利用犬170 K高密度SNP芯片检测16个中国地方犬种全基因组拷贝数变异
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摘要
旨在解析中国地方犬全基因组拷贝数变异(CNV)分布情况,为分析CNV对犬表型变异的影响提供前期基础。本研究采集了16个来自中国不同地方犬品种和9头罗威纳犬共计185头犬的血液样品,使用血液DNA提取试剂盒提取DNA,利用犬170KSNP芯片和PennCNV软件对中国地方犬进行全基因组范围内的CNV分析。参照Illumina芯片标准操作流程进行芯片扫描和基因分型。将质控后的SNP参考PennCNV软件使用说明书,设定分析参数进行CNV检测分析。从分析结果中随机挑选8个CNV区间(CNVRs)使用实时定量PCR进行验证。利用BioMart以及DAVID软件进行基因和功能注释分析。结果表明,在185个个体中共发现了477个CNV,这些CNV随机分布在38条常染色体上,合并后可以得到220个CNVR,总长度约占犬整个基因组序列的1.25%,平均长度为142.24kb。在220个CNVR中,115个为缺失,74个为重复,31个为缺失/重复CNVR。此外,笔者发现53个CNVR为潜在的中国地方犬品种特异性CNVR。在基因和功能注释中,本研究共发现162个基因位于所检测到的CNVR内,这些基因主要富集的功能类别是嗅觉受体活动,嗅觉的感知,对化学刺激物的感知,感官知觉和神经系统过程。这些结果解析了中国地方犬基因组结构变异分布情况,将有助于进一步研究CNV与犬表型变异的关联性。
The main purpose of this study was to decipher the genomic copy number variation(CNV)of Chinese indigenous dog breeds and provide the basic information for elucidating the effect of CNV on canine phenotypic variation.In this study,we collected the blood samples of176 dogs from 16 different Chinese indigenous breeds and 9Rottweiler dogs.DNA was extracted by blood DNA extraction kit.All experimental dogs were genotyped using Illumina CanineHD170 KSNP chip according to the manufacturer's instructions.The CNV detection covering genome-wide was conducted after quality control according to the protocol of PennCNV software.We randomly selected 8CNVRs for validation by real-time quantitative PCR.And the genes andtheir functional annotations were performed with BioMart and DAVID.We totally found 477 CNVs in 185 individuals.These CNVs were randomly distributed on 38 autosomes.We identified220 CNVRs by merging the 477 CNVs.These CNVRs occupied about 1.25% of dog whole-genome sequence,their average length was 142.24 kb.Among the 220 CNVRs,115were deletion,74 were duplication and 31 were deletion/duplication.Furthermore,53 CNVRs observed were specifically identified in Chinese indigenous breeds.We found that 162 genes were located within the CNVRs detected in this study.These CNV-genes were enriched in function terms of olfactory receptor activity,sensory perception of smell,sensory perception of chemical stimulus,sensory perception and neurological system process.The present study illustrate the distribution of CNVs in the genome of Chinese indigenous dogs,and will benefit for further research on the relationship between CNV and canine phenotypic variation.
The main purpose of this study was to decipher the genomic copy number variation(CNV)of Chinese indigenous dog breeds and provide the basic information for elucidating the effect of CNV on canine phenotypic variation.In this study,we collected the blood samples of176 dogs from 16 different Chinese indigenous breeds and 9Rottweiler dogs.DNA was extracted by blood DNA extraction kit.All experimental dogs were genotyped using Illumina CanineHD170 KSNP chip according to the manufacturer's instructions.The CNV detection covering genome-wide was conducted after quality control according to the protocol of PennCNV software.We randomly selected 8CNVRs for validation by real-time quantitative PCR.And the genes andtheir functional annotations were performed with BioMart and DAVID.We totally found 477 CNVs in 185 individuals.These CNVs were randomly distributed on 38 autosomes.We identified220 CNVRs by merging the 477 CNVs.These CNVRs occupied about 1.25% of dog whole-genome sequence,their average length was 142.24 kb.Among the 220 CNVRs,115were deletion,74 were duplication and 31 were deletion/duplication.Furthermore,53 CNVRs observed were specifically identified in Chinese indigenous breeds.We found that 162 genes were located within the CNVRs detected in this study.These CNV-genes were enriched in function terms of olfactory receptor activity,sensory perception of smell,sensory perception of chemical stimulus,sensory perception and neurological system process.The present study illustrate the distribution of CNVs in the genome of Chinese indigenous dogs,and will benefit for further research on the relationship between CNV and canine phenotypic variation.
引文
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[13]NORRIS B J,WHAN V A.A gene duplication affecting expression of the ovine ASIPgene is responsible for white and black sheep[J].Genome Res,2008,18(8):1282-1293.
[14]HILLBERTZ N H C S,ISAKSSON M,KARLSSON E K,et al.Duplication of FGF3,FGF4,FGF19and ORAOV1causes hair ridge and predisposition to dermoid sinus in Ridgeback dogs[J].Nat Genet,2007,39(11):1318-1320.
[15]PARKER H G,KUKEKOVA A V,AKEY D T,et al.Breed relationships facilitate fine-mapping studies:a 7.8-kb deletion cosegregates with Collie eye anomaly across multiple dog breeds[J].Genome Res,2007,17(11):1562-1571.
[16]OLSSON M,MEADOWS J R S,TRUVK,et al.A novel unstable duplication upstream of HAS2predisposes to a breed-defining skin phenotype and a periodic fever syndrome in Chinese Shar-Pei dogs[J].PLoS Genet,2011,7(3):e1001332.
[17]GILL J L,CAPPER D,VANBELLINGHEN J F,et al.Startle disease in Irish wolfhounds associated with a microdeletion in the glycine transporter GlyT2gene[J].Neurobiol Dis,2011,43(1):184-189.
[18]HANDSAKER R E,VAN DOREN V,BERMAN J R,et al.Large multiallelic copy number variations in humans[J].Nat Genet,2015,47(3):296-303.
[19]SUDMANT P H,MALLICK S,NELSON B J,et al.Global diversity,population stratification,and selection of human copy-number variation[J].Science,2015,349(6253):aab3761.
[20]ZARREI M,MACDONALD J R,MERICO D,et al.A copy number variation map of the human genome[J].Nat Rev Genet,2015,16(3):172-183.
[21]CHEN C Y,QIAO R M,WEI R X,et al.A comprehensive survey of copy number variation in 18diverse pig populations and identification of candidate copy number variable genes associated with complex traits[J].BMC Genomics,2012,13(1):733.
[22]RAMAYO-CALDAS Y,CASTELLA,PENA R N,et al.Copy number variation in the porcine genome inferred from a 60kSNP BeadChip[J].BMC Genomics,2010,11(1):593.
[23]GRAUBERT T A,CAHAN P,EDWIN D,et al.A high-resolution map of segmental DNA copy number variation in the mouse genome[J].PLoS Genet,2007,3(1):e3.
[24]FONTANESI L,MARTELLI P L,BERETTI F,et al.An initial comparative map of copy number variations in the goat(Capra hircus)genome[J].BMC Genomics,2010,11(1):639.
[25]LIU G E,HOU Y L,ZHU B,et al.Analysis of copy number variations among diverse cattle breeds[J].Genome Res,2010,20(5):693-703.
[26]BERGLUND J,NEVALAINEN E M,MOLIN A M,et al.Novel origins of copy number variation in the dog genome[J].Genome Biol,2012,13(8):R73.
[27]CHEN W K,SWARTZ J D,RUSH L J,et al.Mapping DNA structural variation in dogs[J].Genome Res,2009,19(3):500-509.
[28]NICHOLAS T J,BAKER C,EICHLER E E,et al.A high-resolution integrated map of copy number polymorphisms within and between breeds of the modern domesticated dog[J].BMC Genomics,2011,12(1):414.
[29]NICHOLAS T J,CHENG Z,VENTURA M,et al.The genomic architecture of segmental duplications and associated copy number variants in dogs[J].Genome Res,2009,19(3):491-499.
[30]MOLIN A M,BERGLUND J,WEBSTER M T,et al.Genome-wide copy number variant discovery in dogs using the CanineHD genotyping array[J].BMC Genomics,2014,15(1):210.
[31]WANG K,LI M Y,HADLEY D,et al.PennCNV:an integrated hidden Markov model designed for highresolution copy number variation detection in wholegenome SNP genotyping data[J].Genome Res,2007,17(11):1665-1674.
[32]PennCNV.http://penncnv.openbioinformatics.org/en/latest/.
[33]DISKIN S J,LI M Y,HOU C P,et al.Adjustment of genomic waves in signal intensities from whole-genome SNP genotyping platforms[J].Nucleic Acids Res,2008,36(19):e126.
[34]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the Method[J].Methods,2001,25(4):402-408.
[35]KORESSAAR T,REMM M.Enhancements and modifications of primer design program Primer3[J].Bioinformatics,2007,23(10):1289-1291.
[36]Ensembl BioMart.www.biomart.org/.
[37]National Institute of Allergy and Infectious Diseases(NIAID),NIH.DAVID bioinformatics resources 6.8[EB/OL].https://david.ncifcrf.gov/.
[38]吴梅花.中国纯种犬资源与保护[J].养犬,2010(4):43-44.WU M H.The resource and protection of Chinese purebred dogs[J].Dog Breeding,2010(4):43-44.(in Chinese)
[39]ASAHINA R,MAEDA S.A review of the roles of keratinocyte-derived cytokines and chemokines in the pathogenesis of atopic dermatitis in humans and dogs[J].Vet Dermatol,2017,28(1):16-e5.
[40]SHORT A D,BOAG A,CATCHPOLE B,et al.A candidate gene analysis of canine hypoadrenocorticism in 3dog breeds[J].J Hered,2013,104(6):807-820.
[41]ARENDT M,CAIRNS K M,BALLARD J W O,et al.Diet adaptation in dog reflects spread of prehistoric agriculture[J].Heredity,2016,117(5):301-306.
[42]ARENDT M,FALL T,LINDBLAD-TOH K,et al.Amylase activity is associated with AMY2Bcopy numbers in dog:implications for dog domestication,diet and diabetes[J].Anim Genet,2014,45(5):716-722.
[43]AXELSSON E,RATNAKUMAR A,ARENDT M L,et al.The genomic signature of dog domestication reveals adaptation to a starch-rich diet[J].Nature,2013,495(7441):360-364.
[2]REDON R,ISHIKAWA S,FITCH K R,et al.Global variation in copy number in the human genome[J].Nature,2006,444(7118):444-454.
[3]ALKAN C,COE B P,EICHLER E E.Genome structural variation discovery and genotyping[J].Nat Rev Genet,2011,12(5):363-376.
[4]杜仁骞,金力,张锋.基因组拷贝数变异及其突变机理与人类疾病[J].遗传,2011,33(8):857-869.DU R Q,JIN L,ZHANG F.Copy number variations in the human genome:their mutational mechanisms and roles in disease[J].Hereditas,2011,33(8):857-869.(in Chinese)
[5]SCHASCHL H,AITMAN T J,VYSE T J.Copy number variation in the human genome and its implication in autoimmunity[J].Clin Exp Immunol,2009,156(1):12-16.
[6]SHA B Y,YANG T L,ZHAO L J,et al.Genomewide association study suggested copy number variation may be associated with body mass index in the Chinese population[J].J Hum Genet,2009,54(4):199-202.
[7]PAGNAMENTA A T,WING K,AKHA E S,et al.A 15q13.3microdeletion segregating with autism[J].Eur J Hum Genet,2009,17(5):687-692.
[8]TAM G W C,REDON R,CARTER N P,et al.The role of DNA copy number variation in schizophrenia[J].Biol Psychiatry,2009,66(11):1005-1012.
[9]JOHANSSON A,PIELBERG G,ANDERSSON L,et al.Polymorphism at the porcine Dominant white/KITlocus influence coat colour and peripheral blood cell measures[J].Anim Genet,2005,36(4):288-296.
[10]PIELBERG G,OLSSON C,SYVNEN A C,et al.Unexpectedly high allelic diversity at the KITlocus causing dominant white color in the domestic pig[J].Genetics,2002,160(1):305-311.
[11]WRIGHT D,BOIJE H,MEADOWS J R S,et al.Copy number variation in intron 1of SOX5causes the Pea-comb phenotype in chickens[J].PLoS Genet,2009,5(6):e1000512.
[12]PIELBERG G R,GOLOVKO A,SUNDSTRM E,et al.Acis-acting regulatory mutation causes premature hair graying and susceptibility to melanoma in the horse[J].Nat Genet,2008,40(8):1004-1009.
[13]NORRIS B J,WHAN V A.A gene duplication affecting expression of the ovine ASIPgene is responsible for white and black sheep[J].Genome Res,2008,18(8):1282-1293.
[14]HILLBERTZ N H C S,ISAKSSON M,KARLSSON E K,et al.Duplication of FGF3,FGF4,FGF19and ORAOV1causes hair ridge and predisposition to dermoid sinus in Ridgeback dogs[J].Nat Genet,2007,39(11):1318-1320.
[15]PARKER H G,KUKEKOVA A V,AKEY D T,et al.Breed relationships facilitate fine-mapping studies:a 7.8-kb deletion cosegregates with Collie eye anomaly across multiple dog breeds[J].Genome Res,2007,17(11):1562-1571.
[16]OLSSON M,MEADOWS J R S,TRUVK,et al.A novel unstable duplication upstream of HAS2predisposes to a breed-defining skin phenotype and a periodic fever syndrome in Chinese Shar-Pei dogs[J].PLoS Genet,2011,7(3):e1001332.
[17]GILL J L,CAPPER D,VANBELLINGHEN J F,et al.Startle disease in Irish wolfhounds associated with a microdeletion in the glycine transporter GlyT2gene[J].Neurobiol Dis,2011,43(1):184-189.
[18]HANDSAKER R E,VAN DOREN V,BERMAN J R,et al.Large multiallelic copy number variations in humans[J].Nat Genet,2015,47(3):296-303.
[19]SUDMANT P H,MALLICK S,NELSON B J,et al.Global diversity,population stratification,and selection of human copy-number variation[J].Science,2015,349(6253):aab3761.
[20]ZARREI M,MACDONALD J R,MERICO D,et al.A copy number variation map of the human genome[J].Nat Rev Genet,2015,16(3):172-183.
[21]CHEN C Y,QIAO R M,WEI R X,et al.A comprehensive survey of copy number variation in 18diverse pig populations and identification of candidate copy number variable genes associated with complex traits[J].BMC Genomics,2012,13(1):733.
[22]RAMAYO-CALDAS Y,CASTELLA,PENA R N,et al.Copy number variation in the porcine genome inferred from a 60kSNP BeadChip[J].BMC Genomics,2010,11(1):593.
[23]GRAUBERT T A,CAHAN P,EDWIN D,et al.A high-resolution map of segmental DNA copy number variation in the mouse genome[J].PLoS Genet,2007,3(1):e3.
[24]FONTANESI L,MARTELLI P L,BERETTI F,et al.An initial comparative map of copy number variations in the goat(Capra hircus)genome[J].BMC Genomics,2010,11(1):639.
[25]LIU G E,HOU Y L,ZHU B,et al.Analysis of copy number variations among diverse cattle breeds[J].Genome Res,2010,20(5):693-703.
[26]BERGLUND J,NEVALAINEN E M,MOLIN A M,et al.Novel origins of copy number variation in the dog genome[J].Genome Biol,2012,13(8):R73.
[27]CHEN W K,SWARTZ J D,RUSH L J,et al.Mapping DNA structural variation in dogs[J].Genome Res,2009,19(3):500-509.
[28]NICHOLAS T J,BAKER C,EICHLER E E,et al.A high-resolution integrated map of copy number polymorphisms within and between breeds of the modern domesticated dog[J].BMC Genomics,2011,12(1):414.
[29]NICHOLAS T J,CHENG Z,VENTURA M,et al.The genomic architecture of segmental duplications and associated copy number variants in dogs[J].Genome Res,2009,19(3):491-499.
[30]MOLIN A M,BERGLUND J,WEBSTER M T,et al.Genome-wide copy number variant discovery in dogs using the CanineHD genotyping array[J].BMC Genomics,2014,15(1):210.
[31]WANG K,LI M Y,HADLEY D,et al.PennCNV:an integrated hidden Markov model designed for highresolution copy number variation detection in wholegenome SNP genotyping data[J].Genome Res,2007,17(11):1665-1674.
[32]PennCNV.http://penncnv.openbioinformatics.org/en/latest/.
[33]DISKIN S J,LI M Y,HOU C P,et al.Adjustment of genomic waves in signal intensities from whole-genome SNP genotyping platforms[J].Nucleic Acids Res,2008,36(19):e126.
[34]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the Method[J].Methods,2001,25(4):402-408.
[35]KORESSAAR T,REMM M.Enhancements and modifications of primer design program Primer3[J].Bioinformatics,2007,23(10):1289-1291.
[36]Ensembl BioMart.www.biomart.org/.
[37]National Institute of Allergy and Infectious Diseases(NIAID),NIH.DAVID bioinformatics resources 6.8[EB/OL].https://david.ncifcrf.gov/.
[38]吴梅花.中国纯种犬资源与保护[J].养犬,2010(4):43-44.WU M H.The resource and protection of Chinese purebred dogs[J].Dog Breeding,2010(4):43-44.(in Chinese)
[39]ASAHINA R,MAEDA S.A review of the roles of keratinocyte-derived cytokines and chemokines in the pathogenesis of atopic dermatitis in humans and dogs[J].Vet Dermatol,2017,28(1):16-e5.
[40]SHORT A D,BOAG A,CATCHPOLE B,et al.A candidate gene analysis of canine hypoadrenocorticism in 3dog breeds[J].J Hered,2013,104(6):807-820.
[41]ARENDT M,CAIRNS K M,BALLARD J W O,et al.Diet adaptation in dog reflects spread of prehistoric agriculture[J].Heredity,2016,117(5):301-306.
[42]ARENDT M,FALL T,LINDBLAD-TOH K,et al.Amylase activity is associated with AMY2Bcopy numbers in dog:implications for dog domestication,diet and diabetes[J].Anim Genet,2014,45(5):716-722.
[43]AXELSSON E,RATNAKUMAR A,ARENDT M L,et al.The genomic signature of dog domestication reveals adaptation to a starch-rich diet[J].Nature,2013,495(7441):360-364.