreeding could be provided by the SSR reaction system.