CN102649956A

CN102649956A - Single nucleotide polymorphic locus of milk goat PITX1 gene, and detection method and application of single nucleotide polymorphic locus

Info

Publication number: CN102649956A
Application number: CN2012101149620A
Authority: CN
Inventors: 蓝贤勇; 赵海谕; 李转见; 潘传英; 陈宏�; 雷初朝
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2012-04-18
Filing date: 2012-04-18
Publication date: 2012-08-29

Abstract

The invention relates to a single nucleotide polymorphic locus of a milk goat PITX1 gene, and a detection method and application of the single nucleotide polymorphic locus, and belongs to the technical field of animal molecular genetic breeding. The gene single nucleotide polymorphic locus comprises the single nucleotide polymorphic locus of the milk goat PITX1 gene with the 201st position is G or A, wherein the genotype is GG, GA and AA. The detection method comprises the following steps: performing polymerase chain reaction (PCR) amplification on the milk PITX1 gene by using the DNA sequence of the PITX1 gene as a template and a primer pair P as a primer; performing enzyme digestion on the PCR product by using restriction endonuclease MspI; and detecting the enzyme-digested product through agarose gel electrophoresis to identify the single nucleotide polymorphic locus of the milk goat PITX1 gene. Through the adoption of the method, the milk goat species with excellent genetic resources can be established quickly in marker assisted selection (MAS) breeding of the milk goat. The method has the advantages of simplicity and quickness in operation, low cost and high detection precision.

Description

Milk goat PITX1 gene mononucleotide polymorphism site and detection method and application

Technical field

The invention belongs to animal molecular genetic breeding technical field; Relate to the detection and the application thereof of gene mononucleotide polymorphism (SNP), be specifically related to milk goat PITX1 (the abnormally-structured territory of homology protein transcription factor 1 in pairs) gene (is reference with sequence A ccession No.NW_003104033) mononucleotide polymorphism site and detection method and application.

Background technology

Compare with milk cow, milk goat more is applicable under severe environmental conditions and raises, because it has strong resistance; Compare with milk, goat milk has higher nutritive value, because its Oil globule is little, does not contain sensibiligen, and acid number is low, more is prone to be described as in international nutrition circle " king in the milk " for absorption of human body than milk.Yet at present, China's milk goat industry relatively lags behind, and utilizes good milk goat colony to obtain the quantity and the quality of goat milk, and gap is huge compared with developed countries, with the dairy development " cannot be mentioned in the same breath " of present domestic main flow.For this reason; As the important component part of milk industry evolutionary operation(EVOP) in China " 12 " planning, the development of milk goat industry is extremely urgent, but at present; This industry development of China lags behind, and its basic reason is that China's milk goat kind provenance is not enough and the population hereditary quality is relatively poor.Therefore, how improving milk goat population hereditary quality, thereby increase the milk yield of milk goat and improve milk-quality, is the key issue that present China milk goat genetic breeding expert waits to solve.

The milk goat milk performance mainly comprises milk yield, milk fat content, fat yield, protein ratio, milk-protein amount, milk solids thing content etc., belongs to the quantitative trait locus (QTL) with important economic worth, and by minor-polygene control, and heritability is lower.Aspect the milk goat breeding; Only depend on the traditional breeding method means that these milk characters are improved; Beyond doubt very slowly, and effect can be very not desirable yet, therefore; The GENERALIZATION OF MODERN BREEDING TECHNIQUE of Application of DNA mark is to accelerate fine-variety breeding and improve the population hereditary quality, thereby increases the milk yield of milk goat and improve the advanced person's of milk-quality effective means.Because dna marker has rich polymorphism, inheritance stability, does not receive plurality of advantages such as tissue, physiological development stage and environmental influence.In addition, along with the kind of dna marker and increasing of number, gene mapping density and precision are improving constantly, and for genetic improvement provides new strategy, make dna marker assisted Selection (MAS) become a kind of molecular breeding technology with bright prospects.This shows that utilize the MAS method and the traditional breeding way of dna marker mediation that the milk goat milk character is improved, improves, greatly the accelerated selection progress improves and selects accuracy.

At present, it is following to utilize dna marker to carry out the key step of MAS breeding: at first, on dna level rapid screening with detect the closely-related gene that influences the milk goat milk character; Secondly, the association analysis of setting up gene polymorphic and milk character is confirmed and remarkable related dna marker or the SNP site of milk character; At last, realize the genetic marker assisted Selection and realize the early diagnosis selection.Particularly, about the research that concerns between dna marker and the milk goat milk character, mainly contain two kinds of basic skills: candidate gene approach and linked marker analysis.In milk goat research, candidate gene approach is more easy to operate than linked marker analysis, and need not carry out specially experimental design, expense is low, the statistics effect is strong, is extensively adopted, and is applicable to any colony that obtains genotype and phenotypic data.This method is set up corresponding mathematical model through statistical method, thereby confirms the relation or the effect of this candidate gene and unknown gene.As a rule, candidate gene is some important gene relevant with milk character, possibly be structure gene, regulatory gene or the gene that in the biochemical metabolism approach, influences this trait expression.

As everyone knows, tethelin (GH) can promote Mammals growth, lactation, cell proliferation.Lactotropin (PRL) also can influence mammiferous growth, lactation and immunity system significantly; And existing research shows that GH and PRL transgenation can mammiferous lactation period of remarkably influenced and milk performances.Therefore, GH gene, PRL gene and their positive regulatory factor gene all can be used as the crucial candidate gene that influences milk performance.The abnormally-structured territory of homology protein transcription factor (PITX) type transcription factor member inspires the cognation research of this genoid and milk performance in gene activation, the developmental key effect of hypophysis.As the important member of PITX family, the abnormally-structured territory of homology protein transcription factor 1 (PITX1) gene is positioned at human No. 5 karyomit(e), mouse and No. 13 karyomit(e) of jungle fowl, No. 7 karyomit(e) of ox respectively, but the corresponding information of goat is not announced as yet.Sequence alignment is the result show; The sequence high conservative of PITX1 in the mankind, chimpanzee, dog, mouse, chicken, zebra fish and ox; In many growth paths, play a significant role; Comprise that influence organ pituitary takes place, influence GH, PRL, TTH (TSH) thus expression influence the milk performance of animal.What deserves to be mentioned is that PITX1 locates foundly at the embryo's of developmental mouse, chicken hind leg, oral epithelium tissue, arm bow etc., it is determining the form of muscle, bone etc.In addition, the mouse that knocks out the PITX1 gene shows defective and the obstacle on hypophysis, hind leg and the sense of taste are grown.Therefore, the PITX1 gene is the important setter of hypophysis-hypothalamus axle associated hormone gene (as: PRL and GH), is one of vital signs gene of known representative hypophysis growth.

So-called SNP (SNP) just is meant in the genomic dna sequence polymorphum that the variation owing to single Nucleotide (A/T/C/G) causes, its variant form mainly contains transversion, conversion, insertion and disappearance etc.In recent years, developed and many methods that are used to seek gene SNP, for example: determined dna sequence method, PCR-SSCP and dna sequencing combined techniques, PCR-RFLP method, AS-PCR method, primer extension and oligonucleotide ligation etc.In these detection techniques, the determined dna sequence method is the most accurately to detect the method for SNP, still; Its testing cost is extremely expensive; And need large-scale instruments such as dna sequencing appearance, simultaneously, in the order-checking process, need very skilled experienced technician; So the determined dna sequence method is not a kind of ideal SNP detection method that is applied to production practice; Utilize PCR-SSCP and dna sequencing combined techniques to detect SNP, can suitably reduce testing cost, still, its experimentation is long, operates more loaded down with trivial detailsly, and has the false negative problem in the experimentation, so, also also nonideal SNP detection means; The AS-PCR method is as a kind of novel SNP detection method; In the Application Areas in future, has boundless prospect; But this method need design special primer, and can only be directed against the special genes site, also has the probability of flase drop simultaneously in the testing process; Therefore, the characteristics that do not have widespread usage at present; And, needing detection platform such as plate reader, gene chip, micro-sphere array technology and mass spectrograph with primer extension and oligonucleotide ligation technology for detection SNP site, exploitativeness is not strong for general molecule laboratory.In sum, the PCR-RFLP method is the ideal genetic marking method of current detection SNP.

Current research shows that biological vivoexpression analysis such as people, mouse is more common in research about the PIX1 gene SNP, and often is used as the organ function defective candidate gene relevant with important disease forecasting, and rarely seen in the livestock and poultry snp analysis.So far, relevant report is not seen in research as yet about milk goat PITX1 gene SNP.The research in China milk goat PITX1 gene SNP field is deficient, and the association study of the functional study of gene locus and heritable variation thereof and milk performance (as: milk yield, milk fat content, fat yield, protein ratio, milk-protein amount, milk solids thing content etc.) is still blank.Because the PITX1 gene SNP site has important effect to forming embryo, regulation and control tethelin and promotion hypophysis secreting hormone etc. in the animal production practice; Detection method provided by the invention is that the foundation that concerns between PITX1 gene SNP and the milk goat milk character is laid a good foundation; Marker assisted selection (MAS) for use in Chinese milk goat milk character; Set up the good milk goat population of genetic resources fast; Thereby improve the population hereditary quality, increase the milk yield of milk goat and improve milk-quality, for the development already of China milk goat provides the theory and practice data.

Summary of the invention

The problem that the present invention solves is; Utilize PCR-RFLP method rapid detection milk goat PITX1 gene SNP; And itself and milk goat milk character carried out association analysis, verify its dna marker, thereby accelerate fine-variety breeding speed as assisted Selection in the milk goat molecular breeding.

In order to realize the purpose of foregoing invention, the present invention takes following technical scheme:

Milk goat PITX1 gene mononucleotide polymorphism site, wherein, this gene mononucleotide polymorphism site comprises the 201st mononucleotide polymorphism site for G or A of milk goat PITX1 gene, genotype is GG, GA, AA.

A kind of method that detects milk goat PITX1 gene mononucleotide polymorphism site comprises the steps:

(1), be template with the dna sequence dna that comprises the PITX1 gene, be primer with primer to P, pcr amplification milk goat PITX1 gene;

(2), the PCR product that utilizes restriction enzyme MspI that step (1) is obtained then carries out enzyme and cuts;

(3), detect enzyme that step (2) obtains through agarose gel electrophoresis again and cut product and can identify that the mononucleotide polymorphism site of milk goat PITX1 gene comprises the 201st mononucleotide polymorphism site for G or A, genotype is GG, GA, AA;

Described primer to P is:

Upstream primer: F:5 '-CCGCCTTCCACCTAGCCCGCAC-3 ';

Downstream primer: R:5 '-TCGTCCATGTCCACGTTCATCG-3 '.

A kind of method that detects milk goat PITX1 gene mononucleotide polymorphism site described in the technique scheme, wherein, described pcr amplification reaction program is:

94 ℃ of preparatory sex change 5min; 35 circulations (72 ℃ are extended 30s for 94 ℃ of sex change 30s, 65.2 ℃ of annealing 30s); 72 ℃ are extended 10min.

A kind of method that detects milk goat PITX1 gene mononucleotide polymorphism site described in the technique scheme, wherein, described agarose gel electrophoresis is 2.0% agarose gel electrophoresis.

The application in the DNA genetic marker of fat yield and milk solids thing content is improved in milk goat PITX1 gene mononucleotide polymorphism site described in the technique scheme in as the marker assisted selection breeding of milk goat milk character.

Application described in the technique scheme, wherein, the GA genotype in milk goat PITX1 gene mononucleotide polymorphism site is as the application of the DNA genetic marker that improves the milk goat milk performance.

Detecting in the present invention in the method in milk goat PITX1 gene mononucleotide polymorphism site, is template with the milk goat genomic dna, at Taq archaeal dna polymerase, Buffer (buffer environment), Mg ²⁺, under the dNTPs situation about existing; Utilize primer that P is carried out pcr amplification; The dna fragmentation that utilizes restriction enzyme that amplification is obtained then carries out enzyme to be cut, and detects through agarose gel electrophoresis and can identify that the 201st of milk goat PITX1 gene is the 41st of introne 1 (NW_003104033:g.201G＞A (SNP of IVS1+41G＞A); Do not contain Mg among the buffer of the present invention ²⁺

The 201st of milk goat PITX1 gene is in the mononucleotide polymorphism site of G or A among the present invention, and A allelotrope shows as 230bp, and G allelotrope shows as: 121bp and 109bp, correspondingly, GG genotype performance: 121bp, 109bp; GA genotype performance: 230bp, 121bp, 109bp; AA genotype performance: 230bp.

The present invention designs primer to P (because NCBI does not announce the goat genome sequence) according to the sequence (GenBank Accession number:NW_003104033) of ox PITX1 gene; With milk goat genomic dna pond, the Central Shanxi Plain is template; Carry out pcr amplification; And the PCR product checked order, obtain the partial sequence of milk goat PITX1 gene after the order-checking.After the sequence of announcing with NCBI compares, find to have SNP at the 201st (introne 1 the 41st).

To above-mentioned the 201st SNP; The invention also discloses its examination and detection method, through designing specific primer, pcr amplification purpose fragment; Identify with specific digestion with restriction enzyme then, can be simply, fast, low cost, accurately detect its SNP.

The present invention has following beneficial effect:

1, the invention discloses the examination and the detection method in milk goat PITX1 gene mononucleotide polymorphism site, through designing specific PCR primer amplification fragment, can enough RFLP methods simple, fast, cost is low, detect the polymorphum of its mononucleotide accurately;

2, the present invention has carried out detection and gene frequency analysis to this SNP genotype of Central Shanxi Plain milk goat; Milk performance (dairy fat content and the milk solids thing content that comprise goat milk) to above-mentioned SNP site and milk goat is carried out association analysis, and the result shows that this site can be used as the molecule marker that improves the milk goat milk performance;

3, detection method disclosed by the invention is that the foundation of milk character relation of SNP and the milk goat of milk goat PITX1 gene is laid a good foundation, and is used for setting up fast the good milk goat population of genetic resources in marker assisted selection (MAS) breeding for use in the milk goat milk character.

Description of drawings

1, Fig. 1 is the individual sequencer map of the 201st GA genotype of milk goat PITX1 gene;

2, Fig. 2 is that MspI PCR-RFLP method detects goat PITX1 gene the 1st intron SNP (the sequential analysis figure of NW_003104033:g.201G＞A or IVS1+41G＞A);

3, Fig. 3 is that MspI PCR-RFLP method detects the 201st (the 41st of introne 1) SNP restriction enzyme digestion and electrophoresis somatotype figure of milk goat PITX1 gene.

Embodiment:

For making technical scheme of the present invention be convenient to understand, the present invention is further described below in conjunction with specific embodiment.

Used main agents and source thereof are as follows among the following embodiment of the present invention:

1, biochemical reagents and biological reagent:

1. Taq archaeal dna polymerase (available from MBI company); 2. restriction enzyme MspI (available from MBI company); 3. dNTPs (available from MBI company); 4. Marker I: (available from sky, Beijing root biochemical technology ltd); 5. Proteinase K (available from Huamei Bio-Engrg Co.).

2, general reagent:

General reagent is bought from Huamei Bio-Engrg Co.,, is import packing product: Hydrocerol A, Trisodium Citrate, glucose, Tris, EDTA, NaCl, NaOH, KCl, Na ₂HPO ₄, KH ₂PO ₄, the saturated phenol of Tris, chloroform, primary isoamyl alcohol, absolute ethyl alcohol, sodium-acetate, sodium laurylsulfonate (SDS), ethidium bromide (EB), tetrabromophenol sulfonphthalein, dimethyl benzene cyanogen FF, acetate, sucrose, boric acid, agarose etc.

3, solution and damping fluid

All solution and damping fluid all adopt the preparation of de-ionized ultrapure water.The autoclaving condition is 15bf/in (1.034 * 10 ⁵Pa), 25min." the molecular cloning experiment guide " that the reagent compound method is all write with reference to Sambrook etc.

(1), the sample used solution of sampling:

Antithrombotics ACD: Hydrocerol A 0.48g; Trisodium Citrate 1.32g; Glucose 1.47g.Their constant volumes in 100mL water, autoclaving.The ACD liquid that adds 1mL in every 6mL fresh blood.This antithrombotics is superior to EDTA, in the blood storage process, can preserve high molecular DNA better, can preserve a couple of days or-70 ℃ of prolonged preservation at 0 ℃ through the blood of its anti-freezing.

(2), the blood sample genomic dna separates used solution:

1., PBS damping fluid: NaCl 8g, KCl 0.2g, Na ₂HPO ₄1.44g, KH ₂PO ₄0.24g, add ultrapure water to 1000mL, transfer pH to 7.4, autoclaving;

2., 10%SDS:10g SDS is dissolved in the ultrapure water of 90mL, 68 ℃ of water-baths dissolvings are transferred pH to 7.2 with HCl, are settled to 100mL;

3., 0.5mol/L EDTA:EDTA 186.1g, be dissolved in the ultrapure water of 800mL, transfer pH to 8.0 with NaOH, be settled to 1000mL, autoclaving, 4 ℃ of preservations;

4., 1mol/LTrisCl:121.14g Tris, be dissolved in the 800mL ultrapure water, HCl regulates pH to 8.0, is settled to 1000mL, autoclaving, 4 ℃ of preservations;

5., 5mol/LNaCl:NaCl 292.2g is dissolved in the 1000mL ultrapure water;

6., DNA extraction buffer: get 0.5mmol/L EDTA 40mL, 1mmol/L TrisCl 10mL;

7., 5mmol/L NaCl 4mL, 10%SDS 10mL is settled to 100mL.Actual concentrations is 200mmol/LEDTA, and pH 8.0; 100mmol/L TrisHCl, pH 8.0; 200mmol/L NaCl, 2%SDS; RNase 20 μ g/mL;

8., NaAc damping fluid: NaAc3H ₂O 20.4g; Ultrapure water 40mL; Rare HAc transfers pH to 7.4; Be settled to 50mL;

9., TE damping fluid: TrisCl damping fluid (pH 8.0) 10mmol/L, edta buffer liquid (pH 8.0) 0.1mmol/L, autoclaving, 4 ℃ of preservations;

10., Proteinase K: be made into 20mg/mL with ultrapure water ,-20 ℃ of preservations;

(3), the used solution of tissue appearance DNA extraction:

The public solution during except extracting genome DNA, also be configured to down reagent:

1., 2mol/L NaCl:11.688g is water-soluble, is settled to 100mL, autoclaving;

2., tissue DNA extracting solution (100mL): 1mol/L Tris-Cl (pH 8.0) l mL, 0.5mol/L EDTA (pH8.0) 20mL, 2mol/L NaCl 5mL is settled to 100mL;

(4), the used solution of agarose electrophoretic analysis

1., 0.5 * tbe buffer liquid: get 10 * TBE 50mL and be settled to 1000mL;

2., sample-loading buffer: 0.25% tetrabromophenol sulfonphthalein, the blue or green FF of 0.25% YLENE, 40.0% (w/v) aqueous sucrose solution.

Embodiment one:The gram of milk goat PITX1 Gene Partial dna sequence dna falls and dna sequencing

One, milk goat sample collecting

Extract 2-4 year Central Shanxi Plain milk goat that used blood sample of DNA (venous blood collection) and ear tissue appearance are all picked up from 658 health and consanguinity-less relation, pick up from Sanyuan County, Shaanxi Province milk goat breeding station.

Two, the extraction of blood sample genomic dna with separate

(1), freezing blood sample (being mainly hemocyte) room temperature is thawed transferase 45 00 μ L to 1.5mL Eppendorf pipe, adding equal-volume PBS liquid; Abundant mixing; The centrifugal 10min of 12000r/min (4 ℃), abandoning supernatant, repetition above-mentioned steps to supernatant is transparent, deposition is faint yellow.

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

(3), add Proteinase K to 30 μ L (20mg/mL) and mixing, 55 ℃ are spent the night to clarification, defecator not can add 10 μ L Proteinase K mixings and continue digestion until clarification as yet.

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

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

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

(7), add the NaAc damping fluid of 0.1 times of volume and the ice-cold absolute ethyl alcohol of 2 times of volumes, mix and rotate centrifuge tube and separate out until the flocks of white, preserve 30～60min for-20 ℃.

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

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

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

Three, the extraction of tissue sample DNA with separate

(1), get the tissue sample of the about 10mg of milk goat ear tissue appearance, be put in the centrifuge tube of 1.5mL, shred with little scissors as far as possible.

(2), add 600 μ L tissue extracts, 10%SDS is 1% to final concentration, Proteinase K to final concentration is 100 μ g/mL, 55.0 ℃ of digestion are spent the night, and guarantee that preferably tissue appearance is evenly distributed in the tissue extract.

(3), solution is cooled to room temperature, add the saturated phenol of isopyknic Tris, the tight pipe lid of lid is put upside down centrifuge tube lentamente back and forth, more than the lasting at least 10min, and the centrifugal 15min of 12000r/min.

(4), get supernatant, add isopyknic phenol: chloroform (1: 1), the tight pipe lid of lid is put upside down centrifuge tube lentamente back and forth, more than the lasting at least 10min, the centrifugal 15min of 12000r/min.

(5), get supernatant, add isopyknic chloroform: primary isoamyl alcohol (24: 1), the tight pipe lid of lid is put upside down centrifuge tube lentamente back and forth, more than the lasting at least 10min, the centrifugal 15min of 12000r/min.

(6), get supernatant, add the ice-cold absolute ethyl alcohol of 2 times of volumes and the 3mol/L sodium acetate of 1/10 volume, the tight pipe lid of lid is put upside down centrifuge tube lentamente back and forth, until white wadding DNA occurring.

(7), choose DNA, put in the centrifuge tube of a 1.5mL, add 500 μ L, 70% ethanol, the tight pipe lid of lid is put upside down centrifuge tube lentamente back and forth, the centrifugal 3～5min of 12000r/min carefully outwells ethanol then, and pipe is inverted on the thieving paper.

(8), again in centrifuge tube, add 500 μ L, 70% ethanol, the tight pipe lid of lid is slowly put upside down, and the centrifugal 3～5min of 12000r/min carefully outwells ethanol then, and pipe is inverted on the thieving paper.

(9), to be dried after, add 60 μ L sterilization ultrapure water, 4 ℃ of preservations are spent the night, and are to be detected.

Four, the purifying of DNA

(1), adding 10%SDS in the dna solution of 500 μ L, to make its final concentration be 0.1%, add Proteinase K to final concentration and reach 100 μ g/mL.

(2), 5 ℃ are incubated about 10h.

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

(4), the centrifugal 5min phase-splitting of 12000r/min, draw the upper strata water to another centrifuge tube.

(5), add 1/10 volume 3mol/L sodium-acetate and the 2 times of ice-cold absolute ethyl alcohol deposit D of volume NA.

(6), outwell liquid, airing after 70% washing with alcohol adds the dissolving of 60 μ L sterilization ultrapure water, 4 ℃ to be detected.

Five, spectrophotometry detects DNA

With the OD value of UV-light photometric determination DNA sample at 260nm, 280nm place.Calculate dna content and OD ₂₆₀/ OD ₂₈₀Ratio.Like OD ₂₆₀/ OD ₂₈₀Ratio contains more protein or phenol less than 1.6 in the interpret sample, then should carry out purifying; If ratio greater than 1.8, then should consider to remove the RNA purifying.

DNA concentration (ng)=50 * OD ₂₆₀Value * extension rate

After DNA detection finishes, get and be diluted to 50ng/ μ L in right amount, be stored in-20 ℃ subsequent use, remaining deposits in-80 ℃.

Six, the structure in DNA pond

After DNA detection finishes, from 100 concentration are the Central Shanxi Plain milk goat DNA sample of 50ng/ μ L, respectively get 10 μ L and mix, make up Central Shanxi Plain milk goat colony DNA pond.

Seven, primer design

Owing to do not announce the sequence of milk goat PITX1 gene at present on u.s. national library of medicine (NCBI) website; For this reason; Going up ox PITX1 gene (the GenBank accession No.NW_003104033) sequence of announcing with NCBI is reference; Utilize 230bp segmental PCR primer that software Primer 5.0 designed amplification milk goat PITX1 gene the 1st intron to P, its primer is following to the sequence information of P:

Upstream primer: F:5 '-CCGCCTTCCACCTAGCCCGCAC-3 ' (nt50-71);

Downstream primer: R:5 '-TCGTCCATGTCCACGTTCATCG-3 ' (nt 259-279)

Eight, PCR clone milk goat PITX1 gene

Central Shanxi Plain milk goat colony DNA pond to build is a template, carries out pcr amplification with above-mentioned designed primer, and the PCR reaction system is seen table 1.

Table 1 PCR reaction system

Sterilization ultrapure water (H ₂O)	15.75μL
		10 * PCR damping fluid (MBI, Fermentas)	2.5μL
MgCl ₂(25mmol/L)	1.5μL
		dNTPs(2.5mmol/L)	2.5μL
Upstream primer (10pmol/L)	0.5μL
		Downstream primer (10pmol/L)	0.5μL
Taq archaeal dna polymerase (0.5U/ μ L)	1.25μL
		Dna profiling (100ng/ μ L)	0.5μL
TV	25μL

The program of PCR reaction is following: 95 ℃ of preparatory sex change 4min, and 35 circulations (94 ℃ of 30s, 65.2 ℃ of 30s, 72 ℃ of 30s), last 72 ℃ are extended 10min, 4 ℃ of preservations.

Nine, PCR product purification and DNA pond order-checking

Carrying out agarose gel electrophoresis after pcr amplification is accomplished detects; The glue of cutting that carries out the PCR product then reclaims and purifying: under uv lamp, contain the segmental gel of purpose from the sepharose cutting-out; Put into the 1.5mL centrifuge tube; Reclaim purification kit (sky, Beijing root biotech firm) purified pcr product with the PCR product then, operate according to the test kit specification sheets.

Send the PCR purified product that with milk goat DNA pond, the Central Shanxi Plain is template Nanjing Jin Sirui ltd to carry out two-way order-checking.Peak figure analyzes to the segmental order-checking of milk goat PITX1 gene the 1st intron 230bp, wherein has two different peaks to represent to have taken place single nucleotide mutation in same site.1 SNP of milk goat PITX1 gene has been arrived in examination of the present invention; As shown in Figure 1; At its 201st is the SNP of G or A; Can be expressed as G＞A, wherein the letter

of band frame representes that the sudden change of G＞A (NW_00314033:g.201G＞A) takes place for this site among Fig. 1; Green line is represented the order-checking peak of A base, and red line is represented the order-checking peak of T base, and blue line is represented the order-checking peak of C base, and black line is represented the order-checking peak of G base).Utilize Blastn software and BioXM (2.6) software that G allelotrope and the allelic corresponding sequence of A are carried out sequence alignment and restriction enzyme site analysis then; Find that (there is the MspI polymorphum in NW_00314033:g.201G＞A) for the sudden change of G＞A; Its analytical results is as shown in Figure 2; Fig. 2 is that MspI PCR-RFLP method detects goat PITX1 gene the 1st intron SNP (the sequential analysis figure of NW_003104033:g.201G＞A or IVS1+41G＞A); Wherein be with the frame sequence to represent the upstream and downstream primer sequence respectively, the letter of runic and underscore or point and following stroke are then represented the SNP site.

Embodiment two:The MspI PCR-RFLP of 201 G of milk goat PITX1 gene＞A polymorphum analyzes

One, polymorphic site analysis

When G＞A sudden change takes place in the 201st of milk goat PITX1 gene; Be that G sports A; Originally CCG

sequence correspondingly becomes CCG

thereby has lost the MspI restriction endonuclease recognition sequence, can not cut by being limited property restriction endonuclease MspI.

Two, PCR reaction conditions

Pcr amplification system and reaction conditions are respectively as noted earlier, and wherein template is used milk goat genes of individuals group DNA, and pcr amplification product detects with 1.0% agarose gel electrophoresis, can see the band of 230bp clearly.

Three, PCR product enzyme is cut (MspI) and RFLP detection

Pcr amplification product is carried out the MspI enzyme at first respectively cut, judge the SNP in this site then according to the agarose gel electrophoresis result.

1, enzyme is cut:

(1), the MspI enzyme is cut system (25 μ L): the individual DNA PCR of 20 μ L milk goats product, 2.5 μ L, 10 * buffer damping fluids (containing BSA), the MspI restriction enzyme of 1.0 μ L (concentration 10U/ μ L), 1.5 μ L sterilization ultrapure water (H ₂O).

(2), enzyme tangent condition: digest 5h in 37 ℃ of waters bath with thermostatic control or the airbath.

2, agarose gel electrophoresis somatotype:

Make 2.0% sepharose, electrophoresis under the 100V voltage, when the different dna fragmentation of molecular weight separates when clear, EB dyeing, with the analysis of taking a picture of BIO-RAD Gel Doc 2000 gel imaging analysis systems, type is declared in manual work.

Electrophoresis result is as shown in Figure 3, and wherein swimming lane 1,2,3,5,9,14 is the GG genotype; Swimming lane 6,8,10,11,12,13 is the GA genotype; Swimming lane 4,7 is the AA genotype; Marker is Marker I, is respectively 600bp, 500bp, 400bp, 300bp, 200bp and 100bp; Because when place, the SNP site of milk goat PITX1 gene PCR amplified production sequence is CCGG, can discern by being limited property restriction endonuclease MspI, the gene fragment of 230bp is cut to 121bp, 109bp two bands; When the sudden change of G＞A takes place in above-mentioned this site, can not discern by being limited property restriction endonuclease MspI, promptly fragment can not be cut by being limited property restriction endonuclease, and the band of a 230bp size is only arranged; Because goat is the diploid animal; When G＞A sudden change takes place when, can form the different gene type, be respectively GG, GA and AA; Electrophorogram after can cutting through its enzyme is differentiated; The AA genotype shows as 230bp one band among Fig. 3, and the GA genotype shows as 121bp, 109bp, 230bp three bands, and the GG genotype shows as 121bp and 109bp two bands.

Embodiment three:The diagnostic use of dna marker in Central Shanxi Plain milk goat colony polymorphum of the present invention's preparation

One, the diagnosis in colony's polymorphum:

Utilize above-mentioned SNP detection method the DNA sample of all Central Shanxi Plain milk goats to be carried out the genotype identification of PCR-RFLP respectively.

Two, the frequency statistics analysis in SNP site:

Genotype frequency is meant the ratio between the range gene type of a certain proterties in the colony.P _AA=N _AA/ N, wherein P _AARepresent the AA genotype frequency in a certain site; N _AAHas the genotypic number of individuals of AA in the expression colony; N is for detecting the total quantity of colony.

Gene frequency is meant its allelic relative ratios of a certain gene pairs in the colony.The formula that calculates can be write as: P _A=(2N _AA+ N _Aa1+ N _Aa2+ N _Aa3+ N _Aa4+ ...+N _Aan)/2N

In the formula, P _AExpression allelotrope A frequency, N _AAHas the genotypic individual amount of AA, N in the expression colony _AaiHave Aai genotype individual amount in the expression colony, a1-an is n the mutually different multiple allelomorphos of allelotrope A.The allelotrope that this institute relates to is A and G, so concrete gene frequency calculation formula is:

P _A＝(2N _AA+N _AG)/2N

P _G＝(2N _GG+N _AG)/2N

In the formula, P _A, P _GThe frequency of representing allelotrope A and G respectively, N _AA, N _GGAnd N _AGRepresent the genotypic individual amount of AA, GG and AG respectively, N representes the total group number.

Gene frequency as shown in table 2 distributes; A gene frequency among the milk goat PITX1 gene M spI site SNP of the Central Shanxi Plain is 0.230; The G gene frequency is 0.770, meets the definition of gene frequency greater than 0.01 (1.0%) SNP, so this site belongs to mononucleotide polymorphic site.

Table 2 Central Shanxi Plain milk goat PITX1-MspI site gene frequency and genotype frequency

Embodiment four:The genetic effect association analysis of milk goat PITX1 gene SNP site

One, genotype data: the genotype (AA, AG and GG) of restriction enzyme MspI identification

Two, production data: Central Shanxi Plain milk goat milk yield, dairy fat content (comprising average dairy fat content, morning milk fat content and late dairy fat content) and milk solids thing content.

Three, association analysis model:

Earlier data are carried out descriptive analysis, determine whether to exist outlier, utilize the least square analysis that data are proofreaied and correct again; According to data characteristics, use the production traits effect between each genotype of SPSS (18.0) software analysis., the genotype effect adopted fixed model when being analyzed:

Y _ijk＝μ+A _i+G _J+(AG) _ij+ _eijk，

Wherein: Y _IjkBe the observed value of milk character, μ is a population mean, A _iBe age effect, G _jBe the genotype effect, (AG) _IjBe age and genotypic interaction, e _IjkBe random error.Because all laboratory animal (Central Shanxi Plain milk goat) are all provided by same plant, so the farm effect is not considered.

The result shows that the genotypic Central Shanxi Plain of (seeing table 3): GA milk goat all is being significantly higher than GG and AA genotype individual (P＜0.05) aspect morning milk fat content, late dairy fat content, the average dairy fat content; GA genotype milk goat is significantly higher than AA genotype individual (P＜0.05) aspect total solids, but with GG genotype difference between individuals not significantly (P＞0.05).This explanation GA genotype can be used as a candidate molecules genetic marker that improves the milk goat milk performance.

Therefore, the present invention first successful use MspI PCR-RFLP method detected the SNP (SNP) of China domestic milk goat PITX1 gene intron 1.And through the association analysis checking, (IVS1+41G＞heterozygous genes type (GA type) A) can be used as the dna marker of raising milk goat milk performance in this site.

The association analysis of table 3 Central Shanxi Plain milk goat P1 site (PITX1-MspI) SNP and milk performance

The above; Being merely preferred embodiment of the present invention, is not that the present invention is done any formal and substantial restriction, allly is familiar with the professional and technical personnel; In not breaking away from technical scheme scope of the present invention; The technology contents that is disclosed more than capable of using, and a little change of making, modify the equivalent variations with differentiation, be equivalent embodiment of the present invention; Simultaneously, all foundations essence technology of the present invention all still belongs in the scope of technical scheme of the present invention change, modification and the differentiation of any equivalent variations that above embodiment did.

Claims

1. milk goat PITX1 gene mononucleotide polymorphism site is characterized in that: this gene mononucleotide polymorphism site comprises the 201st mononucleotide polymorphism site for G or A of milk goat PITX1 gene, and genotype is GG, GA, AA.

2. a method that detects milk goat PITX1 gene mononucleotide polymorphism site comprises the steps:

Described primer to P is:

Upstream primer: F:5 '-CCGCCTTCCACCTAGCCCGCAC-3 ';

Downstream primer: R:5 '-TCGTCCATGTCCACGTTCATCG-3 '.

3. a kind of method that detects milk goat PITX1 gene mononucleotide polymorphism site as claimed in claim 2 is characterized in that described pcr amplification reaction program is:

94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 65.2 ℃ of annealing 30s, 72 ℃ are extended 30s, 35 circulations; 72 ℃ are extended 10min.

4. a kind of method that detects milk goat PITX1 gene mononucleotide polymorphism site according to claim 1, it is characterized in that: described agarose gel electrophoresis is 2.0% agarose gel electrophoresis.

5. the application in the DNA genetic marker of fat yield and milk solids thing content is improved in the described milk goat PITX1 of claim 1 gene mononucleotide polymorphism site in as the marker assisted selection breeding of milk goat milk character.

6. application according to claim 5 is characterized in that: the GA genotype in milk goat PITX1 gene mononucleotide polymorphism site is as the application of the DNA genetic marker that improves the milk goat milk performance.