Genetic variability of the CRC and MYF4 genes in genetic resource, Přeštice Black-Pied pig

The study of molecular genetic variability in genetic resources of farm animals is necessary for optimal conduct of their breeding and for understanding of relations between molecular markers and traits. The determination of variability in the CRC and the MYF4 genes and the evaluation of associations between these genetic markers and the total number born, the number born alive and the number weaned of piglets, weaning weight of piglets and farrowing interval were carried out in population of 102 Přeštice Black-Pied sows. The frequency of the allele CRC (0.083) was found out to be markedly lower than the frequency of the allele CRC and there was observed no sow of homozygous recessive genotype in the investigated sample population. In MYF4 locus, the higher frequency of the MYF allele was detected (0.711). A significant influence of the CRC gene polymorphism on number of piglets alive born in the 1 and the 1 6 litters was discovered. In the total number of born piglets the significant disparity were ascertained between the MYF4/MYF4 and MYF4/MYF4, MYF4/MYF4 and MYF4/MYF4 sows in the 2 6 litters and the 1 6 litters. The obtained data are exploitable in genetic structure mapping, management of breeding and improvement of this breed and comparison with other pig genetic resources.


Introduction
One of goals of animal genetics is to locate and identify the loci that are responsible for economical important traits.Genetic resources of farm animals are very interesting branch of research, because these populations are not influenced by selection as much as the commercial hybrid lines.For preservation and conservation is needful to study genetic polymorphisms and their associations with production traits, as in improved breeds of pigs.
In the Czech Republic, the Přeštice Black-Pied pigs (PBP) have been declared as the genetic resource in 1991.At present, there are approximately 500 sows PBP bred in the Czech Republic, 350 of which are included in the population of genetic resources.These pigs are bred mainly in West Bohemia.The following 16 breeds have participated in their genetic improvement: local Bavarian pigs, Large White, Middle White, Swabian -Hall, Berkshire, Large Black, Suffolk, Sussex, Essex, Mirgorod, Livny, Wessex Saddleback, Pietrain, Welsh, Landrase and Hampshire (FIEDLER and SMITAL, 2001).The Přeštice Black-Pied pigs are characterised by high fertility and excellent maternal properties but their disadvantage is higher back-fat thickness, lower % of lean muscle and worse conversion of fodder.The effect of the CRC gene (ryanodine receptor 1) on the manifestation of malignant hyperthermia syndrome (MHS), carcass traits and meat quality in pigs has been studied in detail (FUJII et al., 1991;FISHER and MELLETT, 1997;de SMET et al., 1998).In the CRC N /CRC N genotype higher pH 1 and pH 24 , backfat thickness and average daily gain and lower carcass lean were discovered (LARZUL et al., 1997;KUHN et al., 1998;KŘENKOVÁ et al., 1999;TOR et al., 2001).The sows of CRC n /CRC n genotype achieve lower total number of born piglets, number of piglets born alive, number of weaned piglets and number of litters per life (WITTMANN et al., 1992;DVOŘÁK, 1994).Likewise , the negative influence of CRC n allele on sperm quality traits (volume, sperm concentration, mobility) in boars has proven (GREGOR and HARDGE, 1995;URBAN and KUCIEL, 2001).The myogenin gene (MYF4), as well as MYF3, MYF5 and MYF6 genes, is a member of MYOD gene family (WEINTRAUB et al., 1991).The MYOD gene family controls a muscle fibre formation during embryonic development in mammals; the myogenin together with the MYF3 gene induce the terminal transformation of myoblasts into myofibers (te PAS and VISSCHER, 1994).In the MYF4 gene three MspI polymorphic sites, in the promoter region, the second intron and the 3´side of the gene, have been observed (ERNST et al., 1993;SOUMILLION et al., 1997).The polymorphism at the 3´side of the MYF4 gene impacts on carcass traits and lean meat content in pigs.For example, CIESĹAK et al. (2000) noted, that MYF4 A /MYF4 A homozygous animals obtain significantly higher half carcass meat weight, meat (%), ham meat weight, ham meat ratio, loin meat weight, loin meat ratio and loin eye area.Not much is known about the relationship between the polymorphism at the 3´side and fertility in pigs.It is associated with birth weight and mortality of piglets (te PAS et al., 1999).KANIAK -POLOK et al. (2001) described differences in several reproductive traits (e.g.number of piglets born alive, number of piglets at the 21 day, farrowing interval) between the sows of MYF4 A /MYF4 A and MYF4 B /MYF4 B genotypes.The primary target of this study is to assess the genetic variation within Přeštice Black-Pied pigs by means of molecular genetic markers CRC and MYF4.A secondary aim is to evaluate the associations between polymorphism of these genes and reproduction traits in sows.

Materials and Methods
In the investigated population, there were included 102 Přeštice Black-Pied sows from several pedigree breedings.The genomic DNA was isolated from blood samples by using QIAamp®DNA Blood Mini Kit (QIAGEN GMBH).
The particular parts of the CRC and MYF4 gene sequences were amplified by polymerase chain reactions as described by BRENIG and BREM (1992) and SOUMILLION et al. (1997), respectively.The predicted sizes of PCR products were 134 bp (the CRC gene) and 353 bp (the MYF4 gene).For the detection of mutant alleles the RFLP method was used.The restriction of the CRC gene PCR product was carried out by endonuclease Hin6I (FUJII et al., 1991).If the restriction site is present (wild allele CRC N ), the PCR product is cut into two fragments (84 and 50 bp).In the presence of the mutant allele (allele CRC n ), the PCR product stays intact.The PCR product of the MYF4 gene is digested by endonuclease MspI into 219 bp and 134 long fragments (allele MYF4 A ).No restriction site is involved in the allele MYF4 B .Therefore, the PCR product is not cleaved (SOUMILLION et al., 1997).The polymorphism degree of these candidate loci was determined by rate of heterozygosity (NEI, 1978) and polymorphism information content -PIC (BOTSTEIN et al., 1980).A general linear model (SAS, 2000) was used for the analysis of the associations between polymorphisms in the CRC and MYF4 genes and total number of born piglets -TNB, number of piglets born alive -NBA, number of weaned piglets -NW, weaning weight of piglets (at the age of 21 days) -WW and farrowing interval -IF.Because of low significance of the 1 st litters, we evaluated the effect of CRC and MYF4 genotypes on reproductive traits in sows for the 1 st , the 2 nd -6 th and the 1 st -6 th litters separately.In the fixed effect were included: genes MYF4, CRC, herd, year of sow birth and litter parity.The models were as follows: for the 1 st litters Y ijkl = µ + MYF4 i + CRC j + H*Y k + e ijkl and for the 2 nd -6 th and the 1 st -6 th litters where: Y ijkl (m) -trait value , µ -general mean, MYF4 i -the effect of MYF4 genotype (i = 1, 2, 3), CRC j -the effect of CRC genotype (j =1, 2) , H*Y k -the effect of the interaction between the herd and the year of sow birth (k = 1, 2, 3, 4, 5, 6, 7, 8, 9 ), L lthe effect of the litter parity (l = 1, 2, 3, 4, 5, 6), e ijkl(m) -random residual

Genetic variability in the CRC and MYF4 gene
In our study a lower frequency of the allele CRC n (0.083) than the frequency of the allele CRC N in PBP sows was observed.There was no sow with genotype CRC n /CRC n in the investigated population.The frequency of dominant homozygous genotypes CRC N /CRC N was very high compared to genotypes CRC N /CRC n (Table 1).The frequency of the allele MYF4 B was found to be higher than the frequency of allele MYF4 A .There were more sows with MYF4 B /MYF4 B genotypes than MYF4 A /MYF4 B heterozygotes in the population of PBP.The frequency of genotypes MYF4 A /MYF4 A was clearly the lowest (Table 1).CRC locus showed lower heterozygosity and polymorphism information content than MYF4 (Table 2), because of missing recessive homozygotes CRC n /CRC n .Associations of the polymorphism in the CRC gene with reproduction traits In the 1 st litters the polymorphism in the CRC gene influenced significantly NBA (P ≤ 0.05).The dominant homozygous sows showed also higher TNB, NW and WW than heterozygous sows (Table 3).No significant differences were found out between CRC N /CRC N and CRC N /CRC n genotypes in the 2 nd -6 th litters, although homozygous sows were better in TNB, NBA, NW and WW as well as in the 1 st litters.However, the sows of CRC N /CRC n genotypes had shorter farrowing interval.The significant associations (P ≤ 0.05) between polymorphism in the CRC gene and NBA were also observed in a joint analysis of the 1 st -6 th litters.
Associations of the polymorphism in the MYF4 gene with reproduction traits The highest TNB, NW and WW in the 1 st litters were recorded in heterozygous sows.The MYF4 B /MYF4 B sows produced most born alive piglets but no differences were significant (Table 3).In the 2 nd -6 th litters, we determined significant differences between sows of MYF4 A /MYF4 A and MYF4 B /MYF4 B genotypes (P ≤ 0.01) and between sows of MYF4 A /MYF4 A and MYF4 A /MYF4 B genotypes (P ≤ 0.001) in TNB.Homozygous MYF4 A /MYF4 A sows excelled in number of born alive piglets.NW and WW from MYF4 B /MYF4 B sows were higher than those from other genotypes.The longest farrowing interval was found out in heterozygous sows.
The similar results we detected in the 1 st -6 th litters, where significant differences (P ≤ 0.01) in TNB between sows of MYF4 A /MYF4 A and MYF4 B /MYF4 B genotypes as well as between sows of MYF4 A /MYF4 A and MYF4 A /MYF4 B genotypes were evaluated.

Discussion
The absence of CRC n /CRC n genotypes and low frequency of the recessive allele in CRC loci were surprising.On the basis of references, we supposed the incidence of the CRC n allele in PBP sows is more frequent.For instance, ČEPICA et al. (1982) described that an occurrence of this allele is 0.348 (more recent data about PBP are not available).The substantial reduction of the recessive allele numerousness must have been caused by efforts of exclusion of CRC N /CRC n and CRC n /CRC n genotypes from reproduction in pedigree breedings that were performed despite declaration of Přeštice Black-Pied pigs as the gene reserve.Thereby, the immigration of CRC n alleles from Landrase breed that was used for the genetic improvement of PBP in 1986(KLUSÁČEK et al., 1991), could not increase the frequency of the recessive allele in PBP sows either.If we compared our data with references about the CRC gene variability in Landrase (L) and Large White (LW) breed in the Czech Republic, we found slight differences only.BEČKOVÁ et al. (2002) andMATOUŠEK et al. (2003) noted the frequency of the recessive allele 0.01 in Landrase sows and 0.025 -0.045 in Large White sows.However, the incidence of the CRC n allele in L and LW breed was far higher a few years ago -0.22 and 0.06, respectively (KAHÁNKOVÁ et al., 1996).Well, just as the frequency of the recessive allele has dropped in L and LW breed, so it has in Přeštice Black-Pied sows.There are no original data about the MspI variability of the MYF4 gene in Přeštice Black-Pied pigs accessible, so we could not assess if changes of genotypes and alleles frequencies have happened within evolution of this breed.It is remarkable that similar results (MYF4 B = 0.68) were obtained in autochthonous breed -Zlotnicka Spotted in Poland (CIESĹAK et al., 2000).In contrast to the PBP breed, there is more frequent occurrence of the MYF4 A allele than the MYF4 B allele in L (0.545 -0.680) and LW (0.750) breed (KOLAŘÍKOVÁ et al., 2002;PUTNOVÁ et al., 2001).These dissimilarities may be explained by more stringent selection to carcass characteristics and meat quality in L and LW breed, because the MYF4 A allele conditions a superior level of these traits (te PAS et al., 1999).
It is obvious from Table 2, that CRC and MYF4 loci demonstrate a different degree of polymorphism measured by heterozygosity and by polymorphism information content in the PBP population.This finding can be elucidated by marker assisted selection implemented in the CRC gene as opposed to the MYF4 gene.The evaluation of associations between the variability in the CRC gene and reproductive traits in PBP sows proved the outcomes in other maternal breeds (DVOŘÁK, 1994;KMIEĆ et al., 2002), because the sows of CRC N /CRC N genotypes showed the higher TNB, NBA and NW than the heterozygous ones.Piglets of MYF4 A /MYF4 A genotypes have greater birth weight, which decreases their mortality (te PAS et al., 1999).Therefore, we expected the sows of MYF4 A /MYF4 A genotypes would achieve more weaned piglets compared to MYF4 B /MYF4 B or MYF4 A /MYF4 B genotypes.These assumptions were not shown to be correct in this study.The homozygous MYF4 A /MYF4 A sows surpassed the sows of MYF4 A /MYF4 B and MYF4 B /MYF4 B genotypes in TNB and NBA in the 2 nd -6 th and the 1 st -6 th litters.Nevertheless, in NW as well as in WW were the sows of MYF4 B /MYF4 B genotypes the best.Some researches point out potential associations of the polymorphism at the 3´side of the MYF4 gene on fertility (KANIAK -POLOK et al., 2001), but this hypothesis neither has been confirmed nor has been disproved so far.That said data could assist in genetic structure assessing, the proposal of measures concerning the breeding and improvement of this breed as well as for comparison with other genetic resources of pigs.The loci described here are utilized for this purpose well.The increment of loci number and inclusion of all chromosomes in the evaluation are essential (WITTMANN and DOHY, 1999) for proper genetic appraisal of Přeštice Black-Pied pigs.

Table 3 Associations
between reproduction traits and genotypes of the CRC gene and the MYF4 gene in Přeštice Black-Pied sows (least-square means LSM ± standard error S E ) (Beziehungen zwischen den Wurfleistungsmerkmalen und Genotypen der Gene CRC und MYF4 der untersuchten Sauen (LSM und S E ))Note: NL-number of investigated litters, TNB-total number of born piglets, NBA-number of piglets born alive, NW-number of weaned piglets, WW -weaning weight of piglets (at the age of 21 days), IF -farrowing interval; Values with superscripts in columns show significant differences: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001