黄皮ISSR-PCR反应体系的优化

Optimization of ISSR-PCR Reaction System in Wampee

为应用ISSR分子标记对黄皮种质资源、分子标记辅助选择育种及其遗传多样性研究,通过单因素和正交试验对ISSR-PCR反应体系进行优化。结果表明,采用改良SDS法提取的DNA条带完整、无RNA污染,适于ISSR-PCR扩增;黄皮ISSR分析最适的扩增体系是:20μl的反应体系中含30ng的模板DNA、1.5UTaqDNA聚合酶、0.4μmol/L引物、0.3mmol/LdNTPs以及引物(gA)8C的最佳退火温度为52.4℃。

To study the extraction method of genomic for wampee. The improved SDS method was used to investigated by a single factor test and orthogonal DNA and optimization of ISSR-PCR reaction conditions extract DNA. The factors influencing ISSR-PCR were design experiment.The researcher used improved SDS method to extract DNA of Wampee, which meets ISSR requirements in quantity and purity. And a better amplification system of ISSR was obtained with the reation system comtaining 30ng template DNA,1.5U Taq DNA polymerase, 0.4 μmol/L primers, 0.3mmol/L dNTPs in the total volume of 20μl.The optimized annealing temperature was 52.4℃ for primer （gA）8C. The stable and reproducible optimal ISSR-PCR reaction system and suitable annealing temperature are established for Wampee, which had laid the good foundation for ISSR analysis on studies of germplasm resources identification, molecular marker assisted breeding and genetic diversity.