多重PCR技术鉴定番茄Ty-3a和Tm-2~a基因
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摘要
本研究利用与抗番茄黄化曲叶病毒病(tomato yellow leaf curl virus, TYLCV)基因Ty-3a紧密连锁的共显性分子标记P6-25和抗番茄烟草花叶病毒病(tomato mosaic virus, ToMV)基因Tm-2~a的功能性显性分子标记,建立了可同时筛选番茄Ty-3a和Tm-2~a基因的多重PCR技术,扩增的特异性片段与常规PCR扩增片段完全吻合。其中P6-25标记在纯合和杂合抗病材料中分别扩增出630 bp、630 bp和320 bp的特异片段,在感病材料中扩增出320 bp的特异片段;Tm-2~a的功能性标记只能在抗病材料中扩增出472 bp的特异片段。利用该技术对19份番茄材料进行基因型鉴定,结果稳定可靠。本研究建立的多重PCR体系提高了抗病基因Ty-3a和Tm-2~a的鉴定效率,为番茄多基因聚合育种提供了技术支持。
In order to establish a multiple PCR technique that could identify tomato Ty-3a and Tm-2~a gene simultaneously, we employed a co-dominant marker P6-25, which tightly linked to tomato yellow leaf curl virus disease-resistant gene Ty-3a, and a functional dominant marker deriving from tomato mosaic virus resistance gene Tm-2~a. The amplified specific fragments were completely in consistent with the regular PCR amplified fragments.Among them, P6-25 primer amplified 630 bp and 630 bp, 320 bp bands, respectively, in tomato homozygous and heterozygous disease resistant materials. While in susceptible lines, it amplified 320 bp band. Tm-2~a functional marker could only produced 472 bp specific fragments in tomato disease resistant materials. Genotype identification of 19 tomato materials was carried out using this technology, and the results are stable and reliable. The multiple PCR technique established in this study improved the identification rate of disease resistant gene Ty-3a and Tm-2~a, which could provide technical support for gene pyramiding in tomato breeding program.
In order to establish a multiple PCR technique that could identify tomato Ty-3a and Tm-2~a gene simultaneously, we employed a co-dominant marker P6-25, which tightly linked to tomato yellow leaf curl virus disease-resistant gene Ty-3a, and a functional dominant marker deriving from tomato mosaic virus resistance gene Tm-2~a. The amplified specific fragments were completely in consistent with the regular PCR amplified fragments.Among them, P6-25 primer amplified 630 bp and 630 bp, 320 bp bands, respectively, in tomato homozygous and heterozygous disease resistant materials. While in susceptible lines, it amplified 320 bp band. Tm-2~a functional marker could only produced 472 bp specific fragments in tomato disease resistant materials. Genotype identification of 19 tomato materials was carried out using this technology, and the results are stable and reliable. The multiple PCR technique established in this study improved the identification rate of disease resistant gene Ty-3a and Tm-2~a, which could provide technical support for gene pyramiding in tomato breeding program.
引文
Chamberlain J.S.,Gibbs R.A.,Ranier J.E.,Nguyen P.N.,and Caskey C.T.,1988,Deletion screening of the duchenne muscular dystrophy locus via multiplex DNA amplification,Nucleic Acids Res.,16(23):11141-11156
Chen M.J.,Fang T.,Ke T.,Xiong Z.Y.,and He G.Y.,2005,Multiplex PCR-a molecular biotechnique of high efficiency and speed,Wuhan Ligong Daxue Xuebao(Journal of Wuhan U-niversity of Technology),27(10):33-36(陈明洁,方倜,柯涛,熊志勇,何光源,2005,多重PCR-一种高效快速的分子生物学技术,武汉理工大学学报,27(10):33-36)
Dax E.,Livneh O.,Aliskevicius E.,Edelbaum O.,Kedar N.,Gavish N.,Milo J.,Geffen F.,Blumenthal A.,Rabinowich H.D.,and Sela I.,1998,A SCAR marker linked to the ToMV resistance gene,Tm22,in tomato,Euphytica,101(1):73-77
Ji Y.F.,and Scott S.J.W.,2007,Ty-3,a begomovirus resistance locus near the Tomato yellow leaf curl virus resistance locus Ty-1 on chromosome 6 of tomato,Mol.Breed.,20(3):271-284
Ji Y.F.,Salus M.S.,Van Betteray B.,Smeets J.,Jensen K.S.,Martin C.T.,Scott J.W.,Havey M.J.,and Maxwell D.P.,2008,Co-dominant SCAR markers for detection of the Ty-3and Ty-3a loci from Solanum chilense at 25 cM of chromosome 6 of tomato,Tomato Genet.Coop.,57:25-28
Jin F.M.,Xue J.,Song J.,Wang S.,and Zhang Y.,2017,Identification of resistance gene of Sw-5 and Ty-3a by multiple PCR in tomato,Beifang Yuanyi(Northern Horticulture),(11):115-118(金凤媚,薛俊,宋建,王姝,张越,2017,番茄抗病基因Sw-5和Ty-3a的多重PCR检测体系的建立,北方园艺,(11):115-118)
Kong F.H.,Li S.,Zhao T.T.,Li D.Y.,Xu X.Y.,Jiang J.B.,and Li J.F.,2015,One-step PCR procedure for identifying Ty-2,Ty-3,and I-2 gene in tomato,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(1):184-189(孔凡慧,李帅,赵婷婷,李冬艳,许向阳,姜景彬,李景富,2015,番茄Ty-2、Ty-3和I-2基因多重PCR鉴定技术,分子植物育种,13(1):184-189)
Lanfermeijer F.C.,Dijkhuis J.,Sturre M.J.,De H.P.,and Hille J.,2003,Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-22 from Lycopersicon esculentum,Plant Mol.Biol.,52(5):1039-1049
Li D.Y.,Xue D.Q.,Li J.F.,Xue X.Y.,and Jiang J.B.,2016,Simultaneous identification of Ty-3,I-2,and Mi genes by multiple PCR and its application on tomato,Dongbei Nongye Kexue(Journal of Northeast Agricultural Sciences),41(2):39-43(李冬艳,薛东齐,李景富,许向阳,姜景彬,2016,番茄Ty-3、I-2和Mi基因多重PCR体系的建立与应用,东北农业科学,41(2):39-43)
Liu C.Q.,Li J.F.,Xu X.Y.,and Jiang J.B.,2013,Study on identification of Ty-1 gene,Ty-2 gene,Mi gene and Cf-5 gene by quadruple PCR in tomato,BeifangYuanyi(Northern Horticulture),(9):119-123(刘超勤,李景富,许向阳,姜景彬,2013,四重PCR技术鉴定番茄Ty-1、Ty-2、Mi和Cf-5基因的研究,北方园艺,(9):119-123)
Sun Y.L.,2008,Development of gene marker for four resistance gene and gene assisted selection in tomato,Thesis for M.S.,Huazhong Agricultural University,Supervisor:Li H.X.,pp.16(孙亚林,2008,番茄四个抗病基因的基因标记的创建与辅助选择,硕士毕业论文,华中农业大学,导师:李汉霞,pp.16)
Verlaan M.G.,Hutton S.F.,Ibrahem R.M.,Kormelink R.,Visser R.G.F.,Scott J.W.,Edwards J.D.,and Bai Y.L.,2013,The tomato yellow leaf curl virus resistance genes Ty-1 and Ty-3are allelic and code for DFDGD-class RNA-dependent RNApolymerases,PLoS Genetics,9(3):e1003399
Xiao H.,2013,Study on co-infections of tomato chlorosis virus and several other viruses,Thesis for M.S.,Shandong Agricultural University,Supervisor:Zhu X.P.,pp.36(肖红,2013,番茄黄花曲叶病毒和其它几种病毒复合侵染的研究,硕士毕业论文,山东农业大学,导师:竺晓平,pp.36)
Yang X.H.,Caro M.,Hutton S.F.,Scott J.W.,Guo Y.M.,Wang X.X.,Rashid M.H.,Szinay D.,Jong H.D.,Visser R.G.F.,Bai Y.L.,and Du Y.C.,2014,Fine mapping of the tomato yellow leaf curl virus resistance gene Ty-2 on chromosome11 of tomato,Mol.Breed.,34(2):749-760
Zamir D.,Ekstein M.I.,Zakay Y.,Navot N.,Zeidan M.,Sarfatti M.,Eshed Y.,Harel E.,Pleban T.,Van O.H.,Kedar N.,Rabinowitch H.D.,and Czosnek H.,1994,Mapping and introgression of a tomato yellow leaf curl virus tolerance gene,Ty-1,Theor.Appl.Genet.,88(2):141-146
Chen M.J.,Fang T.,Ke T.,Xiong Z.Y.,and He G.Y.,2005,Multiplex PCR-a molecular biotechnique of high efficiency and speed,Wuhan Ligong Daxue Xuebao(Journal of Wuhan U-niversity of Technology),27(10):33-36(陈明洁,方倜,柯涛,熊志勇,何光源,2005,多重PCR-一种高效快速的分子生物学技术,武汉理工大学学报,27(10):33-36)
Dax E.,Livneh O.,Aliskevicius E.,Edelbaum O.,Kedar N.,Gavish N.,Milo J.,Geffen F.,Blumenthal A.,Rabinowich H.D.,and Sela I.,1998,A SCAR marker linked to the ToMV resistance gene,Tm22,in tomato,Euphytica,101(1):73-77
Ji Y.F.,and Scott S.J.W.,2007,Ty-3,a begomovirus resistance locus near the Tomato yellow leaf curl virus resistance locus Ty-1 on chromosome 6 of tomato,Mol.Breed.,20(3):271-284
Ji Y.F.,Salus M.S.,Van Betteray B.,Smeets J.,Jensen K.S.,Martin C.T.,Scott J.W.,Havey M.J.,and Maxwell D.P.,2008,Co-dominant SCAR markers for detection of the Ty-3and Ty-3a loci from Solanum chilense at 25 cM of chromosome 6 of tomato,Tomato Genet.Coop.,57:25-28
Jin F.M.,Xue J.,Song J.,Wang S.,and Zhang Y.,2017,Identification of resistance gene of Sw-5 and Ty-3a by multiple PCR in tomato,Beifang Yuanyi(Northern Horticulture),(11):115-118(金凤媚,薛俊,宋建,王姝,张越,2017,番茄抗病基因Sw-5和Ty-3a的多重PCR检测体系的建立,北方园艺,(11):115-118)
Kong F.H.,Li S.,Zhao T.T.,Li D.Y.,Xu X.Y.,Jiang J.B.,and Li J.F.,2015,One-step PCR procedure for identifying Ty-2,Ty-3,and I-2 gene in tomato,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(1):184-189(孔凡慧,李帅,赵婷婷,李冬艳,许向阳,姜景彬,李景富,2015,番茄Ty-2、Ty-3和I-2基因多重PCR鉴定技术,分子植物育种,13(1):184-189)
Lanfermeijer F.C.,Dijkhuis J.,Sturre M.J.,De H.P.,and Hille J.,2003,Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-22 from Lycopersicon esculentum,Plant Mol.Biol.,52(5):1039-1049
Li D.Y.,Xue D.Q.,Li J.F.,Xue X.Y.,and Jiang J.B.,2016,Simultaneous identification of Ty-3,I-2,and Mi genes by multiple PCR and its application on tomato,Dongbei Nongye Kexue(Journal of Northeast Agricultural Sciences),41(2):39-43(李冬艳,薛东齐,李景富,许向阳,姜景彬,2016,番茄Ty-3、I-2和Mi基因多重PCR体系的建立与应用,东北农业科学,41(2):39-43)
Liu C.Q.,Li J.F.,Xu X.Y.,and Jiang J.B.,2013,Study on identification of Ty-1 gene,Ty-2 gene,Mi gene and Cf-5 gene by quadruple PCR in tomato,BeifangYuanyi(Northern Horticulture),(9):119-123(刘超勤,李景富,许向阳,姜景彬,2013,四重PCR技术鉴定番茄Ty-1、Ty-2、Mi和Cf-5基因的研究,北方园艺,(9):119-123)
Sun Y.L.,2008,Development of gene marker for four resistance gene and gene assisted selection in tomato,Thesis for M.S.,Huazhong Agricultural University,Supervisor:Li H.X.,pp.16(孙亚林,2008,番茄四个抗病基因的基因标记的创建与辅助选择,硕士毕业论文,华中农业大学,导师:李汉霞,pp.16)
Verlaan M.G.,Hutton S.F.,Ibrahem R.M.,Kormelink R.,Visser R.G.F.,Scott J.W.,Edwards J.D.,and Bai Y.L.,2013,The tomato yellow leaf curl virus resistance genes Ty-1 and Ty-3are allelic and code for DFDGD-class RNA-dependent RNApolymerases,PLoS Genetics,9(3):e1003399
Xiao H.,2013,Study on co-infections of tomato chlorosis virus and several other viruses,Thesis for M.S.,Shandong Agricultural University,Supervisor:Zhu X.P.,pp.36(肖红,2013,番茄黄花曲叶病毒和其它几种病毒复合侵染的研究,硕士毕业论文,山东农业大学,导师:竺晓平,pp.36)
Yang X.H.,Caro M.,Hutton S.F.,Scott J.W.,Guo Y.M.,Wang X.X.,Rashid M.H.,Szinay D.,Jong H.D.,Visser R.G.F.,Bai Y.L.,and Du Y.C.,2014,Fine mapping of the tomato yellow leaf curl virus resistance gene Ty-2 on chromosome11 of tomato,Mol.Breed.,34(2):749-760
Zamir D.,Ekstein M.I.,Zakay Y.,Navot N.,Zeidan M.,Sarfatti M.,Eshed Y.,Harel E.,Pleban T.,Van O.H.,Kedar N.,Rabinowitch H.D.,and Czosnek H.,1994,Mapping and introgression of a tomato yellow leaf curl virus tolerance gene,Ty-1,Theor.Appl.Genet.,88(2):141-146