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为建立暴马丁香ISSR-PCR最佳反应体系,本研究以暴马丁香基因组DNA为模板,首先通过单因子试验设计筛选出各影响因子比较适宜的浓度范围,再利用正交试验,对暴马丁香ISSR-PCR反应有影响的模板DNA、dNTPs、Mg2+、Taq DNA聚合酶和引物浓度等5种因素4个水平进行了优化试验。暴马丁香的ISSR-PCR最佳反应体系最终确定为:在25μL的反应体系中,DNA模板是40ng,Mg2+浓度为2.0 mmol/L,引物浓度为0.4μmol/L,Taq DNA聚合酶用量为1.5 U,dNTPs浓度为0.2 mmol/L。利用该体系从60条ISSR引物中筛选出了扩增条带清晰、多态性好的10条引物。这一体系的建立及多态性引物的筛选为以后利用ISSR分子标记技术进行暴马丁香的有关研究提供了科学依据。
In order to establish the optimal ISSR-PCR reaction system, we used the genomic DNA of Staphylococcus aureus as a template to screen out the suitable concentration range of each influencing factor through the single factor experiment design. The effects of template DNA, dNTPs, Mg2 +, Taq DNA polymerase and primer concentration on the ISSR-PCR reaction were studied. The optimum reaction system of ISSR-PCR was as follows: the DNA template was 40ng, the concentration of Mg2 + was 2.0mmol / L, the primer concentration was 0.4μmol / L, the amount of Taq DNA polymerase was 1.5 U, dNTPs concentration of 0.2 mmol / L. Sixty ISSR primers were screened out by this system. Ten primers with clear bands and good polymorphism were screened out. The establishment of this system and the selection of polymorphic primers provide a scientific basis for the future research on the use of ISSR molecular marker techniques for the detection of Malvaceae.