长白山杜鹃花基因组DNA提取及RAPD体系的建立

Establishment of RAPD System and DNA Extraction from Rhododendron in Changbai Moutain

采用改良的CTAB法提取杜鹃花的基因组DNA,所得的DNA纯度高、质量好,可用于RAPD分析。筛选出的杜鹃花RAPD反应的最佳体系为25μL反应体系中包括模板DNA20～200 ng,引物10pmol,dNTPs100μmol·L-1,TaqDNA聚合酶0.5U,Mg2+2.5 mmol·L-1,10×buffer缓冲液2.5 mmol·L-1,其余部分用无菌超纯水补充。PCR扩增程序为94℃预变性5 min;94℃变性1 min,37℃退火1min,72℃延伸2min,40次循环;72℃最终延伸7 min。应用优化后的反应体系PCR扩增获得的RAPD指纹图谱带型清晰,重复性好,为长白山杜鹃花品种鉴定、分类的研究提供了一定基础。

High purity and quality DNA extracted from Rhododendron was used for RAPD analysis. The best RAPD reaction system optimized was： total volume 25μL,containing, contained 2 template DNA 10-200ng, primer 10pmol, dNTPs 100μmol·L^-1, MgCl2 2,5 mmol·L^-1,Taq DNA polymerase 0.5unit, 10×buffer 2.5μL, left complement with dd H20. PCR amplification condition was： pre-denaturation at 94℃ for 5min; Denaturation at 94℃ for lmin, annealing at 37℃ for lmin, extension at 72℃ for 2rain, cycle for 40 times. Final extension at 72℃ for 7min. RAPD fingerprinting obtained by the optimized system was clear and repetitive, which provided a certain foundation for Rhododendron variety identification and category in Changbai Mountains.