桃SRAP-PCR反应体系的建立与优化

Optimization of SRAP Reaction System in Peach(Prunus persica)

建立适宜桃基因组DNA的SRAP-PCR扩增体系,为桃基因图谱的构建和分子标记打下基础。以桃基因组DNA为模板,通过正交试验设计,从dNTPs、Mg2+、Taq酶、引物、模板5种因素4个水平对桃SRAP-PCR反应体系进行优化,所建立的体系为25μL:dNTPs为0.12 mmol/L,Mg2+为4 mmol/L,Taq酶2 U,引物为0.3 mmol/L,模板DNA50ng。PCR反应程序为:94℃预变性5 min;94℃变性l min,35℃复性l min,72℃延伸l min,5个循环;94℃变性l min,50℃复性l min,72℃延伸l min,35个循环,72℃延伸10 min。

The aim of the research was to establish SRAP-PCR amplification system which was suitable for Peach（Prunus persica） genome DNA so as to lay foundation for the construction of gene map and molecular marker in Peach（Prunus persica）.Peach（Prunus persica） genome DNA as template,the major components of SRAP,such as concentrations of dNTPs,Mg2＋,Taq DNA polymerase,primers and template,were optimized in this study by orthogonal design in five factors four levels respectively.The results showed that the optimum SRAP reaction system includes dNTPs 0.12 mmol/L,Mg2＋ 4 mmol/L,Taq DNA polymerase 2 U,primer 0.3 mmol/L and DNA template 50 ng in the 25 μL system.The most suitable protocol was initially denaturing at 94℃ for 5 min,then pre-amplifying at 94℃ 1 min,35℃ l min and 72℃ 1 min for five cycles,finally amplifying for 35 cycles when the annealing temperature was adjusted to 50℃.