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[目的]研究SRAP分子标记用于苦荞分析中的条件优化。[方法]采用交互正交设计L27(313)对苦荞SRAP-PCR反应体系进行了5因素(Mg2+、dNTP、Taq酶、模板DNA和引物)3水平优化筛选,比较了非变性和变性PAGE检测方法,并进行了DYCZ-24F与DYCZ-20C2种电泳操作系统的对比试验。[结果]4个单因素(Mg2+、dNTP、Taq酶和引物)和2个交互作用(Mg2+×dNTP、Mg2+×Taq酶)对苦荞SRAP-PCR有极显著影响;最佳反应体系为:20μl总体积,Mg2+1.5mmol/L,dNTP0.2mmol/L,Taq酶1.5U,模板DNA40ng,引物0.25μmol/L,10×缓冲液2μl。运用该体系对7份苦荞材料进行扩增,电泳结果显示扩增条带清晰、多态性高、重复性好。将扩增产物进行非变性与变性PAGE和2种电泳操作系统检测,结果显示,非变性PAGE、DYCZ-24F操作系统更适合于SRAP的分析。[结论]该研究为今后构建苦荞SRAP遗传图谱奠定了基础。
[Objective] The research aimed to study the condition optimization of SRAP molecular marker for tartary buckwheat analysis. [Method] The 5 factors (Mg2 +, dNTP, Taq enzyme, template DNA and primer) 3 levels of SRAP-PCR reaction system of tartary buckwheat were optimized by orthogonal orthogonal design L27 (313). Non-denaturing and denaturing PAGE Method, and DYCZ-24F and DYCZ-20C2 kinds of electrophoresis operating system comparison test. [Result] Four single factors (Mg2 +, dNTP, Taq enzyme and primer) and two interactions (Mg2 + × dNTP, Mg2 + × Taq) had a significant effect on the SRAP-PCR of buckwheat. The optimal reaction system was 20μl Total volume, Mg2 + 1.5 mmol / L, dNTP 0.2 mmol / L, Taq enzyme 1.5 U, template DNA 40 ng, primer 0.25 mol / L, 10x buffer 2 l. The system was used to amplify 7 pieces of buckwheat material. The electrophoresis results showed that the amplified bands were clear, with high polymorphism and good reproducibility. The amplified products were subjected to PAGE and denaturing PAGE and two electrophoresis operating systems. The results showed that the native PAGE and DYCZ-24F operating systems were more suitable for SRAP analysis. [Conclusion] This study laid the foundation for constructing SRAP genetic map of buckwheat in the future.