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本研究利用L16(54)正交试验设计研究了影响冬瓜SSR-PCR的五个因子(10×Buffer(含Mg2+),d NTP,DNA,引物,r Taq),研究结果表明Buffer(含Mg2+)和r Taq对PCR影响最明显,最终筛选出最佳反应体系:10×Buffer 2.0μL(1×Buffer),d NTP 2.0μL(200μmol/μL),DNA 0.6μL(6 ng/μL),引物0.4μL(0.2μmol/L)。体系体积为20μL,循环数为35。引物筛选程序为:94℃预变性3 min,94℃变性45 s,58℃~68℃复性30 s,5个循环,每个循环退火温度降2℃,72℃延伸1 min,94℃变性30 s,50℃~58℃复性1 min,8个循环,每个循环退火温度降低1℃,72℃延伸1 min,94℃变性30 s,50℃复性30 s,72℃延伸1 min,共30个循环,72℃延伸7 min。本研究建立了适合冬瓜的SSR反应体系和引物多态性筛选程序。
In this study, five factors (10 × Buffer (including Mg2 +), dNTP, DNA, primer and r Taq) affecting SSR-PCR of wax gourd were studied using L16 The optimal reaction system was screened by RT-PCR and Taq PCR. The optimal reaction system was screened: 10 × Buffer 2.0 × L, 200μ mol / μL dNTP, 0.6μL DNA (6ng / μL), 0.4 μL (0.2 μmol / L). The volume of the system is 20 μL and the number of cycles is 35. The primer screening procedure was as follows: pre-denaturation at 94 ° C for 3 min, denaturation at 94 ° C for 45 s, refolding at 58 ° C-68 ° C for 30 s for 5 cycles, annealing at 2 ° C per cycle, extension at 72 ° C for 1 min, denaturation at 94 ° C 30 s, renaturation at 50 ℃ ~ 58 ℃ for 1 min, 8 cycles, annealing temperature decreased by 1 ℃ per cycle, extension at 72 ℃ for 1 min, denaturation at 94 ℃ for 30 s, renaturation at 50 ℃ for 30 s, extension at 72 ℃ for 1 min , A total of 30 cycles, 72 ℃ extended 7 min. In this study, SSR reaction system and primer polymorphism screening program for wax gourd were established.