薰衣草高质量DNA提取及ISSR-PCR多重化体系的建立

以伊犁薰衣草‘701’新鲜叶片为材料,对4种基因组DNA提取方法进行比较,并通过单因子梯度比较试验结合L9(34)正交优化设计,对ISSR-PCR扩增体系中退火温度、DNA、Mg2+、dNTP和引物浓度进行最佳条件及配比筛选。结果表明,改良3×CTAB法是薰衣草高质量DNA少量提取的最佳方法 ;建立薰衣草ISSR-PCR扩增优化体系为,20μL反应体系中包括模板DNA 50 ng,Mg2+3 mmol/L,dNTP 0.3 mmol/L,引物0.3 mmol/L,Taq酶1 U;扩增程序退火温度为56℃。运用本试验建立的ISSR-PCR优化体系,对5份薰衣草种质进行了初步验证,获得了良好的多样性扩增条带。

High-quality DNA Extraction of Lavender Leaves and Establishment of Multiple Optimizing of ISSR-PCR System

It was to optimize and establish extractive method of high-quality DNA and stable ISSR-PCR amplified system of lavender.Four genetic DNA-extractive methods were compared by using fresh leaves of YILI Lavender ’701’.The optimum conditions of ISSR-PCR system were compared and screened in different levels of primer,dNTP mixture,Mg2+,Taq DNA polymerase and annealing temperature based on gradient test of single factor and L9(34)orthogonal design.Results showed that the modified 3×CTAB is the best extractive method of high-quality lavender DNA.The optional parameters for 20 μL ISSR-PCR reaction system are DNA 50 ng,Mg2+ 3 mmol/L,dNTP 0.3 mmol/L,random primer 0.3 mmol/L and Taq DNA polymerase 1 U,respectively.The high-quality diversity bands were amplified when applied the optimizing system on five lavender cultivars.The research sets the experimental basis for the future cultivar identification,genetic mapping,and variety breeding and genetic diversity of genus Lavandula.