【摘要】 快速高效的DNA提取是作物大规模分子育种的关键一步。旨为构建一种快速提取大白菜基因组DNA的方法,以大白菜叶片为试验材料,比较了CTAB法、二步CTAB法以及4种碱裂解法提取DNA的质量,分析了不同方法提取的DNA为模板的PCR扩增效果,还对不同方法提取的基因组DNA的保存时间和保存条件进行了比较,选择最优的方法在抗根肿病基因分子标记辅助选择中进行了应用。结果表明,CTAB法和3种碱裂解法提取的基因组DNA作为模板的PCR扩增产物,都可以通过8%的非变性聚丙烯酰胺凝胶电泳检测到清晰的条带。其中碱裂解法Ⅲ,不仅提取质量好,而且提取过程简单、快速,能够满足大白菜高通量DNA提取的需要,提取的DNA在4℃和-20℃的条件下保存,将保存至30 d的基因组DNA作为模板进行PCR扩增,产物仍然可以通过聚丙烯酰胺凝胶电泳检测到清晰的条带,说明这种方法保存时间较长,经验证,该方法在抗根肿病基因分子标记辅助选择中的应用效果也较好。碱裂解法Ⅲ显著提高了大白菜分子标记辅助筛选的效率,可广泛应用于大白菜分子标记辅助选择育种。更多还原

【Abstract】 Rapid and efficient DNA extraction is the key step in large-scale molecular breeding of crop.To construct a method for rapid extraction of genomic DNA from Chinese cabbage,the Chinese cabbage leaves were used as experimental materials,the quality of DNA extracted by the CTAB method,two-step CTAB method and four alkaline lysis methods was compared,the PCR amplification effects of DNA extracted by different methods were analyzed,and the preservation time and preservation conditions of genomic DNA extracted by different methods were compared,the optimal method was selected for the application of molecular marker-assisted selection against clubroot disease.The results showed that genomic DNA extracted by the CTAB and three kinds of alkaline lysis methods could be used as a template for PCR,and the PCR amplification products could be detected by 8% non-denaturing polyacrylamide gel electrophoresis to clear bands.Among them,the alkaline lysis method Ⅲ,not only the extraction quality was good and the extraction process was simple and fast,but also it could meet the needs of high-throughput DNA extraction of Chinese cabbage,the extracted DNA was stored at 4 ℃ and-20 ℃,and the genomic DNA stored for 30 days was used as a template for PCR amplification,the products was still detected by polyacrylamide gel electrophoresis to clear bands,it illustrates that this method has a long preservation time and it has been proved that this method has a good effect on the application of molecular marker-assisted selection against clubroot disease.Alkaline lysis methodⅢhassignificantly improved the efficiency of Chinese cabbage molecular marker-assisted selection,and can be widely used in Chinese cabbage molecular marker-assisted selection breeding.更多还原