烟草航天诱变突变体变异检测及分析
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摘要
为预测控制烟草重要性状的关键基因并研究航天诱变机理,对经航天诱变选育的烟草突变体材料NC89-M与野生型NC89进行全基因组重测序,测序深度30×,并对各种类型变异进行检测注释。NC89中检测到单核苷酸多态性位点(SNP,Single nucleotide polymorphism)1848013个,小片段插入缺失(Indel,insertion-delection)398922个,结构变异(SV,Structure variation)41969个;NC89-M中检测到SNP 1876219个,Indel 402011个,SV 42699个。NC89-M和NC89对比共得到271655个SNP,分布在8378个基因上,23450个Indel造成2156个基因突变。分析结果表明,NC89-M中SNP变异数目最多,其转换类型和颠换类型的比值为2.053,说明航天诱变对烟草基因组的变异以单碱基突变为主,突变类型以转换为主;在Indel中,插入突变数目明显多于缺失突变,证明航天诱变造成的Indel中以插入突变为主;在SV中,航天诱变主要造成了插入、缺失、倒位、染色体内部迁移和染色体间的迁移5种结构变异类型;变异基因KEGG注释表明,与代谢通路和次生代谢产物合成两方面基因突变数目最多;变异基因功能注释表明,突变体中调控开花时间的MADS-box基因,调控侧生器官发育与叶缘形状的KNOX1基因和萜类化合物合成相关基因等发生了变异。
To explore candidate genes of tobacco important traits and provide insights of space mutagenesis,we conducted the whole genome re-sequencing(30 x coverage)in tobacco mutant NC89-M that was generated by space mutagenesis,and wild-type NC89.In comparison to the reference genome,1848013 single nucleotide polymorphisms(SNPs),398922 Indels and 41969 structure variations(SVs)were observed in NC89,while 1876219 SNPs,402011 Indels and 42699 SVs were detected in NC89-M.A total of 271655 SNPs and 23450 Indels differed between NC89-M and NC89,which resulted in sequence modifications on8378 and 2156 genes,respectively.SNPs were often observed in mutant,suggesting that single nucleotide mutation is predominant using spare mutagenesis.The ratio of conversion type to transversion type is 2.053.It is proved that the single-base mutations are the main mutation type in the tobacco genome induced by space and conversion type is the main mutation type among the single base mutation.The number of insertion was significantly more than that of the deletion.It is proved that the insertion is the main mutation type in Indel caused by space mutagenesis.Among the SV,space mutagenesis mainly results in five types of structural variation including insertion,deletion,inversion,intra-chromosomal translocation and inter-chromosomal translocation.KEGG annotation of mutant genes indicated that the number of mutant genes in the metabolic pathway and biosynthesis of secondary metabolites are the highest.Gene function annotation indicated that several functional genes were modified in mutant,including MADS-box genes that control flowering time,KNOX1 gene that regulates development of lateral organ and leaf edge shape and also the genes that associate to the synthesis of terpenoids.
To explore candidate genes of tobacco important traits and provide insights of space mutagenesis,we conducted the whole genome re-sequencing(30 x coverage)in tobacco mutant NC89-M that was generated by space mutagenesis,and wild-type NC89.In comparison to the reference genome,1848013 single nucleotide polymorphisms(SNPs),398922 Indels and 41969 structure variations(SVs)were observed in NC89,while 1876219 SNPs,402011 Indels and 42699 SVs were detected in NC89-M.A total of 271655 SNPs and 23450 Indels differed between NC89-M and NC89,which resulted in sequence modifications on8378 and 2156 genes,respectively.SNPs were often observed in mutant,suggesting that single nucleotide mutation is predominant using spare mutagenesis.The ratio of conversion type to transversion type is 2.053.It is proved that the single-base mutations are the main mutation type in the tobacco genome induced by space and conversion type is the main mutation type among the single base mutation.The number of insertion was significantly more than that of the deletion.It is proved that the insertion is the main mutation type in Indel caused by space mutagenesis.Among the SV,space mutagenesis mainly results in five types of structural variation including insertion,deletion,inversion,intra-chromosomal translocation and inter-chromosomal translocation.KEGG annotation of mutant genes indicated that the number of mutant genes in the metabolic pathway and biosynthesis of secondary metabolites are the highest.Gene function annotation indicated that several functional genes were modified in mutant,including MADS-box genes that control flowering time,KNOX1 gene that regulates development of lateral organ and leaf edge shape and also the genes that associate to the synthesis of terpenoids.
引文
[1]尚志强.中国烟草育种进展及发展对策.内蒙古农业科技,2010(5):1-3Shang Z Q.Advances and development strategies of burley tobacco breeding in China.Inner Mongolia Agricultural Science and Technology,2010(5):1-3
[2]钟波,朱列书,贺鹏,谢慧玲,林跃平.浅谈航天诱变育种.作物研究,2007(S1):511-516Zhong B,Zhu L S,He P,Xie H L,Lin Y P.Discussion on space mutation breeding.Crop Research,2007(S1):511-516
[3]刘敏,李金国,王亚林,张赞,薛淮,张纯花,李社荣,郝连元,李广元.卫星搭载的甜椒87-2过氧化物同工酶检测和RAPD分子检测初报.核农学报,1999,13(5):291-294Liu M,Li J G,Wang Y L,Zhang Z,Xue H,Zhang C H,Li S R,Hao L Y,Li G Y.Preliminary study on peroxidase isoenzyme detection and RAPD molecular verification for sweet pepper87-2 Carried by a recoverable satellite.Journal of Nuclear Agricultural Sciences,1999,13(5):291-294
[4]Cheng Z,Lin J,Lin T,Xu M,Huang Z,Yang Z,Huang X,Zheng J.Genome-wide analysis of radiation-induced mutations in rice(Oryza sativa L.ssp.indica).Molecular Biosystems,2014,10(4):795
[5]Fu C Y,Liu W G,Liu D L,Li J H,Zhu M S,Liao Y L,Liu Z R,Zeng X Q,Wang F.Genome-wide DNA polymorphism in the indica rice varieties RGD-7S and Taifeng B as revealed by whole genome re-sequencing.Genome,2016,59(3):197-207
[6]Maldonado dos Santos J V,Valliyodan B,Joshi T,Khan SM,Liu Y,Wang J,Vuong T D,de Oliveira M F,MarcelinoGuimar?es F C,Xu D,Nguyen H T,Abdelnoor R V.Evaluation of genetic variation among Brazilian soybean cultivars through genome resequencing.Bmc Genomics,2016,17(1):110
[7]Sierro N,Battey J N,Ouadi S,Bakaher N,Bovet L,Willig A,Goepfert S,Peitsch M C,Ivanov N V.The tobacco genome sequence and its comparison with those of tomato and potato.Nature Communications,2014,5(5):3833
[8]Bindler G,Plieske J,Bakaher N,Gunduz I,Ivanov N,Van der Hoeven R,Ganal M,Donini P.A high density genetic map of tobacco(Nicotiana tabacum L.)obtained from large scale microsatellite marker development.Theoretical&Applied Genetics,2011,123(2):219
[9]Li H,Durbin R.Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics,2009,25(14):1754-1760
[10]Li H,Handsaker B,Wysoker A,Fennell T,Ruan J,Homer N,Marth G,Abecasis G,Durbin R;1000 Genome Project Data Processing Subgroup.The Sequence Alignment/Map format and SAMtools.Bioinformatics,2009,25(16):2078-2079
[11]Wang K,Li M,Hakonarson H.ANNOVAR:functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Research,2010,38(16):e164
[12]Chen K,Wallis J W,McLellan M D,Larson D E,Kalicki J M,Pohl C S,McGrath S D,Wendl M C,Zhang Q,Locke D P,Shi X,Fulton R S,Ley T J,Wilson R K,Ding L,Mardis E R.BreakDancer:an algorithm for high-resolution mapping of genomic structural variation.Nature Methods,2009,6(9):677-681
[13]Kanehisa M,Goto S,Kawashima S,Okuno Y,Hattori M.The KEGG resource for deciphering the genome.Nucleic Acids Research,2004,32(Nucleic Acids Research):D277-D280
[14]Altschul S F,Madden T L,Sch?ffer A A,Zhang J,Zhang Z,Miller W,Lipman D J.Gapped BLAST and PSI-BLAST:a new generation of protein database search programs.Nucleic Acids Research,1997,25(17):3389-3402
[15]Hake S,Smith H M,Holtan H,Magnani E,Mele G,Ramirez J.The role of knox genes in plant development.Annual Review of Cell and Developmental Biologyl,2004,20(1):125-151
[16]Messenguy F,Dubois E.Role of MADS box proteins and their cofactors in combinatorial control of gene expression and cell development.Gene,2003,316(1):1-21
[17]Rodr?-Guez-Concepci?3N M,Boronat A.Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids.A metabolic milestone achieved through genomics.Plant Physiology,2002,130(3):1079-1089
[18]张志勇,蒲志刚,王平,向跃武,蔡平钟,张志雄.水稻航天诱变突变体全基因组测序研究.西南农业学报,2014,27(2):469-475Zhang Z Y,Pu Z G,Wang P,Xiang Y W,Cai P Z,Zhang Z X.Sequencing research on whole genome of rice mutant induced by space.Southwest China Journal of Agricultural Sciences,2014,27(2):469-475
[19]张德建,梁发茂,胡刚,邢永忠,李志新.γ射线辐射“9311”水稻突变体的筛选.植物遗传资源学报,2015,16(4):876-882Zhang D J,Liang F M,Hu G,Xing Y Z,Li Z X.Screening of mutant for rice cultivar“9311”byγ-ray irradiation.Journal of Plant Genetic Resources,2015,16(4):876-882
[20]Bowman J L,Alvarez J,Weigel D,Meyerowitz E M,Smyth D R.Control of flower development in Arabidopsis thaliana by APETALA1 and interacting genes.Development,1993,119(3):721-743
[21]Mao L,Begum D,Chuang H W,Budiman M A,Szymkowiak E J,Irish E E,Wing R A.JOINTLESS is a MADS-box gene controlling tomato flower abscission zone development.Nature,2000,406(6798):910-913
[22]Yoo S K,Lee J S,Ahn J H.Overexpression of AGAMOUS-LIKE 28(AGL28)promotes flowering by upregulating expression of floral promoters within the autonomous pathway.Biochem Biophys Res Commun,2006,348(3):929-936
[23]Ori N,Juarez M T,Jackson D,Yamaguchi J,Banowetz G M,Hake S.Leaf senescence is delayed in tobacco plants expressing the maize homeobox gene knotted1 under the control of a senescence-activated promoter.Plant Cell,1999,11(6):1073-1080
[24]Hewelt A,Prinsen E,Thomas M,Van Onckelen H,Meins FJr.Ectopic expression of maize knotted1 results in the cytokininautotrophic growth of cultured tobacco tissues.Planta,2000,210(6):884-889
[25]Frugis G,Mariotti D.Overexpression of KNAT1 in lettuce shifts leaf determinate growth to a shoot-like indeterminate growth associated with an accumulation of isopentenyl-type cytokinins.Plant Physiology,2001,126(4):1370-1380
[26]刘青,汪玉凤,赵韩生,陈颖,高志民.麻竹同源异型盒基因DlKNOX1的克隆及功能初步分析.林业科学,2014,50(2):56-62Liu Q,Wang Y F,Zhao H S,Chen Y,Gao Z M.Molecular characteristics and primary functional analysis of DlKNOX1gene from dendrocalamus latiflorus.Scientia Silvae Sinicae,2014,50(2):56-62
[27]申培林,杨铁钊,张小全,李亚培,贾燕超,宋文峰,吕军朋.烟草类萜生物合成途径中关键酶基因的克隆与表达调控.江苏农业科学,2012,40(1):37-40Shen P L,Yang T Z,Zhang X Q,Li Y P,Jia Y C,Song W F,Lv J P.Cloning and expression regulation of key enzyme genes in tobacco terpenoids biosynthesis pathway.Jiangsu Agricultural Sciences,2012,40(1):37-40
[28]张崇范.烤烟育种目标刍议.中国烟草科学,1986(3):21-23Zhang C F.Discussion on breeding objectives of flue-cured tobacco.Chinese Tobacco Science,1986(3):21-23
[2]钟波,朱列书,贺鹏,谢慧玲,林跃平.浅谈航天诱变育种.作物研究,2007(S1):511-516Zhong B,Zhu L S,He P,Xie H L,Lin Y P.Discussion on space mutation breeding.Crop Research,2007(S1):511-516
[3]刘敏,李金国,王亚林,张赞,薛淮,张纯花,李社荣,郝连元,李广元.卫星搭载的甜椒87-2过氧化物同工酶检测和RAPD分子检测初报.核农学报,1999,13(5):291-294Liu M,Li J G,Wang Y L,Zhang Z,Xue H,Zhang C H,Li S R,Hao L Y,Li G Y.Preliminary study on peroxidase isoenzyme detection and RAPD molecular verification for sweet pepper87-2 Carried by a recoverable satellite.Journal of Nuclear Agricultural Sciences,1999,13(5):291-294
[4]Cheng Z,Lin J,Lin T,Xu M,Huang Z,Yang Z,Huang X,Zheng J.Genome-wide analysis of radiation-induced mutations in rice(Oryza sativa L.ssp.indica).Molecular Biosystems,2014,10(4):795
[5]Fu C Y,Liu W G,Liu D L,Li J H,Zhu M S,Liao Y L,Liu Z R,Zeng X Q,Wang F.Genome-wide DNA polymorphism in the indica rice varieties RGD-7S and Taifeng B as revealed by whole genome re-sequencing.Genome,2016,59(3):197-207
[6]Maldonado dos Santos J V,Valliyodan B,Joshi T,Khan SM,Liu Y,Wang J,Vuong T D,de Oliveira M F,MarcelinoGuimar?es F C,Xu D,Nguyen H T,Abdelnoor R V.Evaluation of genetic variation among Brazilian soybean cultivars through genome resequencing.Bmc Genomics,2016,17(1):110
[7]Sierro N,Battey J N,Ouadi S,Bakaher N,Bovet L,Willig A,Goepfert S,Peitsch M C,Ivanov N V.The tobacco genome sequence and its comparison with those of tomato and potato.Nature Communications,2014,5(5):3833
[8]Bindler G,Plieske J,Bakaher N,Gunduz I,Ivanov N,Van der Hoeven R,Ganal M,Donini P.A high density genetic map of tobacco(Nicotiana tabacum L.)obtained from large scale microsatellite marker development.Theoretical&Applied Genetics,2011,123(2):219
[9]Li H,Durbin R.Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics,2009,25(14):1754-1760
[10]Li H,Handsaker B,Wysoker A,Fennell T,Ruan J,Homer N,Marth G,Abecasis G,Durbin R;1000 Genome Project Data Processing Subgroup.The Sequence Alignment/Map format and SAMtools.Bioinformatics,2009,25(16):2078-2079
[11]Wang K,Li M,Hakonarson H.ANNOVAR:functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Research,2010,38(16):e164
[12]Chen K,Wallis J W,McLellan M D,Larson D E,Kalicki J M,Pohl C S,McGrath S D,Wendl M C,Zhang Q,Locke D P,Shi X,Fulton R S,Ley T J,Wilson R K,Ding L,Mardis E R.BreakDancer:an algorithm for high-resolution mapping of genomic structural variation.Nature Methods,2009,6(9):677-681
[13]Kanehisa M,Goto S,Kawashima S,Okuno Y,Hattori M.The KEGG resource for deciphering the genome.Nucleic Acids Research,2004,32(Nucleic Acids Research):D277-D280
[14]Altschul S F,Madden T L,Sch?ffer A A,Zhang J,Zhang Z,Miller W,Lipman D J.Gapped BLAST and PSI-BLAST:a new generation of protein database search programs.Nucleic Acids Research,1997,25(17):3389-3402
[15]Hake S,Smith H M,Holtan H,Magnani E,Mele G,Ramirez J.The role of knox genes in plant development.Annual Review of Cell and Developmental Biologyl,2004,20(1):125-151
[16]Messenguy F,Dubois E.Role of MADS box proteins and their cofactors in combinatorial control of gene expression and cell development.Gene,2003,316(1):1-21
[17]Rodr?-Guez-Concepci?3N M,Boronat A.Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids.A metabolic milestone achieved through genomics.Plant Physiology,2002,130(3):1079-1089
[18]张志勇,蒲志刚,王平,向跃武,蔡平钟,张志雄.水稻航天诱变突变体全基因组测序研究.西南农业学报,2014,27(2):469-475Zhang Z Y,Pu Z G,Wang P,Xiang Y W,Cai P Z,Zhang Z X.Sequencing research on whole genome of rice mutant induced by space.Southwest China Journal of Agricultural Sciences,2014,27(2):469-475
[19]张德建,梁发茂,胡刚,邢永忠,李志新.γ射线辐射“9311”水稻突变体的筛选.植物遗传资源学报,2015,16(4):876-882Zhang D J,Liang F M,Hu G,Xing Y Z,Li Z X.Screening of mutant for rice cultivar“9311”byγ-ray irradiation.Journal of Plant Genetic Resources,2015,16(4):876-882
[20]Bowman J L,Alvarez J,Weigel D,Meyerowitz E M,Smyth D R.Control of flower development in Arabidopsis thaliana by APETALA1 and interacting genes.Development,1993,119(3):721-743
[21]Mao L,Begum D,Chuang H W,Budiman M A,Szymkowiak E J,Irish E E,Wing R A.JOINTLESS is a MADS-box gene controlling tomato flower abscission zone development.Nature,2000,406(6798):910-913
[22]Yoo S K,Lee J S,Ahn J H.Overexpression of AGAMOUS-LIKE 28(AGL28)promotes flowering by upregulating expression of floral promoters within the autonomous pathway.Biochem Biophys Res Commun,2006,348(3):929-936
[23]Ori N,Juarez M T,Jackson D,Yamaguchi J,Banowetz G M,Hake S.Leaf senescence is delayed in tobacco plants expressing the maize homeobox gene knotted1 under the control of a senescence-activated promoter.Plant Cell,1999,11(6):1073-1080
[24]Hewelt A,Prinsen E,Thomas M,Van Onckelen H,Meins FJr.Ectopic expression of maize knotted1 results in the cytokininautotrophic growth of cultured tobacco tissues.Planta,2000,210(6):884-889
[25]Frugis G,Mariotti D.Overexpression of KNAT1 in lettuce shifts leaf determinate growth to a shoot-like indeterminate growth associated with an accumulation of isopentenyl-type cytokinins.Plant Physiology,2001,126(4):1370-1380
[26]刘青,汪玉凤,赵韩生,陈颖,高志民.麻竹同源异型盒基因DlKNOX1的克隆及功能初步分析.林业科学,2014,50(2):56-62Liu Q,Wang Y F,Zhao H S,Chen Y,Gao Z M.Molecular characteristics and primary functional analysis of DlKNOX1gene from dendrocalamus latiflorus.Scientia Silvae Sinicae,2014,50(2):56-62
[27]申培林,杨铁钊,张小全,李亚培,贾燕超,宋文峰,吕军朋.烟草类萜生物合成途径中关键酶基因的克隆与表达调控.江苏农业科学,2012,40(1):37-40Shen P L,Yang T Z,Zhang X Q,Li Y P,Jia Y C,Song W F,Lv J P.Cloning and expression regulation of key enzyme genes in tobacco terpenoids biosynthesis pathway.Jiangsu Agricultural Sciences,2012,40(1):37-40
[28]张崇范.烤烟育种目标刍议.中国烟草科学,1986(3):21-23Zhang C F.Discussion on breeding objectives of flue-cured tobacco.Chinese Tobacco Science,1986(3):21-23