杜鹃花属植物SRAP-PCR反应体系的优化及其实践应用.pdfVIP

1352 江苏农业学报(JiangsuJ.of Agr.Sci. ),2010,26(6):1352~1356 苏家乐,刘晓宏,刘晓青,等. 杜鹃花属植物SRAP鄄PCR反应体系的优化及其应用[J].江苏农业学报,2010,26(6):1352鄄1356. 杜鹃花属植物SRAP鄄PCR反应体系的优化及其应用 苏家乐,摇 刘晓宏,摇 刘晓青,摇 李摇 畅 (江苏省农业科学院园艺研究所,江苏 南京 210014) 2+ 摇 摇 摘要:摇 采用改良CTAB法提取杜鹃花属植物基因组DNA,对影响SRAP鄄PCR反应体系中的Mg 、dNTPs和引 物浓度进行优化,建立了杜鹃花属植物SRAP分子标记的扩增体系:20 滋l 的PCR体系中含有模板DNA50 ng、10伊 2+ 2+ PCR buffer (不含Mg )、Mg 2郾50 mmol/ L、dNTPs0郾20 mmol/ L、引物0郾40 滋mol/ L、Taq DNA 聚合酶1郾0 U。 并对 10种杜鹃花属植物群体进行扩增验证,共获得261条扩增条带,251个多态性位点,多态性位点比率为96郾17%,结 果表明供试材料间差异明显、多态性较高,该体系适合杜鹃花属植物种间差异性分析,为今后杜鹃花属植物分子生 物学的深入研究奠定了基础。 关键词:摇 杜鹃花属; SRAP; 体系优化; 聚类分析 中图分类号:摇 S685.21摇 摇 摇 文献标识码:摇 A摇 摇 摇 文章编号:摇 1000鄄4440(2010)06鄄1352鄄05 Optimization of SRAP鄄PCR Reaction System in Rhododendron and Its Application SUJia鄄le,摇 LIU Xiao鄄hong,摇 LIU Xiao鄄qing,摇 LI Chang (Institute of Horticulture,JiangsuAcademy of Agricultural Sciences,Nanjing 210014,China) 摇 摇 Abstract:摇 The genomic DNA of Rhododendron was extracted by the revised CTAB method. The concentrations of 2+ Mg ,dNTPsandprimerswhich affectedtheSRAP鄄PCRreactionswereoptimized,andtheSRAPmolecular marker system inRhododendron was established. The optimum system of 20 滋l was as follows:template DNA 50 ng,10伊PCR buffer 2+ 2+ (without Mg ),Mg 2.50 mmol/ L,dNTPs0.20 mmol/ L,primers0.40 滋mol/ L,Taq DNApolymerase1.0 U. Ten genotypes of Rhododendron were amplified and detected under the optimum system. SRAP fingerprinting amplified by 16 pairs of primers revealed that261unambiguous bandswere obtained,of which251 sites were polymorphic,and the poly鄄 morphism frequency was96.17%. Theresultsindicatedthatthesystemwassteady andreliable,whichwouldbehelpfulto study molecular biology of Rhododendron. Key words