CN107574234A - A kind of method and its application for detecting ox ACVR1 gene mononucleotide polymorphisms - Google Patents

Abstract

The invention discloses a kind of method and its application for detecting ox ACVR1 gene mononucleotide polymorphisms.Using the ox complete genome DNA to be measured comprising ACVR1 genes as template, using primer pair 1F as primer, PCR amplification ox ACVR1 genes, it is included in the nucleotide polymorphisms that ox ACVR1 genes the 41793rd are C or T；Enter row agarose gel electrophoresis with digestion with restriction enzyme pcr amplification product and then to the amplified fragments after digestion；The SNP of ox ACVR1 genes the 41793rd is identified according to agarose gel electrophoresis result；It because this method is a kind of examination on DNA level and detects the molecular genetic marker closely related with the ox production traits, therefore, can be used for the marker assisted selection of the meat growth traits of Chinese Cattle, quickly establish the excellent ox population of genetic resources.

Description

A kind of method and its application for detecting ox ACVR1 gene mononucleotide polymorphisms

Technical field

The invention belongs to molecular genetics field, is related to gene mononucleotide polymorphism (SNP) detection, more particularly to one The PCR-RFLP methods of kind detection ox ACVR1 the 41793rd SNP of gene.

Background technology

SNP (SNP) just refers in genomic dna sequence due to single nucleotide acid (A/T/C/G) replacement Caused by polymorphism.Therefore, usually said SNP includes the replacement, insertion, missing of single base.One SNP is represented in base There is the change of a nucleotides on some site because organizing, mainly as caused by the conversion or transversion of single base；With conversion The SNPs of form variation accounts for 2/3.The cytimidine of CpG dinucleotides is the site most easily undergone mutation in genome, wherein mostly Number methylates, and spontaneously can slough amino and form thymidine.

The SNPs of quantity not etc. may all in any known or unknown gene or be nearby found, according to them in gene The position being distributed in group can be divided into gene coding region SNPs (cSNPs), gene periphery SNPs (pSNPs) SNPs between gene (iSNPs) three classes such as.Generally speaking, cSNP is fewer because the aberration rate in extron only account for around sequence 1/5, but It but has significance in the research of hereditary disease and breeding, therefore receives much attention.According to the influence to inhereditary feature, cSNPs Two kinds can be divided into again：One kind is synonymous cSNPs, i.e. the change of coded sequence caused by SNP has no effect on its protein translated Middle amino acid sequence, mutating alkali yl are identical with " implication " of unmutated base；Another kind is non-synonymous cSNPs, i.e. base sequence Change will cause the change of coded amino acid, so as to produce the change of protein sequence, protein may be eventually affected Function.Therefore, for code area SNPs nonsynonymous mutation, they may have direct material impact to gene function.No Only in this way, in population genetic research, these SNPs also have as genetic marker in the research of population genetic and biological evolution It is significant.

In a diplont colony, SNPs is probably to be made up of 2,3 or 4 allele, but actually 3 Individual or 4 allele SNPs are very rare, therefore SNPs is generally referred to simply as two equipotential gene molecule markers.At present, it is main SNP is found using several different routes：That is determined dna sequence method, PCR-SSCP and DNA sequencing combined techniques, AS- PCR method, primer extension and oligonucleotides coupled reaction etc..In these SNP detection techniques, determined dna sequence method is most For accurate SNP detection method, but its testing cost is higher, is not suitable for large-scale groups parting；Surveyed using PCR-SSCP and DNA Sequence combined techniques detection SNP can suitably reduce testing cost, but PCR-SSCP experimentation is long, and operation is comparatively laborious, And false positive issue in experimentation be present；AS-PCR methods are as a kind of new SNP detection method, in the application neck in future There is boundless prospect, still, this method needs to design special primer, and can only be directed to specific gene position in domain Point, meanwhile, the probability of flase drop in detection process also be present, therefore, at present without it is commonly used the characteristics of；And primer extension With oligonucleotides coupled reaction technology for detection SNP site, it is necessary to plate reader, genetic chip, micro-sphere array technology and mass spectrum The detection platforms such as instrument, exploitativeness is not strong in general Molecular Laboratory.

PCR machine (PCR-RFLP) method is a kind of the effective of detection SNP Technology, cut after SNP site is found using restriction enzyme, then carry out agarose, polyacrylate hydrogel electrophoresis point Analysis, can just differentiate SNP site exactly.PCR-RFLP methods not only have the accuracy of DNA sequencing method, overcome expense again and hold high The shortcomings that expensive, troublesome operation, false positive, and the sequence site detected is without particularity requirement.

Bone morphogenetic protein (bone morphogenetic protein, BMP) acceptor belongs to serine/threonine kinase Enzyme acceptor, it is divided into 2 classes, I receptoroid and II receptoroid.The receptors of BMP I (BMPR I) include ALK1, ACVR1 (also known as ALK2, ACTR1 etc.), ALK3 and the receptor of AKL5, BMP II include BMPR2 and ACVR2.The acceptor of type I is responsible for the conduction of signal, type The expression of the acceptor of type I is responsible for ligand binding and regulated and controled to II acceptor.After Type II binding of receptor and ligand, Type II by Body forms stable tetramer compound with the acceptor of type I, and by the receptor phosphorylation of type I, I receptor of activation, and then raise Downstream albumen.Experiment in vitro proves that the tetramer compound can be combined with multiple ligands, for example, BMP4, BMP6, BMP7, BMP9 and activin etc., but its in vivo natural part do not parse also, involved path include BMP, p38/MAPK and The signal paths such as PI3K/Akt.In addition, configuration aspects, ACVR1 has the typical structure domain of ALK acceptors, including extracellular part knot Close glycine-serine (GS) the enrichment domain and protein kinase domain of domain, membrane spaning domain, membrane-proximal region.

ACVR1 genes are that embryonic development and growth are essential, many cells and organization type are relate to, such as muscle, bone Bone and nervous system etc., and have spatial and temporal expression specific in mouse growth course.Research shows that knocking out ACVR1 genes causes Mice embryonic occurs as soon as some retardation in mesoderm growing early stage, therefore it is more in the developmental function of tissue/organ to study ACVR1 genes Using conditional mutation (conditional mutants).The research to ACVR1 genes is concentrated mainly on progressive intermuscular bone at present Change disease (FibrodysplasiaOssificansProgressiva, FOP) and glioma (Diffuse Intrinsic Pontine Glioma, DIPG) etc. in the research of disease, and disclose the crucial mutation related to both.ACVR1 genes 3 ' UTR has miR-148a target site, and miR148a belongs to miR-148 families (miR-148a, miR-148b and miR-152), should Family has the function that regulating cell propagation and apoptosis.In addition, ACVR1 can also be by SOST and DKK1 come indirect adjustments and controls Wnt Signal path.

Ox ACVR1 genes are located at BTA2：39287889-39361502 (UMD 3.1.1), total length 73614bp, includes 10 Extron, 9 intrones, mRNA sequence, which compares, finds that sequence similarity up to 87%, illustrates that the gene has function conservative.But It is that the current research about ACVR1 genes is concentrated mainly on people and mouse, research report is less on domestic animals and fowls.Animal Have 7 QTL (being identified using genome scanning and GWAS) comprising ox ACVR1 genes in QTLdb databases, relate to milk production, Meat and fat deposition correlated traits are produced, but all without the further checking research of progress.This 7 QTL span~17Mb (BTA2：37604171-54426732), 46 genes and 1 miRNA are contained, but this 7 QTL shared region is BTA2： 39293326-39330039, the region only include ACVR1 genes, and it is the important time for controlling the ox production traits to illustrate the gene Select functional gene.

But the candidate gene that ACVR1 genes grow as regulation at present, is chosen, by detecting ox ACVR1 genes Hereditary variation, and analysis is associated with growth traits, to provide important molecule mark for beef cattle marker assisted selection (MAS) The research of note, there is not yet report.

The content of the invention

It is an object of the invention to provide a kind of method for detecting ox ACVR1 gene mononucleotide polymorphisms and its answer With so as to accelerate fine-variety breeding speed.

To reach above-mentioned purpose, present invention employs following technical scheme：

A kind of method for detecting ox ACVR1 gene mononucleotide polymorphisms, comprises the following steps：

Using the ox complete genome DNA to be measured comprising ACVR1 genes as template, using primer pair 1F as primer, PCR amplifications are yellow Ox ACVR1 Gene Partial fragments；Pcr amplification product is digested with restriction enzyme RspRS II (Mse I) and then digestion is disappeared Amplified fragments after change enter row agarose gel electrophoresis；Ox ACVR1 genes the are identified according to agarose gel electrophoresis result The SNP of 41793；

Described primer pair 1F is：

Positive sense primer：5’-TAAAAGGCGCAATCAAGAACGCC-3’；

Reverse anti-sense primer：5’-AACTCACGTAAGCGTGCCAG-3’.

Described pcr amplification reaction program comprises the following steps：95 DEG C of pre-degeneration 5min；38 circulations：94 DEG C of denaturation 30s, 65 DEG C of annealing 30s, 72 DEG C of extension 30s；72 DEG C extend 10min eventually.

The mass concentration of described Ago-Gel is 3.5%.

The SNP for identifying ox ACVR1 genes the 41793rd according to agarose gel electrophoresis result is：CC Genotypic expression is a 264bp band；TT genotypic expressions are a 239bp band；CT genotypic expressions are 264bp With 239bp two band.

A kind of kit for detecting ox ACVR1 gene mononucleotide polymorphisms, including above-mentioned detection ox ACVR1 genes The primer pair 1F of 41793rd SNP.

Compared with prior art, the present invention has technique effect beneficial below：

Ox ACVR1 gene mononucleotide polymorphism detection methods provided by the invention, the 41793rd C to T conversion are dashed forward Become and cause the generation of a natural restriction enzyme site of RspRS II (Mse I), by electrophoresis detection parting can accurately, quickly, conveniently, It is inexpensive, pinpoint accuracy to detect SNP of the ACVR1 genes at the 41793rd.It is easy to utilize in DNA Examination and detection and the closely related genetic marker of the ox production traits, the molecular breeding available for ox in level.

The present invention has carried out detection to the SNP genotype of 3 yellow cattle breeds and gene frequency is analyzed, and finds above-mentioned SNP positions Point associates with ox some growth character, is established for the SNP of ACVR1 genes and the foundation of Chinese Cattle growth traits relation Basis, for use in Chinese Cattle marker assisted selection (MAS), so as to quickly establish the excellent ox population of genetic resources.

Brief description of the drawings

Fig. 1 is the pcr amplification product electrophoretogram of ox ACVR1 genes (primer pair 1F).

Fig. 2 is ox ACVR1 gene PCR amplified production sequencer maps.

Fig. 3 is the electrophoretogram of the digestions of ox ACVR1 gene PCR amplified productions RspRS II；Wherein swimming lane M is Marker I (600bp, 500bp, 400bp, 300bp, 200bp, 100bp), CC genotypic expressions are 264bp band；TT genotypic expressions For 239bp band；CT genotypic expressions are 264 and 239bp band.

Embodiment

The present invention is described in further detail with reference to the accompanying drawings and examples, it is described be explanation of the invention and It is not to limit.

There may be encoding proteins expression to become to the mutation of the bit base of ox ACVR1 genes the 41793rd by the present invention The SNP of change is detected, and the mononucleotide polymorphic of ox ACVR1 genes is detected using PCR-RFLP methods Property, and it is associated analysis with growth traits, verify whether it can be as point of assisted Selection in ox molecular breeding Son mark.

1st, the design of ox ACVR1 genes Exon4 subregions PCR primer

Ox (AC_000159.1) sequence announced using NCBI is designed to amplification bag as reference, using Primer 5.0 The PCR primer (primer pair 1F) of the exon region of the genes of ACVR1 containing ox the 4th, its primer sequence difference are as follows：

Forward primer：5’-TAAAAGGCGCAATCAAGAACGCC-3’

Reverse primer：5’-AACTCACGTAAGCGTGCCAG-3’

With above-mentioned primer pair ox genome amplification, can expand comprising the exon region of ox ACVR1 genes the 4th Genetic fragment；After the fragment progress sequencing identification of amplification, the 41793rd (AC_000159.1) base of ACVR1 genes is worked as in discovery When sporting T by C, coded amino acid codon is caused to sport CTT by CTC, amino acid keeps constant, is still Leu, occurs Same sense mutation；And this mutation forms restriction enzyme RspRS II (Mse I) restriction enzyme site.

2nd, the ACVR1 genetic fragments of performing PCR amplification ox to be measured are entered with primer pair 1F

(1) collection of ox sample

For the present invention specifically using the population of 3 Chinese Cattle kinds as detection object, specific collecting sample is shown in Table 1：Xia Nan Ox (215), Qinchuan Cattle (105), Nanyang cattle (107).

The collection of the ox sample of table 1.

(2.1) separation, the extraction of blood sample genomic DNA

1) blood sample (predominantly haemocyte) thaw at RT is freezed, μ L to the 1.5mL Eppendorf centrifuge tubes of transferase 45 00, is added Enter isometric PBS liquid, fully mix, 12000r/min centrifugation 10min (4 DEG C), abandoning supernatant, repeat the above steps to supernatant Liquid is transparent, precipitation is in faint yellow；

2) the μ L of DNA extraction buffers 500 are added in centrifuge tube, are shaken, departs from haemocyte precipitation and centrifuges tube wall, 37 DEG C water-bath 1h；

3) plus Proteinase K to 3 μ L (20mg/mL) and mixes, and 55 DEG C, overnight to clarification, not yet defecator, can add 1 μ L eggs White enzyme K, which is mixed, continues digestion until clarification；

4) reaction solution is cooled to room temperature, adds the μ L of Tris- saturated phenols 500, it is gentle to shake centrifuge tube 20min, make its abundant Mix；4 DEG C, 12000r/min centrifugation 10min, supernatant is transferred in another 1.5mL centrifuge tubes, is repeated once；

5) chlorination imitates 500 μ L, fully mixes 20min, 4 DEG C, 12000r/min centrifugation 10min, supernatant is transferred to another In 1.5mL centrifuge tubes；

6) chlorination is imitative, isoamyl alcohol mixed liquor (24:1) 500 μ L, 20min is fully mixed, 4 DEG C, 12000r/min is centrifuged 10min, supernatant is transferred in another 1.5mL centrifuge tubes；

7) plus the NaAc buffer solutions of 0.1 times of volume and the ice-cold absolute ethyl alcohol of 2 times of volumes, mixing rotate centrifuge tube, directly Flocculent deposit to white separates out, -20 DEG C of 30~60min of preservation；

8) 4 DEG C, 12000r/min centrifugation 10min, abandoning supernatant, precipitated 2 times with 70% ice cold ethanol rinsing DNA；

9) 4 DEG C, 12000r/min centrifugation 10min, abandoning supernatant, make ethanol volatilization clean at room temperature；

10) dried DNA is dissolved in 80~100 μ L TE liquid, and 4 DEG C of preservations are completely dissolved up to DNA, 0.8% agarose Its quality of detected through gel electrophoresis, -80 DEG C of preservations.

(2.2) purify

11) in 500 μ L DNA solution add 10%SDS make its final concentration of 0.1%, add Proteinase K to final concentration reach To 50 μ g/mL；

12) 5 DEG C are incubated 10h or so；

13) isometric phenol, chloroform, isoamyl alcohol (25:24:1) extracted respectively once with chloroform；

14) 12000r/min centrifuges 5min split-phases, draws upper strata aqueous phase into another centrifuge tube；

15) 1/10 volume 3mol/L sodium acetates and 2 times of volumes ice cold absolute ethyl alcohol precipitation DNA are added；

16) liquid is outwelled, is dried after the washing of 70% ethanol, adds 60 μ L sterilizing ultra-pure water dissolvings, 4 DEG C to be detected.

(3) PCR amplifications and product digestion

PCR reaction systems are using mixing sample-adding method, i.e., the quantity and 1 of the various components according to needed for each reaction system The number of PCR reactions needed for secondary response, calculates the total amount of various reactive components, is added in 1 1.5mL centrifuge tube, fully Brief centrifugation after mixing, then be dispensed into each 0.2mL Eppendorf centrifuge tubes, then add template DNA, then brief centrifugation Laggard performing PCR amplification；Digestions reaction is carried out to pcr amplification product.

1) PCR overall reactions system is 10 μ L, is shown in Table 2；

Table 2.PCR reaction systems

2) PCR response procedures, 3 are shown in Table；

Table 3.PCR response procedures

3) the μ L of RspRS II (Mse I) endonuclease reaction system 25, are shown in Table 4.

Table 4.RspRS II (Mse I) endonuclease reaction system

Note：Digestions condition is 60 DEG C, 5~10h

Referring to Fig. 1, the purpose band of PCR amplifications is 264bp.

3rd, RFLP is detected

1) Ago-Gel of mass concentration 3.5% containing nucleic acid dye is made respectively to produce the PCR after the digestions of RspRS II Thing is detected, 120V electrophoresis 1h after point sample；

2) when the different DNA fragmentation of molecular weight is separated clearly, in the gel imaging systems of BIO-RAD Gel Doc 2000 Imaging；

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

With the gel imaging system PHOTOGRAPHIC ANALYSISs of BIO-RAD Gel Doc 2000, judge and distinguish different banding patterns：

When 41793rd bit base of ACVR1 genes sports T by C, 1F amplification ACVR1 gene outcomes the 41793rd~ 41795 sequences are TTAA, and the sequence is exactly restriction enzyme RspRS II (Mse I) recognition site, when not undergoing mutation When, herein just in the absence of RspRS II (Mse I) recognition site.

Because ox is 2 times of bodies, so the SNP of the 41793rd of the ACVR1 genes of ox genome, by RspRS II After (Mse I) digestion and 3.5% agarose gel electrophoresis, the band of different genotype can be shown, as a result for：

As shown in figure 3, two articles of DNAs of homozygote AA types can not be cut at the 41793rd by RspRS II (Mse I), institute To show as 264bp band；Two articles of DNAs of homozygote GG types are cut in the 41793rd potential energy by RspRS II (Mse I), institute To show as 239bp band；One article of DNA of heterozygote AG types is cut in the 41793rd potential energy by RspRS II (Mse I), institute To show as 264bp and 239bp band, 25bp band can not be shown due to too small on gel.

4th, the purifying and sequencing of different genotype individual PCR primer

After PCR-RFLP detections, different genotype individual is picked out, enters performing PCR amplification, and PCR primer is carried out respectively Gel extraction and purifying：The gel containing purpose fragment is cut from Ago-Gel under uviol lamp, is put into 1.5mL centrifuge tubes In, then with PCR primer recovery purifying kit (Beijing Tiangeng biotech firm) purified pcr product, according to kit specification Operation.

According to agarose gel electrophoresis result, send Nanjing Jin Sirui biological the PCR purified products of different genotype individual Science and Technology Ltd. carries out positive and negative two-way sequencing；Meanwhile carry out SNP position analyses, the results showed that include the individual of 264bp bands For CC genotype, its 41793rd sequencer map is expressed as (C-C) really；The individual of the band containing 239bp is TT genotype, its The sequencer map of the 41793rd is expressed as (T-T) really；The individual of the band containing 264bp and 239bp is CT genotype, and it the 41793rd The sequencer map of position is expressed as (C-T) really, referring to Fig. 2.

5th, the frequency statistics analysis of ox ACVR1 gene SNP sites

1) gene and genotype frequency

Genotype frequency refers to that certain genotype individuals number in a colony accounts for the ratio of total individual number.P_AA=N_AA/ N, its Middle P_AARepresent the AA genotype frequencies in a certain site；N_AARepresent that there is the number of individuals of AA genotype in colony；N is detection colony Total quantity.

Gene frequency refers to that a certain gene number is to the relative ratios of its allele sum in a colony.The formula of calculating It can be write as：P_A=(2N_AA+N_Aa1+N_Aa2+N_Aa3+N_Aa4+……+N_Aan)/2N

In formula, P_ARepresent allele A frequencies, N_AARepresent that there is the individual amount of AA genotype, N in colony_AaiRepresent group There are Aai genotype individuals quantity, the n mutually different multiple alleles that a1~an is allele A in body.

In different the 41793rd SNP allelic A frequencies of yellow cattle breed ACVR1 genes and the change of G frequencies such as the institute of table 5 Show.

Table the 41793rd polymorphic site Gene frequency distribution table of 5. ox ACVR1 genes

6th, the association analysis of ox ACVR1 gene SNP sites gene effect

Genotype data：The genotype (CC, TT and CT) of identification.

Creation data：3 Chinese Cattles (Xia Nanniu, Qinchuan Cattle, Nanyang cattle) amount to 427 individuals for experimental animal and Growth traits (body height, body weight, body length, Body steep length, hip cross height, abdominal circumference, Guan Wei, bust, chest breadth, chest depth, the waist of Chinese Cattle Angular width, point of the buttocks are wide, buttocks length).

Relation analysis model：

First data are described with analysis, it is determined whether outlier be present, recycle Least square analysis to Data correction； According to data characteristics, using in the GLM process analysis procedure analyses genotype and bottle of SPSS (16.0) software to each behavioural effect.To gene Type effect employs fixed model when being analyzed：

Y_ijklm=μ+A_i+G_j+B_k+S_l+e_ijklm,

Wherein：Y_ijklmRecorded for individual phenotype, μ is population mean, A_iFor age effect, G_jFor genotype effects, B_kFor product Kind effect, S_lFor sex-effects, e_ijklmFor random residual effect.

As a result show and (be shown in Table 6)：The 41793rd polymorphic site of ACVR1 genes, the chest depth of CC genotype individuals are higher than TT bases Because of type individual and significant difference (P<0.05).Other characters are not notable with genetic variety.Illustrate that the polymorphic site can be made For the genetic marker of ox chest depth character determination.

The 41793rd variance analysis between polymorphic site and Chinese Cattle growth traits of table 6.ACVR1 genes

Note：Data of going together institute different expression significant difference (a, the b of marking-up parent phase:P<0.05).

Sequence table

<110>Xibei Univ. of Agricultural ＆ Forest Science ＆ Technology

<120>A kind of method and its application for detecting ox ACVR1 gene mononucleotide polymorphisms

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<170> SIPOSequenceListing 1.0

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taaaaggcgc aatcaagaac gcc 23

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aactcacgta agcgtgccag 20

Claims (9)

1. A kind of 1. method for detecting ox ACVR1 gene mononucleotide polymorphisms, it is characterised in that：Comprise the following steps：

   Using ox genomic DNA to be measured as template, using primer pair 1F as primer, PCR amplification ox ACVR1 Gene Partial fragments, Fine jade is carried out with restriction enzyme RspRS II or RspRS II isoschizomers digestion pcr amplification product, then to the fragment after digestion Sepharose electrophoresis, the genotype in ox ACVR1 gene mononucleotide polymorphisms site is identified according to electrophoresis result；

   The primer pair 1F is：

   Positive sense primer：5’-TAAAAGGCGCAATCAAGAACGCC-3’；

   Reverse anti-sense primer：5’-AACTCACGTAAGCGTGCCAG-3’.
2. A kind of 2. method for detecting ox ACVR1 gene mononucleotide polymorphisms as claimed in claim 1, it is characterised in that：Institute The response procedures for stating PCR amplifications comprise the following steps：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 65 DEG C of annealing 30s, 72 DEG C are prolonged Stretch 30s, 38 circulations；72 DEG C of extension 10min.
3. A kind of 3. method for detecting ox ACVR1 gene mononucleotide polymorphisms as claimed in claim 1, it is characterised in that：Institute State ox ACVR1 gene mononucleotide polymorphisms site and be positioned at ox reference sequences AC_000159.1 the 41793rd.
4. A kind of 4. method for detecting ox ACVR1 gene mononucleotide polymorphisms as claimed in claim 1, it is characterised in that：Institute State isoschizomers and be selected from restriction enzyme Mse I.
5. A kind of 5. method for detecting ox ACVR1 gene mononucleotide polymorphisms as claimed in claim 1, it is characterised in that：Institute The mass concentration for the Ago-Gel that electrophoresis uses is stated as 3.5%.
6. A kind of 6. method for detecting ox ACVR1 gene mononucleotide polymorphisms as claimed in claim 1, it is characterised in that：Institute The genotype for stating ox ACVR1 gene mononucleotide polymorphisms site is：CC genotypic expressions are a 264bp band；TT Genotypic expression is a 239bp band；CT genotypic expressions are 264bp and 239bp two band.
7. 7. the method for detection ox ACVR1 gene mononucleotide polymorphisms is auxiliary in ox molecular labeling as claimed in claim 1 The application helped in selection and use.
8. 8. application as claimed in claim 7, it is characterised in that：The ox ACVR1 gene mononucleotide polymorphisms site The DNA marker that CC genotype or C allele can select as ox Growth Traits.
9. A kind of 9. kit for detecting ox ACVR1 gene mononucleotide polymorphisms, it is characterised in that：Including PCR-based-RFLP Method detects the primer pair 1F of ox ACVR1 gene mononucleotide polymorphism loci gene types；

   The primer pair 1F is：

   Positive sense primer：5’-TAAAAGGCGCAATCAAGAACGCC-3’；

   Reverse anti-sense primer：5’-AACTCACGTAAGCGTGCCAG-3’.