Meat Science 80 (2008) 780–787

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Meat Science

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Investigation of candidate genes for glycolytic potential of porcine skeletalmuscle: Association with meat quality and production traitsin Italian Large White pigs

L. Fontanesi a,*, R. Davoli a, L. Nanni Costa a, F. Beretti a, E. Scotti a, M. Tazzoli a, F. Tassone a,M. Colombo a, L. Buttazzoni b, V. Russo a

a DIPROVAL, Sezione di Allevamenti Zootecnici, Faculty of Agriculture, University of Bologna, Via F.lli Rosselli 107, 42100 Reggio Emilia, Italyb Associazione Nazionale Allevatori Suini (ANAS), Via Lazzaro Spallanzani 4/6, 00161 Roma, Italy

a r t i c l e i n f o

Article history:Received 22 December 2007Received in revised form 23 March 2008Accepted 24 March 2008

Keywords:Candidate genesPRKAG3PGAM2PKM2Meat quality and production traits

0309-1740/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.meatsci.2008.03.022

* Corresponding author. Tel.: +39 0522 290516; faxE-mail address: luca.fontanesi@unibo.it (L. Fontan

a b s t r a c t

The objective of this study was to investigate the association of DNA markers in candidate genes for gly-colytic potential on meat quality parameters (pH1, pHu, glycogen and lactate content and glycolyticpotential of semimembranosus muscle) and estimated breeding values (EBVs) for average daily gain, leancuts, back fat thickness, ham weight, and feed:gain ratio in 272 Italian Large White pigs. Three mutationsin the PRKAG3 gene (T30N, G52S and I199V) were investigated as well as single nucleotide polymor-phisms in two other skeletal muscle genes (PGAM2 and PKM2) involved in the glycolytic pathway. Asso-ciation analysis with the PRKAG3 markers showed significant results (P < 0.05) only for pH1 (I199V, withsignificant additive effect) and lactate content (T30N), confirming, at least in part, the effects of this geneon meat quality traits. Significant association (P < 0.05) was also observed for PGAM2 and ham weightEBV with significant additive and dominance effects. PKM2 was associated with average daily gain, leancuts (P < 0.001), back fat thickness and feed:gain ratio (P < 0.05), with significant additive and/or domi-nance effects on these traits. PKM2 encodes for a key enzyme of the muscle glycolytic pathway and mapson porcine chromosome 7 where other studies have reported important QTL for the same traits. Thesedata might suggest an important function of this gene in the mechanisms that produce the observedeffects. The results will be important to evaluate the inclusion of some of these DNA polymorphismsin marker assisted selection programs.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Meat production and quality traits in pigs are largely affected bygenetic factors (for reviews see: Clutter & Brascamp, 1998; Sellier,1998) that have been the matter of an increasing number of studiesaimed at identifying the causative mutations responsible for theirintra and inter line or breed variability (Rothschild, Hu, & Jiang,2007). Genes and mutations affecting economic traits have alreadybeen proposed in marker assisted selection plans in pigs and othersuccessful applications might derive from the identification of newmutations and their inclusion in advanced selection approaches(Rothschild & Plastow, 1999; van der Steen, Prall, & Plastow, 2005).

Two major genes, ryanodine receptor 1 (RYR1; the Halothanelocus) and protein kinase adenosine monophosphate activatedc3-subunit (PRKAG3; the Rendement Napole locus, RN), have beenalready shown to cause the meat defects known as pale, soft, exu-dative (PSE) meat and acid meat, respectively (Fujii et al., 1991;

ll rights reserved.

: +39 0522 290523.esi).

Milan et al., 2000). On the other hand, these loci have positive ef-fects on lean muscle deposition (Enfält, Lundström, Karlsson, &Hansson, 1997; Hamilton, Ellis, Miller, McKeith, & Parrett, 2000;Le Roy et al., 2000).

The RYR1 g.1843C>T mutation has a large impact on pig produc-tion because of its widespread diffusion in many breeds and lines(Houde, Pommier, & Roy, 1993; O’Brien, Shen, Cory, & Zhang,1993; Russo, Dall’Olio, Davoli, Coscelli, & Bigi, 1996) and its influ-ence on several aspects of pig breeding and meat processing (NanniCosta, Lo Fiego, Dall’Olio, Davoli, & Russo, 1999; Nanni Costa, LoFiego, Dall’Olio, Davoli, & Russo, 2002; Pommier & Houde, 1993;Tor et al., 2001). Thus, controlled cross-breeding or eradication ofthe negative recessive allele have been proposed and carried outin several pig populations (Russo & Nanni Costa, 1995).

The PRKAG3 200Q mutation responsible for the RN� allele at theRendement Napole locus has been identified only in the Hampshirebreed or in pig lines with Hampshire blood (Meadus, MacInnis, Du-gan, & Aalhus, 2002; Milan et al., 2000; Moeller, Baas, Leeds, Em-nett, & Irvin, 2003). Then, other mutations of the PRKAG3 gene(T30N, G52S and I199V; nomenclature based on published studies

L. Fontanesi et al. / Meat Science 80 (2008) 780–787 781

on the effects of this gene on pork quality), that are not restrictedto the Hampshire breed, have been suggested to affect muscle gly-cogen content, glycolytic potential (GP), pH, meat colour and driploss (Ciobanu et al., 2001; Fontanesi, Davoli, Nanni Costa, Scotti,& Russo, 2003; Lindahl et al., 2004a, 2004b; Otto et al., 2007).

Mutations in several other genes have been associated withmeat quality parameters (Ciobanu et al., 2004; Otto et al., 2007;Reiner, Heinricy, Müller, Geldermann, & Dzapo, 2002) with sideor direct effects also on lean meat deposition, carcass and growthtraits for some of them (Davoli et al., 2006; Gerbens et al., 1999;Mercadé et al., 2006; Vidal et al., 2006; Wimmers et al., 2007).

We recently investigated twelve candidate genes for GP, chosenamong those involved in the regulation of the energy balance, gly-cogen metabolism and glycolysis of the skeletal muscle and iden-tified a few markers associated with meat quality parameters ina crossbred pig population using a selective genotyping approach(Fontanesi et al., 2003). This study confirmed the effects of twomutations (T30N, G52S) of the PRKAG3 gene on meat colour andshowed significant associations between a single nucleotide poly-morphism (SNP; g.77C>A, EMBL reference sequence: AJ557237)in the coding region of the muscle phosphoglycerate mutase 2(PGAM2) gene and an SNP (g.32T>C, EMBL reference sequence:AJ557235) in the 30-untranslated region of the muscle pyruvate ki-nase (PKM2) gene with drip loss and glycogen content,respectively.

The PGAM2 gene encodes the muscle subunit of the enzymethat catalyzes the interconversion of 2-phosphoglycerate and 3-phosphoglycerate in the glycolytic pathway. This gene is localizedon porcine chromosome 18 (Davoli et al., 2002; Fontanesi et al.,2003) in a region where quantitative trait loci (QTL) for drip loss,meat colour, fat deposition, lean content, muscle fiber diameterand carcass quality have been identified (i.e.: de Koning et al.,2001; Dragos-Wendrich et al., 2003; Malek, Dekkers, Lee, Baas, &Rothschild, 2001a; Malek et al., 2001b; van Wijk et al., 2006).

The PKM2 gene encodes for a muscle isoform of the rate limitingenzyme that catalyses the conversion of phosphoenolpyruvate topyruvate in the last step of the glycolysis pathway. The activityof this enzyme is also involved in muscle glyconeogenesis (Glee-son, 1996). This gene was localized on porcine chromosome 7(Davoli et al., 2002; Fontanesi et al., 2003) where a large numberof reports identified important QTL for meat quality, fat and leandeposition and growth traits (i.e.: Bidanel et al., 2001; Gilbert, LeRoy, Milan, & Bidanel, 2007; Malek et al., 2001a; Nezer, Moreau,Wagenaar, & Georges, 2002; Ovilo et al., 2002; Reiner et al.,2002; Rohrer & Keele, 1998; Yue et al., 2003).

In order to further investigate the effects of the candidate genespreviously shown to influence meat quality traits (Fontanesi et al.,2003), the PRKAG3 (T30N, G52S and I199V), PGAM2 and PKM2mutations were tested for association with glycolytic potentialand related meat quality parameters as well as growth, feed con-version, meat and fat deposition traits in a different pig populationof Italian Large White sib-tested animals.

2. Materials and methods

2.1. Animals and performance traits

The present study was conducted on 272 sib-tested ItalianLarge White pigs (180 females and 92 castrated males coming fromseveral half-sib families) slaughtered on six different days in thesame abattoir in 2002. The pigs were performance tested at theTest Station of the National Association of Pig Breeders (ANAS)beginning at approximately 30 kg live weight and ending at about155 kg live weight. The nutritive level was quasi ad libitum. Dailyfeed intake and bimonthly body weight measures were collected

and daily gain and feed:gain ratio calculated. Then, the animalswere mixed in one group at loading and transported to the slaugh-terhouse located about 25 km far from the Test Station. Afterunloading, the pigs were immediately stunned by CO2 (concentra-tion 87%) using a dip lift system (Butina, Denmark) and bled in alying position.

2.2. Carcass and meat quality traits

At the slaughterhouse, within 3 h post-mortem, back fat thick-ness at the level of M. gluteus medius, weight of lean cuts (necksand loins) and weight of hams were measured.

pH1 (at 2 h post-mortem) and pHu (at 24 h post-mortem) weredetermined on M. semimembranosus using a Crison pH-meterequipped with an Ingold Xerolite electrode (Mettler Toledo, Udorf,Switzerland). Samples of the same muscle were collected at about1 h post-mortem and immediately frozen in liquid nitrogen and la-ter freeze-dried.

GP analyses were performed as described by Monin, Mejenes-Quijano, Talmant, and Sellier (1987). Two separate analyses werecarried out: one for the lactate and the other for the sum of glyco-gen (degraded to glucose with amyloglucosidase from Aspergillusniger), glucose and glucose-6-phosphate using the L-Lactic acidand D-Glucose Enzymatic BioAnalysis kits (Boehringer Mann-heim/R-Biopharma, R-Biopharma GmbH, Darmstadt, Germany),respectively. GP was calculated as the sum of: 2[glycogen + glu-cose + glucose-6-phoshate] + [lactate] according to Monin and Sel-lier (1985) and expressed as lmol of lactic acid equivalent per g offresh muscle.

2.3. DNA markers

A portion of the lyophilised muscle samples collected for theanalysis of GP was used for DNA extraction as described in Sam-brook, Fritsch, and Maniatis (1989). The RYR1 g.1843C>T polymor-phic site (Fujii et al., 1991) was analysed with a DNA test using thepolymerase chain reaction (PCR)-restriction fragment length poly-morphism (RFLP) method described in Russo et al. (1993). ThePRKAG3 T30N, G52S, I199V and R200Q mutations (Ciobanu et al.,2001; Milan et al., 2000) and the PGAM2 single nucleotide poly-morphisms were analysed using the PCR-RFLP protocols alreadydescribed (Fontanesi et al., 2003). A new PCR-RFLP method wasdeveloped to analyse the mutation in the 30-untranslated regionof the PKM2 gene that was previously investigated using a PCR-sin-gle strand conformation polymorphism (SSCP) method (Fontanesiet al., 2003). PCR primers (50-AGGCGGCTGCAGTAGTCG-30; 50-GCTGCAGACAAACACTCCACCCTGC-30) were designed on sequencesAJ557235 and AJ557236 inserting a mismatched nucleotide in thereverse primer (underlined base) in order to obtain an artificialrestriction site for HhaI when allele 2 (C) was present in the ampli-fied fragment of 57 bp. Genotypes obtained by PCR-RFLP were ver-ified on animals already genotyped by PCR-SSCP. PCR was carriedout using a PT-100 (MJ Research, Watertown, MA, USA) thermal cy-cler in a final volume of 20 ll that included 10 pmol of each primer,2.0 mM MgCl2, 2.5 mM each dNTP, 1 U EuroTaq (EuroClone Ltd.,Paington, Devon, UK) DNA polymerase. The PCR profile was the fol-lowing: an initial step of denaturation for 5 min at 95 �C; 35 cyclesof 30 s at 95 �C, 30 s at 67 �C and 30 s at 72 �C; the final extensionstep was for 5 min at 72 �C. Thus, 5 ll of PCR product was digestedovernight at 37 �C with 3 U of HhaI (New England Biolabs, Beverly,MA, USA) in a final volume of 25 ll containing 1X enzyme reactionbuffer and 1X BSA. The resulting DNA fragments were separated byelectrophoresis in 15% polyacrylamide:bis-acrylamide 29:1 gelswith TBE 1X buffer. DNA products were visualized with ethidiumbromide on a UV apparatus (UVP Transilluminator) and imageswere captured with a Kodak EDAS 290 system (Kodak).

782 L. Fontanesi et al. / Meat Science 80 (2008) 780–787

2.4. Statistical analysis

Estimated breeding values (EBV) for five traits (average dailygain, ADG; lean cuts, LC; back fat thickness, BFT; ham weight,HW; and feed:gain ratio, FGR) were calculated using a BLUP-multi-ple trait-animal model. Depending on the trait, the model includedfixed effects of batch, sex, age at the beginning of test, age of sow,weight at slaughter, age at slaughter, inbreeding coefficient and therandom effects of litter, individual permanent environment, andanimal (Russo et al., 2000).

Associations between the genotypes of the candidate genes(PRKAG3 T30N, G52S and I199V; PGAM2; PKM2) and meat qualityparameters (pH1, pHu, glycogen and lactate content and GP) wereassessed using the procedure MIXED in SAS version 8.02 (SAS Insti-tute Inc. Cary, NC, USA), with a model that included sire as a ran-dom effect and the fixed effects of day of slaughtering, sex andgenotype of the analysed DNA markers (one marker for each modelrun). The general linear model (GLM) procedure of SAS, release8.02 (SAS Institute Inc.) was used to test associations betweenthe same genotypes of the considered markers and the EBVs calcu-lated for ADG, LC, BFT, HW and FGR. The model included only fixedeffects of the genotypes of the DNA markers individually analysed.All other factors contributing to the variability of the investigatedtraits were already considered in the calculation of their EBVs.

Additive genetic effect of each locus was estimated as half of thedifference between the 2 homozygous groups: a = 1/2(BB-AA),with A and B that indicate the first and the second allele of the ana-lysed markers, respectively. The dominance effect was estimatedas the difference between the heterozygous group and the averageof the 2 homozygous groups in each locus: d = AB-1/2(AA+BB). Theestimates of effects were tested by t-test on significant deviationfrom zero.

Haplotypes of the PRKAG3 gene including the T30N, G52S andI199V mutations were inferred using the algorithm developed byStephens, Smith, and Donnelly (2001). Phase probabilities of eachsite were calculated for each animal using this software. Haplo-types were analysed considering animals having 0, 1 or 2 copiesof the haplotype in question using the models applied for the singlemarker association tests.

Significant associations were declared when P < 0.05. Sugges-tive associations were considered when P < 0.10.

3. Results and discussion

3.1. Traits

Table 1 reports means and measures of variability of the meatquality traits and considered EBVs in the pigs. Results are compa-

Table 1Means, standard deviations (s.d.; in parenthesis), minimum (Min.) and maximum(Max.) values for meat quality parameters and EBVs

Traitsa Mean (s.d.) Min. Max.

pH1 5.941 (0.240) 5.41 6.80pHu 5.669 (0.212) 5.30 6.54Lactate (lmol/g) 56.374 (15.632) 23.28 116.94Glycogen (lmol/g) 47.123 (22.584) 7.33 135.74GP (lmol/g) 103.501 (22.981) 49.23 182.73ADG EBV (g) +33.321 (26.590) �65.00 +115.00LC EBV (kg) +1.981 (1.861) �2.94 +8.19BFT EBV (mm) �2.044 (3.867) �12.80 +11.30HW EBV (kg) +0.582 (0.612) �1.80 +2.63FGR EBV �0.151 (0.152) �0.64 +0.48

a GP = glycolytic potential; ADG = average daily gain; LC = lean cuts; BFT = backfat thickness; HW = ham weight; FGR = feed:gain ratio.

rable with those reported in previous studies carried out on heavypigs (ANAS, 2004; Nanni Costa et al., 2002).

3.2. RYR1 and PRKAG3 R200Q genotypes

Only one animal out of 272 showed genotype CT at the RYR1g.1843C>T polymorphic site. This pig was not included in the fol-lowing statistical evaluations. All other pigs carried the CC geno-type at this locus, confirming the almost complete eradication ofthe Halothane sensitive allele (g.1843T) in the Italian Large Whitepopulation. As a matter of fact, since the introduction of this genet-ic test in breeding programs (Russo et al., 1993), almost total elim-ination of the g.1843T allele in the Italian pure bred heavy pigpopulations has been reached (Russo et al., 1996).

All analysed pigs showed the RR genotype at the PRKAG3 R200Qsubstitution (RN locus). This is the first large investigation at the RNlocus in the Italian Large White population confirming what wasexpected according to Milan et al. (2000) who indicated that the al-lele causing the acid meat defect is present only in the Hampshirebreed or in synthetic lines in which Hampshire blood wasintroduced.

As the negative alleles, in terms of meat quality, at these twoloci have a very low frequency or are not present at all, the twomentioned mutations do not contribute to the genetic variabilityon quality and production traits in the Italian Large White popula-tion. Thus other genes and mutations may affect these traits in thisbreed.

3.3. Other mutations in the PRKAG3 gene

A few reports have indicated that three other amino acid substi-tutions (T30N, G52S and I199V) in the PRKAG3 gene segregate inmost pig breeds and affect meat quality traits with particularemphasis on GP, glycogen content, pH and colour (Ciobanu et al.,2001; Fontanesi et al., 2003; Lindahl et al., 2004a, 2004b; Ottoet al., 2007).

In the analysed Italian Large White population, the 30N, 52Gand 199I alleles of the three substitution sites were the less fre-quent ones (0.05, 0.27 and 0.17, respectively). These results weresimilar to our previous investigation, based on 37 Large White pigsonly, where frequencies of the same alleles were 0.18, 0.46 and0.20, respectively (Fontanesi et al., 2003). For the same substitu-tions Ciobanu et al. (2001) obtained 0.16, 0.56 and 0.22, and Huanget al. (2004) reported 0.15, 0.52 and 0.21, respectively.

Data obtained in the 271 Italian Large White pigs on the threelinked substitution sites were used for haplotype reconstruction.Five haplotypes (with phase probability of uncertain phases above95%) were reconstructed: haplotype 1 (30N-52G-199V), 2 (30T-52S-199V), 3 (30T-52G-199I), 4 (30T-52G-199V) and 5 (30T-52S-199I) having a frequency of 0.05, 0.70, 0.15, 0.07 and 0.02, respec-tively. Haplotype reconstruction in Large White pigs provided inCiobanu et al. (2001) reported only the first 4 haplotypes with fre-quencies of 0.17, 0.45, 0.22 and 0.16, respectively. Differences be-tween our results and those obtained by others (Ciobanu et al.,2001) may reflect differences in allele frequencies at the three sub-stitution sites between the two Large White populations that couldreflect different selection strategies.

Least square means (LSM) for the PRKAG3 T30N, G52S andI199V substitutions analysed as single markers are reported in Ta-ble 2. Table 4 includes estimated additive and dominance effectswhen differences among LSM showed P < 0.20.

Only two animals showed genotype NN at the T30N substitu-tion, thus the NN and NT genotypes were pooled in the associationtests and additive and dominance effects were not estimated. Forthis substitution, only lactate content showed significant differ-ences (P = 0.016) between the two groups, with genotype TT hav-

Table 2Association analysis between PRKAG3 markers and meat quality parameters and EBVs (GP = glycolytic potential; ADG = average daily gain; LC = lean cuts; BFT = back fatthickness; HW = ham weight; FGR = feed:gain ratio)

DNA markers Traits Genotypes [no. of samples] P

PRKAG3 T30N TT [245] TN+NN [24+2]a NN [2]a

pH1 5.925 (0.018) 5.958 (0.048) – 0.504pHu 5.658 (0.016) 5.666 (0.041) – 0.853Glycogen 48.549 (1.806) 47.599 (4.454) – 0.834Lactate 57.457 (1.027)d 49.928 (3.049)c – 0.019GP 106.070 (1.786)f 97.385 (4.499)e – 0.060EBV ADG +33.294 (1.702) +33.500 (5.225) – 0.970EBV LC +2.006 (0.118) +1.871 (0.363) – 0.724EBV BFT �2.096 (0.243) �1.862 (0.745) – 0.765EBV HW +0.583 (0.039) +0.572 (0.120) – 0.929EBV FGR �0.148 (0.010) �0.150 (0.030) – 0.959

PRKAG3 G52S SS [148] GS [99] GG [24]pH1 5.925 (0.021) 5.929 (0.025) 6.007 (0.049) 0.196pHu 5.666 (0.019) 5.637 (0.022) 5.703 (0.042) 0.285Glycogen 47.744 (2.202) 49.617 (2.449) 47.764 (4.569) 0.794Lactate 57.852 (1.343) 55.884 (1.587) 54.520 (4.204) 0.466GP 105.840 (2.206) 105.330 (2.468) 102.210 (4.643) 0.768EBV ADG +34.527 (2.191) +32.333 (2.679) +29.875 (5.440) 0.657EBV LC +2.059 (0.152) +2.016 (0.186) +1.492 (0.377) 0.375EBV BFT �1.982 (0.312) �2.402 (0.381) �1.279 (0.774) 0.391EBV HW +0.552 (0.050) +0.625 (0.061) +0.595 (0.124) 0.653EBV FGR �0.143 (0.013) �0.160 (0.015) �0.137 (0.031) 0.621

PRKAG3 I199V II [10] IV [72] VV [189]pH1 6.098 (0.076)c,e 5.947 (0.029)f 5.906 (0.020)d 0.034pHu 5.746 (0.065) 5.670 (0.025) 5.647 (0.018) 0.269Glycogen 41.616 (7.032) 48.394 (2.783) 49.011 (2.001) 0.591Lactate 56.714 (4.969) 56.3 06 (1.853) 56.970 (1.192) 0.953GP 97.643 (7.166) 104.630 (2.813) 106.070 (2.008) 0.496EBV ADG +40.200 (8.416) +30.514 (3.137) +34.016 (1.936) 0.451EBV LC +2.384 (0.585) +1.818 (0.218) +2.039 (0.135) 0.548EBV BFT �3.820 (1.198) �2.001 (0.447) �2.008 (0.276) 0.333EBV HW +0.811 (0.192) +0.637 (0.072) +0.540 (0.044) 0.283EBV FGR �0.210 (0.049) �0.139 (0.018) �0.149 (0.011) 0.386

Least square means (LSM) are reported with their standard errors (in parenthesis). Results with P < 0.05 are in bold.a The TN+NN genotype class includes the animals with genotype TN and NN. Only 2 animals showed genotype NN.

b,c LSM different at P < 0.05.d,e LSM different at P < 0.10.

L. Fontanesi et al. / Meat Science 80 (2008) 780–787 783

ing a higher content. Among the other traits, GP (P = 0.06) tendedto be significantly associated following the differences betweenthe genotypic classes already reported for lactate content. The ef-fect on lactate content did not result in a similar effect on pH1. Thiscould be due to the different timing of the sampling of muscle tis-sues for the lactate content analysis (at 1 hour post-mortem) andthe measurements of pH1 (at about 2 h post-mortem). As a matterof fact, the delay between the stunning of the animals and pH1

measurement resulted in a mean for this parameter a little bit low-er than 6.00 (Table 1) that, if taken closer to the death of the ani-mals, may be higher than this value in normal heavy pigs (NanniCosta et al., in press; Russo & Nanni Costa, 1995). No effect was de-tected on the other traits (pHu, glycogen content, EBVs for growth,feed efficiency, meat and fat deposition traits).

The G52S substitution did not show any significant associationwith the analysed traits.

Significant association (P = 0.034) was evident between theI199V substitution and pH1 with significant additive and domi-nance effects (P < 0.05; Table 4). The II and VV genotypes showedthe highest and lowest trait values and the IV genotype had theintermediate LSM. All other traits were not significantly associatedwith this polymorphic site. However, it is worth noting that only10 animals had genotype II and this might have affected the asso-ciation tests. For the other traits there is a general tendency ofgenotype II to be the most favourable in terms of meat qualityparameters (higher pHu, lower glycogen content and GP) as wellas in terms of lean meat deposition and growth traits (higher LC,lower BF, higher HW and higher ADG and lower FGR). A larger

number of animals should be analysed to evaluate these tenden-cies in more detail, both for meat quality parameters and the con-sidered EBVs. However, according to the indications of a possibleeffect on the analysed EBVs, it is possible to speculate that the199I allele might positively influence lean meat deposition, with-out the negative side effect on meat quality (at least for the ana-lysed parameters) of the PRKAG3 200Q allele, that is caused byan amino acid exchange in the protein sequence just one positionafter the I199V substitution. Actually, apart from its detrimentaleffects on meat quality parameters (lower pHu, higher glycogencontent and GP, higher drip loss), the 200Q allele increases leanmeat content, probably as a consequence of its action against try-glyceride accumulation and insulin resistance in skeletal musclethat might be related to an increase in fat oxidation, as evidencedusing transgenic 225Q mice (different nomenclature for the sameposition; Barnes et al., 2004). Moreover, different mechanismsseem to regulate glycogen accumulation in wild type and mutated225Q transgenic mice (Yu et al., 2006). Thus, it could be possiblethat the two close substitutions, I199V and R200Q, affect bothmeat quality traits and lean muscle mass deposition but in differ-ent and opposite way, with the 199I allele affecting positively meatproduction and quality traits. Another possible explanation ofthese putative positive effects of the 199I substitution on bothmeat quality and production traits might be the presence of link-age disequilibrium with another close mutation in the Italian LargeWhite population. These hypotheses might be worthy of furtherinvestigation after having obtained more convincing results interms of association between the 199I allele and the evaluated pro-

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duction traits, considering also that, in crosses between SwedishHampshire and Finnish Landrace, genotype II did not show positiveeffects on meat deposition traits (Enfält et al., 2006). However, inthis study only 7 out of 334 pigs had genotype II.

The I199V polymorphic site has already been shown to be themost important substitution of the PRKAG3 gene after the R200Qmutation, with effects on ham and loin pH and colour, glycogencontent and drip loss in Duroc, Landrace, synthetic and commercialpig lines (Ciobanu et al., 2001; Lindahl et al., 2004a, 2004b; Ottoet al., 2007). However, significant effects on loin pH1 were reportedonly for a commercial undefined population (Otto et al., 2007)while in Large White animals no significant association was evi-dent, probably due to the low number of II individuals present inthis breed (Ciobanu et al., 2001). No significant results were alsoobtained by Stalder, Rothschild, and Lonergan (2005) for severalmeat quality traits in another commercial pig population. Our pre-vious investigation in crossbred pigs (Fontanesi et al., 2003) did notfind any significant association between this substitution and meatquality traits, but in this case this was probably caused by the verylow frequency of the 199I allele in the tested group of animals.However, effects on meat colour were obtained for the T30N andG52S substitutions, also reported in another study, together witheffects on ham and loin ultimate pH, suggesting that their influ-ence might be due to the strong linkage disequilibrium with themore important I199V substitution (Ciobanu et al., 2001).

However, considering the three substitutions together as hapl-otypes, it indicated a certain hierarchy for their effects with haplo-type 3 being the most favourable, haplotype 2 being the secondbest and the remaining haplotypes 1 and 4 being the worst. (Cio-banu et al., 2001). We tried to evaluate the PRKAG3 haplotype ef-fects in the Italian Large White pigs and only in a few cases wasit possible to obtain reliable estimates due to the low number ofcertain haplotype classes. Haplotype 3, carrying the 199I, con-firmed the same direction of its effect on pH1 (P = 0.062) and thegeneral tendency already obtained for the single marker analysisof the I199V substitution (data not shown). This might be expected

Table 3Association analysis between PGAM2 and PKM2 markers and meat quality parameters and Ethickness; HW = ham weight; FGR = feed:gain ratio)

DNA markers Traits Genotypes [n. of samples]

PGAM2 11 [10]pH1 5.925 (0.080)pHu 5.621 (0.067)Glycogen 51.198 (7.257)Lactate 46.291 (5.100)GP 96.407 (7.384)EBV ADG +48.800 (8.386)EBV LC +3.240 (0.581)c

EBV BFT �3.020 (1.198)EBV HW +1.039 (0.191)c

EBV FGR �0.239 (0.048)

PKM2 11 [72]pH1 5.986 (0.030)c,e

pHu 5.673 (0.026)Glycogen 46.877 (2.893)Lactate 54.754 (1.855)GP 101.530 (2.891)EBV ADG +37.250 (3.059)a

EBV LC +2.134 (0.213)a

EBV BFT �2.040 (0.443)EBV HW +0.629 (0.072)EBV FGR �0.175 (0.018)a

Least square means (LSM) are reported with their standard errors (in parenthesis). ResuA,B LSM different at P < 0.001.a,b LSM different at P < 0.01.c,d LSM different at P < 0.05.e,f LSM different at P < 0.10.

as only a few animals carrying the 199I variant are in haplotype 5(whose effect could not be reliably estimated). Haplotype 2 seemedto be the second best haplotype, considering the analysed meatquality parameters, but its effects were not statistically supported.Only pH1 approached the P < 0.10 suggestive threshold (data notshown) following the results obtained for the G52S substitution.Haplotypes 4 and 5 were not associated to any traits (data notshown) but this might be due to the low number of pigs for somehaplotype classes.

3.4. DNA markers in the PGAM2 and PKM2 genes

Allele frequencies of the PGAM2 polymorphic site in the ana-lysed pigs were 0.15 and 0.85 for alleles 1 (g.77C) and 2 (g.77A),respectively. For the PKM2 marker, frequencies of alleles 1(g.32T) and 2 (g.32C) were 0.49 and 051, respectively. Our previoussurvey on allele frequencies at these two loci in the Large Whitebreed (Fontanesi et al., 2003) gave similar results.

Table 3 reports the LSM obtained for the PGAM2 and PKM2markers and Table 4 includes estimated additive and dominanceeffects when differences among LSM showed P < 0.20.

PGAM2 was significantly associated with HW EBV (P = 0.048)with significant dominance effects (P < 0.05; Table 4). Allele 2was dominant over allele 1 in decreasing the weight of the hams.Thus, genotype 11 showed a positive effect on ham weight whosetendency on higher lean meat deposition was also evident on theLC EBV (P = 0.093). Moreover, even though above the statistical sig-nificance level, the PGAM2 marker seemed to affect, in the samedirection, all other EBVs. A similar effect seemed evident for lactatecontent whose test was close to the suggestive threshold(P = 0.111). This might be of particular interest, considering the factthat this gene encodes for a muscle enzyme isoform that is in-volved in one of the last steps of the glycolysis pathway beforethe anaerobic transformation of pyruvate in lactate. If confirmed,there could be a close link between a marker in a gene and the finalproduct of the biochemical reactions in which the coded enzyme is

BVs (GP = glycolytic potential; ADG = average daily gain; LC = lean cuts; BFT = back fat

P

12 [63] 22 [198]5.934 (0.032) 5.926 (0.020) 0.9735.639 (0.027) 5.669 (0.017) 0.516

49.941 (2.997) 47.750 (1.993) 0.74657.528 (1.976) 57.234 (1.201) 0.111

107.240 (3.023) 105.100 (1.988) 0.378+32.460 (3.341) +32.803 (1.885) 0.172

+1.927 (0.231)d +1.951 (0.131)d 0.093�1.490 (0.477) �2.221 (0.269) 0.306+0.535 (0.076)d +0.574 (0.043)d 0.048�0.140 (0.019) �0.147 (0.011) 0.161

12 [125] 22 [74]5.906 (0.022)d 5.917 (0.029)f 0.0755.656 (0.020) 5.652 (0.026) 0.792

48.425 (2.253) 49.911 (2.844) 0.72656.866 (1.411) 58.646 (1.837) 0.313

105.300 (2.236) 108.370 (2.839) 0.202+37.040 (2.321)A +23.189 (3.017)B,b 0.0005

+2.309 (0.162)A +1.323 (0.210)B,b 0.0009�2.631 (0.336)a �1.162 (0.437)b 0.030+0.606 (0.054) +0.497 (0.071) 0.356�0.157 (0.014)c �0.109 (0.018)b,d 0.025

lts with P < 0.05 are in bold.

Table 4Additive and dominance effects (with standard errors in parenthesis: s.e.) obtained for the single marker tests. Results are reported only for association analyses with P < 0.20

DNA markers Traitsa Additive effect (s.e.) P Dominance effect (s.e) P

PRKAG3 I199V pH1 �0.096 (0.039) 0.016 �0.055 (0.048) 0.259PRKAG3 G52S pH1 0.048 (0.027) 0.073 �0.041 (0.036) 0.258PGAM2 Lactate 5.471 (2.634) 0.039 5.765 (3.266) 0.079PGAM2 EBV ADG �7.998 (4.298) 0.064 �8.341 (5.443) 0.127PGAM2 EBV LC �0.644 (0.298) 0.031 �0.669 (0.377) 0.077PGAM2 EBV HW �0.232 (0.098) 0.018 �0.271 (0.124) 0.029PGAM2 EBV FGR 0.046 (0.025) 0.065 0.053 (0.031) 0.097PKM2 pH1 0.034 (0.020) 0.087 0.045 (0.029) 0.129PKM2 EBV ADG �7.030 (2.148) 0.001 6.821 (3.163) 0.032PKM2 EBV LC �0.405 (0.150) 0.007 0.580 (0.220) 0.009PKM2 EBV BFT 0.439 (0.311) 0.159 �1.030 (0.457) 0.025PKM2 EBV FGR 0.033 (0.013) 0.009 �0.015 (0.018) 0.437

a ADG = average daily gain; LC = lean cuts; BFT = back fat thickness; HW = ham weight; FGR = feed:gain ratio.

L. Fontanesi et al. / Meat Science 80 (2008) 780–787 785

involved, opening several hypotheses on the way in which PGAM2might influence this parameter. However, the hypothesized effecton lactate content did not produce a similar effect on pH1, but thiscould be due to the different timing of the measurement of the twoparameters as discussed above. Finally, it is worth considering thatPGAM2 maps on porcine chromosome 18, where QTL for severalmeat quality and production traits, including lean content (Dra-gos-Wendrich et al., 2003), have been reported. Lactate content isnot a usual parameter in QTL studies, only being included in oneinvestigation that did not list chromosome 18 among those havingQTL for this trait (Malek et al., 2001b).

The SNP in the PKM2 gene was associated with EBVs for ADG(P = 0.0005), LC (P = 0.0009), BFT (P = 0.030) and FGR (P = 0.025).Significant additive and dominance effects were obtained forADG and LC, while for BFT and FGR only significant dominance oradditive effects were revealed, respectively. Considering these re-sults, allele 1 was, in general, dominant over allele 2 (ADG, LCand BFT). For FGR an intermediate value of the heterozygous ani-mals was evident (Table 3). Animals with genotype 22 had a lowerADG, lower LC, higher BFT and lower FGR. Suggestive evidence ofassociation with pH1 was also identified (P = 0.075). The PKM2marker was not a source of variability for the other analysed traits,including glycogen content for which our first survey using cross-bred animals gave significant results (Fontanesi et al., 2003). Thisgene is localized on porcine chromosome 7, where several studieshave indicated the presence of QTL affecting the traits for whichPKM2 showed strong significant results. Thus, it is possible that(i) this gene could be linked to the QTL for these production traitsand that the analysed SNP is a close marker to the causative muta-tion(s) in (an)other gene(s), or (ii) PKM2 could be the quantitativetrait gene itself. Regarding the first point, it appears that linkagedisequilibrium between DNA markers seems to be low in the Ital-ian Large White pig breed (Davoli et al., 2006), thus, to show theseeffects, the PKM2 should be very close (0.5-2 cM) to the QTL muta-tion(s). To support the second hypothesis, the functions of thisgene have to be considered. Pyruvate kinase occurs in four isozy-mic forms (L, R, M1 and M2) that are encoded by two genes, PKLRand PKM2, localized on different chromosomes. The M1 and M2isoforms are encoded by the PKM2 gene by alternative splicingand the M2 isoform is highly expressed in skeletal muscle and intumor cells (Noguchi, Inoue, & Tanaka, 1986; Tsutsumi, Tani, Fujii,& Miwa, 1988). The activity of the M2 isoform is regulated by theglycolytic intermediate fructose 1,6-bisphosphate that influencesthe switch between a less active dimeric form and a highly activetetrameric form with a high affinity to its substrate phosphoenol-pyruvate. Channeling of glucose either to synthetic processes (di-meric form) or to glycolytic energy production (tetrameric form)is a mechanism that influences, to a large extent, cell proliferationand differentiation respectively and distinguishes tissues with high

capacity of nucleic acid synthesis from gluconeogenic and lipo-genic tissues (Mazurek, Grimm, Boschek, Vaupel, & Eigenbrodt,2002). The key role of PKM2 in these processes, its map positionand the results obtained in our study might support the hypothesisregarding its direct involvement in the mechanisms that producethe effects observed for several production traits.

4. Conclusions

On the whole, seven DNA markers were analysed. Two (RYR1g.1843C>T and PRKAG3 R200Q) were used to exclude the presencein the Italian Large White animals of alleles with large impacts onmeat quality and production traits. Three other markers wereinvestigated in the PRKAG3 gene (T30N, G52S and I199V): signifi-cant results were obtained only for pH1 (I199V) and lactate content(T30N), confirming, at least in part, the effects of this gene on meatquality traits. SNPs in two other candidate genes (PGAM2 andPKM2), coding for muscle enzyme isoforms involved in the glyco-lytic pathway, were tested for their association with meat qualityparameters and production traits. A significant association was ob-served for PGAM2 and ham weight with a dominance effect of al-lele 2. PKM2 was strongly associated with growth, meat and fatdeposition traits with a general dominance action of allele 1 overallele 2. Its map position on chromosome 7 and its key role inthe regulation of energy balance and glucose uptake might supporta functional involvement of this gene in the mechanisms that pro-duce the observed effects. Confirmation of the results obtained inthis work could be useful to support the latter hypothesis. In con-clusion, the results obtained represent a first step to evaluate if it isworth including some of these DNA polymorphisms in marker as-sisted selection strategies in heavy pig breeding.

Acknowledgement

We thank ANAS for providing data and samples. This work wassupported by Italian MiPAAF SELMOL funds.

References

ANAS (2004) ANAS Notizie numero 3.Barnes, B. R., Marklund, S., Steiler, T. L., Walter, M., Hjälm, G., Amarger, V., et al.

(2004). The 50-AMP-activated protein kinase c3 isoform has a key role incarbohydrate and lipid metabolism in glycolytic skeletal muscle. Journal ofBiological Chemistry, 279, 38441–38447.

Bidanel, J. P., Milan, D., Iannuccelli, N., Amigues, Y., Boscher, M. Y., Bourgeois, F.,et al. (2001). Detection of quantitative trait loci for growth and fatness in pigs.Genetics Selection Evolution, 33, 289–309.

Ciobanu, D., Bastiaansen, J., Malek, M., Helm, J., Woollard, J., Plastow, G., et al.(2001). Evidence for new alleles in the protein kinase adenosinemonophosphate activated c3-subunit gene associated with low glycogencontent in pig skeletal muscle and improved meat quality. Genetics, 159,1151–1162.

786 L. Fontanesi et al. / Meat Science 80 (2008) 780–787

Ciobanu, D. C., Bastiaansen, J. W. M., Lonergan, S. M., Thomsen, H., Dekkers, J. C. M.,Plastow, G. S., et al. (2004). New alleles in calpastatin gene are associated withmeat quality traits in pigs. Journal of Animal Science, 82, 2829–2839.

Clutter, A. C., & Brascamp, E. W. (1998). Genetics of performance traits. In M. F.Rothschild & A. Ruvinski (Eds.), The genetics of the pig (pp. 427–462). Oxon, UK:CAB International.

Davoli, R., Fontanesi, L., Braglia, S., Nisi, I., Scotti, E., Buttazzoni, L., et al. (2006).Investigation of SNPs in the ATP1A2, CA3 and DECR1 genes mapped to porcinechromosome 4: Analysis in groups of pigs divergent for meat production andquality traits. Italian Journal of Animal Science, 5, 249–263.

Davoli, R., Fontanesi, L., Zambonelli, P., Bigi, D., Gellin, J., Yerle, M., et al. (2002).Isolation of porcine expressed sequence tags for the construction of a firstgenomic transcript map of the skeletal muscle in pig. Animal Genetics, 33, 3–18.

de Koning, D. J., Harlizius, B., Rattink, A. P., Groenen, M. A., Brascamp, E. W., & vanArendonk, J. A. (2001). Detection and characterization of quantitative trait locifor meat quality traits in pigs. Journal of Animal Science, 79, 2812–2819.

Dragos-Wendrich, M., Stratil, A., Hojny, J., Moser, G., Bartenschlager, H., Reiner, et al.(2003). Linkage and QTL mapping for Sus scrofa chromosome 18. Journal ofAnimal Breeding and Genetics, 120(Suppl. 1), 138–143.

Enfält, A.-C., Lundström, K., Karlsson, A., & Hansson, I. (1997). Estimated frequencyof the RN-allele in Swedish Hampshire pigs and comparison of glycolyticpotential, carcass composition, and technological meat quality among SwedishHampshire, Landrace, and Yorkshire pigs. Journal of Animal Science, 75,2924–2935.

Enfält, A.-C., von Seth, G., Josell, Å., Lindahl, G., Hedebro-Velander, I., Braunschweig,M., et al. (2006). Effects of a second mutant allele (V199I) at the PRKAG3 (RN)locus on carcass composition in pigs. Livestock Science, 99, 131–139.

Fontanesi, L., Davoli, R., Nanni Costa, L., Scotti, E., & Russo, V. (2003). Study ofcandidate genes for glycolytic potential of porcine skeletal muscle:identification and analysis of mutations, linkage and physical mapping andassociation with meat quality traits in pigs. Cytogenetic and Genome Research,102, 145–151.

Fujii, J., Otsu, K., Zorzato, F., de Leon, S., Khanna, V. K., Weiler, J. E., et al. (1991).Identification of a mutation in porcine ryanodine receptor associated withmalignant hyperthermia. Science, 253, 448–451.

Gerbens, F., van Erp, A. J., Harders, F. L., Verburg, F. J., Meuwissen, T. H., Veerkamp, J.H., et al. (1999). effect of genetic variants of the heart fatty acid-binding proteingene on intramuscular fat and performance traits in pigs. Journal of AnimalScience, 77, 846–852.

Gilbert, H., Le Roy, P., Milan, D., & Bidanel, J. P. (2007). Linked and pleitropic QTLsinfluencing carcass composition traits detected on porcine chromosome 7.Genetical Research, 89, 65–72.

Gleeson, T. T. (1996). Post-exercise lactate metabolism: A comparative review ofsites, pathways, and regulation. Annual Review of Physiology, 58, 565–581.

Hamilton, D. N., Ellis, M., Miller, K. D., McKeith, F. K., & Parrett, D. F. (2000). Theeffect of the Halothane and Rendement Napole genes on carcass and meatquality characteristics of pigs. Journal of Animal Science, 78, 2862–2867.

Houde, A., Pommier, S. A., & Roy, R. (1993). Detection of the ryanodine receptormutation associated with malignant hyperthermia in purebred swinepopulations. Journal of Animal Science, 71, 1414–1418.

Huang, L. S., Ma, J. W., Ren, J., Ding, N. S., Guo, Y. M., Ai, H. S., et al. (2004). Geneticvariations of the porcine PRKAG3 gene in Chinese indigenous pig breeds.Genetics Selection Evolution, 36, 481–486.

Le Roy, P., Elsen, J.-M., Caritez, J.-C., Talmant, A., Juin, H., Sellier, P., et al.(2000). Comparison between the three porcine RN genotypes for growth,carcass composition and meat quality traits. Genetics Selection Evolution, 32,165–186.

Lindahl, G., Enfält, A.-C., von Seth, G., Josell, Å., Hedebro-Velander, I., Andersen, H. J.,et al. (2004a). A second mutant allele (V199I) at the PRKAG3 (RN) locus – I.Effect on technological meat quality of pork loin. Meat Science, 66, 609–619.

Lindahl, G., Enfält, A.-C., von Seth, G., Josell, Å., Hedebro-Velander, I., Andersen, H. J.,et al. (2004b). A second mutant allele (V199I) at the PRKAG3 (RN) locus – II.Effect on colour characteristics of pork loin. Meat Science, 66, 621–627.

Malek, M., Dekkers, J. C., Lee, H. K., Baas, T. J., & Rothschild, M. F. (2001a). Amolecular genome scan analysis to identify chromosomal regions influencingeconomic traits in the pig. I. Growth and body composition. MammalianGenome, 12, 630–636.

Malek, M., Dekkers, J. C., Lee, H. K., Baas, T. J., Prusa, K., Huff-Lonergan, E., et al.(2001b). A molecular genome scan analysis to identify chromosomal regionsinfluencing economic traits in the pig. II. Meat and muscle composition.Mammalian Genome, 12, 637–645.

Mazurek, S., Grimm, H., Boschek, C. B., Vaupel, P., & Eigenbrodt, E. (2002). Pyruvatekinase type M2: A crossroad in the tumor metabolome. British Journal ofNutrition, 87, S23–S29.

Meadus, R., MacInnis, R., Dugan, M. E. R., & Aalhus, J. L. (2002). A PCR-RFLP methodto identify the RN� gene in retailed pork chops. Canadian Journal of AnimalScience, 82, 449–451.

Mercadé, A., Estellé, J., Pérez-Enciso, M., Varona, L., Silió, L., Noguera, J. L., et al.(2006). Characterization of the porcine acyl-CoA synthetase long-chain 4 geneand its association with growth and meat quality traits. Animal Genetics, 37,219–224.

Milan, D., Jeon, J.-T., Looft, C., Amarger, V., Robic, A., Thelander, M., et al. (2000). Amutation in PRKAG3 associated with excess glycogen content in pig skeletalmuscle. Science, 288, 1248–1251.

Moeller, S. J., Baas, T. J., Leeds, T. D., Emnett, R. S., & Irvin, K. M. (2003). RendementNapole gene effects and a comparison of glycolytic potential and DNA

genotyping for classification of Rendement Napole status in Hampshire-siredpigs. Journal of Animal Science, 81, 402–410.

Monin, G., Mejenes-Quijano, A., Talmant, A., & Sellier, P. (1987). Influence of breedand muscle metabolic type on muscle glycolytic potential and meat pH in pigs.Meat Science, 20, 149–158.

Monin, G., & Sellier, P. (1985). Pork of low technological quality with a normal rateof muscle pH fall in the immediate post-mortem period: the case of theHampshire breed. Meat Science, 13, 49–63.

Nanni Costa, L., Lo Fiego, D. P., Dall’Olio, S., Davoli, R., & Russo, V. (1999). Influence ofloading method and stocking density during transport on meat and dry-curedham quality in pigs with different halothane genotypes. Meat Science, 51,391–399.

Nanni Costa, L., Lo Fiego, D. P., Dall’Olio, S., Davoli, R., & Russo, V. (2002). Combinedeffects of pre-slaughter treatments and lairage time on carcass and meat qualityin pigs of different halothane genotype. Meat Science, 61, 41–47.

Nanni Costa, L., Tassone, F., Davoli, R., Fontanesi, L., Dall’Olio, S., Colombo, M., et al.(in press) Glycolytic potential in semimembranosus muscle of Italian LargeWhite pigs. Journal of Muscle Food.

Nezer, C., Moreau, L., Wagenaar, D., & Georges, M. (2002). Results of a wholegenome scan targeting QTL for growth and carcass traits in a Pietran � LargeWhite intercross. Genetics Selection Evolution, 34, 371–387.

Noguchi, T., Inoue, H., & Tanaka, T. (1986). The M1- and M2-type isozymes of ratpyruvate kinase are produced from the same gene by alternative RNA splicing.Journal of Biological Chemistry, 261, 13807–13812.

O’Brien, P. J., Shen, H., Cory, C. R., & Zhang, X. (1993). Use of a DNA-based test for themutation associated with porcine stress syndrome (malignant hyperthermia) in10,000 breeding swine. Journal of the American Veterinary Medicine Association,203, 842–851.

Otto, G., Roehe, R., Looft, H., Thoelking, L., Knap, P. W., Rothschild, M. F., et al. (2007).Association of DNA markers with meat quality traits in pigs with emphasis ondrip loss. Meat Science, 75, 185–195.

Ovilo, C., Clop, A., Noguera, J. L., Oliver, M. A., Barragán, C., Rodrı́guez, C., et al.(2002). Quantitative trait locus mapping for meat quality traits in anIberian � Landrace F2 pig population. Journal of Animal Science, 80,2801–2808.

Pommier, S. A., & Houde, A. (1993). Effect of the genotype for malignanthyperthermia as determined by a restriction endonuclease assay on thequality characteristics of commercial pork loins. Journal of Animal Science, 71,420–425.

Reiner, G., Heinricy, L., Müller, E., Geldermann, H., & Dzapo, V. (2002). Indications ofassociations of the porcine FOS proto-oncogene with skeletal muscle fibre traits.Animal Genetics, 33, 49–55.

Rohrer, G. A., & Keele, J. W. (1998). Identification of quantitative trait loci affectingcarcass composition in swine: I. Fat deposition traits. Journal of Animal Science,76(9), 2247–2254.

Rothschild, M. F., & Plastow, G. S. (1999). Advances in pig genomics and industryapplications. AgBiotechNet, 1, 1–7.

Rothschild, M. F., Hu, Z.-I., & Jiang, Z. (2007). Advances in QTL mapping in pigs.International Journal of Biological Sciences, 3, 192–197.

Russo, V., Buttazzoni, L., Baiocco, C., Davoli, R., Nanni Costa, L., Schivazappa, C., et al.(2000). Heritability of muscular cathepsin B activity in Italian Large White pigs.Journal of Animal Breeding and Genetics, 117, 37–42.

Russo, V., Dall’Olio, S., Davoli, R., Coscelli, M. B., & Bigi, D. (1996). Studio del locusalotano nelle razze suine allevate in Italia mediante test PCR. Zootecnia &Nutrizione Animale, 22, 33–38.

Russo, V., Davoli, R., Tagliavini, J., Dall’Olio, S., Bigi, D., Costosi, E., et al. (1993).Identificazione del genotipo dei suini per la sensibilità all’alotano a livello diDNA mediante PCR. Zootecnia & Nutrizione Animale, 19, 89–93.

Russo, V., & Nanni Costa, L. (1995). Suitability of pig meat for salting and theproduction of quality processed products. Pig News & Information, 16, 17N–26N.

Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratorymanual (2nd ed.). NY: Cold Spring Harbor Laboratory Press.

Sellier, P. (1998). Genetics of meat and carcass traits. In M. F. Rothschild & A.Ruvinski (Eds.), The genetics of the pig (pp. 463–510). Oxon, UK: CABInternational.

Stalder, K. J., Rothschild, M. F., & Lonergan, S. M. (2005). Associations between twogene markers and indicator traits affecting fresh and dry-cured ham processingquality. Meat Science, 69, 451–457.

Stephens, M., Smith, N. J., & Donnelly, P. (2001). A new statistical method forhaplotype reconstruction from population data. American Journal of HumanGenetics, 68, 978–989.

Tor, M., Estany, J., Villalba, D., Cubiló, D., Tibau, J., Soler, J., et al. (2001). A within-breed comparison of RYR1 pig genotypes for performance, feeding behaviour,and carcass, meat and fat quality traits. Journal of Animal Breeding and Genetics,118, 417–427.

Tsutsumi, H., Tani, K., Fujii, H., & Miwa, S. (1988). Expression of L- and M-typepyruvate kinase in human tissues. Genomics, 2, 86–89.

van der Steen, H. A. M., Prall, G. F. W., & Plastow, G. S. (2005). Application ofgenomics to the pork industry. Journal of Animal Science, 83, E1–E8.

van Wijk, H. J., Dibbits, B., Baron, E. E., Brings, A. D., Harlizius, B., Groenen, M. A. M.,et al. (2006). Identification of quantitative trait loci for carcass composition andpork quality traits in a commercial finishing cross. Journal of Animal Science, 84,789–799.

Vidal, O., Varona, L., Oliver, M. A., Noguera, J. L., Sànchez, A., & Amills, M. (2006).Malic enzyme 1 genotype is associated with backfat thickness and meat qualitytraits in pigs. Animal Genetics, 37, 28–32.

L. Fontanesi et al. / Meat Science 80 (2008) 780–787 787

Wimmers, K., Murani, E., Te Pas, M. F. W., Chang, K. C., Davoli, R., Merks, J. W. M.,et al. (2007). Associations of functional candidate genes derived from gene-expression profiles of prenatal porcine muscle tissue with meat quality andmuscle deposition. Animal Genetics, 38, 474–484.

Yu, H., Hirschman, M. F., Fujii, N., Pomerleau, J. M., Peter, L. E., & Goodyer, L. J.(2006). Muscle-specific overexpression of wild type and R225Q mutant

AMP-activated protein kinase –subunit differentially regulates glycogenaccumulation. American Journal of Physiology – Endocrinology andMetabolism, 291, E557–E565.

Yue, G., Stratil, A., Cepica, S., Schröffel, Jr. J., Schröffelova, D., Fontanesi, L., et al.(2003). Linkage and QTL mapping for Sus scrofa chromosome 7. Journal ofAnimal Breeding and Genetics, 120(Suppl. 1), 56–65.