红花檵木SRAP反应体系的建立及优化

Establishment and Optimization of SRAP Reaction System in Loropetalum chinense var.rubrum

为了获得红花檵木清晰的SRAP标记图谱,对红花檵木DNA的提取方法以及SRAP-PCR反应体系的影响因子进行了初步探讨,筛选和建立了扩增多态性高、重复性好、带型清晰的DNA模板和SRAP-PCR反应体系,同时提出了应用SRAP分子标记构建红花檵木遗传图谱的可行性.最佳SPAR-PCR反应体系为:在50μL的反应体系中,Mg2+1.6 mmol/μL,dNTPs 1.0 mmol/μL,DNA模板150 ng,DNA聚合酶3.6 U,上下引物各0.20 mmol/L.扩增程序为:94℃预变性4 min,反应前5个循环在94℃1 min、35℃1 min、72℃1 min条件下运行,随后的30个循环复性温度提高到55℃,最后72℃延伸5 min.

To obtain clear SequenceRelated Amplified Polymorphism （SRAP） fingerprints of Loropetalum chinense var. rubrum, this paper researched on the DNA extraction method and the factors influencing SRAP analysis, and then developed a reliable, effective and reproductive PCR reaction system for detecting SRAP. SRAP technology is a useful molecular marker system for mapping and gene tagging in Loropetalurn chinense var. rubrum, but its optimal SPAR-PCR reaction system is as follows, 50 /μL PCR reaction mixture consisting of 1. 6 mmol/L of MgC12, 1.0 mmol/L of dNTPs, 100 ng of genomie DNA, 3. 6 unit of Taq polymerase and 0. 20 mmol/L of primer. Samples were subjected to a thermal profile for amplification in an oven tbermo-circulator; 4 min of denaturing at 94℃, five cycles of three steps; 1 min of denaturing at 94℃, 1 min of annealing at 35C and 1 rain of elongation at 72℃. In the following 30 cycles the annealing temperature was increased to 55℃, with a final elongation step of 5 min at 72℃.