WO2000006777A2 - Melanocortin-4 receptor gene and use as a genetic marker for fat content, weight gain, and/or feed consumption of animals - Google Patents

Abstract

Genetic markers in the porcine melanocortin-4 receptor (MC4R) gene are disclosed which are associated with fat content, growth rate, and feed consumption. Further, novel sequence data from regions of the gene are disclosed which may be used in a PCR test to screen for the presence of the marker. The genetic marker may be used to screen animals for breeding purposes which have the desired traits regarding fat content, growth rate, and feed consumption. Kits which take advantage of the PCR test are also disclosed.

Description

TITLE: MELANOCORTIN-4 RECEPTOR GENE AND USE AS A GENETIC

MARKER FOR FAT CONTENT, WEIGHT GAIN, AND/OR FEED * CONSUMPTION OF ANIMALS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 60/094,287 filed July 27, 1998 and U.S. Provisional Application Serial No. 60/116,186, filed January 15, 1999, the disclosures of which are hereby incorporated by reference.

GRANT REFERENCE CLAUSE

This invention was supported at least in part by grants from the United States

Department of Agriculture through the Iowa Agriculture and Home Economics Experiment

Station (IaHees) and Project Number IOW03148 (Hatch Funds). The United States government may have certain rights in this invention.

FTELD OF THE INVENTION

The present invention relates to a method of genetically evaluating animals by assaying for the presence of at least one genetic marker which is indicative of one or more ofthe traits of fat content, growth rate, and feed consumption. In particular, the method analyzes for variation in the melanocortin-4 receptor (MC4R) gene which is indicative of these traits. Even more particularly, the method analyzes for a polymorphism in the MC4R gene.

BACKGROUND OF THE INVENTION There is an increasing consumer demand for meat products having low fat content.

This demand is fueled by accumulating evidence in the scientific literature that a high consumption of animal fat, especially fat with a high proportion of saturated fatty acids, represents a significant health hazard, including risk for cardiovascular disease. Other health concerns associated with high fat meats include their high content of cholesterol and the addition of relatively high amounts of salt which are added to improve the binding characteristics since salt aids in extracting the native water binding component myosin from the meat. Furthermore, an increasing number of consumers find meat products containing chemical additives such as phosphates, emulsifying additives, and anti-oxidants less acceptable.

Faced with consumers who seek a healthier meat product, meat producers are continually pressed to offer cheaper and healthier products.

Cheaper products, of course, come from lowering costs of production. Producers are always interested in improving the growth rate and feed conversion of their animals. Lower production costs come from the shorter time to market and lower costs of feeding an animal. This can increase the profit margin in the livestock industry and/or result in lower prices to the consumer.

By being able to select for animals which have the aforementioned traits, producers can raise animals with these desirable characteristics. Selection for desirable traits has traditionally been done using breeding techniques.

Genetic differences exist among individual meat producing animals as well as among breeds which can be exploited by breeding techniques to achieve animals with these desirable characteristics. For example, Chinese breeds are known for reaching puberty at an early age and for their large litter size, while American breeds are known for their greater growth rates and leanness. Thus, it would be desirable to combine the best characteristics of both types of these breeds, thereby improving pork production. Often, however, heritability for desired traits is low, for example, heritability for litter size is around 10%-15%. Standard breeding methods which select individuals based upon phenotypic variations do not take fully into account genetic variability or complex gene interactions which exist. Therefore, there is a need for an approach that deals with selection for leanness, growth rate, and feed consumption at the cellular or DNA level. This method will provide a method for genetically evaluating animals to enable breeders to more accurately select those animals which not only phenotypically express desirable traits but those which express favorable underlying genetic criteria. This has largely been accomplished to date by marker assisted selection.

Restriction fragment length polymorphism (RFLP) analysis has been used by several groups to study pig DNA. Jung et al., Theor. Appl. Genet. 77:271-274 (1989), incorporated herein by reference, discloses the use of RFLP techniques to show genetic variability between two pig breeds. Polymorphism was demonstrated for swine leukocyte antigen (SLA) Class I genes in these breeds. Hoganson et al., Abstract for Annual Meeting of Midwestern Section ofthe American Society of Animal Science. March 26-28, 1990, incorporated herein by reference, reports on the polymorphism of swine major histocompatibility complex (MHC) genes for Chinese pigs, also demonstrated by RFLP analysis. Jung et al, Animal Genetics. 26:79-91 (1989), incorporated herein by reference, reports on RFLP analysis of SLA Class I genes in certain boars. The authors state that the results suggest that there may be an association between swine SLA/MHC Class I genes and production and performance traits. They further state that the use of SLA Class I restriction fragments, as genetic markers, may have potential in the future for improving pig growth performance.

The ability to follow a specific favorable genetic allele involves a novel and lengthy process ofthe identification of a DNA molecular marker for a major effect gene. The marker may be linked to a single gene with a major effect or linked to a number of genes with additive effects. DNA markers have several advantages; segregation is easy to measure and is unambiguous, and DNA markers are co-dominant, i.e., heterozygous and homozygous animals can be distinctively identified. Once a marker system is established selection decisions could be made very easily, since DNA markers can be assayed any time after a tissue or blood sample can be collected from the individual infant animal. The use of genetic differences in receptor genes has become a valuable marker system for selection. For example, United States Patents 5,550,024 and 5,374,526 issued to Rothschild et al. disclose a polymorphism in the pig estrogen receptor gene which is associated with larger litter size, the disclosure of which is incorporated herein by reference. United States application serial number 08/812,208 discloses polymorphic markers in the pig prolactin receptor gene which are associated with larger litter size and overall reproductive efficiency.

It can be seen from the foregoing that a need exists for a method for selecting animals with the improved metabolic traits regarding fat content, growth rate, and feed consumption. SUMMARY OF THE INVENTION

An object ofthe present invention is to provide a genetic marker based on or within - the MC4R gene which is indicative of fat content, growth rate, and/or feed consumption.

Another object ofthe invention is to provide an assay for determining the presence of this genetic marker.

A further object ofthe invention is to provide a method of evaluating animals that increases accuracy of selection and breeding methods for the desired traits.

Yet another object ofthe invention is to provide a PCR amplification test which will greatly expedite the determination of presence ofthe marker. An additional object ofthe invention is to provide a kit for evaluating a sample of animal DNA for the identified genetic marker.

These and other objects, features, and advantages will become apparent after review ofthe following description and claims ofthe invention which follow.

This invention relates to the discovery of a polymorphism within the melanocortin- 4 receptor (MC4R) gene which is associated with fat content, growth rate, and feed conversion traits in animals. According to the invention, the association ofthe MC4R polymorphism with the trait(s) enables genetic markers to be identified for specific breeds or genetic lines. The Taql restriction pattern which identifies the polymorphism is used to assay for the presence or absence of markers associated with the desirable metabolic traits. The breed-dependent marker genotype (i.e., a marker in some breeds and a nonmarker in others) consists of a polymorphism within MC4R, a guanine to adenine transition at position 678 ofthe PCR product (a missense mutation of aspartic acid codon (GAU) into asparagine codon (AAU) at position 298 amino acid ofthe MC4R protein). The invention includes assays for detection ofthe marker as well as the sequence characterization ofthe polymorphism and includes novel sequences in the MC4R gene which may be used to design amplification primers for such an assay. Additionally, the invention includes a method for using the assay in breeding programs for animal selection and a kit for performing the assay. Definitions As used herein, "low fat content" or "leanness" means a biologically significant decrease in body fat relative to the mean of a given population. BRTEF DESCRIPTION OF THE DRAWINGS _>

Figure 1 is the sequence listing for MC4R in pigs (SEQ ID NO:l). "X" represents the site ofthe polymorphism. Figure 2 represents a comparison ofthe DNA sequence between the human (SEQ

ID NO:2) and the porcine (SEQ ID NO:3) MC4R gene.

Figure 3 represents a comparison ofthe amino acid sequence between the human (SEQ ID NO:4) and the porcine (SEQ ID NO:5) MC4R gene.

Figures 4a, 4b, and 4c are linkage reports for MC4R from CRI-MAP. Figure 5 depicts partial nucleotide and amino acid sequences (SEQ ID NO: 12) of the porcine MC4R gene. The amino acid translation shows an amino acid substitution at codon 298.

Figure 6 is an electrophoresis gel of Taql digestion ofthe PCR product. Molecular marker (M) and MC4R genotypes are indicated at the top of each lane. Figure 7 depicts multiple-alignments ofthe putative seventh transmembrane domain of porcine MC4R with other MCRs and GPCRs. The "*" represents the predicted sequence positions for porcine MC4R. The other amino acid sequences were obtained from the GenBank database (accession numbers P32245, P70596, P41983, P56451, P34974, P41968, P33033, Q01718, Q01726, Q28031, AF011466, P21554, P18089, P30680, P47211). The missense variant in porcine MC4R substituted amino acid N for D in the position marked with an arrow. The Asp (D) residue is highly conserved among MCRs, and the Asn (N) residue is well conserved in most other GPCRs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Obesity is a disease affecting energy balance. The control of energy metabolism is simple: store excessive energy as fat and manage the energy to avoid superfluous energy storage, i.e., obesity. Although several genes and signaling systems have been implicated in obesity, there has been little known about the interconnection of energy homeostatic mechanism and genetic polymorphism. The melanocortin-4 receptor (MC4R) has been shown to be an important mediator of long term weight homeostasis. MC4R antagonists can increase food intake and body weight during chronic administration. Skuladottir, G.V., et al., "Long term orexigenic effect of a novel melanocortin 4 receptor selective antagonist", British J. of Pharm.. 126(l):27-34 (1999). » .

Lu et al. (1994) suggested that the melanocortin receptors are involved in controlling food intake and energy balance through studying its antagonism to the agouti obesity syndrome. Huszar et al. (1997) found that inactivation ofthe melanocortin-4 receptor gene (MC4R) resulted in a maturity onset obesity syndrome in mice and demonstrated a major role of MC4R protein in the regulation of energy balance related to the agouti obesity syndrome. In addition, the MC4R protein mediates the effects of leptin, one ofthe important signaling molecules in energy homeostasis (Seeley et al. 1997). According to the present invention, a variant or polymorphism in the MC4R gene has been located, and this genetic variability is associated with phenotypic differences in the metabolic traits of fat content, growth rate, and/or feed consumption.

In one embodiment ofthe invention, an assay is provided for detection of presence of a desirable genotype. The assay involves amplifying the genomic DNA purified from blood, tissue, semen, or other convenient source of genetic material by the use of primers and standard techniques, such as the polymerase chain reaction (PCR), then digesting the DNA with a restriction enzyme (e.g., Taq 7) so as to yield gene fragments of varying lengths, and separating at least some ofthe fragments from others (e.g., using electrophoresis). The fragments may also be detected by hybridizing with a nucleotide probe (e.g., radio-labeled cDNA probes) that contains all or at least a portion ofthe MC4R gene cDNA sequence to the separated fragments and comparing the results ofthe hybridization with assay results for a gene sequence known to have the marker or a sequence known to not have the marker. Selection and use of probes for detection of MC4R sequences based on the known and disclosed MC4R sequences is generally known to those skilled in the art.

The probe may be any sequence which will hybridize to the separated digestion products and allow for detection.

Another embodiment ofthe invention provides a kit for assaying the presence in a MC4R gene sequence of a genetic marker. The marker being indicative of inheritable traits of fat content, growth rate, and/or feed consumption. The kit in a preferred embodiment also includes novel PCR primers comprising 4-30 contiguous bases on either side ofthe polymorphism to provide an amplification system allowing for detection ofthe Taq I polymorphism by PCR and Taq I digestion of PCR products. The preferred primers are « SEQ ID NO:8 and SEQ ID NO:9.

A further embodiment comprises a breeding method whereby an assay ofthe above type is conducted on a plurality of gene sequences from different animals or animal embryos to be selected from and based on the results, certain animals are either selected or dropped out ofthe breeding program.

According to the invention, the polymorphism in the MC4R gene, identifiable by the Taq I restriction pattern, is disclosed. As is known in the art, restriction patterns are not exact determinants ofthe size of fragments and are only approximate. The polymorphism is identifiable by three bands from a Taq I digestion ofthe PCR product, 466, 225, and 76 base pairs (bp) for one homozygous genotype (allele 1); two bands, 542 and 225 bp for another homozygous genotype (allele 2); and four bands for the heterozygous genotype (542, 466, 225, and 76 bp). The marker for leanness and lower feed intake is identifiable by the 466/225/76 bands, except for the Chinese pigs, where the Chinese pigs' marker for leanness is the 542/225 bands. The marker for faster rate of gain is identifiable by the 542/225 bands.

In addition, the polymorphism associated with the pattern has been identified at the nucleotide level. The polymorphic Taq I site was sequenced along with the general surrounding area. See SEQ ID NO: 1. The sequences surrounding the polymorphism have facilitated the development of a PCR test in which a primer of about 4-30 contiguous bases taken from the sequence immediately adjacent to the polymorphism is used in connection with a polymerase chain reaction to greatly amplify the region before treatment with the Taq I restriction enzyme. The primers need not be the exact complement; substantially equivalent sequences are acceptable.

From sequence data, it was observed that in allele 2 the guanine is substituted with an adenine at position 678 ofthe PCR product or at position 298 amino acid ofthe MC4R protein changing the aspartic acid codon (GAU) into an asparagine codon (AAU). The PCR test for the polymorphism used a forward primer of 5'-TGG CAA TAG CCA AGA ACA AG-3' (SEQ. ID NO: 6) and a reverse primer of 5'-CAG GGG ATA GCA ACA

GAT GA-3' (SEQ. ID NO: 7). Pig specific primers used were a forward primer of 5'-TTA AGT GGA GGA AGA AGG-3' (SEQ. ID NO: 8) and a reverse primer of 5 '-CAT TAT GAC AGT TAA GCG G-3' (SEQ ID NO:9). The resulting amplified product of about 7*0 bp, when digested with Taq I, results in allelic fragments of 466, 225, and 76 bp (allele 1) or 542 and 225 bp (allele 2). The marker may be identified by any method known to one of ordinary skill in the art which identifies the presence or absence ofthe marker, including for example, single- strand conformation polymorphism analysis (SSCP), RFLP analysis, heteroduplex analysis, denaturing gradient gel electrophoresis, and temperature gradient electrophoresis, ligase chain reaction or even direct sequencing ofthe MC4R gene and examination for the Taq I RFLP recognition pattern.

One or more additional restriction enzymes and/or probes and/or primers can be used. Additional enzymes, constructed probes, and primers can be determined by routine experimentation by those of ordinary skill in the art.

Other possible techniques include non-gel systems such as TaqMan™ (Perkin Elmer). In this system, oligonucleotide PCR primers are designed that flank the mutation in question and allow PCR amplification ofthe region. A third oligonucleotide probe is then designed to hybridize to the region containing the base subject to change between different alleles ofthe gene. This probe is labeled with fluorescent dyes at both the 5' and 3' ends. These dyes are chosen such that while in this proximity to each other the fluorescence of one of them is quenched by the other and cannot be detected. Extension by Taq DNA polymerase from the PCR primer positioned 5' on the template relative to the probe leads to the cleavage ofthe dye attached to the 5' end ofthe annealed probe through the 5' nuclease activity ofthe Taq DNA polymerase. This removes the quenching effect allowing detection ofthe fluorescence from the dye at the 3' end ofthe probe. The discrimination between different DNA sequences arises through the fact that if the hybridization ofthe probe to the template molecule is not complete, i.e., there is a mismatch of some form, the cleavage ofthe dye does not take place. Thus, only if the nucleotide sequence ofthe oligonucleotide probe is completely complementary to the template molecule to which it is bound will quenching be removed. A reaction mix can contain two different probe sequences each designed against different alleles that might be present, thus, allowing the detection of both alleles in on reaction. Though the use of RFLPs is one method of detecting the polymorphism, other methods known to one of ordinary skill in the art may be used. Such methods include ones that analyze the polymorphic gene product and detect polymorphisms by detecting the resulting differences in the gene product. Though the preferred method of separating restriction fragments is gel electrophoresis, other alternative methods known to one skilled in the art may be used to separate and determine the size ofthe restriction fragments.

It is possible to indirectly select for the polymorphism with alternative DNA markers. It is possible to establish a linkage between specific alleles of alternative DNA markers and alleles of DNA markers known to be associated with the MC4R gene which have previously been shown to be associated with a particular trait. Examples of markers on the published PiGMaP chromosome map which are linked to the MC4R gene include S0331, BHT0433, and S0313.

The reagents suitable for applying the methods ofthe present invention may be packaged into convenient kits. The kits provide the necessary materials, packaged into suitable containers. At a minimum, the kit contains a reagent that identifies the polymorphism in the MC4R gene that is associated with the traits of interest, fat content, growth rate, and feed consumption. Preferably, the reagent that identifies the polymorphism is a PCR set (a set of primers, DNA polymerase, and four nucleoside triphosphates) that hybridize with the MC4R gene or a fragment thereof. Preferably, the PCR set and restriction enzyme that cleaves the MC4R gene in at least one place are included in the kit. Preferably, the kit further comprises additional means, such as reagents, for detecting or measuring the detectable entity or providing a control. Other reagents used for hybridization, prehybridization, DNA extraction, visualization, and similar purposes may also be included, if desired.

The genetic markers, methods, and kits ofthe invention are useful in a breeding program to improve fat content, growth rate, and feed consumption characteristics in a breed, line, or population of animals. Continuous selection and breeding of animals that are at least heterozygous and preferably homozygous for the desired polymorphism associated with the particular trait would lead to a breed, line, or population having those desired traits. Thus, the marker is a selection tool. The following examples are offered to illustrate, but not limit the invention. „

EXAMPLE 1 Melanocortin 4 Receptor PCR-RFLP Test - 7α 7 polymorphism and Genetic Linkage

Mapping of MC4R Gene

Primers:

Primers were designed from homologous regions of human and rat MC4R sequences (Genbank Accession No. s77415 and u67863, respectively). These primers were used to amplify a 750-bp sequence ofthe porcine MC4R gene.

MC4R1: 5' TGG CAATAG CCAAGAACAAG 3' (SEQ ID NO:6)

MC4R4: 5' CAG GGG ATA GCAACA GAT GA 3' (SEQIDNO:7)

PCR Conditions:

Mix 1 : 1 OX Promega Buffer 1.0 μL

25 mM MgCl₂ 0.6 μL dNTPs mix (2.5mM each) 0.5 μL

25 pmol/μL MC4R1 0.1 μL

25 pmol/μL MC4R4 0.1 μL dd sterile H₂O 7.5 μL

Taq Polymerase (5 U/μL) 0.07 μL

Genomic DNA (12.5 ng/μL) 1.0 μL

Ten μL of Mix 1 and DNA were combined in reaction tube, then overlaid with mineral oil. The following PCR program was run: 94°C for 2 min.; 35 cycles of 94°C for 30 sec; 58°C 1 min., and 72°C 1 min. 30 sec; followed by a final extension at 72°C for 15 min. Five μl ofthe PCR reaction product was checked on a standard 1% agarose gel to confirm amplification success and clean negative control. Product size is approximately * 750 base pairs. Digestion was performed by the following procedure.

Tag! Digestion Reaction 10 μL reaction PCR product 5.0 μL 10X 7α?I NE Buffer 1.0 μL BSA (10mg/ml) 0.1 μL Taql enzyme (20 U/μL) 0.5 μL dd sterile H₂O 3.4 μL

A cocktail ofthe buffer, enzyme, BSA, and water was made. Five μL was added to each reaction tube containing the DNA. The mixture was then incubated at 65°C for at least 4 hours to overnight. Loading dye was mixed with the digestion reaction and the total volume was loaded on a 3% agarose gel. The major bands for allele 1 are about 466, 225, and 76 bp. The allele 2 genotype bands are 542 and 225 bp. The heterozygote genotype has both allele 1 and allele 2.

Results

The amplified PCR product is about 750 bp. The sequence ofthe PCR product confirmed that the PCR product is MC4R gene with 97.6%, and 92.2% identities at the amino acid and DNA level, respectively, to corresponding human sequences, (see Figs. 2 and 3).

The Taql digestion ofthe PCR product produced allelic fragments of 466, 225, and 76 bp (allele 1), or 542 and 225 bp (allele 2). The heterozygote genotype has both types of alleles. Mendelian inheritance was observed in three three-generation international reference families, which were used to map this gene by linkage analysis.

The polymorphism between allele 1 and allele 2 resulting from a G - A transition at position 678 ofthe PCR product revealed a missense mutation of Aspartic acid codon (GAU) into Asparagine codon (AAU) at position 298 amino acid of MC4R protein. (See Figure 1, SEQ ID NO: 1). Allele frequencies were determined by genotyping of DNA samples from a small number of animals from different breeds (Table 1). Allele 1 was observed with a » frequency of 1 in Meishan, but was not observed or observed at very low frequency in Hampshire and Yorkshire. The frequencies of allele 1 in Landrace and Chester White were 0.5, respectively.

Figures 2 and 3 illustrate the differences between the DNA and amino acid sequences ofthe human and porcine MC4R gene (SEQ ID NOS:2-5).

TABLE 1 The Frequency of Allele 1 in Different Pig Breeds

Linkage Analyses

Two-point and multi-point linkage analyses were performed on the genotypes of international reference families. See Figs. 4a-4c The data were analyzed by using the CRI-MAP program. MC4R was significantly linked to several markers on porcine chromosome (SSC) 1. The most closely linked markers (recombination fraction and LOD score in parentheses) are SO331 (0.02, 21.97), BHT0433 (0.02, 21.32), and SO313 (0.00, 17.76) by two-point linkage analysis. A multi-point linkage analysis produced the best map order of markers and MC4R (with distance in Kosambi cM): KGF-5.8-CAPN3-2.5- MEF2A-6.1-MC4R-5.6-SO313. Somatic cell hybrid panel of pig and rodent was used to assign MC4R to a cytogenetic region. PCR products from pig specific primers were amplified in clones 7, 8* 16, 18, and 19. MC4R was localized to SSClq 22-27.

EXAMPLE 2

MC4R Receptor PCR-RFLP Test using Pig Specific Primers and Genetic Linkage

Mapping ofthe Porcine MC4R Gene

Pig Specific Primer Sequences Forward primer: 5*-TTA AGT GGA GGA AGA AGG-3' (SEQ ID NO:8)

Reverse primer: 5'-CAT TAT GAC AGT TAA GCG G-3' (SEQ ID NO:9)

Method of Detection

The PCR reaction was performed using

Porcine genomic DNA 12.5 ng lx PCR buffer

MgCl₂ 1.5 mM dNTP 0.125 mM

Forward primer 0.3 μM

Reverse primer 0.3 μM

Taq DNA polymerase (Promega) 0.35 U

in a 10 μL final volume. The PCR profile included 2 min. at 94°C; 35 cycles of 30 sec. at 94°C, 1 min. at 56°C, 1 min. 30 sec. at 72°C; and 15 min. at 72°C in a Robocycler (Statagene, La Jolla, CA). A 5.0 μL aliquot ofthe PCR products was digested in a total volume of 10 μL with 10 U of Taql incubated overnight at 65°C. The digestion products were electrophoresed on a 3% agarose gel. Description of Polymorphism

The Taql digestion ofthe PCR product produced fragments of 466, 225, and 76 bβ in allele 1 and 542 and 225 bp in allele 2. The heterozygous genotype has fragments of both allele 1 and allele 2.

Pattern of Inheritance

Autosomal segregation of Mendelian inheritance was observed in three three- generation European PiGMaP families (Archibald et al., 1995).

Allele Frequencies

Allele frequencies were determined by genotyping the grandparental animals ofthe European PiGMaP families and unrelated animals from ISU reference families. Allele 1 was observed with the following frequencies.

TABLE 2 The Frequency of Allele 1 in Different Pig Breeds

Chromosomal Location Two-point and multi-point linkage analysis were performed on the genotypes of three PiGMaP families using the CRI-MAP program (Green et al. 1990). MC4R was significantly linked to several markers on porcine chromosome 1 (SSC 1). The most closely linked markers (recombination fraction and LOD score in parentheses) are SO331* (0.02, 21.97), BHT0433 (0.02, 21.32), and SO313 (0.00, 17.76) according to two-point linkage analysis. The best map order of MC4R with respect to other linked markers produced by multi-point linkage analysis is as follows (with distance in Kosambi cM): KGF-5.8-CAPN3-2.5-MEF2A-6.1-MC4R-5.6-SO313.

Comments

The Melanocortin-4 Receptor is a G protein-coupled, seven-transmembrane receptor expressed in the brain. Huszar et al. (1997) found that inactivation of MC4R gene resulted in a maturity onset obesity syndrome in mice and demonstrated a major role of MC4R protein in the regulation of energy balance. The MC4R gene has been mapped to human chromosome 18q21.3 (Gantz et al., 1993). The localization of MC4R gene to SSC 1 is consistent with previous chromosome painting data indicating synteny between this chromosome and HSA 18 and 15 (Goureau et al., 1996). However, the gene order of several genes previously mapped from HSA 18 and 15 to SSC 1, including CAPN3, KGF, and MEF2A, is not conserved with MC4R. Therefore, mapping of MC4R to SSC 1 may identify an evolutionary breakpoint between HSA 18 and 15 in relation to SSC 1.

EXAMPLE S

Association of Marker with Enhanced Metabolic Characteristics

In a preliminary study to determine which allele is associated with which trait and in which breeds, the genotypes of several lines of animals were correlated with days to 110 kg, backfat measurements, daily gains, and average daily feed intake. The pigs used in the study were from lines from Pig Improvement Company (PIC).

Data was accumulated using the PCR test described supra for the 1 and 2 allele of the MC4R gene. The collected data is summarized in Tables 3-8 below.

According to the results, allele 1 is the significantly leaner allele (see P2 backfat measurements) in all lines except in Chinese pigs where it is the fat allele. Allele 2 is associated with significantly faster rate of gain (test daily gain) in the tested commercial lines. Overall allele 1 is associated with lower feed intake.

TABLE 3

Number of observations

MC4R genotype:

11 = homozygous allele 1

12 = heterozygous

22 = homozygous allele 2

TABLE 4

Number of observations (males/females)

TABLE 5

Days to 110kg

TABLE 6

P2 backfat Cmm

TABLE 7 Test daily gain (gm/d)

TABLE 8 Average daily feed intake (kg/d). boars only, except L95 which was gilts only

EXAMPLE 4

A Missense Variant ofthe Porcine Melanocortin-4 Receptor (MC4R) Gene is Associated with

Fatness, Growth, and Feed Intake Traits

To determine if there was an association of this MC4R polymorphism with phenotypic variation the mutation was tested in a large number of individual animals from several different pig lines. Analyses of growth and performance test records showed significant associations of MC4R genotypes with backfat, growth rate and feed intake in a number of lines. It is probable that the variant amino acid residue ofthe MC4R mutation causes a significant change ofthe MC4R function. These results support the functional significance of a pig MC4R missense mutation and suggest that comparative genomics based on model species may be equally important for application to farm animals as they are for human medicine.

Identification of mutations in the leptin and the leptin receptor has provided some information on genetic components involved in the regulation of energy balance (Zhang et al. 1994; Tartaglia et al. 1995). Genetic studies using animal models have facilitated the identification of major genetic causes of obesity (Andersson 1996; Pomp 1997; Giridharan 1998). Furthermore, several other genes involved in the neural signaling pathway of energy homeostasis have been identified (Flier and Maratos-Flier 1998; Schwartz et al. 1999). Ofparticular interest among candidate signaling molecules involved in the regulation of energy homeostasis is the melanocortin-4 receptor (MC4R). The MC4R response to leptin signaling is a link between food intake and body weight (Seeley et al. » 1997; Marsh et al. 1999). Neuropeptide Y (NPY) signaling in the central nervous system is also mediated by the MC4R protein (Kask et al. 1998). Several mutations in MC4R including frameshift and nonsense mutations are associated with dominantly inherited obesity in humans (Vaisse et al. 1998; Yeo et al. 1998). Some other MC4R missense mutations in humans have also been identified (Gotoda et al. 1997; Hinney et al. 1999) but the functional significance of these mutations has not been characterized.

Selection based on growth characteristics has been of great importance to the pig industry because of costs associated with feeding and consumer preference for lean meat. Efficient genetic improvement in these quantitative traits may be augmented through the use of marker assisted selection (MAS) using high density genetic maps (Dekkers and van Arendonk 1998; Rothschild and Plastow 1999). An important tool in this process is comparative mapping using the well-developed human and mouse gene maps, which assist in the identification of corresponding genomic regions or major genes controlling growth and performance traits in the pig. Biological understanding of complex traits in human or model species offers an alternative approach to identify genes responsible for the traits of economic interest in livestock. Several quantitative trait loci (QTL) linkage scans using phenotypically divergent breeds and candidate gene analyses have been successfully conducted for fatness and growth traits (Yu et al. 1995; Casas-Carrillo et al. 1997; Knorr et al. 1997; Knott et al. 1998; Rohrer et al. 1998; Wang et al. 1998; Paszek et al. 1999), but no individual genes with major effects on growth and performance traits have yet been established for commercial populations. The role of MC4R in feed intake and obesity suggests it may be an important genetic marker for the growth-related traits in the pig.

Materials and Methods

Animals. Pigs were raised under normal production conditions under the care of PIC employees in nucleus farms in the United States and Europe. Pigs were put on the performance test at approximately 70 days of age and taken off test after 13 weeks. At the end ofthe trial backfat was measured ultrasonically in real time (B mode) at the 10^th rib 2 cm from the centerline. Average daily gain (growth) over the test period was calculated as weight gained divided by days on test. Days to 110 kg market weight was estimated using standard procedures and feed intake was measured using individual electronic » measurement equipment.

PCR amplification of a pig MC4R gene fragment. Primers were designed from homologous regions of human and rat MC4R sequences (GenBank accession no. s77415 and u67863, respectively). The primers were: forward primer: 5'-TGG CAA TAG CCA AGA ACA AG-3' (SEQ. ID NO:6) and reverse primer: 5'-CAG GGG ATA GCA ACA GAT GA-3' (SEQ. ID NO:7). The PCR reaction was performed using 12.5 ng of porcine genomic DNA, lx PCR buffer, 1.5 mM MgCl₂, 0.125 mM dNTPs, 0.3 mM of each primer, and 0.35 U Taq DNA polymerase (Promega) in a lOμL final volume. The conditions for PCR were as follows: 2 min at 94°C; 35 cycles of 30 s at 94°C, 1 min at 56°C, 1 min 30 s at 92°C, and a final 15 min extension at 72°C in a Robocycler (Stratagene, La Jolla, CA).

Sequencing and mutation detection. Sequencing ofthe PCR products from several individual pigs of different breeds was conducted and the sequences were compared to detect any nucleotide change. Sequencing was performed on an ABI sequencer 377 (Applied Biosystems). The porcine MC4R sequence has been submitted to GenBank, and has accession number AF087937. The sequence analysis revealed one nucleotide substitution situated within a Taql restriction enzyme recognition site (Kim et al. 1999). A set of primers was then designed to generate a smaller MC4R gene fragment, which contained only one informative Taql restriction site to specify the polymorphic site and to facilitate the PCR-RFLP test. These primers were: forward 5'-TAC CCT GAC CAT CTT GAT TG-3' (SEQ. ID NO: 10) and reverse: 5'- ATA GCA ACA GAT GAT CTC TTT G-3' (SEQ. ID NO: 11).

Statistical analysis. Analysis of variance procedures were used with a mixed model that accounted for the fixed effects of farm, test period, sex ofthe animal, the MC4R genotype and site (random). All animals in lines of American European descent (Lines A- D) were pooled for the overall analysis and in this analysis line of origin was included. Mean effects were estimated for each genotype and are presented in Tables 9-15. Overall F tests were used to determine level of significance. *

Results Identification of a missense mutation in the pig MC4R gene. The MC4R gene consists of approximately 1 kb of coding sequence contained within a single exon. About 750 bp of a pig MC4R gene fragment was produced by PCR (Kim et al. 1999). The sequence ofthe PCR product confirmed that the PCR product is the MC4R gene with 92.2% and 97.6% identities at nucleotide and the amino acid levels, respectively, to the human MC4R sequence. Multiple alignments ofthe sequences from individual animals of several breeds identified a single nucleotide substitution (G-»A; Fig. 5). The polymoφhism revealed a missense mutation that replaces aspartic acid (GAU) with asparagine (AAU) at the position identical to amino acid 298 of human MC4R protein. To confirm this base change, we designed pig-specific primers flanking the polymorphic site and analyzed the polymoφhism as a TaqlPCR-KF P gel (Fig. 6). Figure 6 shows a Taql digestion ofthe PCR product analyzed by agarose-gel electrophoresis. Allele 1 produced 156 and 70 bp fragments and allele 2 produced a 226 bp fragment as the PCR-RFLP. The heterozygote has both allele 1 and 2 fragments. Molecular marker (M) and MC4R genotypes are indicated at the top of each lane.

The MC4R missense mutation is within a highly conserved region among melanocortin receptors (MCR). The MCR is a subfamily of G-protein coupled receptors (GPCR) containing certain conserved structural elements common to most other GPCRs, but overall amino acid identities between MCR and other GPCRs are low (Tatro 1996). A multiple-alignment ofthe predicted amino acid sequences ofthe pig MC4R with MC4R proteins from other species, other MCR proteins, or representative GPCRs showed that the aspartic acid found at position 298 ofthe seventh transmembrane domain is very highly conserved in the MCR proteins (Fig. 7). It is interesting to note, however, that this position is occupied by asparagine in most other GPCRs. The MCR proteins show 40-80% amino acid identity with each other (Tatro 1996), but the second intracytoplasmic loop and the seventh transmembrane domain are highly conserved among MCR proteins (Gantz et al. 1993). Some ofthe relationships between MCR structure and function have been discovered by the studies of natural and experimental mutations in humans and mice » (Robbins et al. 1993; Valverde et al. 1995; Frandberg et al. 1998). These studies indicate that some mutations in highly conserved regions cause structural changes and alter the function ofthe receptor. The Asp298Asn substitution mutation could have an effect on the function ofthe receptor. However, this will require further testing but it is known that change ofthe homologous residue in MC1R (Asp294His) is associated with fair skin and red hair in humans (Valverde et al. 1995).

The MC4R missense mutation is associated with obesity-related traits. To investigate the effects ofthe missense mutation, the relationship of MC4R genotypes was analyzed for the effects on variation in growth rate, backfat, and feed consumed in over 1,800 animals from several commercial pig lines from PIC, an international pig breeding company. The animals were from closed commercial lines of European/ American breeds (Lines A-D) together with a line originating from a cross between a European and a

Chinese breed (Line E). In lines A-D significant associations ofthe MC4R genotypes were found for all performance traits. The animals homozygous for allele 1 had on average significantly less backfat (P < .001), lower daily gain (P <.001), and lower feed intake(P < .01) than those ofthe homozygous 22 genotype animals (Tables 11, 13, & 15). Overall, pigs with the 11 genotype had approximately 9% less backfat than pigs with the 22 genotype (Table 11), whereas pigs with the 22 genotype grow significantly faster (37g/day) than pigs with the 11 genotype (Table 13). These results appear to be a function of appetite because the 22 genotype animals consume considerably more feed (Table 15). The association between the missense variant ofthe MC4R gene and related performance traits is clearly established in European/ American breeds. Although the number of tested animals is much smaller, these results were not seen in the considerably fatter Chinese crossed line (Line E). Interestingly, line E shows a trend for backfat in the opposite direction to that observed in the other lines (Table 11). Discussion

The present study clearly demonstrates that the porcine MC4R missense mutation*is significantly associated with several performance traits in pigs. Allele 1 representing Asp298, the well conserved amino acid within other MCR subtypes and other species MC4R, was associated with less backfat thickness, slower growth rate, and lower feed intake and allele 2 representing Asn298 was associated with fatter, higher feed intake, and faster growing animals. As the highly conserved residues in the melanocortin receptor proteins have important roles for ligand binding or intracellular signal transmission (Tatro 1996), the MC4R variants might exert functionally distinct abilities in the regulation of food intake and body weight. Further testing of this hypothesis will provide important insights into the structural basis of MCR function and a molecular target for the treatment of human obesity.

Allele 1 was associated with the fattest animals in Line E, which was derived by crossing a Chinese Large White breed with a line of Meishan origin. This is suφrising given that the mutation causes a significant amino acid change in a well-conserved region. The result may be due to sampling. However, if we assume that this result will be significant when more results are added there are several possible explanations. One possibility could be the difference in the background gene effects (epistasis). As growth and fatness are complex polygenic traits, it is certainly possible that the Chinese breed has some distinct allelic interactions derived from several hundred years of isolation and these putative interaction(s) might create variation in polygenic traits within crosses between widely different lines (Frankel and Schork 1996). Several QTL analyses have been conducted for fatness and growth traits using divergent lines (Cases-Carrillo et al. 1997; Knott et al. 1998; Rohrer et al. 1998; Wang et al. 1998; Paszek et al. 1999), but QTL have not been reported near the C4R locus, which maps to chromosome 1 at approximately 80 cm on the linkage map (data not shown). It may mean that the epistatic effects ofthe MC4R alleles suggested in Line E have made it difficult to observe the MC4R locus in most QTL experiments which have involved crosses between Chinese and European/ American lines. It is likely that the effect of some alleles will be variable in the different backgrounds and hard to detect in QTL experiments involving genetically divergent breeds. The effect of MC4R variant will possibly be explained by further studies on the biological effect caused by this mutation in other pig breeds and lines. However, given the strong relationship of MC4R variants to leanness, growth and feed intake, this mutation could be used immediately for merker assisted selection (Meuwissen and Goddard 1996) to develop lines of pigs to satisfy particular customer requirements. For instance, in sow lines where appetite is normally decreased after farrowing, selection for the MC4R 2 allele could help improve feed intake. Furthermore, in some lines deemed to be too fat, selection for allele 1 could be employed and in lines that were considered too slow growth allele 2 selection could be also employed. Therefore, genotyping for the MC4R mutation in pig breeding lines will improve the selection efficiency of feed related production traits including growth and leanness. The candidate gene approach has also been used for investigating the role ofthe porcine leptin gene (Jiang and Gibson 1999). However, in the leptin case, although there was evidence for an association between a leptin polymoφhism and backfat depth in a cross between a commercial breed and an unimproved line, there was no clear association in the different commercial lines tested (Jiang and Gibson 1999). Therefore, it should not be assumed that since one finds a gene that one can assume a relationship exists. In contrast, with MC4R we have determined that variation in this candidate gene can explain significant variation for backfat, growth rate, and feed intake in commercial lines of pigs. These results with MC4R illustrate the potential value of comparative genetic analyses using candidate genes in livestock genomics.

EFFECT OF MC4R GENOTYPE ON SEVERAL PRODUCTION TRAITS IN THE PIG

TABLE 9 Number of observations ( males/females/totals) for Days to 110 kg and backfat

TABLE 10

Days to 110kg

TABLE 11

10^th rib Backfat (mm

TABLE 12 Number of observations (males/females/totals for Test daily gain

TABLE 13

TABLE 14 Number of observations (males/females/totaD for average daily feed intake

TABLE 15 Average daily feed intake (kg/day ). boars only except LINE E which was gilts only

Having described the invention with reference to particular compositions, theories of effectiveness, and the like, it will be apparent to those of skill in the art that it is not intended that the invention be limited by such illustrative embodiments or mechanisms, and that modifications can be made without departing from the scope or spirit ofthe invention, as defined by the appended claims. It is intended that all such obvious modifications and variations be included within the scope ofthe present invention as » defined in the appended claims. The claims are meant to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates to the contrary.

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Claims

What is claimed is:*

1. A method of identifying an animal which possesses a genotype indicative ofthe metabolic traits of fat content, growth rate, and feed consumption, the method comprising: a) obtaining a nucleic acid sample from the animal, and b) identifying a polymoφhism in the MC4R gene ofthe sample.

2. The method of claim 1 wherein the polymoφhism is characterized by a nucleotide position 678 ofthe PCR product ofthe MC4R gene.

3. The method of claim 1 wherein the animal is a pig.

4. The method of claim 2 wherein the polymoφhism at the nucleotide position 678 is associated with fat content.

5. The method of claim 2 wherein a guanine at the nucleotide position 678 is associated with lower feed intake.

6. The method of claim 2 wherein an adenine at the nucleotide position 678 is associated with a faster rate of gain.

7. The method of claim 1 wherein the step of identifying the polymoφhism is a method employing allele specific oligonucleotides.

8. The method of claim 1 wherein the step of identifying the polymoφhism is selected from the group consisting of restriction fragment length polymoφhism (RFLP) analysis, heteroduplex analysis, single strand conformational polymoφhism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE),temperature gradient gel electrophoresis (TGGE), and use of linked genetic markers.

9. The method of claim 8 wherein the step of identifying the polymoφhism comprises RFLP analysis. * -

10. The method of claim 1 further comprising the step of amplifying the MC4R gene sequence.

11. The method of claim 10 further comprising the step of digesting the amplified region with the restriction endonuclease Taq I.

12. The amplified gene sequence of claim 10 wherein primers used in the amplification are selected from the group consisting of SEQ. ID NO:6, SEQ. ID NO:7, SEQ. ID NO:8, SEQ. ID NO:9, SEQ. ID NO: 10, and SEQ. ID NO:l l.

13. A single strand of an oligonucleotide primer useful for detecting nucleotide 678 of the PCR product of a MC4R gene the primer consisting of a nucleotide sequence having about 4-30 contiguous bases from SEQ ID NO:l.

14. The oligonucleotide of claim 13 wherein the oligonucleotide has the nucleotide sequence represented by SEQ ID NO:6.

15. The oligonucleotide of claim 13 wherein the oligonucleotide has the nucleotide sequence represented by SEQ ID NO:7.

16. The oligonucleotide of claim 13 wherein the oligonucleotide has the nucleotide sequence represented by SEQ ID NO:8.

17. The oligonucleotide of claim 13 wherein the oligonucleotide has the nucleotide sequence represented by SEQ ID NO:9.

18. The oligonucleotide of claim 13 wherein the oligonucleotide has the nucleotide sequence represented by SEQ ID NO: 10.

19. The oligonucleotide of claim 13 wherein the oligonucleotide has the nucleotide Γû║ sequence represented by SEQ ID NO: 11.

20. A method of identifying an animal which possess a desired genotype indicative of the metabolic traits of fat content, growth rate, and feed consumption, the method comprising a) obtaining a sample of genomic DNA, b) digesting the sample with Taq I to obtain fragments, c) separating the fragments obtained from the digestion, and d) identifying the presence or absence of a Taq I site at base 678 of the PCR product ofthe MC4R gene.

21. The method of claim 20 further comprising the step of selecting animals with the desired genotype for breeding.

22. The method of claim 20 wherein the site is identifiable by fragments of 466, 225, and 76 bp when a guanine is present at base 678 and fragments of 542 and 225 bp when an adenine is present when a restriction enzyme which cuts at the same recognition site as Taq I is used.

23. The method of claim 20 wherein the step of identifying comprises detecting the Taq I site by amplification.

24. A kit for evaluating a sample of animal DNA comprising a reagent in a container that identifies a polymoφhism in a MC4R gene.

25. The kit of claim 24 wherein the reagent is a primer that amplifies the MC4R gene or a fragment thereof.

26. The kit of claim 24 further comprising a DNA polymerase which cleaves the MC4R gene, a forward primer, and a reverse primer, wherein the primers are capable of * amplifying a region ofthe MC4R gene which contains a polymoφhic site.

27. A primer for assaying the presence of a polymoφhic Taql site in the MC4R gene wherein the primer comprises a sequence selected from the group consisting of SEQ. ID NO:6, SEQ. ID NO:7, SEQ. ID NO:8, SEQ. ID NO:9, SEQ. ID NO: 10, and SEQ. ID NO:l l.

28. A method for selecting animals for the desired traits of lower fat content, faster growth rate, or lower feed consumption comprising the steps of a) obtaining a nucleic acid sample from an animal, b) identifying a polymoφhism characterized by a nucleotide position 678 of a PCR product ofthe MC4R gene, and c) selecting the animals which have the nucleotide associated with the desired traits in position 678.

29. A method for an indirect selection for a polymoφhism in MC4R wherein specific alleles of an alternative DNA marker are used to make the indirect selection wherein the alternative DNA marker is a linked marker near MC4R.

30. The method of claim 29 wherein the linked marker is selected from the group consisting of S0331, BHT0433, and S0313.

31. A method of identifying animals which possess a desired genotype indicative of the metabolic traits of fat content, growth rate, and feed consumption, the method comprising a) determining an association between a MC4R genotype and a trait of interest by obtaining a sample of animals from a line or breed of interest, b) preparing genomic DNA from each animal in the sample, c) determining the genotype ofthe MC4R gene, and d) calculating the association between the MC4R genotype and the trait.

32. A method of selecting animals which possess a desired MC4R genotype indicative* ofthe metabolic traits of fat content, growth rate, and feed consumption, the method comprising a) obtaining a nucleic acid sample from an animal, b) identifying the genotype ofthe MC4R gene ofthe animal, and c) selecting those animals which have the genotype associated with the desired traits.