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以温郁金为试材,运用L25(56)正交设计在5个水平上对影响温郁金SCoT-PCR反应的模板DNA、Mg2+、dNTPs、Taq酶和引物5个因素进行优化试验,对PCR结果进行极差分析。建立并优化温郁金的目标起始密码子多态性-聚合酶链式反应(SCoT-PCR)体系,以期为温郁金的遗传多样性分析及分子鉴定等研究提供技术支持。结果表明:建立了温郁金SCoT-PCR的最佳反应体系(20μL):引物0.8μmol/L,dNTPs 0.4mmol/L,Mg2+1.5mmol/L,Taq酶0.5U,模板DNA 40ng,且确定各因素对温郁金SCoT-PCR反应效果的影响大小依次为:dNTPs>Taq酶>引物>Mg2+>模板DNA,其中dNTPs对体系影响最大。优化的温郁金SCoT-PCR反应体系在多个温郁金品种遗传多样性研究中得到了验证,结果表现出良好的稳定性、重复性和多态性丰富等特点,可用于今后温郁金品种遗传多样性分析、系统发育分析、遗传图谱构建、基因定位和分子标记辅助育种等研究。
The optimum conditions were as follows: temperature 25 ℃, temperature 25 ℃, temperature 25 ℃, temperature 25 ℃, temperature 25 ℃, Poor analysis. To establish and optimize the target start codon polymorphism-polymerase chain reaction (SCoT-PCR) system of warm tulip, in order to provide technical support for the research of genetic diversity analysis and molecular identification of warm tulip. The results showed that the best reaction system (20μL) for SCoT-PCR was established: primer 0.8μmol / L, dNTPs 0.4mmol / L, Mg2 + 1.5mmol / L, Taq enzyme 0.5U and template DNA 40ng, The effect of SCoT-PCR on the effect of SCoT-PCR was as follows: dNTPs> Taq enzyme> primer> Mg2 +> template DNA, of which dNTPs had the most influence on the system. The optimized temperature-tolerant SCoT-PCR reaction system was validated in the study of genetic diversity of a few warm tulip cultivars. The results showed good stability, repeatability and rich polymorphism. It could be used for the analysis of genetic diversity of the cultivated tulip plants in the future, Phylogenetic analysis, genetic map construction, gene mapping and molecular marker assisted breeding.