铁线莲属植物ISSR分子标记体系优化及指纹图谱的构建

Optimization of Clematis ISSR molecular marker system and fingerprint constrution

为进一步利用ISSR分子标记进行铁线莲遗传多样性分析、种质资源鉴定、遗传图谱构建等研究,本试验以卷萼铁线莲DNA为模板,采取L_(9)(3^(4))正交试验设计,对影响扩增反应的模板DNA含量、目标引物浓度、Master Mix、循环数4个主要因素进行优化,从而得出适宜于铁线莲的ISSR-PCR反应体系。并利用筛选出的12条多态性高的引物构建了16种野生铁线莲的指纹图谱。结果表明,在25μL反应体系中,各因素的最佳配比为模板DNA用量40 ng,引物浓度6μmol/L,Master Mix用量14μL,循环数40个。并且利用最优体系构建了16种野生铁线莲的遗传指纹图谱。该体系的建立为ISSR技术在铁线莲遗传多样性研究、遗传指纹图谱构建、分子标记辅助育种等研究提供了理论基础。指纹图谱的构建可以有效的对16种野生铁线莲进行区分和鉴定。

In order to apply ISSR molecular marker to the study of Clematis genetic diversity,germplasm identification and genetic fingerprint construction,the L_(9)(3^(4)) orthogonal experiment design was adopted,four main factors affecting the amplification reaction were optimized,including four factors,DNA template concentration,primer concentration,Master Mix and PCR cycle number,four main factors affecting the amplification reaction were optimized,the ISSR-PCR reaction system suitable for Clematis was obtained.The results showed that in the 25μL reaction system,the optimal ratio of all factors was as followed:Template DNA dosage was 40 ng/25μL,primer concentration was 6μmol/L,Master Mix dosage was 14μL/25μL,and the number of cycles was 40.The genetic fingerprints of 17 Clematis were constructed by using the optimized ISSR system.Optimization of ISSR-PCR reaction system in Clematis would provide a theoretical basis for the evaluation of genetic diversity,genetic fingerprinting construction of Clematis and molecular marker assisted breeding.The construction of fingerprint could effectively distinguish and identify the 17 Clematis.