金铁锁SSR-PCR反应体系的建立与优化

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

【目的】建立和优化金铁锁SSR-PCR反应体系。【方法】以金铁锁嫩叶为试验材料,采用Ezup柱式植物基因组DNA抽提试剂盒、SDS法和CTAB法提取金铁锁DNA,并对提取结果进行比较。利用初筛并合成的20对金铁锁EST-SSR引物,对影响SSR-PCR体系的模板DNA、引物、Taq DNA聚合酶和dNTP用量这4个因素进行L16(4^4)正交实验,从而建立较为稳定的金铁锁SSR-PCR反应体系。【结果】试剂盒法提取得到的金铁锁DNA质量最好且符合SSR分子标记试验;在20对金铁锁EST-SSR引物中,有2对可用于SSR多态性分析;各因素对PCR扩增效果的影响程度为引物>Taq DNA聚合酶>模板DNA>dNTP。试验最终确定的金铁锁SSR-PCR最佳反应体系(20μL):1.0 mL模板DNA(40 ng/μL)、1.5μL引物(10μmol/L)、0.3μL dNTP(10 mmol/L)、0.3μL Taq DNA聚合酶(5U/μL)以及2.0μL 10×PCR Buffer(含Mg^2+15 mmol/L),用ddH2O补足至总体积20μL。【结论】所建立的金铁锁SSR反应体系可为金铁锁不同种群的遗传变异研究及杂交育种时亲缘关系的分子鉴定提供前期研究基础。

[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(4^4) 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(10 mmol/L), 0.3 μL dNTP(10 mmol/L), 0.3 μL Taq DNA polymerase(5 U/μL) and2.0 μL 10×PCR Buffer(containing Mg^2+ 15 mmol/L), supplemented with ddH2 O to 20 μL.[Conclusion]A research basis on genetic variation of different populations and molecular identification of genetic relationship in crossbreeding could be provided by the SSR reaction system.