基于SLAF-seq技术鉴定甘蓝型油菜叶缘裂刻性状候选基因
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摘要
叶缘裂刻性状是甘蓝型油菜遗传育种的理想形态标记。叶缘裂刻性状变异材料的创建对丰富甘蓝型油菜叶片形态和优良品种改良至关重要。本研究以甘蓝型油菜常规品种中双11和甘蓝型油菜叶缘裂刻新品系Y176为亲本(通过甘蓝型油菜与菜远缘杂交获得),从F2群体中分别挑选出叶缘裂刻和正常叶形的植株各50株,构建了两个极端性状混池DNA文库,借助SLAF-seq-BSA技术,挖掘甘蓝型油菜叶缘裂刻性状相关候选基因。共开发出177941个SLAF标签,通过分析SLAF标签的多态性,共获得150168个SNP,其中亲本间共获得128200个SNP。对ED方法和SNP-index方法得到的关联区域进行综合分析,获得1个与目标性状紧密关联的候选区域,位于甘蓝型油菜A10染色体上的14528149～17353693区域,总长度为2.83 Mb,约有666个编码基因。与前人研究定位在同一染色体上,但本研究定位的区间与前人研究不同,说明本研究存在控制叶缘裂刻新基因的可能性大。并对该区域内基因进行功能注释,其中666个注释到NR数据库;517个注释到SwissProt数据库;587个注释到GO数据库;151个注释到KEGG通路;258个注释到COG数据库,对候选基因进行注释有助于进一步分离相关基因。
Lobed-leaf is widely known as an ideal morphological marker in rapeseed breeding.Genetic variation of lobed-leaf plays an important role in enriching the leaf shape diversity and improving genetic breeding of rapeseed.In this study,fifty plants with normal leaf and 50 plants with lobed-leaf were selected from the F2 population respectively,which was derived from a cross of the normal leaf line Zhongshuang No.11 and the leaf-lobed new line Y176.Two bulked DNA pools were generated,which were subjected for SLAF-seq-BSA(specific-locus amplified fragment sequencing and bulked segregant analysis).A total of 177941 labels were obtained and 150168 SNPs were identified,including 128200 SNPs that were polymorphic between both parents.A physical interval expanding 2.83 Mb on chrA10(14528149～17353693 bp)was tightly associated with lobed-leaf trait.Within that,666 candidate genes was found.Since the genetic confidence interval of this locus differed to the physical positions of previously reported genes,there might be a new gene controlling leaf margin cleavage.Furthermore,666,517,587,151 and 258 genes in this region were annotated by NR,Swiss-Prot,GO,KEGG and COG databases,respectively.Thus,this information might be helpful in future map-based gene isolation of lobed-leaf in Brassica napus L..
Lobed-leaf is widely known as an ideal morphological marker in rapeseed breeding.Genetic variation of lobed-leaf plays an important role in enriching the leaf shape diversity and improving genetic breeding of rapeseed.In this study,fifty plants with normal leaf and 50 plants with lobed-leaf were selected from the F2 population respectively,which was derived from a cross of the normal leaf line Zhongshuang No.11 and the leaf-lobed new line Y176.Two bulked DNA pools were generated,which were subjected for SLAF-seq-BSA(specific-locus amplified fragment sequencing and bulked segregant analysis).A total of 177941 labels were obtained and 150168 SNPs were identified,including 128200 SNPs that were polymorphic between both parents.A physical interval expanding 2.83 Mb on chrA10(14528149～17353693 bp)was tightly associated with lobed-leaf trait.Within that,666 candidate genes was found.Since the genetic confidence interval of this locus differed to the physical positions of previously reported genes,there might be a new gene controlling leaf margin cleavage.Furthermore,666,517,587,151 and 258 genes in this region were annotated by NR,Swiss-Prot,GO,KEGG and COG databases,respectively.Thus,this information might be helpful in future map-based gene isolation of lobed-leaf in Brassica napus L..
引文
[1]Nikovics K,Blein T,Peaucelle A,Ishida T,Morin H,Aida M,Laufs P.The balance between the MIR164A and CUC2 genes controls Leaf Margin Serration in Arabidopsis.The Plant Cell,2006,18(11):2929-2945
[2]Blein T,Pulido A,Guiraud A V,Nikovics K,Morin H,Hay A,Johansen I E,Tsiantis M,Larfs P.A conserved molecular framework for compound leaf development.Science,2008,322(5909):1835-1839
[3]祝朋芳,冯馨,程明明,潘志超.羽衣甘蓝裂叶相关性状遗传分析.西北植物学报,2016,36(2):288-295Zhu P F,Feng X,Cheng M M,Pan Z C.Genetic analysis of feathered-leaved related traits in Brassica oleracea var,acephala.Acta Botanica Boreali-Occidentalia Sinica,2016,36(2):288-295
[4]郎丽娜,赵娜,马兵,刘亚萍,田正书,刘璐,卢虹,刘霞,王阳,徐爱遐,黄镇.甘蓝型油菜花叶性状的遗传及基因定位.华北农学报,2016,31(4):39-43Lang L N,Zhao N,Ma B,Liu Y P,Tian Z S,Liu L,Lu H,Liu X,Wang Y,Xu A X,Huang Z.Inheritance and gene mapping of mosaic-leaf in Brassica napus L..Acta Agriculturae BorealiSinica,2016,31(4):39-43
[5]浦惠明,傅寿仲,戚存扣,张洁夫,伍贻美,高建芹,陈新军.花叶性状的遗传及其在杂交油菜育种中的应用.中国油料作物学报,2001,23(3):60-62Pu H M,Fu S Z,Qi C K,Zhang J F,Wu Y M,Gao J Q,Chen X J.Inheritance of divided leaf trait of rapeseed(Brassica napus)and application in hybrid breeding.Chinese Journal of Oil Crop Sciences,2001,23(3):60-62
[6]晏仲元.甘蓝型油菜花叶性状的遗传及基因定位.武汉:华中农业大学,2011Yan Z Y.Inheritance of mosaic-leaf in Brassica napus and its gene mapping.Wuhan:Huazhong Agricultural University,2011
[7]倪西源.甘蓝型油菜“花叶”基因的精细定位及其在杂种优势利用中的应用.杭州:浙江大学,2014Ni X Y.Fine mapping of lobed-leaf gene in rapeseed(Brassica napus L.)and its application in heterosis utilization.Hangzhou:Zhejiang University,2014
[8]Xu F,Sun X,Chen Y L,Huang Y,Tong C,Bao J S.Rapid identification of major QTLs associated with rice grain weight and their utilization.PLoS One,2015,10(3):e122206
[9]Sun X W,Liu D W,Zhang X F,Li W B,Liu H,Hong W G,Jiang C B,Guan N,Ma C X,Zeng H P,Xu C H,Song J,Huang L,Wang C M,Shi J J,Wang R,Zheng X H,Lu CY,Wang X W,Zheng H K.SLAF-seq:an efficient method of large-scale De Novo SNP discovery and genotyping using high-throughput sequencing.PLoS One,2013,8(3):e58700
[10]Chen S Q,Huang Z F,Dai Y,Qin S W,Gao Y Y,Zhang L L,Gao Y,Chen J M.The development of 7E chromosome-specific molecular markers for thinopyrum elongatum based on SLAF-seq technology.PLoS One,2013,8(6):e6512
[11]Qi Z M,Huang L,Zhu R S,Xin D W,Liu C Y,Han X,Jiang H W,Hong W G,Hu G H,Zheng H Z,Chen Q S.Ahigh-density genetic map for soybean based on specific length amplified fragment sequencing.PLoS One,2014,9(8):e104871
[12]Chao X,Chen L L,Rong T Z,Li R,Xiang Y,Wang P,Liu CH,Dong X Q,Liu B,Zhao D,Wei R J,Lan H.Identification of a new maize inflorescence meristem mutant and association analysis using SLAF-seq method.Euphytica,2015,202(1):35-44
[13]Geng X,Jiang C,Yang J,Wang L,Wu X,Wei W.Rapid identification of candidate genes for seed weight using the SLAF-Seq method in Brassica napus.PLoS One,2016,11(1):e147580
[14]Zhao C,Zhao G,Geng Z,Wang Z,Wang K,Liu S,Zhang H,Guo B,Geng J.Physical mapping and candidate gene prediction of fertility restorer gene of cytoplasmic male sterility in cotton.BMC Genomics,2018,DOI:10.1186/S12864-017-4406-Y
[15]Kozich J J,Westcott S L,Baxter N T,Highlander S K,Schloss P D.Development of a dual-index sequencing strategy and curation pipeline for snalyzing smplicon dequence data on the miseq illumina sequencing platform.Applied and Environmental Microbiology,2013,79(17):5112-5120
[16]Hill J T,Demarest B L,Bisgrove B W,Bisgrove B G,Su Y C,Yost J.MMAPPR:mutation mapping analysis pipeline for pooled RNA-seq.Genome Research,2013,23(4):687-697
[17]Fekih R,Takagi H,Tamiru M,Abe A,Natsume S,Yaegashi H,Sharma S,Kanzaki H,Matsumura H,Saitoh H,Mitsuoka C,Utsushi H,Urmura A,Kanzaki E,Kosugi S,Yoshida K,Cano L,Kanoun S,Terauchi R.MutMap+:genetic mapping and mutant identification without crossing in rice.PLoS One,2013,8(7):e68529
[18]Takagi H,Abe A,Yoshida K,Kosugi S,Natsume S,Mitsuoka C,Uemura A,Ursushi H,Tamiru M,Takuno S,Innan H,Cano L M,Kamoun S,Terauchi R.QTL-seq:rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations.The Plant Journal,2013,74(1):174-183
[19]Consortium U P.UniProt:the universal protein knowledgebase.Nucleic Acids Research,2017,45(D1):D158-D169
[20]Altschul S F,Madden T L,Schaffer A A.Gapped BLASTand PSI-BLAST:a new generation of protein database search programs.Nucleic Acids Research,1997,25(17):3398-3402
[21]倪西源,黄吉祥,柳寒,潘兵,赵坚义.花叶性状在油菜杂交种纯度控制中的应用.核农学报,2017,31(6):1070-1075Ni X Y,Huang J X,Liu H,Pan B,Zhao J Y.Application of lobed-leaf trait in controlling hybrid purity of rapeseed.Acta Agriculturae Nucleatae Sinica,2017,31(6):1070-1075
[22]Li R,Song W,Wang B Q,Wang J H,Zhang D M,Zhang Q G,Li X H,Wei J F,Gao Z Y.Identification of a locus conferring dominant resistance to maize rough dwarf disease in maize.Scientific Reports,2018,8(1):3248
[23]葛贤宏.人工合成芸薹属异源六倍体与诸葛菜属间杂种的细胞学及分子生物学研究.武汉:华中农业大学,2007Ge X H.Molecular and cytogenetic characterizations on hybrids between synthetic Brassica allohexaploid and Orychophra gmus violaceus.Wuhan:Huazhong Agricultural University,2007
[24]范成明,田建华,胡赞民,王珏,吕慧颖,葛毅强,魏珣,邓向东,张蕾颖,杨维才.油菜育种行业创新动态与发展趋势.植物遗传资源学报,2018,19(3):447-454Fan C M,Tian J H,Hu Z M,Wang J,Lv H Y,Ge Y Q,Wei X,Deng X D,Zhang L Y,Yang W C.Advances of oilseed rape breeding.Journal of Plant Genetic Resources,2018,19(3):447-454
[2]Blein T,Pulido A,Guiraud A V,Nikovics K,Morin H,Hay A,Johansen I E,Tsiantis M,Larfs P.A conserved molecular framework for compound leaf development.Science,2008,322(5909):1835-1839
[3]祝朋芳,冯馨,程明明,潘志超.羽衣甘蓝裂叶相关性状遗传分析.西北植物学报,2016,36(2):288-295Zhu P F,Feng X,Cheng M M,Pan Z C.Genetic analysis of feathered-leaved related traits in Brassica oleracea var,acephala.Acta Botanica Boreali-Occidentalia Sinica,2016,36(2):288-295
[4]郎丽娜,赵娜,马兵,刘亚萍,田正书,刘璐,卢虹,刘霞,王阳,徐爱遐,黄镇.甘蓝型油菜花叶性状的遗传及基因定位.华北农学报,2016,31(4):39-43Lang L N,Zhao N,Ma B,Liu Y P,Tian Z S,Liu L,Lu H,Liu X,Wang Y,Xu A X,Huang Z.Inheritance and gene mapping of mosaic-leaf in Brassica napus L..Acta Agriculturae BorealiSinica,2016,31(4):39-43
[5]浦惠明,傅寿仲,戚存扣,张洁夫,伍贻美,高建芹,陈新军.花叶性状的遗传及其在杂交油菜育种中的应用.中国油料作物学报,2001,23(3):60-62Pu H M,Fu S Z,Qi C K,Zhang J F,Wu Y M,Gao J Q,Chen X J.Inheritance of divided leaf trait of rapeseed(Brassica napus)and application in hybrid breeding.Chinese Journal of Oil Crop Sciences,2001,23(3):60-62
[6]晏仲元.甘蓝型油菜花叶性状的遗传及基因定位.武汉:华中农业大学,2011Yan Z Y.Inheritance of mosaic-leaf in Brassica napus and its gene mapping.Wuhan:Huazhong Agricultural University,2011
[7]倪西源.甘蓝型油菜“花叶”基因的精细定位及其在杂种优势利用中的应用.杭州:浙江大学,2014Ni X Y.Fine mapping of lobed-leaf gene in rapeseed(Brassica napus L.)and its application in heterosis utilization.Hangzhou:Zhejiang University,2014
[8]Xu F,Sun X,Chen Y L,Huang Y,Tong C,Bao J S.Rapid identification of major QTLs associated with rice grain weight and their utilization.PLoS One,2015,10(3):e122206
[9]Sun X W,Liu D W,Zhang X F,Li W B,Liu H,Hong W G,Jiang C B,Guan N,Ma C X,Zeng H P,Xu C H,Song J,Huang L,Wang C M,Shi J J,Wang R,Zheng X H,Lu CY,Wang X W,Zheng H K.SLAF-seq:an efficient method of large-scale De Novo SNP discovery and genotyping using high-throughput sequencing.PLoS One,2013,8(3):e58700
[10]Chen S Q,Huang Z F,Dai Y,Qin S W,Gao Y Y,Zhang L L,Gao Y,Chen J M.The development of 7E chromosome-specific molecular markers for thinopyrum elongatum based on SLAF-seq technology.PLoS One,2013,8(6):e6512
[11]Qi Z M,Huang L,Zhu R S,Xin D W,Liu C Y,Han X,Jiang H W,Hong W G,Hu G H,Zheng H Z,Chen Q S.Ahigh-density genetic map for soybean based on specific length amplified fragment sequencing.PLoS One,2014,9(8):e104871
[12]Chao X,Chen L L,Rong T Z,Li R,Xiang Y,Wang P,Liu CH,Dong X Q,Liu B,Zhao D,Wei R J,Lan H.Identification of a new maize inflorescence meristem mutant and association analysis using SLAF-seq method.Euphytica,2015,202(1):35-44
[13]Geng X,Jiang C,Yang J,Wang L,Wu X,Wei W.Rapid identification of candidate genes for seed weight using the SLAF-Seq method in Brassica napus.PLoS One,2016,11(1):e147580
[14]Zhao C,Zhao G,Geng Z,Wang Z,Wang K,Liu S,Zhang H,Guo B,Geng J.Physical mapping and candidate gene prediction of fertility restorer gene of cytoplasmic male sterility in cotton.BMC Genomics,2018,DOI:10.1186/S12864-017-4406-Y
[15]Kozich J J,Westcott S L,Baxter N T,Highlander S K,Schloss P D.Development of a dual-index sequencing strategy and curation pipeline for snalyzing smplicon dequence data on the miseq illumina sequencing platform.Applied and Environmental Microbiology,2013,79(17):5112-5120
[16]Hill J T,Demarest B L,Bisgrove B W,Bisgrove B G,Su Y C,Yost J.MMAPPR:mutation mapping analysis pipeline for pooled RNA-seq.Genome Research,2013,23(4):687-697
[17]Fekih R,Takagi H,Tamiru M,Abe A,Natsume S,Yaegashi H,Sharma S,Kanzaki H,Matsumura H,Saitoh H,Mitsuoka C,Utsushi H,Urmura A,Kanzaki E,Kosugi S,Yoshida K,Cano L,Kanoun S,Terauchi R.MutMap+:genetic mapping and mutant identification without crossing in rice.PLoS One,2013,8(7):e68529
[18]Takagi H,Abe A,Yoshida K,Kosugi S,Natsume S,Mitsuoka C,Uemura A,Ursushi H,Tamiru M,Takuno S,Innan H,Cano L M,Kamoun S,Terauchi R.QTL-seq:rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations.The Plant Journal,2013,74(1):174-183
[19]Consortium U P.UniProt:the universal protein knowledgebase.Nucleic Acids Research,2017,45(D1):D158-D169
[20]Altschul S F,Madden T L,Schaffer A A.Gapped BLASTand PSI-BLAST:a new generation of protein database search programs.Nucleic Acids Research,1997,25(17):3398-3402
[21]倪西源,黄吉祥,柳寒,潘兵,赵坚义.花叶性状在油菜杂交种纯度控制中的应用.核农学报,2017,31(6):1070-1075Ni X Y,Huang J X,Liu H,Pan B,Zhao J Y.Application of lobed-leaf trait in controlling hybrid purity of rapeseed.Acta Agriculturae Nucleatae Sinica,2017,31(6):1070-1075
[22]Li R,Song W,Wang B Q,Wang J H,Zhang D M,Zhang Q G,Li X H,Wei J F,Gao Z Y.Identification of a locus conferring dominant resistance to maize rough dwarf disease in maize.Scientific Reports,2018,8(1):3248
[23]葛贤宏.人工合成芸薹属异源六倍体与诸葛菜属间杂种的细胞学及分子生物学研究.武汉:华中农业大学,2007Ge X H.Molecular and cytogenetic characterizations on hybrids between synthetic Brassica allohexaploid and Orychophra gmus violaceus.Wuhan:Huazhong Agricultural University,2007
[24]范成明,田建华,胡赞民,王珏,吕慧颖,葛毅强,魏珣,邓向东,张蕾颖,杨维才.油菜育种行业创新动态与发展趋势.植物遗传资源学报,2018,19(3):447-454Fan C M,Tian J H,Hu Z M,Wang J,Lv H Y,Ge Y Q,Wei X,Deng X D,Zhang L Y,Yang W C.Advances of oilseed rape breeding.Journal of Plant Genetic Resources,2018,19(3):447-454