大白菜软腐病抗性相关基因分子标记的开发
|
摘要
细菌性软腐病对大白菜(Brassica rapa ssp. pekinensis)的危害严重,胡萝卜果胶杆菌(Pectobacterium carotovorum, Pc)是主要致病菌。本研究基于大白菜软腐病防御反应相关基因,拟开发软腐病抗性基因的分子标记。首先利用生物信息学方法对具有自主产权的大白菜抗软腐病相关基因的表达序列标签(expressed sequence tags, ESTs)数据进行分析,设计合成56对特异性引物,在大白菜软腐病抗病材料(A19-2)和感病材料(A32-2)亲本间筛选有多态性的EST-PCR和EST-CAPS (cleaved amplified polymorphic sequence)标记。在两亲本F1代自交产生的F2代群体中(142个单株),统计标记位点的基因型,进行标记的分离分析。56对设计合成的特异性引物均能在亲本中扩增出清晰条带,其中具有多态性的标记共鉴定出30个:11个EST-PCR标记,19个EST-CAPS标记(其中有9对引物和限制性内切酶的组合不唯一);14个显性标记,16个共显性标记。将上述30个标记位点在F2代群体142个单株中的基因型进行统计,结果显示符合3∶1或1∶2∶1分离比例的位点共有24个,频率为80%;表现偏分离(不符合孟德尔分离比, P<0.05)的位点有6个,频率为20%。本研究利用大白菜抗软腐病相关的ESTs资源,开发了EST-PCR和EST-CAPS分子标记,对于加速大白菜ESTs资源的开发利用、大白菜抗性基因定位和分子标记辅助育种技术等研究具有一定的意义。
Bacterial soft rot disease causes severe damage to Chinese cabbage(Brassica rapa ssp. pekinensis),and Pectobacterium carotovorum(Pc) is a major pathogen. Based on defense-related genes in Chinese cabbage to soft rot disease, molecular markers were developed in this study, which would be very important for molecular marker-assisted breeding and studying defense mechanism of Chinese cabbage to soft rot disease.Firstly, the ESTs(expressed sequence tags) database related to soft rot resistance of Chinese cabbage with independent property rights were analyzed by bioinformatics method, and 56 pairs of specific PCR primers were designed and used to test the polymorphism between resistant line(A19-2) and sensitive line(A32-2) of Chinese cabbage to soft rot disease. The genotypes of the F2 population(containing 142 Chinese cabbage individuals) generated by F1 self-breeding were recorded using the markers and the segregation analysis was conducted. All the 56 pairs of specific PCR primers obtained successful PCR-amplification. There were 30 markers which had polymorphism in the resistant and sensitive parents, including 11 EST-PCR markers and 19 EST-CAPS(cleaved amplified polymorphic sequence) markers, and 9 pairs could match with more than one restriction enzymes. There were 14 dominant markers and 16 co-dominant markers. In addition, the genotypes of 142 individuals of the F2 population were statistically analyzed using the above 30 marker loci. The result showed that 24 loci(80%) had the segregation ratio of 3∶1 or 1∶2∶1, and 6 loci(20%) had the genetic distortion(P<0.05) which was inconsistent with Mendelian ratio. This research developed defense-related EST-PCR and EST-CAPS molecular markers of Chinese cabbage to soft rot disease based on the ESTs resources of Chinese cabbage, which have certain significance in the study on utilization of ESTs resources,localization of defense-related genes and molecular marker-assisted breeding.
Bacterial soft rot disease causes severe damage to Chinese cabbage(Brassica rapa ssp. pekinensis),and Pectobacterium carotovorum(Pc) is a major pathogen. Based on defense-related genes in Chinese cabbage to soft rot disease, molecular markers were developed in this study, which would be very important for molecular marker-assisted breeding and studying defense mechanism of Chinese cabbage to soft rot disease.Firstly, the ESTs(expressed sequence tags) database related to soft rot resistance of Chinese cabbage with independent property rights were analyzed by bioinformatics method, and 56 pairs of specific PCR primers were designed and used to test the polymorphism between resistant line(A19-2) and sensitive line(A32-2) of Chinese cabbage to soft rot disease. The genotypes of the F2 population(containing 142 Chinese cabbage individuals) generated by F1 self-breeding were recorded using the markers and the segregation analysis was conducted. All the 56 pairs of specific PCR primers obtained successful PCR-amplification. There were 30 markers which had polymorphism in the resistant and sensitive parents, including 11 EST-PCR markers and 19 EST-CAPS(cleaved amplified polymorphic sequence) markers, and 9 pairs could match with more than one restriction enzymes. There were 14 dominant markers and 16 co-dominant markers. In addition, the genotypes of 142 individuals of the F2 population were statistically analyzed using the above 30 marker loci. The result showed that 24 loci(80%) had the segregation ratio of 3∶1 or 1∶2∶1, and 6 loci(20%) had the genetic distortion(P<0.05) which was inconsistent with Mendelian ratio. This research developed defense-related EST-PCR and EST-CAPS molecular markers of Chinese cabbage to soft rot disease based on the ESTs resources of Chinese cabbage, which have certain significance in the study on utilization of ESTs resources,localization of defense-related genes and molecular marker-assisted breeding.
引文
陈旭,张元跃.2006.分子标记及其在标记辅助选择中的应用[J].畜牧与饲料科学,27(4):29-32.(Chen X,Zhang Y Y.2006.The application of molecular marker in livestock marker-assisted selection[J].Animal Husbandry and Feed Science,27(4):29-32.)
李登科,黄丛林,田建保,等.2005.高质量枣树基因组DNA提取方法的研究[J].分子植物育种,3(4):579-583.(Li D K,Huang C L,Tian J B,et al.2005.Extraction ways of high qualitiful DNA from Z.jujube mill[J].Molecular Plant Breeding,3(4):579-583.)
李巧云,张志刚,赵智中,等.2012.大白菜抗病分子标记研究进展[C].中国园艺学会十字花科蔬菜分会第十届学术研讨会论文集.中国园艺学会十字花科蔬菜分会,21-29.(Li Q Y,Zhang Z G,Zhao Z Z,et al.2012.Research progress of molecular markers for disease resistance in Chinese cabbage(Brassica campestis L.ssp pe?kinensis)[C].The 10th Symposium of the Cruciferous Branch of the Chinese Society for Horticultural Science,21-29.)
李小白,崔海瑞,张明龙.2006.EST分子标记开发及在比较基因组学中的应用[J].生物多样性,14(6):541-547.(Li X B,Cui H R,Zhang M L.2006.Molecular markers derived from EST:Their development and applications in comparative genomics[J].Biodiversity Science,14(6):541-547.)
牟晋华,徐文玲,张一卉,等.2008.大白菜软腐病抗性的分子标记筛选[J].山东农业科学,50(4):1-4.(Mu J H,Xu W L,Zhang Y H,et al.2008.Screening resistance to Chinese cabbage soft rot by molecular marker[J].Shandong Agricultural Sciences,50(4):1-4.)
徐莹莹,崔崇士,屈淑平.2010.大白菜抗软腐病性状的SRAP标记分析[J].东北农业大学学报,41(6):36-40.(Xu Y Y,Cui C S,Qu S P.2010.Analysis of SRAPmarkers for resistance to Chinese cabbage soft rot[J].Journal of Northeast Agricultural University,41(6):36-40.)
远方.2005.大白菜(Brassica rapa ssp.pekinensis L.)软腐病防御反应基因表达分析[D].博士学位论文,首都师范大学,导师:曹鸣庆,马荣才,pp.105-174.(Yuan F.2005.Gene expression analysis of Chinese cabbage(Brassica rapa ssp.pekinensis L.)in the defensive reactions to Er?winia carotovora ssp.carotovora infection[D].Thesis for Ph.D.,Capital Normal University,Supervisor:Cao MQ,Ma R C,pp.105-174.)
张光明,王翠花.1995.大白菜抗软腐病接种鉴定方法的初步研究[J].山东农业科学,(5):39-40.(Zhang G M,Wang C H.1995.Preliminary study for method of resistance identification of Chinese cabbage to soft rot[J].Shandong Agricultural Sciences,(5):39-40.)
张俊华,崔崇士,潘春清.2006.分子标记及其在芸薹属植物育种中的应用[J].东北农业大学学报,37(5):700-705.(Zhang J H,Cui C S,Pan C Q.2006.Molecular marker techniques and their application in Brassica[J].Journal of Northeast Agricultural University,37(5):700-705.)
Cho K H,Park S H,Kim K T,et al.2012.Mapping quantitative trait loci(QTL)for clubroot resistance in Brassica Rapa L[J].The Journal of Horticultural Science and Biotechnology,87(4):325-333.
Chu M,Song T,Falk K C,et al.2014.Fine mapping of Rcr1and analyses of its effect on transcriptome patterns during infection by Plasmodiophora brassicae[J].BMC Genomics,15:1166.
Gao F,Hirani A H,Liu J,et al.2014.Fine mapping a clubroot resistance locus in Chinese cabbage[J].Journal of the American Society for Horticultural Science,139(3):247-252.
Gao L F,Jing R L,Huo N X,et al.2004.One hundred and one new microsatellite loci derived from ESTs(EST-SSRs)in bread wheat[J].Theoretical and Applied Genetics,108(7):1392-1400.
Harry D E,Temesgen B,Neale D B.1998.Codominant PCR-based markers for Pinus taeda developed from mapped cDNA clones[J].Theoretical and Applied Genetics,97(3):327-336.
Kim S,Song Y H,Lee J,et al.2011.Identification of the BrRHP1 locus that confers resistance to downy mildew in Chinese cabbage(Brassica rapa ssp.pekinensis)and development of linked molecular markers[J].Theoretical and Applied Genetics,123(7):1183-1192.
Konishi T,Abe K,Matsuura S,et al.1990.Distorted segregation of the esterase isozyme genotypes in barley(Horde?um vulgare L.)[J].The Japanese Journal of Genetics,65(6):411-416.
Li G L,Qian W,Zhang S J,et al.2016.Development of genebased markers for the Turnip mosaic virus resistance gene retr02 in Brassica rapa[J].Plant Breeding,135(4):466-470.
Li Q,Zhang X,Zeng Q,et al.2015.Identification and mapping of a novel Turnip mosaic virus resistance gene TuR?BCS01 in Chinese cabbage(Brassica rapa L.)[J].Plant Breeding,134(2):221-225.
Qian W,Zhang S,Zhang S,et al.2013.Mapping and candidate-gene screening of the novel Turnip mosaic virus resistance gene retr02 in Chinese cabbage(Brassica rapa L.)[J].Theoretical and Applied Genetics,126(1):179-188.
Scott K D,Eggler P,Seaton G,et al.2000.Analysis of SSRs derived from grape ESTs[J].Theoretical and Applied Genetics,100(5):723-726.
Thiel T,Michalek W,Varshney R K,et al.2003.Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley(Hordeum vul?gare L.)[J].Theoretical and Applied Genetics,106(3):411-422.
Tsuda K,Tsuji G,Higashiyama M,et al.2016.Biological control of bacterial soft rot in Chinese cabbage by Lactoba?cillus plantarum strain BY under field conditions[J].Biological Control,100(9):63-69.
Vanjildorj E,Song S Y,Yang Z H,et al.2009.Enhancement of tolerance to soft rot disease in the transgenic Chinese cabbage(Brassica rapa L.ssp.pekinensis)inbred line,Kenshin[J].Plant Cell Reports,28(10):1581-1591.
Xu Y,Ma R C,Xie H,et al.2004.Development of SSR markers for the phylogenetic analysis of almond trees from China and the Mediterranean region[J].Genome,47(6):1091-1104.
Yu S,Su T,Zhi S,et al.2016.Construction of a sequencebased bin map and mapping of QTLs for downy mildew resistance at four developmental stages in Chinese cabbage(Brassica rapa L.ssp.pekinensis)[J].Molecular Breeding,36(4):1-12.
Yu S,Zhang F,Zhao X,et al.2011.Sequence-characterized amplified region and simple sequence repeat markers for identifying the major quantitative trait locus responsible for seedling resistance to downy mildew in Chinese cabbage(Brassica rapa ssp.pekinensis)[J].Plant Breeding,130(5):580-583.
Zhang T,Zhao Z,Zhang C,et al.2014.Fine genetic and physical mapping of the CRb gene conferring resistance to clubroot disease in Brassica rapa[J].Molecular Breeding,34(3):1173-1183.
李登科,黄丛林,田建保,等.2005.高质量枣树基因组DNA提取方法的研究[J].分子植物育种,3(4):579-583.(Li D K,Huang C L,Tian J B,et al.2005.Extraction ways of high qualitiful DNA from Z.jujube mill[J].Molecular Plant Breeding,3(4):579-583.)
李巧云,张志刚,赵智中,等.2012.大白菜抗病分子标记研究进展[C].中国园艺学会十字花科蔬菜分会第十届学术研讨会论文集.中国园艺学会十字花科蔬菜分会,21-29.(Li Q Y,Zhang Z G,Zhao Z Z,et al.2012.Research progress of molecular markers for disease resistance in Chinese cabbage(Brassica campestis L.ssp pe?kinensis)[C].The 10th Symposium of the Cruciferous Branch of the Chinese Society for Horticultural Science,21-29.)
李小白,崔海瑞,张明龙.2006.EST分子标记开发及在比较基因组学中的应用[J].生物多样性,14(6):541-547.(Li X B,Cui H R,Zhang M L.2006.Molecular markers derived from EST:Their development and applications in comparative genomics[J].Biodiversity Science,14(6):541-547.)
牟晋华,徐文玲,张一卉,等.2008.大白菜软腐病抗性的分子标记筛选[J].山东农业科学,50(4):1-4.(Mu J H,Xu W L,Zhang Y H,et al.2008.Screening resistance to Chinese cabbage soft rot by molecular marker[J].Shandong Agricultural Sciences,50(4):1-4.)
徐莹莹,崔崇士,屈淑平.2010.大白菜抗软腐病性状的SRAP标记分析[J].东北农业大学学报,41(6):36-40.(Xu Y Y,Cui C S,Qu S P.2010.Analysis of SRAPmarkers for resistance to Chinese cabbage soft rot[J].Journal of Northeast Agricultural University,41(6):36-40.)
远方.2005.大白菜(Brassica rapa ssp.pekinensis L.)软腐病防御反应基因表达分析[D].博士学位论文,首都师范大学,导师:曹鸣庆,马荣才,pp.105-174.(Yuan F.2005.Gene expression analysis of Chinese cabbage(Brassica rapa ssp.pekinensis L.)in the defensive reactions to Er?winia carotovora ssp.carotovora infection[D].Thesis for Ph.D.,Capital Normal University,Supervisor:Cao MQ,Ma R C,pp.105-174.)
张光明,王翠花.1995.大白菜抗软腐病接种鉴定方法的初步研究[J].山东农业科学,(5):39-40.(Zhang G M,Wang C H.1995.Preliminary study for method of resistance identification of Chinese cabbage to soft rot[J].Shandong Agricultural Sciences,(5):39-40.)
张俊华,崔崇士,潘春清.2006.分子标记及其在芸薹属植物育种中的应用[J].东北农业大学学报,37(5):700-705.(Zhang J H,Cui C S,Pan C Q.2006.Molecular marker techniques and their application in Brassica[J].Journal of Northeast Agricultural University,37(5):700-705.)
Cho K H,Park S H,Kim K T,et al.2012.Mapping quantitative trait loci(QTL)for clubroot resistance in Brassica Rapa L[J].The Journal of Horticultural Science and Biotechnology,87(4):325-333.
Chu M,Song T,Falk K C,et al.2014.Fine mapping of Rcr1and analyses of its effect on transcriptome patterns during infection by Plasmodiophora brassicae[J].BMC Genomics,15:1166.
Gao F,Hirani A H,Liu J,et al.2014.Fine mapping a clubroot resistance locus in Chinese cabbage[J].Journal of the American Society for Horticultural Science,139(3):247-252.
Gao L F,Jing R L,Huo N X,et al.2004.One hundred and one new microsatellite loci derived from ESTs(EST-SSRs)in bread wheat[J].Theoretical and Applied Genetics,108(7):1392-1400.
Harry D E,Temesgen B,Neale D B.1998.Codominant PCR-based markers for Pinus taeda developed from mapped cDNA clones[J].Theoretical and Applied Genetics,97(3):327-336.
Kim S,Song Y H,Lee J,et al.2011.Identification of the BrRHP1 locus that confers resistance to downy mildew in Chinese cabbage(Brassica rapa ssp.pekinensis)and development of linked molecular markers[J].Theoretical and Applied Genetics,123(7):1183-1192.
Konishi T,Abe K,Matsuura S,et al.1990.Distorted segregation of the esterase isozyme genotypes in barley(Horde?um vulgare L.)[J].The Japanese Journal of Genetics,65(6):411-416.
Li G L,Qian W,Zhang S J,et al.2016.Development of genebased markers for the Turnip mosaic virus resistance gene retr02 in Brassica rapa[J].Plant Breeding,135(4):466-470.
Li Q,Zhang X,Zeng Q,et al.2015.Identification and mapping of a novel Turnip mosaic virus resistance gene TuR?BCS01 in Chinese cabbage(Brassica rapa L.)[J].Plant Breeding,134(2):221-225.
Qian W,Zhang S,Zhang S,et al.2013.Mapping and candidate-gene screening of the novel Turnip mosaic virus resistance gene retr02 in Chinese cabbage(Brassica rapa L.)[J].Theoretical and Applied Genetics,126(1):179-188.
Scott K D,Eggler P,Seaton G,et al.2000.Analysis of SSRs derived from grape ESTs[J].Theoretical and Applied Genetics,100(5):723-726.
Thiel T,Michalek W,Varshney R K,et al.2003.Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley(Hordeum vul?gare L.)[J].Theoretical and Applied Genetics,106(3):411-422.
Tsuda K,Tsuji G,Higashiyama M,et al.2016.Biological control of bacterial soft rot in Chinese cabbage by Lactoba?cillus plantarum strain BY under field conditions[J].Biological Control,100(9):63-69.
Vanjildorj E,Song S Y,Yang Z H,et al.2009.Enhancement of tolerance to soft rot disease in the transgenic Chinese cabbage(Brassica rapa L.ssp.pekinensis)inbred line,Kenshin[J].Plant Cell Reports,28(10):1581-1591.
Xu Y,Ma R C,Xie H,et al.2004.Development of SSR markers for the phylogenetic analysis of almond trees from China and the Mediterranean region[J].Genome,47(6):1091-1104.
Yu S,Su T,Zhi S,et al.2016.Construction of a sequencebased bin map and mapping of QTLs for downy mildew resistance at four developmental stages in Chinese cabbage(Brassica rapa L.ssp.pekinensis)[J].Molecular Breeding,36(4):1-12.
Yu S,Zhang F,Zhao X,et al.2011.Sequence-characterized amplified region and simple sequence repeat markers for identifying the major quantitative trait locus responsible for seedling resistance to downy mildew in Chinese cabbage(Brassica rapa ssp.pekinensis)[J].Plant Breeding,130(5):580-583.
Zhang T,Zhao Z,Zhang C,et al.2014.Fine genetic and physical mapping of the CRb gene conferring resistance to clubroot disease in Brassica rapa[J].Molecular Breeding,34(3):1173-1183.