WO1999014365A1

WO1999014365A1 - The porcine adipocyte fatty acid-binding protein encoding gene and methods to localise, identify or mark genes or alleles or quantitative trait loci of farm animals

Info

Publication number: WO1999014365A1
Application number: PCT/NL1998/000541
Authority: WO
Inventors: Frans Gerbens
Original assignee: Pig Genes B.V.
Priority date: 1997-09-18
Filing date: 1998-09-18
Publication date: 1999-03-25
Also published as: CA2304349A1; EP1015634A1; AU9189998A

Abstract

The present invention provides a novel sequence of the pig A-FABP gene, as well as methods of using said gene and its products. Especially the invention provides methods for detecting different alleles of the pig A-FABP gene, which different alleles are associated with differences in the genotypic and/or phenotypic traits of the pigs having those alleles. Especially the invention provides methods for distinguishing between alleles resulting in different phenotypes, particularly using techniques involving selective amplification of pig A-FABP gene derived materials. These techniques are especially suitable for selecting animals to be used in breeding programmes. Breeding programmes employing such techniques are also disclosed.

Description

Title: The porcine adipocyte fatty acid-binding protein encoding gene and methods to localise, identify or mark genes or alleles or quantitative trait loci of farm animals.

INTRODUCTION

The invention relates to the field of molecular biology as well as to the field of breeding methods for farm animals, in particular pigs. In particular the invention relates to the use of diagnostic methods derived from the field of molecular biology to be applied in breeding programmes that select animals on production traits that improve their breeding value . By selecting animals on their breeding value calculated mainly from phenotypic measurements of production traits, breeding has greatly improved the genotype for production traits of livestock animals. Thus, traditionally, breeding programmes have selected for phenotypic characteristics of animals. However, more recently selection for genotypic characteristics that are associated with improved production traits have gained interest in the field. Selection for phenotypic characteristics entails mainly selection of the respective animal, offspring or siblings or other relatives of the animals to be selected whereas selection of specific genotypic characteristics allows for earlier and specific detection of animals of interest.

Within methods that select on specific genotypic characteristics, one may distinguish between methods that detect genetic variation in genes or quantitative trait loci that are merely associated with production traits of animals and methods that detect genetic variation in functional genes that directly influence those production traits. One of the former methods is a marker assisted selection wherein polymorphisms in markers identified in a random manner are associated with production traits. For instance, meat production is closely linked to embryonic muscle formation, and, consecutively, to the distribution of muscle cells and fat cells. Biologically, production is concentrated in defined tissues of the animal, e.g. muscle tissue for lean meat production. In breeding programmes for optimising porcine lean meat production, various levels of selection pressure have been applied to different tissues (i.e. muscle, fat and bone) . However, when selecting for lean meat, and thus the absence of fat, one may lose certain traits that are wanted after all, i.e. traits that are associated with taste and thus with the consumers perception of the final product.

In pig breeding programs traditionally a lot of emphasis has been put on the fat reduction because of the consumers interest in lean meat, and pig breeding has developed several pig varieties that efficiently can accomodate the client's wishes. Fat reduction is surveyed as a decrease in backfat thickness and a large reduction has been achieved since the establishment of breeding programmes in pigs. However reduction of the backfat depot may also result in less intramuscular fat (IMF) . This last depot is the main fat depot in meat and is positively correlated with the tenderness and taste and thus the acceptance of meat (Wood et al . , 1988) . IMF is located in muscle tissue in fat and muscle cells. To exclude the IMF depot from further reduction a marker or functional gene for this trait is necessary because IMF is hardly measurable in living animals. Recently, it has been statistically shown that a single major gene for IMF deposition in pigs must be present (Janss et al . , 1994), however, the sequence, location and mode of action of the putative gene were not disclosed. Here, with the present invention, we present evidence of a muscle-tissue-specific candidate gene located on porcine chromosome 4 which is the adipocyte fatty acid-binding protein (A-FABP) gene, also known as aP2 or adipocyte lipid binding protein (ALBP) . Fatty acid binding proteins (FABP's) are small intracellular proteins involved in fatty acid transport from the membrane to the sites of β oxidation and/or triacylglycerol or phospholipid synthesis (Veerkamp and Maatman, 1995) . Furthermore, FABP ' s modulate the intracellular fatty acid concentration (Veerkamp et al.,1993). Fatty acid metabolism has historically been linked to insuline resistance (Randle, 1963) , and therefore mutations in FABP genes may be associated with changes in cellular insulin resistance or dependency, fatty acid oxydation and fatty acid binding. FABP ' s are members of a family of intracellular lipid binding proteins comprising at least eight structurally distinct types and named after their first tissue from which they were isolated or identified: adipocytes, brain, epidermal cells, heart, intestinal cells, ileal cells, liver and myelin cells. The present invention provides among others an isolated or recombinant pig A-FABP gene specific nucleic acid molecule or pig A-FABP gene specific fragments thereof comprising or hybridising to the nucleotide sequence as shown in figure 1, or its complementary sequence or the RNA equivalents thereof. The locus of this gene is on porcine chromosome 4. The pig A-FABP gene can be assigned to functions in the regulation of intramuscular fat, thereby changing the ratio of fat deposited within the muscle versus fat deposited outside the muscles, i.e. in backfat depots. Since production and deposition of fat is energy consuming and takes away energy for other purposes, such as muscle growth, the regulation of intramuscular fat is correlated to the regulation of growth, and thus body weight and average daily gain and feed efficiency. Since FABP's are involved in fatty acid transport they can influence fatty acid oxidation rates, the metabolism of fatty acid derivatives in the tissue and the fatty acid composition of cells and thus of meat. Furthermore, FABP's may regulate cellular insulin dependency. Also, in pregnant animals, fat storage has an impact on embryo survival, and regulation of A-FABP will influence birth rates and littersize. Since A-FABP regulates functional differentiation of mammary epithelial cells it is involved in regulating the quantity and composition of the milk available, thus influencing the growth and survival of newborn animals. With the present invention, the genetic variation within the pig A-FABP gene with respect to variation in regulation of expression can now be revealed and analysed for association with above production traits and physiological characteristics.

The present invention further provides a method to generate via recombinant DNA techniques an animal , such as small laboratory animals or farm animals, i.e. a pig, with additional genetic material originating from the pig A-FABP gene. Such animals may than encode wanted alleles of this gene and constitutively or transiently express allelic proteins or fragments thereof that enhance the production or physiological characteristics of those animals. The invention further provides methods to generate proteins or (poly) peptides comprising various allelic proteins or fragments thereof derived from the pig A-FABP gene. Such peptides, or antibodies specifically directed against such peptides, may be used to influence production traits in the live animal, but may also be used in cell- culture systems in vi tro . Such (poly) peptides or proteins, or antibodies specifically directed against these, may also be used in diagnostic test systems to select animals that express wanted forms of allelic proteins or fragments thereof encoded by the pig A-FABP gene.

The invention further provides methods localising, identifying or marking genes or alleles or quantitative trait loci, in particular those corresponding to the pig A-FABP gene, in samples, in particular biological samples, cells or tissues, such as but not limited to hair, skin or blood, of farm animals, in particular pigs, by allowing for specific amplification of genomic fragments of those genes or alleles or quantitative trait loci of pigs. Since marker assisted selection of animals is frequently based upon genetic variation that exists within functional genes that influence a production trait directly, i.e. genes such as the pig A- FABP that regulates fatty acid binding, one of the methods that the invention provides is a method that identifies or marks loci or genes and that can distinguish between characteristics of alleles of those genes which characteristics serve as markers in selection programmes for animals with specific versions of those genes that are directly linked with improved production traits.

The invention further provides a method wherein polymorphic restriction sites within functional genes and thus different alleles of those genes are identified by allowing for specific amplification of genomic fragments of those genes, in particular by allowing for specific amplification of fragments of the A-FABP gene. Amplification methods are well known in the art, the best known being PCR. A short description of the PCR used herein is given in the experimental part. Other primers, enzymes and conditions can of course be applied. After amplification a suitable method of identifying wanted alleles and polymorphic sites related thereto is a restriction endonuclease treatment. Suitable restriction enzymes for identifying pig A-FABP alleles are for example found by amplifying and subsequent digesting A- FABP allele fragments of various breeds of pigs. By these methods large numbers of pigs can be rapidly genotyped for studies in which genotypic variation can be associated with growth characteristics and other production or performance traits of pigs. Such production traits are for example body weight (BW) , back fat thickness (BFT) , intramuscular fat (IMF) and drip loss (DRIP) . Yet another method for identifying wanted alleles is the detection of polymorphic sites such as microsatellites or CA-repeats found in the A- FABP gene. Various alleles, such as the Al to A9 alleles can in this way be detected. Yet another method is the detection of polymorphic sites in porcine short interspersed elements (SINEs) sequences. However, there are many other methods identifying polymorphisms in alleles, both at the nucleic (DNA/RNA) level and at the product (protein) level . The A-FABP protein is encoded by a nucleic acid sequence given in figure 2. In particular at the protein level there are many possibilities using immunoassays, whereas at the nucleic acid levels there are many assays which all include some kind of hybridisation step of for instance primers or labelled nucleic acids. A very good possibility would be mismatch PCR. Primers to be used in the invention can be identified by the person skilled in the art, the sets given in the experimental part are for illustrative purposes only. Furthermore, the methods according to the invention can be developed into diagnostic assays or kits by which selection of pigs with alleles of interest can be performed in routine screening protocols employed in breeding programmes. With such protocols better results of selection can be expected when genes responsible for regulation of commercially interesting body tissues can be rapidly identified and controlled.

In the specific case of the pig A-FABP gene, such testing protocols can be used to identify, select and breed farm animals, such as pigs, which have better production traits, such as IMF% or backfat thickness or average daily weight gain or feed efficiency, than the average animal in the population. Better production traits such as BW or daily weight gain will increase the production per year expressed as amount of meat per animal raised. A population of animals with a higher and less variable IMF% will result in a more homogenous product (meat) which is also better appreciated by putative customers because of a better tenderness or taste. Furthermore, selection for higher IMF% may be possible while at the same time selection against fat deposition in other depots, such as backfat, can be performed.

EXPERIMENTAL

One objective in pig breeding programs is the reduction of fat in the carcass to meet the consumers ' demand for lean meat. This reduction is accomplished by selection for reduced back fat thickness. As a result of this selection the intramuscular fat depot, which is positively correlated with taste and meat acceptance (Wood et al . , 1988) may be reduced. However both fat depots are not or only moderately correlated (Hovenier et al . , 1992) indicating that IMF can be treated independently from BFT at least partially. In order to investigate the role of A-FABP in porcine IMF accretion the respective gene was identified, sequenced and chromosomally localised. Moreover, genetic variation within this gene was identified. To establish the role of A-FABP in porcine IMF accretion this genetic variation was studied in a Duroc pig population.

Materials and methods

Isolation of A-FABP containing phage clones . A porcine genomic DNA EMBL3/SP6/T7 lambda library (Clontech Laboratories Inc. Palo Alto, CA) was screened by plaque hybridization (Sambrook et al . 1989) to mouse A-FABP (ALBP) cDNA (Bernlohr et al . , 1984) in the pGEM vector labeled with [α-³²P]dCTP by nick translation (Sambrook et al . 1989). Briefly, 500,000 plaques were transferred to replica nitrocellulose filters and incubated in denaturation buffer (1.5 M NaCl/0.5 M NaOH) for 2 min, neutralisation buffer (1.5 M NaCl/0.5 M Tris-HCl pH 8.0) for 5 min and fixation buffer (0.2 M Tris-HCl pH 7.5/2X SSC(0.3 M NaCl , 0.03 M sodium citrate)) for 30 s. The filters were air-dried and baked at 80°C for 2 h. The filters were prehybridized (6 X SSC/0.5% (w/v) SDS/5 X Denhardt ' s and 100 mg/ml NaOH-treated salmon sperm DNA) for two h at 67°C and hybridized at 67°C overnight in the same buffer containing the radioactive probe. The filters were washed four times with 2 X SSC, 0.1% (w/v) SDS for 30 min at room temperature. A single plaque that showed positive signals on replicate filters was purified by two additional rounds of low density plaque hybridization. Phage DNA was isolated using the plate lysate method (Sambrook et al. 1989) .

DNA sequence analysis DNA from a positive phage clone was used to subclone the A- FABP gene. Therefore, the BaπHI , HindiII and SacI restriction digestion fragments of the phage DNA were subcloned in pBS . Recombinant plasmid DNA from A-FABP clones was purified with the Wizard Maxiprep kit (Promega, Madison, WI , U.S.A.) . The nucleotide sequence was determined by cycle sequencing

(Perkin Elmer, Foster City, CA, USA) and the analysis was performed on a ABI 373 (Perkin Elmer) . The DNA sequence was analysed by the Genetics Computer Group (University of Wisconsin) software packages.

Polymerase chain reactions

PCR amplifications were performed on 1 μl of a 1:1000 dilution of phage DNA preparations or 50 ng of genomic DNA in

50 μl containing 0.2 units ampliTaq DNA polymerase (Perkin Elmer) in 10 mM Tris-HCl (pH 8.3) /50 mM KC1/1.5 mM MgCl₂/0.5 μM of each primer and 0.2 mM of each dNTP . After 3 min of denaturation at 94°C, 33 cycles of amplification were carried out at: 94°C for 1 min, the indicated annealing temperature for 1 min and 72 °C for the time considering the length of the expected fragment.

H-FABP .RFLP screening

The PCR conditions, restriction digestions with Haelll, Hinfl and spl and the corresponding alleles have been described before (Gerbens et al , 1997) .

Microsatelli te analysis

According to the sequence flanking the CA dinucleotide repeat in the A-FABP gene, primers (example of forward primer GGTACTTTCTGATCTAATGGTG and reverse: GGGAACTCTTGAAGTCTTTCTC) were designed to amplify the corresponding region. Annealing was performed at 56°C. The forward primer was fluorescently labelled and the PCR product was analysed on a denaturing polyacrylamide gel on a ABI 373. The length of the PCR product was estimated according to standard marker fragments (Perkin Elmer) using the GENESCAN software package (Perkin

Elmer) . Observed polymorphic fragments are presented in Table 1.

Chromosomal localization A pig/rodent somatic cell hybrid panel (Rettenberger et al . 1996) was used to assign the A-FABP gene to a specific chromosome by PCR. DNA (100 ng) from each cell hybrid containing porcine chromosomes in various combinations was used in a PCR reaction which unambiguously amplified porcine A-FABP gene exon 3 through exon 4.

Concordancy and correlation (φ) were statistically evaluated as described by Chevalet and Corpet (1986) . The distribution patterns of the PCR signals for porcine A-FABP were compared with the distribution patterns of the reference loci of individual pig chromosomes. A marker is syntenic with a chromosome or reference locus with a probability of 97.5% if φ is >0.74 for 20 hybrid lines. Synteny can be excluded if φ is <0.59.

Animals and data collection

Two Duroc populations from Stamboek and Dumeco Breeding, each housed at separate test stations, were used in this association study. To produce informative offspring, 13 boars and 72 sows were selected based on heterozygosity for each H- FABP RFLP (Gerbens et al . , 1997) . However, some animals were homozygous for the Hinfl RFLP. The offspring was housed in groups and fattened with ad libi tum food access until slaughter weight of about 110 kg. After slaughter, the meat quality traits drip loss (DRIP) and IMF were measured in litter mates belonging to different H-FABP RFLP genotype classes. In total, data from 992 animals, including pedigree, were included in the analysis and for each animal performance traits like age, back fat thickness (BFT) and body weight (BW) were recorded. Furthermore, DRIP and IMF data from animals within the pedigree were also included in the analysis.

Meat quali ty measurements

After slaughter a sample of the M. Longissimus dorsi muscle was isolated at the 3^th lumbar vertebra and divided in three slices. IMF content was measured in one slice using soxleth petroleum-ether extraction. IMF is expressed as the weight percentage of wet muscle tissue. DRIP was measured in duplo in the remaining slices essentially according to Honikel (1985) . However, meat samples were of constant size in stead of constant weight and incubation was performed at 4°C hanging in glass bottles. DRIP is expressed as weight loss over 48 hours.

Statistical analysis

The statistical analysis was performed with the program PEST (Groeneveld, 1991) that uses pedigree information to estimate best linear unbiased estimates of the effect of each A-FABP genotype class on DRIP, IMF, BFT and BW. Pedigree information was included from 3 preceding generations.

Because the data was not sufficient to estimate heritabilities (h²) , the heritability estimates for each trait were taken from Hovenier et al . (1992) . Prior to statistical analysis, BFT was adjusted to a weight of 110 kg and BW was adjusted to a weight at 180 days of age, for each animal .

The effect of the different genotype classes on each trait was estimated with the following model : Trait= int + (test station*test year*test month) + sexe + litter + genotype class + animal + covariate + residual effect

where :

* Trait is BFT, BW, DRIP or IMF.

* All effects were fixed, except the animal and residual effect .

* For IMF content, age at slaughter and BFT were included as covariates in the analysis.

* For DRIP, test station*test year*test month was replaced by slaughter date.

* The animal effect was random, with a relationship matrix that followed from the pedigree of the animals.

Results & Discussion

Sequence analysis

In figure 1 the complete DNA sequence of the porcine A-FABP gene is shown. The A-FABP gene exhibits the four-exon/ three- intron stucture common to all known FABP genes. The size of the introns is 2629 bp, 840 bp and 471 bp, respectively. In addition, 2370 bp of the 5' upstream region and 1435 bp of the 3 ' downstream region are present . The coding region of the porcine A-FABP gene (Fig 2) shows 90% and 83% similarity with the human and mouse A-FABP coding regions (Baxa et al.,1989; Bernlohr et al.,1984).

Chromosomal localization

Analysis of the presence of the porcine A-FABP gene in each cell hybrid showed a single significant correlation of 0.81 with the presence of the S0001 microsatellite marker. All other chromosomes were asyntenic. Therefore, the porcine A- FABP gene was assigned to chromosome 4. This assignment is consistent with the human and mouse A-FABP gene localisation on chromosome 8q21 (Prinsen et al . , 1997) and 3 (Heuckeroth et al.,1987), respectively. Heterologous chromosome painting demonstrated that the short arm and centromeric end of the long arm of porcine chromosome 4 have conserved synteny with human chromosome 8 (Rettenberger et al . , 1995).

Genetic variation

Within the A-FABP gene sequence several potential polymorphic sites were detected. In intron 1 a CA repeat was detected 69 bp before the intronl-exon2 splice junction. Microsatellite analysis of this CA repeat in pigs from several breeds revealed at least 9 alleles (Table 1) . Interestingly, the A- FABP microsatellite was polymorphic for each breed analysed. Furthermore, some alleles seem to be specific to a single breed as for instance the A2 allele for Duroc pigs. Within the A-FABP gene sequence several regions were detected which had high homology with porcine short interspersed elements (SINEs) sequences. In particular, the 3' poly A tract of SINEs could be a source of informative markers (Ellegren et al . , 1993) . In the A-FABP gene two complete copies of porcine SINEs, including the poly A tract, were present in the 5' upstream region and in intron 2. The observed single adenine stretches display genetic polymorphism.

Association analysis The porcine A-FABP gene microsatellite was tested as a potential marker for IMF content, BW, BFT and DRIP in a Duroc pig population previously used for a similar analysis with the porcine H-FABP gene. In this Duroc population three alleles were present Al , A2 and A3 resulting in 6 different genotype classes. Table 2 shows the distribution of the genotype classes for each trait analysed. Because the frequency of the A3 allele is very low in this population, the genotype classes A2A3 and A3A3 can not be included in the analysis with respect to IMF and DRIP. Statistical analysis revealed that the A1A3 genotype class has a significant

(P=0.04) effect on IMF which was not influenced by BFT. In a previous analysis with this dataset the porcine H-FABP gene showed a significant (P<0.05) effect of RFLP genotypes on IMF. To exclude the possibility that the effect of the A1A3 genotype class was the result of this association, a correction for the H-FABP alleles was included in the analysis (Table 6) . Results show that the effect of the A1A3 genotype class is not affected and even more significant . Between the A1A3 and A1A1 genotype classes the contrast was approximately 1% IMF which is very high in relation to the average IMF content of 2.7% (phenotypic standard deviation 1.0%) for this Duroc population. The genotype classes, with respect to the microsatellite in the porcine A-FABP gene, show a considerable and significant difference in IMF content. Moreover, this effect is independent from the genetic variation in the H-FABP gene which was previously demonstrated to affect IMF content . With respect to BW significant contrasts were detected between the A1A2 , A2A3 and A3A3 genotype classes and the A1A1 genotype class.

Table 1: The frequency of A-FABP microsatellite alleles in several pig breeds represented by unrelated animals.

Alleles (bp)

A3 A4 A5 Al A6 A7 A8 A9 A2

Pig breed n 253 255 257 259 261 269 271 273 281

Meishan 8 .56 .44

Hampshire 9 .06 .94

Duroc 7 .00* .79 .21

Dutch 19 .21 .32 .13 .13 .08 .13

Landrace

Great 37 .05 .37 .20 .38

Yorkshire

Pietrain 5 .30 .10 .10 .10 .40

*allele not present in unrelated animals but detected in Duroc population tested for association.

Table 2 : The number of animals for each A-FABP genotype class for each trait as used in the association analysis.

Trait

IMF BW BFT DRIP

A1A1 47 154 154 40

A1A2 126 344 344 110

A1A3 6 33 33 3

A2A2 46 181 181 41

A2A3 2 31 31 2

A3A3 1 4 4 1

REST 212 245 245 222

Total 440 992 992 419

Table 3 : The genotype class mean values and respective standard variation for each trait

IMF BW BFT ukiy mean sd mean sα mean sα mean Sd

A1A1 2.77 0.86 -1.41 y.12 1.71 ±.bϋ 4.3b ^.19

A1A2 3.19 1.08 -0.52 8.99 2.16 1.66 4.07 2.13

A1A3 3.18 1.01 -1.26 7.82 1.59 1.42 4.14 1.73

A2A2 3.32 1.22 0.06 9.09 1.92 1.57 4.23 2.16

A2A3 3.45 1.49 1.59 8.46 1.32 1.47 5.89 4.65

A3A3 2.10 - 14.00 9.14 0.29 0.90 1.60 -

Rest 2.36 0.81 3.93 8.54 2.25 1.68 4.20 2.17

Table 4: Contrasts between A-FABP genotype classes for IMF and IMF adjusted for BFT (IMF/BFT) .

ΪHF IMF/BFT ccoonnttrr .. se contr. Se

A1A2 - 1A1 0.36 o.3y 0.30 0.37

A1A3 -A1A1 1.17** 0.58 1.01* 0.55

A2A2 -A1A1 0.25 0.41 0.19 0.39

A2 3 -A1A1 0.37 0.92 0.31 0.87

A3A3-A1A1 - -

REST-A1A1 0.13 0.29 0.16 0.27

**: P<0.05; *: P<0.10

Table 5: Contrasts between the A-FABP genotype classes for the traits BW, BFT and DRIP

BW BFT DRIP contr se contr se cont . se

A1A -A1A1 3.34 1.67** 0.14 0.27 -1.21 .bi

A1A3 -A1A1 1.92 2.73 0.69 0.45 -0.55 2.07

A2A2 -A1A1 2.32 1.77 0.10 0.29 -0.75 1.55

A2A3 -A1A1 5.71 3.10* 0.18 0.51 2.25 2.57

A3A3 -A1A1 9.41 4.95* 0.37 0.81 - -

REST-A1A1 2.61 1.30** 0.51 0.21** -1.21 1.11

**: P<0. 05; * : P< 0 .10

Table 6: Contrasts between the A-FABP genotype classes for IMF when corrected for each H-FABP RFLP genotype classes.

H-FABP RFLP

Mspl Haelll Hint I contr se contr se contr se

A1A2 -AlAl 0.34 0.39 0.34 0.39 0.36 0.39 A1A3-A1A1 1.23** 0.58 1.22** 0.58 1.21** 0.58 A2A2 -AlAl 0.23 0.41 0.23 0.41 0.25 0.41

** P<0,05

Figure 1. The porcine A-FABP nucleic acid sequence.

Figure 2. The porcine A-FABP nucleic acid sequence encoding the A-FABP protein.

Figure 3. Forward primer flanking CA-repeat.

Figure 4. Reverse primer flanking CA-repeat.

References

1. Andersson, L., Haley, C, Ellegren, H., Knott, S.A., Johansson, M. , Andersson, K. , Andersson-Eklund, L., Edfors- Lilja, I., Fredholm, M. , Hansson, I., Hakansson, J. and Lundstrom K. (1994) Genetic mapping of quantitative trait loci for growth and fatness in pigs. Science 262, 1771-1774.

2. Armstrong, M.K., Bernlohr, D.A., Storch, J. and Clarke, S.D. (1990) The purification and characterization of a fatty acid binding protein specific to pig ( Sus domesticus) adipose tissue. Biochem. J. 267, 373-378. 3. Baxa, C.A., Sha, R.S., Buelt M.K., Smith, A.J.,

Matarese, V., Chinander, L.L., Boundy, K.L. and Bernlohr, D.A. (1989) Human adipocyte lipid binding protein:purification of the protein and cloning of its complimentary DNA. Biochem. 28, 8683-8690. 4. Bernlohr, D.A., Angus, C.W., Lane, M.D., Bolanowski, M.A. and Kelly, T.J. (1984) Expression of specific mRNAs during adipose differentiation: identification of an mRNA encoding a homologue of myelin P2 protein. Proc . Nat. Acad. Sci. USA 81, 5468-5472. 5. Brockmann, G., Timtchenko D., Das, P., Renne, U. ,

Freyer, G., Kuhla, S., Teuscher, F., Wolf, J., Kϋhn, C. and Schwerin, M. (1996) Detection of QTL for body weight and body fat content in mice using genetic markers. J. Anim. Breed. Genet. 113, 373-379. 6. Chevalet, C. and Corpet , F. (1986) Statistical decision rules concerning synteny or independence between markers . Cytogenet. Cell Genet. 43, 132-139.

7. Ellegren, H. (1993) Variable SINE 3' poly (A) sequences: an abundant class of genetic markers in the pig genome. Mamm. Genome 4, 429-434.

8. Falconer, D.S. and Mackay, T.F.C. (1996) Introduction to quantitative genetics, 4th ed. (Longman Group, Essex, England) pp. 1-19. 9. Gerbens, F., Rettenberger, G., Lenstra J.A. , Veerkamp J.H. and te Pas, M.F.W. (1997) Characterization, chromosomal localization, and genetic variation of the porcine heart fatty acid binding protein gene. Mamm Genome 8, 328-332. 10. Groeneveld, E., Bunge R. , Wang, T. and Fernando R.H.

(1991) Hypothesis testing in multivariate mixed models and its implementation in PEST. 42^nd annual meeting of the european association of animal production, Berlin sept 8-12, 1991 11. Janss, L.L.G., Van Arendonk J.A.M., Brascamp, E.W. (1994) Identification of a single gene affecting intramuscular fat in Meishan crossbreds using Gibbs sampling.

12. Honikel, K.O. (1985) How to measure water-holding capacity of meat? Recommendations of standardized methods. Evaluation and control of meat quality in pigs. Martinus

Nijhoff Publishers, Dordrecht, The Netherlands, pp 129-142.

13. Hovenier, R. , Kanis, E., Van Asseldonk, Th. and Westerink, N.G. (1992) Genetic parameters of pig meatquality traits in a halothane negative population. Livest . Prod. Sci . 32, 309-321.

14. Prinsen, C.F.M., de Bruijn, D.R.H., Merkx, G.F.M. and Veerkamp, J.H. (1997) Assignment of the human adipocyte fatty acid binding protein gene (FABP4) to chromosome 8q21 using somatic cell hybrid and fluorescence in si tu hybridization techniques. Genomics 40, 207-209.

15. Rettenberger, G., Klett, C, Zechner, U. , Kunz, J., Vogel, W. and Hameister H. (1995) Visualization of the conservation between pigs and humans by heterologous chromosomal painting. Genomics 26, 372-378. 16. Rettenberger, G., Bruch, J., Fries, R. , Archibald, A.L. and Hameister, H. (1996) Assignment of 19 porcine type I loci by somatic cell hybrid analysis detects new regions of conserved synteny between human and pig. Mamm. Genome 7, 275- 279. 17. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning: a laboratory manual . Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

18. Veerkamp, J.H., Van Kuppevelt , T.H.M. S .M. , Maatman, R.G.H.J. and Prinsen C.F.M. (1993) Structural and functional aspects of cytosolic fatty acid binding proteins. Prostag- landins Leukot . Essent . Fatty Acids 49, 887-906

19. Veerkamp, J.H. and Maatman, R.G.H.J. (1995) Cytoplasmic fatty acid binding proteins: their structure and genes. Prog. Lipid Res. 34, 17-52.

20. Wood, J.D., Enser, M. , Moncrieff, C.B. and Kempster, A.J. (1988) Effects of carcass fatness and sex on the composition and quality of pigmeat . 34^t^^rι International Congress of Meat Science and Technology, august 29- September 2, 1988 Brisbane Australia. 562-564.

Claims

1. An isolated or recombinant pig A-FABP gene specific nucleic acid molecule or pig A-FABP gene specific fragments thereof comprising or hybridising to the nucleotide sequence as listed in figure 1 or its complementary sequence or the RNA equivalents thereof .

2. A method for localising, identifying or marking alleles of pigs, whereby said localisation, identification or marking is carried out using a molecule or a fragment or fragments thereof according to claim 1.

3. A method according to claim 2 whereby alleles are localised, identified or marked that are associated with production traits of pigs.

4. A method according to claim 2, or 3 to identify or mark alleles of the pig A-FABP gene.

5. A method according to claim 4 distinguishing between alleles of the A-FABP gene of pigs.

6. A method according to claim 4 or 5 detecting specific restriction sites in an allele of the A-FABP gene of pigs.

7. A method according to claim 4 or 5 detecting CA-repeat sequences in an allele of the A-FABP gene of pigs.

8. A method according to claim 4 or 5 detecting short interspersed elements sequences (SINEs) in an allele of the A-FABP gene of pigs.

9. A method using a molecule, a fragment or fragments thereof according to claim 1 for localising, identifying or marking genes or alleles or quantitative trait loci in samples, in particular biological samples, cells or tissues, such as but not limited to hair, skin or blood, by allowing for specific amplification of genomic fragments of those genes or alleles or quantitative trait loci.

10. A method according to any of claims 2-8 for localising, identifying or marking genes or alleles or quantitative trait loci in samples, in particular biological samples, cells or tissues, such as but not limited to hair, skin or blood of pigs, by allowing for specific ampli ication of genomic fragments of those genes or alleles or quantitative trait loci .

11. A method according to claims 9 or 10 in which the method of amplification is the polymerase chain reaction.

12. A method according to any of claims 2-11 identifying differences between alleles of the pig that are associated with differences in production traits of pigs.

13. A method according to claim 12 identifying alleles of the pig that are associated with intramuscular fat, backfat thickness, body weight, drip loss, embryo survival and birth weight .

14. Use of the methods according to any of claims 2-13 in marker assisted identification of pigs or in marker assisted selection of pigs.

15. Use of the methods according to claims 12 or 13 in breeding programmes .

16. A diagnostic assay or kit according to any of claims 2- 13.