番茄实用化PCR分子标记反应体系与程序优化
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摘要
为了提高抗病性分子标记检测的效率,为番茄病害检测及多抗品种选育提供技术依据,本研究通过改良CTAB法、96孔深孔板快速提取番茄微量叶片DNA;利用抗根结线虫病Mi标记,在L16(45)正交设计试验基础上,采用直观量化分析和方差分析两种方法,对番茄实用化PCR分子标记PCR反应的5个因素(引物, Mg2+, dNTPs,模板DNA和Taq酶)进行优化;随后分别筛选黄化曲叶病毒病、烟草花叶病毒病、根结线虫病、枯萎病、叶霉病和颈腐根腐病6种病害的8个实用化PCR分子标记PCR程序的退火温度;并对循环次数进行筛选;最后利用优化后的PCR体系与程序对供试的1 442份番茄材料(包括番茄种质资源,群体材料,组合及品种)进行抗病性鉴定。结果表明番茄实用化PCR分子标记的PCR最佳反应体系(20μL)为引物0.5μmol/L、Mg2+1.5 mmol/L、dNTPs 0.3 mmol/L、DNA 480 ng DNA、聚合酶1.0 U Taq;PCR程序中8个分子标记共同最适退火温度为56℃,最佳循环次数为33次;筛选出585份多抗(含2种以上的抗病基因)番茄材料,可作为中间材料或亲本材料进行多抗品种选育。该实用化PCR分子标记的PCR体系的建立为番茄利用抗病基因分子标记检测与筛选多抗种质资源、品种或群体材料提供了标准化的程序。
In order to improve the efficiency of disease-resistant molecular marker detection, provide the technical basis for tomato disease detection and multi resistant cultivar breeding, minimal amount of DNA in tomato leaves was extracted rapidly by modified CTAB method and 96 well plates. By Mi markers for resistance to root-knot nematode disease, based on L16(45) orthogonal experimental design, both visual quantitative analysis method and variance-analysis method were used to optimize 5 factors of practical PCR molecular marker reaction system of tomato(primer, Mg~(2+), dNTPs, template DNA and Taq polymerase). The annealing temperature of 8 practical PCR molecular markers for 6 diseases(yellow leaf curl disease, tobacco mosaic disease, root-knot nematode disease,Fusarium wilt, leaf mold disease, and root rot) and cycle times in PCR program were screened subsequently. Finally,the optimized PCR system and program were used to identify the disease resistance of tested 1 442 tomatoes materials(including tomato germplasm resources, population materials, hybrid combinations and cultivars). The results showed that the optimal PCR reaction system of tomato practical PCR molecular markers(20 μL) was as followed: 0.5 μmol/L primer, 1.5 mmol/L Mg~(2+), 0.3 mmol/L d NTPs, 480 ng template DNA and 1.0 U Taq polymerase. In the PCR program, the optimum annealing temperature of 8 molecular markers was 56℃, and the optimal cycle number was 33 times. In addition, 585 tomato materials with multi resistances(more than 2 kinds of resistant genes) were screened out, which could be used as intermediate materials or parent materials for multiresistant variety breeding. The PCR system of practical PCR molecular markers might provide a standardized procedure for the detection and screening of germplasm resources, varieties, or population materials with multi resistances.
In order to improve the efficiency of disease-resistant molecular marker detection, provide the technical basis for tomato disease detection and multi resistant cultivar breeding, minimal amount of DNA in tomato leaves was extracted rapidly by modified CTAB method and 96 well plates. By Mi markers for resistance to root-knot nematode disease, based on L16(45) orthogonal experimental design, both visual quantitative analysis method and variance-analysis method were used to optimize 5 factors of practical PCR molecular marker reaction system of tomato(primer, Mg~(2+), dNTPs, template DNA and Taq polymerase). The annealing temperature of 8 practical PCR molecular markers for 6 diseases(yellow leaf curl disease, tobacco mosaic disease, root-knot nematode disease,Fusarium wilt, leaf mold disease, and root rot) and cycle times in PCR program were screened subsequently. Finally,the optimized PCR system and program were used to identify the disease resistance of tested 1 442 tomatoes materials(including tomato germplasm resources, population materials, hybrid combinations and cultivars). The results showed that the optimal PCR reaction system of tomato practical PCR molecular markers(20 μL) was as followed: 0.5 μmol/L primer, 1.5 mmol/L Mg~(2+), 0.3 mmol/L d NTPs, 480 ng template DNA and 1.0 U Taq polymerase. In the PCR program, the optimum annealing temperature of 8 molecular markers was 56℃, and the optimal cycle number was 33 times. In addition, 585 tomato materials with multi resistances(more than 2 kinds of resistant genes) were screened out, which could be used as intermediate materials or parent materials for multiresistant variety breeding. The PCR system of practical PCR molecular markers might provide a standardized procedure for the detection and screening of germplasm resources, varieties, or population materials with multi resistances.
引文
Chen G.H.,Ge J.T.,Yuan L.Y.,Zhu S.D.,Su Y.,Liu S.,and Wang C.G.,2017,Extraction of genomic DNA and construction of RAPD molecular marker reaction system in Cucumis melo var.conomon Group,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(4):1575-1580(陈国户,葛继涛,袁凌云,朱世东,苏亚,刘姗,汪承刚,2017,酥瓜(Cucumis melo var.conomon Group)基因组DNA提取及RAPD分子标记反应体系的建立,基因组学与应用生物学,36(4):1575-1580)
Cheng L.,Zhang S.,Li Y.Q.,Chen F.D.,Cheng F.,Zhang X.Y.,Dong T.,and Guo J.J.,2016,Pathogen identification of Fusarium crown root rot and screening for resistant sources in tomato,Yuanyi Xuebao(Acta Horticulturae Sinica),43(4):781-788(程琳,张生,李艳青,陈福东,程斐,张晓艳,董甜,国家进,2016,番茄颈腐根腐病病原菌鉴定与抗病种质材料的筛选,园艺学报,43(4):781-788)
Ge N.P.,Cui L.,Li H.X.,and Ye Z.B.,2014,Double SNP marker of Ty-1 alleles in a co-dominant manner in tomato,Yuanyi Xuebao(Acta Horticulturae Sinica),41(8):1583-1590(葛乃蓬,崔龙,李汉霞,叶志彪,2014,番茄抗黄化曲叶病毒基因Ty-1的双重SNP标记的开发,园艺学报,41(8):1583-1590)
Truong H.T.H.,Choi H.,Cho M.C.,Lee H.E.,and Kim J.H.,2011,Use of Cf-9 gene-based markers in marker-assisted selection to screen tomato cultivars with resistance to Cladosporium fulvum,Hort.Environ.Biotechnol.,52(2):204-210
Lan B.X.,Wang L.,Wu Z.K.,and Jiang B.L.,2015,Rapid miniprep extraction of genomic DNA from micro-endosperm maize with modified CTAB method,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):190-194(蓝碧秀,王凛,吴子恺,姜伯乐,2015,利用改良CTAB法快速小量提取微胚乳玉米基因组DNA,基因组学与应用生物学,34(1):190-194)
Li C.C.,Yang T.Q.,Dai Y.J.,Li J.H.,and Tian C.Y.,2011,Optimization for ISSR-PCR reaction system in lotus root using orthogonal design,Beifang Yuanyi(Northern Horticulture),(1):121-123(李长春,阳天泉,戴余军,李建华,田春元,2011,正交设计优化莲藕ISSR-PCR反应体研究,北方园艺,(1):121-123)
Li F.L.,2010,Molecular markers linked to fusarium wilt resistant genes 1-1 in tomato,Thesis for M.S.,Northeast Agricultural University,Supervisor:Xu X.Y.,pp.1-11(李发玲,2010,番茄枯萎病抗病基因1-1的分子标记及种质资源筛选,硕士学位论文,东北农业大学,导师:许向阳,pp.1-11)
Li W.W.,Wang A.L.,and Nie Z.Z.,2017,Orthogonal design optimization and primer selection of Carlesia sinensis SRAPsystem,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(8):3103-3109(李维卫,王爱兰,聂臻臻,2017,山茴香SRAP反应体系正交设计优化及引物筛选,基因组学与应用生物学,36(8):3103-3109)
Long T.,Huang C.Q.,and Liu G.D.,2016,Optimization of SSR-PCR reaction system for Cynodon dactylon by orthogonal design,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(1):198-205(龙萄,黄春琼,刘国道,2016,正交设计优化狗牙根SSR-PCR反应体系,基因组学与应用生物学,35(1):198-205)
Peng X.Z.,2012,Genetic analysis and molecular marker of tomato yellow leaf curl virus gene Ty-1 in tomato,Thesis for M.S.,Northeast Agricultural University,Supervisor:Li J.F.,pp.1-13(彭祥珍,2012,番茄抗黄化曲叶病基因Ty-1的分子标记及种质资源筛选,硕士学位论文,东北农业大学,导师:李景富,pp.1-13)
Sun Q.X.,Chen J.,Zhang H.,Qi J.M.,Lin Z.P.,Hu Y.L.,and Lin X.J.,2012,Optimization of ISSR's primer and PCR Reaction system for Astragalu siuicus L.,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),13(5):870-878(孙清信,陈坚,张辉,祁建民,林忠平,胡鸢雷,林新坚,2012,紫云英ISSR引物的筛选及PCR反应体系的优化,植物遗传资源学报,13(5):870-878)
Tang Z.H.,Zhu Y.J.,Tan Y.D.,and Tan X.F.,2013,Establishment and optimization of Forsythia suspensa ISSR-PCR reaction system,Zhongnan Linye Keji Daxue Xuebao(Journal of Central South University of Forestry&Technology),33(10):53-56,60(汤正辉,祝亚军,谭运德,谭晓风,2013,连翘ISSR-PCR反应体系的建立及优化,中南林业科技大学学报,33(10):53-56,60)
Wang L.S.,2012,Molecular markers for resistance gene Ty-2and their utilization in marker-assisted selection,Thesis for M.S.,Hebei University of Science and Technology,Supervisor:Song J.J.,pp.1-11(王琳珊,2012,番茄抗病基因Ty-2的分子标记及其在辅助选择中的应用,硕士学位论文,河北科技大学,导师:宋建军,pp.1-11)
Yang H.H.,Zhao T.T.,Liu G.,Xu X.Y.,Jiang J.B.,and Li J.F.,2016,Advanced progress on tomato yellow leaf curl disease resistance genes and disease resistance breeding,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(8):2044-2049(杨欢欢,赵婷婷,刘冠,许向阳,姜景彬,李景富,2016,番茄黄化曲叶病抗病基因与抗病育种的最新进展,分子植物育种,14(8):2044-2049)
Yao Z.P.,Ye Q.J.,Wang R.Q.,Zhou G.Z.,Ruan M.Y.,Li Z.M.,Wan H.J.,ChengY.,and Yang Y.J.,2015,Molecular mapping and identifying pcr-based markers conferring Ty-3a,a resistant gene to Tomato Yellow Leaf Curl Virus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(3):595-600(姚祝平,叶青静,王荣青,周国治,阮美颖,李志邈,万红建,程远,杨悦俭,2015,番茄黄化曲叶病毒病抗性基因Ty-3a分子标记的筛选及定位,分子植物育种,13(3):595-600)
Zhu M.T.,Sun Y.L.,Zheng S.,Zhang X.L.,Wang T.T.,Ye Z.B.,and Li H.X.,2010,Pyramiding disease resistance genes by molecular marker-assisted selection in tomato,Yuanyi Xuebao(Acta Horticulturae Sinica),37(9):1416-1422(朱明涛,孙亚林,郑莎,张晓黎,王涛涛,叶志彪,李汉霞,2010,分子标记辅助聚合番茄抗病基因育种,园艺学报,37(9):1416-1422)
Cheng L.,Zhang S.,Li Y.Q.,Chen F.D.,Cheng F.,Zhang X.Y.,Dong T.,and Guo J.J.,2016,Pathogen identification of Fusarium crown root rot and screening for resistant sources in tomato,Yuanyi Xuebao(Acta Horticulturae Sinica),43(4):781-788(程琳,张生,李艳青,陈福东,程斐,张晓艳,董甜,国家进,2016,番茄颈腐根腐病病原菌鉴定与抗病种质材料的筛选,园艺学报,43(4):781-788)
Ge N.P.,Cui L.,Li H.X.,and Ye Z.B.,2014,Double SNP marker of Ty-1 alleles in a co-dominant manner in tomato,Yuanyi Xuebao(Acta Horticulturae Sinica),41(8):1583-1590(葛乃蓬,崔龙,李汉霞,叶志彪,2014,番茄抗黄化曲叶病毒基因Ty-1的双重SNP标记的开发,园艺学报,41(8):1583-1590)
Truong H.T.H.,Choi H.,Cho M.C.,Lee H.E.,and Kim J.H.,2011,Use of Cf-9 gene-based markers in marker-assisted selection to screen tomato cultivars with resistance to Cladosporium fulvum,Hort.Environ.Biotechnol.,52(2):204-210
Lan B.X.,Wang L.,Wu Z.K.,and Jiang B.L.,2015,Rapid miniprep extraction of genomic DNA from micro-endosperm maize with modified CTAB method,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):190-194(蓝碧秀,王凛,吴子恺,姜伯乐,2015,利用改良CTAB法快速小量提取微胚乳玉米基因组DNA,基因组学与应用生物学,34(1):190-194)
Li C.C.,Yang T.Q.,Dai Y.J.,Li J.H.,and Tian C.Y.,2011,Optimization for ISSR-PCR reaction system in lotus root using orthogonal design,Beifang Yuanyi(Northern Horticulture),(1):121-123(李长春,阳天泉,戴余军,李建华,田春元,2011,正交设计优化莲藕ISSR-PCR反应体研究,北方园艺,(1):121-123)
Li F.L.,2010,Molecular markers linked to fusarium wilt resistant genes 1-1 in tomato,Thesis for M.S.,Northeast Agricultural University,Supervisor:Xu X.Y.,pp.1-11(李发玲,2010,番茄枯萎病抗病基因1-1的分子标记及种质资源筛选,硕士学位论文,东北农业大学,导师:许向阳,pp.1-11)
Li W.W.,Wang A.L.,and Nie Z.Z.,2017,Orthogonal design optimization and primer selection of Carlesia sinensis SRAPsystem,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(8):3103-3109(李维卫,王爱兰,聂臻臻,2017,山茴香SRAP反应体系正交设计优化及引物筛选,基因组学与应用生物学,36(8):3103-3109)
Long T.,Huang C.Q.,and Liu G.D.,2016,Optimization of SSR-PCR reaction system for Cynodon dactylon by orthogonal design,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(1):198-205(龙萄,黄春琼,刘国道,2016,正交设计优化狗牙根SSR-PCR反应体系,基因组学与应用生物学,35(1):198-205)
Peng X.Z.,2012,Genetic analysis and molecular marker of tomato yellow leaf curl virus gene Ty-1 in tomato,Thesis for M.S.,Northeast Agricultural University,Supervisor:Li J.F.,pp.1-13(彭祥珍,2012,番茄抗黄化曲叶病基因Ty-1的分子标记及种质资源筛选,硕士学位论文,东北农业大学,导师:李景富,pp.1-13)
Sun Q.X.,Chen J.,Zhang H.,Qi J.M.,Lin Z.P.,Hu Y.L.,and Lin X.J.,2012,Optimization of ISSR's primer and PCR Reaction system for Astragalu siuicus L.,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),13(5):870-878(孙清信,陈坚,张辉,祁建民,林忠平,胡鸢雷,林新坚,2012,紫云英ISSR引物的筛选及PCR反应体系的优化,植物遗传资源学报,13(5):870-878)
Tang Z.H.,Zhu Y.J.,Tan Y.D.,and Tan X.F.,2013,Establishment and optimization of Forsythia suspensa ISSR-PCR reaction system,Zhongnan Linye Keji Daxue Xuebao(Journal of Central South University of Forestry&Technology),33(10):53-56,60(汤正辉,祝亚军,谭运德,谭晓风,2013,连翘ISSR-PCR反应体系的建立及优化,中南林业科技大学学报,33(10):53-56,60)
Wang L.S.,2012,Molecular markers for resistance gene Ty-2and their utilization in marker-assisted selection,Thesis for M.S.,Hebei University of Science and Technology,Supervisor:Song J.J.,pp.1-11(王琳珊,2012,番茄抗病基因Ty-2的分子标记及其在辅助选择中的应用,硕士学位论文,河北科技大学,导师:宋建军,pp.1-11)
Yang H.H.,Zhao T.T.,Liu G.,Xu X.Y.,Jiang J.B.,and Li J.F.,2016,Advanced progress on tomato yellow leaf curl disease resistance genes and disease resistance breeding,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(8):2044-2049(杨欢欢,赵婷婷,刘冠,许向阳,姜景彬,李景富,2016,番茄黄化曲叶病抗病基因与抗病育种的最新进展,分子植物育种,14(8):2044-2049)
Yao Z.P.,Ye Q.J.,Wang R.Q.,Zhou G.Z.,Ruan M.Y.,Li Z.M.,Wan H.J.,ChengY.,and Yang Y.J.,2015,Molecular mapping and identifying pcr-based markers conferring Ty-3a,a resistant gene to Tomato Yellow Leaf Curl Virus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(3):595-600(姚祝平,叶青静,王荣青,周国治,阮美颖,李志邈,万红建,程远,杨悦俭,2015,番茄黄化曲叶病毒病抗性基因Ty-3a分子标记的筛选及定位,分子植物育种,13(3):595-600)
Zhu M.T.,Sun Y.L.,Zheng S.,Zhang X.L.,Wang T.T.,Ye Z.B.,and Li H.X.,2010,Pyramiding disease resistance genes by molecular marker-assisted selection in tomato,Yuanyi Xuebao(Acta Horticulturae Sinica),37(9):1416-1422(朱明涛,孙亚林,郑莎,张晓黎,王涛涛,叶志彪,李汉霞,2010,分子标记辅助聚合番茄抗病基因育种,园艺学报,37(9):1416-1422)