部分兜兰属植物基于ITS基因的序列分析
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摘要
本研究以兜兰属4个品种为实验材料,对其进行ITS-PCR扩增及测序,并分析其亲缘关系。登入GenBank NBCI数据库进行序列比对,证实为其ITS序列;4个兜兰属品种的ITS序列长度在770～805 bp之间,其中ITS1的序列长度为283～284 bp,ITS2的序列长度为215～257 bp;4个品种共存在157的变异位点,其中ITS1和ITS2的变异位点数较多,5.85区域变异位点较少;4个兜兰属品种的(G+C)含量在49.22%～51.30%之间,其中硬叶兜兰的(G+C)含量最高,长瓣兜兰的(G+C)含量最低。研究认为,ITS基因对植物系统研究、植物资源鉴定有着重要意义,为兜兰属植物亲缘关系和种质资源的鉴定,以及兜兰属植物的保护方面提供了科学依据。
Four species o f Paphiopedilum were used as experimental materials in this study, and ITS-PCR amplification and sequencing were performed, and their genetic relationship was analyzed. Log in the Genbank NBCI database for sequence alignment and confirmed its ITS sequence. The length of ITS sequences of 4 Paphiopedilum species ranged from 770～805 bp, with the sequence length of ITS1 being 283～284 bp and the length of ITS2 being 215～257 bp. There were 157 mutation sites in 4 varieties, among which ITS1 and ITS2 had more mutation sites, and 5.85 region mutation sites was less. The(G+C) content in 4 Paphiopedilum species was between 49.22%and 51.30%, with the highest and the lowest(G+C) content in Paphiopedilum chinensis and Paphiopedilum dianthum,respectively. The research suggested that ITS gene had important significance in plant system research and plant resource identification. It could identify the genetic relationship and germplasm resources of Paphiopedilum. This research could provide scientific reference for the protection of Paphiopedilum plants.
Four species o f Paphiopedilum were used as experimental materials in this study, and ITS-PCR amplification and sequencing were performed, and their genetic relationship was analyzed. Log in the Genbank NBCI database for sequence alignment and confirmed its ITS sequence. The length of ITS sequences of 4 Paphiopedilum species ranged from 770～805 bp, with the sequence length of ITS1 being 283～284 bp and the length of ITS2 being 215～257 bp. There were 157 mutation sites in 4 varieties, among which ITS1 and ITS2 had more mutation sites, and 5.85 region mutation sites was less. The(G+C) content in 4 Paphiopedilum species was between 49.22%and 51.30%, with the highest and the lowest(G+C) content in Paphiopedilum chinensis and Paphiopedilum dianthum,respectively. The research suggested that ITS gene had important significance in plant system research and plant resource identification. It could identify the genetic relationship and germplasm resources of Paphiopedilum. This research could provide scientific reference for the protection of Paphiopedilum plants.
引文
Baldwin B.G.,Sanderson M.J.,Porter J.M.,Wojciechowski M.F.,Campbell C.S.,and Donoghue M.J.,1995,The its region of nuclear ribosomal DNA:a valuable source of evidence on angiosperm phylogeny,Ann.Mo.Bot.Gard.,82(2):247-277
Chen Y.,Zhang Y.J.,Li Y.D.,Jiang H.,Shen W.H.,and Guan P.,2012,Construction and optimization of ITS-PCR reaction system for Paphiopedilum,Guizhou Nongye Kexue(Guizhou Agricultural Sciences),40(7):51-55(陈业,张玉晶,李娅迪,江辉,沈文华,关萍,2012,兜兰ITS-PCR反应体系的建立及优化,贵州农业科学,40(7):51-55)
Cribb P.J.,ed.,1998,The genus paphiopedilum(ed.2),Natural History Publications,Borneo,Kota Kinabalu,pp.22-30
He T.T.,and Chai J.H.,2017,Sequence analysis of Cymbidium based on ITS gene,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(1):52-57(何婷婷,柴军红,2017,基于ITS基因的部分兰属序列分析,分子植物育种,15(1):52-57)
Liu Z.J.,Zhang J.Y.,Ru Z.Z.,Lei S.P.,and Chen L.J.,2004,Conservation biology of Paphiopedilum purpuratum(Orchidaceae),Shengwu Duoyangxing(Biodiversity Science),12(5):509-516(刘仲健,张建勇,茹正忠,雷嗣鹏,陈利君,2004,兰科紫纹兜兰的保育生物学研究,生物多样性,12(5):509-516)
Liu Z.J.,Zhang J.Y.,Xu X.M.,and Ma J.H.,2000,Notes on P-aphiopedilum concolor and its allies,Yunnan Zhiwu Yanjiu(Acta Botanica Yunnanica),22(4):390-339(刘仲健,张建勇,徐向明,马京华,2000,同色兜兰及其亲缘群的研究,云南植物研究,22(4):390-339)
Lu J.S.,Bu Z.Y.,Lv W.L.,He J.Z.,Su J.M.,and Huang C.Y.,2012,Analysis on genetic diversity of 20 plants of Orchidaceae by ISSR molecular marker,Xinan Zhiwu Xuebao(Southwest China Journal of Agricultural Sciences),25(6):2252-2257(卢家仕,卜朝阳,吕维莉,何荆洲,苏建睦,黄昌艳,2012,20份兰科植物的ISSR遗传多样性分析,西南植物学报,25(6):2252-2257)
Luo X.Z.,Nong Z.H.,He F.,Su H.,Zeng X.B.,Wei B.W.,Tang L.F.,Mao Z.Z.,and Wei G.N.,2016,Analysis of genetic relationships of Mlahorcia species in Guangxi region based on nrDNA ITS sequences,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(8):2173-2179(罗小珍,农志欢,何飞,苏华,曾宪彪,韦宝伟,唐毓凡,毛长智,韦桂宁,2016,基于nrDNA ITS序列的广西十大功劳属植物的亲缘关系分析,基因组学与应用生物学,35(8):2173-2179)
Shi Q.L.,an d Zhao W.G.,2001,Preliminary study on ITS sequencing and characteristics of mulberry,Canye Kexue(Acta Sericologica Sinica),27(2):140-141(史全良,赵卫国,2001,桑树ITS序列测定及特点的初步分析,蚕业科学,27(2):140-141)
Shi Z.G.,Ma T.H.,Wan R.,Li Y.L.,and Wang Y.J.,2017,Early screening of in-species hybrids based on ITS barcode sequences,Jiangshu Nongye Kexue(Jiangsu Agricultural Sciences),45(6):138-139(石志刚,马婷慧,万如,李彦龙,王亚军,2017,基于ITS条形码序列早期筛选枸杞种内杂交种,江苏农业科学,45(6):138-139)
Sun C.Y.,Zhang M.Y.,Yie X.,Liang C.Y.,and Xia K.F.,2005,Studies on Relationship between species cultivars of cultivars of Cymbidium uisng RAPD,Yuanyi Xuebao(Acta Horticulturae Sinica),32(6):1121-1124(孙彩云,张明永,叶秀,梁承邺,夏快飞,2005,中国兰属植物种间及品种间亲缘关系的RAPD分析,园艺学报,32(6):1121-1124)
Tian Y.P.,and Long Y.Y.,2006,The study on the application of orchids in positioning of vegetation,Xiandai Yuanlin(Modern Landscape Architecture),(10):46-49(田亦平,龙雅宜,2006,浅谈兰科植物在园林植物造景中的应用,现代园林,(10):46-49)
Tu W.F.,Zhang Z.L.,Wang H.C.Duan Z.Z.,Wu Y.C.,Jia G.H.,Wang W.H.,He Z.Q.,and Yao W.,2017,ITS sequence analysis and identification of wild ancient Malus hupehensis in dajiuhu wetland area of shennongjia,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(4):1484-1491(涂万富,张志麒,汪迎春,段真珍,吴运才,贾国华,王文华,何正权,姚伟,2017,神农架大九湖湿地古野生湖北海棠ITS序列分析及鉴定,基因组学与应用生物学,36(4):1484-1491)
Wang T.X.,Zheng W.,Chen J.,Wang W.,and Xu X.D.,2017,Genetic relationship analysis of Gerbera delavayi from nine populations based on ITS sequences,Guangxi Zhiwu(Guihaia),37(3):329-334(王谈笑,郑伟,陈菁,王炜,徐晓丹,2017,基于ITS序列分析钩苞大丁草九个居群的亲缘关系,广西植物,37(3):329-334)
Zhu G.F.,Li D.M.,and Guo Z.F.,2007,Genetic diversity and relationship of hybrid Cymbidium based on AFLP marker,Yuanyi Xuebao(Acta Horticulturae Sinica),34(2):417-424(朱根发,李冬梅,郭振飞,2007,大花蕙兰遗传多样性及亲缘关系的AFLP分析,园艺学报,34(2):417-424)
Chen Y.,Zhang Y.J.,Li Y.D.,Jiang H.,Shen W.H.,and Guan P.,2012,Construction and optimization of ITS-PCR reaction system for Paphiopedilum,Guizhou Nongye Kexue(Guizhou Agricultural Sciences),40(7):51-55(陈业,张玉晶,李娅迪,江辉,沈文华,关萍,2012,兜兰ITS-PCR反应体系的建立及优化,贵州农业科学,40(7):51-55)
Cribb P.J.,ed.,1998,The genus paphiopedilum(ed.2),Natural History Publications,Borneo,Kota Kinabalu,pp.22-30
He T.T.,and Chai J.H.,2017,Sequence analysis of Cymbidium based on ITS gene,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(1):52-57(何婷婷,柴军红,2017,基于ITS基因的部分兰属序列分析,分子植物育种,15(1):52-57)
Liu Z.J.,Zhang J.Y.,Ru Z.Z.,Lei S.P.,and Chen L.J.,2004,Conservation biology of Paphiopedilum purpuratum(Orchidaceae),Shengwu Duoyangxing(Biodiversity Science),12(5):509-516(刘仲健,张建勇,茹正忠,雷嗣鹏,陈利君,2004,兰科紫纹兜兰的保育生物学研究,生物多样性,12(5):509-516)
Liu Z.J.,Zhang J.Y.,Xu X.M.,and Ma J.H.,2000,Notes on P-aphiopedilum concolor and its allies,Yunnan Zhiwu Yanjiu(Acta Botanica Yunnanica),22(4):390-339(刘仲健,张建勇,徐向明,马京华,2000,同色兜兰及其亲缘群的研究,云南植物研究,22(4):390-339)
Lu J.S.,Bu Z.Y.,Lv W.L.,He J.Z.,Su J.M.,and Huang C.Y.,2012,Analysis on genetic diversity of 20 plants of Orchidaceae by ISSR molecular marker,Xinan Zhiwu Xuebao(Southwest China Journal of Agricultural Sciences),25(6):2252-2257(卢家仕,卜朝阳,吕维莉,何荆洲,苏建睦,黄昌艳,2012,20份兰科植物的ISSR遗传多样性分析,西南植物学报,25(6):2252-2257)
Luo X.Z.,Nong Z.H.,He F.,Su H.,Zeng X.B.,Wei B.W.,Tang L.F.,Mao Z.Z.,and Wei G.N.,2016,Analysis of genetic relationships of Mlahorcia species in Guangxi region based on nrDNA ITS sequences,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(8):2173-2179(罗小珍,农志欢,何飞,苏华,曾宪彪,韦宝伟,唐毓凡,毛长智,韦桂宁,2016,基于nrDNA ITS序列的广西十大功劳属植物的亲缘关系分析,基因组学与应用生物学,35(8):2173-2179)
Shi Q.L.,an d Zhao W.G.,2001,Preliminary study on ITS sequencing and characteristics of mulberry,Canye Kexue(Acta Sericologica Sinica),27(2):140-141(史全良,赵卫国,2001,桑树ITS序列测定及特点的初步分析,蚕业科学,27(2):140-141)
Shi Z.G.,Ma T.H.,Wan R.,Li Y.L.,and Wang Y.J.,2017,Early screening of in-species hybrids based on ITS barcode sequences,Jiangshu Nongye Kexue(Jiangsu Agricultural Sciences),45(6):138-139(石志刚,马婷慧,万如,李彦龙,王亚军,2017,基于ITS条形码序列早期筛选枸杞种内杂交种,江苏农业科学,45(6):138-139)
Sun C.Y.,Zhang M.Y.,Yie X.,Liang C.Y.,and Xia K.F.,2005,Studies on Relationship between species cultivars of cultivars of Cymbidium uisng RAPD,Yuanyi Xuebao(Acta Horticulturae Sinica),32(6):1121-1124(孙彩云,张明永,叶秀,梁承邺,夏快飞,2005,中国兰属植物种间及品种间亲缘关系的RAPD分析,园艺学报,32(6):1121-1124)
Tian Y.P.,and Long Y.Y.,2006,The study on the application of orchids in positioning of vegetation,Xiandai Yuanlin(Modern Landscape Architecture),(10):46-49(田亦平,龙雅宜,2006,浅谈兰科植物在园林植物造景中的应用,现代园林,(10):46-49)
Tu W.F.,Zhang Z.L.,Wang H.C.Duan Z.Z.,Wu Y.C.,Jia G.H.,Wang W.H.,He Z.Q.,and Yao W.,2017,ITS sequence analysis and identification of wild ancient Malus hupehensis in dajiuhu wetland area of shennongjia,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(4):1484-1491(涂万富,张志麒,汪迎春,段真珍,吴运才,贾国华,王文华,何正权,姚伟,2017,神农架大九湖湿地古野生湖北海棠ITS序列分析及鉴定,基因组学与应用生物学,36(4):1484-1491)
Wang T.X.,Zheng W.,Chen J.,Wang W.,and Xu X.D.,2017,Genetic relationship analysis of Gerbera delavayi from nine populations based on ITS sequences,Guangxi Zhiwu(Guihaia),37(3):329-334(王谈笑,郑伟,陈菁,王炜,徐晓丹,2017,基于ITS序列分析钩苞大丁草九个居群的亲缘关系,广西植物,37(3):329-334)
Zhu G.F.,Li D.M.,and Guo Z.F.,2007,Genetic diversity and relationship of hybrid Cymbidium based on AFLP marker,Yuanyi Xuebao(Acta Horticulturae Sinica),34(2):417-424(朱根发,李冬梅,郭振飞,2007,大花蕙兰遗传多样性及亲缘关系的AFLP分析,园艺学报,34(2):417-424)