濒危植物长叶红砂ISSR扩增条件的优化与引物筛选

Optimizing of ISSR Amplifying Condition and Primers Selection of Endangered Plant Reaumuria trigyna Maxim

对濒危植物长叶红砂(Reaumuria trigynaMaxim)的核基因组DNA进行ISSR扩增条件的优化与引物筛选.利用单因素实验,测试了ISSR-PCR反应体系中模板DNA的含量、引物浓度、Master Mix等因素对反应结果的影响,经过优化实验,建立了长叶红砂ISSR-PCR的最佳反应体系,15μL PCR反应体积包括Mix4.5μL,模板DNA 20 ng,引物0.6μmol/L.利用优化反应体系,从60个ISSR引物中筛选出稳定性好、重复性高的14个引物.对长叶红砂5个种群的85个个体进行扩增,共扩增出110个位点,其中多态位点102个,多态位点百分率为92.73%.长叶红砂ISSR反应体系的建立为利用ISSR分子标记技术进一步研究长叶红砂的遗传多样性奠定了良好的基础.

The ISSR amplification conditions of endangered plant Reaurnuria trigyna are optimized. The influencing factors, including Master Mix concentration, DNA templates dosage, primer dosage on iSSR amplification are tested using single factor method. The optimal ISSR-PCR conditions in experiments are as following. 4. 5μL Master Mix, 20 ng templates DNA, 0. 6μmol/L primers in total 15 μL PCR reaction system. Under the optimal reaction system, 14 ISSR primers with high stable and repeatable are selected from 60 primers. 85 individuals from 5 populations were studied, 110 loci are detected by 14 primers,among them 102 were polymorphic and polymorphic loci percentage was 92.73%. Establishment of the reaction system will be the basis for further study on the genetic diversity of R. trigyna by using ISSR marker.