CN108315445A - It is a kind of detection sheep sry gene single nucleotide polymorphism method and application - Google Patents
It is a kind of detection sheep sry gene single nucleotide polymorphism method and application Download PDFInfo
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- CN108315445A CN108315445A CN201810388088.7A CN201810388088A CN108315445A CN 108315445 A CN108315445 A CN 108315445A CN 201810388088 A CN201810388088 A CN 201810388088A CN 108315445 A CN108315445 A CN 108315445A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
- C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a kind of method of single nucleotide polymorphism of detection sheep sry gene and applications.The single nucleotide polymorphism be sheep sry gene 215bp G/A it is polymorphic.This approach includes the following steps：Extract ram complete genome DNA to be measured；Using ram complete genome DNA to be measured as template, PCR amplification sheep sry gene is carried out using forward primer and reverse primer；With restriction enzyme Hpy188I digestion pcr amplification product and then to the amplified fragments after digestion into row agarose gel electrophoresis；Identify the genotype of mononucleotide polymorphism site on sheep sry gene.The nucleotide sequence of forward primer and reverse primer is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.The present invention can be used for Embryo sexing, paternal haplotype identification and improve ram reproductive capacity, and then accelerate sheep stock breeding speed.
Description
Technical field
The invention belongs to molecular genetics fields, specifically, it is more to be related to a kind of mononucleotide of detection sheep sry gene
The method of state property and application.
Background technology
The sex chromosome (X and Y chromosome) of mammal is initially from a pair of original autosome evolution.Property
In the evolutionary process of chromosome, X chromosome tends to conservative, and the quantity and structure of gene are highly conserved between mammal.Y contaminates
Colour solid is then then lost the most gene on ancestors' sex chromosome during evolution.Y chromosome has and other chromosomes
Different characteristics, 95% region do not occur pairing recombination with X chromosome during meiosis, are referred to as Y chromosome
Male special area (Male specific region of Y chromosome, MSY), and only positioned at Y chromosome end
During meiosis with X chromosome weight occurs for pseudoautosomal region (Pseudoautosomal region, PAR)
Group, crossing over.Mammal Y chromosome has the feature of species specificity, and gene content is in different species
There is bigger difference, this species diversity exists in the areas MSY and the areas PAR.
Before the 1950s, because its heterochromatin characteristic and smaller length, Y chromosome are considered as gene
Desert Regions do not contain any function.But by 1976, there is the life of the missing and sperm in some regions of scholar finder's Y chromosome
At related, this region is later known as the azoospermatism factor (AZF).Research shows that the congenital of 10%-18% lacks essence and without essence
Disease is micro-deleted related with what it is positioned at AZF.It is referred to as the discovery of the sex determining gene of testicular determinant with the latter, makes people
More firmly believe that Y chromosome is not without transcriptional activity.Nineteen ninety, Sinclair and his colleague order this testicular determinant
Entitled Y chromosome Sex Determination region (Sex-determining region Y, SRY).Research finds that sry gene can start
The expression of many male specific genes, the transfer of induced male gonadal cell and proliferation, and mammal sex determination is risen
Key effect.Numerous studies show that in addition to a small number of species, sry gene is present in almost all of mammal.Numerous researchs
Show that sry gene is the core gene of Sex Determination, directly induce testis development, critical effect is played to testicualr development,
SRY can trigger the generation of testis, determine male property.Sry gene is relatively conservative in mammals.Mankind's SRY bases
The open reading frame of cause includes 1 exon, the protein of 204 amino acid of codified.The protein can be divided into 3 regions,
79 amino acid therein is known as HMG boxes (high mobility group box, HMG box), the protein tool in this region
The function of having in conjunction with DNA and make DNA bendings, is the major function area of SRY.In addition, HMG boxes also carry 2 nuclear localization signals.Than
Compared with the SRY albumen of the animals such as people and mouse, rabbit, wallaby, sheep, as a result, it has been found that have 70% homology in the regions HMG, and
There is no conserved sequence outside the regions HMG.This shows sry gene other than having the function of Sex Determination, may also have other work(
Can, such as Sperm specific enzyme.Sry gene navigated on sheep Y chromosome it is long-armed on.Mark of the sry gene as paternal inheritance
Note has successfully been applied to Sex Determination, become middle hybridization, descent of man and evolutionary analysis, Phylogenetic Analysis and heredity
Polymorphism analysis.Although many, about domestic animal sry gene, researches show that limited genetic diversities, it is remained able to
Some valuable information are provided in paternal inheritance structure.
The structure of Y chromosome is sufficiently complex, and the gene order and palindrome repeated by a large amount of height forms, and sequencing is very
It is difficult, it is difficult to study.So far, the Y chromosome of only a small number of species such as the mankind, chimpanzee, macaque, mouse, ox completes
Sequencing.About the Y chromosome of sheep, scholars only amplify the Partial Fragment of a small number of genes, still obtain complete Y without research
Chromosomal gene sequence.(Jiang Li, Wang Kanghuan, Wang Hai wait Altai Sheeps, tibetan sheep (Jia Luo monoids, Euler's monoid) to Jiang Li etc.
Sry gene sequence analysis and affiliation research [J] Southwest University for Nationalities journal (natural science edition), 2013,39 (4):487-
494.) goat sry gene primers, amplification is utilized to obtain the entire code area sequence of Altai Sheep, tibetan sheep sry gene
Row, CDS overall length 723bp, and the homology with height between different sheep varieties.Meadows etc. (Meadows J R,
Hawken R J,Kijas J W.Nucleotide diversity on the ovine Y chromosome.[J]
.Animal Genetics,2015,35(5):379-385.) screen to obtain the mutation of an A/G in 5 ' promoter region of sry gene.
(Zhang G, Vahidi S M F, Ma Y H, the et al.Limited polymorphisms of two Y- such as Zhang
chromosomal SNPs in Chinese and Iranian sheep[J].Animal Genetics,2012,43(4):
The mutation of G/T 479-480.) is screened in sry gene.
Up to the present, many researchers have found the sites SNPs in sry gene, and are ground for the evolution of sheep paternal origin
Study carefully and paternal haplotype is built.But sheep sry gene SNPs and the research of Sheep Reproductive Characters association analysis are less.
Invention content
In view of this, the present invention provides a kind of method of single nucleotide polymorphism of detection sheep sry gene and application,
It is detected for the single nucleotide polymorphism on its gene loci using PCR-RFLP methods.
In order to solve the above-mentioned technical problem, the invention discloses a kind of single nucleotide polymorphism of sheep sry gene,
It is described label be sheep sry gene 215bp G/A it is polymorphic.
The invention also discloses the primer used in a kind of single nucleotide polymorphism that detection is above-mentioned, the primer includes
The nucleotide sequence of forward primer and reverse primer, forward primer and reverse primer is respectively such as SEQ ID NO.1 and SEQ ID
Shown in NO.2.
The invention also discloses a kind of methods of the single nucleotide polymorphism of detection sheep sry gene, include the following steps：
1) ram complete genome DNA to be measured is extracted；
2) using ram complete genome DNA to be measured as template, the forward primer and reverse primer described in claim 2 are utilized
Carry out PCR amplification sheep sry gene；
3) it digests pcr amplification product with restriction enzyme Hpy188I and then fine jade is carried out to the amplified fragments after digestion
Sepharose electrophoresis；The genotype of mononucleotide polymorphism site on sheep sry gene is identified according to electrophoresis result.
Optionally, PCR reaction systems are：2 × EasyTaqSuperMix, 12.50 μ L, 0.4 μ L of forward primer, reversely draw
0.4 μ L of object, 10.7 μ L of distilled water, 1 μ L of DNA profiling, with upper volume total amount for 25 μ L.
Optionally, pcr amplification reaction program is：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 62.6 DEG C of 30s that anneal, 72
DEG C extend 30s, 34 cycle；72 DEG C of extension 10min；4 DEG C of preservations.
Optionally, the mass concentration of the Ago-Gel is 2.5%.
Optionally, the mononucleotide polymorphism site shows as the single nucleotide polymorphism of 215bp G or A, tool
There are two kinds of genotype of AA and GG, electrophoresis result to be respectively：AA types show as tetra- bands of 54bp, 63bp, 151bp and 172bp；GG
Type shows as tri- bands of 63bp, 172bp and 205bp.
The invention also discloses a kind of above-mentioned single nucleotide polymorphism, primer, methods in sheep assisted Selection and
Application in molecular breeding.
The invention also discloses a kind of above-mentioned single nucleotide polymorphism, primer, methods in Embryo sexing, silk floss
The application in ram reproductive capacity is identified and improved to sheep paternal line haplotype.
Compared with prior art, the present invention can be obtained including following technique effect：
1) present invention using RFLP-PCR methods to the mutation on the positions 215bp of sheep sry gene segment there may be
The coding changed single nucleotide polymorphism of protein conformation is detected, and is G when site sports A by G>The mutation of A,
The mutation does not cause the change of amino acid, belongs to same sense mutation, makes have albumen coded by the sry gene of important physiological function
Space two, three-level configuration do not change.
2) the SNP polymorphisms of above-mentioned sry gene are directed to, the invention also discloses its detection methods, specific by designing
PCR primer amplified fragments, can it is simple, quick, at low cost with RFLP-PCR methods, accurately detect the polymorphic of its mononucleotide
Property.
Certainly, it implements any of the products of the present invention it is not absolutely required to while reaching all the above technique effect.
Description of the drawings
Attached drawing described herein is used to provide further understanding of the present invention, and constitutes the part of the present invention, this hair
Bright illustrative embodiments and their description are not constituted improper limitations of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 is sheep blood sample genome dna electrophoresis detection figure of the present invention；Wherein, 1-4 is ovine genome DNA；
Fig. 2 is the electrophoretogram of the 440bp segments of sheep sry gene PCR amplification of the present invention；Wherein, 1-3 swimming lanes are ram
DNA cloning product；4-6 swimming lanes are ewe DNA cloning product；7-9 swimming lanes are aqua sterilisa amplified production；M is Marker；
Fig. 3 is the Hpy188I restriction enzyme digestion and electrophoresis detection sry gene polymorphism of sheep sry gene 440bp PCR products of the present invention
Electrophoresis result figure；Wherein, swimming lane 1,3：AA genotype individuals (54bp, 63bp, 151bp and 172bp)；Swimming lane 2,4：GG genes
Type individual (63bp, 172bp and 205bp)；M：Marker (2000bp, 1000bp, 750bp, 250bp and 100bp)；
Fig. 4 is the different genotype sequencing peak figure of 215bp SNP of sheep sry gene segment of the present invention, and wherein Fig. 4 a are
The sequencer map of sheep sry gene 215 GG genotype individuals of segment；Fig. 4 b sheep is 215 AA genotype of sry gene segment
The sequencer map of body.
Specific implementation mode
Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section solves technical problem and reaches the realization process of technical effect to fully understand and implement.
The present invention is with sry gene conserved sequence (Gene ID:100529253) design primer expands sry gene 440bp pieces
Section carries out PCR amplification using ovine genome as template, and the mononucleotide that amplified production finds the amplified fragments after sequencing is more
State.
The detection of 1 sheep sry gene polymorphism of embodiment
1, the acquisition and processing of sheep blood sample
Sheep blood sample 5mL is taken, the 500 μ L anti-freezings of EDTA of 0.5mol/L are added, is slowly put into ice chest after reverse 3 times, -80
It DEG C saves backup.
The present invention uses sheep variety, 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 bloods are transferred in a sterile 2mL centrifuge tube；
(3) PBS buffer solution of isometric (1mL) is added, mildly shakes 15-20min；
(4) 4 DEG C of 12000g centrifuge 10min；
(5) supernatant is abandoned with liquid-transfering gun, repeats step 3,4, precipitate whites transparent to supernatant；
(6) add DNA extracting solution 650-750 μ L, mild shake that cell precipitation is made to suspend in centrifuge tube；
(7) 37 DEG C of water-bath 1h；
(8) 3 μ L of Proteinase K (final concentration of 60ug/mL), mixing is added；
(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 phenols of 1 times of volume (800 μ L-1mL) are added, and is placed mild on ice
Shake 15min；
(11) 4 DEG C, 12000g centrifuges 10min；
(12) upper strata aqueous phase is moved on to liquid-transfering gun in another sterile centrifugation tube；
(13) chloroform of the phenol and 0.5 times (0.5mL) of 0.5 times of volume (0.5mL) is added, places mild on ice shake
15min；
(14) 4 DEG C, 12000g centrifuges 10min；
(15) upper strata aqueous phase is moved on to liquid-transfering gun in another sterile centrifugation tube；
(16) chloroform of 1 times of volume (1mL) is added, places and mildly shakes 15min on ice；
(17) 4 DEG C, 12000g centrifuges 10min；
(18) upper strata aqueous phase is moved on to liquid-transfering gun in another sterile centrifugation tube；
(19) the precooling absolute ethyl alcohol (- 20 DEG C) of 2 times of volumes is added, gently mouth bottom, which is rocked, is repeatedly precipitated to DNA, then-
20 DEG C of placement 30min；
(20) 4 DEG C, 12000g centrifuges 10min；Abandon supernatant
(21) 70% ethyl alcohol 1mL is added, mildly shakes 10min；
(22) 4 DEG C, 12000g centrifuges 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, ultra-pure water 100-300 μ L are added, 4 DEG C of preservations to DNA are completely dissolved, spectrophotometric measurement
After determining concentration, -80 DEG C of preservations.
3, the structure in the ponds DNA
(1) 1% agarose gel electrophoresis detects
Select part DNA sample into row agarose gel electrophoresis detect, select DNA sample band it is uniform, without hangover, without degradation
Sample carry out the structure in DNA ponds.
(2) OD values measure
With OD value of the ultraviolet light photometric determination DNA sample at 260nm, 280nm.Calculate DNA content and OD260/OD280
Ratio.Such as OD260/OD280Ratio is less than 1.6, illustrates to contain more protein or phenol in sample, then should be purified；If
Ratio is more than 1.8, then should consider to remove RNA purifying.
DNA concentration (ng/ μ L)=50 × OD260Value × extension rate
(3) structure in the ponds kind DNA
After DNA is detected, takes out a certain amount and be diluted to 50ng/ μ L, then from a concentration of 50ng/ μ L DNA samples
Take 10 ponds μ L mixing structure DNA；
The testing result of sheep blood sample genomic DNA is shown in Fig. 1, and as can be seen from the figure the quality of ovine genome DNA is non-
Chang Gao.
4, PCR amplification
Using the ponds sheep DNA as masterplate, it is control with ewe DNA and water, PCR amplification, PCR is carried out with the primer pair P of design
Overall reaction system is 25 μ L (table 2), and PCR overall reaction programs are shown in Table 3.
2 PCR reaction systems of table
The best PCR response procedures of 3 primer of table
5, PCR product male specificity verification, purifying and sequencing
Into row agarose gel electrophoresis after PCR amplification completion, electrophoresis result is as shown in Figure 2, here it is apparent that only
PCR product band can be amplified by template of ram DNA, and amplified production length is consistent with expected 440bp, and with mother
Sheep and water, which are template, does not have any PCR product, it may be determined that PCR product is sheep Y chromosome genetic fragment；Then it carries out
The gel extraction of PCR product and purifying：The gel containing target fragment is cut from Ago-Gel in the UV lamp, is put into
In 1.5mL centrifuge tubes, then PCR product recovery purifying kit (Beijing six directions Hua Da Gene Tech. Company Limited) is used to purify
PCR product is operated according to kit specification, is as follows：
(1) 500 μ L equilibrium liquids are added into adsorption column first, 12000r/min centrifuges 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 being cut 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 fully dissolves.
(4) previous step acquired solution is added in an adsorption column, 12000r/min centrifuges 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 centrifuges 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 centrifuges 1min, waste liquid outwelled, by centrifugal adsorbing column
It is put into collecting pipe, 12000r/min centrifuges 2min, removes rinsing liquid as possible.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 centre position
Buffer solution is placed at room temperature for 2 minutes.12000r/min centrifuges 1min and collects DNA solution.
(8) it in order to improve the yield of DNA, can repeat to walk by the obtained solution of centrifugation again add-back centrifugal adsorbing column
Rapid 7.
Bidirectional sequencing is carried out marine growth Engineering Co., Ltd is served as the PCR purified products of template using the ponds sheep DNA.It is continuous
The sequencing result of sheep sry 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；
215bp positioned at sheep sry gene target fragment there is two kinds of testing results of G and A, the sheep SRY that as screening is arrived
The SNP polymorphisms of gene, the site are the nucleotide polymorphisms for G or A.
2 sheep sry gene G of embodiment>The RFLP-PCR of A mutation polymorphisms is detected
Since the nucleotide polymorphisms that screening is arrived are nature restriction enzyme site, can PCR-RFLP mirror be carried out by common restriction endonuclease
It is fixed.215bp when sheep sry gene target fragment G does not occur>When A is mutated, G before being as mutated is expanded using primer pair P
The sry gene sequence TCAGA/TCTGA of increasing is the restriction enzyme enzyme recognition site of Hpy188I；Hpy188I can directly be passed through
Genotyping is carried out to the digestion of target fragment.
1, RFLP-PCR primers
PCR amplification primer is the above-mentioned primer P for being verified as male specificity：
Forward primer：5’-GTCTGCTGCACCTTCATCCT-3’20nt；
Reverse primer：5’-GTTCATGGGTCGCTTGACGT-3’20nt.
The primer can expand sheep sry gene 440bp segments.
2, RFLP-PCR reaction conditions
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 collection of illustrative plates is as shown in Figure 2, it can be seen that the primer pair P of design can expand the segment of 440bp.
3, the Hpy188I digestions of pcr amplification product
(1) digestion system is：For Hpy188I restriction endonucleases 1 μ L, Buffer 5 μ L, ddH215 μ L of O, the production of 4 μ L PCR amplifications
Object amounts to 25 μ L.
(2) it is digested condition：3-4h is digested in 37 DEG C of constant incubators.
(3) agarose gel electrophoresis is analyzed after Hpy188I digests PCR product
With 2.5% Ago-Gel, 80V electrophoresis 1h, nucleic acid staining dye detects digestion as a result, with UVP gels
Imaging system (GelDoc-It TS Imaging System) PHOTOGRAPHIC ANALYSIS, and sentence type, record its genotype；
Due to including others Hpy188I digestion recognition sites in the 440bp segments of PCR-RFLP amplifications, work as sry gene
G does not occur for 215bp of segment>When A is mutated, the sry gene product of PCR amplification is identified by restriction enzyme Hpy188I
Afterwards, 3 sections are cut to by amplified fragments to amplified fragments digestion in TCAGA/TCTGA；And when the of sry gene 440bp segments
Amplified fragments in TCAGA/TCTGA to amplified fragments digestion, are cut to 4 sections by 215bp mutations.
Since the Y chromosome of sheep is monoploid, so when G occurs>When the mutation of A, 2 kinds of different genes can be formed
The gel result of type, respectively AA and GG genotype, PCR-RFLP detections is as shown in Figure 3：
Wherein GG genotype is wild type, the SNP site of its 1 DNA chain can by Hpy188I digestions, show as 63bp,
Three bands of 172bp and 205bp；AA genotype is saltant type, and the SNP site of its 1 DNA chain can be by Hpy188I digestions, table
It is now tetra- band of 54bp, 63bp, 151bp and 172bp.According to the size of the number of band and band, gel as shown in Figure 3 electricity
What swimming testing result can will be apparent that determines whether that point mutation has occurred, and two kinds of genotype are distinguished, more to detect its SNP
State property.
(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 sequenators of ABI
Sequence；Meanwhile carry out SNP position analyses, the results showed that comprising 63bp, 172bp, 205bp three bands GG genotype individuals its
215bp sequencer maps of sry gene 440bp segments are expressed as G really, as shown in fig. 4 a；And AA genotype, such as Fig. 4 b institutes
Show.
215bp SNP of the sry gene target fragment of 3 sheep of embodiment are as molecular labeling in different sheep group
Application in body polymorphism
1, the detection of group's single nucleotide polymorphism
Using above-mentioned SNP pleiomorphism detecting methods to 496 parts of DNA samples of sheep, the identification of SNP polymorphisms is carried out；System
Count the frequency distribution situation of its SNP site.
2, the frequency statistics analysis of SNP site
It 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 a certain character in a group
Certain genotype individuals number accounts for the ratio of total individual number：PAA=NAA/ N, wherein PAARepresent the AA genotype frequencies in a certain site；
NAAIndicate the number of individuals with AA genotype in group；N is the total quantity for detecting group.
Gene frequency refer in a group a certain gene number to the relative ratios of its allele sum.Therefore PA=
PAA。
3, the association analysis of gene effect
Genotype data：The genotype (AA and GG) of Hpy188I identifications
Ram Testicular Size data：White suffolk (12 monthly age) scrotum encloses diameter
The correlation of gene loci and White suffolk Testicular Size is analyzed using 19.0 softwares of SPSS.First to data into
Row descriptive statistical analysis, it is determined whether there are outliers to be visited according to data characteristics using one-way analysis of variance (ANOVA)
Study carefully genotype effects.
The result shows that (being shown in Table 4 and table 5)：For Hpy188I the identifiable 215th SNP site only in Suffolk
With show polymorphism in two kinds of White suffolk, and rise sheep, South African Mutton in Te Kesaier sheep, Du Boyang, Dong Fuli
Polymorphism is not shown then in Merino, sheep, Tibetan sheep and sheep known for its fine thick wool.The GG bases in Suffolk and White suffolk group
Because type is preponderant genotype；Trait associations analysis shows, in White suffolk group, the Testicular Sizes of GG genotype individuals is aobvious
What is write is higher than AA genotype.As a result illustrate that the SNP site of sry gene 215bp can be used as and differentiate Suffolk and White suffolk
Paternal molecular genetic marker, GG genotype can become the molecular genetic marker of a raising White suffolk Testicular Size.Root
According to this result of study, Sheep Breeding worker can carry out Embryo sexing, sheep paternal line haplotype mirror through the invention
Fixed and raising ram reproductive capacity.
SNP Gene frequency distribution tables at 4 sheep sry gene segment 215bp of table
Association analysis at 5 sry gene target fragment 215bp of table between SNP and White suffolk Testicular Size
Above description has shown and described several preferred embodiments of invention, but as previously described, it should be understood that invention is not
It is confined to form disclosed herein, is not to be taken as excluding other embodiments, and can be used for various other combinations, modification
And environment, and can be carried out by the above teachings or related fields of technology or knowledge in the scope of the invention is set forth herein
Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then should all be weighed appended by invention
In the protection domain that profit requires.
Sequence table
<110>Lanzhou University
<120>It is a kind of detection sheep sry gene single nucleotide polymorphism method and application
<130> 2018
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 1
gtctgctgca ccttcatcct 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 2
gttcatgggt cgcttgacgt 20
<210> 3
<211> 440
<212> DNA
<213>Sry gene (Sex-determining region Y gene)
<400> 3
atttgaatac ttccatgaca ccggttgtat ttttaagcag gtattagcgc cttcacaaat 60
tctgattaga tgtaaacaaa gaagaaagca gagcgttaat atcctgttaa gcacctttgg 120
tgggtttggg ctggctgcca ggaggtattg aggggaggta ttgggggcgg agaaataaat 180
atttcactgc aaattttgca ccaagtcagt ctctggtaag aacaacttat gaatagaacg 240
gtgcaatcgt atgcttctgc tatgttcaga gtattgaaag acgatgttta cagtccagcg 300
gtggtacagc aacaaaatac tttcgccttt gggaaaacct cttccttgtg cacagacaat 360
catagcgcaa acgatcagcg tgaaaggggg gaaaatgtta gggagagcag ccagaaccac 420
gtcaagcgcc ccatgaacaa 440
Claims (9)
1. a kind of single nucleotide polymorphism of sheep sry gene, which is characterized in that the label is in sheep sry gene
215bp G/A it is polymorphic.
2. test right requires the primer used in the single nucleotide polymorphism described in 1, which is characterized in that the primer includes
The nucleotide sequence of forward primer and reverse primer, forward primer and reverse primer is respectively such as SEQ ID NO.1 and SEQ ID
Shown in NO.2.
3. a kind of method of the single nucleotide polymorphism of detection sheep sry gene, which is characterized in that include the following steps：
1) ram complete genome DNA to be measured is extracted；
2) using ram complete genome DNA to be measured as template, using described in claim 2 forward primer and reverse primer carry out
PCR amplification sheep sry gene；
3) it digests pcr amplification product with restriction enzyme Hpy188I and then agarose is carried out to the amplified fragments after digestion
Gel electrophoresis；The genotype of mononucleotide polymorphism site on sheep sry gene is identified according to electrophoresis result.
4. according to the method described in claim 3, it is characterized in that, PCR reaction systems are：2×EasyTaqSuperMix
12.50 μ L, 0.4 μ L of forward primer, 0.4 μ L of reverse primer, 10.7 μ L of distilled water, 1 μ L of DNA profiling, with upper volume total amount for 25 μ
L。
5. according to the method described in claim 3, it is characterized in that, pcr amplification reaction program is：94 DEG C of pre-degeneration 5min；94
DEG C denaturation 30s, 62.6 DEG C annealing 30s, 72 DEG C extension 30s, 34 cycle；72 DEG C of extension 10min；4 DEG C of preservations.
6. according to the method described in claim 3, it is characterized in that, the mass concentration of the Ago-Gel is 2.5%.
7. according to the method described in claim 3, it is characterized in that, the mononucleotide polymorphism site shows as
The single nucleotide polymorphism of 215bp G or A, with two kinds of genotype of AA and GG, electrophoresis result is respectively：AA types are shown as
Tetra- bands of 54bp, 63bp, 151bp and 172bp；GG types show as tri- bands of 63bp, 172bp and 205bp.
8. any in the primer, claim 3-7 described in single nucleotide polymorphism described in claim 1, claim 2
Application of the method in sheep assisted Selection and molecular breeding described in claim.
9. any in the primer, claim 3-7 described in single nucleotide polymorphism described in claim 1, claim 2
Application of the method in Embryo sexing, the identification of sheep paternal line haplotype and raising ram reproductive capacity described in claim.
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