道地药材咸丰白术ISSR反应体系优化及多态性引物筛选

Optimization of ISSR Reaction and Primer Screening for Genuine Medicinal Materials Xian-feng Atractylodes macrocephala Koidz.

为了得到适宜的咸丰白术ISSR反应体系,采用两轮U12(62×4)均匀设计的方法,对ISSR反应中各组分设置不同的梯度,分析反应体系各组分用量对扩增结果的影响。结果表明,在18μL的ISSR反应体系中,引物0.29μmol/L,2×Taq Master Mix 9.6μL,模板DNA 47 ng时,扩增条带较清晰,多态性好。根据得到的优化体系,进一步开展白术的引物筛选,并从中得到了9条扩增条带相对清晰稳定、多态性较好的引物。对筛选的多态性引物开展了温度梯度PCR实验,发现温度对ISSR-PCR扩增结果影响显著。本实验通过采用均匀设计的方法,降低了常规体系优化实验所需次数,节省了时间和试剂的用量,为快速建立ISSR反应体系和后续咸丰白术遗传多样性分析提供了便利与帮助。

In order to obtain optimized ISSR reaction system for Xian-feng A tractylodes macrocephala Koidz., Two rounds of uniform design method U12 （62×4） were adopted to analyze the effect of different components on ISSR-PCR reaction, and different gradients were set for each factor in ISSR-PCR reaction. Our results indicated the optimal reaction system contains 0.29 μmol/L primer, 9.6 μL 2×Taq Master Mix and 47 ng DNA template in a total volume of 18 μL, which showed clear amplification bands and fine polymorphism. Primer screening was further conducted according to the optimal system, and 9 primers showed relative clear, stable amplification bands with desirable polymorphism. Subsequently, thermal gradient test was used to validate the effect of temperature on ISSR-PCR reaction, which suggest the amplification results were significantly affected by melting temperature. In this study, the application of uniform design method not only greatly reduces the number of experiments, but also saves time and reagent usage, which is intended to facilitate the development of optimal ISSR reaction system and genetic diversity analysis in Xian-fengA tractylodes macrocephala Koidz.