Comparative muscle transcriptome of Mali and Hampshire breeds of pigs: a preliminary study

Abstract

Muscle development is an important priority of pig breeding programs. There is a considerable variation in muscularity between the breeds, but the regulation mechanisms of genes underlying myogenesis are still unclear. Transcriptome data from two breeds of pigs with divergent muscularity (Mali and Hampshire) were integrated with histology, immunofluorescence and meat yield to identify differences in myogenesis during the early growth phase. The muscle transcriptomics analysis revealed 17,721 common, 1413 and 1115 unique transcripts to Hampshire and Mali, respectively. This study identified 908 differentially expressed genes (p < 0.05; log₂FC > ±1) in the muscle samples, of which 550 were upregulated and 358 were downregulated in Hampshire pigs, indicating differences in physiological process related to muscle function and development. Expression of genes related to myoblast fusion (MYMK), skeletal muscle satellite cell proliferation (ANGPT1, CDON) and growth factors (HGF, IGF1, IGF2) were higher in Hampshire than Mali, even though transcript levels of several other myogenesis-related genes (MYF6, MYOG, MSTN) were similar. The number of fibers per fascicle and the expression of myogenic marker proteins (MYOD1, MYOG and PAX7) were more in Hampshire as compared to Mali breed of pig, supporting results of transcriptome studies. The results suggest that differences in muscularity between breeds could be related to the regulation of myoblast fusion and myogenic activities. The present study will help to identify genes that could be explored for their utility in the selection of animals with different muscularities.

Introduction

Growth and muscularity are considered to be the most important economic traits in meat animals that determine the productivity and profitability of the farming enterprise. Global pork production has increased about four-folds in past five decades and is expected to continue primarily due to increase in production capabilities and consumption concomitant to human population growth.¹ The increase in the pig production has resulted from intense breeding experiments to continuously improve growth, muscle and fertility traits. Previous studies have examined development of muscle mass, fiber characteristics (number, diameter and type), color, metabolism and marbling fat in farm animals.^2–4 Development of genomics tools such as next-generation sequencing and large data analysis have provided insights into muscle development in various breeds of pigs^5–10 that could be useful for selection and breeding.

Skeletal muscle tissue accounts for about 40% of the body mass and plays a major role in locomotion and energy metabolism.¹¹ Increasing skeletal muscle growth in food animals is important as it provides meat for direct consumption and provides extensive scope of value addition. In porcine species, most of the muscle fiber development occurs after completion of second wave of myogenesis toward the end of gestation period.¹² There is a third wave of myogenesis by third week after birth, during which the number of muscle fibers are fixed.¹³ During myogenesis, the progenitor cell determines to form myoblasts, exit cell cycle, and differentiate to myotubes, which fuses to form muscle fibers. Postnatally, an increase in the skeletal muscle mass in young and adult animals is achieved by hypertrophy of existing cells after the fusion of resident stem cells, the satellite cells. These cells can also undergo differentiation during muscle repair process following an injury.¹⁴ In the adult skeletal muscle, the satellite cells apparently remain quiescent and play a significant role in maintenance, repair and remodeling.¹⁵ The myogenesis involves orchestrated expression of several genes and transcription factors, including paired box protein (PAX3/7), sine oculis homeobox (SIX1/4), paired-like homeodomain (PITX), myogenic regulators (MYOD, MYF5, MYF6, MYOG), and myocyte enhancer family (MEF2A, MEF2B, MEF2C) in a favorable nutritional and epigenetic environment.^16,17 Recently, several new factors with key role in myogenesis have been identified,^18–20 whose role in farm animals is currently less known.

In India, there are 13 registered indigenous breeds of pigs; most of them are smaller in size as compared to exotic breeds such as Hampshire. Mali is a medium-sized breed of pig with its native tract in North Eastern region of India. The objective of the present study was to obtain insights into muscle development by comparing muscle transcriptome of two pig breeds differing in muscularity and growth rate (Mali and Hampshire). We combined transcriptome data with immunofluorescence, histology and carcass trait evaluation to understand postnatal myogenesis in these different breeds of pigs. The studies were conducted using semimembranosus muscle, used for preparation of ham and has been shown to be distinct from longissimus dorsi.²¹ In the present study, we used Sus scrofa 11.1 genome build, as compared to earlier studies,^21–24 a substantial update from the previous Sus scrofa 10.2 genome build²⁵ for analysis of transcriptome.

Materials and methods

Ethical statement

All the experiments were conducted as per the guidelines of the Committee for the purpose of control and supervision of experiment on animals (CPCSEA) and were approved by institutional animal ethics committee (NRCP/CPCSEA/1658/IAEC-33).

Animals and tissue collection

Muscle samples were collected from two different breeds of pigs (Hampshire, n = 15 and Mali, n = 12), aged 35–40 days, maintained in the institutional farm of the ICAR-National Research Center on Pig, Guwahati, Assam. All the pigs were apparently healthy and maintained under uniform feeding and management practices. Semimembranosus muscle samples were obtained using biopsies under local anesthesia with 2% lignocaine. The muscle tissues were cleaned in normal saline and part of tissue was transferred to Qiazol (Qiagen, Germany) for RNA isolation and rest was placed in 10% neutral buffered formaldehyde for histology and immunofluorescence studies. The growth data of animals were recorded from birth to 8 months of age. The mean age of slaughter of animals (n = 6, three from each breed) was 377.5 days and weight of major pork cuts (ham, loin, bacon, spare ribs, boston butt and picnic shoulder) was obtained for estimating carcass traits for the two pig breeds.

RNA isolation and RNA-sequencing

Total RNA was isolated using a commercial product Qiazol (Qiagen, Germany) from all the muscle samples for RNA-sequencing (RNA-seq) experiments. RNA from individual animal (n = 4, two from each breed) was processed for sequencing. The RNA quality and quantity was initially checked using a small volume spectrophotometer (Picodrop, UK) and subsequently using Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). From 1 μg of total RNA, the mRNA was selectively purified using oligo-dT beads (TruSeq RNA Sample Preparation Kit, Illumina). The mRNA was fragmented at 90 °C and reverse transcribed using random hexamers and Superscript II Reverse Transcriptase (Invitrogen, USA). Later, second strand cDNA was synthesized on the first strand template using RNaseH and DNA polymerase I and were purified further using AgencourtAmpure XP SPRI beads (Beckman Coulter, USA). Sequencing libraries were generated using a commercially available kit (TruSeq RNA Library Prep Kits for Illumina, NEB, USA) according to the manufacturer’s instructions, and index codes were added to attribute sequences to each sample. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina, USA). Finally, paired-end sequence reads of 100 bp were generated in the Illumina Hi-Seq 2500 platform. The RNA-seq data have been deposited in the NCBI Gene Expression Omnibus under the GEO accession number GSE184970 (GSM5602638, GSM5602639, GSM5602640, GSM5602641).

Data analysis and identification of differentially expressed transcripts

The raw data obtained in FASTQ format were subjected to quality check using FastQC program available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. The raw data was then processed to remove adaptor sequences, ambiguous reads (reads with unknown nucleotides ‘N’) and low-quality sequences (reads with <30 Phred score) using Trimmomatic utility.²⁶ The high-quality clean reads were then aligned against reference pig genome (Sus scrofa 11.1) available at ftp\\\ftp.ncbi.nlm.nih.gov\genomes\all\GCF\000\003\025\GCF_000003025.6_Sscrofa11.1\GCF_000003025.6_Sscrofa11.1_genomic.gff.gz using Hisat2.²⁷ The expression levels of mapped reads were normalized as fragments per kilobase of exon per million mapped fragments (FPKM), which was used to calculate the fold change (FC) values as FPKM_Hampshire/FPKM_Mali. Differentially expressed genes (DEGs) between Hampshire and Mali muscle samples were identified by Cuffdiff program of Cufflink package with a statistically significant Student’s t test p value threshold of 0.05 (p < 0.05) and log₂FC ≥1. The DEGs were generated from samples in duplicates by changing number of default samples to two in the CuffDiff program,²⁸ similar to previous studies describing analysis of limited number of RNA-seq samples.^29,30 The DEGs with log₂FC greater than +1 were considered upregulated, whereas those less than −1 were considered downregulated.²⁹

Functional profiling of DEGs and hub gene identification

The functional profiling of the DEGs for gene ontology (GO) enrichment, KEGG and reactome pathway analysis was conducted using g:Profiler online tool.³¹ Protein–protein interaction (PPI) network for upregulated and downregulated DEGs were performed using STRING database accessed from URL: https://string-db.org/.³² The PPI network was then exported into Cytoscape³³ and hub gens were identified along with their subnetwork using betweenness centrality with default settings in Cytohubba application.³⁴ The hub genes and the DEGs in the subnetwork were further explored by Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/tools.jsp) online tool.³⁵

Histology and immunofluorescence

The muscle samples from twenty animals (ten from each breed) were fixed in 10% neutral buffered formalin for 24 h and were processed for preparation of multiple sets of 8 μm thick sections as per standard histological procedures.³⁶ One set of sections was stained with hematoxylin and eosin and the other set was used for antigen retrieval and immunofluorescence studies.³⁷ Before antibody tagging, the sections were dewaxed and heat treated in a microwave in 0.1 M sodium citrate buffer (pH 6.0) for antigen retrieval. Slides after antigen retrieval were washed in phosphate-buffered saline (PBS, Invitrogen, USA) (pH 7.4), incubated for 1 h at room temperature with 5% bovine serum albumin (Sigma Aldrich, USA) in PBS without Ca²⁺ and Mg²⁺, followed by overnight incubation at 4 °C with primary antibody. Later, slides were washed three times with PBS and incubated with secondary antibody at room temperature for 1 h in dark. The slides were washed again for three times each in dark, followed by nuclear staining with H33342 (5 μg/mL, Invitrogen) and examined under a fluorescent microscope (TS100 Eclipse, Nikon, Japan). The percentages of cells expressing the specific marker (MYOD1, MYOG and PAX7) were calculated by dividing the number of cells with total number of cells (nucleus stained by H33342). ImageJ (RRID:SCR_003070) was used to count the muscle cells, analyze the diameter of muscle fibers and mark fluorescent microscopic images.³⁸ The details of antibodies and working dilutions used for immunofluorescence are as follows: Anti-MyoD1: monoclonal antibody (1:100 dilution; Sigma Aldrich, Cat # WH0004654M1; Lot #07289-1A7); Anti-Myog (c-terminal): (1:100 dilution; Sigma Aldrich, Cat# SAB2501587; Lot # 9955P1); Anti-Pax7: monoclonal antibody Pax7 (Sigma Aldrich, Cat# WH0005081M3; Lot # 12073-3C9); Myf-5: rabbit polyclonal antiserum (1:200 dilution; catalogue # sc-302, Santa Cruz Biotechnology); Anti-mouse IgG tagged with FITC produced in rabbit) (1:1000, Sigma Aldrich, Cat# F9137; Lot # 102M4801V), Anti-mouse IgG tagged with texas red produced in rabbit) (1:1000, Sigma Aldrich, Cat# SAB3701026; Lot # RI2542), Anti-goat IgG tagged with FITC produced in rabbit) (1:1000, Santa Cruz Biotechnology, Cat# SC-2024; Lot # L0513).

Validation of DEGs with quantitative real-time PCR

To validate DEGs, expression analysis of selected genes related to myogenesis was conducted using quantitative real-time polymerase chain reaction (PCR). Total RNA was isolated from muscle tissue using Qiazol Reagent (Qiagen, Germany), according to the manufacturer’s instructions. Total of 5 μg RNA was used for cDNA synthesis, using MMLV Reverse Transcriptase (Fermentas, Thermo Scientific, USA) with 1 μm of oligo-dT18 primer (Qiagen, Germany) in a 20 μL reaction. Subsequently, real-time PCR amplification of 1 μL of cDNA was performed using 0.5 μM specific primers (details of primers and methods used are shown in Supplementary Table 1) and a commercial real-time PCR master mix (SYBR Green JumpStart Taq Ready Mix, Sigma Aldrich, USA) in a real-time PCR machine (StepOne Plus, Applied Biosystems, USA). Beta actin and hypoxanthine-guanine phosphoribosyltransferase1 (HPRT1) were used as endogenous control genes based on a previous study.³⁹ All PCR experiments included a nontemplate negative control, and each sample was analyzed in triplicate. The real-time PCR data were normalized to mean of the data from beta actin and HPRT values. The relative expression levels of the target mRNAs were calculated using the 2^−ΔΔCt method.⁴⁰ The Pearson correlation coefficient (r) and the coefficient of determination (R²) between the relative gene expression data from quantitative reverse transcription PCR (RT-qPCR) and log₂FC values from RNA-seq experiment were determined using GraphPad PRISM v. 8.0 and XY correlation data graph were drawn.

Statistical analysis

Growth rate, carcass yield and RT-qPCR data analysis were conducted using SPSS 17.0 software (SPSS Inc., USA) by one-way ANOVA followed by Duncan multiple comparison tests. The graphs were drawn using MS-excel and GraphPad PRISM v. 8.0 (GraphPad Software, Inc., San Diego, CA, USA). Data are expressed as Mean ± SEM and p < 0.05 was considered to be statistically significant.

Results

Growth rate and carcass traits of animals

There was significant difference (p < 0.05) in growth rate between two breeds of pigs, with Hampshire reaching about twice the body weight of Mali (Fig. 1A, B). We examined the carcass yield at an average age of 377.5 days after humane slaughter of experimental pigs (n = 3/breed). Overall, yield of major pork cuts (ham, loin, bacon, spare ribs, Boston butt and picnic shoulder) was 16.69% more in Hampshire than in Mali pigs. Except bacon (1:0.93), yield of all other cuts was more in Hampshire than in Mali (ham – 1.24, loin – 1.04; spare ribs – 1.22, Boston butt – 1.09; picnic shoulder 1.40 times) (carcass yields of specific pork cuts are shown in Supplementary Table 2).

Figure 1.

Growth rate and muscle fiber characteristics of Hampshire and Mali breed of pigs. (A) Growth rate of pigs from Hampshire and Mali breed of pigs. (B) Grower animals of Hampshire and Mali. (C) Muscle fiber diameter and number of fibers per fascicle. (D) Negative correlation between Muscle fiber diameter and number of fibers per fascicle. Relatively number of fibers per fascicle was higher in Hampshire as compared to Mali breed.

Histology and immunofluorescence

The diameter of the muscle fiber in Mali breed was 1.20 times more (80.18 ± 0.87 µm) than that in Hampshire (67.05 ± 1.22 µm). On the other hand, the number of fibers per fascicle was more in Hampshire (1.25 times) than Mali breed of pig (Fig. 1C, D). Hampshire pigs had 4.74% greater muscle fiber area than Mali pigs. The percentage of cells expressing specific myogenic marker proteins (MYOD1, MYOG and PAX7) was calculated after immunofluorescence microscopy (Fig. 2). There were more cells expressing myogenesis-related proteins—MYOD1 (9.40 vs. 6.96%), MYOG (8.18 vs. 5.71%) and PAX7 (10.19 vs. 10.00%) in Hampshire muscle samples compared to Mali pigs.

Figure 2.

Expression of MYOD1, PAX7 and MYOG proteins in skeletal muscle from Hampshire and Mali breeds of pigs. Histology section (H&E) was stained with hematoxylin and eosin to show muscle fibers and fascicles. Respective myogenic protein is indicated in figure and nuclei was stained with H33342. Scale bar – 100 µm.

RNA-seq studies

The average number of filtered paired-end high-quality read used for mapping in Hampshire and Mali was 66.48 (96.06% mapping) and 65.89 million (95.46% mapping), respectively (Alignment statistics in Supplementary Table 3). These statistics indicated that our data were suitable for further analysis. The numbers of transcripts in Hampshire and Mali muscle samples were 19,134 and 18,836, respectively. A total of 17,721 transcripts were common to both the breeds, whereas 1413 and 1115 were unique to Hampshire and Mali, respectively (Fig. 3A). The highest percentage of transcripts (96.88 and 97.41% in Mali and Hampshire breeds, respectively,) had FPKM values below 100, of which approximately 25% transcripts had FPKM values below 0.1, suggesting advantages of next generation RNA sequencing in detecting transcripts with low abundance.⁴¹ The details of transcript abundance based on FPKM are presented in Supplementary Table 4.

Figure 3.

Distribution of muscle transcripts in Hampshire (HA) and Mali (MA) breed of pigs. (A) Distribution of transcripts in samples of Hampshire and Mali pigs. (B) The number of DEGs in Hampshire (HA) vs. Mali (MA) muscle comparison. (C) Heatmap shows differentially expressed genes in Hampshire (HA1 and HA2) and Mali (MA1 and MA2) muscle. (D) Volcano plot showing the distribution of fold changes in the expression of transcripts in muscle. The myogenic genes have been highlighted.

Differential gene expression analysis

This study identified 908 DEGs (p < 0.05) in the muscle samples, of which 550 were upregulated and 358 were downregulated in Hampshire pigs (Fig. 3B). The upregulated DEGs in Hampshire muscle had log₂FC ranging from 5.617 to 1.068, whereas the downregulated DEGs log₂FC ranged from −6.563 to −1.035. The top 10 known upregulated and downregulated DEGs based on significance level is listed in presented in Table 1. The details of differential gene expression analysis are provided in Supplementary Table 5). The hierarchical heatmap of highly significant DEGs (q < 0.05) as shown in Fig. 3C divides the samples into two groups, showing differences in physiological process related to muscle function and development between the two divergent breeds under study. The DEGs in Hampshire vs. Mali pigs muscle comparison (shown in Fig. 3D), were found to be associated with the different physiological process like muscle development, fatty acid synthesis and metabolism, cell growth and proliferation.

Table 1.

Top 10 known upregulated and downregulated DEGs in Hampshire vs. Mali pigs muscle sample comparison.

Gene ID	Log2(fold change)	q Value	Gene description
Upregulated DEGs
PHGDH	5.61774	0.0179329	Phosphoenolpyruvate carboxykinase 2, mitochondrial
PCK2	4.43046	0.043286	Radical S-adenosyl methionine domain containing 2
RSAD2	4.2402	0.017933	MX dynamin-like GTPase 2
MX1	3.89694	0.017933	Phosphoserine aminotransferase 1
PSAT1	3.86522	0.017933	Peptidase inhibitor 15
PI15	3.78884	0.036921	Relaxin family peptide receptor 1
RXFP1	3.77444	0.017933	HECT and RLD domain containing E3 ubiquitin protein ligase 5
HERC5	3.77239	0.017933	Guanylate binding protein 2
GBP2	3.75677	0.017933	Collagen-type VI alpha 6 chain
COL6A6	3.62707	0.017933	Insulin-like growth factor 2
Downregulated DEGs
TMEM51	−4.82468	0.017933	Transmembrane protein 51
NAT8L	−4.48566	0.048281	N-acetyltransferase 8 like
SLCO1A2	−4.44733	0.017933	Solute carrier organic anion transporter family member 1A2
SMPDL3A	−4.38001	0.017933	Sphingomyelin phosphodiesterase acid like 3A
GALNT15	−4.2444	0.017933	Polypeptide N-acetylgalactosaminyltransferase 15
ZDHHC9	−3.67753	0.043286	Zinc finger DHHC-type palmitoyltransferase 9
LOC110257572	−3.59367	0.036921	Uncharacterized
FBXO32	−3.55167	0.017933	F-box protein 32
SLC26A7	−3.45187	0.043286	Solute carrier family 26 member 7
MICAL2	−3.09338	0.017933	Microtubule associated monooxygenase, calponin and LIM domain containing 2

Muscle development

The myogenic DEGs MYMK encoding for myomaker, myoblast fusion factor, MSC encoding for musculin, ELN encoding for elastin, MYO1B encoding for myosin IB, LOC100736765 encoding for myosin heavy chain 6, LOC106505299 encoding for myosin heavy chain 7, CAV3 encoding for caveolin 3 and MYL3 encoding for myosin light chain 3 were upregulated, whereas MYBPH encoding for myosin binding protein H and MYLIP encoding for myosin regulatory light chain interacting protein were downregulated in Hampshire as compared to Mali. Interestingly, the expression of genes related to the regulation of collagen fibril organization (AEBP1 encoding for adipocyte enhancer-binding protein 1, COL1A2), actin cytoskeleton reorganization (ANTXR1, encoding for ANTXR cell adhesion molecule 1), collagen formation (COL6A6, COL3A1, COL1A2, COL1A1, COL15A1, COL5A2, COL5A1, COL21A1, ADAMTS2, COL8A1, LOXL2, COL14A1, COL4A5, PCOLCE, COL4A1, COL6A3, CTSS, COL6A2, COL5A3, PXDN, SERPINH1), extracellular matrix structural constituent (FBN3 encoding for fibrillin 3), protein binding (KCNE5 encoding for potassium voltage-gated channel subfamily E regulatory subunit 5, KCP encoding for kielin cysteine-rich BMP regulator), and developmental process (PLAC8 encoding for placenta associated 8) were upregulated in Hampshire, indicating their putative role in muscle development process.

Fatty acid synthesis and metabolism

The DEGs related to fatty acid biosynthesis and metabolic process like HACD1 encoding for 3-hydroxy-acyl-CoA dehydratase, AOAH encoding for acyloxy-acyl hydrolase, ADIPOQ adiponectin C1Q and collagen domain containing, AIG1 androgen induced 1, LPL lipoprotein lipase and PRKAR2B protein kinase cAMP-dependent type II regulatory subunit beta were upregulated in Hampshire indicating differential metabolism of fatty acid in two pig breeds. However, the DEG SCD encoding for stearoyl-CoA desaturase involved in synthesis of monounsaturated fatty acids was downregulated in Hampshire in comparison with Mali pigs.

Growth and cell proliferation

The expression of DEGs encoding for growth factors like, HGF encoding for hepatocyte growth factor, IGF1 encoding for insulin-like growth factor 1, IGF2 encoding for insulin-like growth factor 2, FGF6 encoding for fibroblast growth factor 6, PDGFD encoding for platelet-derived growth factor D, TGFBI encoding for transforming growth factor beta induced and their receptors PDGFRL, PDGFRA encoding for platelet-derived growth factor receptors, FGFR4, FGFRL1 encoding for fibroblast growth factor receptors, which are associated with skeletal muscle proliferation and growth were higher in Hampshire as compared to Mali. The growth factor binding proteins genes like, IGFBP5 encoding for insulin like growth factor binding protein 5 and LTBP1 encoding for latent transforming growth factor beta binding protein 1 were also significantly upregulated in Hampshire, indicating their key role in muscular growth. Moreover, the DEGs associated with positive regulation of cell population proliferation (PDGFD, PLAC8, PLBD1, CNTFR encoding for ciliary neurotrophic factor receptor), muscle cell cellular homeostasis and skeletal muscle tissue growth (CHRNA1 encoding for cholinergic receptor nicotinic alpha 1 subunit), regulation of skeletal muscle satellite cell proliferation (ANGPT1 encoding for angiopoietin 1 and CDON encoding for cell adhesion associated, oncogene regulated), positive regulation of fibroblast proliferation (E2F encoding for transcription factor 1 and PDGFRA), regulation of cell proliferation (STAT2 encoding for signal transducer and activator of transcription 2, SFRP2 encoding for secreted frizzled-related protein 2, PTK2B encoding for protein tyrosine kinase 2 beta) act in synergism with growth factors to regulate the overall muscle development and growth.

GO analysis

The functional analysis for GO which included enrichment analysis for biological process (BP), molecular function (MF) and cellular component (CC) revealed enrichment of genes possibly responsible for differences in muscle mass and growth in between Hampshire and Mali pigs. The upregulated DEGs were enriched in 208 GO terms (p_adj. <0.05) consisting of 173 BP, 17 MF and 18 CC terms, whereas downregulated DEGs were enriched in 42 GO terms (p_adj. <0.05) consisting of 28 BP, 4 MF and 10 CC terms. The details of GO analysis of upregulated and downregulated DEGs are presented in Supplementary Table 6. The upregulated DEGs were enriched in BPs like extracellular matrix (ECM) organization, anatomical structure development, developmental process, cell migration and adhesion, response to stimulus, collagen fibril organization, cell surface receptor signaling pathway and animal organ development. There MFs terms enriched in Hampshire primarily consisted of ECM structural constituent and binding with ECM, calcium, collagen, PDGF, heparin, fibronectin, metal ion, cation, proteoglycan and protein containing complex binding. The upregulated and enriched CC terms consisted of extracellular region, collagen trimer, cell surface, plasma membrane, collagen containing ECM and external encapsulating structure, indicating the role of ECM and collagen in higher muscularity of Hampshire pigs. Figure 4A shows the enriched GO terms of upregulated DEGs. The downregulated BPs in Hampshire pigs consisted of protein catabolic process, cellular catabolic process and autophagy whereas ubiquitin binding and peptidase activator activity were downregulated MFs. The CCs like proteasome complex, endopeptidase complex, proteosome regulatory complex, cytoplasm and proteasome accessory complex were downregulated in Hampshire, indicating the possible role of autophagy inhibition mechanism behind greater muscling in Hampshire pigs. Figure 4B shows the enriched GO terms in downregulated DEGs.

Figure 4.

Gene ontology of differentially expressed genes indicating differences in physiological process related to muscle function and development. (A) GO profile of upregulated DEGs in Hampshire vs. Mali comparison and (B) GO profile of downregulated DEGs in Hampshire vs. Mali comparison.

Pathway analysis

The KEGG and Reactome pathway analysis of upregulated and downregulated DEGs provided insights into various significantly enriched (p_adj. <0.05) cellular pathways possibly associated with the differences in the myogenic process between the two breeds. The details of pathway analysis of upregulated and downregulated DEGs are presented in Supplementary Table 7. Among these, the upregulated KEGG pathways consisted of protein digestion and absorption, PI3K-Akt signaling, ECM–receptor interaction, focal adhesion, complement and coagulation cascades, calcium signaling pathway and cell adhesion molecules. However, the upregulated Reactome pathways included ECM organization, ECM proteoglycans, assembly of collagen fibrils and other multimeric structures, collagen formation and degradation, integrin cell surface interactions, nonintegrin membrane–ECM interactions, elastic fiber formation, signaling by receptor tyrosine kinases and regulation of IGF transport and uptake by IGFBPs. The downregulated KEGG pathways consisted of proteasome and protein processing in endoplasmic reticulum, whereas Reactome pathways that were downregulated in Hampshire pigs comprised p53-independent G1/S DNA damage checkpoint, ubiquitin-dependent degradation of cyclin D, autodegradation of the E3 ubiquitin ligase COP1, regulation of apoptosis, hedgehog ligand biogenesis, negative regulation of NOTCH4 signaling, regulation of HMOX1 expression and activity, MAPK1/MAPK3 signaling and signaling by WNT and interleukins. The differential expression of these biological pathways most probably relates to the differences in muscularity and growth patterns in exotic Hampshire and indigenous Mali pigs. Figure 5 shows the enriched KEGG and Reactome pathways enriched in the upregulated and downregulated DEGs. The results of transcriptome analysis were validated through real-time PCR of selected genes, which showed similar trends as that of RNA-seq in the expression between Hampshire and Mali muscle samples (Fig. 6A). The relative gene expression values obtained from RT-qPCR were significantly correlated (p < 0.0001) with those obtained from RNA-seq (r = 0.9493) and exhibited a high coefficient of determination (R² = 0.9011; Fig. 6B).

Figure 5.

KEGG and REACTOME pathway analysis. (A) Pathway enrichment analysis of upregulated DEGs in Hampshire vs. Mali comparison and (B) Pathway enrichment analysis of downregulated DEGs in Hampshire vs. Mali comparison.

Figure 6.

Validation of transcriptome data using real time PCR in Hampshire and Mali muscles.

Protein–protein interaction and hub gene analysis

The PPI network of upregulated DEGs in STRING database resulted in a dense network of interconnected proteins, with 479 nodes and 1399 edges (Supplementary Fig. 1). The average node degree was 5.84 with average local clustering coefficient of 0.403 and PPI enrichment p < 1.0 × 10⁻⁶. However, the PPI network of downregulated DEGs (Supplementary Fig. 2) was less dense with 297 nodes, 406 edges, 2.73 average node degree and average local clustering coefficient of 0.371 with PPI enrichment p < 1.0 × 10⁻⁶. The hub genes regulating the upregulated PPI network (Fig. 7) consisted of FN1 (fibronectin 1), CD4, ITGB3 (integrin subunit beta 3), ACTC1 (actin alpha cardiac muscle 1) and PTPRC (protein tyrosine phosphatase receptor type C), whereas the hub genes of downregulated PPI network (Fig. 8) consisted of PSMD4 (proteasome 26S subunit ubiquitin receptor), SQSTM1 (sequestosome 1), PNO1 (partner Of NOB1 homolog), SSR1 (signal sequence receptor subunit 1) and UBC (ubiquitin C). These hub genes can be regarded as probable regulator of differential muscularity in the two pig breeds under study.

Figure 7.

PPI network analysis for upregulated hub genes. Interaction network showing relationship between hub genes and their subnetwork influencing muscle development. Hub genes are indicated in red color.

Figure 8.

PPI network analysis for downregulated hub genes. Interaction network showing relationship between hub genes and their subnetwork influencing muscle development. Hub genes are indicated in red color.

Discussion

To obtain insights into genetic differences in muscularity, we concurrently examined muscle transcriptome in two breeds of pigs at molecular level, histology and expression of key myogenic markers using immunofluorescence at cellular level and meat yield at animal level.

Muscle development in animals is influenced by diverse factors such as environment, breed, species and age.⁴² Myogenesis is a well-regulated event leading to differentiation of myogenic progenitors (satellite cells) to myoblast, then to a committed myocyte and subsequent formation of myofibers following the fusion of myotube developed from myocytes.^43–45 We selected semimembranosus, an external muscle, for the studies as it is one of the most important muscles contributing to weight of the ham, a primal cut of pork.

Broadly, the results of the present study regarding DEGs are in line with the earlier reports comparing muscle transcriptome of different breeds of pigs.^{10,22–24,46} We identified 550 upregulated and 358 downregulated DEGs in association with animals with differences in muscularity with significant FCs (p < 0.05) in Hampshire as compared to Mali, many of them not reported previously.^10,22,24,47 Even though the transcript levels of several myogenesis-related genes (MYF6, MYOG, MYMX, SIX1/4, LBX1, MEOX1, MEOX2, MSTN) were similar, significant differences (p < 0.05) in expression levels of MYMK gene, related to myoblast fusion and generation of large myotubes was higher in Hampshire than Mali. Myoblast fusion driven by the union of plasma membrane is involved in skeletal muscle regeneration and the process is largely associated with the regulation of skeletal muscle hypertrophy.⁴⁸ Hence, the expression of MYMK is essentially required for myoblast fusion, critical to myofibers development, which forms the basic cellular and functional units of skeletal muscles.⁴⁹ It has been reported that reduction in the fusion of myogenic cells in cattle^50,51 could be associated with less muscle mass.⁵² In the present study, higher number of fibers observed during histology of Hampshire muscle further supports the gene expression studies.

The biological processes and molecular functions related to muscle physiology were enriched in Hampshire pig, suggesting its role in the muscle development. The genes regulating skeletal muscle cell differentiation (SOX8, ATF3, HLF, COL6A6), muscle fiber development (RIPOR2, SHOX2) and skeletal muscle tissue development (MSC, CDON, CHRNA1, ELN) were upregulated in Hampshire indicating greater ability of this breed for acquiring more muscle mass. Moreover, the upregulation of genes regulating other muscle functionalities like muscle filament sliding (LOC100736765), striated muscle contraction (PTPRD, TMOD2, ARG2, CAV3, CHRNA1, MYL3) and cellular homeostasis (CHRNA1) along with the skeletal muscle satellite cell proliferation (ANGPT1, CDON) indicates that functionality of muscle fibers is differentially regulated in Hampshire and Mali. Also, the upregulation of ANXA1 and PTGFRN encoding for prostaglandin F2 receptor inhibitor, which regulates myoblast migration and fusion involved in skeletal muscle regeneration, indicates greater regenerative capacity of Hampshire muscles in comparison with Mali pigs.⁵³

Skeletal muscle formation, proliferation, differentiation and regeneration requires both growth factor and extracellular matrix components such as collagen, which aid as a scaffold for cell growth.⁴⁸ The upregulation of growth factor gene HGF, IGF1, IGF2, TGFBI and FGF6 in Hampshire pigs indicates differential regulation of muscle cell differentiation leading to greater muscularity in Hampshire breed in comparison to Mali pigs. These growth factors along with the DEGs RIPOR2 and SHOX2 associated with muscle fiber development can act synergistically in bringing greater muscle mass in Hampshire pigs. Unlike muscle development, significant differences in expression of few genes related to fatty acid metabolism were present between the two breeds of pigs. Fat deposition usually happens during 90–280 days in pigs⁵⁴ and animals were growers at the time of sampling, hence explicit differences large number of DEGs related to fatty acid metabolism were not anticipated, however, the DEGs HACD1, AOAH, AIG1, ADIPOQ, LPL and PRKAR2B may be related to difference in carcass quality between the two breeds. Higher ratio of LDHB:CS gene expression in Hampshire as compared to Mali muscle suggests an elevated glycolytic potential,⁵⁵ a dominant trait of Hampshire breed.⁵⁶

Pathway analysis and PPI network of DEGs revealed significant enrichment of several pathways associated with cellular function and metabolism in addition to muscle development. PI3-Akt signaling pathway, calcium signaling, syndecan interactions, ECM–receptor interaction and interferon signaling shown to regulate cell fate decisions,⁵⁷ myogenesis, connecting muscle fiber to the extracellular matrix and homeostasis⁵⁸ were upregulated. However, hedgehog signaling, which plays an important role in intercellular communication and MyoD transcription and activity, thus regulating myogenesis⁵⁹ were upregulated in Mali pigs. Significant enrichment of phosphoinositide 3-kinase PI3K-Akt signaling with diverse cellular effects⁶⁰ suggests difference in the muscle metabolism between Hampshire and Mali breeds of pig. Differences in immune-related pathway, especially related to interleukin-1 and interferon alpha-beta signaling were significantly enriched in the muscle of Hampshire as compared to Mali pigs. The present study confirms a previous report⁶¹ suggesting relationship between DEGs associated with metabolism and immune response pathways besides myogenesis. Cell adhesion plays a major role in myogenesis and satellite cell function.⁶² Enrichment of adhesion pathways associated with DEGs in Hampshire as compared to Mali, further supports the importance of cell adhesion in muscle development.

The phenotypic variations in the muscle development and growth in pigs are determined by genetics, epigenetic factors and environment. In the present study, all the animals were maintained at same environment with a similar feed regime. Hence, the observed variations in musculature between breeds could have been predominantly resulted from underlying genetic differences. Moreover, the hub analysis in this study revealed the regulatory roles of diverse pathways regulating muscle development, which have not been previously associated with muscle physiology. The role of postnatal myogenesis in the determination of fiber number and final meat yield in domestic animals needs further studies. The results of the study are influenced by the following considerations: (a) samples obtained from fast glycolytic semimembranosus muscle tissue with higher proportion of type IIB fibers^55,63 and there is a considerable difference between transcriptome across porcine muscle tissue⁶⁴; (b) single time sampling of muscle tissue during postnatal period, around 30–35 days of age; (c) muscle specific variations in the gene expression, which is currently less known. As the weight of inter-muscular fat was not accounted separately, the yield of major pork cuts described in the present study includes the lean meat as well as adipose tissue.

Taken together, it is suggestive that an increased fusion with the concomitant expression of genes associated with muscle regulatory proteins, cell proliferation and other cellular function contribute to the development of higher muscle mass in Hampshire breeds of pig as compared to Mali, which could be observed also at animal level (reduced meat yield), cellular level (less number of fibers per fascicule) as well as molecular level (reduced expression of myogenesis and muscle regulatory genes). In nutshell, the transcriptome analysis, supported by other observations, suggests dissimilarity in muscularity between breeds that could be due to DEGs, associated with myogenesis, supported by modulation of genes associated with growth factors, cell proliferation, extracellular matrix and cell adhesion. The findings of the present study will help to identify genes that could be further explored for their utility in selection of animals exhibiting different muscularity.

Funding Statement

The study was supported by Indian Council of Agricultural Research through institute funded and the ICAR-National Fellow Projects.

Ethical approval

All the experiments were conducted as per the guidelines of the Committee for the purpose of control and supervision of experiment on animals and were approved by institutional animal ethics committee (NRCP/CPCSEA/1658/IAEC-33).

Author contributions

Conceptualization, NHM; Data curation, JD and JB; Formal analysis, JB, PP and RT; Funding acquisition, NHM and DKS; Investigation, PP, LB, AKD, RT and RS; Project administration, NHM, DKS and VKG; Software, JD and BCD; Validation, LB; Visualization, JB and BCD; Writing – original draft, NHM, JB and PP; Writing – review & editing, NHM, JB and PP. Revision- NHM and JB.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All data generated or analyzed during this study are included in supplementary files. The RNA sequencing data have been deposited in the NCBI Gene Expression Omnibus under the GEO accession number GSE184970 (GSM5602638, GSM5602639, GSM5602640, GSM5602641).