The orthogonal design table of L16(4 )was used to optimize the five factors( Taq DNA polymerase, Mg2+ , DNA template, dNTPs and primer concentrations) of SCoT-PCR( start codon targeted polymorphism) system in tree peony. The results showed that the optimized system was as follows; a total volume of 20 μL including 1. 0 ng/μL DNA template,0. 50 U Taq polymerase,0. 60 μmol/L primer,2. 50 mmol/L Mg2+ ,0. 25 mmol/L dNTPs and 1 PCR Buffer. Each factor had different effect on the results. The concentration of Mg * was the key factor affecting the SCoT-PCR system. The optimized SCoT-PCR system was tested on seventeen peony genus,and the result was stable and reliable. From the 36 primer combinations tested,Twenty-four were selected with clear band patterns and abundant polymorphism. The optimized SCoT-PCR system and polymorphism primmer combinations provided the basis for further research on Paeonia suffruticosa.%以牡丹基因组DNA为模板,采用L16(45)正交试验设计对影响目标起始密码子多态性-聚合酶链式反应( SCoT-PCR)的五因素(Taq酶用量、Mg2+浓度、模板DNA用量、dNTPs浓度和引物浓度)进行优化试验,建立了优化的牡丹SCoT-PCR反应体系:Mg2+ 2.50 mmol/L、dNTPs 0.25 mmol/L、引物0.60 μmol/L、Taq DNA聚合酶0.50 U、模板DNA 1.00 ng/μL,1×PCR-Buffer,总体积20.00 μL.比较各因素对扩增反应的结果,其中以Mg2+浓度的影响最大,DNA模板用量的影响最小.运用牡丹17个品种验证了该体系稳定可靠,并从36个SCoT引物中筛选出扩增条带清晰、多态性丰富的24个引物.这一体系的建立及多态性引物的筛选为今后利用SCoT标记技术对牡丹进行相关研究提供了科学依据.
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