贵州野生淡黄花百合RAPD反应体系的建立

Establishment of RAPD Reaction System for Wild Lilium sulphureum in Guizhou Province

以贵州林下野生淡黄花百合(Lilium sulphureum Baker)幼嫩叶片为材料,采用改良的CTAB法提取百合基因组DNA,得到了较高质量的DNA。并对影响随机扩增多态DNA性(RAPD)反应的主要因素进行了优化,建立并优化了野生淡黄花百合的RAPD反应体系及程序。优化的20μL反应体系中包含20 ng模板DNA,2.0 mmol/L Mg2+、0.25 mmol/L dNTPs、1.0 U Taq DNA聚合酶、10×Buffer 2μL、0.4μmol/L随机引物、ddH2O补足至20μL。优化的RAPD扩增程序为94℃3 min;94℃50 s,37℃40 s,72℃80 s,40个循环;72℃10 min,4℃保存。该反应体系具有较好的稳定性及可重复性,适合贵州野生淡黄花百合遗传多样性研究。

Genomic DNA with good quality from tender leaves of wild Lilium sulphureum sampled from undergrowth in Guizhou province was extracted by modified CTAB method.And the RAPD reaction system for L.sulphureum was optimized.The results showed that the optimized 20 μL reaction systerm contained 20 ng DNA,2.0 mmol/L Mg2＋,0.25 mmol/L dNTPs,1.0 U Taq DNA polymerase,10×Buffer 2 μL,0.4 μmol/L primer,and the rest volume was filled with ddH2O.And PCR procedure was 94 ℃ 3 min;94 ℃ 50 s,37 ℃ 40 s,72 ℃ 80 s,40 cycles;72 ℃ 10 min,4 ℃ forever.The reaction system was well reproducible and highly reliable,and was suitable for studying the genetic diversity of wild L.sulphureum in Guizhou province.