CN103320429A

CN103320429A - Method for detecting Qinchuan cattle Wnt7a gene single nucleotide polymorphism, and application thereof

Info

Publication number: CN103320429A
Application number: CN201310185665XA
Authority: CN
Inventors: 陈宏�; 薛璟; 孙加节; 蓝贤勇; 雷初朝
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2013-05-20
Filing date: 2013-05-20
Publication date: 2013-09-25

Abstract

The invention discloses a method for detecting Qinchuan cattle Wnt7a gene single nucleotide polymorphism (SNP). According to the invention, genome DNA of a Chinese local cattle breed Qinchuan cattle is adopted as a template; Wnt7a gene sequence is subjected to PCR amplification by using Taq DNA polymerase, Buffer (a buffer solution), Mg<2+>, dNTPs and primers M; a PCR product is subjected to enzyme digestion by using restriction endonuclease Alw44I; a fragment is subjected to separation and detection through agarose gel electrophoresis, and Qinchuan cattle Wnt7a gene single nucleotide polymorphism is finally determined. The Wnt7a gene function is related to growth traits such as weight and body length, such that the detection method provided by the invention establishes a basis for establishing a relationship between the SNP of the Wnt7a gene and growth traits. Therefore, marker-assisted selection of meat-purpose growth traits of Chinese Qinchuan cattle is facilitated, and a Qinchuan cattle population with excellent genetic resource can be rapidly established.

Description

A kind of method and application that detects Qinchuan Cattle Wnt7a gene mononucleotide polymorphism

Technical field

The invention belongs to the molecular genetics field, relate to the detection of gene mononucleotide polymorphism (SNP), particularly a kind of method that detects the 63777th single nucleotide polymorphism of Qinchuan Cattle Wnt7a gene.

Background technology

Gene pleiomorphism refers to that polymorphism (polymorphism) refers to be in the colony of random mating, and can there be the genotype more than 2 kinds in the same gene site.Biotic population gene pleiomorphism phenomenon is very general, wherein, the structure of Human genome, expression and function, research and comparison is deep.The Human genome polymorphism had both derived from the difference of tumor-necrosis factor glycoproteins copy number in the genome, also derived from the variation of single-copy sequence, and diallelic conversion or replacement.By causing the priority of paying close attention to and studying, usually be divided into 3 large classes: dna fragmentation length polymorphism, repetitive dna sequence polymorphism, single nucleotide polymorphism.

Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) refer to change on a certain specific nucleotide position in the genome, the variation such as transversion, insertion or disappearance, and the frequency of any allelotrope in colony is not less than 1%.

Can be divided into two large classes according to the form of SNP in genome, the one, be dispersed throughout genomic a large amount of single nucleotide variation; The 2nd, be distributed in gene coding region (coding region), be called cSNP, function of dominant sudden change.SNP is inhomogeneous at individual gene or whole genomic distribution, and the non-transcribed sequence will be more than transcription sequence, and is in the frequency of transcriptional domain nonsynonymous mutation, more much lower than the frequency that other modes are suddenlyd change.

SNP mainly contains following several large characteristics:

(1) density is high: SNP the mean density of human genome be estimated as 1 1000bp, reach 3 * 10 in whole genomic distribution ⁶Individual, genetic distance is 2～3cM, and the density ratio microsatellite marker is higher, can the inside of any one gene to be studied or near a series of marks are provided.

(2) typical: some SNP that is positioned at gene inside might directly affect protein structure or expression level.Therefore, they may represent some influencing factor in the disease genetic mechanism.

(3) genetic stability: compare with polymorphic repetitive sequence marks such as little satellites, SNP has higher genetic stability.

(4) the easily automatization of Realization analysis: SNP is marked at only has two kinds of allelotypes (allele) among the crowd.Only need like this mode of "+-" or " complete without " when detecting, and need not resemble the detection limit fragment length polymorphism, little satellite is made measurement to the length of fragment like that, and this is so that easily realize automatization based on the determination method of SNP.

SNP mainly contains two large class detection methods.The first kind is take the detection method of gel electrophoresis as the traditional classical on basis, such as restriction fragment length polymorphism method PCR-RFLP; Single strand conformation polymorphism PCR-SSCP; Denaturing gradient gel electrophoresis (denaturing gradient gel eletrophoresis DGGE); Allele-specific PCR (allele specific PCR, ASPCR) etc.Second largest class is the method for the higher detection SNPs of high-throughput, level of automation, and comparatively commonly used has: the dna sequencing method; The DNA chip detection; Flight mass spectrometer (MALDI-TOFMS) detects; Sex change high performance liquid chromatography (DH PLC) method etc.

Molecular breeding, be molecular marker assisted selection breeding (Molecular Mark-Assist Selection, MAS), this technology is by dna molecular marker genetic resources or breeding material to be selected, Comprehensive Traits to livestock and poultry carries out breed improvement, it is the method for utilizing modern molecular biology and traditional genetic breeding to combine, carries out breeding of new variety.In Genetic Improvement of Beef Cattle, people expectation, by closely related to growth traits, and with the selection of the closely linked dna marker of quantitative character, reach early stage seed selection and improve the purpose of breeding value accuracy, thereby in Animal Breeding, obtain larger genetic progress.

The molecular genetic marker assisted Selection combines modern biotechnology exactly with conventional system of selection, by the selection of genetic marker being selected indirectly the quantitative trait locus (QTL) of certain proterties of control, enable to utilize simultaneously the phenotype information of marker site information and quantitative character, more accurately estimate the breeding value of animal individual, improve efficiency of selection, accelerate Advances in Breeding.Marker assisted selection has mainly experienced three phases: the fs is the genetic analysis between each proterties of domestic animal; Subordinate phase is that protein (enzyme) mark is to the marking phase of quantitative character; Three phases is the molecular genetic marker stage.Along with molecular marking technique is day by day ripe and abundant, make to cover whole genomic mark and become possibility, by and QTL between linkage analysis, realize the target of molecular marker assisted selection.Single nucleotide polymorphism (single nucleotide polymorphism, SNP) has been widely used in the assignment of genes gene mapping, clone, genetic breeding and multifarious research as new genetic marker.

Wnt7a is an important member of Wnt gene family, and it is positioned on the Human Chromosome 3 karyomit(e).Encode a kind of signal protein of secretor type of Wnt7a.Wnt7a can bring into play its regulating effect in many biological processess.In adult animals, Wnt7a can directly affect the prenatal growth of female repro ductive system and keep normal uterine function.Also it acts on particularly aspect the nervous disorder Wnt7a in nervous system development.Wnt7a has also played vital role in cancer.Aspect the flesh generation, Wnt7a brings into play the rise effect and can stimulate the symmetry division of muscle satellite cell in anathreptic process.Cross expressing of Wnt7a can strengthen muscle regeneration and increase ratio and the quantity of muscle satellite cell., can obviously reduce muscle the quantity of muscle satellite cell if lacking Wnt7a.This effect of Wnt7a is then regulated by regulating planar cell polarity (PCP) approach.

Recently the research of Wnt7a mainly concentrated on its function and the upstream and downstream regulatory factor.Aspect Research on Genetic Variation, some polymorphisms of Wnt7a can affect the function of its gene.Isozygotying on the Wnt7a (c.664C〉T) can cause Deformities of Limbs, comprise George Foreman syndromes and thalidomide syndrome.1 special same sense mutation (c.342C〉T) and 3 known SNPs (rs3749319 in Chinese han population Miller pipe heteroplasia (MDAs) patient's Wnt7a gene, have been found, rs12639607 and rs3762719), but these 4 SNPs are on not impact of MDAs.Remove to have found a single nucleotide mutation (rs13896353) at congenital talipes equinovarus patient's Wnt7a upstream promoter, but this is not the main cause of disease.In a word, less to the research of Wnt7a gene so far, aspect myogenesis, more rarely have bibliographical information.The research of Wnt7a gene pleiomorphism aspect also mainly concentrates on the disease aspect, the research in China Qinchuan Cattle Wnt7a gene genetic variation field is deficient, and the functional study of this gene locus and heritable variation thereof the research related with growth traits (as: proterties such as body weight, body length) is still blank.

Summary of the invention

The problem that the present invention solves is to provide a kind of method of Qinchuan Cattle Wnt7a gene mononucleotide polymorphism of detection, seeks the SNP related with economic characters as molecule marker, and quickening has the foundation of high-quality economic characters Qinchuan Cattle population.

One object of the present invention is to provide Qinchuan Cattle Wnt7a gene mononucleotide polymorphism site, and wherein said gene mononucleotide polymorphism site is the 63777th mononucleotide polymorphism site for C or T of Qinchuan Cattle Wnt7a gene.

A further object of the present invention provides a kind of method that detects Qinchuan Cattle Wnt7a gene mononucleotide polymorphism site, and the method for the invention is to be achieved through the following technical solutions:

A kind of method that detects Qinchuan Cattle Wnt7a gene mononucleotide polymorphism take the Qinchuan Cattle complete genome DNA to be measured that comprises the Wnt7a gene as template, is carried out pcr amplification Qinchuan Cattle Wnt7a gene with primer pair M; After restriction enzyme A lw44I digestion pcr amplification product, the fragment after again enzyme being cut is carried out agarose gel electrophoresis; Identify the single nucleotide polymorphism of the 63777th of Qinchuan Cattle Wnt7a gene according to the agarose gel electrophoresis result;

Described primer pair M is:

Upstream primer: ATCCTGGAGGAAAACATGAAGC 22bp

Downstream primer: TCACTTGCACGTGTAGACCTCGGTGC 26bp.

Described pcr amplification reaction program is:

94 ℃ of denaturation 5min; 94 ℃ of sex change 45s, the 62 ℃ of 1min that anneal, 72 ℃ are extended 45s, 32 circulations; 72 ℃ are extended 10min.Described agarose gel electrophoresis is that mass concentration is 3% agarose gel electrophoresis.

Describedly identify that according to the agarose gel electrophoresis result single nucleotide polymorphism of the 63777th of Qinchuan Cattle Wnt7a gene is: A ₃A ₃Genotype shows as the 480bp band; G ₃G ₃Genotype shows as 435bp and 45bp band.The 45bp band is too short and invisible, but does not affect the identified gene type.

Compared with prior art, the present invention has following useful technique effect:

The present invention is according to the primers of Wnt7a gene, respectively take the genomic dna of Qinchuan Cattle as template, carry out pcr amplification, and the PCR product checked order, the sequence of the Qinchuan Cattle Wnt7a gene that obtains after the order-checking is compared with the sequence that NCBI announces, and there is polymorphism in discovery in the 63777th site of Wnt7a gene.Form the missense codom sudden change when the C of the 63777th of Wnt7a gene sports T, namely the 326th amino acids sports Cys by Arg.

For above-mentioned the 63777th SNP polymorphism, the invention also discloses its examination and detection method, by after designing specific primer and carrying out pcr amplification, identify with specific digestion with restriction enzyme, can be simply, quick, cost is low, detect accurately the polymorphism of its mononucleotide:

When 63777bp sports T by C, formed the restriction enzyme site GTGCAC of restriction enzyme A lw44I, if not sudden change, restriction enzyme A lw44I just can not identify; So just can detect this site SNP polymorphism.

Because the Wnt7a gene function relates to the growth traitss such as body weight, height, body length, chest breadth, chest measurement, detection method provided by the invention is that SNP and the Qinchuan Cattle some growth proterties (body weight, height, body length, chest measurement etc.) of Wnt7a gene carried out association analysis, and the result shows that this site can be as improving Qinchuan Cattle body weight, body length, chest breadth and the high molecule marker of hip cross.For use in the marker assisted selection (MAS) of the meat growth traits of Chinese Qinchuan Cattle, the Qinchuan Cattle population that the Rapid Establishment genetic resources is good.

Description of drawings

Fig. 1 is the sequencer map of the 63777th SNP of Qinchuan Cattle Wnt7a gene.Wherein second base C/T represents that namely this site has single nucleotide polymorphism: the 63777th, and C〉T.

Fig. 2 is Qinchuan Cattle Wnt7a gene PCR product electrophoresis.Swimming lane 1 to 5 is Wnt7a gene PCR product; Swimming lane 6 is Marker I.

Fig. 3 is the restriction enzyme digestion and electrophoresis result of PCR product that Qinchuan Cattle Wnt7a gene comprises the 63777th polymorphic site.Swimming lane 5 is Marker I;

Swimming lane

2,4 is A ₃A ₃Genotype;

Swimming lane

1,3 is G ₃G ₃Genotype.

Embodiment

The present invention is by the 63777th site missense mutation may produce the single nucleotide polymorphism that the proteins encoded conformation changes and detects to Qinchuan Cattle Wnt7a gene, for use in the marker assisted selection of the meat growth traits of Chinese Qinchuan Cattle, the Qinchuan Cattle population that the Rapid Establishment genetic resources is good.Related with proterties the present invention is described in further detail below in conjunction with the detection of concrete sample, and the explanation of the invention is not limited.

A, all round design of aobvious subregion PCR primer of Qinchuan Cattle Wnt7a gene

Take ox Wnt7a gene (NC_007320.5) sequence that NCBI was announced as reference, utilize the Primer5.0 design to increase to comprise all round PCR primer of aobvious subregion of Qinchuan Cattle Wnt7a gene the, the sequence of its primer M is as follows:

Upstream primer: ATCCTGGAGGAAAACATGAAGC 22bp

Downstream primer: TCACTTGCACGTGTAGACCTCGGTGC 26bp

With above-mentioned primer pair Qinchuan Cattle genome amplification, can increase comprises all round gene fragment of aobvious subregion of Qinchuan Cattle Wnt7a gene (NC_007320.5 sequence) the, after the amplification electrophoresis detection of fragment as shown in Figure 2, wherein, swimming lane 1～5 is for detecting fragment, and swimming lane 6 is Marker; To the fragment of amplification check order identify after, wherein, the sequence of the 63771bp～63780bp is as follows:

AGCGAG

A

By analysis, find Wnt7a gene all round aobvious the 63777th of subregion have the SNP polymorphism: when the C of the 63777th of Wnt7a gene sports T, cause encoding the 326th codon by CGC(sequence shown in the frame line) sport TGC, thereby form the missense codom sudden change, namely sport Cys by Arg.

When 63777bp sports T by C, formed the restriction enzyme site of restriction enzyme A lw44I, so just can detect this site SNP polymorphism.

B, carry out the Wnt7a gene fragment of pcr amplification Qinchuan Cattle to be measured with primer M

1, the collection of Qinchuan Cattle sample

The present invention specifically with the population of Chinese Qinchuan Cattle kind as detected object, concrete collecting sample sees Table 1:

The collection of table 1 Qinchuan Cattle sample

2, the separation of blood sample genomic dna, extraction, purifying

1) freezing blood sample (being mainly hemocyte) room temperature is thawed, and transferase 45 00 μ L to 1.5mL Eppendorf centrifuge tube adds equal-volume PBS liquid, abundant mixing, the centrifugal 10min(4 of 12000r/min ℃), abandoning supernatant, the repetition above-mentioned steps is transparent to supernatant liquor, precipitation is faint yellow;

2) in centrifuge tube, add DNA extraction buffer 500 μ L, shake, make the hemocyte precipitation break away from centrifuge tube tube wall, 37 ℃ of water-bath 1h;

3) add Proteinase K to 3 μ L(20mg/mL) and mixing, 55 ℃ are spent the night to clarification, and not yet the defecator can add 1 μ L Proteinase K mixing and continue digestion until clarify;

4) reaction solution is cooled to room temperature, adds the saturated phenol 500 μ L of Tris-, gentleness is shaken centrifuge tube 20min, makes its abundant mixing; 4 ℃, the centrifugal 10min of 12000r/min changes supernatant liquor in another 1.5mL centrifuge tube over to, repeats once;

5) add chloroform 500 μ L, abundant mixing 20min, 4 ℃, the centrifugal 10min of 12000r/min changes supernatant liquor in another 1.5mL centrifuge tube over to;

6) add chloroform, primary isoamyl alcohol mixed solution (24:1) 500 μ L, abundant mixing 20min, 4 ℃, the centrifugal 10min of 12000r/min changes supernatant liquor in another 1.5mL centrifuge tube over to;

7) add the NaAc damping fluid of 0.1 times of volume and the ice-cold dehydrated alcohol of 2 times of volumes, mix and rotate centrifuge tube, until the flocks of white is separated out, preserve 30～60min for-20 ℃;

8) 4 ℃, the centrifugal 10min of 12000r/min, abandoning supernatant precipitates 2 times with 70% ice-cold ethanol rinsing DNA;

9) 4 ℃, the centrifugal 10min of 12000r/min makes the ethanol volatilization clean under the abandoning supernatant, room temperature;

10) dried DNA is dissolved in the TE liquid of 80～100 μ L, preserves until DNA dissolves fully for 4 ℃, and 0.8% agarose gel electrophoresis detects its quality ,-80 ℃ of preservations.

11) adding 10%SDS in the dna solution of 500 μ L, to make its final concentration be 0.1%, adds Proteinase K to final concentration and reach 50 μ g/mL;

12) 5 ℃ are incubated about 10h;

13) equal-volume phenol, chloroform, primary isoamyl alcohol (25:24:1) and the extracting of chloroform difference are once;

14) the centrifugal 5min phase-splitting of 12000r/min is drawn the upper strata water to another centrifuge tube;

15) add 1/10 volume 3mol/L sodium-acetate and the 2 times of ice-cold dehydrated alcohol precipitation of volume DNA;

16) outwell liquid, dry after 70% washing with alcohol, add the dissolving of 60 μ L sterilization ultrapure water, 4 ℃ to be detected.

3, pcr amplification

The PCR reaction system adopts mixes the application of sample method, namely according to the number of the required PCR reaction of the quantity of the required various components of each reaction system and 1 secondary response, calculate the total amount of various reactive components, join in 1 1.5mL centrifuge tube, fully instantaneous centrifugal behind the mixing, divide again to install in each 0.2mLEppendorf PCR pipe, then add template DNA, more instantaneous centrifugal laggard performing PCR amplification;

The PCR reaction system sees Table 2:

Table 2PCR reaction system

15 μ L reaction systems comprise 0.375U Taq archaeal dna polymerase (sky, Beijing root Science and Technology Ltd.), and 2 * Buffer7.50 μ L(includes Mg ²⁺, dNTPs etc.) (Mix of sky, Beijing root Science and Technology Ltd.), 50ng/ μ L contains the Qinchuan Cattle genomic dna 0.50 μ L of Wnt7a gene, each 0.30 μ L of 10pmol/ μ L upstream and downstream primer;

The PCR response procedures:

94 ℃ of denaturation 5min; 94 ℃ of sex change 45s, the 62 ℃ of 1min that anneal, 72 ℃ are extended 45s, 32 circulations; 72 ℃ are extended 10min.4 ℃ of preservations.

Genomic dna to 488 samples of Qinchuan Cattle kind carries out pcr amplification, obtains to comprise in the Qinchuan Cattle Wnt7a gene of 488 individualities the dna fragmentation of the 480bp in this SNP site.

C, enzyme are cut the Wnt7a gene fragment of digestion pcr amplification

1, endonuclease reaction digestion system (25～30 μ L): 10～15 μ L PCR products, 1 * damping fluid, 2.5～3.0 μ L, ALW44I(10U/ μ L) 1.0～1.5 μ L, sterilization pure water (H ₂O) 11.5～16.5 μ L.

2, enzyme is cut digestion condition: digest 5～10h in 37 ℃ of constant incubators.

Agarose gel electrophoresis analysis behind d, the enzymic digestion PCR product

1) sepharose of making 3%, 120V voltage electrophoresis 60min behind the point sample, EB dyeing after electrophoresis finishes;

2) treat that the different dna fragmentation of molecular weight separates when clear, in the imaging of BIO-RAD Gel Doc2000 gel imaging system;

3) according to agarose gel electrophoresis interpretation of result SNP polymorphism:

Analyze with the photograph of BIO-RAD Gel Doc2000 gel imaging system, judge the polymorphism of SNP:

When the 63777bp of Wnt7a gene sports T by C, formed the restriction enzyme site GTGCAC of restriction enzyme A lw44I, if not sudden change, restriction enzyme A lw44I just can not identify; So just can detect this site SNP polymorphism.

Because Qinchuan Cattle is 2 times of bodies, so the agarose gel electrophoresis result of the 63777th single nucleotide polymorphism of the genomic Wnt7a gene of Qinchuan Cattle is:

As shown in Figure 3: A ₃A ₃Genotype shows as the 480bp band; G ₃G ₃Genotype shows as 435bp and 45bp band.The 48bp band is too short and invisible, but does not affect genotypic evaluation.

The frequency statistics analysis of e, Qinchuan Cattle Wnt7a gene SNP site

1) gene and genotype frequency

Genotype frequency refers to that certain genotype number of individuals of a certain proterties in the colony accounts for the ratio of total individual number.P _TT=N _TT/ N, wherein P _TTRepresent the TT genotype frequency in a certain site; N _TTHas the genotypic number of individuals of TT in the expression colony; N is for detecting the total quantity of colony.

Gene frequency refers to that a certain gene number is to the relative ratios of its allelotrope sum in the colony.The formula that calculates can be write as: P _T=(2N _TT+ N _Ta1+ N _Ta2+ N _Ta3+ N _Ta4+ ... + N _Tan)/2N

In the formula, P _TExpression allelotrope T frequency, N _TTHas the genotypic individual amount of TT, N in the expression colony _TaiHave Tai genotype individual amount in the expression colony, a1-an is n the mutually different multiple allelomorphos of allelotrope T.

The allelotrope that this institute relates to is A ₃And G ₃So concrete gene frequency calculation formula is:

P _G3=（2N _G3G3+N _A3G3）/2N

P _A3=（2N _A3A3+N _A3G3）/2N

In the formula, P _A3, P _G3Represent respectively allelotrope A ₃And G ₃Allelic frequency, N _A3A3, and N _G3G3Represent respectively A ₃A ₃And G ₃G ₃Genotypic individual amount, N represent the total group number.

Gene frequency distribution table as shown in table 3, the A in different Qinchuan Cattle kind Wnt7a gene SNPs ₃Gene frequency is 61.3%, G ₃Gene frequency is 38.7%.

The 63777th SNP Gene frequency distribution table of table 3 Qinchuan Cattle Wnt7a gene

The association analysis of f, Qinchuan Cattle Wnt7a gene SNP site genetic effect

Genotype (the A of genotype data: Alw44I identification ₃A ₃And G ₃G ₃)

Production data: the body weight at 48 monthly ages of Qinchuan Cattle, height, body length, chest measurement, chest breadth, hip cross is high, buttocks is long and the significant data of circumference of cannon bone.

Relation analysis model:

First data are described analysis, determine whether to exist outlier, the analysis of recycling least square is proofreaied and correct data; According to data characteristics, use SAS(9.1) in the GLM process analysis genotype of software and the bottle to each behavioural effect.When being analyzed, the genotype effect adopted fixed model:

Y _ijkl=μ+BF _i+Month _j+G _k+e _ijkl

Wherein: Y _IjklBe the character observation value, μ is population mean, BF _iBe the fixedly effect on i kind and farm, Month _jBe the fixed effect of observation in j month, G _kBe the fixed effect of k single SNP marker genetype, e _IjklBe random error.

The result shows (seeing Table 4): G in Qinchuan Cattle ₃G ₃Genotype individuality and A ₃A ₃It is long that the genotype individuality is compared body, body weight, high significantly increase (P＜0.01 or P＜0.05) of chest breadth and hip cross.G is described ₃G ₃Genotype can be used as and improves Qinchuan Cattle body length, body weight, the candidate molecules genetic marker that chest breadth and hip cross are high.

Variance analysis between table the 63777th polymorphic site of 4Wnt7a gene and the Qinchuan Cattle growth traits

A and b represent significant difference (P＜0.05), and A and B represent difference extremely significantly (P＜0.01).

Claims

1. Qinchuan Cattle Wnt7a gene mononucleotide polymorphism site is characterized in that, described gene mononucleotide polymorphism site is the 63777th mononucleotide polymorphism site for C or T of Qinchuan Cattle Wnt7a gene.

2. method that detects Qinchuan Cattle Wnt7a gene mononucleotide polymorphism, it is characterized in that, take the Qinchuan Cattle complete genome DNA to be measured that comprises the Wnt7a gene as template, carry out pcr amplification with primer pair M, fragment after the amplification is carried out enzyme with Restriction Enzyme ALW44I cut, the fragment after again enzyme being cut is carried out agarose gel electrophoresis; Identify the 63777th single nucleotide polymorphism for C or T of Qinchuan Cattle Wnt7a according to the agarose gel electrophoresis result;

Described primer pair M is:

Upstream primer: ATCCTGGAGGAAAACATGAAGC,

Downstream primer: TCACTTGCACGTGTAGACCTCGGTGC.

3. the method for detection Qinchuan Cattle Wnt7a gene mononucleotide polymorphism as claimed in claim 2 is characterized in that, described pcr amplification reaction program is:

94 ℃ of denaturation 5min; 94 ℃ of sex change 45s anneal 1min62 ℃; 72 ℃ are extended 45s, 32 circulations; 72 ℃ are extended 10min.

4. the method for detection Qinchuan Cattle Wnt7a gene mononucleotide polymorphism as claimed in claim 2 is characterized in that, described agarose gel electrophoresis is that mass concentration is 3% agarose gel electrophoresis.

5. the method for detection Qinchuan Cattle Wnt7a gene mononucleotide polymorphism as claimed in claim 2 is characterized in that, identifies that according to the agarose gel electrophoresis result single nucleotide polymorphism of the 63777th of Qinchuan Cattle Wnt7a gene is: A ₃A ₃Genotype shows as the 480bp band; G ₃G ₃Genotype shows as 435bp and 45bp band.

6. the application of the described Qinchuan Cattle Wnt7a of claim 1 gene mononucleotide polymorphism site in the molecule marker of the early stage body weight of conduct raising Qinchuan Cattle.

7. the application of the described method of arbitrary claim in the molecule marker of the early stage body weight of conduct raising Qinchuan Cattle among the claim 2-5.