Effect of a c-MYC Gene Polymorphism (g.3350G>C) on Meat Quality Traits in Berkshire

Animals and DNA isolation

First, to detect breed-specific variations in the MYC gene, 20 blood samples from each of four types of purebred pigs in Korea – Berkshire, Duroc, Landrace, and Yorkshire – were collected in anticoagulant containing vacutainers. Genomic DNA was isolated from the blood samples using the G-DEX IIb Genomic DNA Extraction Kit (IntronBio Inc., Seoul, Korea).

To measure meat quality traits studied in this study, muscle samples were obtained from 378 Berkshire carcasses (138 sows, 240 boars) shipped from a slaughter house located in Namwon city of Chonbuk province, Republic of Korea. Genomic DNA was extracted from muscle samples using the same extraction kit mentioned above. All procedures were followed according to the manufacturer’s instructions.

Primer design and polymerase chain reaction

The DNA sequence of porcine MYC gene was retrieved from NCBI database (accession number: NC_010446.4). As shown in Table 1, Primer 3 program (http://frodo.wi.mit.edu/primer3/) was used to design 8 primer sets (P1-P8) for full length sequencing of the MYC gene by polymerase chain reaction (PCR).

Table 1

Primer sets for sequencing of porcine MYC gene

The PCR reaction mixture was composed of 1.5 μL of genomic DNA (25 ng), 0.2 pmole of forward and reverse primers, 200 nM of dNTP, 2. 5 unit of Taq polymerase, and 1× reaction buffer of 10 mM of Tris-HCl (pH 9.0), 50 mM of KCl, 1.4 mM of MgCl₂ and 1% of Triton X-100 with a total volume of 20 μL. The thermal cycling condition of the mixture included denaturation at 94ºC for 5 min, and 35 cycles of PCR amplification conducted for 30 s at different annealing temperatures for each primer set (Table 1) with primer extension at 72ºC for 40 s. Final extension was performed at 72ºC for 5 min. Electrophoresis of the amplified products was conducted on 1.5% agarose gels stained with ethidium bromide (EtBr).

Sequencing and genotyping

Twenty DNA samples pooled from each of the four breeds (Berkshire, Duroc, Landrace, and Yorkshire) were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems, Foster City, CA, USA) and the ABI 3130 Genetic Analyzer (Applied Biosystems, USA). The genotyped sequences were subject to peak and alignment analyses using the SeqMan 7.0 program (DNASTAR, Madison, WI, USA) to detect polymorphism in the gene and determine breed-specificity.

To determine genotypes from Berkshire-specific polymorphism that was detected in the earlier step (Table 2), polymerase chain reaction-restriction length polymorphism (PCR-RFLP) analysis was performed on genomic DNA from muscle samples amplified by PCR. The amplified products of PCR (2 μL) were treated with 1 unit of HhaI restriction enzyme, 1 μL of 10× restriction enzyme buffer and sterilized distilled water, giving a total volume of 10 μL. After digestion reactions took place for restriction enzyme-specific temperature and time, the DNA fragments were separated on 1.5% agarose gel in 1× TAE buffer containing EtBr for genotyping.

Table 2

Information of PCR-RFLP for polymorphic site of c-MYC gene in Berkshire

Meat quality measurement

After slaughter, a section of loin at the 10th rib of the carcass was taken to assess meat quality based on the four meat quality traits – pH/color, drip loss, cooking loss, and shear force.

Statistical analysis

SAS 9.1 (SAS, USA) program was used to estimate the genotype effect of MYC gene on meat quality traits by general linear model procedure. The least significance difference test was implemented to determine significance between mean values. The model used in this study to estimate the genotype effect on the traits is as follows:

Where,

Detection of Berkshire-specific polymorphism

Porcine MYC sequences were retrieved from the NCBI database (accession number: NC_0104464.4). The MYC gene is located on chromosome 4 comprising three exons and 2,464 base pairs with coding regions of 1,359 bp, and 5′ untranslated region (UTR) and 3′ UTR at lengths of 591 bp and 514 bp respectively. Full length sequencing of MYC gene was performed for pooled DNAs from each four pig breeds (Berkshire, Duroc, Landrace, Yorkshire) commonly bred in Korea by using eight primer sets (P1-P8; Table 1). When compared with the MYC SNPs deposited in dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?geneId=448810), sequencing results showed that the g.3350G>C polymorphism in the intron 2 of porcine MYC gene was found only in Berkshire pigs, but not in other breeds (Duroc, Landrace, and Yorkshire, Table 2).

Genotyping of the MYC g.3350G>C SNP in Berkshire

Once the polymorphic site (g.3350G>C) was detected in the MYC gene and identified to be Berkshire-specific, genotyping was carried out using the genomic DNA of muscle samples of Berkshire. Genotypes were determined with PCR-RFLP using HhaI restriction enzyme (Table 2). Digestion of PCR product with HhaI restriction enzyme yields two fragments with 480 bp and 201 bp respectively for allelotype G, whereas for allelotype C one fragment with 681 bp is produced (Figure 1). Allele frequencies of GG, GC, and CC genotypes of 378 Berkshire pigs were 0.399, 0.508, and 0.093 respectively (Table 3).

Figure 1

Genomic structure and polymorphism of the MYC gene in Berkshire. Coding exons are marked by shaded blocks and 5′ and 3′ UTR by white blocks. (A) agarose gel displaying a HhaI restriction digest on an amplified portion of c.3350 G>C. M: 100 bp ladder; GG: 480 bp and 201 bp; GC: 681 bp, 480 bp and 201 bp; CC: 681 bp.

Table 3

Genotype and allele frequencies of polymorphic site of MYC gene in Berkshire

Association between the MYC g.3350G>C SNP and meat quality traits

The meat quality measurements obtained from 378 Berkshire pigs in this study are shown in Table 4. Although the muscle samples used for this study were obtained from 138 sows and 240 boars, a significant difference between sexes was not observed across the meat quality traits. The pH of muscle, which affects freshness of meat due to postmortem metabolism, was 6.07±0.30 at 45 min and 5.79±0.22 at 24 h postmortem respectively. Meat color which was evaluated for lightness (L*), redness (a*), and yellowness (b*), was based on CIE LAB. Lightness (L*) was 50.49±3.00, redness (a*) was 16.36±1.16, and yellowness was 5.28±1.19 respectively. As for the water-holding capacity of meat, drip loss was 2.51±2.20 and cooking loss was 16.79±3.99. Shear force was 2.66±0.69. Based on the results concerning meat quality traits, the g.3350G>C SNP effect was significant for pH_45min and cooking loss (Table 5, p<0.05). Berkshire pigs with the homozygous genotype C had the highest pH_45min and smallest cooking loss (Table 5). Except two traits, the g.3350G>C SNP had no effects on other trait including pH₂₄ h, meat colors and drip loss, and shear force (Table 5, p>0.05). There are several physical and chemical factors known to affect pork quality (Stalder et al., 1998). A previous study reported that postmortem pH in muscle and meat quality characteristics correlated (Huff-Lonergan et al., 2002). According to Jung et al. (2011), pH_{45 min} and pH_{24 h} were positively correlated whereas both were negatively correlated with drip loss and shear force in Berkshire pigs.

Table 4

Means and standard deviations for meat quality measurements in Berkshire

Table 5

Association analyses between the polymorphisms and individual traits in Berkshire

Porcine MYC gene was mapped on SSC4 (Reiner et al., 1998; Cepica et al., 2003), where particularly major quantitative trait locus regions for backfat at tenth rib (Liu et al., 2007; Edwards et al., 2008) and average daily gain (de Koning et al., 2001) reside. Porcine chromosome SSC 4p13 contains a synteny group, with F13B, ATP1B1, GBA, ATP1A1, IVL, and two microsatellites S0001, S0067 (Andersson et al., 1994; Marklund et al., 1999).

Given the fact that MYC plays a role in the cellular processes as a molecular hub that receives multiple signals and integates them, either negatively or positively, to control other sets of genes. Thus, MYC has not only as a single function, but also a multitude of different categories of cellular functions (Potter and Marcu, 1997). In this regard, MYC expression that is regulated by external cellular stimulus including hormones, growth factors, cytokines, lymphokines, nutritional status, development and differentiation (Levens et al., 1997), is connected to various physiological processes. More specifically, MYC is involved in myogenesis (Miner and Wold, 1991; Whitelaw and Hesketh, 1992), muscle hyperplasia (Kipshidze et al., 2002) and adipogenesis (Ninomiya-Tsuji et al., 1993; MacDougald and Lane 1995), hence the proto-oncogene could be a candidate gene contributing to variability of fatness in pigs. In addition, previous study has suggested the influence of MYC gene expression on glucose metabolism (Collier et al., 2003), whereby lactic acid production from glycolysis reaction might have an effect on the pH of meat.

Significant difference was also noted between GG, CG, and CC genotypes at pH_45min, with measurements of 5.96±0.04, 5.97±0.04, and 6.13±0.07 respectively (Table 5). Muscle pH is an important economic factor that is associated with water-holding capacity of muscle’s, which in turn has a marked influence on the appearance of meat (Barge et al., 1991). According to Jung et al. (2011), pH_45min and pH_24min were positively correlated whereas both were negatively correlated with drip loss and shear force in Berkshire pigs. Previous studies have suggested the influence of MYC gene on glucose metabolism (Collier et al., 2003), whereby lactic acid production from glycolysis reaction has an effect on the pH of meat. Because pH_45min was significantly higher in the animals with CC genotype of g.3350G>C polymorphism than the levels in the animals having other genotypes in this study, it is suggested that this genotype may play an influential role in improving pork quality.

Muscle structure could (or may) be affected by heat technique, composition of meat, and degree of heating meat. Heating of meat contracts muscle fibers that can cause decrease in water-holding capacity and cooking loss irrespective to the method of heating (Cho et al., 2008). High cooking loss is associated with poor meat quality (Schmidt et al., 2010) and is reported that the higher the marbling score is, the lower the cooking loss (Kim and Lee, 2003). Therefore, low cooking loss is regarded as a favorable trait of meat quality. Significant difference in genetic effect of GG, CG, and CC genotypes was identified with respect to loss, with measurements 16.38±0.63, 15.48±0.84, and 15.50±0.67 respectively (Table 5).