目的 建立黄芪ISSR分析最佳反应体系, 并分析内蒙古地区黄芪遗传多样性。方法 采用单因素梯度浓度实验和正交试验相结合的优化方法建立稳定、可靠的ISSR反应体系;对内蒙古9个盟市30个不同居群的黄芪样本进行遗传多样性分析, 用NTSYS2.1软件进行数据统计。结果 ISSR-PCR最佳反应体系(20 μL):10×PCR 缓冲液 2.0 μL、MgCl₂ 1.5 mmol/L、dNTP 0.4 mmol/L、引物0.5 μmol/L、Taq DNA聚合酶1.5 U、模板DNA 40 ng;15条ISSR引物共检测到169个扩增位点, 多态性位点157个, 多态位点百分率为75%～100%, 遗传距离变幅范围0.242 7～0.730 8, 聚类分析可知, 30个居群黄芪样本可分为2大类, 大多数呈现地域分布规律。结论 建立的黄芪ISSR-PCR反应体系稳定可靠;内蒙古地区黄芪具有较高的遗传多样性, 种质间的亲缘关系与地理位置具有一定的关系。

Objective To establish an optimum reaction system suitable for ISSR analysis of Astragalus membranaceus and to analyze the genetic diversity of wild populations in Inner Mongolia. Methods A stable and reliable ISSR reaction system was set up combining the concentration gradient of the single factor test and orthogonal test. The genetic diversity of 30 A. membranaceus populations in nine zones of Inner Mongolia was analyzed using NTSYS2.1 software. Results The optimal ISSR reaction system (20 μL) contained 10 × PCR buffer 2.0 μL, 1.5 mmol/L MgCl₂, 0.4 mmol/L deoxyribonucleotide triphosphate (dNTP), 1.5 U Taq DNA polymerase, 0.5 μmol/L primer, and 40 ng template DNA. A total of 169 amplified loci were detected by 15 ISSR primers, in which 157 loci were polymorphic loci with the percentage of 75%～100%. The genetic distance amplitude ranged between 0.242 7—0.730 8. The clustering analysis showed that 30 A. membranaceus populations could be divided into two categories, and most of them corresponded to the geographical distribution. Conclusion ISSR-PCR reaction system for A. membranaceus is stable and reliable. Wild resources of A. membranaceus in Inner Mongolia have higher genetic diversity. The genetic relationship of the populations is correlated with its geographic location.

内蒙古自然科学基金资助项目(2012MS0529)