Associations of MYF 5 gene polymorphisms with meat quality traits in different domestic pig ( Sus scrofa ) populations

The MYF5 gene is first inducibly expressed in muscle cell during embryonic muscle development and plays an important role in regulating the differentiation of skeletal muscle precursors. In this study we used PCR-RFLP to investigate two pig (Sus scrofa) populations (n = 302) for two MYF5 gene polymorphisms, a previously unreported novel Met-Leu shift single nucleotide polymorphism (SNP) MYF5/Hsp92II located on exon 1 and the previously identified intron 1 MYF5/HinfI SNP. Haplotype and association analysis showed that haplotypes of the two SNPs were significantly associated with drip loss rate (DLR, p < 0.05), water holding capacity (WHC, p < 0.05), biceps femoris meat color value (MCV2, p < 0.05), biceps femoris marbling score (MM2, p < 0.01), longissimus dorsi intramuscular fat percentage (IMF, p < 0.01) and longissimus dorsi Water moisture content (WM, p < 0.01) in the population 2. However, further studies are needed to confirm these preliminary results.


Introduction
Continued genetic improvement of domestic pigs (Sus scrofa) requires molecular markers to assist selection.In some cases both genes and the underlying causal mutation have been identified by the candidate gene approach (Harlizius and van der Lende, 2001;Li et al., 2002;Xu et al., 2005).Animal breeders have started applying markerassisted selection to improve the quality and performance of livestock (van der Steen et al., 2005) and genetic polymorphisms (marker loci) significantly associated with important traits have become very useful tools.
After slaughtering an animal muscle tissue becomes meat and meat quality traits, controlled by multiple genes, are economically important traits in all animals reared for meat production, including pigs (Zhao et al., 2004).Myogenic regulatory factors (MRF) are involved in muscle development from commitment and proliferation through muscle fiber formation and postnatal muscle maturation and function (Hughes et al., 1999).The known myogenic regulatory factors are the MYOD1, MYF5, MYOG (myogenin) and MYF6 (MRF4) genes which encode highly conserved basic helix-loop-helix (bHLH) proteins (Olson and Klein, 1994).The MYF5 gene (Ott et al., 1991) is first inducibly expressed in muscle cells during embryonic muscle development, with many discrete regulatory elements being involved in the activation and maintenance of MYF5 gene expression in the various muscle precursor populations (Teboul et al., 2002).The effects of MYF5 gene restriction fragment length polymorphism (RFLP) on carcass traits have been described by various authors (Stratil and Cepica, 1999;te Pas et al., 1999;Cieslak et al., 2002;Urbanski and Kuryl, 2004) and this gene has been considered a candidate gene for meat production and meat quality (te Pas, 2004;Carmo et al., 2005).However, few studies on the effect of MYF5 gene polymorphisms on meat quality have, in fact, been published and before such polymorphisms can be used efficiently in breeding and management decisions studies with different polymorphisms in different populations are required to properly characterize any associations of this gene with economically important traits across pig populations.
During the study described in this paper we used the polymerase chain reaction (PCR) and RFLP to identify novel MYF5 gene polymorphisms in pure and crossbred pig populations with the aim of elucidating the relationship between MYF5 genotypes and meat quality traits.
At slaughter the pigs were stunned using a head-only electric stun-tong apparatus (SFK Meat Systems, a.m.b.a, Kolding, Denmark) after at least two hours rest, slaughtered by the sticking method, exsanguinated, scalded, mechanically dehaired, eviscerated and weighed.The left side of each carcass was used to assess meat quality.At 45 min postmortem we used a portable digital pH-Meter (model 646, Knick, Berlin) to measure the pH of the last thoracic vertebral longissimus dorsi (LD), the biceps femoris (BF) and the semispinalis capitis (SC) muscles.At the same time, we measured the drip loss rate (DLR, %) and water holding capacity (WHC, %) by the press technique (Wierbicki and Deatherage, 1958) using a 2.523 cm diameter 1 cm high columnar meat sample pressed for 5 min between 36 medium-speed filter papers (Xinhua Paper Industry Co., LTD, China) using a swelling press (Qinchuan Electric Apparatus Factory, China) and an applied force of 35 kg (Xiong and Deng, 1999).
At one to two hours postmortem we used a reflectometer (Model 43, Diffusion Systems Ltd, UK) to measure the objective meat color value (MCV, normal color value is 15 to 25) of the LD muscle (MCV1) on the freshly cut surface of a one cm thick chop removed from the thorax-waist LD (Hornsey, 1956) and the same chop was also given a subjective color score using a standard 1 to 5 color scale (NPPC, 1991) in which 1 represented a very bright color, 3 normal quail-meat color and 5 a very dark color.The same system was used to score the BF MCV (MCV2) of muscle from the core of a hind leg.A subjective meat marbling (MM) score was also given to the thorax-waist LD (MM1) and BF (MM2) muscles using the international marbling standard (American system) 5-grade marking system scale of 1 to 5 in which 1 indicates that the muscle is devoid of marbling, 2 that it is practically devoid of marbling, 3 that marbling is moderately abundant, 4 that marbling is abundant and 5 that marbling is overly abundant (NPPC, 1991).
One day after slaughter, intramuscular fat percentage (IMF, %) of the last thoracic vertebral LD was determined by chloroform-methanol extraction (Bligh and Dyer, 1959).Water moisture content (WM, %) was measured in a drying oven at 102 °C for 18 h (Bourke et al., 1970).Genomic DNA was isolated from blood samples using a standard phenol: chloroform extraction method (Blin and Stafford, 1976).
The PCR amplification was carried out in a 20 μL final volume containing 25 ng of genomic DNA as template, 0.25 μM of each dNTP (MBI Fermentas, Lithuania), 0.25 μM of each primer and 1 unit of Taq polymerase (Biostar Internation, Canada) in 1PCR reaction buffer (Biostar Internation, Canada).The PCR conditions consisted of an initial denaturation at 95 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 57 °C for MYF5-p1 or 63 °C for MYF5-p2 for 45 s and extension at 72 °C for 1 min, with a final 72 °C extension for 10 min.
The MYF5 alleles were analyzed using a RFLP protocol in which 8.5 μL of the PCR products were digested with 5 units of restriction enzyme (Hsp92II for the MYF5-p1 primer, HinfI for the MYF5-p2 primer) at 37 °C for 4 h in 10 μL of 1 buffer and the digestion products separated by electrophoreses on 1.5% (w/v) agarose gels using 1TAE buffer (Sambrook et al., 1989), the gels being stained with ethidium bromide.

Statistical analysis
Associations between the MYF5 gene haplotypes and meat quality traits were evaluated using the least square method of the GLM (General Linear Models) procedure and the Statistical Analysis Software version 8.0 (SAS Institute Inc., Cary, USA).The model used to analyze the data was assumed to be: where Y ijklm is the observation of the trait; i is the population mean, S i is the effect of ith sex (i = 1 for male or 0 for female), B j is the effect of j th breed, G k is the effect of kth haplotype, D l is the effect of boars, P m is the batch effect, b ijklm is the regression coefficient of the slaughter age and e ijklm is the random residue.vidual samples (2 Yorkshire, 2 Landrace and 2 Meishan pigs) allowed the identification of a novel single nucleotide polymorphism (SNP) adenine to cytosine (methionine (ATG) to leucine (CTG) shift) substitution (MYF5/ Hsp92II, detected with the MYF5-p1 primer) in exon 1 and a previously described cytosine/guanine mutation (MYF5/ HinfI, detected with the MYF5-p2 primer) in intron 1 (te Pas et al., 1999).We applied the Hsp92II and HinfI PCR-RFLP protocols to genotyping the exon 1 and intron 1 mutations in 302 pigs with phenotypic records and designated the MYF5-p2 primer MYF5/HinfI genotypes as AA (1.19 kb), AB (1.19 kb + 729 bp + 464 bp) and BB (729 bp + 464 bp), while the MYF5-p1 primer MYF5/Hsp92II genotypes as CC (243 bp), CD (243 bp + 210 bp + 33 bp) and DD (210 bp + 33 bp).

Genotype frequencies in the different populations
The combined effects of the exon 1 and intron 1 substitutions were estimated as haplotype substitution effects.The combined genotype and haplotype frequencies of the Hsp92II and HinfI MYF5 gene polymorphisms for pigs with records in the two populations studied are shown in Table 1.The HinfI locus had three genotypes in the population 1 and two genotypes (AA and AB) in population 2. The Hsp92II locus was monomorphic in population 1, where only the DD genotype was found, but polymorphic in population 2 where all three genotypes (CC, CD and DD) were detected (Table 1).In population 2 the values in Table 1 suggest the presence of six haplotypes (AA/CC, AA/CD, AA/DD, AB/CC, AB/CD and AB/DD) but since the AB/CC haplotype was detected in only one pig we excluded this pig and the AB/CC haplotype from the association analyses.Moreover, only three of the nine possible genotypic combinations of the two polymorphisms were recorded in population 1. Table 1 also shows that the AA/DD haplotype was present at high frequencies in both population 1 (0.785) and 2 (0.529).

Relationship between MYF5 genotypes and meat quality traits
In population 1 there were only three MYF5 haplotypes (AA/DD, AB/DD and BB/DD) but these did not differ for any meat quality trait analyzed (data not shown).We focused our association analysis on the five population 2 haplotypes (AA/CC, AA/CD, AA/DD, AB/CD and AB/DD) and found statistically highly significant associations with the MM2, IMF and WM traits and significant associations with the DLR, WHC and MCV2 traits (Table 2).For the DLR and WHC traits the effect for the AA/CD haplotype was significantly different (p < 0.05) to the AA/DD haplotype, replacing the AA/DD haplotype by the AA/CD haplotype would significantly decrease WHC by 0.78% and increase the DLR by 0.583%.For the MCV2 trait, the effect of the AB/CD haplotype was significantly different (p < 0.05) to the AB/DD haplotype in population 2 and replacing the AB/DD with AB/CD would significantly decrease the MCV2 by 0.597.The AA/CC haplotype was associated with the highest significant IMF value (p < 0.01) and the lowest significantly WM value (p < 0.01).In contrast, pigs carrying the AB/CD haplotype had the lowest IMF and MM2 values but the highest WM values of all the haplotypes.

Discussion
Research on mutations in targeted functional genes (candidate genes) and their association with economic traits has been performed to ascertain the genetic basis of production traits and to develop DNA tests as selection tools in pig breeding schemes (de Vries et al., 1998).This approach is a very promising route for the improvement of meat quality, since direct meat quality records are not available for potential breeding animals (Óvilo et al., 2006).A well known example is the gene tests used to remove the Halothane (HAL) and ryanodine receptor (RN) mutations which have undesirable effects on meat quality, these tests having resulted in a clear improvement of the technological quality of the pork produced in many countries (Fujii et al., 1991;Le Roy et al., 2000).In order to deal with growing market segmentation new genetic techniques are needed to adapt technological and sensory qualities to the requirements of processors and consumers (Monin, 2003;Óvilo et al., 2006).
The MYF5 gene plays a key regulatory role in the initiation and development of skeletal muscle and the maintenance of its phenotype, and is thus a candidate gene for involvement in traits related to growth and meat quality (Maak et al., 2006).In our study we constructed and used the MYF5-p1 primer to identify a novel SNP, the Hsp92II polymorphic loci, an adenine to cytosine shift resulting in a methionine (ATG) to leucine (CTG) amino acid substitu-  (1999) investigated the HinfI polymorphic site in the first intron of the MYF5 gene and found that the A-allele predominated in the all pig breeds tested (including those tested by us), which is supported by our results (Table 1).However, Te Pas et al. (1999) found no genotypically associated differences for any of the traits investigated (birth weight, weight at slaughter, growth rate, meat weight and subcutaneous fat thickness) in 1216 Yorkshire pigs and concluded that the porcine MYF5 gene lacks a significant causal mutation affecting these traits, or that the linkage phase of the MYF5/HinfI polymorphism is not in phase with the MYF5 causal mutation.However, Cieslak et al. (2002) analyzed the MYF5/HinfI locus in 333 unrelated (Pietrain, Zlotnicka Spotted, Polish Landrace, Pietrain (Pietrain Zlotnicka Spotted), Pietrain (Polish Large White Polish Landrace) and Dutch Large White Dutch Landrace) pigs with an equal proportion of young female pigs (gilts) and castrated male (barrows) pigs and found that gilts with the TT RYR1 genotype and AA or AB genotype at the MYF5/HinfI locus had a significantly higher loin eye area and carcass meat content than pigs without this genotype.Thus, it is still difficult to evaluate the effect of the MYF5/HinfI polymorphism and the different results may depend on the pig breed investigated and statistical model used.
Traditionally, one single nucleotide polymorphism (SNP) is used for genotyping and association analysis.However, using haplotypes, which are specific combinations of nucleotides on the same chromosome, will provide more information on the complex relationship between DNA variation and phenotypes than any single SNP can provide (Stephens et al., 2001;Grindflek et al., 2004).Thus, we used the haplotype information to evaluate the relationship between MYF5 polymorphisms and meat quality traits.Our results showed that two haplotypes, AA/CD and AA/DD, were highly frequent in population 2 and showed significant difference in their effects on drip loss rate (DLR) (p < 0.05) and water holding capacity (WHC) (p < 0.05).This supports the results of Carmo et al. (2005), who found that MYF5 gene allelic variants had a significant effect on DLR, cooking properties and total cooking loss in a divergent F2 pig population (n = 359) of Brazilian Piau boars and commercial white females and that an insertion (I) variation in the MYF5 gene is associated with water-holding capacity in the same population.In our work, we also found that there were significant differences in population 2 between the different haplotypes in respect of the biceps femoris meat color value (MCV2, p < 0.05), biceps femoris marbling score (MM2, p < 0.05), longissimus dorsi intramuscular fat percentage (IMF, p < 0.01) and longissimus dorsi moisture content (WM, p < 0.01).Similarly, the differences for MM2 (p < 0.01), IMF (p < 0.01) and WM (p < 0.01) were also found among different MYF5/Hsp92II genotypes in population 2 (data not shown).Thus, the mutation (MYF5/Hsp92II) in coding regions could be responsible for changes in muscle protein structure or function and lead to changes in meat quality.Of course, the mutation is also possible to link to a quantitative trait locus (QTL) or loci.Because MYF5 has been localized to the Sus scrofa chromosome 5 (SSC5) (Soumillion et al., 1997) and a meat quality QTL has been mapped between the MYF5 and SW967 regions of this chromosome in the W x M family (Lee et al., 2003).In addition, the insulin-like growth factor I (IGFI) and MYF6 genes have been located on the same chromosome as the MYF5 gene (Wintero et al., 1994;Vykoukalova et al., 2003).
To better assess the real impact of the effects of the MYF5 gene on meat quality, further investigations are needed to confirm our results, such as an appropriate area for research being the possible effects of other genes in linkage disequilibrium with the MYF5 SNPs.
Two fragments, MYF5-p1 (243 bp) and MYF5-p2 (1193 bp) were PCR amplified and sequenced from genomic DNA.Multiple alignments of the sequences of six indi-Liu et al.

Table 1 -
Genotypic frequency distributions for the MYF5 gene HinfI and Hsp92II polymorphic loci haplotypes for two pig populations.The number of pigs with records for each genotype frequency is shown in parentheses.Population 1 (n = 130), composed of 28 Yorkshire, 46 Landrace, 21 Yorkshire¡ x Landrace and 35 Landrace x Yorkshire pigs; and population 2 (n = 172), consisting of 50 Meishan, 78 Yorkshire x Meishan and 44 Meishan x Yorkshire pigs.

Table 2 -
Association of haplotypes in the MYF5 gene with the phenotype value of partial meat quality traits in population 2 (n = 172), consisting of 50 Meishan, 78 Yorkshire x Meishan and 44 Meishan x Yorkshire pigs.Within-trait (i.e.within the same row) significant differences between the genotype classes are indicated by superscripts, lower case at p < 0.05 and upper case at p < 0.01.