马铃薯SSR标记多重PCR反应体系优化研究

Multiplex PCR reaction system optimization with potato SSR marker

以马铃薯品种克新18为试验材料,探讨了多重PCR反应体系主要成分、引物不同比例关系及退火温度对SSR标记扩增的影响。在不改变其他成分浓度的条件下,对PCR反应体系的4个组分(Taq酶、MgCl2、模板DNA、dNTPs)进行浓度或用量梯度试验;利用正交设计L(934)对反应体系的4对引物组合(STM0014、Pat、SSI、UGP)在3个水平上进行优化;最终确立了马铃薯SSR标记多重PCR反应优化体系,总体积为20μL;2.5μL 25 mmol.L-1 MgCl2,0.6μL 10 mmol.L-1 dNTPs,Taq酶0.8 U,模板DNA 80 ng;4 mmol.L-1的4对引物之间的用量比为2:1:2:3,退火温度为54.7℃。优化后的反应体系重复性好,扩增结果稳定可靠,能够明显区分不同的马铃薯品种。为进一步探讨马铃薯品种资源遗传多样性、构建DNA指纹图谱打下了坚实的基础。

Potato variety Kexin18 was used as testing materials in the research to study the impacts from main components in multiplex PCR reaction system, different primer ratios and annealing temperature in SSR marker amplification. Concentration and gradient experiments for four components （Taq enzyme, MgC12, DNA template and dNTPs） in PCR reaction system were used in the research with the concentration of the other component remain the same; the orthogonal design 1_9（34） was applied in the optimization of four primer combinations （STM0014, Pat, SSI, UGP） in the reaction system at three levels; the temperature gradient selection was used to find out the optimum annealing temperature for the primer. The optimized multiplex PCR reaction system of potato SSR marker with a total volume of 20 μL： 2.5 μL 25 mmol. L-1 MgCI2, 0.6 μL 10 mmol. L-1 dNTPs, 0.8 U Taq, 80 ng DNA template was ultimately established through the comparison and analysis of test results; the ratio of four pairs of 4 mmol. L-1 primers was 2：1 ： 2：3, and the annealing temperature was 54.7 ℃. The optimized reaction system was of good reproducibility; and the stable and reliable amplification results was able to clearly distinguish different potato varieties. This research built the solid foundation for the further study of genetic diversity of potato germplasm and construction of DNA fingerprint.