暴马丁香ISSR-PCR反应体系优化及引物筛选

Optimization of ISSR-PCR System and Selection of Primers for Syringa reticulata var.amurensis

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 an optimal reaction system oflSSR-PCR in Syringa reticulata var. amurensis, the suitable concentration ranges of the different influential factors were firstly screened by the single factor experiments design with genome DNA as the templates. And then the 5 factors （DNA template, Mg2＋, dNTPs, Taq polymerase and primer） that affected the ISSR-PCR amplification system were optimized through the orthogonal experiment design （L16（4~5））. The results showed that a suitable ISSR-PCR reaction system established： 40 ng DNA template, 2.0 mmol/L Mg2＋, 0.4 μmol/L primer, 1.5 U Taq polymerase, 0.2 mmol/L dNTPs were contained in 25 μL reaction solution. Using this system, 10 primers with high resolution and multiple polymorphic bands were selected from 60 primers. The optimal ISSR-PCR system and polymorphism primers could be provide the scientific basis for related research using ISSR molecular marker technology in Syringa reticulata var. araurensis.