拷贝数变异全基因组关联分析及数量性状基因座定位联合鉴定猪体高性状候选基因

doi:10.11843/j.issn.0366-6964.2020.07.005

摘要/Abstract

摘要： 旨在利用全基因组拷贝数变异区域（copy number variation regions，CNVRs）关联分析以及全基因组数量性状基因座（quantitative trait locus，QTLs）定位联合筛选出影响猪体高性状的候选基因。本研究利用快速检测基因组拷贝数变异软件CNVcaller对本实验室构建的大白×民猪F2代资源群体的重测序数据进行拷贝数变异检测。利用混合线性模型（mixed-linear model，MLM）将性别和胎次作为固定效应对体高性状进行拷贝数变异全基因组关联分析（CNVR-GWAS）。采用软件R/qtl进行QTL分析，并使用置换检验（permutation test，PT）进行检验。将CNVR-GWAS与QTL结果进行联合注释，结合GO富集和KEGG通路分析，对影响猪体高的位点和基因进行挖掘。利用实时荧光定量PCR（qPCR）方法验证候选基因。结果表明，本群体在全基因组范围内共有3 099个CNVRs，其中有两个CNVRs与体高性状在全基因组范围内显著相关，分别位于7号染色体的25 358 001~26 696 400 bp处（CNVR1）和54 087 201~54 090 000 bp处（CNVR2）。在混合线性模型分析的结果中发现，CNVR1拷贝数增加（P<0.01）和CNVR2拷贝数缺失（P<0.01）对猪的体高性状具有显著影响。基因组显著水平可找到2个显著影响猪体高的QTLs，分别为BH-1和BH-2，其中BH-2对体高性状的影响较大。CNVR1和BH-2重叠区存在1个嗅觉受体基因OR12D3和18个未被注释的基因。qPCR验证OR12D3的拷贝数变异与利用混合线性模型统计推断出的结果一致。初步推测，OR12D3基因的拷贝数变异可能与猪体高性状相关。

关键词: 大白×民猪F2代猪群体, 拷贝数变异, 数量性状基因座, 体高, 全基因组关联分析

Abstract: The purpose of this study was to screen candidate genes affecting pig body height traits by using genome-wide copy number variation regions (CNVRs) association analysis and genome-wide quantitative trait loci (QTLs) mapping. CNVcaller software was used for rapid detection of genome copy number variations based on resequencing data of the Large White×Min pig F2 population constructed. Mixed-linear model (MLM) with gender and parity as fixed effects was used to perform copy number variation genome-wide association study (CNVR-GWAS) of body height traits. The software R/qtl was used for QTL analysis and the permutation test (PT) was used for inspection. The results of CNVR-GWAS and QTL were jointly annotated, combined with GO enrichment and KEGG pathway analysis, the loci and genes affecting pig body height were excavated. Candidate genes were verified by real-time quantitative polymerase chain reaction (qPCR) method. The results showed that there were 3 099 CNVRs in the whole genome of the population. Among them, two CNVRs were significantly associated with body height trait in the whole genome level, and they located at 25 358 001-26 696 400 bp (CNVR1) and 54 087 201-54 090 000 bp(CNVR2) on chromosome 7, respectively. The results of mixed linear model analysis showed that the increase of copy number of CNVR1 (P<0.01) and the loss of copy number of CNVR2 (P<0.01) had significant effects on pig body height. Two significant QTLs (BH-1 and BH-2, respectively)affecting pig body height were also found at genomic level, of which BH-2 had a greater impact on body height trait. There were one olfactory receptor gene OR12D3 and 18 unannotated genes in the overlap region of CNVR1 and BH-2. And the verification results of qPCR indicated that the copy number variation of OR12D3 was consistent with the results inferred by mixed linear model statistics. The copy number variation of the OR12D3 gene was inferred to be associated with pig body height trait.

Key words: Large White×Min pig F2 population, copy number variation, quantitative trait loci, body height, genome-wide association study

中图分类号:

S828.2

欧阳峰正, 王立刚, 岳静伟, 颜华, 张龙超, 侯欣华, 刘欣, 王立贤. 拷贝数变异全基因组关联分析及数量性状基因座定位联合鉴定猪体高性状候选基因[J]. 畜牧兽医学报, 2020, 51(7): 1515-1524.

OUYANG Fengzheng, WANG Ligang, YUE Jingwei, YAN Hua, ZHANG Longchao, HOU Xinhua, LIU Xin, WANG Lixian. Genome-wide Association Study of Copy Number Variations and Quantitative Trait Loci Mapping to Identify Candidate Genes for Body Height Trait in Pigs[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1515-1524.

参考文献 41

[1]	FEUK L,MARSHALL C R,WINTLE R F,et al.Structural variants:changing the landscape of chromosomes and design of disease studies[J].Hum Mol Genet,2006,15(Suppl1):R57-R66.
[2]	ZHANG F,GU W L,HURLES M E,et al.Copy number variation in human health,disease,and evolution[J].Annu Rev Genom Hum Genet,2009,10(1):451-481.
[3]	ZHANG L Z,JIA S G,YANG M J,et al.Detection of copy number variations and their effects in Chinese bulls[J].BMC Genomics, 2014,15(1):480.
[4]	XU L Y,HOU Y L,BICKHART D M,et al.Population-genetic properties of differentiated copy number variations in cattle[J].Sci Rep,2016,6(1):23161.
[5]	PINTO D,DARVISHI K,SHI X H,et al.Comprehensive assessment of array-based platforms and calling algorithms for detection of copy number variants[J].Nat Biotechnol,2011,29(6):512-520.
[6]	CURTIS C,LYNCH A G,DUNNING M J,et al.The pitfalls of platform comparison:DNA copy number array technologies assessed[J].BMC Genomics,2009,10(1):588.
[7]	HOU Y L,LIU G E,BICKHART D M,et al.Genomic characteristics of cattle copy number variations[J].BMC Genomics, 2011, 12(1):127.
[8]	XU L Y,COLE J B,BICKHART D M,et al.Genome wide CNV analysis reveals additional variants associated with milk production traits in Holsteins[J].BMC Genomics,2014,15(1):683.
[9]	DI GERLANDO R,SUTERA A M,MASTRANGELO S,et al.Genome-wide association study between CNVs and milk production traits in Valle del Belice sheep[J].PLoS One,2019,14(4):e0215204.
[10]	WANG L G,XU L Y,LIU X,et al.Copy number variation-based genome wide association study reveals additional variants contributing to meat quality in Swine[J].Sci Rep,2015,5:12535.
[11]	REVILLA M,PUIG-OLIVERAS A,CASTELLÓ A,et al.A global analysis of CNVs in swine using whole genome sequence data and association analysis with fatty acid composition and growth traits[J].PLoS One,2017,12(5):e0177014.
[12]	WRIGHT D,BOIJE H,MEADOWS J R S,et al.Copy number variation in intron 1 of SOX5 causes the Pea-comb phenotype in chickens[J].PLoS Genet,2009,5(6):e1000512.
[13]	VAN LAERE A S,NGUYEN M,BRAUNSCHWEIG M,et al.A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig[J].Nature,2003,425(6960):832-836.
[14]	ZHU M J,ZHAO S H.Candidate gene identification approach:progress and challenges[J].Int J Biol Sci,2007,3(7):420-427.
[15]	FUJII J,OTSU K,ZORZATO F,et al.Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia[J].Science,1991,253(5018):448-451.
[16]	STRATIL A,GELDERMANN H.Analysis of porcine candidate genes from selected QTL regions affecting production traits[J].Anim Sci Pap Rep,2004,22(1):123-125.
[17]	GRISART B,COPPIETERS W,FARNIR F,et al.Positional candidate cloning of a QTL in dairy cattle:identification of a missense mutation in the bovine DGAT1 gene with major effect on milk yield and composition[J].Genome Res,2002,12(2):222-231.
[18]	CLOP A,MARCQ F,TAKEDA H,et al.A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep[J].Nat Genet,2006,38(7):813-818.
[19]	HÉRAULT F,DAMON M,CHEREL P,et al.Combined GWAS and LDLA approaches to improve genome-wide quantitative trait loci detection affecting carcass and meat quality traits in pig[J].Meat Sci,2018,135:148-158.
[20]	FANG Z H,PAUSCH H.Multi-trait meta-analyses reveal 25 quantitative trait loci for economically important traits in Brown Swiss cattle[J].BMC Genomics,2019,20(1):695.
[21]	MEBRATIE W,REYER H,WIMMERS K,et al.Genome wide association study of body weight and feed efficiency traits in a commercial broiler chicken population,a re-visitation[J].Sci Rep,2019,9(1):922.
[22]	MARTIN P,PALHIÈRE I,MAROTEAU C,et al.Genome-wide association mapping for type and mammary health traits in French dairy goats identifies a pleiotropic region on chromosome 19 in the Saanen breed[J].J Dairy Sci,2018,101(6):5214-5226.
[23]	WANG X,ZHENG Z,CAI Y,et al.CNVcaller:highly efficient and widely applicable software for detecting copy number variations in large populations[J].Gigascience,2017,6(12):1-12.
[24]	BRADBURY P J,ZHANG Z,KROON D E,et al.TASSEL:software for association mapping of complex traits in diverse samples[J].Bioinformatics,2007,23(19):2633-2635.
[25]	BROMAN K W,WU H,SEN Ś,et al.R/qtl:QTL mapping in experimental crosses[J].Bioinformatics,2003,19(7):889-890.
[26]	BAUM L E,PETRIE T,SOULES G,et al.A maximization technique occurring in the statistical analysis of probabilistic functions of Markov chains[J].Ann Math Statist,1970,41(1):164-171.
[27]	LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitativePCR and the 2-ΔΔCT method[J]. Methods,2001,25(4):402-408.
[28]	叶健,胡晓湘,屈胜丽,等.大白猪体型性状的遗传参数及其与生产性能的关系[J].中国畜牧杂志,2016,52(19):6-8.YE J,HU X X,QU S L,et al.Estimation of genetic parameters of type traits and it's correlation with production traits in large white pigs[J].Chinese Journal of Animal Science,2016,52(19):6-8.(in Chinese)
[29]	王立刚,张跃博,颜华,等.拷贝数变异全基因组关联鉴定猪骨率性状候选基因[J].畜牧兽医学报,2019,50(11):2208-2214.WANG L G,ZHANG Y B,YAN H,et al.Identification of candidate genes for porcine bone rate traits by genome-wide association of copy number variation[J].Acta Veterinaria et Zootechnica Sinica,2019,50(11):2208-2214.(in Chinese)
[30]	GOSHU H A,WU X Y,CHU M,et al.Copy number variations of KLF6 modulate gene transcription and growth traits in Chinese Datong Yak (Bos Grunniens)[J].Animals,2018,8(9):145.
[31]	LIU M,LI B,HUANG Y Z,et al.Copy number variation of bovine MAPK10 modulates the transcriptional activity and affects growth traits[J].Live Sci,2016,194:44-50.
[32]	YANG M J,LV J Q,ZHANG L Z,et al.Association study and expression analysis of CYP4A11 gene copy number variation in Chinese cattle[J].Sci Rep,2017,7:46599.
[33]	ZHANG G M,ZHENG L,HE H,et al.Associations of GBP2 gene copy number variations with growth traits and transcriptional expression in Chinese cattle[J].Gene,2018,647:101-106.
[34]	RAN X Q,PAN H,HUANG S H,et al.Copy number variations of MTHFSD gene across pig breeds and its association with litter size traits in Chinese indigenous Xiang pig[J].J Anim Physiol Anim Nutr (Berl),2018,102(5):1320-1327.
[35]	JI J X,ZHOU L S,GUO Y M,et al.Genome-wide association study identifies 22 new loci for body dimension and body weight traits in a white Duroc×Erhualian F2 intercross population[J].Asian-Austral J Anim Sci,2017,30(8):1066-1073.
[36]	GONG H F,XIAO S J,LI W B,et al.Unravelling the genetic loci for growth and carcass traits in Chinese Bamaxiang pigs based on a 1.4 million SNP array[J].J Anim Breed Genet,2019,136(1):3-14.
[37]	MAO H R,GUO Y M,YANG G C,et al.A genome-wide scan for quantitative trait loci affecting limb bone lengths and areal bone mineral density of the distal femur in a White Duroc×Erhualian F 2 population[J].BMC Genetics,2008,9(1):63.
[38]	MAGALHAES A F B,DE CAMARGO G M F,FERNANDES G A,et al.Genome-wide association study of meat quality traits in Nellore cattle[J].PLoS One,2016,11(6):e0157845.
[39]	OLIVIERI B F,MERCADANTE M E Z,CYRILLO J N D S G,et al.Genomic regions associated with feed efficiency indicator traits in an experimental Nellore cattle population[J].PLoS One,2016,11(10):e0164390.
[40]	WANG X G,CAO X K,WEN Y F,et al.Associations of ORMDL1 gene copy number variations with growth traits in four Chinese sheep breeds[J].Arch Anim Breed,2019,62(2):571-578.
[41]	NICOLAE D,COX N J,LESTER L A,et al.Fine mapping and positional candidate studies identify HLA-G as an asthma susceptibility gene on chromosome 6p21[J].Am J Hum Genet,2005,76(2):349-357.