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以玫瑰为试材,采用L16(45)正交设计和单因素试验2种方法,研究模板DNA、Mg2+、dNTPs、引物和Taq酶5个因素对玫瑰SCoT-PCR反应体系的影响,建立最优化的反应体系并筛选合适引物。结果表明:模板DNA浓度为1.50ng/μL,Mg2+浓度为2.00mmol/L,dNTPs浓度为0.35mmol/L,引物浓度为0.70μmol/L,Taq酶用量为0.50U时,可建立玫瑰SCoT-PCR最佳反应体系,并筛选出20条扩增条带清晰、多态性丰富的SCoT引物。反应体系的优化及引物的筛选,为日后利用SCoT分子标记技术对玫瑰进行相关研究提供理论依据和技术支持。
The effects of template DNA, Mg2 +, dNTPs, primers and Taq enzyme on the SCoT-PCR reaction system of roses were studied by using L16 (45) orthogonal design and single-factor experiments with rose as test material. The reaction system and screening appropriate primers. The results showed that when the DNA concentration of template was 1.50ng / μL, the concentration of Mg2 + was 2.00mmol / L, the concentration of dNTPs was 0.35mmol / L, the concentration of primer was 0.70μmol / L and the amount of Taq enzyme was 0.50U, Rose SCoT-PCR The optimal reaction system was screened and 20 SCoT primers with clear amplification bands and abundant polymorphisms were screened out. The optimization of the reaction system and the selection of the primers provided the theoretical basis and technical support for the future research on the rose by using SCoT molecular marker technology.