CARCASS AND MEAT QUALITY IN RELATION TO THE POLYMORPHISM IN PORCINE MYF4 GENE

The aim of this study was to analyse the associations between polymorphism in porcine myogenin gene (MYF4) and economically important traits in relation to the carcass and meat quality of pigs. The genomic DNA samples were taken from in total 180 crossbreeds (Large White x Landrace). The detection of polymorphism in MYF4 gene were performed in order to evaluate its effect on back fat thickness, proportion of valuable meat parts, MLT area and proportion of thigh. The genotyping of analysed individuals was carried out by means PCR-RFLP method and restriction endonuclease MspI. The allele frequencies were as follows: A 0.75 and B 0.25. A prevalence of AA genotype (59%) compared to AB (33%) and BB genotypes (8%) were detected in analysed crossbreed population. The observed average value of heterozygosity (0.33) and positive value of the Wright's FIS index (0.12) similarly reflected the higher proportion of homozygous genotype in populations. The effect of MYF4 gene polymorphism on selected phenotypic traits has been tested using one-way ANOVA procedure. The statistical analysis showed only non-significant results. Due to the polygenic character of selected phenotypic traits the involvement of other candidate genes and increase of sample size could clarify the role of MYF4 gene in porcine carcass and meat quality regulation. The study, which is based on molecular variability of livestock genetic resources, is necessary for the genetic improvement and understanding of relations between markers and trait of interest.


INTRODUCTION
The pig breeding plays currently a key role in meat production, and the high consumption of pork corresponds to this. The high efficiency of pig breeding is absolutely necessary for competitiveness under today's difficult economic conditions (Weisz et al., 2011). One of goals of animal genetics is to locate and identify loci that are responsible for economical important traits such as meat performance, reproduction, and product qualities which are the most significant factors for pork production efficiency (Bulla et al., 2007).
Variation in meat quality is likely to be caused by differences in many genetic and non-genetic (environmental) factors, which interact together and determine the course of metabolic processes in muscle tissue and in the postmortem conversion of muscle into meat (Kapelañski et al., 2005). Intensive selection for lean growth in pigs may have caused a considerable genetic change in fibre type composition, which resulted in a higher proportion of glycolytic fibres and in an increase in fibre diameter in domestic pigs compared to native breeds. Changes in fibre type ratios affect metabolic properties of a muscle and thereby meat quality (Kłosowska et al., 2004).
Histochemical and biochemical muscle fibre properties are the factors that influence the quantitative and qualitative characteristics of pork meat (Stupka et al., 2014). Muscle fibre formation takes place during embryonic development in mammals and is controlled by the MyoD gene family, which consists of four genes, MyoD1 (MYF3), myogenin (MYOG or MYF4), MYF5 and MYF6 (MRF4) (Horák et al., 2004;Zhang et al., 2007;Ujan et al., 2011). The expression of the MYF5 and MYOD1 (MYF3) genes plays a fundamental role during myoblast proliferation, while the expression of the MYOG (MYF4) and the MYF6 (MRF4) genes is linked to the differentiation and maturation of myofibres (Stupka et al., 2012). The myogenin (MYF4) gene, which controls the start of myoblast fusion and myofibres formation, was identified on the 9 th autosome, in the 9q2.1-2.6 area (Soumillion et al., 1997;Ernst et al. 1998). The myogenin is a transcription factor specific to skeletal muscle and fulfils a key function in muscle differentiation by controlling the onset of myoblast fusion and the establishment of myofibres. Genetic variation in the MYF4 gene can be associated with differences in myoblast and myofibril numbers (Weisz et al., 2011). Ernst et al. (1993) and Soumillion et al. (1997) detected using endonuclease MspI gene three polymorphic sites in the MYF4, in the promoter region, the second intron and the 3' side of the gene.
The aim of this study was to analyse the effect of polymorphism in porcine MYF4 gene on economically important traits in relation to the carcass and meat quality in population of Large White x Landrace crossbreeds.

MATERIAL AND METHODS
The biological samples were collected in 2014 from in total of 180 crossbreeds of Large White x Landrace (86 boars and 94 sows) from the Experimental Centre of Farm Animals (Department of Animal Husbandry, Slovak University of Agriculture in Nitra, Slovakia). Each of selected individuals included in present study have been farmed in the same conditions and fed with standard feed mixtures.
To extract of genomic DNA from blood samples the protocol according to Miller et al. (1988) has been used. Subsequently, the concentration and purity of genomic DNA were analysed based on the spectrophotometry measurements by the optical density at wave length of 260 nm. The polymorphism in MYF4 gene was analysed according to Soumillion et al. (1997) using restriction endonuclease MspI. The products of PCR reaction and restriction fragments have been separated and visualised using horizontal electrophoresis in 2% agarose gels (130 V for 50 min) and stained with day GelRed.
The genotypic structure of population and allele frequencies have been determined using Genalex version 6.1 (Peakall and Smouse, 2012). The Hardy-Weinberg equilibrium in population was tested based on the significance between observed and expected genotype frequencies using Chi-square test. The diversity indices including observed (H o ) and expected heterozygosity (H e ), effective allele numbers (N e ), polymorphic information content (PIC), and F IS index was calculated using Genalex version 6.1 (Peakall and Smouse, 2012).
The effect of MYF4 has been studied in relation to the back fat thickness (BFT), lean meat percentage (LM), thigh percentage (TP) and MLT area. Each of these carcass and meat quality indicators was measured according to Slovak standard technical norm (STN 466164). The associations between locus in MYF4 gene and selected production parameters have been analysed using One-way ANOVA procedure incorporated in SAS software environment (SAS Institute Inc., 2009).

RESULTS AND DISCUSSION
In analysed population of Large White x Landrace crossbreeds the prevalence of homozygous AA genotype was found (59%), whereas the AB heterozygotes reached the level of 33%. The lowest proportion was observed for BB homozygotes (8%). The A allele was more frequent (0.75) than B allele (0.25). The higher proportion of homozygotes across all evaluated individuals was reflected in the decrease of H o and H e heterozygosities (0.33 and 0.39). The differences between observed and expected genotype frequencies have been only non-significant (P<0.05) that indicated the Hardy-Weinberg equilibrium in population. The value of N e (1.59) signalised unbalanced alleles activity and observed value of polymorphic information content (0.38) showed medium level of polymorphism according to Botstein et al. (1980). The positive value of F IS index (0.12) and also relative high value of observed homozygosity (0.67) indicated the deficiency of heterozygous animals compared to the Hardy-Weinberg expectations. The value of F IS index, which is also considered as molecular equivalent of individual inbreeding coefficient with respect to the population, reflected in analysed population the increase of homozygosity resulting probably from the higher animal's relatedness and signalized the risk of inbreeding increase in the future generations.
The effect of MYF4 gene polymorphism has been analysed in association to four indicators reflected carcass and quality of pork meat: back fat thickness, lean meat percentage, thigh percentage and MLT area. Table 1 shows the observed average values of analysed traits in relation to the MYF4 genotypes. Contrary to the expectations resulting from previously published studies (Verner et al., 2007;Civaňova and Knoll, 2007;Stupka et al., 2012) all of the MYF4 genotypes showed only non-significant (P<0.05) impact on selected production traits. The associations of MYOD genes family with pork quality has been previously investigated in various pig populations by several studies (Soumillion et al., 1997;Kapelañski et al., 2005;Cinar et al., 2012;Stupka et al., 2014). The MyoD genes were tested as candidate genes with an expected, significant effect principally on the muscle deposition (Kłosowska et al., 2004). The genetic variation in the MYF4 gene has been associated mainly with differences in myoblast and myofibre numbers (Soumillion et al., 1997;Kim et al., 2009). Te pass et al. (2004 revealed that the level of MYF4 gene mRNA expression is localized mainly in red muscles in animals at slaughter and is associated with muscle fibre type. Gerześ et al. (2010) and Stupka et al. (2014) reported significant effect of MYF4 polymorphism on the number of muscle fibres per area unit in pigs. Some authors also showed that the MYF4 gene had also a tendency to regulate backfat thickness (Civaňova and Knoll, 2007). The animals with AA genotype were associated with increase birth weight, growth rate and content of lean meat (Te Pass et al., 1999). In the Czech Large White population was found the significant association between MYF4 gene polymorphism and back fat thickness (Verner et al., 2007). Based on this and due to the polygenic character of selected phenotypic traits the involvement of other candidte genes mainly from MYOD family and increase of sample size is in the future studies needed in order to clarify the role of MYF4 gene in porcine carcass and meat quality regulation.

CONCLUSIONS
In analysed population of Large White x Landrace crossbreeds the prevalence of homozygous AA genotype was found, whereas the lowest proportion was observed for BB homozygotes. The lower proportion of heterozygotes within population was reflected in the decrease of genetic variability represented by FIS index and heterozygosity. Moreover, the FIS index as molecular equivalent of inbreeding coefficient signalized for the analysed population the risk of animal's relatedness increase in the next generations. In the contrary to the expectations resulted from the previously reported studies the MYF4 genotypes showed only non-significant (P<0.05) impact on all of the analysed production traits. In the future, the involvement of other candidate genes and increase of sample size could clarify the role of MYF4 gene in porcine carcass and meat quality regulation.

ACKNOWLEDGEMENT
This work has been supported by the Slovak Research and Development Agency (Contract No. APVV-14-0054 and No. APVV-0636-11).