Meat Science 94 (2013) 328335

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Meat Science
journal homepage: www.elsevier.com/locate/meatsci

Association of bovine meat quality traits with genes included in the PPARG and
PPARGC1A networks
N. Sevane a, E. Armstrong b, O. Corts a, P. Wiener c, R. Pong Wong c,
S. Dunner a,, and the GemQual Consortium
a
Dpto. de Produccin Animal, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain
b
Dpto. Gentica y Mejora Animal, Facultad de Veterinaria, UdelaR., Uruguay
c
The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9PS, Scotland, UK

a r t i c l e i n f o a b s t r a c t

Article history: Understanding which are the genetic variants underlying the nutritional and sensory properties of beef, enables
Received 13 July 2012 improvement in meat quality. The aim of this study is to identify new molecular markers for meat quality
Received in revised form 8 February 2013 through an association study using candidate genes included in the PPARG and PPARGC1A networks given their
Accepted 19 February 2013
master role in coordinating metabolic adaptation in fat tissue, muscle and liver. Amongst the novel associations
found in this study, selection of the positive marker variants of genes such as BCL3, LPL, PPARG, SCAP, and SCD will
Keywords:
Candidate genes
improve meat organoleptic characteristics and health by balancing the n6 to n3 fatty acid ratio in meat. Also
Meat quality previous results on GDF8 and DGAT1 were validated, and the novel ATF4, HNF4A and PPARGC1A associations, al-
SNP though slightly under the signicance threshold, are consistent with their physiological roles. These data contrib-
Bos ute insights into the complex gene-networks underlying economically important traits.
2013 Elsevier Ltd. All rights reserved.

1. Introduction Apart from meat quality aspects such as tenderness, avour, juic-
iness or colour, health concerns are of particular interest given the re-
Many economically important traits in cattle production, such as lationship found between incidence of lifestyle diseases and dietary
those related to meat quality, dened by the nutritional and sensory intake of saturated fatty acids (SFA) and the ratio of n 6 to n 3
properties of beef, are very complex, involve many genes and are greatly fatty acids, currently far from the recommended 14:1 (Scollan et
inuenced by a variety of environmental factors (Hocquette et al., 2012). al., 2006). Understanding the genetic variation underlying economi-
Being difcult and expensive to measure (Simm, Lambe, Bnger, Navajas, cally important traits will enable us to improve production efciency
& Roehe, 2009), they are not usually included in selection programs and meat quality. For this purpose, we performed an association
based on phenotypic performance. However, the identication of molec- study between 26 single nucleotide polymorphisms (SNP) located
ular markers linked to economically important traits has evolved sub- in 20 candidate genes and different production traits measured in
stantially in the last years and provides an alternative way to evaluate 314 muscle samples of individuals belonging to 11 European bovine
the genetic merit of livestock (Hocquette et al., 2010). Genomic Selection breeds. Amongst the genes associated so far with production traits,
(GS) strategies focus on the incorporation of molecular information in we focussed on those related to energetic metabolism and specically
breeding programs in order to directly select the benecial genetic vari- several genes linked to the peroxisome proliferator activated receptor
ants underlying those complex traits (Pimentel & Knig, 2012). However, (PPARG) and its coactivator the peroxysome proliferator-activated
GS will not likely be extended in the short term to beef cattle populations receptor- coactivator-1 (PPARGC1A) networks, given their key
due to small population sizes and lack of high accuracy of estimated role in coordinating metabolic adaptation in fat tissue, muscle and
breeding values, so a candidate gene approach is currently useful to liver (Fig. 1).
extend the panel of associated SNP and estimate better SNP effects in
these breeds. 2. Materials and methods

2.1. Animals

A total of 314 muscle samples from unrelated bulls belonging to 11

Corresponding author at: Av. Puerta de Hierro, s/n, Dpto. Produccin Animal, Facultad
de Veterinaria, Universidad Complutense de Madrid, 28040, Madrid, Spain. Tel.: +34 91
European cattle breeds and fed from weaning to adult weight on a sim-
394 3765; fax: +34 91 394 3772. ilar diet were genotyped (Albert et al., 2008). The panel of animals
E-mail address: [email protected] (S. Dunner). consisted of one highly selected dairy breed (n = 26 Holstein); eight

0309-1740/$ see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.meatsci.2013.02.014
 N. Sevane et al. / Meat Science 94 (2013) 328335 329

Glucose Glucose
Hepatocytes Adipocytes

18:2 n-6 PKA SIRT1

ATF4
22:4 n-6
SCAP K/S

18:0
12:0 14:0
22:5 n-3; 20:5 n-3
BCL3 pH
22:6 n-3 PPARGC1A
PPARG
SREBF1

ACACA
ICDH
FOXO1 HNF4A CPT1 MEF2C PPARA

18:2 n-6
9c18:1 LPL
SCD
20:3 n-6
20:4 n-6
16:1
ICDH
MGAT1 DGAT1 GDF8 SLC2A4
Muscle fibre type
Collagen, Fat
Colour, pH

Lipogenesis Adipogenesis Muscle Gluconeogenesis FA oxidation Mitochondrial Glucose

atrophy biogenesis uptake

In blue: traits found to be associated to the nearby gene in this study Down-regulation Up-regulation (and/or eventual participation of the gene(s)
in the path way sat the bottom)
Liver networks Adipose tissue networks Different tissue networks, including liver and muscle

Fig. 1. Associations found in this study incorporated into the PPARG-PPARGC1A gene-network and energy metabolism. Apart from the master energy regulator PPARG, which function is
tissue dependent, two categories of genes are included in the network whether they are up-regulated in the presence of glucose ATF4, SCAP, SREBF1, ACACA, SCD, MGAT1, and DGAT1
enhancing lipogenic and adipogenic metabolic pathways; or up-regulated in the absence of glucose PKA, SIRT1, PPARGC1A, BCL3, FOXO1, HNF4A, CPT1, LPL, MEF2C, PPARA, SLC2A4, and
GDF8 increasing the availability of glucose through different metabolic processes, such as gluconeogenesis, fatty acid oxidation, mitochondrial biogenesis, glucose uptake or muscle at-
rophy. Interactions between these two main gene categories have also been described as these may drive the cell machinery towards glucose production (e.g., FOXO1 is up-regulated in
absence of glucose and, apart from inhibiting the lipogenic and adipogenic effect of PPARG in adipocytes and hepatocytes, also directly down-regulates the lipogenic pathway) or towards
expenditure (e.g., SREBF1 and ACACA genes are up-regulated in the presence of glucose and down-regulate HNF4A and CPT1 respectively, diminishing indirectly gluconeogenesis and fatty
acid oxidation).

beef breeds, some of them well distributed throughout the world (30 regions (UTR) were chosen to search for causative mutations. Polymor-
Charolais, 31 Limousin, and 18 Simmenthal and 30 Piedmontese) whilst phisms belong to one of the following categories and genes:
others more locally used (30 Asturiana de los Valles, 31 Pirenaica, 29
Danish Red, 28 Marchigiana,); and two unimproved local breeds (31 Polymorphisms from literature (8): diacylglycerol O-acyltransferase
Asturiana de la Montaa, and 30 Avilea-Negra Ibrica). (DGAT1) ss77831745 (Grisart et al., 2002); myostatin (GDF8)
ss77831865, ss77831863, ss77831864 (Grobet et al., 1997, 1998);
peroxisome proliferator-activated receptor gamma coactivator 1
2.2. Phenotypic data
alpha (PPARGC1A) c.1892 + 19T>C, c.5314C>T, c.920G>A
(Weikard, Khn, Goldammer, Freyer, & Schwerin, 2005); and
A comprehensive range of phenotypes were measured which fell
stearoyl-CoA desaturase (SCD) g.10329TbC AY241932 (Taniguchi
into three categories: physical variables, lipid traits and sensory analysis
et al., 2004).
(Table S1). Fat was extracted as described by Christensen et al. (2011).
Polymorphisms from GenBank database (17): acetyl coenzyme A
Total lipid content, was taken as the sum of the neutral lipid and
carboxylase (ACACA) ss64381883; B-cell CLL/lymphoma 3 (BCL3)
phospholipid fractions. Some additional phenotypes were set as are
ss65392310; carnitine palmitoyltransferase-1 (CPT1) ss65363345;
PUFA, n6n3 ratios, P:S ratios and antithrombotic potential (ATT),
DnaJ (Hsp40) homologue subfamily A member 1 (DNAJA1)
which is the ratio between the sum of the antithrombogenic fatty acids,
ss65351307; forkhead box O1 (FOXO1) ss65611802; solute carri-
eicosatrienoic acid (C20:3n6) and C20:5n3, and the thrombogenic
er family 2 (facilitated glucose transporter) member 4 (SLC2A4
fatty acid, C20:4n6 ((C20:3 + C20:5)/C20:4) (Enser, Hallett, Hewitt,
or GLUT4) ss62538460; hepatocyte nuclear factor 4 (HNF4A)
Fursey, & Wood, 1996). Sensory panel tests assessed meat using a
ss61961144; lipoprotein lipase (LPL) ss65478732; myocyte enhancer
nine-point scale as described in Christensen et al. (2011). Briey the
factor 2C (MEF2C) ss65449641, ss38329156; protein Kinase cAMP-
criteria assessed were: avour and abnormal avour intensity, tender-
dependent regulatory typeII (PKA) ss62837667, ss62837580; perox-
ness, and juiciness.
isome proliferator activated receptor (PPARA) ss65362714; perox-
isome proliferator activated receptor (PPARG) ss62850198; sirtuin
2.3. SNP selection and genotyping 1 (SIRT) ss61550598; SREBP cleavage activating protein (SCAP)
ss62839002; and sterol regulatory element binding transcription fac-
Twenty-six SNPs located in 20 candidate genes known to be involved tor 1 (SREBF1) ss62543518.
in beef quality were selected from the literature or the GenBank Polymorphism inferred from GenBank sequence alignments (1): ac-
database (http://www.ncbi.nlm.nih.gov). Whenever possible, non- tivating transcription factor 4 or cyclic AMP response element-
synonymous polymorphisms or those located in 5 or 3 untranslated binding protein 2 (ATF4 or CREB2) ss244244311.
330 N. Sevane et al. / Meat Science 94 (2013) 328335

Polymorphisms were genotyped with Multiplex-Capillary Primer found according to the literature. This summary is not exhaustive,
Extension as described in Sevane, Crespo, Can, and Dunner i.e. other genes not analyzed here are not included even if they are
(2011). Table S2 details the multiplex and Primer Extension primers known to play a role in this pathway. After eliminating SNPs with
and PCR conditions for those polymorphisms not previously recorded. MAF under 0.05 (Table 1), 19 polymorphisms belonging to 17 differ-
Replication of SNP genotyping was performed in 5% of the samples ent genes were analysed and 10 SNP located in 10 candidate genes in-
for repeatability purposes and Mendelian inheritance was checked in cluded in the energy metabolism network were found associated
four trios for reliability. with different live, carcass and meat quality traits through linear
regression analysis (Table 2). Signicant as well as suggestive
2.4. Statistical analysis (F Reg > 8) associations are shown. Frequencies of the analysed
SNP per breed are shown in Table 1, and mean and standard deviation
Many phenotypic data had to be transformed to comply with nor- for the traits associated to different genes in Table S3.
mality conditions underlying the linear model, either by log(1 + Y) or There is a clear partition of the whole sample, formed by the dif-
Y transformation (Table S1). SNPs with minor allele frequency (MAF) ferent breeds, and this information was taken into account to avoid
less than 0.05 were excluded from the association analysis to avoid false positives by including the breed effect in the linear model
bias of the data (Table 1). Linear regression analysis was then applied used. In any case, the 11 different breeds and the relatively few indi-
to test associations between genotypes and phenotypes using R pro- viduals within each population, does possibly miss some positive re-
gramming (http://www.r-project.org) and the lme4 statistical package, sults and lowers the success of this candidate gene approach, but
which ts the linear models and generalized linear mixed models allows a view on the issues that should be addressed when starting
(GLMM) to data (Bates & Maechler, 2008). The main assumptions in this kind of association studies.
this study were that the SNP effect on any of the traits is completely The genes evaluated in this study are all connected to the energy
additive and there is no interaction between SNP genotype and breed metabolism and specically to the PPARG and PPARGC1A networks.
(some preliminary analyses allowing interaction between breed and PPARG is a critical transcriptional regulator of genes controlling ener-
SNP effect were carried out, the results were unreliable as expected getic metabolism, adipogenesis and maintenance of the differentiated
from the relatively small number of records within each breed, and there- state (Memisoglu et al., 2003; Rosen & MacDougald, 2006; Xu et al.,
after no interaction between SNP genotype and breed was assumed). 1999). Regarding energetic regulation, apparently contradictory func-
The effect of the SNP on each of the traits was estimated by includ- tions have been described for this gene depending on the tissue
ing them as a covariate into a linear model. The model used in this where it is expressed (Fig. 1). Thus, whereas PPARG has a lipogenic
study was: and adipogenic effect in adipocytes and hepatocytes (Kersten,
2001), it promotes FA oxidation in the muscle, which eventually
y breed farm season g e leads to decreased lipid availability (Lapsys et al., 2000). In the pres-
ence of glucose, PPARG activates genes such as SREBF1 in the liver,
where y is the trait in question, breed is the effect of breed, farm_season and ACACA, SCD and DGAT1 both in hepatocytes and adipocytes, all
is the combined effect of farm and slaughter date, g is the SNP genotype, of them with a direct impact on lipogenesis/lipolysis balance,
and is the additive effect of the SNP. Traits were analyzed by groups: adipogenesis and gluconeogenesis. In contrast, when glucose levels
physical variables and sensory analysis group, total lipids, phospho- are low, activation of PPARG in muscle through genes like SIRT1 and
lipids, and neutral lipids. PPARGC1A promotes the expression of LPL and SLC2A4 and leads to in-
In order to correct for multiple testing in each group a permuta- creased FA oxidation, glucose uptake and mitochondrial biogenesis
tion analysis was carried out to calculate the experiment-wise signif- (Fig. 1). PPARGC1A, a coactivator of PPARG, has a key function in acti-
icance threshold within each trait (Churchill & Doerge, 1994). For vating a variety of nuclear hormone receptors and transcription fac-
each permutation, SNP genotypes were randomized across all ani- tors regulating energy homeostasis (Puigserver & Spiegelman,
mals. The effect of each SNP was then estimated and maximum F sta- 2003). In particular, this gene has been shown to mediate the expres-
tistic across all SNP was used to calculate the distribution of the null sion of genes involved in oxidative metabolism, adipogenesis, and
hypothesis. A total of 10,000 permutations were used to calculate gluconeogenesis, such as HNF4A, CPT1, LPL, PPARA, MEF2C or PPARG
the null distribution from which the 5% experiment-wise signicance (Fig. 1). Consistent with these roles, different genotypes of PPARG
thresholds were inferred. seem to have important effects in physiological responses to dietary
fat in humans (Memisoglu et al., 2003), and genetic variation in the
2.5. Gene pathways human PPARGC1A gene were found to be associated with insulin re-
sistance, susceptibility to type II diabetes, indicators for obesity, and
Gene pathways were built (Fig. 1) using the association results of altered lipid oxidation (Esterbauer et al., 2002; Hara et al., 2002;
this study along with previously published gene functions and associa- Muller, Bogardus, Pedersen, & Baier, 2003).
tions (Alaynick, 2008; Allen & Unterman, 2007; Bassel-Duby & Olson, In the current study, allele A of SNP ss62850198 in the PPARG gene
2006; Bionaz & Loor, 2008; Brennan, Michal, Ramsey, & Johnson, was found associated with a considerable increase of several omega-3
2009; Corton & Brown-Borg, 2005; Erkens, Vandesompele, Van PUFA in the muscle: docosapentaenoic acid (DPA, 22:5n 3),
Zeveren, & Peelman, 2009; Finley & Haigis, 2009; Glass, 2005; eicosapentaenoic acid (EPA, 20:5n 3), and docosahexaenoic acid
Graugnard et al., 2009; Kamei et al., 2003; Kersten, 2001; Konno, (DHA, 22:6n 3), with increases of 9%, 15% and 18%, respectively
Negishi, & Kodama, 2008; Kousteni, 2012; Lange et al., 2007; McAinch for the AA genotype compared to GG homozygous. As omega-3
et al., 2003; Scarpulla, 2008; Seo et al., 2009; Soyal, Krempler, PUFA and their metabolites are natural ligands for PPARG (Edwards
Oberkoer, & Patsch, 2006; Wang et al., 2010). & O'Flaherty, 2008), the inuence of PPARG on omega-3 levels is
clearly consistent with its known physiological roles. Consistent
3. Results and discussion with the current results, Oh, Lee, Lee, Chung, and Yeo (2011) found
an exonic SNP of PPARG associated with both SFA and MUFA in
We studied a specic network of genes related to energy metabo- Korean cattle. Many studies have reported the benecial effects of
lism and specically to PPARG and PPARGC1A pathways, to nd asso- omega-3 FA in the prevention and treatment of coronary artery dis-
ciations between 20 genes and traits inuencing meat physical ease, hypertension, diabetes, arthritis, cancer, and inammatory, au-
variables, lipid traits and organoleptic characteristics. Fig. 1 shows toimmune and psychiatric disorders (Berquin et al., 2007; Calder,
the network studied, where connections between genes are those 2006; De Caterina, Madonna, Bertolotto, & Schmidt, 2007; De
Table 1
Twenty-six polymorphisms genotyped, dbSNP accession number or location, and allele frequencies per breed.

Locus symbol GenBank Allele1/Allele2 Frequency of allele 1

dbSNP/Locationa
HOLb DRb SMb LIMb CHAb PIEb MARb ASTb CASb AVIb PIb Overall
(n = 26) (n = 29) (n = 18) (n = 31) (n = 30) (n = 30) (n = 28) (n = 30) (n = 31) (n = 30) (n = 31) (314)

ACACA ss64381883 G/A 0.827 0.759 1 1 1 0.900 0.982 0.983 1 0.967 0.984 0.946
ATF4 ss244244311 G/T 0.167 0 0 0.139 0.023 0.420 0 0 0 0 0.750 0.161
BCL3 ss65392310 T/C 0.154 0.035 0.278 0.016 0.067 0.067 0 0.017 0 0 0.194 0.067
CPT1*c ss65363345 G/C 1 0.983 1 0.983 0.983 1 0.981 1 1 0.914 0.977 0.983

N. Sevane et al. / Meat Science 94 (2013) 328335

DGAT1 ss77831745 A/G 0.442 0.121 0.094 0.097 0.100 0.017 0.463 0.328 0.250 0.267 0.333 0.228
FOXO1* ss65611802 T/C 0 0 0 0 0 0 0 0.020 0.113 0.093 0.048 0.026
GDF8_del11 ss77831865 G/del 1 1 1 1 1 1 1 0.317 0.984 1 0.887 0.921
GDF8_F94L ss77831863 C/A 1 1 1 0.016 0.983 1 1 1 1 1 0.694 0.869
GDF8_Q204X* ss77831864 C/T 1 1 1 0.984 0.833 1 1 1 1 1 1 0.973
SLC2A4 ss62538460 G/A 0.827 0.793 1 1 1 0.900 0.982 0.983 1 0.967 0.984 0.949
HNF4A ss61961144 T/C 0.039 0.017 0.083 0 0 0.267 0.071 0.138 0.210 0.035 0.161 0.095
LPL ss65478732 T/C 0.096 0.052 0.056 0 0.100 0.050 0 0 0.083 0.086 0.016 0.048
MEF2C* ss65449641 G/T 1 1 1 1 1 1 1 1 1 1 1 1
MEF2C ss38329156 G/T 0.423 0.397 1 0.710 0.650 0.600 0.500 0.648 0.677 0.638 0.694 0.621
MGAT1* ss65425229 T/C 1 1 1 1 1 1 0.929 0.983 0.968 1 1 0.989
PPARGC1A c.1892 + 19T>C A/G 0.173 0.121 0.028 0.032 0.167 0.183 0.304 0.276 0.371 0.383 0.016 0.192
PPARGC1A c.5314C>T T/C 0.154 0.224 0.667 0.194 0.083 0.300 0.054 0.120 0.016 0.069 0.113 0.164
PPARGC1A c.920G>A G/A 0.077 0 0.028 0.050 0.017 0.183 0.071 0.021 0.167 0.250 0.117 0.092
PKA ss62837667 T/C 0.135 0.138 0.083 0.387 0.190 0.517 0.196 0.304 0.516 0.357 0.355 0.301
PKA ss62837580 T/C 0.962 0.839 0.917 0.694 0.828 0.483 0.804 0.625 0.500 0.828 0.661 0.730
PPARA* ss65362714 C/T 1 1 1 1 1 1 1 1 1 1 1 1
PPARG ss62850198 G/A 0.885 0.810 0.861 0.823 0.750 0.883 0.893 0.900 0.897 0.944 0.839 0.861
SCAP ss62839002 G/A 0.846 1 0.971 1 0.983 0.917 0.911 0.850 0.887 0.850 0.968 0.925
SCD g.10329TbC T/C 0.385 0.173 0.306 0.419 0.467 0.350 0.429 0.333 0.742 0.667 0.436 0.437
SIRT* ss61550598 G/A 1 1 1 1 0.967 1 1 1 1 1 1 0.997
SREBF1 ss62543518 T/C 0.500 0.293 0.708 0.161 0.233 0.350 0.232 0.233 0.400 0.345 0.258 0.314
a
GenBank dbSNP accession numbers or location for the interrogated SNP.
b
Complete breed names: Holstein (HOL), Danish Red (DR), Simmental (SM), Limousin (LIM), Charolais (CHA), Piedmontese (PIE), Marchigiana (MAR), Asturiana de los Valles (AST), Asturiana de la Montaa (CAS), Avilea-Negra Ibrica
(AVI), Pirenaica (PI).
c
Superscript (*): SNP with minor allele frequency (MAF) less than 0.05 excluded from the association analysis.

331
332 N. Sevane et al. / Meat Science 94 (2013) 328335

Table 2
Signicant and suggestive associations between SNP and different traits.

Locus symbol GenBank dbSNPa Trait associationsb Mean Stand. Dev. F Thc Alleled F Rege SE p-value Effect Effect/
s.d.

ATF4 ss244244311 FA N % 18:2 n6 3.006 1.533 10.493 G 8.205 0.014 0.005 0.042 0.027
BCL3 ss65392310 pH thaw 5.576 0.089 9.886 C 12.554* 0.001 0.0005 0.002 0.023
Exon 3-S pH 3 h 6.422 0.319 10.046 11.729* 0.002 0.0007 0.008 0.025
DGAT1 ss77831745 FA % 16:1 3.053 0.725 10.741 G 13.333* 0.005 0.0003 0.018 0.025
FA N % 16:1 3.666 0.588 10.757 10.624 0.004 0.001 0.014 0.024
GDF8 ss77831865 L 10d 42.235 3.668 10.113 del 11 13.785* 0.721 0.0002 2.676 0.730
3UTR L 48 h 40.430 3.372 9.817 12.533* 0.671 0.0005 2.377 0.705
nt821(del11) MHCIIX 42.107 12.837 9.956 9.351 0.202 0.002 0.383 0.030
A610 48 h 23.590 3.571 10.305 10.321* 0.737 0.0015 2.368 0.663
A670 48 h 30.590 4.252 10.481 11.175* 0.809 0.0009 2.705 0.636
A670 10d 33.272 5.520 10.103 10.685* 1.029 0.0012 3.363 0.609
Collagen total 3.398 0.711 10.034 G 19.778* 0.012 0.00001 0.055 0.077
MHCI 16.615 4.115 9.844 13.666* 0.108 0.0003 0.161 0.039
pH thaw 5.576 0.089 9.886 9.608 0.001 0.002 0.003 0.034
K/S610 10d 1.132 0.349 10.445 13.974* 0.068 0.0002 0.255 0.732
K/S670 10d 0.715 0.255 10.323 11.820* 0.048 0.0007 0.166 0.652
HNF4A ss61961144 ICDH 1.324 0.406 10.294 C 8.865 0.019 0.003 0.003 0.007
LPL ss65478732 FA N W 20:3n6 0.812 0.880 10.682 T 16.704* 0.018 0.00006 0.078 0.089
Exon 2-S FA N W 20:4n6 0.898 0.712 10.518 9.371 0.023 0.006 0.073 0.103
PPARGC1A c.5314C>T FA % 18:0 15.095 1.937 10.674 T 9.861 0.006 0.002 0.018 0.009
3UTR FA N % 12:0 0.066 0.018 10.640 C 9.332 0.001 0.006 0.003 0.167
FA N % 14:0 2.837 0.463 10.653 8.113 0.006 0.005 0.016 0.035
PPARG ss62850198 FA % 22:5n3 0.544 0.337 10.641 A 13.499* 0.007 0.0003 0.025 0.074
5UTR FA % 20:5n3 0.233 0.188 10.815 11.013* 0.005 0.001 0.017 0.091
FA % 22:6n3 0.055 0.040 10.860 10.920* 0.001 0.001 0.005 0.124
SCAP ss62839002 FA P % 22:4n6 1.030 0.265 10.677 A 8.220 0.007 0.004 0.020 0.075
K/S600 10d 1.574 0.471 10.365 13.664* 0.051 0.0003 0.173 0.368
SCD g.10329 T b C FA P % 18:2n6 25.332 4.978 10.641 T 11.516* 0.005 0.0008 0.018 0.004
Exon 5-NS 293aa FA % 18:2 n6 10.142 6.129 10.733 9.575 0.012 0.002 0.036 0.006
Ala Val FA N % 18:2 n6 3.006 1.533 10.493 8.011 0.008 0.005 0.022 0.014
FA % 9c18:1 29.076 6.039 10.684 C 8.023 0.006 0.005 0.018 0.003
a
GenBank dbSNPs accession number and SNP location. S: synonymous SNP; NS: non-synonymous SNP.
b
pH thaw: pH on thawed samples at 10 days post mortem; pH 3 h: pH at 3 h post mortem; L: physical colour measured as lightness at 10 days or 48 h; MHCIIX: myosin heavy
chain isoform IIX (%); A: wavelength absorbance; collagen total: total amount of collagen (mg/g meat); MHCI: myosin heavy chain isoform I (%); K/S: ratio of light absorption (K) to
light scattering (S); ICDH: isocitrate dehydrogenase activity (mol/min for g of muscle); FA: fatty acid; W: mg/100 g muscles; %: percentage regarding total FA; P: phospholipid;
P %: percentage regarding total phospholipids; N: neutral FA; N %: percentage regarding total neutral lipids.
c
Trait signicant thresholds.
d
Allele positively correlated with the trait.
e
F regression statistics. *: signicant associations.

Caterina, Madonna, Zucchi, & La Rovere, 2003; Ross, Seguin, & Sieswerda, up-regulation of lipogenic genes such as SREBF1. In the current study,
2007; Simopoulos, 1999; Von Schacky, 2000), so selection of animals a SNP in the 5UTR of the ATF4 gene was found associated with the in-
carrying AA or AG genotypes in the PPARG SNP ss62850198 may crease of neutral linoleic acid (LA, 18:2n6) in the muscle. In parallel,
help to balance the n 6 to n 3 ratio and improve meat health- SREBP cleavage activating protein (SCAP) neutralizes SREBP precursors,
fulness. The PPARGC1A gene has been found to be responsible for which controls the nutritional activation of lipogenic genes and sup-
variation in milk fat synthesis in cattle (Weikard et al., 2005), and oxida- presses expression of gluconeogenic genes through the competitive
tive energy metabolism in equine skeletal muscle during exercise (Eivers inhibition of PPARGC1A recruitment, a requirement for HNF4A activa-
et al., 2012). In the present study, three polymorphisms previously pub- tion (Fig. 1) (Yamamoto et al., 2004). Consistent with these functions,
lished by Weikard et al. (2005) were analyzed (c.1892 + 19T>C, several polymorphisms in SREBF1 were previously associated with
c.5314C>T, c.920G>A). The intronic c.1892 + 19T>C polymor- meat FA composition in cattle (Hoashi et al., 2007) and intramuscular
phism linked previously with a QTL for fat in milk was not associated fat (Chen et al., 2008) and leg weight (Renaville et al., 2010) in pigs,
with any trait included in this study. However, another PPARGC1A SNP, and SNPs in SCAP were correlated with lean percentage, back-fat thick-
c.5314C>T, was associated with the amount of stearic acid (18:0), neu- ness, fat colour and salting losses in pigs (Renaville et al., 2010). Al-
tral lipid lauric acid (12:0), and neutral lipid myristic acid (14:0) in though one SNP in SREBF1 was included in the current analysis, no
muscle. association was detected with any trait. However, the SNP located in
Apart from the master energy regulators PPARG and PPARGC1A, two SCAP was associated with the amount of the phospholipid fraction of
categories of genes are included in the network depending on their adrenic acid (22:4n6) and the ratio of light absorption (K) to light
up-regulation in the presence ATF4, SCAP, SREBF1, ACACA, SCD, scattering (S) (K/S) at 600 nm at 10 days post mortem, such that the
MGAT1, and DGAT1 or the absence PKA, SIRT1, BCL3, FOXO1, HNF4A, individuals with the AA genotype had greater trait values by 4% and
CPT1, LPL, MEF2C, PPARA, SLC2A4, and GDF8 of glucose (Fig. 1). 22% respectively, compared to GG homozygous. The trait S is known
Amongst them, ATF4, SCAP, SCD, DGAT1, HNF4A, LPL, BCL3 and GDF8 to be inuenced by pH (when pH falls, S increases) and is related to pro-
were found associated with different production traits (Table 2). tein denaturation amongst other processes (Kubelka & Mink, 1995;
Activating transcription factor 4 (ATF4), also known as CREB2, is a Swatland, 2004). Thus, an increase in the ratio K/S implies low protein
bZIP class transcription factor and, amongst its large number of regula- denaturation and elevated pH, giving rise to tougher meat. pH ultimate-
tory roles, a link between this gene and lipid metabolism has been ly depends on ATP availability, and connects SCAP's role in the regula-
reported (Seo et al., 2009; Wang et al., 2010). ATF4-decient mice tion of SREBF1 and energetic metabolism with meat pH.
were used in both studies, revealing increases in lipolysis and decreases Further downstream in the pathway, PPARG over-expression has a
in expression of lipogenic genes, thus pointing at a role of ATF4 in the direct inuence on the activity of SCD and DGAT1 when glucose intake
 N. Sevane et al. / Meat Science 94 (2013) 328335 333

is increased (Fig. 1). Stearoyl-CoA desaturase (SCD) is the enzyme re- and PPARGC1A (Corton & Brown-Borg, 2005; Kousteni, 2012). FOXO1
sponsible for conversion of SFA into MUFA in mammalian adipocytes, transcription factors also regulate the expression of myostatin
either synthesized de novo or derived from diet (Ntambi, 1999). (GDF8) and contribute to the control of muscle cell growth and differ-
Moreover, SCD activity seems to be essential for lipogenic capacity entiation (Allen & Unterman, 2007). Although no association was
and development of subcutaneous adipose tissue (Hausman et al., detected between the polymorphism in FOXO1 and any trait in the
2009), and it is regulated by SREBF1 (Rahmouni & Sigmund, 2008). current study, the 11-bp deletion in GDF8 (nt821-del11) was found
In this study, the SNP g.10329TbC described by Taniguchi et al. (2004), associated with several carcass and meat quality traits. Three poly-
which causes a valine to alanine substitution in the fth exon, was morphisms in GDF8 gene were genotyped, all of them previously asso-
analysed. The C allele has previously been positively correlated with ciated with increased muscularity: an 11-bp deletion (nt821-del11)
MUFA content and lower melting point in beef cattle (Taniguchi et al., resulting in the truncation of the bioactive carboxyterminal domain of
2004), with higher intramuscular fat content in bovine M. longissimus the protein (ss77831865); a transition C T at bp 610 that yields a
and M. semimembranosus (Reardon, Mullen, Sweeney, & Hamill, 2010), premature stop codon at amino acid position 204 (Q204X); and a con-
and with MUFA prole in milk (Kgwatalala, Ibeagha-Awemu, Mustafa, servative phenylalanine to leucine substitution at amino acid position
& Zhao, 2009). The results obtained here show the association of T 94 (F94L, C A) (Grobet et al., 1998). Only nt821(del11) and F94L mu-
allele with the increase of the amount of LA (18:2n6) in muscle, relat- tations had a MAF exceeding 0.05 (Table 1) and were included in the as-
ed both to phospholipid and neutral lipids, whereas the C allele is asso- sociation analysis, and only the rst one was found associated with
ciated with an increase in oleic acid (9c18:1n9). The inuence of different traits. In agreement with previous results (Gil et al., 2001),
this gene on the index C18:1/(C18:0 + C18:1), as well as on C14:1/ the nt821-del11 allele responsible for the double muscle phenotype
(C14:0 + C14:1), has been recently reported by Baeza et al. (2012). was associated with colour parameters related to paler meat, increasing
Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) utilizes diacylglycerol lightness (L*), and absorbance at several wavelengths (Fig. S1), but
and fatty acyl-CoA as substrates in order to catalyze the nal stage of especially between 670 and 700 nm red spectrum , all of them at
triacylglycerol synthesis, and is known to affect fat content in milk 48 h and 10 days. Apart from these, nt821-del11 was associated with
(Grisart et al., 2002). The SNP ss77831745 analyzed here is the A G the increase in myosin heavy chain isoform IIX (MHCIIX), and the
polymorphism of the ApA to GpC dinucleotide substitution in exon 8 de- wild-type allele with an increase in collagen content, MHCI, pH on
scribed by Grisart et al. (2002), causing a lysine to alanine substitution at thawed samples at 10 days post mortem (pH thaw), and the ratio K/S
aa 232 with documented effects on milk fat content and marbling at several wavelengths, and specially between 670 and 700 nm at
(Grisart et al., 2002; Thaller et al., 2003). Also, Dunner, Sevane, Garca, 10 days (Fig. S1). The inuence of this polymorphism on the muscle
Levziel, and Williams (in press) recently described an effect of this bre prole is consistent with previous data (Gil et al., 2001) showing
SNP on beef avour, and 16:1 and 12:0 muscle content in cattle. In agree- that the hypertrophic allele increases MHCIIX bres.
ment with these data, the G allele was found to be associated with the
amount of palmitoleic acid (16:1) in the muscle in the current study. 4. Conclusions
The second category of genes of the PPARG-PPARGC1A network in-
cludes genes activated when glucose is decreased. Hepatocyte nuclear The candidate gene approach performed has revealed a total of 42
factor 4 (HNF4A) is a highly conserved member of the nuclear re- associations involving 10 different genes, some of them suggesting
ceptor superfamily (Sladek, Zhong, Lai, & Darnell, 1990). Specically, new relationships between genes and meat quality traits. Most of
the HNF4A/PPARGC1A pathway plays a crucial role in the transcrip- these associations have an overall low effect probably due to the fact
tional regulation of hepatic gluconeogenic genes that are activated that the traits measured are inuenced by multiple genes and the
at fasting and inhibited by SREBP1 in a fed state (Yamamoto et al., genes detected only account for a small amount of the total effect, and
2004). In the present study, the association analysis suggests that in addition, the SNP screened may not be causative mutations but in
one SNP near HNF4A (ss61961144) inuences the activity of the en- linkage disequilibrium with them. However, amongst the novel associ-
zyme isocitrate dehydrogenase (ICDH), which is related to the oxida- ations found in this study, it is worth highlighting the considerable ef-
tive potential of muscle bres to catabolize FA (Beer et al., 2007). fect of PPARG on the benecial omega-3 PUFA DPA, EPA and DHA, and
Lipoprotein lipase (LPL) plays a key role in lipid metabolism by hy- LPL on DGLA and AA. Also, the associations found here between the
drolyzing triglyceride-rich particles, thereby generating free FA and genes ATF4, BCL3, HNF4A, PPARGC1A, SCAP, and SCD, and meat organo-
glycerol for energy utilization and storage (Merkel, Eckel, & Goldberg, leptic characteristics and lipid prole, despite having small effects, are
2002). Several studies have reported associations of this gene with plas- described here for the rst time and may bring insights into the com-
ma lipid levels (Sagoo et al., 2008), and with milk fat content and dry plex gene-networks underlying economically important traits. Regard-
weight in goat (Badaoui et al., 2007). Here, the T allele of the exonic ing GDF8 and DGAT1, the results obtained conrm previously described
SNP ss65478732 was found associated with an increase of both neutral associations. All these data offer scientic community a starting point
dihomo-gamma-linolenic acid (DGLA, 20:3n6) and arachidonic acid from which to study some complex gene-networks underlying eco-
(AA, 20:4n6) in muscle. The large effects of this SNP, for which the nomically important traits.
TT genotype increased the amount of DGLA in muscle by 16% and of ar- Supplementary data to this article can be found online at http://
achidonic acid by 19% compared to CC homozygous, are consistent with dx.doi.org/10.1016/j.meatsci.2013.02.014.
its documented physiological role and previous associations.
B-cell CLL/lymphoma 3 (BCL3) is a transcriptional regulator of genes Acknowledgements
controlling energetic metabolism through the activation of diverse
pathways, such as the coactivation of the nuclear receptors ERR and This work was supported in part by an EC grant QLK5CT2000-0147.
PPARA synergistically with PPARGC1A (Yang, Williams, & Kelly, 2009).
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