农业科学与技术（英文版）
農業科學與技術（英文版）
농업과학여기술（영문판）
AGRICULTURAL SCIENCE & TECHNOLOGY
2009年
6期
40-43,46
,共5页
李娟玲%刘国民%宫庆龙%翟丽艳
李娟玲%劉國民%宮慶龍%翟麗豔
리연령%류국민%궁경룡%적려염
鹧鸪茶%ISSR标记%引物筛选
鷓鴣茶%ISSR標記%引物篩選
자고다%ISSR표기%인물사선
Mallotus oblongiolius(Miq.)Muell.-Arg.%ISSR marker%Primer screening
[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/Hind Ⅲ+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OD260和OD280,并计算出OD260/OD280值,每份样品的OD260/OD280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.
[目的]為快速和準確地對鷓鴣茶種質材料進行ISSR分析提供有效引物.[方法]基因組DNA的提取採用改良的CTAB法:先用0.8%瓊脂糖凝膠電泳檢測DNA完整性,EB染色,以Lambda DNA/Hind Ⅲ+Eco RⅠMarkers為參照,電泳結果用Gel Doc XR型凝膠成像分析繫統下照相併記錄.再用Biophotometer紫外分光光度計分彆測定OD260和OD280,併計算齣OD260/OD280值,每份樣品的OD260/OD280比值均要求在1.8～2.0範圍內.測定每份樣品DNA濃度(ng/μl),併將其稀釋至20 ng/μl,分裝後置-20℃保存.利用99箇ISSR引物,對來自海南島境內的10箇居群中共20份鷓鴣茶種質材料進行PCR擴增,以篩選齣適閤于所有鷓鴣茶種質材料ISSR分析的有效引物.[結果]從99箇供試引物中共篩選齣15箇多態性豐富、條帶清晰且可重複性良好的有效引物.用篩選齣的15箇引物對66份鷓鴣茶種質材料進行ISSR-PCR擴增,均可穫得帶型豐富和清晰可辨的DNA指紋圖譜;15箇引物共擴增齣286條DNA譜帶,其中231條為多態性帶,佔總擴增帶數的80.77%,平均每箇引物擴增齣19.1條譜帶.[結論]所篩選的15條引物可以有效地應用于鷓鴣茶種質資源材料的ISSR分析.
[목적]위쾌속화준학지대자고다충질재료진행ISSR분석제공유효인물.[방법]기인조DNA적제취채용개량적CTAB법:선용0.8%경지당응효전영검측DNA완정성,EB염색,이Lambda DNA/Hind Ⅲ+Eco RⅠMarkers위삼조,전영결과용Gel Doc XR형응효성상분석계통하조상병기록.재용Biophotometer자외분광광도계분별측정OD260화OD280,병계산출OD260/OD280치,매빈양품적OD260/OD280비치균요구재1.8～2.0범위내.측정매빈양품DNA농도(ng/μl),병장기희석지20 ng/μl,분장후치-20℃보존.이용99개ISSR인물,대래자해남도경내적10개거군중공20빈자고다충질재료진행PCR확증,이사선출괄합우소유자고다충질재료ISSR분석적유효인물.[결과]종99개공시인물중공사선출15개다태성봉부、조대청석차가중복성량호적유효인물.용사선출적15개인물대66빈자고다충질재료진행ISSR-PCR확증,균가획득대형봉부화청석가변적DNA지문도보;15개인물공확증출286조DNA보대,기중231조위다태성대,점총확증대수적80.77%,평균매개인물확증출19.1조보대.[결론]소사선적15조인물가이유효지응용우자고다충질자원재료적ISSR분석.
[Objective]To provide effective primers for the rapid and accurate ISSR analysis of the germplasm materials of Mallotus oblongiolus(Miq.)Muell.-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.
