基于全基因组差异甲基化分析鉴别影响苏姜猪体重的候选基因

doi:10.16431/j.cnki.1671-7236.2021.08.021

摘要/Abstract

摘要： 本研究旨在分析不同体重苏姜猪的全基因组DNA甲基化差异，以期筛选出影响苏姜猪体重的差异甲基化基因（differentially methylated genes，DMGs）。利用全基因组重亚硫酸盐测序（whole genome bisulfite sequencing，WGBS）技术分析了3头180日龄高体重和3头180日龄低体重苏姜猪的全基因组DNA的甲基化程度、差异甲基化区域（differentially methylated regions，DMRs）和DMGs，对DMGs进行GO和KEGG分析，鉴别影响苏姜猪体重的候选基因。结果显示，苏姜猪全基因组范围内胞嘧啶（C）的平均甲基化率为4.1%，胞嘧啶（C）甲基化平均98.41%发生在CG序列上，CG序列环境下外显子、内含子和3'UTR的甲基化水平高于启动子和5'UTR。本研究共检测出1 657个DMRs和575个DMGs，8号染色体上DMRs分布最多，88.89%的DMRs的长度在500 bp以内，62.78%的DMRs分布在远端基因间区，98个DMGs显著富集于TOR信号的负调控、碳水化合物衍生物分解代谢过程、脂肪细胞因子信号通路、FoxO信号通路等53个GO条目和29个信号通路，鉴别到5个与苏姜猪体重相关的候选基因：瘦素受体（leptin receptor，LEPR）基因、肿瘤坏死因子受体相关因子6（tumor necrosis factor receptor associated factor 6，TRAF6）基因、生肌因子6（myogenic factor 6，MYF6）基因、钙依赖性分泌激活因子2（calcium dependent secretion activator 2，CADPS2）基因和表皮生长因子（epidermal growth factor，EGF）基因。本研究利用WGBS绘制了高、低体重组苏姜猪的全基因组DNA甲基化图谱，为深入研究苏姜猪体重差异的分子机制奠定了基础。

关键词: 苏姜猪; DNA甲基化; 全基因组重亚硫酸盐测序; 体重; 候选基因

Abstract: The study was aimed to analyze the difference of genome-wide DNA differential methylation in Sujiang pigs with different body weight, as to screen the differentially methylated genes (DMGs) that affect the body weight of Sujiang pigs.In the study, three 180-day-old Sujiang pigs with high body weight and three 180-day-old Sujiang pigs with low body weight were detected by the whole genome bisulfite sequencing (WGBS) method.The degree of genome-wide DNA methylation, differentially methylated regions (DMRs) and DMGs were analyzed.The candidate genes affecting the body weight of Sujiang pig were identified by GO and KEGG pathway enrichment analysis of DMGs.The results showed that average 4.1% cytosine (C) were methylated in the whole genome of Sujiang pigs.Average 98.41% methylcytosines occurred on CG sequences.The DNA methylation level of exon, intron and 3'UTR was higher than that of promoter and 5'UTR at CG sequence context.A total of 1 657 DMRs and 575 DMGs were detected in this study.The distribution of DMRs on chromosome 8 was the most, 88.89% of the DMRs were within the length of 500 bp, and 62.78% of the DMRs were distributed in the distal intergenic.98 DMGs were significantly enriched in 53 GO terms and 29 signal pathways, including negative regulation of TOR signaling, carbohydrate derivative catabolic process, adipocytokine signaling pathway and FoxO signaling pathway.Five candidate genes related to body weight of Sujiang pigs were identified:leptin receptor(LEPR), tumor necrosis factor receptor associated factor 6(TRAF6), myogenic factor 6(MYF6), calcium dependent secretion activator 2(CADPS2) and epidermal growth factor(EGF).In this study, the whole genome DNA methylation map of Sujiang pigs with high and low body weight was drawn by WGBS, which laid a foundation for the further study of the molecular mechanism of body weight difference in Sujiang pigs.

Key words: Sujiang pigs; DNA methylation; whole genome bisulfite sequencing; body weight; candidate gene

中图分类号:

S813.1

徐盼, 仲德, 马政, 刘林雨, 王怡晖, 赵旭庭. 基于全基因组差异甲基化分析鉴别影响苏姜猪体重的候选基因[J]. 中国畜牧兽医, 2021, 48(8): 2889-2900.

XU Pan, ZHONG De, MA Zheng, LIU Linyu, WANG Yihui, ZHAO Xuting. Identification of Candidate Genes for Body Weight in Sujiang Pigs Based on Genome-wide Differential Methylation Analysis[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 2889-2900.

参考文献

[1] JONES P A.Functions of DNA methylation:Islands,start sites,gene bodies and beyond[J].Nature Reviews Genetics,2012,13(7):484-492.
[2] LLUIS F,COSMA M P.Resetting epigenetic signatures to induce somatic cell reprogramming[J].Cellular and Molecular Life Sciences,2013,70(8):1413-1424.
[3] LAENOI W,UDDIN M J,CINAR M U,et al.Molecular characterization and methylation study of matrix gla protein in articular cartilage from pig with osteochondrosis[J].Gene,2010,459(1-2):24-31.
[4] LIU X,YANG J,ZHANG Q,et al.Regulation of DNA methylation on EEF1D and RPL8 expression in cattle[J].Genetica,2017,145(4-5):387-395.
[5] 吴贤锋.山羊STAT3和STAT5A基因SNPs,mRNA表达及甲基化对生产性能的影响[D].杨凌:西北农林科技大学,2015. WU X F.SNPs,mRNA expression and methylation of STAT3 and STAT5A genes and their influences on production traits in goats[D].Yangling:Northwest A&F University,2015.(in Chinese)
[6] XU P,NI L,TAO Y,et al.Genome-wide association study for growth and fatness traits in Chinese Sujiang pigs[J].Animal Genetics,2020,51(2):314-318.
[7] KRUEGER F,ANDREWS S R.Bismark:A flexible aligner and methylation caller for Bisulfite-Seq applications[J].Bioinformatics,2011,27(11):1571-1572.
[8] HAO F,CONNEELY K N,HAO W.A Bayesian hierarchical model to detect differentially methylated loci from single nucleotide resolution sequencing data[J].Nucleic Acids Research,2014,42(8):e69.
[9] WU H,XU T L,FENG H,et al.Detection of differentially methylated regions from whole-genome bisulfite sequencing data without replicates[J].Nucleic Acids Research,2015,43(21):e141.
[10] PARK Y,WU H.Differential methylation analysis for BS-seq data under general experimental design[J].Bioinformatics,2016,32(10):1446-1453.
[11] YU G C,WANG L G,HAN Y Y,et al.clusterProfiler:An R package for comparing biological themes among gene clusters[J].OMICS,2012,16(5):284-287.
[12] 蒋曹德,邓昌彦,熊远著.猪个体DNA甲基化百分差异与胴体性状的关系[J].农业生物技术学报,2005,13(2):179-185. JIANG C D,DENG C Y,XIONG Y Z.Relationship between individual DNA methylation percentage difference and carcass traits in pig[J].Journal of Agricultural Biotechnology,2005,13(2):179-185.(in Chinese)
[13] LI M,WU H,LUO Z,et al.An atlas of DNA methylomes in porcine adipose and muscle tissues[J].Nature Communications,2012,3:850.
[14] YANG Y,ZHOU R,MU Y,et al.Genome-wide analysis of DNA methylation in obese,lean,and miniature pig breeds[J].Scientific Reports,2016,6:30160.
[15] 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 Science,2016.(in Chinese)
[16] 郭添福,张志燕,陈冬,等.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[J].畜牧兽医学报,2018,49(11):2326-2339. GUO T F,ZHANG Z Y,CHEN D,et al.High resolution and single base genome-wide methylation variance analysis of muscle of Large White pigs with differnent sexes[J].Acta Veterinaria et Zootechnica Sinica,2018,49(11):2326-2339.(in Chinese)
[17] 刘会敬.中外猪种胚胎不同发育时期骨骼肌转录组和全基因组甲基化分析[D].武汉:华中农业大学,2018. LIU H J.Integration of DNA methylome and transcriptome analysis of prenatal skeletal muscles between Tongcheng and Yorkshire pigs[D].Wuhan:Huazhong Agricultural University,2018.(in Chinese)
[18] 张效川.发情期和间情期奶山羊卵巢全基因组甲基化差异分析[D].杨凌:西北农林科技大学,2020. ZHANG X C.The analysis on methylation differential of ovarian genome between oestrous and dioestrous phases of dairy goats[D].Yangling:Northwest A&F University,2020.(in Chinese)
[19] MEISSNER A,GNIRKE A,BELL G W,et al.Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis[J].Nucleic Acids Research,2005,33(18):5868-5877.
[20] CHOI M,LEE J,LE M T,et al.Genome-wide analysis of DNA methylation in pigs using reduced representation bisulfite sequencing[J].DNA Research,2015,22(5):343-355.
[21] 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].Scientific Reports,2016,6:22138.
[22] JACINTO F V,BALLESTAR E,ESTELLER M.Methyl-DNA immunoprecipitation (MeDIP):Hunting down the DNA methylome[J].Biotechniques,2008,44(1):35,37,39 passim.
[23] 董辉.基于MeDIP-Seq研究皖南花猪和大约克猪背最长肌基因差异甲基化[D].合肥:安徽农业大学,2015. DONG H.Study on differential methylation of genes in longissimus dorsi muscle of Wannanhua pig and Yorkshire pig based on MeDIP-Seq technology[D].Hefei:Anhui Agricultural University,2015.(in Chinese)
[24] CLEMENT K,VAISSE C,LAHLOU N,et al.A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction[J].Nature,1998,392(6674):398-401.
[25] MUNOZ G,OVILO C,SILIO L,et al.Single- and joint-population analyses of two experimental pig crosses to confirm quantitative trait loci on Sus scrofa chromosome 6 and leptin receptor effects on fatness and growth traits[J].Journal of Animal Science,2009,87(2):459-468.
[26] PEREZ-MONTARELO D,FERNANDEZ A,FOLCH J M,et al.Joint effects of porcine leptin and leptin receptor polymorphisms on productivity and quality traits[J].Animal Genetics,2012,43(6):805-809.
[27] ASAGIRI M,TAKAYANAGI H.The molecular understanding of osteoclast differentiation[J].Bone,2007,40(2):251-264.
[28] ZHANG C Y,WANG Z,BRUCE H L,et al.Associations between single nucleotide polymorphisms in 33 candidate genes and meat quality traits in commercial pigs[J].Animal Genetics,2014,45(4):508-516.
[29] DO D N,STRATHE A B,OSTERSEN T,et al.Genome-wide association study reveals genetic architecture of eating behavior in pigs and its implications for humans obesity by comparative mapping[J].PLoS One,2013,8(8):e71509.
[30] WANG X,ZHENG F,LIU O S,et al.Epidermal growth factor can optimize a serum-free culture system for bone marrow stem cell proliferation in a miniature pig model[J].In Vitro Cellular & Developmental Biology-Animal,2013,49(10):815-825.