CN1328602A - Melanocortin-4 receptor gene and use as genetic marker for fat content, weight gain, and/or feed consumption 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

Melanocortin-4 receptor gene and as the purposes of the genetic marker of animal tallow content, weight increase and/or feed intake

Related application intersection document

The application has required the U.S. Provisional Application No.60/094 of submission on July 27th, 1998, the U.S. Provisional Application No.60/116 that on January 15th, 287 and 1999 submitted to, and 186 right of priority, the disclosure of these documents is included this paper in as a reference.

Subsidy is with reference to clause

The present invention obtains USDA to small part and passes through the support that Iowa agricultural and household economy experiment centre (IaHees) and IOW03148 project (Hatch Funds) are appropriated funds.United States Government enjoys certain right in the present invention.

Invention field

The present invention relates to the method for genetic evaluation animal, this method is to measure the existence of the genetic marker of at least one one or more feature such as indication lipid content, the speed of growth and feed intake.Specifically, this method is to analyze the difference of melanocortin-4 receptor (MC4R) gene of these features of indication.More particularly, this methods analyst is the polymorphism of MC4R gene.

Background of invention

The human consumer increases the demand of low-fat content meat product.The evidence that accumulates in the scientific literature has more stimulated this demand, because these evidences show that mass consumption animal tallow (especially a high proportion of saturated fatty acid) has tangible Health hazard, comprises the danger of suffering from cardiovascular disorder.Other health problem relevant with higher fatty acid meat comprises: its cholesterol level is higher, and has added the salt (adding salt is in order to improve binding characteristic, because salt helps to extract natural water associativity component myosin from meat) of higher amount.And a large amount of human consumers find that meat product contains such as not too receptible chemical additives such as phosphoric acid salt, emulsifying property additive and antioxidants.

In the face of seeking the human consumer of more healthy meat product, the meat product manufacturer constantly is forced to the meat product that will provide more cheap and healthy.

More cheap goods are certainly come from reducing production costs.The manufacturer is very interested in improving animal growth rate and feed intake.Reducing production costs, it is lower to derive from the cost that arrives market and nutrition purposes, especially for feeding animals in shorter time.This can improve the marginal profit of livestock industry, and/or causes reducing to human consumer's price.

By selecting the animal with above-mentioned feature, the manufacturer can raise out the animal with these required features.The traditional method of selecting required feature is to adopt breeding technique.

There is hereditary difference in indivedual producing in meat animal and the fauna, and breeding technique can utilize this difference to obtain to have the animal of required feature.For example, known Chinese variety has and early to arrive pubescence and the large-framed characteristics of cub, and u.s. variety has the higher and thin characteristics of the speed of growth.Therefore, wish the best features of these two kinds of kinds is combined, to improve the production of pork.

Yet the heritability of required feature is very low usually.For example, the heritability of cub physique is about 10-15%.The breeding method of standard is selected individual according to phenotypic difference, this technology does not fully take into account the hereditary difference or the complicated gene interaction of existence.Therefore, need a kind of method is selected lean meat, the speed of growth and feed intake on cell or dna level feature.This method will provide a kind of and estimate the method for animal from gene, thus those animals that the raiser can be selected more accurately not only on phenotype, show required feature but also show favourable latent gene standard.At present, this selects to realize by marker auxiliary to a great extent.

Several research groups have used restriction fragment length polymorphism (RFLP) pig DNA of having analyzed and researched.People such as Jung (Theor.Appl.Genet, 77:271-274 (1989) includes this paper in as a reference) disclose with the RFLP technology and have shown gene difference between the pig of two kinds of kinds.Confirm that swine leukocyte antigen in these kinds (SLA) I genoid has polymorphism.People such as Hoganson (Abstract for Annual Meeting of Midwestem Section of theAmerican Society of Animal Science, 26-28 day March nineteen ninety, include this paper in as a reference) reported that this also obtains the confirmation of rflp analysis about the polymorphism of Chinese Pigs major histocompatibility complex (MHC) gene.People such as Jung (Animal Genetics, 26:79-91 (1989) includes this paper in as a reference) have reported the rflp analysis about SLA I genoid in some boar.This article the author claim, these results show between pig SLA/MHC I genoid and production and the behavior performance characteristic relevant.They claim that also available SLAI class restriction fragment improves the behavior of pig growth as genetic marker in the future.

Follow the tracks of concrete favourable allelic ability and relate to a kind of very long novel method consuming time, this method is to identify the molecule marker of main effects gene DNA.Mark can be with individual gene with main effects chain or with a plurality of gene linkages with additive effect.Dna marker has several advantages: gene isolation is measured easily and is clear and definite, and dna marker is codominant, promptly can identify heterozygosis distinctively and animal that isozygoty.In case set up Mk system, the decision that just can make one's options easily is because dna marker can be measured after collection obtains single animal child's tissue or blood sample at any time.

The gene difference of acceptor gene has been used as a kind of valuable Mk system that is used to select.For example, United States Patent (USP) 5,550,024 and 5,374,526 (authorizing people such as Rothschild) discloses the polymorphism (it is relevant with bigger cub physique) in pig female hormone receptor gene, fits into this paper in this article as a reference.U.S. Patent application 08/812,208 discloses the polymorphism mark in pig prolactin antagonist acceptor gene, and they are relevant with bigger cub physique and whole reproductive efficiency.

From aforementioned content as can be seen, need a kind of method of selecting the animal of metabolic characteristics (as lipid content, the speed of growth and feed intake) with improvement.

Summary of the invention

An object of the present invention is to provide a kind of based on the MC4R gene or at the intragenic genetic marker of MC4R, the indication of this MC4R gene lipid content, the speed of growth and/or feed intake.

Another object of the present invention provides a kind of measuring method of determining that this genetic marker exists.

A further object of the invention provides a kind of method of estimating animal, and this method has improved selection and the breeding method accuracy at required feature.

A further object of the invention provides a kind of pcr amplification test, and this test will be quickened the mensuration that mark exists greatly.

A further object of the invention provides a test kit that is used for estimating genetic marker to be identified in the animal DNA sample.

After the description and claim below having read the present invention, these and other objects, feature and advantage will be conspicuous.

The present invention relates to a discovery, that is, exist in melanocortin (melanocortin) 4 acceptors (MC4R) gene and animal tallow content, polymorphism that the speed of growth is relevant with the feed intake feature.According to the present invention, getting in touch of MC4R polymorphism and feature makes that the genetic marker of specific kind or hereditary system can be identified.Measuring mark whether relevant with required metabolic characteristics with the TaqI estriction map of identifying this polymorphism exists.Kind dependent form marker gene type (is a mark in some kinds promptly, be not mark in other kind) to form by the polymorphism in the MC4R, the guanine that the PCR product is 678 becomes VITAMIN B4 (missense mutation from aspartic acid codon (GAU) to l-asparagine codon (AAU) takes place proteic 298 amino acids of MC4R).The present invention includes the test of the sequence signature of certification mark and polymorphism, also comprise the new sequence of the amplimer that can be designed for this test in the MC4R gene.In addition, the present invention also comprise a kind of in the procedure of breeding with method of this test and Selection animal and the test kit that carries out this test.

Definition

Term used herein " low-fat content " or " thin " refer to that body fat is biologically obviously reducing for the mean value of given colony.

The accompanying drawing summary

Fig. 1 is the sequence table (SEQ ID NO:1) of MC4R in the pig." X " represents pleomorphism site.

Fig. 2 represents that the dna sequence dna between people (SEQ ID NO:2) and pig (SEQ ID NO:3) the MC4R gene compares.

Fig. 3 represents that the aminoacid sequence between people (SEQ ID NO:4) and pig (SEQ ID NO:5) the MC4R gene compares.

Fig. 4 a, 4b and 4c are the reports of lock repeatedly of the MC4R of CRI-MAP.

Fig. 5 has shown the part Nucleotide and the aminoacid sequence (SEQ ID NO:12) of pig MC4R gene.The amino acid translation demonstrates 298 of codons amino-acid substitution.

Fig. 6 is the running gel of the TaqI digestion of PCR product.Molecule marker (M) and MC4R genotype have been pointed out in each swimming lane top.

Fig. 7 has shown the multiple sequence contrast of the 7th membrane spaning domain of inferring and other MCR and the GPCR of pig MC4R." * " represents the sequence location of the expectation of pig MC4R.Other aminoacid sequence from the GenBank database obtain (accession number is P32245, P70596, P41983, P56451, P34974, P41968, P33033, Q01718, Q01726, Q28031, AF011466, P21554, P18089, P30680, P47211).Amino acid N is marked with arrow by D metathetical missense change location among the pig MC4R.Asp (D) residue is high conservative in MCR, and Asn (N) residue is a high conservative in other most of GPCR.

Preferable embodiment describes in detail

Obesity is the disease that influences energy balance.The control of energy metabolism is very simple: with too much energy storages be fat and control energy in order to avoid too much energy storages, promptly fat.Although several genes and signal transduction system relate to obesity, know little about it for connecting each other also of energy homeostasis mechanism and gene pleiomorphism.Show that now melanocortin-4 receptor (MC4R) is the important attemperator of long term weight stable state.The MC4R antagonist can increase food ration and body weight during giving for a long time.Skuladottir, G.V waits people's " new melanocortin 4 receptor-selective antagonists increase for a long time appetite effect ", British J.of Pharm.126 (1): 27-34 (1999).

People such as Lu (1994) to the fat syndromic antagonism of agouti, point out that it participates in the control feed and takes in and energy balance by the research melanocortin receptor.People such as Huszar (1997) find that the inactivation of melanocortin-4 receptor gene (MC4R) causes mouse to produce the fat syndrome of ripening stage morbidity, and confirm that MC4R albumen plays a major role in regulating the energy balance relevant with the fat syndrome of agouti.In addition, MC4R albumen is regulated the effect (a kind of important signal transducers in the energy homeostasis) also transmit modest albumen (leptin) people such as (, 1997) Seeley.

The present invention has determined the variation in the MC4R gene or the position of polymorphism, and this heredity (gene) difference is relevant with the phenotype difference of metabolism features such as lipid content, the speed of growth and/or feed intake.

In one embodiment of the invention, provide a test to detect required genotypic existence.This test comprises and utilizes primer and standard technique (as polymerase chain reaction (PCR)) to increase from the genomic dna of other conventional source purifying of blood, tissue, seminal fluid or genetic material, use Restriction Enzyme (as TaqI) to digest this DNA then, thereby produce the different gene fragment of length, at least some fragments are separated (for example using electrophoresis) with other fragment.

Also can detect fragment like this, make and contain all or to the nucleotide probe (for example radiolabeled cDNA probe) of small part MC4R gene cDNA sequence and isolating fragment hybridization, then with results of hybridization and known gene order or the known test-results comparison that does not have the gene order of mark with mark.According to known and disclosed MC4R sequence select and use the probe that detects the MC4R sequence be those skilled in the art usually known to.Probe can be any and isolating digestion product hybridization and the detected sequence of energy.

Another embodiment of the invention provides a kind of test kit that genetic marker exists in the MC4R gene order that is used for measuring.This mark can indicate heritable features such as lipid content, the speed of growth and/or feed intake.In a preferable embodiment, this test kit also comprises new PCR primer, this primer has 4-30 base of adjoining at polymorphism (site) either side, thereby an amplification system is provided, and this system can digest by the TaqI of PCR and PCR product and detect the TaqI polymorphism.Preferable primer is SEQID NO:8 and SEQ ID NO:9.

Another embodiment comprises a kind of breeding method, and this method is a plurality of gene orders of different animals to be selected or animal embryo to be carried out the test of the above-mentioned type, and some animal is selected or is got rid of outside the procedure of breeding according to the result.

The invention discloses the polymorphism in the MC4R gene that available TaqI estriction map identifies.Road as known in the art, estriction map can not determine clip size definitely, it is proximate.For homozygous genotype (allelotrope 1), polymorphism can be identified by three bands (466,225 and 76 base pair (bp)) that PCR product TaqI digestion obtains; For another homozygous genotype (allelotrope 2), can identify by two bands (542 and 225 base pair); For the heterozygous genes type, can identify by four bands (542,466,225 and 76 base pair).The less mark of lean meat species and food ration can identify by 466/225/76 base pair, but except the Chinese Pigs, its lean meat species be labeled as 542/225 band.Weight increase mark faster can bring evaluation by 542/225.

In addition, on nucleotide level, identified the polymorphism relevant with collection of illustrative plates.Checked order in polymorphic sequence TaqI site and entire circumference zone.See SEQ ID NO:1.Polymorphism sequence on every side helps to develop PCR to be tested, from adjoining base as the primer that is used for polymerase chain reaction near taking out about 4-30 the sequence of polymorphism, so that increase these zones in a large number before the TaqI restriction enzyme treatment.Primer need not complete complementation; Basically Deng Jia sequence is acceptable.

Observe from sequence data, in allelotrope 2, the guanine that the PCR product is 678 is replaced by VITAMIN B4, or proteic 298 amino acids of MC4R become l-asparagine codon (AAU) from aspartic acid codon (GAU).The PCR of polymorphism test adopt forward primer 5 '-TGG CAA TAG CCA AGA ACA AG-3 ' (SEQ IDNO:6) and reverse primer 5 '-CAG GGG ATA GCA ACA GAT GA-3 ' (SEQ ID NO:7).Used pig Auele Specific Primer is: forward primer 5 '-TTA AGT GGA GGA AGA AGG-3 ' (SEQ ID NO:8) and reverse primer 5 ' CAT TAT GAC AGT TAA GCG G-3 ' (SEQ ID NO:9).The amplified production that obtains has 750 base pairs approximately, after with TaqI digestion, obtains the allelotrope fragment of 466,225 and 76 base pairs (allelotrope 1), or the allelotrope fragment of 542 and 225 base pairs (allelotrope 2).

Mark can be identified with the method whether known identifying mark of any those of ordinary skills exists, these methods for example comprise single-strand conformation polymorphism analysis (SSCP), rflp analysis, heteroduple analysis, denaturing gradient gel electrophoresis and temperature gradient electrophoresis, ligase chain reaction or or even directly the MC4R gene is checked order, and check TaqIRFLP identification collection of illustrative plates.

Also can adopt one or more additional restriction enzymes and/or probe and/or primer.The probe of additional enzyme, structure and primer can be determined by normal experiment by those of ordinary skills.

Other technology that may adopt comprises non-gel systems, as TaqMan ^TM(Perkin Elmer).In this system, design side joint sudden change interested and can pcr amplification should the zone oligonucleotide PCR primer.Then, design the 3rd oligonucleotide probe and an area hybridization, the base that isoallele not changes is contained in this zone.5 of this probe ' and 3 ' end fluorochrome label.Select such dyestuff, when they were closer to each other, the fluorescence of one of them can not be detected by another cancellation.PCR primer with Taq archaeal dna polymerase 5 ' end on the template extends with respect to probe, and the result causes rupturing with the 5 ' nuclease of the annealing dyestuff that probe 5 ' end links to each other by the Taq archaeal dna polymerase.This has removed quenching effect, makes that the dye fluorescence of probe 3 ' end can be detected.Can distinguish different dna sequence dnas by the following fact: if the hybridization of probe and template molecule is incomplete, the mispairing of certain form is arranged promptly, then dyestuff can not rupture.Therefore, have only when the nucleotide sequence of oligonucleotide probe with treat that with its bonded template molecule quenching effect just can be removed when complementary fully.Reaction mixture can contain two kinds of different probe sequences, and each sequence designs at the different allelotrope that may exist, thereby makes two kinds of allelotrope to be detected in a reaction.

Although adopting RFLP is a kind of method that detects polymorphism, known other method of also available those of ordinary skills.These methods comprise by the difference of the gene product that detect to form to be analyzed the polymorphism gene product and detects the method for polymorphism.

Although the segmental preferred approach of separation limit is gel electrophoresis, also can adopts other method well known by persons skilled in the art to separate and measure restricted segmental size.

Available other dna marker is selected polymorphism indirectly.Can the specific alleles of other dna marker with known and above-mentioned show and the allelotrope of the dna marker of the MC4R gene-correlation that special characteristic is relevant between set up linkage relationship.Comprise S0331, BHT0433 and S0313 with the mark example on the disclosed PiGMaP chromosome map of MC4R gene linkage.

Can adopt the pack of the inventive method to dress up suitable test kit with being fit to.This test kit provides required material, and these materials are packaged in the suitable containers.The minimum reagent of identifying polymorphism in the MC4R gene relevant that contains of this test kit with interested feature (lipid content, the speed of growth and feed intake).Preferable, the reagent of identifying polymorphism is the PCR series (a cover primer, archaeal dna polymerase and four kinds of nucleoside triphosphates) of hybridizing with MC4R gene or its fragment.Preferable, comprise PCR series and the restriction enzyme of cutting the MC4R gene at least one place in the test kit.Preferable, this test kit also comprises other means, but for example is used for detecting or measure recognizate or the reagent of contrast is provided.If desired, also can comprise be used to hybridize, other reagent of prehybridization, DNA extraction, colour developing and similar purpose.

Genetic marker of the present invention, method and test kit can be used for the procedure of breeding and improve features such as lipid content, the speed of growth and feed intake in animal varieties, strain or the colony.The animal that the required polymorphism relevant with special characteristic is at least heterozygosis (preferably isozygotying) carries out Continuous Selection and breeding, will produce kind, strain or colony with required feature.Therefore this mark is a selection tool.

The purpose that the following example is provided is in order to describe, and unrestricted the present invention.

Embodiment 1

Melanocortin 4 acceptor PCR-RFLP test-MC4R genes

TaqI polymorphism and gene linkage collection of illustrative plates

Primer:

Use homologous region (the GenBank accession number is respectively s77415 and u67863) design primer from people and rat MC4R sequence.With the increase 750bp sequence of pig MC4R gene of these primers.

MC4R1：5′-TGG?CAA?TAG?CCA?AGA?ACA?AG-3′(SEQ?ID?NO：6)

MC4R4：5′-CAG?GGG?ATA?GCA?ACA?GAT?GA-3′(SEQ?ID?NO：7)

The PCR condition:

Mixture 1:10X Promega damping fluid 1.0 microlitres

25mM MgCl ₂0.6 microlitre

DNTP mixture (respectively being 2.5mM) 0.5 microlitre

25 picomole/microlitre MC4R1 0.1 microlitre

25 picomole/microlitre MC4R4 0.1 microlitre

Dd sterilized water 7.5 microlitres

Taq polysaccharase (5U/ microlitre) 0.07 microlitre

Genomic dna (12.5 nanogram(ng)s/microlitre) 1.0 microlitres

10 microlitre mixtures 1 are blended in the reaction tube with DNA, cover then and go up mineral oil.Carry out following PCR program: 94 ℃ 2 minutes; 35 94 ℃ of taking turns 30 seconds, 58 ℃ of 1 minute and 72 ℃ 1 minute and 30 seconds; Extended 15 minutes down at 72 ℃ at last.

On 1% sepharose of standard, check 5 microlitre PCR reaction product, increase successfully, and remove negative contrast with affirmation.The product size is about 750 base pairs.Digest with follow procedure.

TaqI digestion reaction 10 microlitre reactants

PCR product 5.0 microlitres

10XTaqINE damping fluid 1.0 microlitres

BSA (10 mg/ml) 0.1 microlitre

TaqI enzyme (20U/ microlitre) 0.5 microlitre

Dd sterilized water 3.4 microlitres

Make the mixture of damping fluid, enzyme, BSA and water.Contain at each and to add 5 microlitres in the reaction tubes of DNA.Cultivate mixture at least 4 hours to spending the night for 65 ℃ then.To load dyestuff and mix, whole volumes will be splined on 3% sepharose with the digestion reaction thing.The main band of allelotrope 1 is about 466,225 and 76bp.Allelotrope 2 genotypic bands are 542 and 225bp.The heterozygous genes type has allelotrope 1 and 2.

The result

Amplification PCR products is about 750bp.The true PCR product of the sequence of PCR product is the MC4R gene, with corresponding human sequence 97.6% and 92.2% homogeny (seeing Fig. 2 and 3) is arranged respectively on amino acid and dna level.

The TaqI of PCR product digestion has produced 466,225 and the allelotrope fragment (allelotrope 1) of 76bp, or 542 and the fragment (allelotrope 2) of 225bp.The genome of heterozygosis has two kinds of allelotrope.Observe Mendelian inheritance in three kinds of three generations worlds that are used for describing this gene mapping in reference to family by linkage analysis.

The G that the PCR product is 678 has explained the missense mutation (see Fig. 1, SEQ ID NO:1) of MC4R protein 29 8 amino acids from aspartic acid codon (GAU) to l-asparagine (AAU) to allelotrope that conversion caused 1 and the polymorphism between the allelotrope 2 of A.

The genotype of the DNA sample of the animal by the minority different varieties is determined gene frequency (table 1).Find that the frequency of allelotrope 1 in Meishan is 1, and in Hampshire and Yorkshire, do not have or frequency very low.The frequency of allelotrope 1 in Landrace and Chester White is respectively 0.5.

Fig. 2 and Fig. 3 have described the DNA and the difference between the aminoacid sequence (SEQ IDNO:2-5) of people and pig MC4R gene.

Table 1

The frequency of allelotrope 1 in the different pig varieties

Kind	Number of animals	The frequency of allelotrope 1
			????Meishan	??????8	???????????1
??Large?White	??????8	?????????0.56
			???Yorkshire	??????6	?????????0.08
???Hampshire	??????5	??????????0
			???Landrace	??????4	?????????0.5
Chester?White	??????4	?????????0.5
			????Minzu	??????2	??????????1
???Wild?Boar	??????2	??????????1

Linkage analysis

The world is carried out and multiple spot linkage analysis at 2 with reference to the genotype of family.See Fig. 4 a-4c.With CRI-MAP programanalysis data.MC4R obviously and the several marks on the pig karyomit(e) (SSC) 1 chain.By 2 linkage analysises, chain immediate mark (recombination fraction is the LOD scoring in the bracket) is S0331 (0.02,21.97), BHT0433 (0.02,21.32) and S0313 (0.00,17.76).The multiple spot linkage analysis has produced the best collection of illustrative plates order (distance is Kosambi cM) of mark and MC4R: KGF-5.8-CAPN3-2.5-MEF2A-6.1-MC4R-5.6-S0313.

Somatic hybridization body with one group of pig and rodent is that MC4R distributes a cytogenetics zone.The PCR product of pig Auele Specific Primer increases in clone 7,8,16,18 and 19.The position of MC4R is at SSClq22-27.

Embodiment 2

Gene linkage collection of illustrative plates with pig Auele Specific Primer and pig MC4R gene

Carry out MC4R acceptor PCR-RFLP test

The pig specific primer sequence

Forward primer: 5 '-TTA AGT GGA GGA AGA AGG-3 ' (SEQ ID NO:8)

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

Digestion method

The following material that is used in the 10 microlitre final volumes carries out the PCR reaction.

Pig genomic dna 12.5ng

The lxPCR damping fluid

MgCl ₂??????????????????1.5mM

dNTP????????????????????0.125mM

Forward primer 0.3 μ M

Reverse primer 0.3 μ M

Taq archaeal dna polymerase (Promega) 0.35U

The PCR process comprises, Robocycler (Statagene, La Jolla, CA) in, 94 ℃ 2 minutes; 35 94 ℃ of taking turns 30 seconds, 56 ℃ 1 minute, 72 ℃ 1 minute 30 seconds; And 72 ℃ 15 minutes.TaqI65 ℃ of cultivation with 10U in 10 microlitre cumulative volumes spent the night, and digests a 5.0 microlitre PCR products.On 3% sepharose, digestion product is carried out electrophoresis.

The description of polymorphism

The Taq I of PCR product digestion has produced 466,225 and the fragment (in allelotrope 1) of 76bp, and 542 and the fragment (in allelotrope 2) of 225bp.The genotype of heterozygosis has the fragment of allelotrope 1 and 2.

Mode of inheritance

The euchromosome of finding Mendelian inheritance in the PiGMaP families of three kinds of three generations Europe separates people such as (, 1995) Archibald.

Gene frequency

Animal for generations by European PiGMaP family and ISU with reference to the incoherent animal of family genotype, determine gene frequency.Find that allelotrope 1 has following frequency.

Table 2

The frequency of allelotrope 1 in the different pig varieties

Kind	Number of animals	The frequency of allelotrope 1
			????Meishan	?????8	??????????1
??Large?White	?????8	????????0.56
			???Yorkshire	????10	????????0.15
??Hampshire	????12	??????????0
			??Landrace	?????8	????????0.56
?Chester?White	?????8	????????0.56
			????Minzu	?????2	??????????1
???Wild?Boar	?????2	??????????1

Chromosome position

With CRI-MAP program people such as (, 1990) Green the genotype of three PiGMaP families is carried out and multiple spot linkage analysis at 2.MC4R obviously and the several marks on the pig karyomit(e) 1 (SSC1) chain.By 2 linkage analysises, chain immediate mark (recombination fraction is the LOD scoring in the bracket) is S0331 (0.02,21.97), BHT0433 (0.02,21.32) and S0313 (0.00,17.76).The MC4R that the multiple spot linkage analysis produces is with respect to the best collection of illustrative plates order of other linked marker following (distance is Kosambi cM): KGF-5.8-CAPN3-2.5-MEF2A-6.1-MC4R-5.6-S0313.

Estimate

Melanocortin 4-acceptor is to be expressed in the brain and 7 acceptors of the striding film G-albumen coupling.People such as Huszar (1997) find that the inactivation of MC4R gene causes the fat syndrome of the ripening stage morbidity in the mouse, and confirm that MC4R albumen plays a major role in regulating energy balance.Determine that through collection of illustrative plates the MC4R gene is positioned at human chromosome 18q21.3 people such as (, 1993) Gantz.The position that the MC4R gene is positioned at SSC1 conforms to chromosome dyeing data in the past, shows colinearity between this karyomit(e) and HSA18 and 15 people such as (, 1996) Goureau.Yet in the past the gene order of several genes (comprising CAPN3, KGF and MEF2A) that record from HSA18 and 15 to SSC1 was conservative to MC4R.Therefore, the figure spectral position of MC4R in SSC1 may identify that HSA18 and 15 is with respect to the evolution breakpoint between the SSC1.

Embodiment 3

Mark is relevant with the enhanced metabolic characteristics

In determining which allelotrope and preliminary study in which kind relevant, with the genotype of several animal strains and grow to 110 kilograms fate, back fat test, every day weight increase and on average every day, food ration associated with which feature.Be used for the strain of the pig of this research from Pig Improvement Company (PIC).

With the allelotrope 1 of above-mentioned PCR test accumulation MC4R gene and 2 data.The data of collecting are summarized among the following table 3-8.

According to the result, allelotrope 1 is evident as lean meat species allelotrope (seeing the fat test of P2 back) in all strains, and removing is lard type allelotrope in Chinese Pigs.In the commercial strain of test, allelotrope 2 is with obviously speed of weight increment (test day weightening finish) is relevant faster.Whole allelotrope 1 is relevant with lower food ration.

Table 3

Observed number

The MC4R genotype	???L02	??L03	??L19	??L65	Altogether	??L95
							????11	???88	???30		??32	???150	??20
????12	???57	???54	??56	??74	???241	??67
							????22	???12	???31	??254	??33	???330	??37

721MC4R genotype: the 11=allelotrope 112=heterozygosis 22=allelotrope 2 that isozygotys that isozygotys altogether

Table 4 observed number (male/female)

The MC4R genotype	???L02	???L03	???L19	??L65	Altogether		??L95
								????11	??9/79	??12/18		?15/17	??36/114		?0/20
????12	??9/48	??37/17	??12/44	?44/30	?102/139		?0/67
								????22	??3/9	??28/3	??89/165	?21/12	?141/189		?0/37

Table 5

Fate to 110kg

The MC4R genotype	??L02	??L03	??L19	??L65	Altogether		????L95
								????11	?169.7	?172.4		?169.6	????168.5		???219.1
????12	?170.2	?171.5	?165.0	?171.2	????168.7		???212.2
								????22	?165.3	?173.4	?162.9	?170.3	????167.1		???211.4
	?p＜.23	?p＜.75	?p＜.15	?p＜.76	???p＜.31		??p＜.27

Table 6

P2 back fat (millimeter)

The MC4R genotype	???L02	???L03	???L19	???L65	Altogether		???L95
								????11	??10.8	??11.9		???9.7	??11.1		???22.8
????12	??11.3	??12.5	??12.2	???10.5	??11.8		???21.5
								????22	??12.1	??12.7	??12.6	???10.7	??12.1		???20.3
	?p＜.10	?p＜.43	?p＜.34	??p＜.17	?p＜.006		??p＜.17

Table 7

Test day weightening finish (gram/sky)

The MC4R genotype	??L02	??L03	??L19	??L65	Altogether		???L95
								????11	?882.2	?811.0		?881.8	???871.9		??688.8
????12	?891.2	?820.5	?875.6	?873.0	???876.3		??676.2
								????22	?969.1	?819.5	?906.7	?906.2	???906.9		??692.5
	p＜.01	p＜.96	p＜.05	p＜.24	?p＜.006		?p＜.66

Table 8

Average food ration every day (kg/day), boar only is except that only being the L95 of gilt

The MC4R genotype	???L02	???L03	???L19	???L65	Altogether		????L95
								????11	??2.31	??1.78		??1.75	???1.89		???2.05
????12	??2.11	??1.90	??1.97	??1.90	???1.96		???2.03
								????22	??2.15	??1.97	??2.00	??1.97	???2.02		???2.08
	?p＜.84	?p＜.14	?p＜.56	?p＜.14	??p＜.16		??p＜.36

Embodiment 4

The missense variant of pig melanocortin-4 receptor (MC4R) gene

With fat, growth is relevant with the food ration feature

In order to determine whether this MC4R polymorphism is relevant with phenotypic difference, tested the sudden change in a large amount of animal individuals of several different pig strains.The analysis revealed of growth and performance testing record, the back fat of MC4R genotype and many strains, the speed of growth and food ration significant correlation.The different aminoacids residue of MC4R sudden change may make the noticeable change of MC4R function.These results have confirmed the functional meaning of pig MC4R missense mutation, and the contrast of the genome of hints model species is applied to farm-animals and human medical no less important.

The information that relates to some genetic constitutions that energy balance regulates (people such as Zhang, 1994 are provided to the evaluation that in leptin and leptin acceptor, suddenlys change; People such as Tartaglia, 1995).Carry out gene studies with animal model and help to identify fat main genetic cause (Andersson1996; Pomp1997; Giridharan, 1998).In addition, gene (Flier and the Maratos-Flier1998 of other several participation energy homeostasis nerve signal transduction pathway have been identified; People such as Schwartz, 1999).In the candidate signal molecule that participates in the energy homeostasis adjusting, that most interested is melanocortin-4 receptor (MC4R).MC4R for replying of leptin signal be food ration with body weight between related (Seeley waits the people, 1997; People such as Marsh, 1999).Neuropeptide tyrosine in the central nervous system (NPY) signal is also by MC4R protein mediated (people such as Kask, 1998).Several sudden changes among the MC4R (comprising frameshit and nonsense mutation) relevant (people such as Vaisse, 1998 with the obesity of people's dominant inheritance; Yeo waits the people, and 1998).Some other MC4R missense mutation of people also identifies out (people such as Gotoda, 1997; People such as Hinney, 1999), but the functional meaning of these sudden changes is not also determined.

Select for the industry of raising pigs according to growth characteristics extremely important because cost is relevant with raising, and consumer preference lean meat.The efficient gene of these quantitative characters improves and can and strengthen (Dekkers and van Arendonk1998 with the high-density gene mapping by applying marking assisted Selection (MAS); Rothschild and Plastow1999).In the method, important instrument is to compare mapping with people and the mouse gene mapping be familiar with, and it can help to identify the control growing corresponding in the pig and the genome area or the oligogene of behavioural characteristic.The biology knowledge of complex characteristic provides other method for the gene identification of feature that economic worth is arranged in the livestock in human or animal's model.For fat and growth characteristics, successfully carried out several with the kind of phenotype divergence and the chain scanning of quantitative trait locus (QTL) of candidate gene approach (people such as Yu, 1995; People such as Casas-Carrillo, 1997; People such as Knorr, 1997; People such as Knott, 1998; People such as Rohrer, 1998; People such as Wang, 1998; People such as Paszek, 1999), but commercial fauna is not also had to determine growth and behavioural characteristic are had the individual gene of main effect.The effect prompting of MC4R in food ration and obesity, it may be the important gene mark of the relevant proterties of growth in the pig.

Material and method

Animal.Pig is grown up by the nursing of the PIC employee in the nucleus farm of US and European under normal production conditions.When big pig is carried out performance testing in about 70 days, and stopped test after 13 weeks.Last what test, measure back fat with rib apart from center line 2 centimeters the 10th with ultrasonic wave real-time (B pattern).The weight that increases divided by the test fate, is calculated average day weight gain (growth) in the testing period.Estimate to grow to the fate of 110 kilograms of market weight with standard program, each measures food ration with electronics metering equipment.

Pcr amplification pig MC4R gene fragment.From people and rat MC4R sequence (the GenBank accession number is respectively the homologous region of s77415 and u67863) design primer.Primer is a forward primer: 5 '-TGG CAA TAG CCA AGAACA AG-3 ' (SEQ ID NO:6) and reverse primer: 5 '-CAG GGG ATA GCA ACA GAT GA-3 ' (SEQ ID NO:7).With 12.5ng pig genomic dna, 1xPCR damping fluid, 1.5mM MgCl ₂, each primer of 0.125mM dNTP0.3mM and 0.35U Taq archaeal dna polymerase (Promega) carry out the PCI reaction in the final volume of 10 microlitres.The condition of PCR is as follows: Robocycler (Stratagene, La Jolla, CA) in, 94 ℃ 2 minutes, 35 94 ℃ of taking turns 30 seconds, 56 ℃ 1 minute, 92 ℃ 1 minute 30 seconds, last 72 ℃ were extended 15 minutes.

Order-checking and sudden change detect.Several pigs to different varieties carry out the order-checking of PCR product, and comparative sequences changes to detect any Nucleotide.Order-checking is carried out on ABI sequenator 377 (Applied Biosystems).Pig MC4R sequence has been submitted to GenBank, and accession number is AF087937.Sequential analysis discloses a nucleotide subsitution and is positioned at TaqI restriction enzyme recognition site people such as (, 1999) Kim.Design a series of primers then, to produce less MC4R gene fragment, this fragment only contains a TaqI restriction site that information is arranged, to specify pleomorphism site and to help the PCR-RFLP test.These primers are: forward primer 5 '-TAC CCT GAC CAT CTT GAT TG 3 ' (SEQ ID NO:10) and reverse primer: 5 '-ATA GCA ACA GAT GAT CTC TTT G-3 ' (SEQ IDNO:11).

Statistical analysis.The program of variance analysis adopts the mixture model of the fixed effect at explanation farm, testing period, animal sex, MC4R genotype and position (at random).The animal of gathering all U.S./European strain (strain A-D) is used for whole analysis, in this is analyzed, comprises primitive variety.Estimate each genotypic average effect, the results are shown among the table 9-15.Check with overall F and to measure significance level.

The result

The evaluation of missense mutation in the pig MC4R gene.The MC4R gene is made up of the encoding sequence that is included in an about 1kb in the exon.PCR has produced about 750 base pairs (people such as Kim, 1999) of pig MC4R gene fragment.The sequence conclusive evidence PCR product of PCR product is the MC4R gene, and it has 92.2% and 97.6% homogeny respectively with people MC4R sequence on Nucleotide and amino acid levels.The multiple contrast of the sequence of the single animal of several kinds has identified a nucleotide subsitution (G → A; Fig. 5).Polymorphism discloses a missense mutation, with 298 identical positions of the proteic amino acid of people MC4R, aspartic acid (GAU) is replaced by l-asparagine (AAU).In order to confirm this sequence change, we design the pig Auele Specific Primer of side joint pleomorphism site, with TaqIPCR-RFLP gel analysis polymorphism (Fig. 6).Fig. 6 has shown the TaqI digestion result of the PCR product that obtains with the agarose gel electrophoresis analysis.Allelotrope 1 produced 156 and the fragment of 70bp as PCR-RFLP, allelotrope 2 has produced the fragment of 226bp as PCR-RFLP.Heterozygote has allelotrope 1 and 2 fragments.Molecule marker (M) and MC4R genotype are marked on the top of each swimming lane.

The MC4R missense mutation is in the conservative zone of melanocortin receptor (MCR) camber.MCR is the subtribe of coupling G protein receptor (GPCR), and it contains some common conservative structural element of other GPCR of great majority, but the whole amino acid homogeny between MCR and other GPCR very low (Tatro 1996).Aminoacid sequence and other animal species, other MCR albumen or the typical GPCR of the proteic expection of pig MC4R are done the multiple sequence contrast, and the result shows that the aspartic acid of 298 of the 7th membrane spaning domains is at MCR albumen camber conservative (Fig. 7).Yet, notice that with interest this position is a l-asparagine among other GPCR of great majority.MCR albumen demonstrates the amino acid homogeny (Tatro 1996) of 40-80% to each other, but ring and the 7th membrane spaning domain are high conservative people such as (, 1993) Gantz in MCR albumen in second kytoplasm.Research by natural in people and mouse and experiment sudden change (people such as Robbins, 1993; Valverde waits the people, and 1995; Frandberg waits the people, and 1998), have been found that some relations between MCR structure and the function.These studies show that some sudden changes in the high conservative zone cause structural changes and change the function of acceptor.The Asp298Asn replacement mutation is influential to the function of acceptor.Yet this needs further test, but known homology residue in MC1R changes (Asp294His) and people's white skin and red hair relevant (Valverde waits the people, 1995).

The MC4R missense mutation is relevant with fat correlated characteristic.In order to study the influence of missense mutation, in the several commercial pig strain of PIC (a tame world raise pigs company) altogether in 1800 pigs, analyze the MC4R genotype and to the relation of the influence of the speed of growth, back fat and feed intake difference.Animal comes the commercial strain (strain A-D) of self-enclosed Europe/u.s. variety, also has the strain (strain E) from Europe and Chinese variety hybridization.In strain A-D, find that all behavioural characteristics are all obviously relevant with the MC4R genotype.(((P＜.01) obviously is less than the 22 genotype animals (table 11,13 and 15) of isozygotying to the average back fat of the animal that allelotrope 1 isozygotys for P＜.001) and food ration for P＜.001), day weight gain.In a word, the back fat with 11 genotypic pigs lacks about 9% (table 11) than 22 genotype pigs, and 22 genotypic pigs obviously grow soon (37 gram/sky) (table 13) than 11 genotypic pigs.These results it seems relevant with appetite, because much more (table 15) that the ratio of 22 genotype animals consumings is eaten.In Europe/u.s. variety, getting in touch between the missense mutation of clearly having established the MC4R gene and the relevant behavioural characteristic.Though the quantity of test animal is much smaller, in quite fat China hybridization system (strain E), do not find these results.Interesting is that strain E demonstrates the trend direction and other strain viewed opposite (table 11) of back fat.

Discuss

This research confirms that clearly pig MC4R missense mutation is obviously relevant with several behavioural characteristics of pig.The allelotrope 1 that shows as Asp298 (at the conservative amino acid of the MC4R camber of other MCR hypotype and other animal species) and less respective thickness of back fat, the slower speed of growth and less food ration are associated, 2 in the allelotrope that shows as Asn298 with than fertilizer, food ration is higher and grow that animal is relevant faster.Since the residue of the high conservative in the melanocortin receptor albumen for part in conjunction with or the intracellular signal transmission play an important role (Tatro 1996), the MC4R variant may be in regulating food intake and body weight different ability on the performance function.Further this hypothesis of checking will provide important information for the architecture basics of MCR function and the molecular target of treatment people obesity.

Animal the most fat among allelotrope 1 and the strain E is relevant, and this animal is that Chinese Large White kind and Meishan incross obtain.If sudden change causes that significant amino acid takes place in the high conservative zone to be changed, this is wondrous so.The possibility of result is caused by sampling.Yet if our hypothesis is when that more result adds is fashionable, this result is significant, and this has several explanations.A possibility perhaps is the difference of background genetic effect (epistasis).Because growth and obesity are complicated polygenic features, therefore very possible Chinese variety has and separated some different allelotrope of deutero-in hundreds of years and interact, and the interaction that these are inferred may and the strain that differs greatly between hybridization in make polygenic feature change (Frankel and Schork 1996).Fat and growth characteristics several qtl analysis (people such as Cases-Carrillo, 1997 have been carried out with divergent strain; People such as Knott, 1998; People such as Rohrer, 1998; People such as Wang, 1998; People such as Paszek, 1999), but do not report that QTL is near C4R locus (its figure spectral position on karyomit(e) 1 apart from the about 80cm of linkage map (data not shown)).It may mean, the allelic epistatic effect of MC4R of strain E prompting makes and is difficult to observe the MC4R locus in great majority relate to the QTL experiment of Chinese strain and Europe/America interlinear cross.Might some allelic effect can under different backgrounds, change, and be difficult in the QTL experiment that relates to the gene divergence kind, detect.

The effect of MC4R variant can be explained by the further research of biological effect that this sudden change is caused in other pig variety and strain.Yet if MC4R variant and lean meat, growth and food ration have confidential relation, this sudden change can be used for marker assisted selection (Meuwissen and Goddard 1996) immediately, to develop the pig strain that satisfies the particular consumer demand.For example, in the sow strain that appetite goes down usually behind farrowing, the selection of MC4R allelotrope 2 can help to improve food ration.In addition, in some think too fertile strain, can utilize the selection of allelotrope 1, think the selection that can utilize allelotrope 2 in the too slow strain of growth.Therefore, in the pig breeding strain, select the genotype of MC4R sudden change to select and the production feature of the ingesting relevant efficient of (comprising growth and lean meat) with improving.Candidate gene approach also is used to investigate the effect (Jiang and Gibson 1999) of pig leptin gene.Yet, with regard to leptin, although commercial breed with do not improve the kind of hybridizing between the strain in have the leptin polymorphism evidence relevant with respective thickness of back fat, in the different commercial strain of test but not clearly related (Jiang and Gibson 1999).Therefore, should not think, just can suppose a kind of relation that exists owing to found a gene.On the contrary, for MC4R, we determine that the variation in this candidate gene can be explained the notable difference of back fat, the speed of growth and food ration in the commercial strain of pig.These results of MC4R have described the potential value that adopts the comparative genetic analysis of candidate gene in the livestock genome.

The MC4R genotype is to the influence of the several production features of pig

Table 9

Grow to the fate of 110kg and the observed number of back fat (male/female/sum)

The MC4R genotype	Strain A	Strain B	Strain C	Strain D	Altogether	Strain E
							????11	?9/212/221	?12/94/106		??37/17/54	??58/323/381	???0/20/20
????12	?9/150/159	?37/96/133	?12/158/170	?152/30/182	??210/434/644	???0/67/67
							????22	??3/16/19	?28/36/64	?89/356/445	?155/12/167	??275/420/695	???0/37/37

Table 10

Grow to the fate of 110kg

The MC4R genotype	Strain A	Strain B	Strain C	Strain D	Altogether	Strain E
							????11	?166.3+/-0.8	?168.4+/-1.4		?170.0+/-2.4	???167.9+/-0.9	???219.1+/-4.8
????12	?165.6+/-0.9	?166.8+/-1.1	?163.9+/-1.0	?170.2+/-1.8	???166.9+/-0.8	???212.2+/-3.4
							????22	?162.3+/-2.3	?166.8+/-1.5	?161.5+/-0.8	?167.0+/-1.9	???164.6+/-0.9	???211.4+/-4.0
	?????p＜.24	?????p＜.47	?????p＜.007	?????p＜.10	???????p＜.001	???????p＜.27

Table 11

The back fat (millimeter) of the 10th root bone

The MC4R genotype	Strain A	Strain B	Strain C	Strain D	Altogether	Strain E
							????11	?10.7+/-0.2	?12.1+/-0.2		??9.8+/-0.5	?11.1+/-0.2	?22.8+/-1.2
????12	?11.2+/-0.2	?12.5+/-0.2	?12.3+/-0.2	?10.5+/-0.4	?11.6+/-0.2	?21.5+/-0.9
							????22	?12.5+/-0.5	?12.6+/-0.3	?12.7+/-0.2	?10.9+/-0.4	?12.0+/-0.2	?20.3+/-1.0
	????p＜0.2	????p＜.31	????p＜.06	????p＜.05	????p＜.00l	????p＜.17

Table 12

The observed number (male/female/sum) of test day weightening finish

The MC4R genotype	Strain A	Strain B	Strain C	Strain D	Altogether	Strain E
							????11	???9/105/114	???12/38/50		????37/17/54	?????58/160/218	???0/20/20
????12	????9/65/74	???37/35/72	???12/97/109	???152/30/182	????210/227/437	???0/67/61
							????22	????3/13/15	???28/15/43	??89/225/314	???155/12/167	????275/265/539	???0/37/37

Table 13

Test day weightening finish (gram/sky)

The MC4R genotype	Strain A	Strain B	Strain C	Strain D	Altogether	Strain E
							????11	?892.6+/-l0.4	?841.7+/-13.8		?882.2+/-18.4	?871.9+/-10.2	??688.8+/-24.5
????12	?913.3+/-11.6	?868.4+/-12.1	?882.2+/-12.9	?883.7+/-14.3	?885.1+/-8.9	??676.2+/-17.6
							????22	?982.8+/-22.8	?862.4+/-15.1	?913.4+/-10.5	?904.6+/-15.1	?908.8+/-9.3	??692.5+/-20.4
	?????p＜.001	?????p＜.28	?????p＜.006	?????p＜.20	?????p＜.001	??????p＜.66

Table 14

The observed number (male/female/sum) of average day food ration

The MC4R genotype	Strain A	Strain B	Strain C	Strain D	Altogether	Strain E
							????11	???7/0/7	??11/0/11		??13/0/13	??31/0/31	??0/18/18
????12	???8/0/8	??31/0/31	???9/0/9	??34/0/34	??82/0/82	??0/63/63
							????22	???3/0/3	??25/0/25	??74/0/74	??16/0/16	?118/0/118	??0/32/32

Table 15

Average day food ration (kg/day) only is a boar, except that strain E only is the gilt

The MC4R genotype	Strain A	Strain B	Strain C	Strain D	Altogether	Strain E
							????11	?2.31+/-0.2	?1.78+/-0.09		?1.75+/-0.06	?1.94+/-0.07	?2.05+/-0.10
????12	?2.11+/-0.3	?1.90+/-0.07	?1.97+/-0.10	?1.90+/-0.07	?2.03+/-0.06	?2.03+/-0.07
							????22	?2.15+/-0.4	?1.97+/-0.06	?2.00+/-0.07	?1.97+/-0.08	?2.11+/-0.06	?2.08+/-0.08
	????p＜.84	????p＜.14	????p＜.56	????p＜.14	????p＜.01	????p＜.36

After reference specific composition, effect theory etc. has been described the present invention, those skilled in the art obviously can understand, the present invention is not limited to these descriptive embodiment or mechanism, can not break away from the defined scope of the invention of claims or spirit and change.Estimate that all these are significantly changed and variation includes in the scope of the invention of claims.Claim means any steps in order that has covered desired component and can effectively satisfy its purpose, unless this paper has opposite particular content.

Following citing document is all included this paper in as a reference:

Andersson LB (1996) " gene and obesity ", Ann.Med., 28:5-7.

Arden KC (1990). " prolactin antagonist and growth hormone receptor are positioned at the same area of human chromosome 5 ", Cytogenet.Cell Genet.53:161-165.

Barinaga M (1996). " the clear and definite leptin acceptor of investigator ", Science271:913.

Bray?GA(1978).Physiol.Rev.59，719-809.

Casas-Carillo E (1997) " makes collection of illustrative plates to the genome area relevant with growth speed of pigs ", J.Anim Sci75:2047-2053

Chajlani V, " molecular cloning of new people's melanocortin receptor ", Biochem.Biophys.Res.Commun., 195,866-873.

Chen H (1995) " evidence of diabetes genes encoding leptin acceptor: the evaluation of the sudden change of leptin acceptor gene in the db/db mouse ", Cell 84,491-495.

Chevalet C (1996) .CABIOS. publishes.

Chua?SC(1996).Science?271，994-996.

Chung?WK(1996).Genome?Res，6，431-438.

Cioffi?JA(1996).Nature?Med.2，585-589。

Cybulsky MI (1991). " gene structure of people VCAM1 gene, chromosome position and mRNA alternative splicing " Proc.Natl.Acad.Sci.88:7859-7863.

Dekkers JCM (1998) " with the selection of the quantitative proterties of genes identified seat information optimization in the colony of outbreeding ", Genet Res 71:257-275.

Flier JS (1998), " obesity and hypothalamus: the new peptide of new way ", Cell 92:437-440.

Frandberg P (1997) " glutamine 235 in people's melanocortin 5 acceptors and arginase 12 72 have been determined its low affinity to MSH ", Biochem Biophys Res Commun236:489-492.

Frankel WN (1996) " who fears epistasis? " Nat Genet14:371-373.

GantzI (1993a) " molecular cloning of new melanocortin receptor ", J.Biol.Chem.268,8246-8250.

Gantz (1993) " molecular cloning, the expression of the gene location of the 4th kind of melanocortin receptor ", J.Biol.Chem.268,15174-15179.

Gantz (1994) " molecular cloning of the 5th kind of melanocortin receptor is expressed and identified " Biochem BiophysRes Commun., 200,1214-1220.

Giridharan NV (1998) " fat animal model and the application in the obesity molecular method thereof ", IndianJ Med res 108:225-242.

Gotoda T (1997) " molecular screening of people's melanocortin-4 receptor gene: the evaluation of missense variant demonstrates with obesity, blood sugar or Regular Insulin irrelevant " Diabetologia 40:976-979.

Goureau A (1996) .Genomics. publishes.

Griffon N (1994) " molecular cloning and the evaluation of the 5th melanocortin receptor of rat ", BiochemBiophys Res Comm., 200,1007-1014.

Helm J (1994) " news flash: have the segmental SacI restriction fragment length polymorphism of pig vascular cell adhesion molecule 1 (VCAM1) cDNA ", J.Anim.Science72:2764.

Hinney A (1999) " the several sudden changes in the melanocortin-4 receptor gene comprise the meaningless and phase shift mutation fat relevant with people's dominant inheritance " J Clin Endocrinol Metab 84:1438-1486.

Huszar (1997) " orientation of melanocortin-4 receptor is destroyed and caused the mouse obesity ", Cell 88:131-141.

Kask A (1998) " the appetizing effect of melanocortin 4 receptor antagonist HS014 is by the evidence of neuropeptide tyrosine mediation ", Biochem Biophys Res Commun 248:245-249.

Kim KS (1999) " news flash: the chain and physical map of pig melanocortin-4 receptor (MC4R) gene " J AnimSci (submission).

Knorr C (1997) " behavioural characteristic in GH genetic mutation and aper, Pietrain and Meishan pig F2 generation related " Anim Genet28:124-128.

Knott SA (1998) " the multiple labeling collection of illustrative plates of quantitative trait locus in outbreeding wild boar and the Large White filial generation ", Genetics149:1069-1680.

Labbe O (1994) " molecular cloning of mouse melanocortin-5 acceptor gene of wide expression in peripheral tissues " Biochemistry, 33,4543-4549.

Lee?GH(1996).Nature379，632-635.

Ju Dongsi " agouti albumen is the antagonist of msh receptor " Letters to Nature.

Marsh DJ (1999) " the melanocortin-4 receptor deficient mice is replied apocleisis and appetizing property peptide " Nat Genet21:119-122.

Messer L., (1996a) " collection of illustrative plates and the investigation of the candidate gene of French Large White son size " ISAG Procedings (submitting to).

Messer L (1996b) " retinoic acid receptor (RAR) γ (RARG) gene is measured through linkage map and is positioned at pig karyomit(e) 5 " .Anim.Genet (publishing).

Messer L (1996c) " retinol conjugated protein 4 (RBP4) gene is measured through linkage map and is positioned at pig karyomit(e) 14 " .Mammal.Genome (publishing).

Meuwissen THE (1996) " monoploid is marked at the purposes in the improvement of breed scheme " Genet Sel Evol28:161-176.

Montjoy KG (1992) " clone of the gene family of coding melanocortin receptor ", Science 257,1248-1251.

Montjoy KG (1994) " melanocortin-4 receptor (MC4-R) in brain in main control loop the position in the neuroendocrine " Mol.Endocrinol, 8,1298-1308.

Paszek A (1996) " to the genome scanning of pig quantitative trait locus ", Proceed.Midwest.ASASMeeting, 24 pages.

Paszek AA (1999) " the growth spacing collection of illustrative plates in the divergent pig hybridization " Mamm Genome 10:117-122.

Phillips M (1996) " leptin acceptor missense mutation in the fat Zucker rat ", Nature Genetics13:18-19.

Pomp D (1997) " the gene dissection and analysis of obesity in the polygene animal model " Behav Genet 27:285-306.

Robbins LS (1993) " point mutation that changes the MSH function of receptors produces the allelic pigmentation phenotype of variant extension gene seat " Cell72:827-834.

Robic?A(1996).Mamm.Genome?7，438-445.

Rohrer GA (1998) " influence the evaluation of the quantitative trait locus that the pig corpse forms: I. fatty deposits feature " J Anim Sci76:2247-2254.

Rothschild MF (1996). " the female hormone receptor gene seat is relevant with the oligogene of pig cub size " .PNAS93:201-205.

Rothschild MF (1999) " progress of pig genome and industrial application " AgBiotechNet 1:1-8.

Schwartz MW (1999) " central nervous system is regulated the model of energy balance and obesity " Am J ClinNutr69:584-96.

SeeleyRJ (1997) " melanocortin receptor in the leptin effect ", Nature390:349.

Tartaglia LA (1995) " evaluation of leptin acceptor and cloning by expression " Cell 83:1263-1271.

Tatro JB (1996) " nerve immunity is regulated the receptor biological of peptide family melanocortin ", Neuroimmunomodulation3:259-284.

Truett?GE(1995).Mamm.Genome6，25-30.

Vaisse C (1998) " phase shift mutation among the people MC4R is relevant with fat dominant form " Nat Genet20:113-114.

Valverde P (1995) " variant of melanocortin stimulation hpr gene is relevant with the red hair of people and Baise skin " Nat Genet 11:328-330.

Wang L (1998) " directly searches the quantitative trait locus on pig karyomit(e) 4 and 7 " J Anim Sci76:2560-2567.

Warner CM (1991). " the main histocompatibility complex of pig (SLA) ", " immunogenetics of MHC ", VCH publishes, NY, NY, 368-397 page or leaf.

Winick?JD(1996).Genomics36，221-222.

Xy Y (1994) " identify the Ped gene at molecular level: Q9 MHC I class transgenosis changes the slow phenotype of Ped into Ped rapid phenotype " Biol.Reprod.51:695-699.

Yeo GS (1998) " phase shift mutation among the MC4R fat relevant " Nat Genet20:111-112. with the people of dominant inheritance

Yerle?M(1996).Cytogenet.Cell?Genet?73，194-202.

Youngs CR (1993) " research of control pig cub size: the ectogenetic comparative study of I.Meishan and Yorkshire pre-implantation embryos " J.Anim.Sci., 1561-1565.

Yu TP (1995) " relation of PIT1 polymorphism and pig growth and corpse feature " J Anim Sci73:1282-1288.

Zhang Y (1994) " positional cloning of mouse ob gene and people's homologue thereof ", Nature 372:425-432.

Ziang ZH (1999) " gene pleiomorphism in the leptin gene and with the relation of four boar obesities " MammGenome 12:191-193.

Sequence table＜110〉Iowa State Univ. Research Foundation, Inc.＜120 the melanocortin-4 receptor gene and increase and/or the purposes of the genetic marker of food consumption＜130〉rothshcild mc4r2＜140 as animal tallow content, body weight PCT/US99/16862＜141 1999-07-26＜150 60/094; 287＜151〉1998-07-27＜150〉60/116,196＜151〉1999-01-15＜160〉26＜170〉PatentIn Ver.2.0＜210〉1＜211〉745＜212〉DNA＜213〉＜220〉＜221〉＜222〉 ( 678 )＜223〉G/A＜400〉1acaagaatct gcattcaccc atgtactttt tcatctgtag cctggctgtg gctgatatgc 60tggtgagcgt ttccaatggg tcagaaacca ttgtcatcac cctattaaac agcacggaca 120cggacgcaca gagtttcaca gtgaatattg ataatgtcat tgactcagtg atctgtagct 180ccttactcgc ctcaatttgc agcctgcttt cgattgcagt ggacaggtat tttactatct 240tttatgctct ccagtaccat aacattatga cagttaagcg ggttggaatc atcatcagtt 300gtatctgggc agtctgcacg gtgtcgggtg ttttgttcat catttactca gatagcagtg 360ctgttattat ctgcctcata accgtgttct tcaccatgct ggctctcatg gcttctctct 420atgtccacat gttcctcatg gccagactcc acattaagag gatcgccgtc ctcccaggca 480ctggcaccat ccgccaaggt gccaacatga agggggcaat taccctgacc atcttgattg 540gggtctttgt ggtctgctgg gcccccttct tcctccactt aatattctat atctcctgcc 600cccagaatcc atactgtgtg tgcttcatgt ctcactttaa tttgtatctc atcctgatca 660tgtgtaattc catcatcgat cccctgattt atgcactccg gagccaagaa ctgaggaaaa 720ccttcaaaga gatcatctgt tgctat 746＜210〉2＜211〉840＜212〉DNA＜213〉＜400〉2atatcttagt gattgtggca atagccaaga acaagaatct gcattcaccc atgtactttt 60tcatctgcag cttggctgtg gctgatatgc tggtgagcgt ttcaaatgga tcagaaacca 120ttatcatcac cctattaaac agtacagata cggatgcaca gagtttcaca gtgaatattg 180ataatgtcat tgactcggtg atctgtagct ccttgcttgc atccatttgc agcctgcttt 240caattgcagt ggacaggtac tttactatct tctatgctct ccagtaccat aacattatga 300cagttaagcg ggttgggatc agcataagtt gtatctgggc agcttgcacg gtttcaggca 360ttttgttcat catttactca gatagtagtg ctgtcatcat ctgcctcatc accatgttct 420tcaccatgct ggctctcatg gcttctctct atgtccacat gttcctgatg gccaggcttc 480acattaagag gattgctgtc ctccccggca ctggtgccat ccgccaaggt gccaatatga 540agggagcgat taccttgacc atcctgattg gcgtctttgt tgtctgctgg gccccattct 600tcctccactt aatattctac atctcttgtc ctcagaatcc atattgtgtg tgcttcatgt 660ctcactttaa cttgtatctc atactgatca tgtgtaattc aatcatcgat cctctgattt 720atgcactccg gagtcaagaa ctgaggaaaa ccttcaaaga gatcatctgt tgctatcccc 780tgggaggcct ttgtgacttg tctagcagat attaaatggg gacagagcac gcaatatagg 840＜210〉3＜211〉311＜212〉＜213〉＜400〉3Gln Leu Phe Val Ser Pro Glu Val Phe Val Thr Leu Gly Val Ile Ser 1 5 10 15Leu Leu Glu Asn Ile Leu Val Ile Val Ala Ile Ala Lys Asn Lys Asn

20??????????????????25??????????????????30Leu?His?Ser?Pro?Met?Tyr?Phe?Phe?Ile?Cys?Ser?Leu?Ala?Val?Ala?Asp

35??????????????????40??????????????????45Met?Leu?Val?Ser?Val?Ser?Asn?Gly?Ser?Glu?Thr?Ile?Ile?Ile?Thr?Leu

50??????????????????55??????????????????60Leu?Asn?Ser?Thr?Asp?Thr?Asp?Ala?Gln?Ser?Phe?Thr?Val?Asn?Ile?Asp?65??????????????????70??????????????????75??????????????????80Asn?Val?Ile?Asp?Ser?Val?Ile?Cys?Ser?Ser?Leu?Leu?Ala?Ser?Ile?Cys

85??????????????????90??????????????????95Ser?Leu?Leu?Ser?Ile?Ala?Val?Asp?Arg?Tyr?Phe?Thr?Ile?Phe?Tyr?Ala

100?????????????????105?????????????????110Leu?Gln?Tyr?His?Asn?Ile?Met?Thr?Val?Lys?Arg?Val?Gly?Ile?Ser?Ile

115?????????????????120?????????????????125Ser?Cys?Ile?Trp?Ala?Ala?Cys?Thr?Val?Ser?Gly?Ile?Leu?Phe?Ile?Ile

130?????????????????135?????????????????140Tyr?Ser?Asp?Ser?Ser?Ala?Val?Ile?Ile?Cys?Leu?Ile?Thr?Met?Phe?Phe145?????????????????150?????????????????155?????????????????160Thr?Met?Leu?Ala?Leu?Met?Ala?Ser?Leu?Tyr?Val?His?Met?Phe?Leu?Met

165?????????????????170?????????????????175Ala?Arg?Leu?His?Ile?Lys?Arg?Ile?Ala?Val?Leu?Pro?Gly?Thr?Gly?Ala

180?????????????????185?????????????????190Ile?Arg?Gln?Gly?Ala?Asn?Met?Lys?Gly?Ala?Ile?Thr?Leu?Thr?Ile?Leu

195?????????????????200?????????????????205Ile?Gly?Val?Phe?Val?ValCys?Trp?Ala?Pro?Phe?Phe?Leu?His?Leu?Ile

210????????????????215?????????????????220Phe?Tyr?Ile?Ser?Cys?Pro?Gln?Asn?Pro?Tyr?Cys?Val?Cys?Phe?Met?Ser225?????????????????230?????????????????235??????????????????240His?Phe?Asn?Leu?Tyr?Leu?Ile?Leu?Ile?Met?Cys?Asn?Ser?Ile?Ile?Asp

245?????????????????250?????????????255Pro?Leu?Ile?Tyr?Ala?Leu?Arg?Ser?Gln?Glu?Leu?Arg?Lys?Thr?Phe?Lys

260?????????????????265?????????????????270Glu?Ile?Ile?Cys?Cys?Tyr?Pro?Leu?Gly?Gly?Leu?Cys?Asp?Leu?Ser?Ser

275?????????????????280?????????????????285Arg?Tyr?Ala?Pro?Pro?Glu?Asn?Asp?Ile?Xaa?Val?Ile?Cys?Asn?Phe?Ile

290 295 300Asp Glu Asn Thr Ile Ala Leu305,310＜210〉4＜211〉248＜212〉protein＜213〉pig＜400〉4 Lys Asn Leu His Ser Pro Met Tyr Phe Phe Ile Cys Ser Leu Ala Val, 15 10 15 Ala Asp Met Leu Val Ser Val Ser Asn Gly Ser Glu Thr Ile Val Ile

20??????????????????25??????????????????30?Thr?Leu?Leu?Asn?Ser?Thr?Asp?Thr?Asp?Ala?Gln?Ser?Phe?Thr?Val?Asn

35??????????????????40??????????????????45?Ile?Asp?Asn?Val?Ile?Asp?Ser?Val?Ile?Cys?Ser?Ser?Leu?Leu?Ala?Ser

50??????????????????55??????????????????60?Ile?Cys?Ser?Leu?Leu?Ser?Ile?Ala?Val?Asp?Arg?Tyr?Phe?Thr?Ile?Phe??65??????????????????70??????????????????75??????????????????80?Tyr?Ala?Leu?Gln?Tyr?His?Asn?Ile?Met?Thr?Val?Lys?Arg?Val?Gly?Ile

85??????????????????90??????????????????95?Ile?Ile?Ser?Cys?Ile?Trp?Ala?Val?Cys?Thr?Val?Ser?Gly?Val?Leu?Phe

100?????????????????105?????????????????110?Ile?Ile?Tyr?Ser?Asp?Ser?Ser?Ala?Val?Ile?Ile?Cys?Leu?Ile?Thr?Val

115?????????????????120?????????????????125Phe?Phe?Thr?Met?Leu?Ala?Leu?Met?Ala?Ser?Leu?Tyr?Val?His?Met?Phe

130?????????????????135?????????????????140Leu?Met?Ala?Arg?Leu?His?Ile?Lys?Arg?Ile?Ala?Val?Leu?Pro?Gly?Thr145?????????????????150?????????????????155????????????????160Gly?Thr?Ile?Arg?Gln?6ly?Ala?Asn?Met?Lys?6ly?Ala?Ile?Thr?Leu?Thr

165?????????????????170?????????????????175Ile?Leu?Ile?Gly?Val?Phe?Val?Val?Cys?Trp?Ala?Pro?Phe?Phe?Leu?His

180?????????????????185?????????????190Leu?Ile?Phe?Tyr?Ile?Ser?Cys?Pro?Gln?Asn?Pro?Tyr?Cys?Val?Cys?Phe

195?????????????????200?????????????????205Met?Ser?His?Phe?Asn?Leu?Tyr?Leu?Ile?Leu?Ile?Met?Cys?Asn?Ser?Ile

210?????????????????215?????????????????220Ile?Asn?Pro?Leu?Ile?Tyr?Ala?Leu?Arg?Ser?Gln?Glu?Leu?Arg?Lys?Thr225?????????????????230?????????????????235?????????????????240Phe?Lys?Glu?Ile?Ile?Cys?Cys?Tyr

245＜210〉5＜211〉20＜212〉DNA＜213〉＜400〉5tggcaatagc caagaacaag 20＜210〉6＜211〉20＜212〉DNA＜213〉＜400〉6caggggatag caacagatga 20＜210〉7＜211〉18＜212〉DNA＜213〉＜400〉7ttaagtggag gaagaagg 18＜210〉8＜211〉19＜212〉DNA＜213〉＜400〉8cattatgaca gttaagcgg 19＜210〉9＜211〉20＜212〉DNA＜213〉＜400〉9taccctgacc atcttgattg 20＜210〉10＜211〉22＜212〉DNA＜213〉＜400〉10atagcaacag atgatctctt tg 22＜210〉11＜211〉24＜212〉＜213〉＜400〉1lMet Ser His Phe Asn Leu Tyr Leu Ile Leu Ile Met Cys Asn Ser Ile 1 5 10 15Ile Asp Pro Leu Ile Tyr Ala Leu。

20＜210〉12＜211〉24＜212〉protein＜213〉homo sapiens＜400〉12Met Ser His Phe Asn Leu Tyr Leu Ile Leu Ile Met Cys Asn Ser Ile, 15 10 15Ile Asp Pro Leu Ile Tyr Ala Leu

20＜210〉13＜211〉24＜212〉protein＜213〉rat＜400〉13Met Ser His Phe Asn Leu Tyr Leu Ile Leu Ile Met Cys Asn Ala Val, 15 10 15Ile Asp Pro Leu Ile Tyr Ala Leu

20＜210〉14＜211〉23＜212〉protein＜213〉homo sapiens＜400〉14Met Ser His Phe Asn Met Tyr Leu Ile Leu Ile Met Cys Asn Ser Val, 15 10 15Ile Asp Pro Leu Ile Tyr Ala

20＜210〉15＜211〉23＜212〉protein＜213〉Mammals＜400〉15Met Ser His Phe Asn Met Tyr Leu Ile Leu Ile Met Cys Asn Ser Val, 15 10 15Ile Asp Pro Leu Ile Tyr Ala

20＜210〉16＜211〉24＜212〉protein＜213〉homo sapiens＜400〉16Met Ser Leu Phe Gln Val Asn Gly Val Leu Ile Met Cys Asn Ala Ile, 15 10 15Ile Asp Pro Phe Ile Tyr Ala Leu

20＜210〉17＜211〉22＜212〉protein＜213〉cow adenovirus 1 class＜400〉17Ala His Phe Asn Thr Tyr Leu Val Leu Ile Met Cys Asn Ser Val Ile 15 10 15Asp Pro Leu Ile Tyr Ala

20＜210〉18＜211〉22＜212〉protein＜213〉homo sapiens＜400〉18Ala His Phe Asn Thr Tyr Leu Val Leu Ile Met Cys Asn Ser Val Ile, 15 10 15Asp Pro Leu Ile Tyr Ala

20＜210〉19＜211〉23＜212〉protein＜213〉homo sapiens＜400〉19Met Ser His Phe Asn Met Tyr Leu Ile Leu Ile Met Cys Asn Ser Val, 15 10 15Met Asp Pro Leu Ile Tyr Ala

20＜210〉20＜211〉22＜212〉protein＜213〉homo sapiens＜400〉20Ser Tyr Phe Asn Leu Phe Leu Ile Leu Ile Ile Cys Asn Ser Val Val, 15 10 15Asp Pro Leu Ile Tyr Ala

20＜210〉21＜211〉25＜212〉protein＜213〉cow adenovirus 1 class＜400〉21Leu Ala Tyr Glu Lys Phe Phe Leu Leu Leu Ala Glu Phe Asn Ser Ala 15 10 15Met Asn Pro Ile Ile Tyr Ser Tyr Arg

20 25＜210〉22＜211〉19＜212〉protein＜213〉homo sapiens＜400〉22Phe Leu Leu Leu Ala Glu Ala Asn Ser Leu Val Asn Ala Ala Val Tyr, 15 10 15Ser Cys Arg＜210〉23＜211〉22＜212〉protein＜213〉homo sapiens＜400〉23Val Phe Ala Phe Cys Ser Met Leu Cys Leu Leu Asn Ser Thr Val Asn, 15 10 15Pro Leu Ile Tyr Ala Leu

20＜210〉24＜211〉21＜212〉protein＜213〉homo sapiens＜400〉24Phe Gln Phe Phe Phe Trp Ile Gly Tyr Cys Asn Ser Ser Leu Asn Pro, 15 10 15Val Ile Tyr Thr Ile

20＜210〉25＜211〉22＜212〉protein＜213〉rat＜400〉25Phe Asp Phe Val Val Ile Leu Thr Tyr Ala Asn Ser Cys Ala Asn Pro, 15 10 15Ile Leu Tyr Ala Phe Leu

20＜210〉26＜211〉16＜212〉protein＜213〉homo sapiens＜400〉26Leu Ala Tyr Ser Asn Ser Ser Val Asn Pro Ile Ile Tyr Ala Phe Leu 15 10 15

Claims (32)

1. an evaluation has the method for the genotypic animal of the metabolic characteristics that indicates lipid content, the speed of growth and feed intake, and this method comprises:

A) obtain this animal nucleic acid samples and

B) polymorphism in the evaluation sample MC4R gene.

2. method according to claim 1, wherein polymorphism is characterised in that 678 in the Nucleotide of MC4R gene PCR product.

3. method according to claim 1, wherein animal is a pig.

4. method according to claim 2, wherein the polymorphism of 678 in Nucleotide is relevant with lipid content.

5. method according to claim 2, wherein the guanine of 678 in Nucleotide is relevant with lower food ration.

6. method according to claim 2, wherein the VITAMIN B4 of 678 in Nucleotide is with weight increase speed is relevant faster.

7. method according to claim 1, the step of wherein identifying polymorphism are to adopt the method for allele specific oligonucleotide.

8. method according to claim 1 identifies that wherein the step of polymorphism is selected from restriction fragment length polymorphism rflp analysis, heteroduplex analysis, single strand conformation polymorphism sscp analysis, denaturing gradient gel electrophoresis DGGE, temperature gradient gel elec-trophoresis (TGGE) TGGE and adopts chain genetic marker.

9. method according to claim 8 identifies that wherein the step of polymorphism comprises rflp analysis.

10. method according to claim 1, this method also comprise the step of amplification MC4R gene order.

11. method according to claim 10, it also comprises the step with the zone of restriction endonuclease TaqI digest amplification.

12. the gene order of amplification according to claim 10, the primer that wherein is used to increase are selected from SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11.

13. a single stranded oligonucleotide primer that is used to detect MC4R gene PCR product Nucleotide 678, this primer is made up of the 4-30 with SEQ ID NO:1 nucleotide sequence that adjoins base.

14. oligonucleotide according to claim 13, wherein oligonucleotide has the nucleotide sequence of SEQ ID NO:6 representative.

15. oligonucleotide according to claim 13, wherein oligonucleotide has the nucleotide sequence of SEQ ID NO:7 representative.

16. oligonucleotide according to claim 13, wherein oligonucleotide has the nucleotide sequence of SEQ ID NO:8 representative.

17. oligonucleotide according to claim 13, wherein oligonucleotide has the nucleotide sequence of SEQ ID NO:9 representative.

18. oligonucleotide according to claim 13, wherein oligonucleotide has the nucleotide sequence of SEQ ID NO:10 representative.

19. oligonucleotide according to claim 13, wherein oligonucleotide has the nucleotide sequence of SEQ ID NO:11 representative.

20. a method of identifying animal, this animal have the required genotype of the metabolic characteristics of indication lipid content, the speed of growth and feed intake, this method comprises:

A) obtain genome DNA sample,

B) mirror digests this sample with TaqI, obtains fragment,

C) fragment that separating digesting obtained and

D) whether 678 of the bases of identification of M C4R gene PCR product exist the TaqI site.

21. also comprising, method according to claim 20, this method select to have the step that required genotypic animal is used for breeding.

22. method according to claim 20, wherein when using cutting the Restriction Enzyme of identical recognition site to be arranged with TaqI, when base 678 has guanine, the site can be identified by the fragment of 466,225 and 76 base pairs, when having VITAMIN B4, the site can be identified by the fragment of 542 and 225 base pairs.

23. method according to claim 20, wherein authentication step comprises by amplification and detects the TaqI site.

24. a test kit of estimating the animal DNA sample, this test kit are included in the reagent of polymorphism in the interior identification of M C4R gene of container.

25. test kit according to claim 24, wherein reagent is amplification MC4R gene or its segmental primer.

26. test kit according to claim 24, it also comprises archaeal dna polymerase, forward primer and the reverse primer of cutting MC4R gene, wherein the primer MC4R gene region that can increase and contain pleomorphism site.

27. a primer that is used for measuring the existence in polymorphism TaqI site in the MC4R gene, wherein primer comprises the sequence that is selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ IDNO:11.

28. a selection has the method than the animal of the required feature of low-fat content, faster growing speed or low feed intake, this method comprises the following steps

A) nucleic acid samples of acquisition animal,

B) identification mark be MC4R gene PCR product 678 Nucleotide polymorphism and

C) select 678 animals with Nucleotide relevant with required feature.

29. a method of selecting polymorphism among the MC4R is indirectly wherein selected indirectly with the specific alleles of other dna marker, wherein other dna marker is the linked marker near MC4R.

30. method according to claim 29, wherein chain mark is selected from S0331, BHT0433 and S0313.

31. a method of identifying animal, this animal have the required genotype of the metabolic characteristics of indication lipid content, the speed of growth and feed intake, this method comprises

A) by obtaining the sample of interested animal strain or kind, determine the cognation between MC4R genotype and the feature of interest,

B) genomic dna of every kind of animal in the preparation sample,

C) measure the MC4R gene genotype and

D) cognation between calculating MC4R genotype and this feature.

32. a method of selecting animal, this animal have the required MC4R genotype of the metabolic characteristics of indication lipid content, the speed of growth and feed intake, this method comprises

A) nucleic acid samples of acquisition animal,

B) identify animal MC4R gene genotype and

C) select and have genotypic those animals relevant with required feature.

Images