广西莪术ISSR-PCR反应体系优化及引物筛选

Optimization of ISSR-PCR system reaction and primer selection of Curcuma kwangsiensis S.G·Lee et C·F.Liang

[目的]建立和优化广西莪术(Curcuma kwangsiensis S.G·Lee et C·F.Liang)的ISSR-PCR反应体系。[方法]通过单因素及正交试验设计研究模板DNA含量、Mg^(2+)浓度、dNTP浓度、TaqDNA聚合酶浓度、引物浓度对广西莪术ISSR-PCR反应的影响。[结果]25μL广西莪术的ISSR-PCR最佳反应体系为模板DNA用量30 ng,Mg^(2+)2.25 mol/L,dNTPs 2.5 mol/L,引物浓度0.5μmol/L,Taq DNA聚合酶用量1.25 U;利用该体系从100条ISSR引物中进行初筛和复筛,筛选出14条引物。[结论]优化建立的广西莪术ISSR-PCR反应体系稳定可靠,可用于广西莪术遗传多样性分析及种质资源保护研究。

[Objective]To establish and optimize the ISSR-PCR reaction system of Curcuma kwangsiensis.[Method] The single-factor experiment and orthogonal experiment was used to optimize the template DNA content, Mg^(2+)concentration, dNTPs concentration, TaqDNA polymerase concentration and primer concentration in the ISSR-PCR reaction system of Curcuma kwangsiensis.[Result] Optimum ISSR-PCR reaction system(25 μL) template DNA dosage of 30 ng, Mg^(2+) of 2.25 mol/L,dNTPs of 2.5 mol/L,primer concentration of 0.5 μmol/L,TaqDNA polymerization of 1.25 U.Using this system for primary screening and rescreening from 100 ISSR primers, 14 primers were selected.[Conclusion] The optimized ISSR-PCR reaction system can be used for the study of the genetic diversity analysis and germplasm resources protection in Curcuma kwangsiensis.