珍稀植物浙江雪胆SCoT-PCR体系的建立及优化

Establishment and Optimization of the SCoT-PCR Reaction System of a Rare Plant Species Hemsleya zhejiangensis

为建立浙江雪胆SCoT-PCR的最佳反应体系,本研究采用正交实验对dNTPs、引物、模板DNA及Taq聚合酶等的浓度进行筛选,并对采自景宁畲族自治县的材料进行验证。结果表明,PCR体系中各因素用量对多态性结果的影响大小依次是引物>dNTPs=Taq酶>模板DNA,最佳反应体系(20μL)为0.7μL dNTPs(10 mmol/L),0.3μL引物(20μmol/L),0.4μL Taq酶(2 U/μL),1.2μL DNA模板(20 ng/μL),2μL 10×缓冲液(含20 mmol/L Mg^(2+)),dd H_(2)O 15.4μL;筛选出11条稳定性好、多态性高和重复性好的引物,同时优化了它们的最佳退火温度;对10份种质资源进行验证,结果表明该体系扩增的结果稳定、扩增条带清晰。浙江雪胆SCoT-PCR体系的建立和优化,为后续开展遗传多样性研究、资源保护和分子育种提供了依据。

To establish an optimum SCoT-PCR reaction system of Hemsleya zhejiangensis,the orthogonal design was applied to screen the concentrations of dNTPs,primers,template DNA and Taq polymerase.It was then tested by using materials collected from Jingning She Autonomous County.The factors affecting polymorphism were listed as follows,primers>dNTPs=Taq polymerase>DNA,and the suitable reaction system in 20μL was 0.7μL dNTPs(10 mmol/L),0.3μL primer(20μmol/L),0.4μL Taq polymerase(2 U/μL),1.2μL DNA template(20 ng/μL),2μL 10×buffer(with 20 mmol/L Mg^(2+))and 15.4μL ddH_(2)O.Totally eleven primers with clear band patterns,high polymorphisms,and good repeatability were selected.Meanwhile,the optimum annealing temperatures were obtained.Ten germplasm resources were tested to verify the stability of the reaction system,and as a result,clean band patterns were observed.Establishment and optimization of the SCoT-PCR system provide insights into studies on genetic diversity,resource conservation,and molecular breeding of Hemsleya zhejiangensis in the future.