早熟马铃薯 ISSR-PCR反应体系优化及引物筛选

为得出马铃薯ISSR-PCR最佳的反应体系,采用正交试验设计,对rTaq DNA聚合酶、Mg2+、模板DNA、dNTPs和引物进行5因素4水平筛选.结果表明,马铃薯ISSR-PCR的最佳反应体系为:总体积25 μL的反应体系中含有rTaq DNA聚合酶0.5 U、Mg2+浓度为2.5 mmol/L、模板DNA用量为75 ng、dNTPs浓度为200 μmol/L、引物浓度为0.8 μmol/L.这些因素对PCR反应的影响大小顺序为Mg2+浓度＞rTaq DNA聚合酶用量＞dNTPs浓度=引物浓度＞模板DNA用量.应用该最佳反应体系对39条ISSR引物进行筛选,得出15条条带清晰、条带数多、重复性好的引物.并通过退火温度梯度试验得出筛选引物的最佳退火温度.

Optimization of ISSR-PCR reaction system and ISSR primer screening in early potato

To screen the optimum ISSR-PCR reaction system for potato, the experiment was conducted by using orthogonal design with 4 levels of 5 factors (i.e. rTaq DNA polymerase, Mg2+, DNA, dNTPs and primers). Results showed that the optimal reaction system of ISSR-PCR was as follows: 0.5 U rTaq DNA polymerase, 2.5 mmol/L Mg2+, 75 ng DNA, 200 mol/L dNTPs and 0.8 mol/L primer in 25 L reaction volume. And the PCR reaction effects was in the order of Mg2+ > rTaq DNA polymerase> dNTPs = primer> DNA. Then, 39 ISSR primers were screened by using the optimal reaction system, and 15 primers with clear band, more band number and good repeatability were selected. Finally, the optimized annealing temperatures of the 15 primers were obtained by gradient PCR.