南酸枣ISSR-PCR反应体系的建立及优化

Establishment and optimization of ISSR-PCR system for Choerospondias axillaris

以南酸枣叶片基因组DNA为模板，采用单因子实验方法对影响ISSR反应体系的dNTP、Mg2＋、引物浓度、模板DNA、Taq酶等5个因子进行优化，结果表明，在20 L ISSR反应体系中，各反应物的最适含量为：2.5 mmol/L Mg2＋、0.2 mmol/L dNTP、0.25～0.5 mol/L引物、20 ng DNA和0.5 U Taq聚合酶。利用该优化体系扩增85条ISSR引物，有65条有扩增产物，38条具有多态性，并且利用842引物扩增不同南酸枣DNA模板，扩增的目的条带清晰、多态性好、稳定性强，可以用于后期的南酸枣种质资源鉴定及遗传多样性研究。

In order to optimize ISSR-PCR system for Choerospondias axillaris, different levels of concentration of dNTP, Mg2＋, primer, template DNA and Taq DNA polymerase were trailed by single factor experiment. The results showed that the optimal ISSR-PCR system for Ch. axillaris contained 2.5 mmol/L Mg2＋、0.2 mmol/L dNTP、0.25~0.5 mol/L primer、20 ng DNA和0.5 U Taq polymerase in 20 L PCR system. Furthermore, 65 primers could produce target bands among 85 ISSR primers, and including 38 polymorphic primers. With this optimal ISSR-PCR amplification system, high resolution, multiple polymorphic and good repeatability bands were gained on 6 different DNA templates, which indicated the stability and availability of this system. The ISSR-PCR system, which was established in this paper, could be used to study germplasm identification and genetic diversity of Ch. axillaris.