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菰是一种稻族野生资源,水生或沼生,在我国有着广泛的分布。野生菰群体不仅是一种重要的育种基因资源,同时还有着巨大的生态作用。为建立适宜于菰的ISSR反应体系,以菰DNA基因组为模板对ISSR反应体系中的主要影响因子进行优化。采用单因素变量法在12个不同梯度水平上设计正交试验针对体系中d NTPs浓度、Mg2+浓度、Taq聚合酶浓度、引物浓度、模板DNA浓度及引物退火温度等6个因子进行优化,确定了在20μL ISSR反应体系中各组分的最适因素水平分别为:3.0 mmol Mg2+(10×Buffer),0.5 mmol d NTPs、1.0μmol引物浓度、0.24 U/μL Taq聚合酶、1.5 ng/μL DNA模板以及55℃退火温度。应用该优化体系对80个菰材料进行扩增,证实了该体系的适用性和稳定性,为菰遗传资源的鉴定、评价与利用提供了技术支撑。
菰 is a wild rice resource, aquatic or marsh, has a wide distribution in our country. Wild boars are not only an important genetic resource for breeding, but also have a huge ecological role. In order to establish a suitable ISSR reaction system, the main influencing factors in the ISSR reaction system were optimized using the DNA genome as a template. Six factors (dNTPs concentration, Mg2 + concentration, Taq polymerase concentration, primer concentration, template DNA concentration and primer annealing temperature) were optimized by single factor method at 12 different gradient levels. The optimal factors of each component in 20μL ISSR reaction system were: 3.0 mmol Mg2 + (10 × Buffer), 0.5 mmol dNTPs, 1.0 μmol primer, 0.24 U / μL Taq polymerase, 1.5 ng / μL DNA template And an annealing temperature of 55 ° C. The application of the optimization system to 80 pieces of material to expand, confirmed the applicability and stability of the system, for 菰 identification, evaluation and utilization of genetic resources provided technical support.