CN107641657A - A kind of detection method of ox ACVR1 gene insertion/deletions and its application - Google Patents

Abstract

The invention discloses a kind of detection method of ox ACVR1 gene insertion/deletions and its application.Using ox complete genome DNA to be measured as template, using primer pair P1 as primer, ACVR1 genes are expanded by round pcr, then enter row agarose gel electrophoresis, identify ACVR1 genes in AC_000159.1 according to electrophoresis result:Different genotype be present in the sites of g33007 33023.As a result find, the different type and Chinese Cattle pipe of the bp insertion/deletions of ACVR1 genes 16 enclose exist between the Growth Traits such as bust it is significantly correlated, the bp insertion/deletions of ACVR1 genes 16 are prompted to be advantageous to quickly establish the excellent ox genetic resources colony of Growth Traits as the DNA marker for improving Chinese Cattle Growth Traits.

Description

A kind of detection method of ox ACVR1 gene insertion/deletions and its application

Technical field

The invention belongs to modern biotechnology and cattle breeding field, is related to the detection of gene insertion/deletion, more particularly to A kind of method for detecting Chinese Cattle ACVR1 gene 16-bp insertion/deletions.

Background technology

Animal breeding technology mainly includes traditional breeding method based on phenotype and phenotypic number and polymorphic for base with DNA The molecular breeding technology of plinth.As the important component of molecular breeding technology system, molecular marker assisted selection (marker- Assisted selection, MAS) breeding technique detects the DNA polymorphism of important gene first, then analyze DNA it is polymorphic with Correlation between inhereditary feature, finally carry out character determination further according to the significantly correlated DNA marker of inhereditary feature.As existing For the new technology to be arisen in Protocols in Molecular Biology evolution, MAS breeding techniques can be on DNA level quick and precisely The genetic constitution of ground analysis individual, target gene is transferred in the parent for needing to improve by this method by sexual hybridization, by mesh The identification of mark genotype is combined with traditional breeding method, and genotype is directly selected so as to realize, improves the orientation of breeding objective Property.This method has superiority in the harmless Character Evaluation of Seedling selection, progress and selection and raising breeding efficiency etc..

In MAS breeding technique systems, critical function gene, examination important gene hereditary variation site are found, and analyze Critical function gene genetic variant sites and the correlation of growth performance, be Molecular Marker Assisted Selection Technology application premise and It is crucial.One of important way as molecular genetic marker, insertion/deletion (indel) are a kind of New molecular markers.Indel Refer to the insertion of nucleotide fragments or missing on DNA sequence dna and cause the change of DNA sequence dna, cause species including humans Between DNA sequence diversity.Indel refers to insertion or missing of the fragment length between 1-50bp in genome, can Small CNV (Copy number variations) can be included, can preferably be explained using indel analyses genes of individuals group individual The phenotypic difference of body, therefore indel detections are carried out in Animal molecular breeding to the difference of small fragment nucleotides from DNA level It is significant in MAS systems.

Insertion/deletion is that the evolution that DNA or the horizontal upper occurrence frequency of protein sequence are only second to residue replacement changes, Indel polymorphisms are a kind of two equipotential gene genetics marks of specific type in genome, show as inserting or lacking in genome Different size of small fragment DNA is lost.Indel is roughly divided into following 5 major class：(1) insertion/deletion of single base pair；(2) it is single The insertion/deletion of base；(3) repeat unit is more base-pair insertion/deletions of 2~15 bases；(4) transposons insertion/deletion； (5) insertion/deletion of any DNA sequence.With the further investigation of comparative genomics, indel be theoretical research and Genetic breeding application study provides substantial amounts of biological information, its science of heredity identification marking as a new generation, has the excellent of SNP concurrently Point, compared with SNP, single mutational events being derived from, its frequency of mutation is relatively low, and about 10^-8, it is relatively stable.In structure Belong to two polymorphic alleles, allele it is all fixed and, it is known that can be expanded by the amplicon of very little (< 50bp), the success rate of amplification height degradation of dna is improved.As a kind of important genetic marker, indel research gathers earliest Jiao is in molecular biology and biomedical sector.The indel frequencies for being dispersed throughout whole gene group are only second to SNP, wherein about three points One of in known gene region, also have some positioned at the critical regions for determining gene function, such as promoter region and outer Aobvious sub-district.Molecular biologist uses it for the related research of gene phenotype earliest, it is desirable to by by mankind's character, disease symptomses Or neurological susceptibility is contacted, so as to reach the purpose of gene diagnosis and treatment.2005, Bhangale etc. reported one kind The method that indel variations can be identified comprehensively from target gene, and find 2393 from 330 alternative genes using this method Individual indel variant sites, show that the frequency that insertion/deletion state occurs in human genome is higher than the conclusion for inserting polymorphism, together When propose, indel and SNP Forming Mechanism may be different, but its evolutionary history is similar.

With economic development and people's living standards continue to improve, society constantly strengthens the demand of ox product, but Due to the product critical shortage such as beef, milk in recent years, thus breeding expert it is expected earlier, more preferably, quickly obtain productivity The excellent yellow cattle breed of shape.On high yield, high-quality and efficient ox breeding objective, ox breeding expert pays close attention to growth hair always Fertility shape, but be only not all right by traditional breeding method means, it is also necessary to by molecular breeding technology.I.e. screening grows with ox first The DNA marker of the closely related important candidate gene of development character, then carries out the detection of gene pleiomorphism, is finally to carry out base Because of the association analysis of polymorphism and Growth Traits, so as to realize MAS and realize early diagnosis selection.

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 (Fibrodysplasia Ossificans Progressiva, FOP) and glioma (Diffuse Intrinsic Pontine Glioma, DIPG) etc. in the research of disease, and disclose the crucial mutation related to both. The UTR of ACVR1 genes 3 ' has miR-148a target site, and miR148a belongs to miR-148 families (miR-148a, miR-148b And miR-152), the family has the function that regulating cell propagation and apoptosis.In addition, ACVR1 can also by SOST and DKK1 carrys out indirect adjustments and controls Wnt signal paths.

Ox ACVR1 genes are located at BTA2：39287889-39361502 (UMD 3.1.1), total length 73614bp, includes 10 Individual extron, 9 intrones, mRNA sequence, which compares, finds that sequence similarity up to 87%, illustrates that the gene has function conservative. But the research about ACVR1 genes at present is concentrated mainly on people and mouse, research report is less on domestic animals and fowls. There are 7 QTL (being identified using genome scanning and GWAS) comprising ox ACVR1 genes in Animal QTLdb databases, be related to Milk production, production meat and fat deposition correlated traits, but all without carrying out further checking research.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 control ox production traits to illustrate the gene Important candidate's 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 detection method of ox ACVR1 gene insertion/deletions and its application.

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

A kind of detection method of ox ACVR1 gene insertion/deletions, comprises the following steps：

Using the complete genome DNA of Chinese Cattle to be measured as template, using primer pair P1 as primer, by PCR expand it is to be measured in State's ox ACVR1 genetic fragments, the fragment include the ACVR1 genes 16-bp insertion/deletion polymorphic sites；PCR is expanded again Obtained fragment enters row agarose gel electrophoresis；Chinese Cattle ACVR1 genes to be measured are identified according to agarose gel electrophoresis result The insertion/deletion of 16-bp insertion/deletion polymorphic sites；

Described primer pair P1 is：

Sense primer：5’-CACGGAAGAGACAGACCAGTATT-3’；

Anti-sense primer：5’-TGGGGGAGAGTTCCATCTGTTT-3’.

The reaction system of the PCR includes 2 × Taq PCR SuperMix (including Taq archaeal dna polymerases, dNTPs and anti- Buffer solution is answered, concentration is 2 ×) 5~12.5 μ L；The μ L of sense primer 0.3~1；μ L (the upstream and downstream primer concentration of anti-sense primer 0.3~1 It is respectively 10pmol/ μ L)；Template DNA (concentration is 50ng/ μ L oxes genomic DNA) 0.3~1 μ L；The μ of deionized water 4.1~9.5 L；The PCR amplification system of totally 10~25 μ L volumes.

The amplified reaction program of the PCR comprises the following steps：1) 95 DEG C of pre-degeneration 5min, subsequently into step 2)； 2) Circulation：94 DEG C of denaturation 30s, 59.1 DEG C of renaturation 30s, 72 DEG C of extension 30s；Totally 36 circulations, subsequently into step 3)；3) 72 DEG C are prolonged Stretch 10min.

The mass concentration for the Ago-Gel that the agarose gel electrophoresis uses is 3.5%.

The insertion/deletion is：Insertion/insertion genotypic expression is the band lines of 166bp mono-；Insertion/deletion base Because type shows as 166bp and the band lines of 150bp two；Missing/deletion Genotype shows as the band lines of 150bp mono-.

The detection method of above-mentioned ox ACVR1 gene insertion/deletions is in Chinese Cattle molecular marker assisted selection breeding Application.Missing/deletion Genotype (DD) of the insertion/deletion polymorphic site can be used as Chinese Cattle bust and pipe to enclose index DNA marker.

A kind of detection kit of ox ACVR1 gene insertion/deletions, the kit include above-mentioned be used in PCR amplifications The primer pair P1 of 16-bp insertion/deletion polymorphic sites in state's ox ACVR1 genes.

Beneficial effects of the present invention are embodied in：

Primers of the invention according to ACVR1 genes, respectively using the genomic DNA of 5 yellow cattle breeds as template, Enter performing PCR amplification, and enter row agarose gel electrophoresis to PCR primer, the insertion of the ACVR1 genes of ox/lack is obtained after electrophoresis Lose polymorphism (AC_000159.1:G33007-33023 insertion/deletion).

The present invention has carried out detection and gene frequency point to the insertion/deletion loci gene type of 5 yellow cattle breeds Analysis, analysis is associated to above-mentioned insertion/deletion site and ox some growth character (such as pipe encloses, bust), as a result Show that the site can enclose the molecular labeling of (P=0.014) and bust (P=0.024) as raising Chinese Cattle pipe.

The invention provides the detection method for the insertion/deletion site, is expanded by the primer of design through PCR Identified again through agarose gel electrophoresis after increasing, can simply, quick, low cost, accurately detect the polymorphic of its insertion/deletion Property, so as to provide foundation for ox fine-variety breeding, be advantageous to quickly establish the excellent ox genetic resources group of Growth Traits Body, accelerate fine-variety breeding speed.

Brief description of the drawings

Fig. 1 is ox ACVR1 gene 16-bp insertion/deletion (indel) polymorphic sites (AC_000159.1: g33007- 33023ins sites) PCR primer electrophoresis result (WW, WD and DD genotype).

Fig. 2 is ox ACVR1 gene PCR product sequencing result figures：At the insertion/deletion site of 33007-33023 positions, 16 Bp insetion sequences (Insert DNAsequences) are shown in inframe, and " △ " is shown in insertion position such as figure.

Embodiment

The present invention is elaborated with reference to the accompanying drawings and examples, described is explanation of the invention rather than limit It is fixed.

The present invention is using PCR amplification method to ox ACVR1 genes in AC_000159.1:G33007-33023ins sites It is mutated issuable insertion/deletion to be detected, and itself and growth traits is associated analysis, whether verifies it Can be as the molecular labeling of assisted Selection in ox molecular breeding, so as to accelerate fine-variety breeding speed.

(1) experimental drug and reagent

1. biochemical reagents and biological reagent：1. Proteinase K (is purchased from Huamei Bio-Engrg Co.)；2. Marker I (purchases From TIANGEN Biotech (Beijing) Co., Ltd.).

2. general reagent：General reagent is bought from Huamei Bio-Engrg Co., and product is dispensed for import：Tris、EDTA、 NaCl、NaOH、KCl、Na₂HPO₄、KH₂PO₄, Tris saturated phenols, chloroform, isoamyl alcohol, absolute ethyl alcohol, sodium acetate, dodecyl sulphur Sour sodium (SDS), ethidium bromide (EB), bromophenol blue, dimethyl benzene cyanogen FF, acetic acid, sucrose, boric acid, agarose etc..

3. solution and buffer solution：All solution are prepared with buffer solution using deionization ultra-pure water.Autoclave conditions are 15 bf/in(1.034×10⁵Pa), 25min.What preparation of reagents method was write with reference to Sambrook etc.《Molecular Cloning: A Laboratory Guide》.

1) solution used in tissue sample DNA is extracted：In addition to public solution when extracting genome DNA, following try also is prepared Agent：①2mol/L NaCl：11.688g is dissolved in water, is settled to 100mL, autoclaving.2. tissue DNA extract solution (100mL)：l Mol/L Tris-Cl (pH 8.0) l mL, 0.5mol/L EDTA (pH 8.0) 20mL, 2mol/L NaCl 5mL, are settled to 100mL。

2) solution used in agarose electrophoretic analysis:1. 1 × tbe buffer liquid：10 × TBE 100mL are taken to be settled to 1000mL. 2. sample-loading buffer：0.25% bromophenol blue, 0.25% dimethylbenzene green grass or young crops FF, and 40.0% (w/v) aqueous sucrose solution.

(2) ox ACVR1 genes indel site (AC_000159.1:G33007-33023ins sites) PCR primer Design

The sequence of ox ACVR1 genes is retrieved on NCBI, and is designed to amplification using Premier 5.0 and includes ox The PCR primer that ACVR1 genes the 22nd include subregion 16-bp indel sites is as follows to P1, its primer sequence：

Sense primer：5’-CACGGAAGAGACAGACCAGTATT-3’；

Anti-sense primer：5’-TGGGGGAGAGTTCCATCTGTTT-3’；

Above-mentioned primer pair P1 can be expanded comprising ox ACVR1 genes in AC_000159.1 to ox genome amplification: The indel in g33007-33023ins sites different genotype fragments.In theory, when between 33007 and 33023nt When GCAGTAGACTTCATTT is lacked, PCR primer is a band line of 150bp sizes after agarose gel electrophoresis detection； When GCAGTAGACTTCATTT between 33007 and 33023nt is inserted, PCR products are after agarose gel electrophoresis detection One band line of 166bp sizes.During GCAGTAGACTTCATTT insertion/deletions between 33007 and 33023nt, PCR primer It is the band lines of 150bp+166 bp two after agarose gel electrophoresis detection.Therefore, according to theoretical analysis result, insertion/insert Entering genotype (WW) and show as the band lines of 166bp mono-, insertion/deletion genotype (WD) shows as the band lines of 150bp+166bp two, Missing/deletion Genotype (DD) shows as the band lines of 150 bp mono-.

(3) the ACVR1 genetic fragments of ox to be measured are expanded with primer pair P1PCR

1st, the collection of ox sample

Experiment animal used amounts to 840 samples for 5 Breeds of Yellow Cattle, and the specific situation that gathers is referring to table 1：

The collection of the ox sample of table 1.

2nd, tissue sample DNA extraction is with separating

1) about 10mg ear tissue samples are taken, are put in 1.5mL centrifuge tube, are shredded as far as possible with small scissors.

2) 600 μ L tissue DNA extract solutions, 10%SDS to final concentration of 1%, Proteinase K to final concentration of 100 μ are added G/mL, 55.0 DEG C of digestion overnight, preferably ensure that tissue sample is relatively evenly distributed in tissue extract.

3) solution is cooled to room temperature, adds isometric Tris saturated phenols, cover tightly lid, slowly overturn centrifugation back and forth Pipe, at least persistently more than 10min, 12000r/min centrifugation 15min.

4) supernatant is taken, adds isometric phenol:Chloroform (1:1) lid, is covered tightly, slowly overturns centrifuge tube back and forth, extremely Continue more than 10min, 12000r/min centrifugations 15min less.

5) supernatant is taken, adds isometric chloroform:Isoamyl alcohol (24:1) lid, is covered tightly, slowly overturns centrifugation back and forth Pipe, at least persistently more than 10min, 12000r/min centrifugation 15min.

6) supernatant is taken, the 3mol/L sodium acetates of 1/10 volume and the ice-cold absolute ethyl alcohol of 2 times of volumes is added, covers tightly pipe Lid, centrifuge tube is slowly overturned back and forth, until liquid is limpid, white flock DNA occur.

7) choose DNA, in the centrifuge tube for putting a 1.5mL into, add the ethanol of 500 μ L 70%, cover tightly lid, slowly Centrifuge tube is overturned back and forth, and then 12000r/min centrifuges 3~5min, carefully outwells ethanol, pipe is inverted on blotting paper.

8) ethanol of 500 μ L 70% is added into centrifuge tube again, covers tightly lid, slowly overturns centrifuge tube back and forth, so 12000r/min centrifuges 3~5min afterwards, carefully outwells ethanol, pipe is inverted on blotting paper.

9) after to be dried, 60 μ L sterilizing ultra-pure waters are added, to be completely dissolved it, 4 DEG C preserve overnight, to be detected.

3rd, agarose gel electrophoresis detection DNA

1) it is gel electrophoresis trough washery is clean, both ends are sealed with adhesive tape, plug comb.

2) 2.8g agarose is weighed, is transferred in triangular flask, adding 1 × TBE 80mL makes its suspension, and microwave ingle adds Heat, wait to seethe with excitement 2 times and take out, final concentration of 0.5 μ g/mL nucleic acid dye is added when it is cooled to non-scald on hand.Then quickly will Agarose solution gentle agitation, prevents bubble.

3) after mixing (about 60 DEG C), immediately by agarose solution to entering in groove.Such as there is bubble, moved immediately with pipettor Go out.

4) completely after cooled and solidified (about 25~40min), comb is pulled out, removes both ends adhesive tape.

5) 1 × tbe buffer liquid is added into electrophoresis tank, liquid level is higher by 2~5mm of glue surface.

6) the μ L of DNA sample 2~4 are taken, are mixed after adding 2 μ L sample-loading buffers, unified loading (notices that the order of pipette tips should be front and rear It is corresponding), and DNAMarker is added on one side.

7) 120V voltages, electrophoresis 1h.

8) observed on uv analyzer, if RNA then needs to purify, if obvious degradation, phase need to be extracted again Answer the DNA of sample.

4th, DNA purifying

1) in 500 μ L DNA solution add 10%SDS make its final concentration of 0.1%, add Proteinase K to final concentration reach To 100 μ g/mL.

2) 55 DEG C are incubated 10h or so.

3) isometric phenol:Chloroform:Isoamyl alcohol (25:24:1) extracted respectively once with chloroform.

4) 12000r/min centrifuges 5min split-phases, draws upper strata aqueous phase into another centrifuge tube.

5) 1/10 volume 3mol/L sodium acetates and 2 times of volumes ice cold absolute ethyl alcohol precipitation DNA are added.

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

5th, spectrophotometry DNA

With OD value of the ultraviolet light photometric determination DNA sample at 260nm, 280nm.Calculate DNA content and OD₂₆₀/ OD₂₈₀Ratio.Such as OD₂₆₀/OD₂₈₀Ratio is less than 1.6, illustrates to contain more protein or phenol in sample, then should carry out pure Change；If ratio is more than 1.8, should consider to remove RNA purifying.

DNA concentration (ng/ μ L)=50 × OD₂₆₀Value × extension rate

DNA detect after, take out a certain amount be diluted to 50ng/ μ L, be stored in -20 DEG C it is standby, remaining deposits in -80 ℃。

6th, PCR is expanded

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 PCR pipes, then add template DNA, then brief centrifugation Laggard performing PCR amplification；PCR reaction systems include 2 × Taq PCR SuperMix (including Taq DNA polymerases, dNTPs and excellent The reaction buffer of change, concentration are 2 ×) 12.5 μ L；The μ L of sense primer 1.0；(upstream and downstream primer concentration is equal by the μ L of anti-sense primer 1.0 For 10pmol/ μ L)；Template DNA (concentration is 50ng/ μ L oxes genomic DNA) 1.0 μ L；The μ L of deionized water 9.5；Totally 25 μ L bodies Long-pending PCR amplification system.

7th, the program of PCR reactions

Pcr amplification reaction program is：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 59.1 DEG C of renaturation 30s, 72 DEG C extend 30s, 36 circulations；72 DEG C of extension 10min, 12 DEG C of preservation amplified productions.

(4) agarose gel electrophoresis is analyzed after expanding PCR primer

1) 3.5% Ago-Gel added with nucleic acid dye, 120V voltages after point sample, electrophoresis 1h are made；

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 imaging analysis indel polymorphisms.

With the gel imaging system PHOTOGRAPHIC ANALYSISs of BIO-RAD Gel Doc 2000, the polymorphic of insertion/deletion (indel) is judged Property：

The ACVR1 genes of ox genome are in AC_000159.1:The insertion/deletion in g33007-33023ins sites (indel) the agarose gel electrophoresis result of polymorphism is：Insertion/deletion genotype (WD) shows as 150bp and 166bp Two band, insertion/insertion genotype (WW) show as the band of 166bp mono-, and missing/deletion Genotype (DD) is shown as The band of 150bp mono-, referring to Fig. 1；Sequence verification is referring to Fig. 2.

(5) the frequency statistics analysis in ox ACVR1 genes indel 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_TT=N_TT/ N, its Middle P_TTRepresent the TT genotype frequencies in a certain site；N_TTRepresent that there is the number of individuals of TT 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_T=(2N_TT+N_Ta1+N_Ta2+N_Ta3+N_Ta4+……+N_Tan)/2N

In formula, P_TRepresent allele T frequencies, N_TTRepresent that there is the individual amount of TT genotype, N in colony_TaiRepresent group There are Tai genotype individuals quantity, the n mutually different multiple alleles that a1~an is allele T in body.

Genotype frequency and equipotential in different yellow cattle breed ACVR1 gene 16-bp insertion/deletions (indel) sites Gene frequency is as shown in table 2.Pi Nanniu, Qaidam ox, Xia Nanniu, Qinchuan Cattle and Nanyang cattle allele " W " frequency point Not Wei 0.424,0.683,0.614,0.767 and 0.692, the frequency of corresponding allele " D " is 0.576,0.317, 0.386th, 0.233 and 0.308, allele " W " and " D " frequency are all higher than 1%, therefore to be stabilized insertion/deletion (indel) allelic gene type, referring to table 2.

The ox ACVR1 gene indel frequency distribution tables of table 2.

(6) association analysis of ox ACVR1 genes indel locus gene effects

Genotype data：The genotype that agarose gel electrophoresis identifies after PCR amplifications.

Creation data：Pi Nanniu, Chaidamu Yellow Cattle, Xia Nanniu, the body height of Qinchuan Cattle and Nanyang cattle, body weight, body is long, body is oblique The body measurement trait data such as length, hip cross height, abdominal circumference, Guan Wei, bust, chest breadth, chest depth, hip width, point of the buttocks are wide, buttocks length.

Relation analysis model：Kind, different factors and growth form correlation are analyzed using SPSS (18.0) softwares.It is first Statistical analysis that first will be descriptive to the data obtained, to determine whether outlier be present.Then according to the characteristic of data, utilize Variance analysis, multivariate linear model or t are analyzed and then to be analyzed the effect of genotype.During data processing, foundation Influence the difference of the factor for the index that body weight, body chi grow, it is contemplated that individual effect, interaction and base between gene Because of the effect of type, correlation analysis is carried out using fixed model.In addition, accepted or rejected according to physical condition, complete model It is as follows：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 the age Effect, G_jFor genotype effects, B_kFor variety effect, S_lFor sex-effects, e_ijklmFor random residual effect.

As a result show：In the research to 840 Chinese Cattle colonies, the 16-bp insertion/deletions of ACVR1 genes (indel) polymorphism encloses (P=0.014) to the pipe of Chinese Cattle and bust (P=0.024) has a significant impact (P<0.05). Therefore, ACVR1 genes 16-bp insertion/deletions (indel) are that Chinese Cattle is grown the insertion/deletion that bust and pipe enclose (indel) genetic marker, referring to table 3.

Influence of the table 3.ACVR1 gene indel polymorphisms to Chinese Cattle growth traits

Note：Data of going together institute different expression significant difference (a, the b of marking-up parent phase:P<0.05) or extremely significantly (A, B:P<0.01).

Sequence table

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

<120>A kind of detection method of ox ACVR1 gene insertion/deletions and its application

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<170> SIPOSequenceListing 1.0

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<211> 23

<212> DNA

<213>Artificial synthesized ()

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cacggaagag acagaccagt att 23

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<212> DNA

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tgggggagag ttccatctgt tt 22

Claims (8)

1. A kind of 1. detection method of ox ACVR1 gene insertion/deletions, it is characterised in that：Comprise the following steps：

   Using ox genomic DNA to be measured as template, using primer pair P1 as primer, ox ACVR1 genes to be measured are expanded by PCR Partial Fragment；Row agarose gel electrophoresis are entered to the PCR fragments for expanding to obtain；Ox ACVR1 to be measured is identified according to electrophoresis result The genotype of gene 16-bp insertion/deletion polymorphic sites；

   The primer pair P1 is：

   Sense primer：5’-CACGGAAGAGACAGACCAGTATT-3’；

   Anti-sense primer：5’-TGGGGGAGAGTTCCATCTGTTT-3’.
2. A kind of 2. detection method of ox ACVR1 gene insertion/deletions according to claim 1, it is characterised in that：It is described PCR amplification reaction system include the μ L of 10pmol/ μ L sense primers 0.3~1, the μ L of 10pmol/ μ L anti-sense primers 0.3~1 and The μ L of 50ng/ μ L template DNAs 0.3~1.
3. A kind of 3. detection method of ox ACVR1 gene insertion/deletions according to claim 1, it is characterised in that：It is described The response procedures of PCR amplifications comprise the following steps：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 59.1 DEG C of renaturation 30s, 72 DEG C are prolonged Stretch 30s；Totally 36 circulations；72 DEG C of extension 10min.
4. A kind of 4. detection method of ox ACVR1 gene insertion/deletions according to claim 1, it is characterised in that：The fine jade The mass concentration for the Ago-Gel that sepharose electrophoresis uses is 3.5%.
5. A kind of 5. detection method of ox ACVR1 gene insertion/deletions according to claim 1, it is characterised in that：It is described to insert Enter/genotype in deletion polymorphism site is：Insertion/insertion genotypic expression is the band lines of 166bp mono-；Insertion/deletion genotype Show as 166bp and the band lines of 150bp two；Missing/deletion Genotype shows as the band lines of 150bp mono-.
6. 6. a kind of detection method of ox ACVR1 gene insertion/deletions as claimed in claim 1 aids in ox molecular labeling Application in selection and use.
7. 7. application according to claim 6, it is characterised in that：Missing/missing gene of the insertion/deletion polymorphic site Type can enclose the DNA marker of index as ox bust and pipe.
8. A kind of 8. detection kit of ox ACVR1 gene insertion/deletions, it is characterised in that：Including including ACVR1 for expanding The primer pair P1, the primer pair P1 of the ox genomic DNA fragment of gene 16-bp insertion/deletion polymorphic sites be：

   Sense primer：5’-CACGGAAGAGACAGACCAGTATT-3’；

   Anti-sense primer：5’-TGGGGGAGAGTTCCATCTGTTT-3’.