火龙果EST-SSR分子标记反应体系的建立与优化

Establishment and Optimization of EST-SSR Marker Methodology in Pitaya

基于火龙果转录组测序序列设计引物,以生物学性状差异明显的11份火龙果种质DNA为材料,采用正交试验设计,对影响火龙果EST-SSR-PCR扩增的2×Taq PCR Master Mix、引物及模板DNA浓度等因素进行了优化,在此基础上对变性聚丙烯酰胺凝胶质量浓度及上样量进行筛选确定。以期建立适合火龙果的EST-SSRPCR最佳反应体系。结果表明,优化后的火龙果EST-SSR-PCR扩增的最佳反应体系为:10μL混合反应体系含基因组DNA 30 ng、6μL 2×PCR Mix和0.6μL(10-5mol/L)的EST-SSR引物。在聚丙烯酰胺凝胶电泳检测中,10%的变性聚丙烯酰胺凝胶质量浓度、2.5μL上样量扩增效果最佳。该体系的建立可为今后利用EST-SSR标记对火龙果遗传多样性分析、系统发育研究、遗传图谱构建、基因定位和分子标记辅助育种等研究提供基础。

Based on the RNA-Seq of Hylocereus polyrhizuse,EST-SSR markers were investigated. SSR primers were designed and synthesized. Genomic DNA of pitaya germplasms from 11 obviously distinguishable phenotypes were used to carry out PCR. To optimize the experimental system,the effect degree of 2 × Taq PCR Master Mix,primers and DNA template concentration was analyzed respectively through orthogonal test.Furthermore,the suitable loading quantity and concentration for de-naturing polyacrylamide gel electrophoresis were determined. The results showed that the optimal amplification system was 10 μL mixture containing 30 ng DNA template,6μL 2 × PCR Mix,and 0. 6 μL（ 10^-5 mol/L） EST-SSR primer. The optimal concentration for de-naturing polyacrylamide gel electrophoresis was 10%,and the loading quantity was 2. 5μL. Our results indicated that the optimized SSR-PCR system on pitaya germplasms would provide theoretic and technical support for analysis of genetic diversity,establishment of genetic maps,gene localizations and molecular marker assisted breeding.