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相关序列扩增多态性(Sequence-Related Amplified Polymorphism,SRAP)是对ORFs(OpenReading Frames)进行扩增的一种新型的基于PCR的标记方法。以CTAB法提取的红松针叶DNA为模板,应用单因子试验及L16(45)正交试验系统分析了DNA模板浓度、Mg2+浓度、dNTPs浓度、引物浓度、Taq酶浓度等对SRAP-PCR扩增结果的影响。确定了PCR的最佳反应体系为:20μL体系中,1×PCRbuffer 2μL、DNA模板40~100 ng,Mg2+2.5mmol/L,dNTPs 0.15 mmol/L,引物0.15μmol/L,Taq酶1.5 U。PCR最优的扩增程序为:进行35个循环,前5个循环中94℃变性1 min,35℃复性1 min,延伸30 s;后30个循环中94℃变性1 min,50℃复性1 min,72℃延伸1 min;最后72℃延伸7 min,4℃保存。在此反应体系下,所扩增谱带清晰、稳定、多态性高,为进行红松不同种源间遗传分化以及构建遗传图谱等内容的研究奠定基础。
Sequence-Related Amplified Polymorphism (SRAP) is a novel PCR-based method for the amplification of ORFs (OpenReading Frames). Using DNA extracted from pine needles DNA as template, single factor experiments and L16 (45) orthogonal test system were used to analyze the effects of DNA template concentration, Mg2 + concentration, dNTPs concentration, primer concentration and Taq polymerase concentration on SRAP-PCR amplification Effect of the result. The optimum reaction system for PCR was determined as follows: 1 × PCR buffer 2 μL, DNA template 40 ~ 100 ng, Mg2 + 2.5 mmol / L, dNTPs 0.15 mmol / L, primer 0.15 μmol / L and Taq 1.5 U in 20 μL system. The optimal PCR procedure was as follows: 35 cycles of denaturation at 94 ° C for 1 min in the first 5 cycles, refolding at 35 ° C for 1 min and extension for 30 s, denaturation at 94 ° C for 1 min followed by 30 cycles in 30 cycles 1 min at 72 ° C, 1 min at 72 ° C, 7 min extension at 72 ° C, and storage at 4 ° C. Under this reaction system, the amplified bands were clear, stable and highly polymorphic, which lays the foundation for the research on the genetic differentiation and construction of genetic maps between different provenances of Korean pine.