Epigenome-wide DNA methylation analysis reveals differentially methylation patterns in skeletal muscle between Chinese Chenghua and Qingyu pigs

WANG Kai ,WU Ping-xian ,WANG Shu-jie ,Jl Xiang ,CHEN Dong ,JlANG An-anXlAO Wei-hangJlANG Yan-zhi,ZHU LiZENG Yang-shuang,XU Xu,QlU Xiao-tian,Ll Ming-zhouLl Xue-weiTANG Guo-qing

1 Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province,Sichuan Agricultural University,Chengdu 611130,P.R.China

2 College of Life Science,Sichuan Agricultural University,Ya’an 625014,P.R.China

3 Sichuan Animal Husbandry Station,Chengdu 610041,P.R.China

4 National Animal Husbandry Service,Beijing 100125,P.R.China

5 Chongqing Academy of Animal Science,Chongqing 402460,P.R.China

Abstract Chenghua (CH) pig and Qingyu (QY) pig are typical Chinese native fatty breeds. CH pig is mainly distributed in Chengdu Plain,while QY pig is widely distributed throughout the mountain areas around the Sichuan Basin. There are significant differences in their phenotypic traits,including body image,growth performance,and meat quality. This study compared several meat quality traits of CH and QY pigs and conducted a genome-wide DNA methylation analysis using reduced representation bisulfite sequencing (RRBS). It was observed that the pH at 45 min (pH45min,P=5.22e-13),lightness at 45 min (L＊45min,P=4.85e-5),and lightness at 24 h (L＊24h,P=3.57e-5) of CH pigs were higher than those of QY pigs. We detected 10 699 differentially methylated cytosines (DMCs) and 2 760 differentially methylated genes (DMGs) associated with these DMCs. Functional analysis showed that these DMGs were mainly enriched in the AMPK signaling pathway,Type II diabetes mellitus,Insulin signaling pathway,mTOR signaling pathway,and Insulin resistance. Furthermore,15 DMGs were associated with fat metabolism (ACACA,CAB39,CRADD,CRTC2,FASN,and GCK),muscle development (HK2,IKBKB,MTOR,PIK3CD,PPARGC1A,and RPTOR),or meat quality traits (PCK1,PRKAG2,and SLC2A4). The findings may help to understand further the epigenetic regulation mechanisms of meat quality traits in pigs and provide new basic data for the study of local pigs.

Keywords:DNA methylation,reduced representation bisulfite sequencing (RRBS),Chenghua pig,Qingyu pig

1.lntroduction

Pigs are an important food source and a medical model for humans (Schooket al.2005). Chenghua (CH) pig and Qingyu (QY) pig are typical Chinese native fatty breeds with good meat quality performance. CH pig is mainly distributed in Chengdu Plain,while QY pig is widely distributed throughout the mountain areas around the Sichuan Basin (Megenset al.2008). Under the different living conditions for a long time,there are considerable differences between CH and QY pigs in the meat quality traits,including pH,meat color,and drip loss (DL),which suggests genetic and epigenetic differences between CH and QY pigs’ meat quality traits. Besides,the slow growth rate,low slaughter rate,and low meat yield of these two breeds need to be solved to increase their pork production on a large scale. Some studies have reported the genetic architecture of meat traits in CH and QY pigs (Wuet al.2020),but the epigenetic regulation mechanisms of meat traits are still unclear.

The yield and quality of pork are closely related to skeletal muscle development. Muscle growth and development are affected not only by genotype but also by epigenetic regulatory mechanisms,including DNA methylation. Although the genetic mechanisms,signaling pathways,and transcriptional regulation of muscle growth and development have been extensively studied,the epigenetic mechanisms associated with muscle development remain unclear.

DNA methylation is an essential genetic modification in mammals that regulates many key biological processes(Smith and Meissner 2013),including gene transcription,genomic imprinting,tissue differentiation,and phenotypic variation. Genome-wide DNA methylation analysis of skeletal muscle and adipose tissue of three pig breeds revealed differences in methylation among different breeds and tissues (Liet al.2012). Genome-wide DNA methylation analysis of skeletal muscle of pigs at different ages suggested that DNA methylation plays a key role in protein degradation associated with muscle function (Jinet al.2014). Moreover,DNA methylation has been shown to play an important role in muscle development in other domestic animals,including cattle (Fanget al.2017),sheep (Fanet al.2020),and ducks (Jiet al.2019).

CH and QY pig are typical Chinese native fatty breeds.However,there is still a great difference between them in the meat quality traits. DNA methylation plays an important role in muscle development and meat quality.Therefore,this study hypothesized that CH and QY pigs would have epigenetic differences that reflect their specific phenotypic characteristics. This study aimed to investigate the epigenetic differences underlying the meat quality traits in skeletal muscle between CH and QY pigs by epigenome-wide DNA methylation analysis using reduced representation bisulfite sequencing (RRBS) data and identify key genes related to these differences of meat quality traits. It systemically studied the whole-genome DNA methylation patterns of CH and QY pigs,which provides new insights into a better understanding of the epigenetic regulation mechanisms of meat quality traits and enriches the epigenetic research of the two local pig breeds.

2.Materials and methods

2.1.Animals and DNA samples

Fifty healthy pigs were used in this study,including CH pigs(n=20) and QY pigs (n=30). The population ratio between CH and QY pigs depends on the actual conditions of the farm. These pigs were maintained in a similar environment to avoid the effects of other confounders. There are 10 males and 10 females in the CH pigs,and 14 males and 16 females in the QY pigs. The pigs with an initial weight of about 30 kg were group-housed in cement-floor pens.The pigs were fed twice daily using the same diet,withad libitumaccess to water,using nipple drinkers. The diets,based on corn and soybean meal,were formulated with crude protein,trace mineral and vitamin concentrations that met or exceeded the National Research Council (NRC 1998) recommendations for different growth phases (Luoet al.2021). The pigs were slaughtered at a commercial slaughterhouse when they reached the slaughter weight of 105 kg. After slaughter,muscle pH values were measured at 45 min and 24 h postmortem using a portable pH meter (model 720A;Orion Research Inc.,Boston,MA,USA). Meat color,including lightness (L＊),redness (a＊),and yellowness (b＊),were assessed at 45 min and 24 h postmortem on thelongissimus dorsimuscle using a Minolta CR-300 Colorimeter (Minolta Camera,Osaka,Japan).Subsequently,tissue samples from muscle were collected from each breed for DNA isolation. Tissue samples were frozen in liquid nitrogen and stored at ?20°C until analysis.

2.2.Library construction

The genomic DNA was isolated from flash frozen muscular tissue. Then,the construction of RRBS libraries and paired-end sequencing using Illumina HisSeq Analyzer was performed at Novogene Technology Co.,Ltd.(Beijing,China). Raw sequencing data were processed by an Illumina base-calling pipeline. Clean reads were obtained from the raw data after removing reads containing adaptor sequences,unknown,or low-quality bases.

2.3.Data analysis

Clean reads were aligned to the pig reference genome(Sscrofa11.1,https://asia.ensembl.org/Sus_scrofa/Info/Index) using a default setting of Bismark v0.22.1 (Krueger and Andrews 2011). This progress includes three steps:genome preparation,alignment using Bowtie 2 v2.3.5.1(Langmead and Salzberg 2012),and methylation extractor.Bismark methylation extractor outputs read coverage and methylation percentage of detected methylated or unmethylated reads at one genomic position. The differential methylation analysis was done using the R package v4.0.3. CpG sites covering less than 10× were removed using the R package MethyKit v1.14.2 (Akalinet al.2012). Then,MethyKit was used to identify differentially methylated cytosines (DMCs) (q-value＜0.01,methylation difference≥0.25) based on the Bismark coverage file.Differentially methylated genes (DMGs) associated with DMCs were annotated using the Ensembl database. The R package genomation v1.20 (Fanget al.2017) was used to perform annotation of cytosine and guanine dinucleotide(CpG) sites and DMCs. The porcine reference genome was obtained from the NCBI (Suscrofa11.1). The porcine gene annotation was downloaded from the Ensembl (http://www.ensembl.org/,protein-coding genes). The region from the transcription start site (TSS) to the transcription termination site was regarded as the gene body region,and the promoter was defined as the 2-kb region upstream of the TSS.

2.4.Enrichment analysis

Genes annotated from the DMCs (q-value＜0.01,methylation difference≥0.25) were included in the enrichment analysis. The Gene Ontology (GO)enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were analyzed in DAVID v6.8 (Database for Annotation,Visualization and Integrated Discovery) (Huanget al.2009). Significant GO terms and KEGG pathways were selected after filtering withP＜0.01. R package ggplot2 v3.3.2 was used to visualize the significant GO terms and KEGG pathways for the DMGs associated with DMCs.

2.5.Statistical analyses

The data in meat quality traits of the two breeds were analyzed by Student’st-test using R v4.0.3. The results were presented as the mean±standard deviation (SD).The individual pig served as the experimental unit,and differences between groups were statistically significant ifP＜0.01.

3.Results

3.1.Summary of meat quality traits

The meat quality traits of CH and QY pigs were shown in Table 1. As Table 2 shown,the pH45min(P=5.22e-13),pH24h(P=1.78e-4),L＊45min(P=4.85e-5),b＊45min(P=8.53e-4)and L＊24h(P=3.57e-5) of the CH pigs were higher than those of QY pigs,while the a＊45min(P=5.66e-4) of the CH pigs were lower than those of QY pigs.

3.2.Summary of RRBS data

A total of 50 muscle samples from CH and QY pigs were used in this study. Approximately 687.90 Gb raw data were generated by RRBS (approximately 13.76 Gb raw data per individual),and approximately 508.20 Gb clean data were obtained (approximately 10.16 Gb clean data per individual). Moreover,the average bisulfite conversion efficiencies of CH and QY pigs were 99.55 and 99.26%,respectively. Besides,63.60 and 59.84% of the reads in CH and QY pigs were mapped to the porcine reference genome,respectively (Table 2).

3.3.DMCs and annotation

A total of 2 075 875 CpG sites were covered in these 50 pigs,and the number of DMCs identified by CHvs.QY comparison was 10 699 (Table 3). When considering the 2 075 875 CpG sites,the distribution of 2 075 875 CpG sites annotation within the promoter,exon,intron,and intergenic regions was 28.37,15.33,35.73,and 20.57%,respectively (Fig.1-A). Additionally,the percentages of 2 075 875 CpG sites annotated within CpG islands,CpG island shores,and other regions were 49.69,18.59,and 31.72%,respectively (Fig.2-A). However,of these 10 699 DMCs,3.02% were annotated within promoter regions,12.74% were annotated within exon regions,56.94% were annotated within intron regions,and 27.30%were annotated within intergenic regions (Fig.1-B). The distribution of the 10 699 annotated DMCs within CpG islands,CpG island shores,and other regions was 27.96,22.66,and 49.38%,respectively (Fig.2-B). Besides,of these DMCs,much fewer (34.03% in CHvs.QY comparison) were hypermethylated in CH pigs (Table 3).Among 18Sus scrofachromosomes (SSC),CpG sites occupied SSC6 (12.90%) mostly,and nearly no CpG sites occupied SSC16 and SSC10 with the percentages of 2.25 and 2.77%,respectively (Fig.3-A). Similarly,DMCs occupied SSC6 (9.89%) mostly,and nearly no DMCs occupied SSC16 and SSC10 with the percentages of 2.07 and 3.06%,respectively (Fig.3-B).

Fig.1 The distribution of cytosine and guanine dinucleotide (CpG) sites (A) and differentially methylated cytosines (DMCs) (B)annotated within gene in Chenghua (CH) pig vs.Qingyu (QY) pig.

Fig.2 The distribution of cytosine and guanine dinucleotide (CpG) sites (A) and differentially methylated cytosines (DMCs) (B)annotated within CpG island in Chenghua (CH) pig vs.Qingyu (QY) pig.

Fig.3 The distribution of cytosine and guanine dinucleotide (CpG) sites (A) and differentially methylated cytosines (DMCs) (B) on the pig genome. SSC,Sus scrofa chromosomes.

Table 1 Summary of meat quality traits of the two breeds1)

Table 2 Mapping results of reduced representation bisulfite sequencing (RRBS) data in the two breeds

3.4.DMGs associated with DMCs and their functional annotation

We annotated 2 760 DMGs associated with DMCs(Table 4;Appendix A). Fifteen genes associated with DMCs related to fat metabolism,muscle development,or meat quality traits were also reported by other studies(Table 5). Six genes,includingACACA(Stachowiak and Flisikowski 2019),CAB39(Qiet al.2019),CRADD(Ramoset al.2009),CRTC2(Xuet al.2015),FASN(Grzeset al.2016),andGCK(Xinget al.2019),were associated with fat metabolism and deposition,while geneHK2(Shenet al.2016),IKBKB(Bakkaret al.2008),MTOR(Wanget al.2018),PIK3CD(Duet al.2018),PPARGC1A(Leeet al.2012),andRPTOR(Suryawan and Davis 2011) were associated with muscle development.In addition,other three genesPCK1(Jianget al.2018),PRKAG2(Demeureet al.2004),andSLC2A4(Chenet al.2019) were found to be related to meat quality traits.

Table 3 Summary of cytosine and guanine dinucleotide (CpG) sites and differentially methylated genes (DMCs) identified by Chenghua pig (CH) vs.Qingyu pig (QY) group

Table 4 Summary of differentially methylated genes (DMGs)associated with differentially methylated cytosines (DMCs) in this study

Table 5 Comparisons between differentially methylated cytosines (DMCs) related genes of this study and identified genes for muscle development,fat metabolism or meat quality traits of other studies

Whereafter,2 760 DMGs were enriched in 17 GO terms (Appendices B and C) and 16 KEGG pathways(P＜0.01) (Appendices D and E). These GO terms included 5 GO terms of biological process,8 GO terms of cellular component and 4 GO terms of molecular function. The GO term (GO:0005737～cytoplasm) in thecellular component contained the genes mostly,that was 230 DMGs. The most significant GO term was GO:0005524～ATP binding (P=1.41e-8) containing 141 genes. And the most significant pathway was AMPK signaling pathway (P=2.05e-5) containing 24 genes namelyRPS6KB2,PIK3CD,PPARGC1A,PRKAG2,RPTOR,LOC100511937,ACACA,PIK3R5,CAB39,SLC2A4,MTOR,PCK1,FASN,CRTC2,PFKL,HNF4A,PFKFB3,CFTR,CAMKK1,EEF2K,PPP2R2B,ADRA1A,PPP2R1A,andSCD5.

Moreover,this study observed several interesting pathways associated with muscle development,glycometabolism,lipogenesis,and that contained AMPK signaling pathway (P=2.05e-5),Type II diabetes mellitus(P=2.39e-4),Insulin signaling pathway (P=3.48e-4),mTOR signaling pathway (P=1.23e-3),and Insulin resistance (P=1.97e-3).

4.Discussion

This study performed a genome-wide DNA methylation analysis between CH and QY pigs using RRBS. It then identified 10 699 DMCs and 2 760 DMGs associated with these DMCs. Functional annotation analysis found that these DMGs were mainly enriched in AMPK signaling pathway,Type II diabetes mellitus,Insulin signaling pathway,mTOR signaling pathway,and Insulin resistance.Finally,it highlighted that 15 DMGs were associated with fat metabolism,muscle development,and meat quality traits. These results comfirmed our hypothesis that CH and QY pigs would have epigenetic differences that reflect their specific phenotypic traits. These DMCs and DMGs contribute to the in-depth analysis of the molecular genetic mechanism of pigs,and will be given more attention in epigenetic analysis of complex traits for CH and QY pigs.

DNA methylation is a complex form of epigenetic regulation that plays an important role in gene expression and tissue development (Smith and Meissner 2013).Bisulfite sequencing is an ideal and practical technique for studying DNA methylation of different species and tissues (Fenget al.2010). Compared with whole-genome bisulfite sequencing (WGBS),RRBS is an effective method that can detect the DNA methylation level at each base position of the whole genome (Guet al.2011).Although WGBS can generate more data and genome coverage,it produces many reads in non-coding DNA regions and thus has a lower mapping rate than the RRBS approach (Doherty and Couldrey 2014). Overall,RRBS is a more suitable method for this study based on the cost of sequencing,data coverage,and experimental procedures. Therefore,RRBS was used in this study to explore DNA methylation patterns in different pig breeds.

This study identified 2 075 875 CpG sites and 10 699 DMCs. The percentages of DMCs annotation within promoter and exon region decreased dramatically,while DMCs annotation within intron and intergenic regions increased when comparing DMCs with CpG sites annotation within genic features. And similarly,the percentage of DMCs annotation within CpG island shores and other regions increased,whereas DMCs annotation within CpG islands decreased. Wang and Kadarmideen(2019) also found a similar trend in their study.

This study revealed the role of DMGs through functional annotation,and the results showed that DMGs between the two breeds may be related to muscle development,including the AMPK signaling pathway,Type II diabetes mellitus,Insulin signaling pathway,mTOR signaling pathway,and Insulin resistance. AMPK signaling pathway plays a key role in skeletal muscle metabolism control by regulating many downstream targets (Thomson 2018). In mammals,the mTOR signaling pathway plays a crucial role in regulating protein synthesis to maintain muscle protein turnover and nutrition (Ilhaet al.2018). Muscle is a major metabolic tissue,and thus metabolic pathways,including Type II diabetes mellitus,Insulin signaling pathway,and Insulin resistance,were enriched in this study. The results showed that DMGs associated with these metabolic processes show significant differences between CH and QY pigs,which suggested that they may be important for muscle metabolism. Pork pH is closely related to the products of muscle metabolism (Shenet al.2006;Luoet al.2017). DMGs involved in muscle metabolism were identified in CH and QY pigs,which were consistent with differences in pH between the two breeds.

Many DMGs between CH and QY pigs identified in this study are also related to lipid metabolism,fat deposition,and glucose metabolism. TheACACAgene catalyses the first committed step in the biosynthesis of longchain fatty acids by converting acetyl-CoA into malonyl-CoA (Penaet al.2019) and plays a key role in fatty acid composition in pigs (Gallardoet al.2009;Stachowiak and Flisikowski 2019). TheCAB39gene,a key regulator factor upstream of AMPK/mTOR pathway (Alessiet al.2006),plays a regulatory role in lipid production through the AMPK signaling pathway (Qiet al.2019). TheCRADDgene is significantly associated with fat deposition in pigs(Ramoset al.2009). In addition,genomic analyses in native chickens of China revealed that theCRADDgene with functions related to cell differentiation and apoptosis(Guet al.2020). TheCRTC2gene has been found to be involved in fat deposition in cattle (Xuet al.2015). And studies of Hanet al.(2015) reported that the CRTC2 controls hepatic lipid metabolism by regulating sterol regulatory element-binding protein 1 (SREBP1). TheFASNgene plays an important role inde novolipogenesis through catalyzing the reductive synthesis of long-chain fatty acids and is a potential marker for the accumulation of subcutaneous adipose tissue (Grzeset al.2016). Besides,the promoter and first exon methylation are significantly and negatively correlated withFASNgene expression (Longet al.2015). TheGCKgene is associated with fatty acid synthesis (Xinget al.2019). As the primary glucose sensor,GCK plays a crucial role in glucose metabolism (Sternisha and Miller 2019). In addition,GCK acts to maintain glucose homeostasis within pancreatic β-cells,while GCK participates in glycogen synthesis in the liver (Ferreet al.1996;Wilson 2003). Pork from CH has high L＊ and b＊,which may be related to differential methylation patterns of genes involved in fat deposition and fatty acid synthesis.

So far,several studies have reported the genomewide methylation of skeletal muscle in pigs. Choiet al.(2015) analysed the DNA methylome profile of five different tissues and a cell line originated from Landrace×Yorkshire crossbreed pigs. Ponsuksiliet al.(2019) performed genomewide DNA methylation analysis of muscle tissue using RRBS while this study analysed RRBS data further. This study used two different breeds (Chinese CH and QY pigs)with a larger sample size and focused on the differences in the methylation patterns of skeletal muscle between the two breeds. As a result,different DMCs and DMGs were detected. Yanget al.(2021) generated more paired-end reads with higher mapping rate. Furthermore,they profiled the dynamic DNA methylome and transcriptome landscape of skeletal muscle across 27 developmental stages.

5.Conclusion

This study performed epigenome-wide DNA methylation analysis of muscle tissue in Chinese CH and QY pigs using RRBS technology. It detected 10 699 DMCs in total.Of these 10 699 DMCs,3.02% were annotated within promoter regions,12.74% were annotated within exon regions,56.94% were annotated within intron regions,and 27.30% were annotated within intergenic regions.The distribution of the 10 699 annotated DMCs within CpG islands,CpG island shores,and other regions was 27.96,22.66,and 49.38%,respectively. Fifteen annotated genes were associated with fat metabolism (ACACA,CAB39,CRADD,CRTC2,FASN,andGCK),muscle development (HK2,IKBKB,MTOR,PIK3CD,PPARGC1A,andRPTOR),or meat quality traits (PCK1,PRKAG2,andSLC2A4). The findings provide new insights into the epigenetic regulation of pig muscle development,which will be helpful to understand muscle-related traits in pigs,and thus contribute to the development of the local pork industry and food processing industry.

Acknowledgements

The study was supported by the grants from the Sichuan Science and Technology Program,China (2020YFN0024),the China Agriculture Research System of MOF and MARA (CARS-35-01A),the National Key R&D Program of China (2018YFD0501204),the National Natural Science Foundation of China (C170102),and the Sichuan Innovation Team of Pig,China (sccxtd-2021-08). This study supported by the High-performance Computing Platform of Sichuan Agricultural University,China.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

All experimental procedures and sample collection were approved by the Institutional Animal Care and Use Committee of the College of Animal Science and Technology of Sichuan Agricultural University,China,under permit No.DKY-B20121403.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm