Relations between the polymorphism in the coding and 5 ’-flanking regions of the porcine MYOD 1 and MYF 5 genes and productive traits in pigs *

The MyoD family genes were considered as candidate genes for growth rate and carcass meatiness in pigs. Gene mutations (SNPs, single nucleotide polymorphisms) discovered in the coding and 5’-flanking regions of the MYOD1 and MYF5 were analysed as possible causal mutations for these traits. Studies of the relation between polymorphisms in both those genes and productive traits were performed on Polish Landrace and Polish Large White gilts. A total of 401 animals (185 Polish Large White and 216 Polish Landrace) was encompassed by the analyses. Homozygotes of the “wild” allele as regards all three mutations of the MYOD1 taken into consideration in the present study appeared to be more profitable for traits characterizing carcass meatiness than the two remaining genotypes. In turn, heterozygotes for mutations identified in the MYF5 gene proved to be most favourable in terms of carcass meatiness. The effect of MyoD genotypes on performance traits was observed to be similar irrespective of the animals’ breed. This may suggest that new mutations, identified in the coding and 5’-flanking regions of MYOD1 and MYF5 genes, could be more useful for selection than the earlier known mutations in the non-coding regions of these genes. In our opinion, two mutations identified in exon 1 of the MYOD1 gene could be most beneficial and useful in selection and breeding of pigs.


INTRODUCTION
Given the critical function of myogenic regulatory factors (MRFs) in skeletal muscle cell specification, their genes might be considered as candidate genes affecting the meat content of pig carcasses (te Pas and Visscher, 1994).The MRF family consists of four factors: MyoD1 (Myf-3), , coded by genes MYOD1 (MYF3), MYF5, MYOG and MRF4 (MYF6), respectively, known as the MyoD family genes.The expression of MyoD genes takes place exclusively in skeletal muscles.Thus it has been suggested that the MRF transcription factors play key regulatory roles in the development of the skeletal muscle lineage (Weintraub, 1993).Investigations of the MRF family members during myogenesis have been performed predominantly on rodents.Proteins Myf-3 and Myf-5 are thought to play an important role at the level of myoblast determination and proliferation, whereas myogenin and Myf-6, at the level of myoblast differentiation (Buckingham, 1994).The functional cooperation and redundancy between Myf-3 and Myf-5 factors was investigated by Kablar et al. (1997).Mice lacking a functional MYOD1 gene were found to have no overt abnormalities in skeletal muscle but expressed about a four-fold higher level of Myf-5.Similarly, newborn Myf-5 deficient mice display apparently normal skeletal muscle but die because of severe rib abnormalities.Newborn animals deficient for both Myf-3 and Myf-5 factors are devoid of skeletal myoblasts and muscle (see review, Megeney et al., 1996).
Postnatal expression of the MYOD1, MYF5 and MYOG genes in muscle is much lower than prenatal expression and has been found only in satellite cells (Koishi et al., 1995).Satellite cells proliferate and differentiate, thereby enabling postnatal muscle growth (Beilharz et al., 1992).te Pas and Visscher (1994) also suggested that the MyoD genes could have a major effect on muscularity and growth.
A relation between the polymorphism identified in non-coding regions of MYOD1 and MYF5 genes (region 3' and intron 1, respectively) and carcass traits has already been reported (te Pas et al., 1999;Cieślak et al., 2000Cieślak et al., , 2002;;Kurył et al., 2002).Moreover, it was shown that the effect of genotype at these loci on carcass quality depended on pig breed.This indicated that mutations in noncoding regions of both genes could not be viewed as causal mutations but as markers of other mutations within MYOD1 and MYF5 or other genes linked to them.
The objective of this study was to evaluate the effect of new mutations, which have already been reported (Urbański and Kurył, 2004a,b) and identified in the coding and 5'-flanking regions of the MYOD1 and MYF5 genes, on growth rate and traits characterizing meat deposition in pig carcasses.URBAŃSKI P.. ET AL.

MATERIAL AND METHODS
The analysis was conducted on 401 gilts of two breeds, Polish Landrace (PL) and Polish Large White (PLW).Between 25 and 100 kg of body weight they were fed a commercial mixed feed ad libitum.Right carcass sides were dissected according to the procedure described by Różycki (1996).For the analyses the average daily gain (ADG) was taken into consideration as well as nine carcass traits: weight of right carcass side (WCS), weight of ham (HW), loin (WL) and sirloin (WSL), loin eye width (LW), height (LH) and area (LA), meat content in valuable cuts (MCVC) and in carcass (MCC).
Genomic DNA was isolated from leukocytes according to Kawasaki (1990).The RYR1/HinP1 genotypes were identified using a sequence of primers according to Kamiński et al. (2001), whereas those at the MYOD1 and MYF5 loci, according to Urbański and Kurył (2004a, b).The sequences of the MYOD1 and MYF5 genes presented by Chang et al. (1995) and te Pas et al. (1999), respectively, were termed the "wild" allele.
Association analyses were carried out for each breed separately using the least squares method of the GLM procedure (SAS 8.2, 2002).The model included a fixed effect of the RYR1 genotype (Table 1) and the boar effect (sire groups ranged from 1 to 27 animals).Age at slaughter and weight of right carcass side were included as covariates.
Differences between pig groups (with highest and lowest meat content in carcass and greatest and smallest loin eye area) for the frequency of alleles at the MYOD1 and MYF5 loci were evaluated according to Weber (1986).

RESULTS
Three point mutations (SNPs, single nucleotide polymorphisms) in each of the MYOD1 and MYF5 genes were taken into consideration.They were located in the 5'UTR region (G302A) and exon 1 (C489T and G566C) of the MYOD1 gene, the 5'-flanking region (A65C and C613T) and exon 3 (C2931T) of the MYF5 gene (Urbański and Kurył, 2004 a,b).
Table 1 shows the frequency of genotypes at loci MYOD1 and MYF5 concerning the mutations identified within these genes as well as RYR1 genotypes in both breeds.The frequency of genotype CC was low at locus MYOD1 (mutation G566C) in both PLW and PL breeds (1.4 and 0.7 %, respectively).For this reason it was not taken into consideration in a further analysis on association with traits.Absence of genotype TT at the MYF5 locus (mutation C613T) was observed in both of the tested breeds, as was a low frequency of heterozygotes in PL gilts.The effect of the RYR1 genotype on productive traits within PLW and PL gilts analysed in this study appeared to be insignificant.
The results of the association analyses between genotypes at the MYOD1 and MYF5 loci and growth rate and carcass traits of PLW gilts are presented in Table 2.
A significant relation was found between the genotype at the MYOD1 locus and several carcass traits.A comparison of carcass sides from animals with genotypes GG, GA and AA as regards 302nt located in the 5'UTR region of exon 1 showed the highest weight of carcass side (WCS) and ham (WH), as well as a higher value of loin eye height (LH) and area (LA) in GG homozygotes.A similar tendency was observed when analysing the relationship between carcass traits and genotype at the two remaining MYOD1 gene mutations.Homozygotes of the "wild" C allele regarding 489nt showed a significantly higher weight of ham (WH) and carcass meat content (MCC) than TT homozygotes; this difference amounted to 0.4 kg and 2.15%, respectively.Homozygotes of the "wild" G allele as regards 566nt also proved more profitable than heterozygotes for weight of loin (WL) and size of loin eye (width -LW, height -LH and area LA), meat content in valuable cuts (MCVC) and in carcass (MCC).CC homozygotes were not included in the analysis because this genotype was found in only two gilts of the PLW breed.Mutations identified in the 5' flanking region and in exon 3 of the MYF5 gene also affected some carcass traits characterizing carcass meat deposition.All of these mutations affected loin eye area (LA).Moreover, loin weight (WL) and carcass meat content (MCC) were significantly affected by genotype at the 65nt (5'-flanking region).In all cases, the value of traits was lowest in heterozygotes (compared with both homozygous genotypes).Of interest is the relation between the genotype at the MYF5 locus as regards mutation A65C and loin eye area -as it was observed that in CC gilts it was 5.33 cm 2 greater than that observed in heterozygotes.
The associations between genotype at the MYOD1 and MYF5 loci and productive traits in PL gilts were similar to those described in PLW pigs (Table 3).As a rule, homozygotes of the "wild" allele regarding individual mutations of the MYOD1 gene showed the highest value of carcass traits (excluding width of loin eye in animals with the GG genotype as regards 302nt) of all the animals analysed.On the other hand, heterozygotes as regards mutations of the MYF5 gene, proved to have the lowest weight and size of loin (WL, LW, LA) as well as carcass meat content (MCC).Of particular interest was the effect of an individual mutation in the MYF5 gene on the loin eye area because it led to a difference between individual genotypes that ranged from 2.26 to 5.92 cm 2 , depending on the mutation (A65C and C613T, respectively).
Groups of animals with the highest (>63%) and lowest (56%) carcass meat content were selected from gilts of each breed separately, basing on the mean value of the trait (x±SD).The frequency of genotypes regarding individual mutations within the MYOD1 and MYF5 genes was compared between these groups of pigs.They did not differ in frequency of MYF5 genotypes.The frequency of individual MYOD1 genotypes did, however, differ between groups of PL and PLW pigs (Table 4).The share of homozygotes of the "wild" allele of all mutations identified within the MYOD1 gene was significantly higher among pigs showing a carcass meat content exceeding 63% when compared with those with the lowest value of this trait (<56 %).A similar relation was observed for mutation G566C within the PLW breed.
An analogous analysis was performed to evaluate the share of individual MYOD1 and MYF5 genotypes within two groups of pigs with the greatest (> 62 cm 2 ) or smallest (<49 cm 2 ) loin eye area (Table 4).Each of the two breeds examined in the present study was analysed separately.No significant differences in the frequency of individual MYF5 genotypes were identified between groups of pigs differing in loin eye area.Homozygotes of "wild" alleles for all mutations within the MYOD1 gene showed a significantly higher frequency in the group of  PL pigs with loin eye area exceeding 62 cm 2 as compared with animals in which the value of this trait lower than 49 cm 2 .A similar tendency was also observed among PLW pigs.

DISCUSSION
The MYOD1 and MYF5 genes belong to the MyoD family, coding for myogenic regulatory factors.Both of those genes were selected as potential candidate genes affecting carcass meatiness due to their role in muscle development.Moreover, on pig chromosome 2, QTL for lean meat content was mapped in the region encompassing the MYOD1 locus (Lee et al., 2003).In our earlier study, an analysis was made of the relation between the polymorphism in non-coding regions of both genes MYOD1 and MYF5 and carcass traits (Cieślak et al., 2000(Cieślak et al., , 2002;;Kurył et al., 2002).The observed significant or highly significant effect of genotype at these loci on the meat content in ham, loin and carcass was shown to depend on the pig breed or line.Thus, it was suggested that mutations of the analysed genes could not be accepted as causing the differences in the value of carcass traits observed between various MYOD1 or MYF5 genotypes.On the other hand, those results rendered it possible to assume that causative mutations could be localized in other regions of the MYOD1 and MYF5 genes.te Pas et al. (1999) failed to find a significant relation between the polymorphism identified in intron 1 of the porcine MYF5 gene and several performance traits obtained for Yorkshire pigs (birth weight, growth rate, weight at slaughter corrected for slaughter age, carcass meat weight, mean back fat thickness from 4 ultrasonic measurements between shoulder and last rib).
Recently, we identified new polymorphisms (SNPs, single nucleotide polymorphisms) in the coding and 5'-flanking regions of the porcine MYOD1 and MYF5 genes (Urbański and Kurył, 2004a,b).The G566C mutation in exon 1 of the MYOD1 gene leads to a replacement of arginine with proline, whereas the C2931T transition in exon 3 of the MYF5 gene results in a change of the amino acid sequence Leu→Pro, which may affect the functional properties of both proteins.Another four SNPs were found in the 5'-flanking regions of both genes.The analysis presented here shows a significant association between individual MYOD1 and MYF5 genotypes and the recorded carcass traits.Homozygotes of the "wild" alleles for mutations of the MYOD1 gene appeared to be more profitable than the remaining genotypes.These relations were similar in all the pig breeds analysed.On the other hand, homozygotes of the wild allele for transition A65C in the 5'-flanking region of MYF5 gene seemed to be unprofitable as regards loin eye size, but beneficial for loin weight and carcass meat content in both analysed pig breeds.The share of homozygotes of the "wild" alleles for all SNPs identified in the MYOD1 gene was significantly higher in the group of PLW and PL pigs showing a high carcass meat content (>63%) and greatest loin eye area (>62cm 2 ) than in the group with a low value of these traits (<56 and 49 cm 2 , respectively).
In the present study, heterozygotes in terms of a mutation of the MYOD1 and MYF5 genes demonstrated the highest or lowest values for given carcass traits when compared with both homozygous genotypes.This phenomenon has been observed for certain human genes and is termed negative-or positive heterosis (Comings and MacMurray, 2000).These authors suggested that if the regulation of the gene is dose dependent, the presence of a regulatory sequence in a heterozygous state could modify the gene function.
In summarizing the results presented in this study, it should be emphasized that if a particular MYOD1 or MYF5 gene variant had a beneficial effect on the value of any trait, it was observed in both tested breeds.No opposite effects of the same gene variant on a particular trait in different breeds were observed, as has been reported in an earlier study concerning the relation between slaughter traits and polymorphisms in non-coding regions of the MyoD genes (Cieślak et al., 2000).This makes it possible to conclude that the effect of the mutations in the MYOD1 and MYF5 genes analysed in this study is similar to that characteristic for causal mutations.Linkage between the analysed SNPs herein and an unknown gene mutation affecting carcass traits should also be taken into consideration.

CONCLUSIONS
The presented results indicate that identifying the genotype concerning point mutations in the coding and 5'-flanking regions of the porcine MYOD1 and MYF5 genes may be useful for selection aimed at improving the value of traits characterizing carcass meat deposition.Moreover, the fact that the relationship between genotype and trait value showed a similar tendency irrespectively of the breeds tested indicates that the associations observed in the present study may also be true for other pig breeds.

Table 1 .
Frequency of genotypes at loci MYOD1, MYF5 and RYR1 in Polish Large White and Polish Landrace pigs

Table 2 .
Relation between SNPs in the porcine genes MYOD1 and MYF5 and carcass traits of Polish Large White pigs -contrasts ± SE WCS -weight of right carcass side; WH -weight of ham; WSL -weight of sirloin; WL -weight of loin; LW -width of loin eye; LH -height of loin eye; LA -loin eye area; MCVC meat content in valuable carcass cuts; MCC -meat content in carcass

Table 3 .
A relations between SNPs in the porcine genes MYOD1 and MYF5 and carcass traits of Polish Landrace pigs -contrasts ± SE