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本研究采用L_(16)(4~5)正交试验设计方法,对朱顶红SRAP-PCR反应体系中的5种因素(Mg~(2+)浓度,d NTPs浓度,引物浓度,DNA模板量和Taq DNA聚合酶用量)进行优化,结果表明各因素对朱顶红SRAP-PCR扩增反应影响大小依次为:d NTPs浓度>引物浓度>Mg~(2+)浓度>DNA模板量>Taq DNA聚合酶用量。优化获得的朱顶红最佳SRAP-PCR反应体系为:1×PCR Buffer、Mg~(2+)浓度2.0 mmol/L、d NTPs浓度0.2 mmol/L、引物浓度0.25μmol/L、20μL体系中DNA模板量80 ng以及Taq DNA聚合酶用量0.5 U。用10个朱顶红品种基因组DNA对所得最优体系进行验证,证明该体系具有较高的稳定性和重复性,能够为朱顶红遗传多样性研究和遗传图谱构建等提供重要技术支持。
In this study, five factors (Mg 2+ concentration, d NTPs concentration, primer concentration, amount of DNA template and DNA template) of Hippeastrum SRAP-PCR reaction system were determined by L_ (16) (4 ~ Taq DNA polymerase), the results showed that the factors affecting the amplification reaction of Hippeastrum by SRAP-PCR were as follows: d NTPs concentration> Primer concentration> Mg 2+ concentration> DNA template amount> Taq DNA polymerase dosage . The optimized system of Hippeastrum by SRAP-PCR was as follows: 1 × PCR Buffer, 2.0 mmol / L Mg 2+, 0.2 mmol / L d NTPs, 0.25 μmol / L primer and 20 μL DNA template Amount of 80 ng and Taq DNA polymerase dosage of 0.5 U. The optimized system was verified by genomic DNA of 10 Hippeastrum varieties, which proved that this system has high stability and repeatability and could provide important technical support for the research on genetic diversity and genetic map construction of Hippeastrum.