用于长绿2号苦瓜种子纯度鉴定的SSR分子标记的开发
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摘要
苦瓜新品种的推广和种业的提质增效需要建立配套的苦瓜品种种子纯度鉴定方法。本研究利用SSR分子标记技术,对苦瓜新品种"长绿2号"及其父母本进行多态性分析,发掘出2对能够区分该杂交一代新品种及其父母本的共显性引物SSR-04和SSR-08;通过对差异条带的DNA回收和测序鉴定,明确了相应的多态性片段的序列。利用SSR-04与SSR-08分别对"长绿2号"苦瓜杂交种的纯度进行分子鉴定,准确性皆与田间性状鉴定结果一致。本研究成功开发出两对可用于"长绿2号"杂种F1代种子纯度鉴定的SSR分子标记,同时也为SSR标记在苦瓜品种鉴定和分子育种中的精准应用提供技术支撑。
Establishment a method for seed purity identification matching a bitter gourd variety is necessary for promotion of the variety and improvement of the seed industry. In this study, SSR molecular marker technique was used to analyze the polymorphism in new bitter gourd variety Changlü No.2 hybrids and its parents, and two pairs of co-dominant primers SSR-04 and SSR-08 were screened, which can distinguish between the parents and their hybrid F1 generation. The DNA sequences of the SSR primers covered polymorphic fragments were further identified by DNA recovery of the polymorphic bands and sequencing. Molecular identification of the seeds purity of Changlü No.2 hybrids were carried out by SSR-04 and SSR-08 respectively, and the accuracy was consistent with the traits identification in the field. Together, this study successfully developed two pairs of SSR primers for the purity identification of F1 generation seeds of Changlü No.2 hybrids, and also provides technical basis for molecular breeding and identification of bitter gourd varieties acurately with SSR markers.
Establishment a method for seed purity identification matching a bitter gourd variety is necessary for promotion of the variety and improvement of the seed industry. In this study, SSR molecular marker technique was used to analyze the polymorphism in new bitter gourd variety Changlü No.2 hybrids and its parents, and two pairs of co-dominant primers SSR-04 and SSR-08 were screened, which can distinguish between the parents and their hybrid F1 generation. The DNA sequences of the SSR primers covered polymorphic fragments were further identified by DNA recovery of the polymorphic bands and sequencing. Molecular identification of the seeds purity of Changlü No.2 hybrids were carried out by SSR-04 and SSR-08 respectively, and the accuracy was consistent with the traits identification in the field. Together, this study successfully developed two pairs of SSR primers for the purity identification of F1 generation seeds of Changlü No.2 hybrids, and also provides technical basis for molecular breeding and identification of bitter gourd varieties acurately with SSR markers.
引文
Cui J.J.,Cheng J.W.,Nong D.G.,Peng J.Z.,Hu Y.F.,He W.M.,Zhou Q.J.,Dhillon N.P.S.,and Hu K.L.,2017,Genome-wide analysis of simple sequence repeats in bitter gourd(Momordica charantia),Front.Plant Sci.,8:1103-1112
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-SSRmarker 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)
Guo D.L.,Zhang J.P.,Xue Y.M.,and Hou X.G.,2012,Isolation and characterization of 10 SSR markers of Momordica charantia(Cucurbitaceae),Am.J.Bot.,99(5):e182-e183
Li M.,Zhang B.,Liu L.,Liu X.,Wen F.Y.,Luo Z.M.,Liu X.H.,Wang C.N.,and Huang Z.Y.,2015,Identification on purity of bitter gourd hybrid'Yuanmeng 08''by SSR marker,Zhongzi(Seed),34(4):113-114,117(李梅,张斌,刘莉,刘翔,闻凤英,罗智敏,刘晓晖,王超楠,黄志银,2015,“圆梦08”苦瓜杂交种纯度的SSR鉴定,种子,34(4):113-114,117)
Liu Z.J.,Zhan Y.F.,and Yang Y.,2014,Hybrid purity identification of Reyan No.2 using SSR markers,Shengwu Jishu Tongbao(Biotechnology Bulletin),45(2):75-78(刘子记,詹园凤,杨衍,2014,利用SSR标记鉴定热研二号油绿苦瓜杂交种纯度,生物技术通报,45(2):75-78)
Mi J.H.,2013,Powdery mildew-resistant gene in bitter gourd(Momordica charantia L.),Thesis for M.S.,Guangxi University,Supervisor:Liang H.,pp.17-18(米军红,2013,苦瓜抗白粉病基因的分子标记筛选及初步定位,硕士学位论文,广西大学,导师:梁和,pp.17-18)
Salgado K.,Pinho T.V.,Garciasouza R.,and De L.O.V.,2006,Genetic purity certificate in seeds of hybrid maize using molec ular markers,Revista Brasileira de Sementes,28(1):169-175
Saxena S.,Singh A.,Archak S.,Behera T.K.,John J.K.,Meshram S.U.,and Gaikwad A.B.,2015,Development of novel simple sequence repeat markers in bitter gourd(Momordica charantia L.)through enriched genomic libraries and their utilization in analysis of genetic diversity and cross-species transferability,Appl.Biochem.Biotechnol.,175(1):93-118
Urasaki N.,Takagi H.,Natsume S.,Aiko U.,Naoki T.,Norimichi M.,Mai F.,Shouta S.,Kazuhiko T.,Moritoshi T.,Moriaki S.,Ryohei T.,and Hideo M.,2017,Draft genome sequence of bitter gourd(Momordica charantia),a vegetable and medicinal plant in tropical and subtropical regions,DNA Res.,24(1):51-58
Wang G.L.,Xu Y.X.,and Huang M.H.,2016a,Identification and genetic relationship analysis of bitter gourd varieties based on SSR and SPAP,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(2):501-510(王国莉,徐毓璇,黄梅花,2016a,基于SSR和SRAP标记的苦瓜品种鉴定及亲缘关系分析,分子植物育种,14(2):501-510)
Wang G.L.,Chen Y.Z.,Ran M.L.,He F.F.,She M.C.,Chen L.S.,Gong G.P.,Feng R.H.,and Liu X.S.,2016b,Screening and analysis of SSR and SRAP markers related to sex expression in bitter gourd,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(7):1764-1771(王国莉,陈勇智,冉梦莲,何凤芳,佘美纯,陈丽珊,龚冠平,冯荣华,刘晓诗,2016b,苦瓜性别相关SSR和SRAP分子标记的筛选及分析,分子植物育种,14(7):1764-1771)
Wang S.Z.,Li Z.L.,Yang G.L.,Ho C.T.,and Li S.M.,2017,Momordica charantia:a popular health-promoting vegetable with multifunctionality,Food and Funct.,8(5):1749-1762
Wang S.Z.,Pan L.,Hu K.,Chen C.Y.,and Ding Y.,2010,Development and characterization of polymorphic microsatellite markers in Momordica charantia(Cucurbitaceae),Am.J.Bot.,97(8):e75-78
Zhang T.F.,Ge M.,Wei Y.C.,and Zhao H.,2012,Discovery for maize function Insertion/Deletion(InDel)polymorphic marker and its implication in purity identification of maize hybrid seeds,Yumi Kexue(Journal of Maize Sciences),20(2):64-68(张体付,葛敏,韦玉才,赵涵,2012,玉米功能性Insertion/Deletion(InDel)分子标记的挖掘及其在杂交种纯度鉴定中的应用,玉米科学,20(2):64-68)
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-SSRmarker 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)
Guo D.L.,Zhang J.P.,Xue Y.M.,and Hou X.G.,2012,Isolation and characterization of 10 SSR markers of Momordica charantia(Cucurbitaceae),Am.J.Bot.,99(5):e182-e183
Li M.,Zhang B.,Liu L.,Liu X.,Wen F.Y.,Luo Z.M.,Liu X.H.,Wang C.N.,and Huang Z.Y.,2015,Identification on purity of bitter gourd hybrid'Yuanmeng 08''by SSR marker,Zhongzi(Seed),34(4):113-114,117(李梅,张斌,刘莉,刘翔,闻凤英,罗智敏,刘晓晖,王超楠,黄志银,2015,“圆梦08”苦瓜杂交种纯度的SSR鉴定,种子,34(4):113-114,117)
Liu Z.J.,Zhan Y.F.,and Yang Y.,2014,Hybrid purity identification of Reyan No.2 using SSR markers,Shengwu Jishu Tongbao(Biotechnology Bulletin),45(2):75-78(刘子记,詹园凤,杨衍,2014,利用SSR标记鉴定热研二号油绿苦瓜杂交种纯度,生物技术通报,45(2):75-78)
Mi J.H.,2013,Powdery mildew-resistant gene in bitter gourd(Momordica charantia L.),Thesis for M.S.,Guangxi University,Supervisor:Liang H.,pp.17-18(米军红,2013,苦瓜抗白粉病基因的分子标记筛选及初步定位,硕士学位论文,广西大学,导师:梁和,pp.17-18)
Salgado K.,Pinho T.V.,Garciasouza R.,and De L.O.V.,2006,Genetic purity certificate in seeds of hybrid maize using molec ular markers,Revista Brasileira de Sementes,28(1):169-175
Saxena S.,Singh A.,Archak S.,Behera T.K.,John J.K.,Meshram S.U.,and Gaikwad A.B.,2015,Development of novel simple sequence repeat markers in bitter gourd(Momordica charantia L.)through enriched genomic libraries and their utilization in analysis of genetic diversity and cross-species transferability,Appl.Biochem.Biotechnol.,175(1):93-118
Urasaki N.,Takagi H.,Natsume S.,Aiko U.,Naoki T.,Norimichi M.,Mai F.,Shouta S.,Kazuhiko T.,Moritoshi T.,Moriaki S.,Ryohei T.,and Hideo M.,2017,Draft genome sequence of bitter gourd(Momordica charantia),a vegetable and medicinal plant in tropical and subtropical regions,DNA Res.,24(1):51-58
Wang G.L.,Xu Y.X.,and Huang M.H.,2016a,Identification and genetic relationship analysis of bitter gourd varieties based on SSR and SPAP,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(2):501-510(王国莉,徐毓璇,黄梅花,2016a,基于SSR和SRAP标记的苦瓜品种鉴定及亲缘关系分析,分子植物育种,14(2):501-510)
Wang G.L.,Chen Y.Z.,Ran M.L.,He F.F.,She M.C.,Chen L.S.,Gong G.P.,Feng R.H.,and Liu X.S.,2016b,Screening and analysis of SSR and SRAP markers related to sex expression in bitter gourd,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(7):1764-1771(王国莉,陈勇智,冉梦莲,何凤芳,佘美纯,陈丽珊,龚冠平,冯荣华,刘晓诗,2016b,苦瓜性别相关SSR和SRAP分子标记的筛选及分析,分子植物育种,14(7):1764-1771)
Wang S.Z.,Li Z.L.,Yang G.L.,Ho C.T.,and Li S.M.,2017,Momordica charantia:a popular health-promoting vegetable with multifunctionality,Food and Funct.,8(5):1749-1762
Wang S.Z.,Pan L.,Hu K.,Chen C.Y.,and Ding Y.,2010,Development and characterization of polymorphic microsatellite markers in Momordica charantia(Cucurbitaceae),Am.J.Bot.,97(8):e75-78
Zhang T.F.,Ge M.,Wei Y.C.,and Zhao H.,2012,Discovery for maize function Insertion/Deletion(InDel)polymorphic marker and its implication in purity identification of maize hybrid seeds,Yumi Kexue(Journal of Maize Sciences),20(2):64-68(张体付,葛敏,韦玉才,赵涵,2012,玉米功能性Insertion/Deletion(InDel)分子标记的挖掘及其在杂交种纯度鉴定中的应用,玉米科学,20(2):64-68)