Association of osteopontin gene with intramuscular fat content and fatty acid composition traits in pigs

Osteopontin (OPN) is a secreted phosphoprotein that is involved in the development of skeletal muscle and fat deposition. The objectives of this study were to identify the polymorphism of the OPN gene and to analyze the association of the OPN gene with intramuscular fat (IMF) content and fatty acid (FA) composition in pigs. Longissimus thoracis (LT) muscle samples taken from the 10-11 th rib were collected from a total of 328 Duroc pigs. Genomic DNA samples were extracted from LT muscle tissues using the phenol-chloroform method. IMF content was measured using the ether extraction method and FA composition was measured by gas chromatography. The porcine OPN polymorphisms were identified by DNA sequencing and were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The association analysis of the OPN gene with IMF and FA composition traits was performed using a general linear model (GLM). Two polymorphic sites ( OPN g.2442-2471indel and g.3836A>G) were found in the 5´-flanking region and intron 1 of the porcine OPN gene. The OPN g.2442-2471indel polymorphism was found to be significantly associated with IMF content and ω3 FA levels (P<0.05). Moreover, OPN g.3836A>G polymorphism was significantly associated with the linolenic acid levels in the muscles of pigs (P<0.05). The results of this study indicate that the OPN gene is important to IMF content, as well as linolenic and ω3 FA levels in pigs, and could be used as a candidate gene to improve fat deposition and fatty acid composition in the muscles of pigs.


INTRODUCTION
Intramuscular fat (IMF) content is an economically important trait for the determination of meat quality and is strongly related to the eating quality of meat (Ros-Freixedes et al., 2014;Wood et al., 2004). Moreover, fatty acid (FA) composition is considered an indicator of lipid quality and is closely related to the nutritional value of pork (Sanchez et al., 2007;Wood et al., 2004). The IMF is defined as the total lipid associated with all cells present in a meat sample, excluding adipocytes from the intermuscular fat (Gao and Zhao, 2009) whereas, the FA composition of muscle is amount of fatty acid components in total lipid of IMF. High levels of IMF content and FA composition especially, palmitoleic, oleic, and monounsaturated fatty acid (MUFA) are positively associated with the flavor of pork (Cameron et al., 2000;Wood et al., 2004;Zhang et al., 2019). Currently, the genome-wide association study (GWAS) has been used to identify candidate genes for IMF and FA content in the muscle tissue of pigs (Ding et al., 2019;Zhang et al., 2016;Zhang et al., 2019). Moreover, the entire transcriptomic approach has been analyzed along with numerous genes that are related to IMF content and FA composition in various pig breeds (Ropka-Molik et al., 2015). One of these genes is porcine osteopontin (OPN), which is known to be differently expressed in muscle and adipose tissues (Ropka-Molik et al., 2015).
The OPN or secreted phosphoprotein 1 (SPP1) gene is a member of the cytokine family and is expressed in macrophage, skeletal muscle, and adipose tissues (Gómez-Ambrosi et al., 2007;O'Brien et al., 1994;O'Regan et al., 1999;Xu et al., 2005). It plays a major role in immune responses, apoptosis, and cell-cell signaling (Ropka-Molik et al., 2014). The porcine OPN gene has been mapped on Sus scrofa chromosome 8 (SSC8). The coding sequence of the porcine OPN gene is 1507 bp in length. It is composed of seven exons and six introns and was found to encode a peptide of 303 amino acids (ENSSSCT00000010091.5; Ensembl data base; https://asia.ensembl. org/). Previous studies have reported that the polymorphism of porcine OPN is associated with reproductive and litter size traits in pigs (Knoll et al., 1999;Korwin-Kossakowska et al., 2002;Kumchoo and Mekchay, 2015;Niu et al., 2008). However, it has been demonstrated that the OPN gene is up-regulated in the muscle tissues of lean pigs (Muráni et al., 2009;Ropka-Molik et al., 2014). Moreover, OPN is also expressed in backfat and intramuscular fat tissues in pigs (Sun et al., 2013;Wu et al., 2013). The polymorphism of the porcine OPN gene is associated with the loin eye area and backfat thickness (Han et al., 2012). Thus, the OPN gene may play an important role in adipogenesis in skeletal muscle tissues and backfat tissues (Han et al., 2012). However, available information on the association of the porcine OPN gene with IMF content and FA composition traits in pigs has been limited. In the present study, polymorphisms of the porcine OPN gene have been identified and their association with IMF and FA composition traits has also been assessed in commercial Duroc pigs.

Animals and phenotypic determination
A total of 328 pigs were obtained from a group of purebred Duroc pigs (158 barrows and 170 gilts) that were reared under the commercial conditions of the Betagro Hybrid International Company, Thailand. All the pigs were fed the same corn-soybean-based diet containing 3200 kcal/kg digestible energy and 16% crude protein. The pigs were slaughtered according to standard commercial procedures at a body weight of about 90 kg (153±1.98 days of age). Thus, ethical approval was not required for this investigation. Longissimus thoracis (LT) muscle tissues obtained from the 10-11 th rib were taken to analyze IMF content and FA composition ( Figure 1). The genomic DNA samples were isolated from LT muscle tissues using the standard phenol-chloroform protocol. The degree of concentration of the DNA sample was measured with a Nanodrop 2000c spectrophotometer (Thermo Scientific, USA). The LT muscle tissues (10 g) were freeze-dried and pulverized prior to IMF analysis. IMF content was determined using the ether extraction method (AOAC, 2000) and expressed as the percentage of IMF of the dry matter of muscle tissues. Fatty acid composition was measured with the method according to a previous study (Zappaterra et al., 2020) by using a gas chromatography-flame ionization detector (SCION 456-GC, Bruker Daltonics Inc., Fremont, USA) with an RT-2560 capillary column (100 m length, 0.25 mm internal diameter, 0.2µm film thickness, RESTEK, Bellefonte, PA, USA). Injector and detector temperatures were held at 250°C. The column oven temperature program was increased from 50 to 220°C at a rate of 10°C/min, held for 35 min, then increased from 200 to 230°C at a rate of 5°C/min and held at 230°C for 20 min. The sample volume of 1 μl was injected. A 37-component standard FAME mix (Food Industry Fame Mix, RESTEK, Bellefonte, PA, USA) was used for the identification of FAMEs. FA composition was expressed as the percentage of total fatty acids.

Polymorphisms of porcine OPN gene and genotyping
To identify polymorphisms in the porcine OPN gene, specific primers were designed based on the available sequence information (GenBank accession number: M84121.1). Ten DNA samples of the 5-highest and 5-lowest IMF content levels (11.96±0.26% vs 6.28±0.19%) were selected and used to amplify the DNA fragments of the porcine OPN genes using each primer (Table 1). The amplicons of the porcine OPN gene were sequenced using the CEQ 8000 Genetic Analysis System (Beckman-Coulter, USA) in order to identify the SNP variants in this pig population. Two single nucleotide polymorphisms (SNPs) of the porcine OPN gene (g.2442-2471indel and g.3836A>G) were genotyped in 328 Duroc pigs. Polymerase chain reaction (PCR) amplification was carried out in a total volume of 20 µL consisting of 50 ng of genomic DNA sample, 1×(NH 4 ) 2 SO 4 buffer, 1.5 mM MgCl 2 , 0.2 mM dNTPs, 0.4 mM each primer (Table 2), and 0.2 U Taq DNA polymerase (Fermentas, USA). The PCR conditions were as follows: 95°C for 3 min, followed by 32 cycles of denaturation at 95°C for 30 sec, annealing at 58-60°C for 30 sec, elongation at 72°C for 45 sec, and then 5 min at 72°C to complete the reaction. The PCR products of the porcine OPN g.3836A>G gene were digested with restriction enzymes ( Table 2). The PCR products and digested products were electrophoresed on 6% polyacrylamide gels and stained with ethidium bromide for visualization.

Statistical analysis
The genotype and allele frequencies of the porcine OPN polymorphisms were estimated. Effects of the porcine OPN polymorphisms on IMF content and FA composition traits were analyzed with a general linear model as expressed below: Y ijk = µ+S i +G j +e ijk where Y ijk represents the observations of the phenotype values, μ is representative of the overall mean for each trait, S i represents the fixed effect of sexes, G j represents the fixed effect of the OPN genotypes, and e ijk represents the residual error. The effects were estimated by t-tests on significant deviations from zero (P<0.05). Table 1 Primer sequences used for polymorphism identification in the porcine OPN gene.

Polymorphisms of porcine OPN gene
Two SNP loci of the porcine OPN gene were found to be segregated among this Duroc population. An indel of GT polymorphisms of the porcine OPN gene was found in the OPN-2 DNA fragments of the 5´-flanking region at position g.2442-2471indel. This fragment length polymorphism was detected with PCR and gel electrophoreses (Figure 2). Four motif alleles were presented as (GT) 14 , (GT) 20 , (GT) 35 , and (GT) 46 with PCR product sizes of 142, 154, 184, and 206-bp, respectively. A polymorphic site of g.3836A>G was found in the OPN-3 DNA fragments of intron 1 of the porcine OPN gene. This was detected with the restriction enzyme Tsp509I (Figure 3). Two specific alleles exhibited a 149-bp fragment for allele A and two fragments of 125 and 24-bp for allele G.   Table 3 Genotype frequencies of the porcine OPN g.2442-2471indel and the porcine OPN g.3836A>G in pigs.

Genotype frequencies
The genotype frequencies of the porcine OPN gene are shown in Table 3. At the OPN g.2442-2471indel locus, five genotype patterns were presented as 142/142, 154/154, 142/154, 142/206, and 184/206-bp. The OPN g.2442-2471indel with 142/154 genotype was found to be more frequent in this pig population. At the OPN g.3836A>G locus, three genotypes were observed. The OPN g.3836A>G polymorphism with GG genotype was found to be more frequent in these Duroc pigs.

DISCUSSION
Duroc pigs are the most popular breed in the pig production due to its good growth and show superior IMF deposition. In this present study, we identified polymorphisms in the porcine OPN gene and assessed their effects on IMF content and FA composition in the LT muscle tissues of Duroc pigs. Two polymorphic sites of the porcine OPN g.2442-2471indel and g.3836A>G loci were segregated in these Duroc pigs. These two polymorphisms were located in a non-coding sequence (5´-flanking region and intron 1) and this corresponded with the uncovered SNPs of the previous study (Muráni et al., 2009). The porcine OPN g.2442-2471indel with the 142/154 and 154/154-bp genotypes was more frequent in this Duroc population. In contrast, the porcine OPN g.3836A>G locus revealed a similar genotype distribution among three genotypes (AA, AG, and GG) in this pig population. These results indicate that the porcine OPN g.2442-2471indel locus might be related to selective pressures on some desirable production traits. On the other hand, the porcine OPN g.3836A>G was under homeostasis accompanied by the effect of artificial selection. Moreover, the SNP variants at g. 1804C>T, g.1999A>G, and g.2011A>G loci in the 5´-flanking region of the porcine OPN gene have been identified (Korwin-Kossakowska et al., 2013;Muráni et al., 2009). Currently, a IMF: intramuscular fat content, SFA: saturated fatty acids (C14:0+C16:0+C18:0+C20:0), MUFA: monounsaturated fatty acids (C16:1n-7+C18:1n-9+C20:1n-9), PUFA: polyunsaturated fatty acids (C18:2n-6+C18:3n-6+C20:2n-6+C20:3n-6+C20:4n-6), ω3 fatty acids (C18:3n-3+C20:5n-3+C22:6n-3), ω6 fatty acids (C18:2n-6+C18:3n-6+C20:3n-6) and ω9 fatty acids (C18:1n-9). IMF was expressed as the percentage (%) of IMF content of the dry matter of muscle tissues. FA composition was expressed as the percentage (%) of total fatty acids. The results of this study also indicate that the porcine OPN g.2442-2471indel significantly affected IMF content and ω3 FA levels. The porcine OPN g.2442-2471indel with 142 and 154-bp alleles seem to be the favorable alleles for IMF and ω3 FA deposition in the LT muscle tissues of Duroc pigs. Moreover, the porcine OPN g.3836A>G polymorphism was significantly associated with linolenic acid levels. Furthermore, the porcine OPN g.3836A>G with AG genotype was positively correlated with linolenic acid levels. Although, the role of the porcine OPN gene on IMF content and FA composition in muscle tissue was scarcely studied, the porcine OPN gene is an up-regulated expression in the LT muscle tissues of the porcine fetus (at 49-91 days post-conception). It has been reported that the OPN g.3836A>G polymorphism is related to myogenesis by regulating CCAAT/enhancer-binding protein beta (C/EBPβ) in pigs (Muráni et al., 2009). Moreover, the short interspersed nuclear element (SINE) variation in intron 6 of the porcine OPN gene is associated with backfat thickness and loin muscle area (Han et al., 2012). Additionally, the expression levels of the porcine OPN gene are related to IMF content in the LT muscle tissues. These pieces of evidence indicate that the OPN gene plays an important role in adipogenesis in skeletal muscle and adipose tissues (Han et al., 2012). The present study showed a significant association of the porcine OPN polymorphisms with IMF content and FA composition traits, especially with regard to linolenic and ω3 FA levels. Indeed, monogastric animals are not able to de novo synthesize a significant amount of essential FA, hence their diet would need to be supplemented (Zappaterra et al., 2020). Therefore, the observed genetic association could be indicated by an alteration in the gene that is related to absorption, transformation, or transport of these FAs resulting in modulation of the accumulated FAs in muscle tissue (Muñoz et al., 2012;Supakankul and Mekchay, 2016). All this information indicates that the OPN gene might be involved in lipid and FA deposition in the muscle tissue of pigs. In this study, our results indicated the effects of OPN polymorphisms on IMF content and FA composition in the LT muscle tissue of pigs. Further studies will be required to confirm the association of the OPN gene with IMF content and FA composition traits in various pig populations.

CONCLUSION
In the present study, we have identified the polymorphisms of the porcine OPN gene and elucidated their effects on IMF content and FA composition in the LT muscles of pigs. Two polymorphic sites of the porcine OPN gene (g.2442-2471indel and g.3836A>G) revealed an association with IMF content and FA composition of ω3 and linolenic acid levels. These results indicate that the OPN gene is essential for lipid deposition and FA accumulation in the muscle tissue of pigs. Thus, the porcine OPN gene may be used as candidate marker for the selection of IMF and FA deposition in the muscles of pigs.