CN109735630A - The detection method and label application of sheep ZFY gene mononucleotide polymorphism label - Google Patents

Abstract

The invention discloses the detection method of the single nucleotide polymorphism of sheep ZFY gene and label applications.The label is the single nucleotide polymorphism of sheep ZFY genetic fragment the 403rd G or A.Detection primer includes forward primer and reverse primer, the respectively nucleotide sequence as shown in SEQ ID NO.1 and SEQ ID NO.2.Detection method includes: to extract ram complete genome DNA to be measured；Using ram complete genome DNA to be measured as template, using ewe DNA as negative control, using water as blank control, PCR is carried out using detection primer and expands sheep ZFY gene；With restriction enzyme Bsp1286It digests pcr amplification product and then agarose gel electrophoresis is carried out to the amplified fragments after digestion；The genotype of mononucleotide polymorphism site on sheep ZFY gene is identified according to electrophoresis result.

Description

The detection method and label application of sheep ZFY gene mononucleotide polymorphism label

Technical field

The present invention relates to molecular genetics fields, specifically the detection side of sheep ZFY gene mononucleotide polymorphism label Method and label application.

Background technique

Mammal sex chromosome originates from reptile autosome, is being evolved by a pair of of autosome.Often Chromosome forms X chromosome by constantly breaking up and evolving.

Y chromosome is the smallest chromosome, accounts for about the 2%-3% of haploid genome, and may include 70 to 200 Gene.During evolution, some functional gene missings, Y chromosome are degenerated, multicopy base related with testis and Sperm specific enzyme Cause largely in long-armed rich product and remains, and forms the special area MSY of a male (male-specific region on The Y), account for about the 95% of Y chromosome.And only positioned at the pseudoautosomal region of Y chromosome end (Pseudoautosomal region, PAR) is recombinated during meiosis with X chromosome, crossing over. Mammal Y chromosome, consists of two parts: pseudoautosomal region and the special district's groups of Y chromosome male at.MSY Qu Youchang Chromatin area and heterochromatic zone composition, euchromatic region is by X indexing area (X-transposed), the degenerate region X (X- Degenerate it) is formed with amplification region (ampliconic).The degenerate region X is made of single copy gene, although they are in subtrahend point Do not recombinated with X chromosome during splitting, but with the homologous gene homology with higher on X chromosome.The area MSY Genetic marker, the i.e. haplotype of paternal inheritance, be used to study that the paternal origin of animal population, the migrating of animal, population expands And the research of gene transgression.The gene of the male specific regions of mammal Y chromosome (MSY) is in Sex Determination, male Breeding and development have important function.Pseudoautosomal region forms (PAR1 and PAR2) by 2 parts, accounts for about entire dyeing The 5% of body, on X chromosome with homology region；The special area of Y chromosome male, also known as Y chromosome non-recombinant region (non- Recombining region on Y, NRY), it is not recombinated with X chromosome.PAR1 is located at the terminal region (Yp) of galianconism, It is about 2600kb；PAR2 is about 320kb on long-armed top (Yq).

ZFY gene, that is, y linkage zinc finger protein factor (zinc finger protein, Y-linked) is located at Y and dyes It is dynamic in male by 13 " zinc-refers to " structure compositions of 11 exons and 1 random repeat region on body galianconism (Yp11.3) It plays a role in the growth and development process of object testis,

The DNA homolog gene ZFX gene enters Meiosis and starts to express, until round spermatid expression quantity Highest.It is considered as Sex Differentiation, and spermatogenesis and reproductive growth are leavened dough important gene.ZFX gene includes nuclear location Signaling zone and DNA binding site, energy specific bond target gene, guidance target gene pass through nuclear membrane, are located in Sperm nuclei.

Nucleotides sequence between 9 species ZFX/ZFY such as hair ability and political integrity research discovery yak, orangutan, people, ox is shown compared with high homology Property.Xiong Yong etc. between other species such as breeds differentiation ZFY gene and pig, mouflon, wolf, the mankind research shows that have very high Homology.LAWSON etc. is had found by phylogenetic analysis: sheep ZFY is closer with blue sheep, goat, the affiliation of ox ZFY. ARKADI clones to have obtained the entire code area of entire ox ZFY gene and horse and the gene coding region pig ZFY partial sequence, passes through comparison It was found that the gene coding region ZFY is higher in the homology of 3 species.Early-stage study find sheep ZFY3 and ZFY6 segment and blue sheep, Homology size is in 89.11%-99.6% between the species such as goat, ox, yak, show the gene also have between different plant species compared with High homology, it is almost the same with the research of forefathers.AASEN et al. is identifying people using the amplification and digestion of ZFY segment, Ox, the embryo gender of sheep and goat, the result of the research can also be used for the early stage identification of sheep embryo gender.

There is not been reported for the research screened about the amplification of sheep ZFY gene order and SNPs.

Summary of the invention

It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of mononucleotide polymorphics of sheep ZFY gene Property label detection method and its application, it is special that amplification to the gene specific sequence and SNPs screening will be helpful to discovery kind Anisotropic SNPs and molecular labeling relevant to ram reproductive trait, and then for the identification of the early stage of sheep embryo gender, kind father It is building and the early screening of prolificacy stud ram of haplotype.

The purpose of the present invention is achieved through the following technical solutions: a kind of mononucleotide polymorphic of sheep ZFY gene Property label detection method, the label for ZFY genetic fragment the 403rd G or A single nucleotide polymorphism, it is described Detection method includes the following steps:

S1. ram complete genome DNA to be measured is extracted；

S2. using ram complete genome DNA to be measured as template, using ewe DNA as negative control, using water as blank control, PCR amplification sheep ZFY gene is carried out using detection primer, obtains amplified production, and in cryo-conservation；

S3. pcr amplification product is digested with restriction enzyme Bsp1286I and then the amplified fragments after digestion are carried out Agarose gel electrophoresis；The genotype of mononucleotide polymorphism site on sheep ZFY gene is identified according to electrophoresis result.

Preferably, the detection primer includes forward primer and reverse primer, the forward primer and the reverse primer The respectively nucleotide sequence as shown in SEQ ID NO.1 and SEQ ID NO.2.

Preferably, it in the S2, when carrying out PCR amplification sheep ZFY gene using the detection primer, need to first prepare Reaction system:

Tks Gflex DNA Polymerase 12.50 μ L, 0.2 μM of 0.5 μ L of forward primer, 0.2 μM of reverse primer 0.5 μ L, sterile purified water 10.5 μ L, DNA profiling 50ng.

Preferably, in the S2, the PCR amplification control process are as follows: 94 DEG C of initial denaturation 1min；98 DEG C of denaturation 10s, 58 DEG C annealing 15s, 72 DEG C of extensions 30s, 30 recycle；72 DEG C extend 1min eventually.

Preferably, in the S2,4 DEG C of cryo-conservation environmental selection.

Preferably, in the S3, the mass concentration of the Ago-Gel that the agarose gel electrophoresis process uses for 1%.

A kind of application of the single nucleotide polymorphism of sheep ZFY gene, the label are ZFY genetic fragment the The single nucleotide polymorphism of 403 G or A apply the label in sheep assisted Selection and molecular breeding.

The beneficial effects of the present invention are: the present invention utilizes polymerase chain reaction-restriction fragment length polymorphism (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction, RFLP-PCR) Method carries out parting to the mutation of the 403rd A > G of sheep ZFY genetic fragment, because the site is located at sheep ZFY gene intron 1, mutation does not cause the change of amino acid, and the space two of ZFY coded by said gene albumen, three-level configuration do not change；And it is directed to The SNP polymorphism of ZFY gene, detection method of the invention can use RFLP- by designing specific PCR primer amplified fragment PCR method polymorphism that is simple, quick, at low cost, accurately detecting its mononucleotide, and sequence site detected is without spy Different property requirement.

Detailed description of the invention

Fig. 1 is the ram blood sample genomic DNA agarose gel electrophoresis figure in the embodiment of the present invention；

Fig. 2 is the agarose gel electrophoresis of the sheep ZFY gene PCR amplified production in the embodiment of the present invention；

Fig. 3 is Ago-Gel of the ram ZFY gene PCR product after Bsp1286I digestion in the embodiment of the present invention Electrophoresis；

Fig. 4 is the different genotype sequencing peak of ram ZFY genetic fragment the 403rd SNP in the embodiment of the present invention Figure.

Specific embodiment

Technical solution of the present invention is described in further detail with reference to the accompanying drawing, but protection scope of the present invention is not limited to It is as described below.

Embodiment

The detection of ram ZFY gene pleiomorphism

1, the acquisition and processing of sheep blood sample

Sheep blood sample 5mL is taken, the EDTA500 μ L that 0.5mol/L is added is anticoagulant, slowly it is put into ice chest after reverse 3 times, -80 DEG C It saves backup.

The sheep variety that the present embodiment uses, it is specific as shown in table 1.

1 sheep sample message of table

2, the extraction of blood sample genomic DNA

(1) blood sample freezed melts in room-temperature water bath；

(2) 1mL whole blood is transferred in a sterile 2mL centrifuge tube；

(3) PBS buffer solution of 1mL is added, mildly shakes 15-20min；

(4) 4 DEG C, 12000rpm is centrifuged 10min；

(5) supernatant is abandoned with liquid-transfering gun, repetition step (3), (4) are transparent to supernatant, precipitate white；

(6) add DNA extracting solution 650-750 μ L in centrifuge tube, mild shake makes cell precipitation suspend；

(7) 37 DEG C of water-bath 1h；

(8) 3 μ L of Proteinase K is added, mixes；

(9) in constant water bath box 55 DEG C be incubated overnight (16h or so), until cell precipitate is digested completely, solution is clear Clearly；

(10) reaction solution is cooled to room temperature, and the Tris saturated phenol of 800 μ L-1mL is added, and is placed and is mildly shaken 15min on ice；

(11) 4 DEG C, 12000rpm is centrifuged 10min；

(12) upper strata aqueous phase is moved on in another sterile centrifugation tube with liquid-transfering gun；

(13) phenol of 0.5mL and the chloroform of 0.5mL is added, places and mildly shakes 15min on ice；

(14) 4 DEG C, 12000rpm is centrifuged 10min；

(15) upper strata aqueous phase is moved on in another sterile centrifugation tube with liquid-transfering gun；

(16) chloroform of 1mL is added, places and mildly shakes 15min on ice；

(17) 4 DEG C, 12000rpm is centrifuged 10min；

(18) upper strata aqueous phase is moved on in another sterile centrifugation tube with liquid-transfering gun；

(19) the pre-cooling dehydrated alcohol (dehydrated alcohol temperature is -20 DEG C) of 2mL is added, jiggles and is repeatedly precipitated to DNA, Then 30min is placed under the conditions of -20 DEG C；

(20) 4 DEG C, 12000rpm is centrifuged 10min；Abandon supernatant

(21) 70% ethyl alcohol 1mL is added, mildly shakes 10min；

(22) 4 DEG C, 12000rpm is centrifuged 10min, abandons ethyl alcohol, repeats to rinse primary；

(23) it is dried in vacuo or makes ethyl alcohol volatilization clean at room temperature；

(24) according to the amount of DNA, ultrapure water 100-300 μ L is added, 4 DEG C of preservations to DNA are completely dissolved, spectrophotometric measurement After determining concentration, -80 DEG C of preservations.

3, the building in the pond DNA

The detection of (1) 1% agarose gel electrophoresis

To DNA sample carry out agarose gel electrophoresis detection, select DNA sample band it is uniform, without hangover, the sample without degradation The building in the product progress pond DNA.

(2) OD value measures

With OD value of the ultraviolet light photometric determination DNA sample at 260nm, 280nm.Calculate DNA content and OD260/ The ratio of OD280.If OD260/OD280 ratio is less than 1.6, illustrate to contain more protein or phenol in sample, then Ying Jinhang Purifying；If ratio is greater than 1.8, should consider to remove RNA purifying.

DNA concentration (ng/ μ L)=50 × OD260 value × extension rate

(3) building in the pond kind DNA

A certain amount is taken out after DNA is detected, in every sheep DNA stoste and is diluted to 50ng/ μ L, and 10 μ L is then taken to construct The pond DNA, each pond DNA are mixed by the genomic DNA of 10 different sheep；

The testing result of ram blood sample genomic DNA is shown in Fig. 1, wherein swimming lane 1-4 is ram genomic DNA, from It can be seen that the quality of ram genomic DNA is very high in figure.

4, PCR amplification

Using the pond ram DNA as template, it is control with ewe DNA and water, carries out PCR amplification with the primer pair P of design, PCR overall reaction system is 25 μ L (table 2), and PCR overall reaction program is shown in Table 3.

2 PCR reaction system of table

3 PCR response procedures of table

5, PCR product male specificity verification, purifying and sequencing

PCR amplification carries out agarose gel electrophoresis after completing, and electrophoresis result is as shown in Figure 2, wherein 1-3 swimming lane is public affairs Sheep DNA cloning product (547bp)；4-6 swimming lane is ewe DNA cloning product；7-9 swimming lane is aqua sterilisa amplified production；M is Marker (label), here it is apparent that only can amplify PCR product band, and amplified production by template of ram DNA Length is consistent with expected 547bp, and does not have any PCR product using ewe and water as template, can determine that PCR product is Sheep Y chromosome genetic fragment；Then the gel extraction and purifying of PCR product are carried out: in the UV lamp from Ago-Gel The gel containing target fragment is cut, is put into 1.5mL centrifuge tube, is then recycled with the Ago-Gel DNA of PCR product recycling Kit (Tiangeng biochemical technology Co., Ltd) purified pcr product, operates according to kit specification, the specific steps are as follows:

(1) 500 μ L equilibrium liquids are added into adsorption column first, 12000r/min is centrifuged 1min, outwells useless in collecting pipe Liquid places back in adsorption column in collecting pipe.

(2) single target DNA band is put into clean centrifuge tube from cutting in Ago-Gel, weighs weight.

(3) isometric solution PC is added into blob of viscose, 10min or so is placed in 60 DEG C of water-baths, constantly leniently upper and lower therebetween Centrifuge tube is overturn, to ensure that blob of viscose sufficiently dissolves.

(4) previous step acquired solution is added in an adsorption column, 12000r/min is centrifuged 1min, outwells in collecting pipe Adsorption column is reentered into collecting pipe by waste liquid.

(5) 700 μ L rinsing liquids are added into adsorption column, 12000r/min is centrifuged 1min, outwells waste liquid, again by adsorption column It is put into collecting pipe.

(6) 500 μ L rinsing liquids are added into adsorption column, 12000r/min is centrifuged 1min, waste liquid is outwelled, by centrifugal adsorbing column It is put into collecting pipe, 12000r/min is centrifuged 2min, as far as possible removing rinsing liquid.Adsorption column is placed in room temperature or 50 DEG C of incubator numbers point Clock thoroughly dries.

(7) adsorption column is put into a clean centrifuge tube, suitable elution is vacantly added dropwise to adsorbed film middle position Buffer is placed at room temperature for 2 minutes.12000r/min is centrifuged 1min and collects DNA solution.

(8) in order to improve the yield of DNA, the solution that can obtain centrifugation in add-back centrifugal adsorbing column, repeats to walk again Rapid 7.

Bidirectional sequencing is carried out marine growth Engineering Co., Ltd is served as the PCR purified product of template using the pond ram DNA. The sequencing result of sheep ZFY gene target fragment as shown in figure 4, its nucleotide sequence as shown in SEQ ID NO.3.

Sequencing peak figure is analyzed, wherein there are two different peaks being single nucleotide mutation has occurred in same site； Occur two kinds of testing results of A and G positioned at sheep ZFY genetic fragment the 403rd, the as sheep ZFY gene SNP that arrives of screening is more State property, the site are the nucleotide polymorphisms for A or G.

Sheep ZFY Gene A > G mutation polymorphism RFLP-PCR detection

Since the nucleotide polymorphisms that screening is arrived are nature restriction enzyme site, PCR-RFLP mirror can be carried out by common restriction endonuclease It is fixed.When the 403rd generation A > G mutation of sheep ZFY genetic fragment, G after being as mutated utilizes the ZFY gene sequence of primer amplification GDGCHC/GHCGDG is arranged, is the restriction enzyme enzyme recognition site of Bsp1286I；It can be directly by Bsp1286I to target fragment Digestion carry out Genotyping.

1, RFLP-PCR primer

PCR amplification primer is the above-mentioned primer P for being verified as male specificity:

Forward primer: 5 '-CAACTGTGAACTTGGTGAAG-3 '；20nt(SEQ ID NO.1)；

Reverse primer: 5 '-GTAGGCATGAATGACAATCTC-3 '；21nt(SEQ ID NO.2).

The primer can expand sheep ZFY gene 547bp segment.

2, RFLP-PCR reaction condition

Respectively as described in table 2 and table 3,1% agarose of pcr amplification product is solidifying for PCR product amplification system and reaction condition Gel electrophoresis map is as shown in Figure 2, it can be seen that the primer of design can expand the segment of 547bp.

3, the Bsp1286I digestion of pcr amplification product

(1) digestion system are as follows: expand for 1 μ L, Buffer 2.5 μ L, ddH2O 17.5 μ L of Bsp1286I restriction endonuclease, 4 μ LPCR Increase production object, amounts to 25 μ L.

(2) it is digested condition: digesting 3-4h in 37 DEG C of constant incubators.

(3) agarose gel electrophoresis is analyzed after Bsp1286I digests PCR product

With 1% Ago-Gel, 160V electrophoresis 30min, nucleic acid staining dye detects digestion as a result, solidifying with UVP Glue imaging system (GelDoc-It TS Imaging System) PHOTOGRAPHIC ANALYSIS, and sentence type, record its genotype；

Due to including others Bsp1286I digestion recognition site in the 547bp segment of PCR-RFLP amplification, when ZFY gene When the 403rd generation A > G mutation of segment, after the ZFY gene product of PCR amplification is identified by restriction enzyme Bsp1286I, GDGCHC/GHCGDG is cut to 2 sections to amplified fragments digestion, by amplified fragments；And it does not dash forward when ZFY genetic fragment the 403rd Become, restriction endonuclease Bsp1286I cannot cut amplified fragments.

Since the Y chromosome of sheep is monoploid, so 2 kinds of different genes can be formed when the mutation of C > G occurs The gel result of type, respectively AA and GG genotype, PCR-RFLP detection is as shown in Figure 3, wherein swimming lane 1-3:AA genotype Individual (547bp)；Swimming lane 4-6:GG genotype individuals (403bp and 144bp)；M: label (2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp)；Wherein AA genotype is wild type, by Bsp1286I digestion, cannot show as one of 547bp Band；GG genotype is saltant type, can show as two band of 403bp and 144bp by Bsp1286I digestion.According to the number of band With strip segments size, what detected through gel electrophoresis result as shown in Figure 3 can will be apparent that determines whether to have occurred point mutation, will Two kinds of genotype distinguish, to detect its SNP polymorphism.

(4) sequence verification of different genotype individual PCR product

Positive and negative two-way survey is carried out respectively to different genotype individual PCR product using ABI 5019 and 3730 sequenator of ABI Sequence；Meanwhile carrying out SNP position analysis, the results showed that its ZFY gene piece of the AA genotype individuals of the band comprising 547bp bp The 403rd sequencer map of section is expressed as A really, as shown in fig. 4 a；The GG genotype of two bands comprising 403bp bp and 144bp Body the 403rd sequencer map of its ZFY genetic fragment is expressed as G really, as shown in Figure 4 b.Wherein in Fig. 4, Fig. 4 a is ram ZFY The sequencing result of the 403rd AA genotype individuals of genetic fragment；Fig. 4 b is the 403rd GG genotype of ram ZFY genetic fragment The sequencing result of individual.

Polymorphism of the SNP that sheep ZFY genetic fragment is the 403rd as molecular labeling in different sheep varieties

1, the detection of group's single nucleotide polymorphism

Using above-mentioned SNP pleiomorphism detecting method to 594 parts of DNA samples of sheep, the identification of SNP polymorphism is carried out；System Count the frequency distribution situation of its SNP site.

2, the frequency statistics analysis of SNP site

It is single times since the special area of the male of Y chromosome does not recombinate during meiosis with X chromosome Body, only there are two types of genotype, therefore its gene frequency and genotype are equal.

Genotype frequency refers to that certain genotype individuals number of a certain character in a group accounts for the ratio of total individual number.

Gene frequency refers to that a certain gene number is to the relative ratios of its allele sum in a group.Therefore, base Because frequency and genotype are equal.

3, the association analysis of gene effect

Genotype data: the genotype (AA and GG) of Bsp1286I identification

Ram Testicular Size data: the scrotum of Suffolk and SA Meat Merino encloses (6 monthly age).

Utilize the correlation of 19.0 software of SPSS analysis gene loci and sheep Testicular Size.First data are described Property statistical analysis, it is determined whether there are outliers, according to data characteristics, probe into gene using one-way analysis of variance (ANOVA) Type effect.

The result shows that (being shown in Table 4, table 5 and table 6): can recognize ZFY genetic fragment the 403rd position SNP for Bsp1286I Point only shows polymorphism in two kinds of Suffolk and SA Meat Merino, and in white Suffolk, Te Kesaier Sheep, Du Boyang, Dong Fuli rise in sheep, hiding sheep, sheep known for its fine thick wool and sheep and do not show polymorphism then.Suffolk, Te Kesaier sheep are continuous There was only AA genotype in sheep；Du Boyang, Dong Fuli, which rise in sheep, hiding sheep, sheep known for its fine thick wool and sheep, only has GG genotype.

Trait associations analysis shows, in the Sheep Populations of Suffolk, the testis of AA genotype individuals has than GG genotype It is individual (table 5) to show that the site can be used for screening the biggish sheep of screening testis for the big trend of body (0.05 < P < 0.1).

4 the 403rd SNP Gene frequency distribution table of sheep ZFY gene segment of table

Association analysis of the 5 ZFY genetic fragment of table the 403rd between SNP and Suffolk reproductive trait

Association analysis of the 6 ZFY genetic fragment of table the 403rd between SNP and SA Meat Merino reproductive trait

To sum up, in the specific embodiment of the invention, firstly, utilizing polymerase chain reaction-restriction fragment length polymorphism (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction, RFLP-PCR) Method carries out parting to the mutation of the 403rd A > G of sheep ZFY genetic fragment, because the site is located at sheep ZFY gene intron 1, mutation does not cause the change of amino acid, and the space two of ZFY coded by said gene albumen, three-level configuration do not change.Secondly, needle To the SNP polymorphism of above-mentioned ZFY gene, detection method is by designing specific PCR primer amplified fragment, Neng Gouyong RFLP-PCR method polymorphism that is simple, quick, at low cost, accurately detecting its mononucleotide, and sequence position detected Point is without particularity requirement.In addition, the present invention will screen the obtained site SNPs and 6 monthly age of Suffolk sheep Testicular Size and epididymis Size correlated traits is associated analysis, it is found that the site SNPs is closely related with correlated traits, can be used for screening testis and attached The biggish Suffolk sheep individual of testis.

Since ZFY gene is located at Y chromosome male special zone, the gene in the region does not dye during meiosis with X Body recombinates.Therefore ZFY gene is not present in female individuals, sheep gender can be identified using the principle.

Sheep, hiding sheep, sheep known for its fine thick wool belong to place of china sheep variety.Gene frequency deducibility: there was only GG genotype in China Individual.Sheep Y chromosome haplotype can be identified using the Y-SNP and other identified Y-SNPs；It can be first using the result Step identifies the paternal origin of sheep.

The above is only a preferred embodiment of the present invention, it should be understood that the present invention is not limited to described herein Form should not be regarded as an exclusion of other examples, and can be used for other combinations, modifications, and environments, and can be at this In the text contemplated scope, modifications can be made through the above teachings or related fields of technology or knowledge.And those skilled in the art institute into Capable modifications and changes do not depart from the spirit and scope of the present invention, then all should be in the protection scope of appended claims of the present invention It is interior.

Claims (7)

1. a kind of detection method of the single nucleotide polymorphism of sheep ZFY gene, it is characterised in that: the label is The single nucleotide polymorphism of ZFY genetic fragment the 403rd G or A, described detection method includes the following steps:

S1. ram complete genome DNA to be measured is extracted；

S2. it using ram complete genome DNA to be measured as template, using ewe DNA as negative control, using water as blank control, utilizes Detection primer carries out PCR and expands sheep ZFY gene, obtains amplified production, and in cryo-conservation；

S3. pcr amplification product is digested with restriction enzyme Bsp1286I and then fine jade is carried out to the amplified fragments after digestion Sepharose electrophoresis；The genotype of mononucleotide polymorphism site on sheep ZFY gene is identified according to electrophoresis result.

2. a kind of detection method of the single nucleotide polymorphism of sheep ZFY gene according to claim 1, special Sign is: the detection primer includes forward primer and reverse primer, and the forward primer and the reverse primer are respectively such as Nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2.

3. a kind of detection method of the single nucleotide polymorphism of sheep ZFY gene according to claim 2, special Sign is: in the S2, when carrying out PCR amplification sheep ZFY gene using the detection primer, need to first prepare reactant System:

Tks Gflex DNA Polymerase 12.50 μ L, 0.2 μM of 0.5 μ L of forward primer, 0.2 μM of reverse primer 0.5 μ L, sterile purified water 10.5 μ L, 50 ng of DNA profiling.

4. a kind of detection method of the single nucleotide polymorphism of sheep ZFY gene according to claim 2 or 3, Be characterized in that: in the S2, the PCR expands control process are as follows: 94 DEG C of 1 min of initial denaturation；98 DEG C of denaturation 10 s, 58 DEG C Anneal 15 s, 72 DEG C of 30 s of extension, 30 circulations；72 DEG C extend 1 min eventually.

5. a kind of detection method of the single nucleotide polymorphism of sheep ZFY gene according to claim 2, feature It is: in the S2,4 DEG C of cryo-conservation environmental selection.

6. a kind of detection method of the single nucleotide polymorphism of sheep ZFY gene according to claim 1, feature Be: in the S3, the mass concentration of the Ago-Gel that the agarose gel electrophoresis process uses is 1%.

7. a kind of application of the single nucleotide polymorphism of sheep ZFY gene, it is characterised in that: the label is ZFY The single nucleotide polymorphism of genetic fragment the 403rd G or A apply the label in sheep assisted Selection and molecular breeding.