J. Anim Sci. 2006. 84:2907-2913. doi:10.2527/jas.2005-663
© 2006 American Society of Animal Science
Adipocyte fatty-acid binding protein is closely associated to the porcine FAT1 locus on chromosome 41
A. Mercadé*,2,
M. Pérez-Enciso
,*,
L. Varona
,
E. Alves
,
J. L. Noguera,
A. Sánchez* and
J. M. Folch*
* Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain;
and
Institut Català de Recerca i Estudis Avançats, 08010, Barcelona, Spain;
and
Àrea de Producció Animal, Centre UdL-IRTA, 25198, Lleida, Spain;
and
Departamento de Mejora Genética Animal SGIT-INIA, 28040, Madrid, Spain
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Abstract
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We identified 22 polymorphisms in the adipocyte fatty-acid binding protein (FABP4) gene, a strong positional candidate gene for the FAT1 locus in porcine chromosome 4. The most informative polymorphism, an insertion/deletion in intron 1, together with a single nucleotide polymorphism in intron 3, was genotyped in a cross between Iberian and Landrace pigs. After performing QTL, single marker, and haplotype analyses, we showed that there were at least 2 quantitative trait genes in the FAT1 region and that the FABP4 polymorphism was tightly associated to fatness. A comparison of allelic frequencies in a panel of pig breeds suggested that the Del2634C polymorphism was under indirect selection. We also showed that FABP4 is tightly associated to fatness but not growth. Furthermore, a haplotype analysis suggests that there is genetic heterogeneity at the FAT1 locus within the Landrace breed.
Key Words: FABP4 • FAT1 • fatness • pig • QTL
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INTRODUCTION
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The first highly significant QTL in animals was reported by Andersson et al. (1994)
. It was located on chromosome 4 and had an important effect on fatness and on growth. This locus was named FAT1 after Marklund et al. (1999) and has been confirmed in a large number of crosses involving different breeds (Paszek et al., 1999, De Koning et al., 2001, Milan et al., 2002). But despite the high repeatability across experiments and that it is the "oldest" QTL identified in animals, its precise molecular nature has remained elusive. This situation is in stark contrast with other loci that have been cloned already, like RN (Milan et al., 2000) or IGF2 (Van Laere et al., 2003).
Why FAT1 is still unidentified is not clear at this moment, but one reasonable explanation is that there are at least 2 loci involved, complicating the search for causal mutations. Recently, we have obtained additional statistical evidence in the Landrace x Iberian (IBMAP) experiment that reinforces the 2-locus hypothesis (Mercadé et al., 2005), with one of the QTL mapping to the adipocyte fatty-acid binding protein (FABP4) gene. The FABP4 facilitates transport of fatty acids from the plasma membrane to sites of oxidation or esterification (Haunerland and Spener, 2004). A previous report described that a microsatellite within the first intron of the pig FABP4 gene was associated with differences in intramuscular fat content in a Duroc population (Gerbens et al., 1998). To our knowledge, no further associations between FABP4 and pig fatness have been reported.
Thus, the recent results of Mercadé et al. (2005) and the fact that, because of its physiological properties, the FABP4 gene is a logical candidate gene for fat metabolism motivated us to further analyze FABP4 polymorphisms in the IBMAP cross.
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MATERIALS AND METHODS
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All animals of study were cared for according to the guidelines of IRTA.
Animal Material and Traits Analyzed
The population used consisted of a cross between 3Iberian Guadyerbas boars and 31 Landrace sows. Complete details of the cross are given in Varona et al. (2002) and Mercadé et al. (2005). The pedigree was formed by 79 F1, 321 F2, 87 F3, and 85 backcross animals obtained after mating 4 F2 and 22 Landrace gilts.
We analyzed the traits with largest QTL effects in the FAT1 region: backfat thickness measured with a ruler at the shoulder level (BF1), backfat thickness measured with a ruler at the last rib (BF2), live weight recorded 1 or 3 d before slaughter (LW), carcass length (CL), and shoulder weight (SW). The measures of these phenotypic traits are described elsewhere (Óvilo et al., 2000, Pérez-Enciso et al., 2000).
Amplification and Sequencing of the Pig FABP4 Gene
All 4 exons and intron 3, as well as fragments of the promoter region, and of intron 1, 2, and 3'-UTR region were amplified and sequenced in the 3 Iberian Guadyerbas boars and 7 Landrace sows (primers in Table 1). The PCR were performed in a 25-µL final volume containing 1.5 mM MgCl2, 200 µM dNTP, 300 nM of each primer, 60 ng of genomic DNA, and 1.3 U of Expand High Fidelity PCR (Roche Molecular Biochemicals, Barcelona, Spain). Thermocycling was at 95°C for 3 min., followed by 10 cycles (95°C for 30 s, 56°C 1 min, and 72°C 2.5 min), by 25 cycles with increasing the extension step by 20 s in each cycle, and a final extension (72°C for 10 min). The amplified products were sequenced using the BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit in an ABI PRISM 3100 Avant sequencer (Applied Biosystems, Foster City, CA). The sequences obtained were analyzed using the SeqScape v2.1 software (Applied Bio-systems).
Genotyping
We implemented a pyrosequencing protocol to genotype a total of 56 founders, 79 F1, 321 F2, 87 F3, and 85 backcross pigs of the IBMAP pedigree for loci Del2634C and C6252T. For the insertion/deletion polymorphism at locus Del2634C, a 186 bp-long fragment of intron 1 was amplified by PCR using FABPpyroFw and FABP4in1R primers (Table 1
). For the C6252T locus, a 197 bp-long fragment of intron 3 was amplified by PCR using FABPpyro2Fw and FABPpyro2Rv primers (Table 1). The PCR were performed in a 25-µL final volume containing 1.5 mM MgCl2, 200 µM dNTP, 500 nM of each primer, 45 ng of DNA, and 0.6 U of Taq DNA polymerase (Invitrogen, Barcelona, Spain). Thermocycling was 95°C for 3 min, 45 cycles of 95°C for 1 min, 60°C for the Del2634C or 64°C for the C6252T for 1 min, 72°C for 1.5 min, and a final extension of 72°C for 5 min.
The genotyping of these polymorphisms was done in a PSQ HS 96 system (Pyrosequencing AB, Uppsala, Sweden) using FABPpyroRv and FABPpyro2seq as the sequencing primers for the Del2634C and the C6252T polymorphisms, respectively (Table 1). Microsatellite genotyping was carried out as detailed previously (Mercadé et al. 2005). Briefly, microsatellite PCR reactions were carried out in an automatic PCR ABI PRISM 877 integrated thermal cycler (Perkin Elmer, Foster City, CA) and analyzed with fluorescent detection in an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The genotypes were determined using the GeneScan 3.7 Analysis software (Applied Bio-systems).
Statistical Analyses
We employed 3 models to analyze the data: a QTL model [1], an association model [2], and a QTL + association model [3], as follows:
 | [1] |
 | [2] |
 | [3] |
where yi is the ith individual record; batch is the slaughter batch (14 in total); ßc is a covariate coefficient with c being carcass weight (except for LW, which was corrected by age at slaughter); a is the QTL additive effect; d is the dominance effect; Pa and Pd are the additive and dominance coefficients, respectively (Pa is the probability of the individual being homozygous for alleles of Iberian origin minus the probability of being homozygous for alleles of Landrace origin, and Pd is the probability of the individual being heterozygous); u is the infinitesimal genetic effect; and e is the residual. In [2] and [3] 
ikh is a 0 or 1 indicator variable, which is 1 when the allele at the hth haplotype (h = 1, 2) of the ith individual is k and 0 otherwise; and g represents each allelic effect. A dominance effect (d) was included when significant (P < 0.05); i.e., only for BF2 and LW. Model [2] was fitted at the Del2634C and the C6252T polymorphisms and the FAT1 region markers, whereas models [1] and [3] were fitted every centimorgan across SSC4, and model [3] included the Del2634C locus.
To gain more insight, we studied the haplotypic effects of the polymorphisms in FABP4 and SW35 using model [2]. Thereafter, we concentrated on BF1 for the sake of conciseness and because it was the QTL with the greatest significance. First, we considered loci Del2634C and C6252T and next the haplotypes that included the Del2634C and the FABP4 and SW35 microsatellites. A disadvantage of haplotype analyses is that it can reduce power and make interpretation difficult when the number of haplotypes is large. To improve upon this, we devised a sequential pooling strategy in which the goal was to find an allele partition with minimum P value. Initially, we fitted all haplotypes, next we identified the 2 haplotypes that had the most similar effects and pooled them. We computed the P value associated with the newly pooled haplotypes. We repeated the pooling strategy until the P value could not be decreased.
Finally, we confirmed the haplotype analysis using a transmission test. We selected the 4 heterozygous F2 boars that sired the F3 and backcross generation, and we applied model [2], excluding batch and infinitesimal effect only to the offspring where the allele origin could be unambiguously determined. We computed the P value of the transmitted effect of one or the other haplotype for the offspring.
Nominal P values were obtained via likelihood ratio tests and the 
2 approximation. All statistical analyses were carried out with Qxpak software (Pérez-Enciso and Misztal, 2004), which is freely available for download at: http://www.icrea.es/pag.asp?id=Miguel.Perez (last accessed 2 June 2006).
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RESULTS AND DISCUSSION
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Polymorphisms
After sequencing the 3 Iberian founder boars and 7 Landrace sows, 22 polymorphisms were found (Table 2). Nevertheless, none of these polymorphisms were in the exons. All Landrace sows were homozygous and identical to the reference sequence GenBank Y16039, while 1 Iberian Guadyerbas boar was homozygous for the alternate alleles found (fourth column of Table 2), and the 2 remaining boars were heterozygous for all positions except 2634, where all boars were homozygous for the C allele. Thus, the only polymorphism that showed extreme frequencies between the parental lines (Iberian Guadyerbas and Landrace) was position Del2634C.
QTL and Association Analyses
A linkage reanalysis showed that SW35 was wrongly positioned in our current map, and the new order was SW317 – FABP4 – SW35, whereas the order used in Mercadé et al. (2005) was SW317 – SW35 – FABP4. This new order coincides with RH mapping data.
The classical QTL analysis (model 1) is shown in Table 3. These analyses have already been reported (Mercadé et al., 2005), except that the new marker order was used here. The only difference with Mercadé et al. (2005) is that here we also included the Del2634C locus as a simple marker. Regarding the association study (models 2 and 3), the first remarkable result is that the Del2634C locus is tightly associated with fatness and shape traits (shoulder weight and carcass length), but not with growth. Note that the growth QTL maps to a shifted position relative to the fatness QTL and that the growth QTL is significant after fitting the Del2634C locus (P = 5 x 10–3), and the QTL significance decreases dramatically for the remaining traits. This would confirm that there are at least 2 quantitative trait genes in the FAT1 region, one affecting fatness and shape, the other with an effect on growth. It is interesting to recall that the FAT1 locus is located between S0073 and S0214 (Moller et al., 2004), which falls outside of the strongest association with the fatness QTL detected here. Although the QTL for BF2 showed dominance, we did not find any significant dominance effect for the Del2634C locus itself (results not presented). Linkage disequilibrium measures in the overall population were not very illustrative (shown in Table 4 for selected markers). Values for the correlation (r) were relatively small because a high r is only possible if disequilibrium is high and allele frequencies are similar (Ardlie et al., 2002) whereas D’, computed as in Farnir et al. (2000), was greater than r.
To gain more insight, we studied the haplotypic effects of the polymorphisms in FABP4 and SW35 using model [2]. First, we considered Del2634C and C6252T. Only 3 haplotypes were found in the parental population. Effects between haplotypes CC and CT were not significantly different, and it follows that a haplotype analysis is not necessary here because all variability was explained by the Del2634C polymorphism. This can also be inferred by noticing that P values for the haplotype models are not more significant than the single marker analyses (Table 5). Next, we considered the Del2634C, FABP4, and SW35 microsatellites. A total of 14 haplotypes were found in the founder animals; Iberian boars had 2 haplotypes. Using the pooling strategy described, we ended up with 4 haplotypes named A, B, C, and D in Table 6. This final pooled haplotype had a P value of 1 x 10–12, better than any of the markers taken individually (Table 3). Two relevant facts appear: 1) there is genetic heterogeneity within haplotypes segregating exclusively in Landrace (all Iberian boars were C-19-20 and C-20-22), and 2) there exists an interaction in the statistical sense between the 3 markers, and it was uncovered only after allele pooling. One must be cautious, though, in raising genetic interpretations from this interaction. It does not follow that epistasis exists (although it cannot be ruled out); it simply could be due to incomplete linkage disequilibrium between the haplotype and the causal mutation or to the presence of several causal mutations. Finally, we repeated model [3] analysis including the haplotype partition instead: now the PQTL was 0.24, i.e., adding a QTL does not improve model fitting significantly and the haplotype explains all variability for BF1.
Transmission Test (Half-Sib Analysis)
Additional evidence about the effect of a given allele can be obtained by comparing the phenotypes of offspring that have received alternative alleles from heterozygous parents, i.e., equivalent with a half-sib analysis because we considered only paternal haplotypes. Results are shown in Table 7. All estimates were in the expected direction, with the haplotype A increasing fatness, in agreement with results in Table 6. Although not all were significant at the 5% level, this was probably because of the small size of each family. The combined estimate across boars was 0.32 ± 0.08 (P < 0.001) and 0.36 ± 0.10 considering the last 3 boars. This confirms that the haplotype partition reported in Table 6 is associated with fatness.
Allelic Frequencies in Pig Breeds
A panel of different pig breeds was also genotyped for the Del2634C locus (Table 8). We also included wild boar of European and Tunisian origin, Iberian from 6 different strains, plus babirusa (Babyrousa babyrussa) and collared peccary (Pecari tajacu) as outgroups. Out-group genotypes permit us to conclude unambiguously that the mutant allele is actually the deletion, i.e., the reference sequence GenBank Y16039 contains the derived allele. But the most interesting result from Table 8 is that there is a clear gradient in allele frequencies: intermediate in wild boar and "unimproved" breeds of Asiatic (Meishan) and European (Iberian) origin, through extreme for the mutant allele in breeds selected for leanness and growth (Pietrain, Landrace and Large White). Although the wild type allele seems to be fixed in the Vietnamese pig, these results are not conclusive because of the small sample size. The fact that the deletion is segregating at intermediate frequencies in wild boar and is common in Meishan and Iberian pigs suggests that this is a very ancient mutation. It should be recalled that there is substantial evidence that the Iberian breed has not been introgressed with Asiatic alleles, as supported by analysis of mitochondrial DNA (Alves et al., 2003).
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IMPLICATIONS
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We have shown that FABP4, or a nearby gene, is tightly linked to the FAT1 locus. The haplotype association model is able to explain all variability for backfat thickness measured with a ruler at the shoulder level. Also importantly, we have demonstrated that there exist at least 2 functional mutations: one, close or within the FABP4 gene with a strong effect on fatness, and the second, about 20 cM away, with a smaller effect on growth. Moreover, there is evidence of genetic heterogeneity for fatness within the FABP4 region itself, and further studies will be needed to disentangle its exact genetic nature.
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Footnotes
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1 In addition to the IBMAP cross, carried out in Nova Genètica facilities (Lleida, Spain), many people and institutions provided samples: we would like to thank specially C. Talavera (Madrid’s zoo), Copaga, L. Silió (Instituto Nacional de Investigaciones Agrarias), E. v Eckhardt, C. Renard (Institut National de la Recherche Agronomique), M. Cumbreras (Diputación de Huelva), A. Angiolillo, J. Garrido and Diputación de Córdoba, Paulino Martínez, J. Jaume (Instituto de Biología Animal de Baleares), and Associació de Criadors del Porc Negre, C. Lemús (Mexico), and E. Grinflek (Norway). A. Mercadé is funded by a Formació Personal Investigador (FI) fellowship from the Generalitat de Catalunya. Work funded by INIA acción especial (CPE03-010-C3) and in part by grant AGF2004-00103/GAN (Ministerio de Educación y Ciencia, Spain). 
2 Corresponding author: Anna.mercader{at}uab.es
Received for publication November 15, 2005.
Accepted for publication May 22, 2006.
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Abstract
INTRODUCTION
