CN1260369C - Retinol binding protein 4 as genetic marker for increased litter size - Google Patents

Abstract

The present invention discloses a genetic marker for good animal reproduction performance (such as each litter whelp number and weaning body weight), a method for identifying the genetic marker, an animal screening method for determining animals which are possibly to produce good reproduction performance and a method for preferentially selecting the animals for future reproduction. The genetic marker is based on whether retinol binding protein 4 of pig reproduction gene has polymorphism.

Description

Retinol conjugated protein 4 is as the genetic marker of litter size

Invention field

The present invention relates generally to the hereditary difference that detects the animal reproductive efficiency, the genetic marker that more particularly the present invention relates to identify in several genes, show the genetic phenotype relevant with high reproductive performance.The invention also discloses and utilize these marks to carry out the method and composition that animal gene is identified and selected.

Background of invention

Reproductive efficiency, relevant with litter size specifically reproductive efficiency is the major limitation sexual factor of high efficiency production pork and other family's livestock product of great majority.There is hereditary difference in measurement demonstration to several reproductive performance indexs.4 to 16 of the average litter sizes of farrowing sow, the average developmental age 3, it was possible that the heredity of this heredity difference prompting fecundity in the pig variety improves to 7 months.The piglet alive of U.S.'s birth is counted about 9.5 in the every nest of average out to.Litter size purpose hereditary potency low (10%-15%), and it is no longer valid to select the standard genetic method of feeding mother pig according to a farrowing number in past.So the method that needs a kind of cell levels or dna level is handled the selection problem of reproductive performance.

Known Chinese Pigs kind reaches the morning at age of growth, and the litter size order is many.Know U.S.'s pig variety fast growth again, lean meat is many.Therefore it is desirable to the good characteristic of this two boar is combined, thereby improve the efficient that U.S.'s pork is produced.These effort greatly get help because of having found the genetic marker relevant with high reproductive performance (increasing as every nest piglet number).

Several research groups have adopted rflp analysis to study the DNA of pig.Jung etc. (Theor.Appl.Genet.77:271-274 (1989)) disclose and have adopted the PFLP technology to show gene difference between the two product boars to prove that there is polymorphism in swine leukocyte antigen (SLA) I genoid in this two kind.People such as Hoganson have reported the polymorphism of main histocompatibility complex (MHC) gene of Chinese Pigs on (annual meeting summary in Westbound in the U.S. animal science association, March 26～28 nineteen ninety), and are proved with rflp analysis.People such as Jung (Animal Genetics, 26:79-91,1989) have reported the rflp analysis result of some boar SLAI genoids.These authors claim they result of study prompting pig SLA/MHC I genoid and output and reproductive performance between may be correlated with.They claim that also adopting SLAI class restriction fragment may improve from now on as genetic marker has potential use on the pig growth performance.

In addition, the United States Patent (USP) 5,550,024 of granting people such as Rothschild discloses the polymorphism with the pig female hormone receptor gene of the more heterogeneous pass of litter size.

Messer, people such as L (no.Suppl 2,1996 for " location and the investigation of the candidate gene of the young number of every nest in the French white pig " AnimalGenetics, vol.27) disclose the increase that retinol conjugated protein 4 may cause the young number of every nest.Messer, and people such as L (" retinol conjugated protein 4 (RBP4) gene linkage is positioned pig karyomit(e) 14 " Mammalian Genome, vol.7, no.5,1996, p396) primer in the pig RBP4 zone that contains pleomorphism site of being used to increase is disclosed.

Another hormone of the pig relevant with good reproductive performance is prolactin (PRL), and it is a kind of prepituitary gland peptide hormone, participates in many different endocrine activities, by reproductive success essential.U.S. Patent No. 5,935,784 have described and disclose the mark that adopts the polymorphic locus in the prolactin receptor gene to increase as litter size.

Genetic marker provided by the invention is the discovery according to the RBP4 gene pleiomorphism, and this polymorphism is relevant with the young number that goes out to live with raising reproductive performance such as litter size.This makes can do gene type assay to the reproductive gene of pig, and determines the mutual relationship of concrete gene and reproductive performance mark.Also will identify the individual boar and the sow of carrying the excellent genes type.

With regard to sow, this makes that the every nest piglet number that can hope the sow fertility is more than the mean number of this product boar, grows more early, and is more healthy.With regard to boar, can hope that its female offspring has this kind premium properties.Therefore, these marks selection tool that will become the breeding program can be given birth to the pig with good reproduction phenotype piglet with exploitation.

An object of the present invention is to provide a kind of screening method of determining which pig may give birth to more piglets.

Another object of the present invention provides the method for the genetic marker of a kind of evaluation pig reproductive performance (as litter size).

A further object of the invention provides the genetic marker of the litter size of pig.

A further object of the invention provides a kind of test kit of estimating unique genetic marker relevant with good reproductive performance in the pig DNA sample.

Other purpose of the present invention and advantage will be made part in the following description and set forth, and also can partly understand from describe, and maybe can understand by practice of the present invention.Objects and advantages of the present invention are by the method for pointing out in the appended claims and combination thereof and reach.

Summary of the invention

As this paper enumerate with broadly described, in order to realize and meet the purpose of invention, the invention provides a kind of method of screening pig and other animals, those may have good reproduction phenotype (manys' as every nest farrowing) animal to determine raising, or select eliminating to have the allelic pig of the bad phenotype of demonstration." every nest farrowing is more " used herein expression litter size is on colony's mean value.

Term used herein " reproductive performance " comprises any performance that can show that reproductive efficiency improves, and includes but not limited to testis size, seminal fluid volume, sperm concentration, sperm quality, sexual desire, breeding, litter size order, the young number that goes out to live, piglet birth weight, wean age, developmental age, weans to growing pitch time, farrowing internal time, ovulation rate, uterus capacity and embryo survival.

Term used herein " reproductive gene " means any gene that can influence good or bad reproductive performance behind the gene product expression of its coding.The example of this genoid includes but not limited to female hormone receptor gene, prolactin receptor gene and retinol conjugated protein 4 genes, and other gene of this paper disclosure and description.

Therefore, the invention provides a kind of screening method, determine which pig more may produce good reproductive performance (more as the litter size order) and/or the unlikely every nest of which pig is given birth to less piglet.This method may further comprise the steps: the genome DNA sample that 1) obtains pig or other animals; 2) genomic dna that obtains of analytical procedure 1 is to determine to exist that or that several allelotrope of retinol conjugated protein 4.In brief, obtain the also sample of analyzing gene material, to determine to exist the polymorphism that still lacks the gene relevant with required reproductive performance.

In a preferred embodiment, this polymorphism is the polymorphism of restriction fragment length.This test comprises: identify the reproductive gene in the isolating genetic material; Make this gene contact restriction enzyme produce the different restriction fragment of this mrna length; Separate the restriction enzyme digestion mode chart that these fragments form with methods such as electrophoresis or HPLC; Relatively whether the restriction fragment mode chart of animal reproductive gene generation has or does not have known required mark.If this mark positive of animal testing can be considered this animal is included in the breeding and propagation program.This animal can not rejected from used colony if this genotype mark of this animal testing is not the positive, otherwise then use.

In a most preferred embodiment, with the specific region of containing polymorphism in primer and the archaeal dna polymerase amplification gene, separate its gene fragment, then direct the separation or zone that order-checking is increased or the fragment that is produced with digestion with restriction enzyme and separation.By these segmental simple dyeing or in amplification the primer or the nucleoside triphosphate of applying marking manifest isolating fragment or RFLP pattern.

In another embodiment, the present invention includes the method for the genetic marker of a kind of evaluation reproductive performance (as the litter size in certain specific population).Raise the male and jenny of same kind or Hybrid or similar genetic strain, and measure offspring's number that every jenny produces.Identify the polymorphism of every animal reproductive gene, and it is associated with required reproductive performance.Preferred employing PCR-RFLP analyzes and measures polymorphism.

Linkage relationship between the allelotrope of the dna marker thing that the unique allelotrope that also can set up other dna marker and known and certain specific gene (before proved relevant with certain specific trait, reproductive gene) as discussed herein are relevant.In this case, it should be possible obtaining specific reproductive gene, at least at short notice, every nest can be selected and more young babies' pig or other animal can be given birth to, perhaps select allelotrope indirectly, reject the pig that every nest may be given birth to less young baby with mark relevant with certain specific reproductive gene by unique allelotrope of selecting to have other chromosomal marker.For example, the mark of retinol conjugated protein 4 gene linkages comprises S0007 on known and the pig karyomit(e) 14, S0116 and SW210, and they all are microsatellite DNAs.

Whether the present invention also comprises a kind of test kit, be used for detecting the DNA sample and exist and be arranged in the required genetic marker that reproductive gene shows inheritability reproductive performance (more as litter size).Bottom line, this test kit are containers, and the reagent that one or more identify the RBP4 gene pleiomorphism is housed, and preferably, this reagent is a cover Oligonucleolide primers, its can increase fragment of the selected reproductive gene that contains polymorphism.Preferably, this test kit also contains a kind of restriction enzyme, and it can cut this reproductive gene at least one position, thereby is separated these fragments and detect polymorphic locus.

The present invention also comprises a kind of method of screening pig, give birth to less porkling in order to more possible every nest more porklings of fertility of definite which pig and/or the unlikely every nest of which pig, this method comprises the steps: to measure the allelotrope of retinol conjugated protein 4 genes that exist in the pig; Described allelotrope contains the MspI polymorphism; Measure the allelotrope of other mark in the known gene that can influence litter size; Select animal and reject those animals of carrying bad combination with allelotrope fine combination.The preferred allelic mensuration that wherein can influence the gene of litter size comprises: measure whether there is at least a allelotrope that is associated with the linked at least a dna marker of described retinol conjugated protein 4 genes.Preferred dna marker is a microsatellite marker.

The accompanying drawing that adds this paper has constituted the part of this specification sheets, is used to set forth example of the present invention, and is used from explanation principle of the present invention with specification sheets one.

Description of drawings

The expection fragment schema figure that Fig. 1 is produced for second kind of PCR scheme adopting embodiment 5 and primer.

Fig. 2 is the comparison diagram of the sequence of the human retinol-binding protein 4 delivered and new pig retinol-binding protein 4 sequences.

Detailed Description Of The Invention

With reference to preferred embodiment provided by the invention, these preferred embodiments and following examples illustrate principle of the present invention together in detail.

The present invention relates to the genetic marker of pig and the good reproductive performance of other animals (as litter size).A kind of method of screening animal is provided, is tested and appraised the middle existence of the reproductive gene relevant with some reproductive performance (being the RBP4 gene) or lacks certain polymorphism, those may more early give birth to the animal in determining to raise, and more healthy, every nest is farrowed more.

Therefore, the present invention relates to the pig or the genetic marker in other animals of concrete kind, strain, population, family and identify the method for these marks.Rely on this genetic marker, can determine in the pig of above-mentioned concrete kind, strain, population, family, the every nest fertility of possibility number is obviously more, more healthy, the sow of more precocious piglet.Identify that the method whether this mark exists all can use, comprise: for example, single strand conformation polymorphism (SSCP) is analyzed, rflp analysis, heteroduple analysis, denaturing gradient gel electrophoresis and temperature gradient electrophoresis, ligase chain reaction, or even directly measure the sequence of reproductive gene and check certain recognition mode.

Other technology that may adopt comprise non-gel systems, as TaqMan ^TM(Perkin Elmer).In this system, designed the PCR Oligonucleolide primers of this sudden change of side joint, and carried out this regional pcr amplification.Design the 3rd oligonucleotide probe then and contain the not area hybridization of variable choline base between the isoallele of this gene.With two kinds of fluorescence dyes at 5 ' and 3 ' these probes of two ends mark.When selected two kinds of fluorescence dyes should be adjacent to each other, the fluorescence of an end can not be detected by the institute's cancellation of the fluorescence of the other end.When the TagDNA polysaccharase when the PCR primer that is positioned at template 5 ' end begins to extend with respect to probe, 5 ' nuclease of this enzyme causes cutting off the fluorescence dye that is incorporated into annealing probe 5 ' end, eliminate the cancellation effect like this, thereby can detect the fluorescence that probe 3 ' end dyestuff sends.

If the hybridization of probe and template molecule is incomplete, the mispairing of certain form is promptly arranged, it is cut just can not dyestuff to take place, and so just can distinguish different dna sequence dnas.Therefore, have only nucleotide sequence and the complementation fully of its bonded template molecule when oligonucleotide probe, the cancellation effect just can be eliminated.Reaction mixture can contain two kinds of different probe sequences, respectively at the not isoallele that may exist, thereby can detect these two kinds of allelotrope in a reaction.

Using RLFP is the preferred method that detects polymorphism, and most preferably PCR-RFLP analyzes.Yet,, also can adopt the additive method that detects polymorphism because the application of rflp analysis finally depends on the polymorphism and the DNA restriction site of nucleic acid molecule.These methods comprise to be analyzed the polymorphism gene product and detects polymorphism by detecting the difference that produces in this gene product.

RFLP is a kind of current techique well-known to those skilled in the art.For example, see following United States Patent (USP): authorized the No.4 of Erlich on April 15th, 1986, authorized the No.4 of Gusella, 666 on May 19th, 582,788 and 1987,828, authorize the No.4 of Frossard on September 20th, 1988, authorized the No.4 of Frossard on August 29th, 772,549 and 1989,861,708.Put it briefly, this technology comprises: obtain DNA to be studied, digest this DNA with restriction endonuclease, separate fragment that produces and the fragment that detects range gene.

Among the present invention, the sample of genetic material is available from animal.Can obtain sample from blood, tissue, sperm etc.Usually adopt peripheral blood cells to originate, genetic material is DNA.The cell that obtains q.s is analyzed with the DNA that q.s is provided.Those skilled in the art will know that or be not difficult to determine aequum.From hemocyte, isolate DNA with technology well known by persons skilled in the art.

Next step contains the zone of polymorphism with the amplification of primer and standard technique (as polymerase chain reaction).This technology has description in following United States Patent (USP): authorized people's such as Mullis Nos.4 on July 28th, 1987,683,195 and 4,583, authorized people's such as Mullis No.4 on January 24th, 202,1989,800,159, authorized people's such as Gelfaud No.4 on December 26th, 1989,889,818 and authorize people's such as Clumbus No.4 February 20 nineteen ninety, 902,624.In the document that these are mentioned, selection of primers has been discussed.These primers should be able to increase and contain the zone of polymorphism.To openly increase several primers of specific polymorphic regions of this paper.Those skilled in the art can be in conjunction with other primer of technical project described herein, and these should comprise in the present invention.

The DNA of analytical separation then, and can be randomly with restriction endonuclease digestion, this enzyme can or cut off DNA in the specific nucleotide sequence place cutting that is called restriction site.This endonuclease is also referred to as Restriction Enzyme, is well known to those skilled in the art.For the present invention, should select and to produce at least two fragments of this gene at the Restriction Enzyme of at least one place of selected reproductive gene cutting.Whether with the method for technology known in the art in conjunction with this paper, measuring these fragments is whether being associated with needed reproductive performance (as litter size) with its polymorphism of polymorphism.Join the amount that contains this kind of enzyme in the pig DNA sample, and other suitable conditions of handling sample, those skilled in the art are not doubt according to the instruction of this paper.

Then, analyze restriction fragment with known technology.These technology generally include: fragment is separated, dye or remake trace and hybridization shows, obtaining specific mode chart, or measure segmental different size.The latter will be identified one or more fragments (mark) that litter size increases.Preferred isolation technique is gel electrophoresis.

In this technology, the digestion fragment in the Supporting Media is separated by size.Usually adopt gel film or plate (as agarose or agarose one allylamine) as Supporting Media, the sample that will contain restriction fragment is added to an end of gel.Race is electrophoretic on same clotting glue has one or more markers that vary in size in contrast, so that estimate the size of restriction fragment.The common distinguishable size of this method differs few isolated fragment to 100 base pairs each other.

In another embodiment, make the fragment sex change, it is transferred on the solid phase carrier (preferred nylon membrane) from gel by physical method.Method is under promoting the felicity condition that DNA shifts and when having suitable reagent, and gel is contacted with film.These reagent and condition are well known to those skilled in the art.The relative position that so, has just kept each dna fragmentation of this separation method generation.

Next step comprises the fragment that the detected magnitude scope is different, or detects certain fragment of specific size.The latter may be meaningful especially, because a kind of genetic marker that this fragment is associated with required reproductive performance.Preferably with ethidium bromide etc. with fragment dyeing, detect then.

Another technology is to adopt hybridization probe.This probe is a kind of oligonucleotide or polynucleotide, and itself and abundant complementation of fragment or homology to be hybridized form probe one fragment mixture.The preferred cDNA probe of probe.But oligonucleotide or polynucleotide are with the detection molecules mark, thereby are detected the restriction fragment with this probe hybridization.The marking method of available standards is as using label probes such as radio-labeling, enzyme labelling, fluorescent mark, vitamin H one avidin mark.See United States Patent (USP): authorized people's such as Ward No.4 on December 8th, 1987, authorized people's such as Stavrianopoulos No.4 on September 19th, 711,955 and 1989,868,103.

Under the hybridization conditions that is fit to the hybridization of probe and fragment, make probe contact the sufficiently long time with the nylon membrane that contains restriction fragment.The washing nylon membrane is removed unconjugated probe and other materials of not wanting.

Detect and nylon membrane bonded probe one fragment mixture with known technology then.For example, if probe with radio-labeling ( ³²P), detect and to comprise nylon membrane is contacted with a radiosensitivity film.After the appropriate time exposure, demonstrate interested fragment and contrast fragment.

This detects step provides a fragment to separate by size and the mode chart that produces.These fragments are compared with the contrast fragment of running electrophoretic known dimensions on same gel, just can estimate and respectively organize segmental size.By to the similar analysis from the DNA of different pigs, relatively the mode chart of Chan Shenging is determined the various polymorphisms in the reproductive gene then.For some individual animals, its mode chart will be different from the normal mode figure that other animals of great majority are produced.This is because due to the length polymorphism of one or more restriction fragments, promptly since endonuclease cutting reproductive gene produce due to the different restriction fragment of length.This has reflected in this boar to have different base-pair sequences.

In case identify specific RFLP, i.e. the restriction fragment of length-specific, available currently known methods makes up this segmental probe.This be able to other faster mode detect this polymorphism.For example, the DNA of digestion can detect with sandwich hybridization.This method such as following United States Patent (USP) are disclosed: authorized people's such as RanKi No.4 on December 4th, 1984, authorized people's such as RanKi No.4 on January 7th, 486,539 and 1986,563,419.The probe of capturing enemy personnel on making sample and being fixed on solid phase carrier contacts, and probe combines with this fragment, wash vehicle, the detection probes of adding mark.After the washing, measure detection probes, thereby prove required segmental existence once more.

In another embodiment,, it is knownly compared with RFLP mode chart or fragment that the litter size increase is associated with second kind in case determined RFLP pattern or specific polymorphic fragment.Also can under similarity condition, adopt and for the first time identical restriction endonuclease and identical probe or its Equivalent second kind of pattern determining that reproductive gene produces or fragment.

In another embodiment of the present invention, can come the detection limit fragment by solution hybridization.In this method, fragment elder generation and probe hybridization separate again, then detection probes one fragment mixture as mentioned above.Usually on the gel and need not transfer on the filter paper and to detect this mixture.

In an optimum implementation, need not this polymorphism of any probe in detecting with pcr amplification, those skilled in the art will know that this method, and can be referring to U.S. Patent No. 4,795,699, be entitled as " archaeal dna polymerase " and U.S. Patent No. 4,965,188, be entitled as " with the method for a kind of thermostable enzyme amplification, detection and/or cloning nucleic acid sequences ".

This method need make up the primer of energy amplification polymorphism region.So should design the primer of 4-30 base according to the sequence around this polymorphism, comprise the forward primer of this polymorphic regions 5 ' and 3 ' antisense (oppositely) primer.These primers need not be accurately complementary, and essentially identical sequence also can be accepted.When having 4 kinds of nucleoside triphosphates and often buffer reagent being arranged, add archaeal dna polymerase then, as Taq polysaccharase (polysaccharase so a lot of be known be not that commercially available product is arranged).Use such as the ethidium bromide separated product that simply dyes to help detecting, to measure the fragment that whether has the size that institute's amplification region length estimates.Reaction times, reagent and design of primers all those skilled in the art will know that in the document that this paper quotes description is arranged.Pcr amplification can with single strand conformation polymorphism (SSCP) combined utilization.See people such as Orita " polymorphism of usefulness detected through gel electrophoresis people DNA: single strand conformation polymorphism ", people such as PNAS 86 (8) Apr.1989 (2766-70) and Lessa " triage techniques that allelotrope changes in the detection dna sequence dna " Mol Ecol2 (2) 119-29 Apr.1993.

Though method described herein has adopted an a kind of Restriction Enzyme and a cover primer, this method is not limited thereto.If desired, can adopt one or more other Restriction Enzymes and/or probe and/or primer.Can pass through normal experiment,, determine other enzyme, structure probe and primer in conjunction with technology provided herein.

With reproductive gene, particularly the genetic marker of RBP4 gene or other gene linkages can followingly be measured.Make the similar male and jenny mating of same kind, Hybrid or genetic strain, measure offspring's number, measure every nest offspring number of every jenny fertility with good reproductive performance.Carry out the rflp analysis of parental DNA as mentioned above, to determine the polymorphism in every selected reproductive gene of animal.This polymorphism is associated with reproductive performance.Carry out thisly adopting 20 at least when definite, be preferably at least 40 jennies.Every jenny gave birth to a tire at least.Preferable breed and the farrowing cycle repeats secondary at least, best 3 times.

When carrying out this analysis and analyzing to determine polymorphism with RFLP or other, the known array of available people of the kind or other closely related animals comes design of amplification primers.Many reproductive gene sequences have homology.The known sequences Design primer that also available gene pool (Genbank) exemplifies, or even design primer from the sequence that closely obtains around the chain data of these genes.The present invention has selected some cover primers and has identified polymorphic regions in the reproductive gene.Shown that these polymorphic bandses are allelotrope fragments.Each fragment demonstration is associated with the good reproductive performance (increasing as litter size) of various kinds.The genotype that is associated with this performance is usually different in other different varietiess.This result is similar to U.S. Patent No. 5,374, situation described in 523 " allelotrope of bovine somatotropin gene changes, the genetic marker of super milking cow ".This patented invention person finds that a kind of polymorphic allele is the somatotropin gene, and a kind of allelic form is useful to the Jersey cow, and another kind of form is useful to the Holstein cow.

The suitable agent of implementing the inventive method can be packaged in the conventional test kit.Test kit provides the necessary material that is packaged in the appropriate containers.Test kit contains the reagent that can identify reproductive gene polymorphism selected, that be associated with certain reproductive performance (increasing as litter size) at least.This reagent is preferably complete PCR reagent (a cover primer, archaeal dna polymerase and 4 kinds of nucleoside triphosphates), can hybridize with reproductive gene or its fragment.Test kit should comprise PCR series and cut the reproductive gene restriction enzyme at least one site.Test kit also should comprise other materials, but as the reagent of detection or mensuration detection molecules or reference substance.If need, also can comprise be used to hybridize, other reagent of prehybridization, DNA extraction, demonstration etc.

Material of the present invention and method can be used to estimate animal DNA more generally, measure the genotype of individual animals and the gene difference in the detection animal.Can estimate with reference to one or more contrasts specifically ,-genome DNA sample to be to determine whether to exist the polymorphism of reproductive gene.Should carry out rflp analysis and compared with the control to reproductive gene with the result.Contrast is the result of the reproductive gene rflp analysis of the known different animals of reproductive gene polymorphism.Similarly,, carry out the rflp analysis of reproductive gene among this DNA, with the result compared with the control, can determine this animal reproduction genotype by obtaining the genomic dna of animal.Also have, contrast is the result of different animals reproductive gene rflp analysis.This result determines the genotype of this pig by the polymorphism of the selected reproductive gene of definite pig.Finally, by obtaining at least two animal gene group DNA samples, whether the existence of evaluation reproductive gene polymorphism, and comparative result, can detect the hereditary difference between the animal.Fig. 2 is the comparison diagram of human retinol-binding protein 4 sequences of publishing and new pig retinol-binding protein 4 sequences.

As mentioned above, these tests can be used for identifying the genetic marker relevant with litter size, other polymorphisms of the reproductive gene that evaluation may be relevant with other characteristics, and to genotype and the overall scientific analysis of phenotype do.

Genetic marker of the present invention, method and test kit also can be used for improving animal varieties, strain and population litter size breeding in the works.For with the relevant polymorphism of good reproductive performance (increasing) as litter size, constantly select and breed the animal of heterozygosis (being preferably the animal of isozygotying) at least, will produce the more kind of dam litter size, strain and population.Like this, these marks have just become selection tool.

The embodiment of this paper and method disclose certain reproductive gene that has polymorphism through evaluation, and this polymorphism is positive or be associated with certain the good reproductive performance (litter size) that influences the animal reproductive efficiency negatively.Polymorphism in the gene (RBP4) that identifies usually is that the single base that causes producing restriction site in certain allelotrope changes.Yet as this paper proof with discussing, a certain allelotrope can have many sequence changes that are associated with it.Can test those bases and show identical polymorphism, also other genetic markers or gene can be connected with polymorphism as herein described, test can comprise other genes of evaluation or gene fragment like this, but test finally depends on the hereditary feature of identical polymorphism animal.Any test according to selection of allelotrope difference and evaluation animal all comprises within the scope of the invention.Also proof is relevant with certain specified property in case identify polymorphism, and each technician of this area will be understood that this polymorphism genotype that has or not counting method to determine animal.As this paper fully as described in.Parameter well known by persons skilled in the art is just optimized in the design of the test of these changes, should be included in the scope of the present invention.

The present invention has identified the polymorphism in the RBP4 gene that is associated with the litter size increase.Blastocyst has been expressed retinol conjugated protein 4 (RBP4) in the extension process, infer its transhipment and regulated the Vogan-Neu amount that the embryo accepted.The present invention has identified a kind of polymorphism and RBP4 has been drawn gene map.SacI digestion product and RBP4 hybridization have shown that has two allelic polymorphisms (fragment), and its linkage analysis discovery is obviously chain with the several locus on the pig karyomit(e) 14.Further scrutiny comprises with primer tests the polymorphism of measuring MSPI as secondary PCR, and test shows that the difference between the homozygous genotype is 1.05 pigs of every nest farrowing.

Should be understood that according to content described herein instruction of the present invention is implemented on particular problem or environment, is the thing in those of ordinary skills' limit of power.Product of the present invention and method example shown in the following example are not to mean scope of the present invention and content are limited.

Embodiment 1

The linkage map of retinol conjugated protein 4 (RBP4) gene and pig karyomit(e) 14

Shown position: the loci sequence of karyomit(e) (Chr) 14 distal portions :-ACTN2-1.7-ACTAl-2.7-PLAU-0-SW210-8.2-S0169-9.9-S0072-11. 1-S0007-7.3-RBP4-16.2-S0116-20.0-Sw761-36.1-S0015.

Drafting method: the three generations PiGMaP family of six Meishan x Large Whites and European wild boar x Large White, Archibald etc., 1995.Mamm.Genome 6:157-175

Molecular agents: pig RBP4 gene probe adopts the 311-bp fragment according to the primer amplification pig blastocyst on the 12nd of pig cDNA sequences Design to obtain.5 ' primer (5 '-TTCCGAGTCAAAGAGAACTTCG-3 ', SEQ ID NO:1) is represented Nucleotide 79-100.3 ' primer (5 '-TCATAGTCCGTGTCGATGATCC-3 ' SEQ IDNO:2) is represented Nucleotide 368-389.People such as TroutW, 1991, Mol.Endocrinol.5:1533-1540.The purifying amplified production, with random priming with ³²The P radio-labeled.、

Allelotrope detects: detected SacI polymorphism in the pig genomic dna by making the Southern blot hybridization with the 011bp pig RBP4 fragment of mark.The rigorous degree of final washing is decided to be 65 ℃, 0.7xSSC and 0.2%SDS.In two polymorphic bandses of 12.1kb that is detected and 7.8kb, found Mendelian inheritance (rule).Measured the RBP4 genotype of 62 affinity-less relation pigs of 8 kinds.12.1kb the frequency of section Landrace pig (n=10) is 0.55, (n=10) is 0.75 the Duroc pig.Yorkshire pig (n=5) is 1.0, and the white pig of Chester (n=4) is 0.5, and Large White (n=11) is 0.59, and Hampshire pig (n=5) is 1.0, and Meishan pig (n=15) is 0.80, and wild boar (n=2) is 1.0.

The previous homology of identifying: people RBP4 is positioned 10q23-24 (people 1989 such as Rocchi.M, Somat.Cell Mol.Genet., 15:185-190) and mouse Rbp-4 be positioned at far-end (Chainani, the people such as M. of karyomit(e) 19, Genomics.1991,9:376-379).

Discuss: retinol conjugated protein is a kind of main secretory product before the pig conceptus is implanted.The rapid morphology that extends at the pig blastocyst between the growth period (this is the critical period of embryo survival) RBP4 produces increases, pointing out RBP4 may be a kind of interesting candidate gene that pig reproduction quantitative trait locus (QTL) is studied.

Carried out linkage analysis with 2.4 editions software packages of CRIMAP.2 linkage analysises have obtained locus S0007 on RBP4 and the pig karyomit(e) 14, the significance Lod scoring (＞3.0) (table 1) of S0116 and SW210.On our collection of illustrative plates on the order of this locus and KapKe and colleague's thereof the karyomit(e) 14 the new locus of PiGMaP figure arrange consistent people 1995.Anim Genet. such as (, wait to deliver) KapKe.P, exception is the rearrangement of locus S0007 and S0072.RBP4 is added to the length that has further increased this sex state diagram among the PiGMaP figure that revises again, from 193cM to 202cM.RBP4 on the displacement karyomit(e) 14 has strengthened the homology between pig karyomit(e) 14 and the human chromosome 10.(people 1995 such as Johansson, Genomics, 25:682-690).

The result of 2 linkage analysises of table 1:RBP4

Mark 1	Mark 2	Recombinant fragment	The Lod scoring
				RBP4 RBP4 RBP4	S0007 S0116 SW210	0.07 0.21 0.27	17.02 4.63 3.50

Embodiment 2

The dependency of polymorphism and litter size

On the Southern film of the DNA of family that contains digestion, carried out RFLP mensuration with the Partial cDNA fragment of RBP4 gene with reference to product.Survey the SacI film with RBP4, shown 12.1kb and two allelotrope fragments of 7.8kb.The RBP4 figure of linkage analysis karyomit(e) 14.For the effect of this gene is described, measured the genotype of two kinds of these locus of Fronch Large White.Last strain is made up of the high delivery and feed infant of France (LMH) pig (216 piglet records of 32 sow fertilities), and back one strain is made up of France contrast (LW) pig (242 piglet records of 27 sow fertilities).Assessed the genotypic average interpolation effect of this gene pairs with the linear regression of litter size.RBP4 is 0.52 ± 0.30 in the LMH pig to the allelic interpolation genetic effect of 7.8kb, is 0.45 ± 0.43 in the LW pig.Allelotrope is 5～17% of phenotype STD to the substitution effect scope of litter size

Embodiment 3 (RBP4)

The PCR-RFLP test of MSDI polymorphism

According to the evaluation of polymorphism in the RBP4 gene, developed a kind of PCR test

Primer

LM24195’-GAGCAAGATGGAATGGGTT-3’SEQ ID NO：4

LM24205’-CTCGGTGTCTGTAAAGGTG-3’SEQ ID NO：5

The PCR condition 25 μ L reactants

No. 1 mixed solution

10X Promega damping fluid 2.5 μ L

25mM MgCl ₂ 1.5μL

10mM dNTP’s 0.5μL

16pMol LM2419(100ng/μL) 0.5μL

16pMol LM2420(100ng/μL) 0.5μL

Two steaming sterilized water 12.5 μ L

25ng genomic dna (12.5ng/ μ L) 2.0 μ L

The Taq mixed solution

Two steaming sterilized water 4.9 μ L

0.6U Taq polysaccharase (Promega) 0.12 μ L

In reaction tubes, mix No. 1 mixed solution of 18 μ l and DNA.Mineral oil above superposes.85 ℃ of preheatings on the heating cycle instrument.Add 5 μ l Tag mixed solutions, move following PCR program:

93 ℃ of steps 13 minutes

93 ℃ of steps 2 30 seconds

56 ℃ of steps 3 45 seconds

72 ℃ of steps 4 45 seconds

Step 5 is got back to step 2,39 take turns above

72 ℃ of steps 65 minutes

4 ℃ of maintenances of step 7

On 1% sepharose of standard, verify 5 μ l PCR reactants, to confirm to increase successfully and negative control clearly.About 550 base pairs of product size can directly digest in the PCR pipe.

The MSPI digestion reaction 30 μ L reactants

Residue PCR product 20.0 μ L

10X NE damping fluid 2 3.0 μ L

10U MSP I enzyme (20U/ μ L) 0.1 μ L

Two steaming sterilized water 6.9 μ L

The mixture of preparation damping fluid, enzyme and water is got 10 μ l adding and is contained in each reaction tubes of DNA.37 ℃ are incubated overnight.The application of sample dye liquor is directly added in the digestion reaction thing, with whole volume application of sample on the 3%NuSieve gel.AA genotype master band is 190bp, 154bp and 136bp.BB genotype band is 154bp, 136bp and 125bp.

Analyze French pig strain and show, MSPI allelotrope A=(old) the SacI allelotrope 2 of this new PCR test.

Adopt PCR-RFLP to detect the high yield pig strain and the check clone of Nebraska university, the result is as follows:

	A	B
			The index contrast	.50 .47	.50 .53

Therefore, the frequency of A is higher in this index.

With the MSPI analysis of experiments 19 animals (having suitable record) of PIC strain the results are shown in Table 2 and table 3.

Table 2

AVE NB	Genotype N	Average	Standard deviation
				AA AB BB	93 98 18	10.178 9.292 9.722	0.237 0.275 0.511

Table 3

EBV TNB	Genotype N	Average	Standard deviation
				AA AB BB	93 98 18	0.160 0.034 0.108	0.062 0.049 0.112

Data show below by the litter size grouping

	AA	AB	BB	A	B
						The many every nest farrowing of every nest farrowing are few	.62 .24	.36 .64	.04 .12	.78 .56	.22 .44

Embodiment 4

Developed second kind of RCR test, as follows.

PIC RBP4 testing program

RBP4-FOR2 5’ACT GTG CTC TTT GTG CTG 3’SEQ ID NO：6

RBP4-REV 5’CTC GGT GTC TGT AAA GGT G 3’SEQ ID NO：7

Method

10x PCT damping fluid 1.2 μ l

2Mm dNTP’s 1.2μl

25mM Mg ²⁺ 1.2μl

RBP4-FOR 2(5μM) 1.2μl

RBP4-REV(5μM) 1.2μl

Amplitag Gold 0.12μl

QH ₂O 4.88μl

Cut substrate 1.0 μ l

12.0μl

PE9700

94 ℃ 30 seconds

94 ℃ 12 minutes 58 ℃ 30 seconds x30 → 72 ℃ 4 minutes → 6 ℃

72 ℃ 30 seconds

(9600Ramp)

Digestion: 37 ℃ 3 hours

PCR product 12.01

MSP1(10μ/l) 0.5μl

QH ₂O 1.0μl

14.0μl

Behind the application of sample, on the 5%NuMe agarose, assign to 1 hour in 150V electrophoresis about 45.Fig. 1 shows the stripe size of expectation.

Embodiment 5

The Europe result

To 264 Landrace sows that European PIC breeding farm has litter size to write down, measured the RBP4 genotype with embodiment 4 schemes.Identify two allelotrope 1 and 2.Marker genotypes distributes and follows Hardy Weinerg equilibrium principle in all strains.

Analyze two kinds of performances----and given birth to total young number (TNB) and fertility young number (NBA) alive

Adopt with the mixed mode of male animal and analyzed data (h2=0.09) as stochastic effect

The fixed effect comprises: the year that farrows, the moon, and the sow strain, the boar strain is planted pig farm (3A1 boar) cycle 1,2,3 ⁺Boar and insemination of sows (1,2,3 ⁺).All breedings all are A1 boar and insemination of sows.

RBP4 influence between all parities and the sow strain

Genotype 11 12 22	Nest several 212 223 87	TNB 10.84 10.28 9.93	NBA 9.93 9.66 9.36
				a d *P＜0.1		+.46 * -.11	+0.29 +0.02

Surpass the RBP4 results suggest of 7509 nest piglets, the difference between the homozygote genotype is 1.05 pigs of litter size.Expection fragment schema when Fig. 1 has shown with above-mentioned second kind of PCR scheme and primer.

Embodiment 6

There are 154 Dam strain sows (forming) of litter size record to carry out the RBP4 genotype detection to U.S. breeding farm by Landrace and Large White/Duroc synthetic line.

Genotype 11 12 22	Nest several 297 350 103	TNB 10.74 10.04 9.66
			P＜0.02

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Claims (17)

1. one kind is screened pig to determine the more method of possibility litter size increase of which pig, it is characterized in that, this method comprises: whether be determined in the genetic stocks sample in retinol conjugated protein 4 genes existence of polymorphism, described polymorphism is associated with the litter size increase

Whether wherein measure described polymorphism exists and comprises step:

A certain amount of reproductive gene or its fragment that contains described polymorphism increases;

Be used in the MspI Restriction Enzyme that position, at least one place cuts off described retinol conjugated protein 4 genes and digest described genetic stocks;

The fragment that separating digesting produced;

Measure the restricted mode chart that fragment that described digestion produces produces;

This mode chart and another restricted mode chart of the reproductive gene that obtains with described Restriction Enzyme are made comparisons, and wherein said another mode chart is associated with the litter size increase.

2. method according to claim 1, wherein, the step of described mensuration restriction sexual norm figure is selected from: direct sequencing analysis, restriction fragment length polymorphism is analyzed, heteroduplex is analyzed, single-strand conformation polymorphism analysis, denaturing gradient gel electrophoresis and temperature gradient gel elec-trophoresis (TGGE).

3. method according to claim 1, wherein, described separation is gel electrophoresis.

4. method according to claim 1, wherein, the step of more described restricted mode chart comprises fragment and the more described segmental size of identifying specific size.

5. method according to claim 4, wherein, the step of measuring described clip size comprises:

When having the contrast dna fragmentation of known dimensions, separate described fragment by size with gel electrophoresis;

Described isolating fragment is contacted with a probe, and this probe can form probe-fragment mixture with described fragment hybridization;

Whether existence by detection probes-fragment mixture comes to determine isolating segmental size;

Determine the relative position of they and described contrast dna fragmentation.

6. method according to claim 1 wherein, is carried out described amplification with the Taq polysaccharase.

7. method according to claim 1, wherein, described amplification comprises the step of selecting forward and reverse primer sequence, the described reproductive gene zone that these primers can increase and contain pleomorphism site.

8. method according to claim 7, wherein, described primer is SEQ ID NO:4 and SEQ IDNO:5.

9. method according to claim 1, wherein, described polymorphism is the restricted length polymorphism of 190bp.

10. method according to claim 1, wherein, described polymorphism is the restricted length polymorphism of 125bp.

11. method according to claim 7, wherein, described primer is SEQ ID NO:6 and SEQID NO:7.

12. measure the primer whether pleomorphism site that is associated with the litter size increase in pig retinol-binding protein 4 genes exists for one kind, wherein, described primer is selected from down group: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

13. genetic marker that is associated with the increase of pig litter size, wherein, described mark comprises retinol conjugated protein 4 genes, and described mark can be identified by the MSP I polymorphism in the described gene region that amplifies with SEQ ID NO:6 and SEQ ID NO:7.

14. genetic marker that is associated with the increase of pig litter size, wherein, described mark comprises retinol conjugated protein 4 genes, and described mark can be identified by the MSP I polymorphism in the described gene region that amplifies with SEQ ID NO:4 and SEQ ID NO:5.

15. method of screening pig, give birth to less porkling in order to determine more possible every nest more porklings of fertility of which pig and/or the unlikely every nest of which pig, it is characterized in that this method comprises the steps: to measure the allelotrope of retinol conjugated protein 4 genes that exist in the pig; Described allelotrope contains the MspI polymorphism; Measure the allelotrope of other mark in the known gene that can influence litter size; Select pig and reject those pigs of carrying bad combination with allelotrope fine combination.

16. method according to claim 15, wherein, the allelic mensuration that can influence the gene of litter size comprises: measure whether there is at least a allelotrope that is associated with the linked at least a dna marker of described retinol conjugated protein 4 genes.

17. method according to claim 16, wherein, dna marker is a microsatellite marker.