漆树EST-SSR引物开发
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摘要
利用NCBI数据库进行漆树EST-SSR引物开发,从NCBI数据库中共下载漆树EST序列87 856条。利用MISA软件进行序列处理、拼接及聚类后,从87 856条漆树EST序列中拼接组装成3 979条非冗余序列,含SSR位点的EST序列出现频率占EST序列总数的4.5%,从3 979条非冗余序列中检测到487个SSRs微卫星位点,出现频率为12.2%。这些SSR位点中,三核苷酸和二核苷酸重复基元所占比例较高。采用Primer5.0软件,共成功设计50对EST-SSR引物,50对EST-SSR引物在25个漆树个体上均能扩增出清晰的电泳条带,其中18对引物检测出了多态性条带,扩增率达36%。
The objective of this study was to develop simple sequence repeat(SSR) markers from expressed sequence tags(ESTs) of Toxicodendron vernicifluum based on the NCBI database.A total of 87 856 Toxicodendron vernicifluum ESTs from NCBI database were used to search for SSR and 3 954 SSR loci were identified(4.5% of the total number of EST sequences) after the sequences were handled,jointed and clustered by MISA software.487 microsatellite markers were detected from the non-redundant sequences with a frequency of 12.2%.A higher proportion of dinucleotide and trinucleotide repeats were found in the SSR sites.50 pairs of EST-SSR primers were successfully designed by using Primer 5.0 software,and clear electrophoresis bands could be amplified in 25 individuals.There were EST-SSR polymorphic bands in 18 pairs of primers,with the effective amplification rate of 36%.
The objective of this study was to develop simple sequence repeat(SSR) markers from expressed sequence tags(ESTs) of Toxicodendron vernicifluum based on the NCBI database.A total of 87 856 Toxicodendron vernicifluum ESTs from NCBI database were used to search for SSR and 3 954 SSR loci were identified(4.5% of the total number of EST sequences) after the sequences were handled,jointed and clustered by MISA software.487 microsatellite markers were detected from the non-redundant sequences with a frequency of 12.2%.A higher proportion of dinucleotide and trinucleotide repeats were found in the SSR sites.50 pairs of EST-SSR primers were successfully designed by using Primer 5.0 software,and clear electrophoresis bands could be amplified in 25 individuals.There were EST-SSR polymorphic bands in 18 pairs of primers,with the effective amplification rate of 36%.
引文
Bozhko M.,Riegel R.,Schubert R.,and Mller-Starck G.,2003,A cyclophilin gene marker confirming geographical differentiation of Norway spruce populations and indicating viability response on excess soil-born salinity,Mol.Ecol.,12(11):3147-3155
Brown G.R.,Kadel E.E.,Bassoni D.L.,Kiehne K.L.,Temesgen B.,van Buijtenen J.P.,Sewell M.M.,Marshall K.A.,and Neale D.B.,2001,Anchored reference loci in loblolly pine(Pinus taeda L.)for integrating pine genomics,Genetics,159(2):799-809
Cordeiro G.M.,Casu R.,Mcintyre C.L.,Manners J.M.,and Henry R.J.,2001,Microsatellite markers from sugarcane(Saccharum spp.)ESTs cross transferable to erianthus and sorghum,Plant Sci.,160(2001):1115-1123
Cui H.R.,Liu J.Y.,Tong Z.G.,Wang X.W.,Zhang Z.,and Qiao Y.S.,2010,Development and application of EST-SSRs in sand pear,Xibei Zhiwu Xuebao(Acta Bot.Boreal.-Occident.Sin.),30(8):1551-1556(崔海荣,刘金义,佟兆国,王新卫,章镇,乔玉山,2010,砂梨EST-SSR引物开发及其应用,西北植物学报,30(8):1551-1556)
Deng C.L.,Gao J.,Cao Y.,Qin R.Y.,Gao W.J.,and Lu L.D.,2012,Development and application of sex-specific EST-SSR marker in spinach(Spinacia oleracea L.),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),31(5):467-472(邓传良,高俊,曹莹,秦瑞云,高武军,卢龙斗,2012,菠菜性别相关EST-SSR标记的开发及应用,基因组学与应用生物学,31(5):467-472)
Eujayl I.,Sorrells M.E.,Baum M.,Wolters P.,and Powell W.,2002,Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat,Theoretical and Applied Genetics,104(2-3):399-407
Gupta P.K.,Rustgi S.,Sharma S.,Singh R.,Kumar N.,and Balyan H.S.,2003,Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat,Molecular Genomics and Genetics,270(4):315-323
Jin J.Q.,Li S.F.,Gong X.C.,Lu M.Z.,Yao Y.L.,Xin Y.,and Cui H.R.,2006,Analysis of SSR information in EST resource of tea plants(Camellia sinensis),Keji Tongbao(Bulletin of Science and Technology),22(4):471-476(金基强,李素芳,龚晓春,卢美贞,姚艳玲,忻雅,崔海瑞,2006,茶树EST资源中SSR的信息分析,科技通报,22(4):471-476)
Li G.P.,and Cao F.L.,2012,Development of EST-SSR primer from Ginkgo biloba,Xibei Nongye Xuebao(Acta Agriculturae Boreali-Occidentalis Sinica),21(12):149-152(李广平,曹福亮,2012,银杏EST-SSR引物开发,西北农业学报,21(12):149-152)
Morgante M.,Hanafey M.,and Powell W.,2002,Microsatellites are preferentially associated with non-repetitive DNA in plant genomes,Nature Genetics,30(2):194-200
Murray M.G.,and Thompson W.F.,1980,Rapid isolation of high molecular weight plant DNA,Nuclear Acid Research,8(19):4321-4325
Rder M.S.,Korzun V.,Wendehake K.,Plaschke J.,Tixier M.H.,Leroy P.,and Ganal M.W.,1998,A microsatellite map of wheat,Genetics,149(4):2007-2023
Schubert R.,Mller-Starck G.,and Riegel R.,2001,Development of EST-PCR markers and monitoring their intrapopulational genetic variation in Picea abies(L.)Karst.,Theor.Appl.Genet.,103(8):1223-1231
Varshney R.K.,Graner A.,and Sorrells M.E.,2005a,Genic microsatellite markers in plants:features and applications,Trends in Biotechnology,23(1):48-55
Varshney R.K.,Sigmund R.,Brner A.,Korzun V.,Stein N.,Sorrells M.E.,Langridge P.,and Graner A.,2005b,Interspecific transferability and comparative mapping of barley EST-SSRmarkersin wheat,rye and rice,Plant Sci.,168(1):195-202
Wang J.Y.,Chen Y.Y.,Liu W.L.,and Wu Y.T.,2008,Development and application of EST derived SSR markers for ba nanas(Musa nana Lour.),Yichuan(Hereditas),30(7):933-940(王静毅,陈业渊,刘伟良,武耀廷,2008,香蕉EST-SSRs标记的开发与应用,遗传,30(7):933-940)
Wei S.N.,Li L.,Zhao X.P.,Zhao M.,and Hu Z.H.,2011,HPLCfingerprint analysis of eight varieties and five wild populations of Toxicodendron vernicifluum,Xibei Zhiwu Xuebao(Acta Bot.Boreal.-Occident.Sin.),31(3):620-627(魏朔南,李林,赵喜萍,赵猛,胡正海,2011,漆树8个品种和5个野生居群HPLC指纹图谱分析,西北植物学报,31(3):620-627)
Wei S.N.,Zhao X.P.,Tian M.J.,Li L.,and Hu Z.H.,2010,Application of plant morphology and AFLP molecular markers to identify Toxicodendron vernicifluum varieties of Shaanxi,Xibei Zhiwu Xuebao(Acta Bot.Boreal.-Occident.Sin.),30(4):0645-0651(魏朔南,赵喜萍,田敏爵,李林,胡正海,2010,应用植物形态学和AFLP分子标记鉴别陕西漆树品种,西北植物学报,30(4):0645-0651)
Wen Y.,Lu X.P.,Ren R.,Mi F.G.,Han P.A.,and Xue C.L.,2013,Development of EST-SSR marker and genetic diversity analysis in Sorghum bicolor伊Sorghum sudanenes,Yichuan(Hereditas),35(2):225-232(温莹,逯晓萍,任锐,米富贵,韩平安,薛春雷,2013,高丹草EST-SSR标记的开发及其遗传多样性,遗传,35(2):225-232)
Yang C.J.,Wang J.,Mu L.Q.,Li S.C.,Liu G.J.,and Hu C.Q.,2008,Development of EST-SSR marker in Panax ginseng,Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),16(1):114-120(杨成君,王军,穆立蔷,李绍臣,刘关君,胡长群,2008,人参EST-SSR标记的建立,农业生物技术学报,16(1):114-120)
Zhao G.F.,and Hu Z.H.,1991,Studies on the relation between the ultrastructure of secretory cells and their synthesis and secretion of lacquer in laticiferous canals of Rhus vernicifluua,Zhiwu Xuebao(Acta Botanica Sinica),33(1):26-30(赵桂仿,胡正海,1991,漆树乳汁道分泌细胞的超微结构及其与生漆产生和分泌关系的研究,植物学报,33(1):26-30)
Zhao M.,Wei S.N.,and Hu Z.H.,2012,Structure and development of phloem in Toxicodendron vernicifluum,Linye Kexue(Scientia Silvae Sinicae),48(9):36-43(赵猛,魏朔南,胡正海,2012,漆树韧皮部的结构与发育,林业科学,48(9):36-43)
Zhao X.P.,and Wei S.N.,2007,Genetic evaluation of Toxicodendron vernicifluum cultivars using amplified fragment length polymorphism markers,Fenzixibao Shengwu Xuebao(Journal of Molecular Cell Biology),40(4):262-266(赵喜萍,魏朔南,2007,漆树品种的AFLP分析及评价(简报),分子细胞生物学报,40(4):262-266)
Brown G.R.,Kadel E.E.,Bassoni D.L.,Kiehne K.L.,Temesgen B.,van Buijtenen J.P.,Sewell M.M.,Marshall K.A.,and Neale D.B.,2001,Anchored reference loci in loblolly pine(Pinus taeda L.)for integrating pine genomics,Genetics,159(2):799-809
Cordeiro G.M.,Casu R.,Mcintyre C.L.,Manners J.M.,and Henry R.J.,2001,Microsatellite markers from sugarcane(Saccharum spp.)ESTs cross transferable to erianthus and sorghum,Plant Sci.,160(2001):1115-1123
Cui H.R.,Liu J.Y.,Tong Z.G.,Wang X.W.,Zhang Z.,and Qiao Y.S.,2010,Development and application of EST-SSRs in sand pear,Xibei Zhiwu Xuebao(Acta Bot.Boreal.-Occident.Sin.),30(8):1551-1556(崔海荣,刘金义,佟兆国,王新卫,章镇,乔玉山,2010,砂梨EST-SSR引物开发及其应用,西北植物学报,30(8):1551-1556)
Deng C.L.,Gao J.,Cao Y.,Qin R.Y.,Gao W.J.,and Lu L.D.,2012,Development and application of sex-specific EST-SSR marker in spinach(Spinacia oleracea L.),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),31(5):467-472(邓传良,高俊,曹莹,秦瑞云,高武军,卢龙斗,2012,菠菜性别相关EST-SSR标记的开发及应用,基因组学与应用生物学,31(5):467-472)
Eujayl I.,Sorrells M.E.,Baum M.,Wolters P.,and Powell W.,2002,Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat,Theoretical and Applied Genetics,104(2-3):399-407
Gupta P.K.,Rustgi S.,Sharma S.,Singh R.,Kumar N.,and Balyan H.S.,2003,Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat,Molecular Genomics and Genetics,270(4):315-323
Jin J.Q.,Li S.F.,Gong X.C.,Lu M.Z.,Yao Y.L.,Xin Y.,and Cui H.R.,2006,Analysis of SSR information in EST resource of tea plants(Camellia sinensis),Keji Tongbao(Bulletin of Science and Technology),22(4):471-476(金基强,李素芳,龚晓春,卢美贞,姚艳玲,忻雅,崔海瑞,2006,茶树EST资源中SSR的信息分析,科技通报,22(4):471-476)
Li G.P.,and Cao F.L.,2012,Development of EST-SSR primer from Ginkgo biloba,Xibei Nongye Xuebao(Acta Agriculturae Boreali-Occidentalis Sinica),21(12):149-152(李广平,曹福亮,2012,银杏EST-SSR引物开发,西北农业学报,21(12):149-152)
Morgante M.,Hanafey M.,and Powell W.,2002,Microsatellites are preferentially associated with non-repetitive DNA in plant genomes,Nature Genetics,30(2):194-200
Murray M.G.,and Thompson W.F.,1980,Rapid isolation of high molecular weight plant DNA,Nuclear Acid Research,8(19):4321-4325
Rder M.S.,Korzun V.,Wendehake K.,Plaschke J.,Tixier M.H.,Leroy P.,and Ganal M.W.,1998,A microsatellite map of wheat,Genetics,149(4):2007-2023
Schubert R.,Mller-Starck G.,and Riegel R.,2001,Development of EST-PCR markers and monitoring their intrapopulational genetic variation in Picea abies(L.)Karst.,Theor.Appl.Genet.,103(8):1223-1231
Varshney R.K.,Graner A.,and Sorrells M.E.,2005a,Genic microsatellite markers in plants:features and applications,Trends in Biotechnology,23(1):48-55
Varshney R.K.,Sigmund R.,Brner A.,Korzun V.,Stein N.,Sorrells M.E.,Langridge P.,and Graner A.,2005b,Interspecific transferability and comparative mapping of barley EST-SSRmarkersin wheat,rye and rice,Plant Sci.,168(1):195-202
Wang J.Y.,Chen Y.Y.,Liu W.L.,and Wu Y.T.,2008,Development and application of EST derived SSR markers for ba nanas(Musa nana Lour.),Yichuan(Hereditas),30(7):933-940(王静毅,陈业渊,刘伟良,武耀廷,2008,香蕉EST-SSRs标记的开发与应用,遗传,30(7):933-940)
Wei S.N.,Li L.,Zhao X.P.,Zhao M.,and Hu Z.H.,2011,HPLCfingerprint analysis of eight varieties and five wild populations of Toxicodendron vernicifluum,Xibei Zhiwu Xuebao(Acta Bot.Boreal.-Occident.Sin.),31(3):620-627(魏朔南,李林,赵喜萍,赵猛,胡正海,2011,漆树8个品种和5个野生居群HPLC指纹图谱分析,西北植物学报,31(3):620-627)
Wei S.N.,Zhao X.P.,Tian M.J.,Li L.,and Hu Z.H.,2010,Application of plant morphology and AFLP molecular markers to identify Toxicodendron vernicifluum varieties of Shaanxi,Xibei Zhiwu Xuebao(Acta Bot.Boreal.-Occident.Sin.),30(4):0645-0651(魏朔南,赵喜萍,田敏爵,李林,胡正海,2010,应用植物形态学和AFLP分子标记鉴别陕西漆树品种,西北植物学报,30(4):0645-0651)
Wen Y.,Lu X.P.,Ren R.,Mi F.G.,Han P.A.,and Xue C.L.,2013,Development of EST-SSR marker and genetic diversity analysis in Sorghum bicolor伊Sorghum sudanenes,Yichuan(Hereditas),35(2):225-232(温莹,逯晓萍,任锐,米富贵,韩平安,薛春雷,2013,高丹草EST-SSR标记的开发及其遗传多样性,遗传,35(2):225-232)
Yang C.J.,Wang J.,Mu L.Q.,Li S.C.,Liu G.J.,and Hu C.Q.,2008,Development of EST-SSR marker in Panax ginseng,Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),16(1):114-120(杨成君,王军,穆立蔷,李绍臣,刘关君,胡长群,2008,人参EST-SSR标记的建立,农业生物技术学报,16(1):114-120)
Zhao G.F.,and Hu Z.H.,1991,Studies on the relation between the ultrastructure of secretory cells and their synthesis and secretion of lacquer in laticiferous canals of Rhus vernicifluua,Zhiwu Xuebao(Acta Botanica Sinica),33(1):26-30(赵桂仿,胡正海,1991,漆树乳汁道分泌细胞的超微结构及其与生漆产生和分泌关系的研究,植物学报,33(1):26-30)
Zhao M.,Wei S.N.,and Hu Z.H.,2012,Structure and development of phloem in Toxicodendron vernicifluum,Linye Kexue(Scientia Silvae Sinicae),48(9):36-43(赵猛,魏朔南,胡正海,2012,漆树韧皮部的结构与发育,林业科学,48(9):36-43)
Zhao X.P.,and Wei S.N.,2007,Genetic evaluation of Toxicodendron vernicifluum cultivars using amplified fragment length polymorphism markers,Fenzixibao Shengwu Xuebao(Journal of Molecular Cell Biology),40(4):262-266(赵喜萍,魏朔南,2007,漆树品种的AFLP分析及评价(简报),分子细胞生物学报,40(4):262-266)