Primer Screening on Germplasm Resources of Mallotus oblongiolus by ISSR Molecular Marker  

鹧鸪茶种质资源ISSR分子标记中的引物筛选(摘要)(英文)

作　　者：李娟玲[1,2] 刘国民[1,2] 宫庆龙[2] 翟丽艳[2] 

机构地区：[1]海南大学热带生物资源教育部重点实验室,海南海口570228 [2]海南大学苦丁茶研究所,海南海口570228

出　　处：《Agricultural Science & Technology》2009年第6期40-43,46,共5页农业科学与技术（英文版）

基　　金：Supported by Special Fund for Key Disciplines Program of HainanUniversity~~

摘　　要：[ Objective] To provide effective primers for the rapid and accurate ISSR analysis of the germplasm materials of Mallotus oblongiolus （Miq.） Muello-Arg.. [Method] The modified CTAB method was used in the extraction of the genomic DNA. 99 ISSR primers were used in the ISSR-PCR amplification for 20 germplasm materials from 10 populations in Hainan Island, so that some primers, which were suitable to all gerplasm materials of M. oblongiolu, could be selected. [ Result] 15 effective primers with characteristics of rich polymorphism, clear bands, and good repeatability were selected from 99 test primers. The 15 primers selected were used in the ISSR-PCR amplification for 66 germplasm materials of M. oblongiolus. From all of which the abundant and distinct DNA fingerprintings could be obtained. 286 DNA bands were obtained, and of which 231 bands were polymorphic, which amounted to 80.77% of the total bands amplified. And 19.1 bands could be obtained with each primer, averagely. [ Conclusion] The 15 primers selected could be effectively applied to ISSR analysis of the germplasm resources of M. oblongiolus.[目的]为快速和准确地对鹧鸪茶种质材料进行ISSR分析提供有效引物。[方法]基因组DNA的提取采用改良的CTAB法:先用0.8%琼脂糖凝胶电泳检测DNA完整性,EB染色,以Lambda DNA/HindⅢ+Eco RIMarkers为参照,电泳结果用Gel Doc XR型凝胶成像分析系统下照相并记录。再用Biophotometer紫外分光光度计分别测定OD_(260)和OD_(280),并计算出OD_(260)/OD_(280)值,每份样品的OD_(260)/OD_(280)比值均要求在1.8～2.0范围内。测定每份样品DNA浓度(ng/μl),并将其稀释至20 ng/μl,分装后置-20℃保存。利用99个ISSR引物,对来自海南岛境内的10个居群中共20份鹧鸪茶种质材料进行PCR扩增,以筛选出适合于所有鹧鸪茶种质材料ISSR分析的有效引物。[结果]从99个供试引物中共筛选出15个多态性丰富、条带清晰且可重复性良好的有效引物。用筛选出的15个引物对66份鹧鸪茶种质材料进行ISSR-PCR扩增,均可获得带型丰富和清晰可辨的DNA指纹图谱;15个引物共扩增出286条DNA谱带,其中231条为多态性带,占总扩增带数的80.77%,平均每个引物扩增出19.1条谱带。[结论]所筛选的15条引物可以有效地应用于鹧鸪茶种质资源材料的ISSR分析。

关 键 词：Mallotus oblongiolius （ Miq. ） Muell. - Arg. ISSR marker Primer screening 

分 类 号：S571.1[农业科学—茶叶生产加工]