不同性别大白猪肌肉全基因组高分辨率单碱基甲基化差异分析
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摘要
旨在通过不同性别大白猪的全基因组高分辨率单碱基甲基化差异分析,检测差异甲基化区域(DMR)和差异甲基化基因(DMG),为进一步解析公母猪骨骼肌发育差异奠定基础。本研究采用全基因组重亚硫酸盐测序(WGBS)技术分析了4头210日龄长白猪(公、母各半)背最长肌全基因组DNA的甲基化程度和差异甲基化区域,探讨性别间DNA甲基化水平的差异。结果表明,全基因组范围约有5%的胞嘧啶(C)发生了甲基化(mC);母猪和公猪的总体甲基化水平基本一致。共检测到1 629个DMRs和841个DMGs。母猪的DMRs平均甲基化水平明显高于公猪。通过基因本体分析(GO)和相关信号通路(KEGG)分析,共检测到171个GO条目和10个信号通路,显著富集在轴突导向、细胞连接、细胞粘附、ECM受体互作等相关过程中;筛选出5个与不同性别肌肉调节相关的候选基因。本研究绘制了不同性别大白猪的高分辨率单碱基全基因组甲基化图谱,为不同性别表观遗传研究以及肌肉发育和肉质相关候选基因的筛选提供了信息参考。
The aims of this study were to detect the differentially methylated region(DMR)and differentially methylated gene(DMG)and lay a foundation for further analyzing the skeletal muscle development difference between male and female pigs by high resolution and single base genome-wide methylation variance analysis for Large White pigs of different genders.In this study,four 210-day-old Large White pigs(two males and two females)were scanned by the whole genome bisulfite sequencing method(WGBS).The degree of methylation and differentially methylated region in the whole genome DNA of longissimus dorsi(LD)muscle were studied to explore the difference of DNA methylation level between male and female pigs.The results showed that about 5% Cytosine(C)were methylated in the whole genome.The overall methylation level of sows and boars was basically the same.A total of 1 629 DMRs and 841 related DMGs were detected.The DMRs methylation level of sows was higher than that of boars.One hundred and sev-enty one GO terms and 10 related signal pathways were detected by GO and KEGG analysis,respectively,which were showed to be significantly enriched in cell junction,axon guidance,cell adhesion molecule binding,ECM-receptor interaction,and so on.Five candidate genes were screened out in muscle tissues of male and female pigs.This study provided the single base and high resolution genome-wide methylation pattern of male and female Large White pigs.The results provide reference information for the epigenetic study of different genders and screening candidate genes related to muscle development and meat quality.
The aims of this study were to detect the differentially methylated region(DMR)and differentially methylated gene(DMG)and lay a foundation for further analyzing the skeletal muscle development difference between male and female pigs by high resolution and single base genome-wide methylation variance analysis for Large White pigs of different genders.In this study,four 210-day-old Large White pigs(two males and two females)were scanned by the whole genome bisulfite sequencing method(WGBS).The degree of methylation and differentially methylated region in the whole genome DNA of longissimus dorsi(LD)muscle were studied to explore the difference of DNA methylation level between male and female pigs.The results showed that about 5% Cytosine(C)were methylated in the whole genome.The overall methylation level of sows and boars was basically the same.A total of 1 629 DMRs and 841 related DMGs were detected.The DMRs methylation level of sows was higher than that of boars.One hundred and sev-enty one GO terms and 10 related signal pathways were detected by GO and KEGG analysis,respectively,which were showed to be significantly enriched in cell junction,axon guidance,cell adhesion molecule binding,ECM-receptor interaction,and so on.Five candidate genes were screened out in muscle tissues of male and female pigs.This study provided the single base and high resolution genome-wide methylation pattern of male and female Large White pigs.The results provide reference information for the epigenetic study of different genders and screening candidate genes related to muscle development and meat quality.
引文
[1] HE X J,CHEN T P,ZHU J K.Regulation and function of DNA methylation in plants and animals[J].Cell Res,2011,21(3):442-465.
[2] EL-MAARRI O,BECKER T,JUNEN J,et al.Gender specific differences in levels of DNA methylation at selected loci from human total blood:A tendency toward higher methylation levels in males[J].Hum Genet,2007,122(5):505-514.
[3] FUKE C,SHIMABUKURO M,PETRONIS A,et al.Age related changes in 5-methylcytosine content in human peripheral leukocytes and placentas:An HPLC-based study[J].Ann Hum Genet,2004,68(3):196-204.
[4] SHIMABUKURO M,SASAKI T,IMAMURA A,et al.Global hypomethylation of peripheral leukocyte DNA in male patients with schizophrenia:A potential link between epigenetics and schizophrenia[J].J Psychiatr Res,2007,41(12):1042-1046.
[5] XIAO Y M,WORD B,STARLARD-DAVENPORT A,et al.Age and gender affect DNMT3aand DNMT3bexpression in human liver[J].Cell Biol Toxicol,2008,24(3):265-272.
[6] GOLBUS J,PALELLA T D,RICHARDSON B C.Quantitative changes in T cell DNA methylation occur during differentiation and ageing[J].Eur J Immunol,1990,20(8):1869-1872.
[7]尹慧,黄代新,翟仙敦,等.HPLC法检测人外周血5-mC含量及其与年龄相关性研究[J].中国法医学杂志,2007,22(1):8-11.YIN H,HUANG D X,ZHAI X D,et al.Age related changes in 5-methylcytosine content in human peripheral leukocytes by HPLC[J].Chinese Journal of Forensic Medicine,2007,22(1):8-11.(in Chinese)
[8]白小青,王金勇,陈英,等.荣昌猪仔猪性别间DNA甲基化水平的差异研究[J].中国畜牧杂志,2010,46(13):12-13,80.BAI X Q,WANG J Y,CHEN Y,et al.The study on gender specific differences in levels of DNA methylation of Rongchang pigs[J].Chinese Journal of Animal Science,2010,46(13):12-13,80.(in Chinese)
[9] XI Y X,LI W.BSMAP:Whole genome bisulfite sequence MAPping program[J].BMC Bioinform,2009,10:232.
[10] LIU H B,LIU X J,ZHANG S M,et al.Systematic identification and annotation of human methylation marks based on bisulfite sequencing methylomes reveals distinct roles of cell type-specific hypomethylation in the regulation of cell identity genes[J].Nucleic Acids Res,2016,44(1):79-94.
[11] HUANG D W,SHERMAN B T,LEMPICKI R A.Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[J].Nat Protoc,2009,4(1):44-57.
[12] DOI A,PARK I H,WEN B,et al.Differential methylation of tissue-and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells,embryonic stem cells and fibroblasts[J].Nat Genet,2009,41(12):1350-1353.
[13] JONES P A.Functions of DNA methylation:Islands,start sites,gene bodies and beyond[J].Nat Rev Genet,2012,13(7):484-492.
[14]楼平儿.猪不同年龄段骨骼肌的差异甲基化组分析[D].雅安:四川农业大学,2013.LOU P E.Aging-related DNA methylomes in porcine skeletal muscles[D].Ya′an:Sichuan Agricultural University,2013.(in Chinese)
[15]张小丽.猪背部浅层和背部深层脂肪组织全基因组甲基化研究[D].雅安:四川农业大学,2013.ZHANG X L.Genome-wide DNA methylation changes between the superficial and deep backfat tissues of the pig[D].Ya′an:Sichuan Agricultural University,2013.(in Chinese)
[16] JIN L,JIANG Z,XIA Y D,et al.Genome-wide DNA methylation changes in skeletal muscle between young and middle-aged pigs[J].BMC Genomics,2014,15:653.
[17] SU Y Y,FAN Z P,WU X S,et al.Genome-wide DNA methylation profile of developing deciduous tooth germ in miniature pigs[J].BMC Genomics,2016,17:134.
[18] WANG H F,WANG J Y,NING C,et al.Genomewide DNA methylation and transcriptome analyses reveal genes involved in immune responses of pig peripheral blood mononuclear cells to poly I:C[J].Sci Rep,2017,7(1):9709.
[19] KIM W,PARK H,SEO P S,et al.Characterization and functional inferences of a genome-wide DNA methylation profile in the loin(longissimus dorsi)muscle of swine[J].Asian-Australas J Anim Sci,2018,31(1):3-12.
[20] LI M Z,WU H L,LUO Z G,et al.An atlas of DNA methylomes in porcine adipose and muscle tissues[J].Nat Commun,2012,3:850.
[21] YANG Y L,LIANG G M,NIU G L,et al.Comparative analysis of DNA methylome and transcriptome of skeletal muscle in lean-,obese-,and mini-type pigs[J].Sci Rep,2017,7:39883,doi:10.1038/srep39883.
[22] KWAK W,KIM J N,KIM D,et al.Genome-wide DNA methylation profiles of small intestine and liver in fast-growing and slow-growing weaning piglets[J].Asian-Australas J Anim Sci,2014,27(11):1532-1539.
[23] SCHACHTSCHNEIDER K M,MADSEN O,PARK C,et al.Adult porcine genome-wide DNA methylation patterns support pigs as a biomedical model[J].BMC Genomics,2015,16:743.
[24] CHOI M,LEE J,LE M T,et al.Genome-wide analysis of DNA methylation in pigs using reduced representation bisulfite sequencing[J].DNA Res,2015,22(5):343-355.
[25] YUAN X L,GAO N,XING Y,et al.Profiling the genome-wide DNA methylation pattern of porcine ovaries using reduced representation bisulfite sequencing[J].Sci Rep,2016,6:22138,doi:10.1038/srep22138.
[26] HAO Y,CUI Y J,GU X H.Genome-wide DNA methylation profiles changes associated with constant heat stress in pigs as measured by bisulfite sequencing[J].Sci Rep,2016,6:27507.
[27]张恬.猪肌内脂肪全基因组甲基化差异分析及候选基因研究[D].北京:中国农业科学院,2016.ZHANG T.Study on genome-wide methylation differential analysis and candidate genes in pig IMF[D].Beijing:Chinese Academy of Agricultural Sciences,2016.(in Chinese)
[28] JIN L,MAO K,LI J,et al.Genome-wide profiling of gene expression and DNA methylation provides insight into low-altitude acclimation in Tibetan pigs[J].Gene,2018,642:522-532.
[29] ANTHONY K,CIRAK S,TORELLI S,et al.Dystrophin quantification and clinical correlations in Becker muscular dystrophy:Implications for clinical trials[J].Brain,2011,134(12):3547-3559.
[30] HEALD A,ANDERSON L V B,BUSHBY K M D,et al.Becker muscular dystrophy with onset after 60years[J].Neurology,1994,44(12):2388-2390.
[31] FERREIRO V,GILIBERTO F,MUNIZ G M N,et al.Asymptomatic Becker muscular dystrophy in a family with a multiexon deletion[J].Muscle Nerve,2009,39(2):239-243.
[32] MONACO A P,BERTELSON C J,LIECHTI-GALLATI S,et al.An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus[J].Genomics,1988,2(1):90-95.
[33] BUSHBY K M D,CLEGHORN N J,CURTIS A,et al.Identification of a mutation in the promoter region of the dystrophin gene in a patient with atypical Becker muscular dystrophy[J].Hum Genet,1991,88(2):195-199.
[34] HELLER K N,MONTGOMERY C L,SHONTZ K M,et al.Humanα7integrin gene(ITGA7)delivered by adeno-associated virus extends survival of severely affected dystrophin/utrophin-deficient mice[J].Hum Gene Ther,2015,26(10):647-656.
[35] QUIJANO-ROY S,MBIELEU B,BONNEMANN C G,et al.De novo LMNA mutations cause a new form of congenital muscular dystrophy[J].Ann Neurol,2008,64(2):177-186.
[36] BONNE G,DI BARLETTA M R,VARNOUS S,et al.Mutations in the gene encoding lamin A/C cause autosomal dominant Emery-Dreifuss muscular dystrophy[J].Nat Genet,1999,21(3):285-288.
[37] JIMENE Z-ESCRIG A,GOBERNADO I,GARCIAVILLANUEVA M,et al.Autosomal recessive EmeryDreifuss muscular dystrophy caused by a novel mutation(R225Q)in the lamin A/C gene identified by exome sequencing[J].Muscle Nerve,2012,45(4):605-610.
[38] DE SANDRE-GIOVANNOLI A,CHAOUCH M,KOZLOV S,et al.Homozygous defects in LMNA,encoding lamin A/C nuclear-envelope proteins,cause autosomal recessive axonal neuropathy in human(Charcot-Marie-Tooth disorder type 2)and mouse[J].Am J Hum Genet,2002,70(3):726-736.
[39] CAO H,HEGELE R A.Nuclear lamin A/C R482Q mutation in Canadian kindreds with Dunnigan-type familial partial lipodystrophy[J].Hum Mol Genet,2000,9(1):109-112.
[40] HERSHBERGER R E,SIEGFRIED J D.Update2011:Clinical and genetic issues in familial dilated cardiomyopathy[J].J Am Coll Cardiol,2011,57(16):1641-1649.
[41] LIU D,HOU J,HU X,et al.Neuronal chemorepellent slit2inhibits vascular smooth muscle cell migration by suppressing small GTPase rac1activation[J].Circ Res,2006,98(4):480-489.
[42] QU X H,WEI H D,ZHAI Y,et al.Identification,characterization,and functional study of the two novel human members of the semaphorin gene family[J].J Biol Chem,2002,277(38):35574-35585.
[43] TOYOFUKU T,ZHANG H,KUMANOGOH A,et al.Dual roles of Sema6Din cardiac morphogenesis through region-specific association of its receptor,Plexin-A1,with off-track and vascular endothelial growth factor receptor type 2[J].Genes Dev,2004,18(4):435-447.
[2] EL-MAARRI O,BECKER T,JUNEN J,et al.Gender specific differences in levels of DNA methylation at selected loci from human total blood:A tendency toward higher methylation levels in males[J].Hum Genet,2007,122(5):505-514.
[3] FUKE C,SHIMABUKURO M,PETRONIS A,et al.Age related changes in 5-methylcytosine content in human peripheral leukocytes and placentas:An HPLC-based study[J].Ann Hum Genet,2004,68(3):196-204.
[4] SHIMABUKURO M,SASAKI T,IMAMURA A,et al.Global hypomethylation of peripheral leukocyte DNA in male patients with schizophrenia:A potential link between epigenetics and schizophrenia[J].J Psychiatr Res,2007,41(12):1042-1046.
[5] XIAO Y M,WORD B,STARLARD-DAVENPORT A,et al.Age and gender affect DNMT3aand DNMT3bexpression in human liver[J].Cell Biol Toxicol,2008,24(3):265-272.
[6] GOLBUS J,PALELLA T D,RICHARDSON B C.Quantitative changes in T cell DNA methylation occur during differentiation and ageing[J].Eur J Immunol,1990,20(8):1869-1872.
[7]尹慧,黄代新,翟仙敦,等.HPLC法检测人外周血5-mC含量及其与年龄相关性研究[J].中国法医学杂志,2007,22(1):8-11.YIN H,HUANG D X,ZHAI X D,et al.Age related changes in 5-methylcytosine content in human peripheral leukocytes by HPLC[J].Chinese Journal of Forensic Medicine,2007,22(1):8-11.(in Chinese)
[8]白小青,王金勇,陈英,等.荣昌猪仔猪性别间DNA甲基化水平的差异研究[J].中国畜牧杂志,2010,46(13):12-13,80.BAI X Q,WANG J Y,CHEN Y,et al.The study on gender specific differences in levels of DNA methylation of Rongchang pigs[J].Chinese Journal of Animal Science,2010,46(13):12-13,80.(in Chinese)
[9] XI Y X,LI W.BSMAP:Whole genome bisulfite sequence MAPping program[J].BMC Bioinform,2009,10:232.
[10] LIU H B,LIU X J,ZHANG S M,et al.Systematic identification and annotation of human methylation marks based on bisulfite sequencing methylomes reveals distinct roles of cell type-specific hypomethylation in the regulation of cell identity genes[J].Nucleic Acids Res,2016,44(1):79-94.
[11] HUANG D W,SHERMAN B T,LEMPICKI R A.Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[J].Nat Protoc,2009,4(1):44-57.
[12] DOI A,PARK I H,WEN B,et al.Differential methylation of tissue-and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells,embryonic stem cells and fibroblasts[J].Nat Genet,2009,41(12):1350-1353.
[13] JONES P A.Functions of DNA methylation:Islands,start sites,gene bodies and beyond[J].Nat Rev Genet,2012,13(7):484-492.
[14]楼平儿.猪不同年龄段骨骼肌的差异甲基化组分析[D].雅安:四川农业大学,2013.LOU P E.Aging-related DNA methylomes in porcine skeletal muscles[D].Ya′an:Sichuan Agricultural University,2013.(in Chinese)
[15]张小丽.猪背部浅层和背部深层脂肪组织全基因组甲基化研究[D].雅安:四川农业大学,2013.ZHANG X L.Genome-wide DNA methylation changes between the superficial and deep backfat tissues of the pig[D].Ya′an:Sichuan Agricultural University,2013.(in Chinese)
[16] JIN L,JIANG Z,XIA Y D,et al.Genome-wide DNA methylation changes in skeletal muscle between young and middle-aged pigs[J].BMC Genomics,2014,15:653.
[17] SU Y Y,FAN Z P,WU X S,et al.Genome-wide DNA methylation profile of developing deciduous tooth germ in miniature pigs[J].BMC Genomics,2016,17:134.
[18] WANG H F,WANG J Y,NING C,et al.Genomewide DNA methylation and transcriptome analyses reveal genes involved in immune responses of pig peripheral blood mononuclear cells to poly I:C[J].Sci Rep,2017,7(1):9709.
[19] KIM W,PARK H,SEO P S,et al.Characterization and functional inferences of a genome-wide DNA methylation profile in the loin(longissimus dorsi)muscle of swine[J].Asian-Australas J Anim Sci,2018,31(1):3-12.
[20] LI M Z,WU H L,LUO Z G,et al.An atlas of DNA methylomes in porcine adipose and muscle tissues[J].Nat Commun,2012,3:850.
[21] YANG Y L,LIANG G M,NIU G L,et al.Comparative analysis of DNA methylome and transcriptome of skeletal muscle in lean-,obese-,and mini-type pigs[J].Sci Rep,2017,7:39883,doi:10.1038/srep39883.
[22] KWAK W,KIM J N,KIM D,et al.Genome-wide DNA methylation profiles of small intestine and liver in fast-growing and slow-growing weaning piglets[J].Asian-Australas J Anim Sci,2014,27(11):1532-1539.
[23] SCHACHTSCHNEIDER K M,MADSEN O,PARK C,et al.Adult porcine genome-wide DNA methylation patterns support pigs as a biomedical model[J].BMC Genomics,2015,16:743.
[24] CHOI M,LEE J,LE M T,et al.Genome-wide analysis of DNA methylation in pigs using reduced representation bisulfite sequencing[J].DNA Res,2015,22(5):343-355.
[25] YUAN X L,GAO N,XING Y,et al.Profiling the genome-wide DNA methylation pattern of porcine ovaries using reduced representation bisulfite sequencing[J].Sci Rep,2016,6:22138,doi:10.1038/srep22138.
[26] HAO Y,CUI Y J,GU X H.Genome-wide DNA methylation profiles changes associated with constant heat stress in pigs as measured by bisulfite sequencing[J].Sci Rep,2016,6:27507.
[27]张恬.猪肌内脂肪全基因组甲基化差异分析及候选基因研究[D].北京:中国农业科学院,2016.ZHANG T.Study on genome-wide methylation differential analysis and candidate genes in pig IMF[D].Beijing:Chinese Academy of Agricultural Sciences,2016.(in Chinese)
[28] JIN L,MAO K,LI J,et al.Genome-wide profiling of gene expression and DNA methylation provides insight into low-altitude acclimation in Tibetan pigs[J].Gene,2018,642:522-532.
[29] ANTHONY K,CIRAK S,TORELLI S,et al.Dystrophin quantification and clinical correlations in Becker muscular dystrophy:Implications for clinical trials[J].Brain,2011,134(12):3547-3559.
[30] HEALD A,ANDERSON L V B,BUSHBY K M D,et al.Becker muscular dystrophy with onset after 60years[J].Neurology,1994,44(12):2388-2390.
[31] FERREIRO V,GILIBERTO F,MUNIZ G M N,et al.Asymptomatic Becker muscular dystrophy in a family with a multiexon deletion[J].Muscle Nerve,2009,39(2):239-243.
[32] MONACO A P,BERTELSON C J,LIECHTI-GALLATI S,et al.An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus[J].Genomics,1988,2(1):90-95.
[33] BUSHBY K M D,CLEGHORN N J,CURTIS A,et al.Identification of a mutation in the promoter region of the dystrophin gene in a patient with atypical Becker muscular dystrophy[J].Hum Genet,1991,88(2):195-199.
[34] HELLER K N,MONTGOMERY C L,SHONTZ K M,et al.Humanα7integrin gene(ITGA7)delivered by adeno-associated virus extends survival of severely affected dystrophin/utrophin-deficient mice[J].Hum Gene Ther,2015,26(10):647-656.
[35] QUIJANO-ROY S,MBIELEU B,BONNEMANN C G,et al.De novo LMNA mutations cause a new form of congenital muscular dystrophy[J].Ann Neurol,2008,64(2):177-186.
[36] BONNE G,DI BARLETTA M R,VARNOUS S,et al.Mutations in the gene encoding lamin A/C cause autosomal dominant Emery-Dreifuss muscular dystrophy[J].Nat Genet,1999,21(3):285-288.
[37] JIMENE Z-ESCRIG A,GOBERNADO I,GARCIAVILLANUEVA M,et al.Autosomal recessive EmeryDreifuss muscular dystrophy caused by a novel mutation(R225Q)in the lamin A/C gene identified by exome sequencing[J].Muscle Nerve,2012,45(4):605-610.
[38] DE SANDRE-GIOVANNOLI A,CHAOUCH M,KOZLOV S,et al.Homozygous defects in LMNA,encoding lamin A/C nuclear-envelope proteins,cause autosomal recessive axonal neuropathy in human(Charcot-Marie-Tooth disorder type 2)and mouse[J].Am J Hum Genet,2002,70(3):726-736.
[39] CAO H,HEGELE R A.Nuclear lamin A/C R482Q mutation in Canadian kindreds with Dunnigan-type familial partial lipodystrophy[J].Hum Mol Genet,2000,9(1):109-112.
[40] HERSHBERGER R E,SIEGFRIED J D.Update2011:Clinical and genetic issues in familial dilated cardiomyopathy[J].J Am Coll Cardiol,2011,57(16):1641-1649.
[41] LIU D,HOU J,HU X,et al.Neuronal chemorepellent slit2inhibits vascular smooth muscle cell migration by suppressing small GTPase rac1activation[J].Circ Res,2006,98(4):480-489.
[42] QU X H,WEI H D,ZHAI Y,et al.Identification,characterization,and functional study of the two novel human members of the semaphorin gene family[J].J Biol Chem,2002,277(38):35574-35585.
[43] TOYOFUKU T,ZHANG H,KUMANOGOH A,et al.Dual roles of Sema6Din cardiac morphogenesis through region-specific association of its receptor,Plexin-A1,with off-track and vascular endothelial growth factor receptor type 2[J].Genes Dev,2004,18(4):435-447.