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为了对杜仲种质资源的遗传多样性研究奠定理论和技术基础,采用正交试验设计和单因素分析,建立并优化了杜仲的SSR-PCR反应体系。结果表明,杜仲SSR-PCR最适反应体系为正反向引物(10μmol.L-1)各0.2μL,Taq DNA聚合酶(5 U.μL-1)0.1μL,dNTP(10 mmol.L-1)0.2μL,模板DNA(5～10 ng.μL-1)1.0μL,Mg2+(25 mmol.L-1)0.6μL,10×PCR缓冲液1.0μL,HPLC超纯水6.7μL,总体积10.0μL。同时筛选出了3对多态性有效引物,研究结果证明了微卫星引物在不同物种间的通用性受物种间亲缘关系远近的影响,并初步揭示了杜仲的遗传多样性,表明了微卫星标记在遗传多样性研究上的优越性。
In order to lay a theoretical and technical foundation for the genetic diversity of germplasm resources of Eucommia ulmoides, SSR-PCR reaction system of Eucommia ulmoides was established and optimized by orthogonal experimental design and single factor analysis. The results showed that the optimal reaction system of SSR-PCR for Eucommia ulmoides Oliv. Was 0.2μL each of the positive and negative primers (10μmol.L-1), 0.1μL Taq DNA polymerase (5μ.μL-1), dNTP (10mmol.L-1 ), 1.0 μL of template DNA (5~10 ng.μL-1), 0.6 μL of Mg2 + (25 mmol.L-1), 1.0 μL of 10 × PCR buffer, 6.7 μL of HPLC ultrapure water and a total volume of 10.0 μL . At the same time, three pairs of polymorphic primers were screened. The results showed that the commonality of microsatellite primers among different species was influenced by the genetic relationship among species, and the genetic diversity of Eucommia ulmoides was revealed initially, which indicated that microsatellite markers Superiority in the study of genetic diversity.