CN101705289A - Single nucleotide polymorphism of GHRHR genes in dairy goat and detection method thereof - Google Patents

Abstract

The invention discloses single nucleotide polymorphism of GHRHR genes in dairy goat and a detection method thereof. The single nucleotide polymorphism of the genes comprises C/G base polymorphism which is the 231st in the sequence table of the GHRHR gene in the dairy goat shown in SEQ ID NO.1. The detection method comprises the following steps: taking the DNA of the whole genome of the dairy goat to be detected containing the GHRHR genes as a template and a primer pair P as a primer to carry out PCR amplification on the GHRHR genes in the dairy goat; after using restriction enzyme MspI to digest the PCR amplified product, carrying out agarose gel electrophoresis on amplified fragments after restriction enzyme; and identifying the polymorphism of the genes according to the electrophoresis results. The method screens and detects the molecular genetic markers closely related to the milk production traits of the dairy goat on the DNA level to be used for assisted selection and molecular breeding of the dairy goat and speed up find breeding of the dairy goat.

Description

The single nucleotide polymorphism of GHRHR genes in dairy goat and detection method thereof

Technical field

The invention belongs to the molecular genetics field, relate to single nucleotide polymorphism (SNP) with the functional gene of milk goat as molecular genetic marker, particularly the single nucleotide polymorphism of GHRHR genes in dairy goat and detection method thereof.

Background technology

Goat milk has very high nutritive value, does not contain anaphylactogen, and the nutritive ingredient equilibrium is is easily digested and assimilated, and forms similar to people lactoprotein's matter; The fat particle volume of goat milk is 1/3rd of a milk, and the protein in the goat milk, mineral substance, especially calcium, phosphorus, VITAMIN and content of elements are slightly higher than milk, and are more conducive to absorption of human body, are described as in international nutrition circle " king in the milk ".Goat milk is compared with the drink milk of distinguishing the flavor of owing to having a strong smell, and the people of goat milk is less.Along with high-tech is applied to bio-pharmaceuticals, food-processing, takes off the The Application of Technology of having a strong smell and give the flourish possibility of bringing of goat milk circle.Though one of milk goat dairy stock that to be China main, it is huge that the production of China's goat milk is compared gap with developed country, mainly is because Chinese elite milk goat kind provenance is not enough and the population hereditary quality is relatively poor.

The phenotype of traditional breeding method application testing animal and its parental generation, reach the genetic information of other relatives in little animal model for generations, the imagination proterties is subjected to the influence of many gene genetic differences, each gene pairs proterties has less relatively contribution, therefore to inevitable some deviation of the estimation of proterties.Molecular genetic marker is one of modern genetics field with fastest developing speed in recent years, and new method continues to bring out, and oriented marker gene number grows with each passing day.This will be for quantitative genetics provides molecular level information, and cattle breeding also will stride into the molecular breeding stage.The GENERALIZATION OF MODERN BREEDING TECHNIQUE of using molecular genetic marker 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.

The molecular genetic marker assisted Selection combines modern biotechnology exactly with conventional system of selection, by the selection of genetic marker being selected indirectly the quantitative trait locus (QTL) of certain proterties of control, enable to utilize simultaneously the phenotype information of marker site information and quantitative character, the breeding value of more accurate estimation animal individual, improve efficiency of selection, accelerate the breeding progress.Marker assisted selection has mainly experienced three phases: the fs is the genetic analysis between each proterties of domestic animal; Subordinate phase is the marking phase of protein (enzyme) mark to quantitative character; Three phases is the molecular genetic marker stage.Along with molecular marking technique is day by day ripe and abundant, make the mark that covers whole genome become possibility, by and QTL between linkage analysis, realize the target of molecular marker assisted selection.

(single nucleotide polymorphism SNP) has been widely used in the assignment of genes gene mapping, clone, genetic breeding and multifarious research as new genetic marker to single nucleotide polymorphism.SNP is the profuse variant form of a kind of quantity that exists in the genome, accounts for more than 90% of genetic polymorphism in the human genome.SNP is different with rare variation, usually is equal to or less than this kind variation of 1% at the population medium frequency and is called as sudden change, and have only frequency greater than just being called as single nucleotide polymorphism at 1% o'clock.

At present, several diverse ways of main employing are found SNPs:DNA sequencing method, PCR-SSCP and dna sequencing combined techniques, the AS-PCR method, the PCR-RFLP method, TaqMam technology and molecular beacon (Molecular Beacons) etc. in these SNP detection techniques, (1) the determined dna sequence method is a SNP detection method the most accurately, but, its testing cost is extremely expensive, and need large-scale instruments such as dna sequencing instrument, simultaneously, in the order-checking process, need very those skilled in the art and experience, so the determined dna sequence method is not a kind of actual desirable SNP detection method that is applied to produce; (2) PCR-SSCP and dna sequencing combined techniques detect SNP and can suitably reduce testing cost, and still, the experimentation of PCR-SSCP is long, operate more loaded down with trivial detailsly, and have the false positive problem in the experimentation, so, also also nonideal SNP detection means; (3) 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 simultaneously, also there be the probability of flase drop in the testing process at the special genes site, therefore, the characteristics that do not have widespread usage at present; (4) TaqMam technology and molecular beacon (Molecular Beacons) all are that (Fluorescence Resornance Energ Transfer FRET) sets up on the basis, utilizes fluorescent mark and instrument to reach testing goal in fluorescence energy transfer.Tetra-sodium order-checking (Pyrosequencing) then is to utilize releasable tetra-sodium and enzyme generation fluorescence in the order-checking process, is the fluorescent mark technology that does not rely on dull and stereotyped glue or capillary electrophoresis, do not rely on DNA, becomes following mainstream technology probably.(5) the RFLP-PCR method is the effective technology of a kind of SNP of detection, introduces restriction enzyme and cut after finding the SNP site, carries out agarose, polyacrylate hydrogel electrophoretic analysis then, just can differentiate the SNP site exactly.The RFLP-PCR method not only has the accuracy of dna sequencing method, overcome expense costliness, troublesome operation, false-positive shortcoming again, and the sequence site of being detected does not have the singularity requirement.

Ghrh receptor (Growth hormone-releasing hormone receptor, GHRHR) be a kind of film associating acceptor, with G albumen coupling activated adenyl cyclase, make cAMP increase in the cell, thereby bring out the activity of protein kinase A path, cause the propagation and the secretion of growth hormone of cell, and then influence proterties such as the growing of animal, lactation.Hypothalamus, kidney, placenta animal have all been found the GHRHR expression of gene, GHRHR plays critical effect in normal adjusting and controlling growth, but it can not play a role separately, the on the one hand secretion of GHRHR by combining regulation and control tethelin (GH) with GHRH and synthetic, it is grown the factor as one play specific action in hypophysis somatotroph propagation on the other hand.

At present, many on the mankind, mouse and ox for the research of GHRHR gene, mainly in preceding brain development, animal growth, made a large amount of research.Animals such as mouse, ox are more common in research about the variation of animal GHRHR gene genetic both at home and abroad, do not see the report of GHRHR genes in dairy goat heritable variation and proterties association study.At present related research is still blank about the functional study of this gene locus and heritable variation thereof and economic characters (as: different lactation period milk yield).

Summary of the invention

The problem that the present invention solves is to provide the single nucleotide polymorphism and the detection method thereof of GHRHR genes in dairy goat, utilize the polymorphism of target DNA sequence of the method examination GHRHR genes in dairy goat of DNA pond order-checking, seek the SNP relevant as molecule marker, accelerate milk goat stock breeding speed with the milk goat milk character.

The present invention is achieved through the following technical solutions:

A kind of single nucleotide polymorphism of GHRHR genes in dairy goat, its gene mononucleotide polymorphism comprises:

The 231st at the GHRHR gene order table of the milk goat shown in SEQ ID NO.1 is the nucleotide polymorphisms of C or G;

The detection method of the single nucleotide polymorphism of above-mentioned GHRHR genes in dairy goat is:

With the milk goat complete genome DNA to be measured that comprises the GHRHR gene is template, is primer with primer to P, the pcr amplification GHRHR genes in dairy goat; After restriction enzyme MspI digestion pcr amplification product, the amplified fragments after again enzyme being cut carries out agarose gel electrophoresis; Identify the 231st bit base polymorphism of the milk goat GHRH gene order table shown in the SEQ ID NO.1 according to electrophoresis result;

Described primer to P is:

Upstream primer P1:acgccaccct ctttcaccc 19;

Downstream primer P2:catcctgggt gcttcttgaa g 21.

Described pcr amplification reaction program is:

94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 57 ℃ of annealing 35s, 72 ℃ are extended 50s, 35 circulations; 72 ℃ are extended 10min.

Described agarose gel electrophoresis is that mass concentration is 2% agarose gel electrophoresis.

Describedly identify that according to the agarose gel electrophoresis result the 231st bit base polymorphism of milk goat GHRH gene order table is: the CC genotype shows as 291bp and 134bp band; The CG genotype shows as 21bp, 195bp, 134bp and 96bp band; The GG genotype shows as 195bp, 134bp and 96bp band.

Compared with prior art, the present invention has following beneficial technical effects:

The invention discloses the nucleotide polymorphisms of the functional gene GHRHR relevant with the milk goat lactation, this nucleotide polymorphisms can be as a molecular genetic marker, utilize the phenotype information of marker site information and quantitative character, the breeding value of more accurate estimation animal individual, improve efficiency of selection, accelerate the breeding progress.

SNP polymorphism at above-mentioned GHRHR gene the invention also discloses its detection method, by designing specific PCR primer amplification fragment, can enough RFLP methods simple, fast, cost is low, detect the polymorphism of its mononucleotide accurately.

The present invention has carried out gene type and gene frequency analysis to the SNP of GHRHR gene, and and Sa can milk goat carry out the proterties association analysis between each parity annual lactation amount; The result shows: the CG genotype is a preponderant genotype, has its triplet annual lactation amount of the genotypic milk goat of CG and all is higher than CC, GG genotype; The Nucleotide polymorphic site of GHRHR gene can become the mark of molecular genetic assistant breeding.

Because the GHRHR gene function relates generally to difference milk yield lactation period, detection method provided by the invention is that the SNP of GHRHR gene and the foundation of milk yield relation are laid a good foundation, for use in the marker assisted selection of Chinese milk goat breast, set up the good milk goat population of genetic resources fast with economic characters.

Description of drawings

Fig. 1 is that milk goat blood sample genome dna electrophoresis detects figure;

Fig. 2 is a GHRHR genes in dairy goat PCR product electrophoresis;

Fig. 3 is the electrophoresis result figure that the MspI restriction enzyme digestion and electrophoresis of the PCR product of GHRHR genes in dairy goat the 6th exon and part the 6th intron 425bp detects the C/G polymorphism of the 7307th of GHRHR gene; The CC genotype shows as 291bp and 134bp band; The CG genotype shows as 21bp, 195bp, 134bp and 96bp band; The GG genotype shows as 195bp, 134bp and 96bp band;

Fig. 4 is the different genotype order-checking peak figure of GHRHR genes in dairy goat SNP.

Embodiment

The present invention according to the GHRHR gene conservative sequences Design primer of the isogenic animal ox of milk goat, genomic dna pond with 2 kinds of milk goat kinds is a template respectively, carry out pcr amplification, and, after checking order, seek the mononucleotide polymorphic of this amplified fragments the PCR product purification; Carry out the proterties correlation analysis at the mononucleotide polymorphic of finding, and provide its detection method, make the nucleotide polymorphisms of GHRHR gene become a kind of can be fast, the convenient molecular genetic marker that detects, provide foundation for accelerating to set up milk goat population with high-quality economic characters.

The clone of a, GHRHR genes in dairy goat partial dna sequence and the detection of polymorphism thereof

1, the collection of milk goat blood sample and processing

Get goat blood sample 10mL, add the EDTA 500 μ L anti-freezings of 0.5mol/L, put into ice chest after slowly putting upside down 3 times ,-80 ℃ of preservations are standby.

The present invention has adopted 2 milk goat kinds, is specially:

(1) Central Shanxi Plain milk goat blood sample picks up from three former Ta Nanbao kind fields, Xian City, Shanxi Province for 350 parts;

(2) Sa energy milk goat blood sample picks up from Baoji, Shaanxi province city Qianyang County Sa for 268 parts and can breed center (201 parts) and Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology milk goat field (67 parts) by milk goat;

2, the extraction of blood sample genomic dna, purifying

(1) freezing blood sample room temperature is thawed, transferase 45 00 μ L to 1.5mL Eppendorf pipe adds equal-volume PBS damping fluid, abundant mixing, and the centrifugal 10min of 12000r/min (4 ℃), abandoning supernatant, the repetition above-mentioned steps is transparent to supernatant liquor, precipitation is faint yellow.

(2) in centrifuge tube, add DNA extraction buffer 500 μ L, shake, make the hemocyte precipitation break away from centrifuge tube tube wall, 37 ℃ of water-bath 1h.The SDS of the Tris of the preparation of DNA extraction buffer: 0.6057g, the EDTA of 18.612g and 2.5g adds ultrapure water 500mL, sterilization, accent pH to 8.0, and 4 ℃ of preservations are standby.

(3) add Proteinase K 3 μ L (20mg/mL) and mixings, 55 ℃ are spent the night to clarification, and defecator not can add 1 μ L Proteinase K mixing and continue digestion to clarification as yet.

(4) reaction solution is cooled to room temperature, adds 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 liquor in another 1.5mL centrifuge tube over to, repeats once.

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

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

(7) 4 ℃, the centrifugal 10min of 12000r/min, abandoning supernatant, the ice-cold ethanol rinsing DNA precipitation with 70% 2 times.

(8) 4 ℃, the centrifugal 10min of 12000r/min makes the ethanol volatilization clean under the abandoning supernatant, room temperature.

(9) dried DNA is dissolved in TE-damping fluid or the ultrapure water of 80～100 μ L, and 4 ℃ of preservations are dissolved fully until DNA, and 0.8% agarose gel electrophoresis detects its quality ,-80 ℃ of preservations.

Adding 10%SDS in the dna solution of (10) 500 μ L, to make its final concentration be 0.1%, adds Proteinase K to final concentration and reach 50 μ g/mL;

About (11) 5 ℃ of insulation 10h;

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

(13) the centrifugal 5min phase-splitting of 12000r/min is drawn the upper strata water to another centrifuge tube;

(14) add 1/10 volume 3mol/L sodium-acetate and the 2 times of ice-cold dehydrated alcohol deposit D of volume NA;

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

3, the structure in DNA pond

(1) 1% agarose gel electrophoresis detects

Select part DNA sample to carry out agarose gel electrophoresis and detect, the structure that select DNA sample strip homogeneous, do not have hangover, no degradation samples is carried out the DNA pond.

(2) OD pH-value determination pH

With the OD value of UV-light photometric determination DNA sample at 260nm, 280nm place.Calculate dna content and OD ₂₆₀/ OD ₂₈₀Ratio.As 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 quality (ng)=50 * OD ₂₆₀Value * extension rate

(3) structure in kind DNA pond

After DNA detection finishes, taking out certain amount and be diluted to 50ng/ μ L, is to get 10 μ L mixing the 50ng/ μ L DNA sample to be built into kind DNA pond from 50 concentration of Central Shanxi Plain milk goat kind then;

Also making up Sa after the same method can milk goat kind DNA pond.

4, pcr amplification primer design

Because the goat genome sequence is not announced as yet, so is reference with the higher ox of the homology that NCBI was announced (NC_007302) with part sheep sequence, utilize Primer 5.0 designs to increase and comprise the PCR primer in GHRHR genes in dairy goat the 6th exon and part intron zone, its primer is as follows to sequence P:

Upstream primer P1:acgccaccct ctttcaccc 19;

Downstream primer P2:catcctgggt gcttcttgaa g 21;

5, PCR clone GHRHR genes in dairy goat

DNA pond with 2 milk goat kinds is a masterplate respectively, with the primer that designs P is carried out pcr amplification, and PCR total reaction system is 25 μ L, sees Table 1; PCR total reaction program sees Table 2.

Table 1PCR reaction system

The system composition	Volume (μ L)
		10 * PCR damping fluid (MBI)	??2.5
??MgCl 2(25mmol/L)	??1.5
		??dNTPs(2.5mmol/L)	??2.5
Upstream primer (10pmol/L)	??0.25
		Downstream primer (10pmol/L)	??0.25
Taq archaeal dna polymerase (0.5U/ μ L)	??2.0
		Dna profiling (50ng/ μ L)	??1.0
Sterilization ultrapure water (H 2O)	??15.0
		Cumulative volume	??25.0

Table 2PCR response procedures

6, PCR product purification and order-checking

Pcr amplification carries out agarose gel electrophoresis after finishing, and electrophoresis result can be known the band of seeing 425bp as shown in Figure 1, illustration purpose gene clone success; The glue of cutting that carries out the PCR product then reclaims and purifying: contain the segmental gel of purpose from the sepharose cutting-out under ultraviolet lamp, put into the 1.5mL centrifuge tube, reclaim purification kit (sky, Beijing root biotech firm) purified pcr product with the PCR product then, according to the operation of test kit specification sheets, concrete steps are as follows:

(1) at first add 500 μ L balance liquid BL in adsorption column, centrifugal 1 minute of 12000rpm outwells the waste liquid in the collection tube, adsorption column is relay reclaim in the collector.

(2) single target DNA band is downcut from sepharose put into clean centrifuge tube, take by weighing weight.

(3) add equal-volume solution PC in blob of viscose, 60 ℃ of water-baths were placed about 10 minutes, constantly leniently spun upside down centrifuge tube therebetween, fully dissolved to guarantee blob of viscose.

(4) previous step gained solution is added in the adsorption column, centrifugal 1 minute of 12000rpm outwells the waste liquid in the collection tube, and adsorption column is reentered in the collection tube.

(5) add 700 μ L rinsing liquids in adsorption column, centrifugal 1 minute of 12000rpm outwells waste liquid, and adsorption column is reentered in the collection tube.

(6) add 500 μ L rinsing liquids in adsorption column, centrifugal 1 minute of 12000rpm outwells waste liquid, and centrifugal adsorption column is put into collection tube, and centrifugal 2 minutes of 12000rpm removes rinsing liquid as far as possible.Adsorption column is placed room temperature or 50 ℃ of incubator numbers minute, thoroughly dry.

(7) adsorption column is put in the clean centrifuge tube, to an amount of elution buffer of the unsettled dropping in adsorption film mid-way, room temperature was placed 2 minutes.12000rpm collected dna solution in centrifugal 1 minute.

(8) in order to improve the yield of DNA, can be with in the centrifugal solution that the obtains centrifugal adsorption column of add-back again, repeating step 7.

Above two kind DNA ponds PCR purified product that is template is served marine life Engineering Co., Ltd carry out two-way order-checking.The sequencing result of GHRHR genes in dairy goat purpose fragment 425bp is shown in SEQID NO.1.

Peak figure analyzes to order-checking, and what wherein in same site two different peaks are arranged is that single nucleotide mutation has taken place; Two kinds of detected results of C, G have appearred in the GHRHR gene the 7307th (the 231st of SEQ ID NO.1) that is positioned at milk goat, are the SNP polymorphism of the GHRHR genes in dairy goat that examination arrives, and this site is the nucleotide polymorphisms for C or G.

The PCR-RFLP of b, GHRHR genes in dairy goat C＞G mutation polymorphism detects

C＞when G suddenlyd change, promptly C sported G, makes original sequence C CCG also correspondingly become CCGG, thereby becomes the restriction enzyme enzyme recognition site of MspI when the generation of the 7307th site; Can directly cut the segmental enzyme of purpose and carry out gene type by MspI.

1, RFLP-PCR design of primers

With primer to the primer of P as pcr amplification.

2, PCR reaction conditions

PCR product amplification system and reaction conditions are described as table 1 and table 2 respectively, and 1.0% agarose gel electrophoretogram of pcr amplification product can be seen the band of 425bp as shown in Figure 2 clearly.

3, the MspI enzyme of pcr amplification product is cut

(1) 20 μ LMsp I endonuclease reaction system: 10 μ L PCR products, 10 * damping fluid (containing BSA), 2.5～3.0 μ L, Msp I (10U/ μ L) is 1.0 μ L, 8.0 μ L sterilization pure water (H ₂O);

(2) enzyme is cut digestion condition: digest 5～10h in 37 ℃ of constant incubators.

(3) agarose gel electrophoresis analysis behind the Msp I digestion PCR product

Sepharose with 2.0%, 120V voltage electrophoresis 1 hour, EB dyeing detects enzyme and cuts the result, takes a picture with Kodak DC 120 gel imaging analysis systems and analyzes, and declare type, write down its genotype;

Cut recognition site owing to also comprise other a Msp I enzyme in the 425bp fragment of pcr amplification, when the 7307th of GHRHR gene sports G by C, the 7305bp of the GHRHR gene product of pcr amplification～7308bp sequence is ccgg, cut the amplified fragments enzyme at cc/gg restriction enzyme MspI identification back, and amplified fragments is cut to 4 sections; And do not undergo mutation as the 7307th of GHRHR gene, can not form new restriction enzyme MspI enzyme and cut recognition site, amplified fragments is cut to 2 sections;

Because milk goat is 2 times of body animals, so when the sudden change of C＞G takes place, can form 3 kinds of different genotype, be respectively CC, CG, GG, figure is as shown in Figure 3 as a result for the gel of its PCR-RFLP detection:

Wherein, the CC genotype is a wild-type, and the SNP site of its two DNA chains all can not be cut by the MspI enzyme, shows as 291bp and 134bp band; The SNP site of two chains of the wild-type GG after undergoing mutation all can be digested, shows as 291bp, 134bp and 96bp band; One SNP site in two chains of heterozygote CG can be identified and another can not be identified, and shows as 291bp, 195bp, 134bp and 96bp band; According to the number of band and the size of band, detected through gel electrophoresis result shown in Figure 3 can judge very clearly whether point mutation has taken place, and three kinds of genotype is distinguished, thereby detect its SNP polymorphism.

(4) sequence verification of the individual PCR product of different genotype

Utilize ABI 377 and ABI 3730 sequenators that the individual PCR product of different genotype is carried out positive and negative two-way order-checking respectively; Simultaneously, carry out the SNP position analysis, the result shows that individual its 7307 the sequencer map of the heterozygote CG genotype that comprises 291bp, 195bp, 134bp and 96bp band is expressed as C or G really, shown in Fig. 4 b, the 6th peak is two peaks from left to right, and CC genotype, GG genotype are respectively C, G, respectively shown in Fig. 4 a, 4c.

The SNP that c, GHRHR gene are the 7307th is as the application of molecule marker in different goat colony polymorphism

1, the diagnosis in colony's polymorphism

Utilize above-mentioned SNP pleiomorphism detecting method to 268 parts of DNA samples of Sa energy milk goat, 440 parts of DNA samples of Central Shanxi Plain milk goat, carry out the evaluation of SNP polymorphism respectively; Add up its SNP site frequency and with the related situation of proterties.

2, the frequency statistics analysis in SNP site

Genotype frequency is meant that certain genotype number of individuals of a certain proterties in the colony accounts for the ratio of total individual number.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 that a certain gene number is to the relative ratios of its allelotrope sum in the colony.The formula that calculates can be write as: P _A=(2N _AA+ N _Aa1+ N _Aa2+ ...+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 different multiple allelomorphos of allelotrope A; Statistics sees Table 3.

The 7307th SNP gene frequency distribution table of table 3 GHRHR genes in dairy goat

As can be seen from Table 3: the C gene frequency of Sa energy milk goat and Central Shanxi Plain milk goat is far above T allelotrope, and this shows that allele C is relevant with milk character.

3, the association analysis of genetic effect

The genotype (CC, CG and GG) of genotype data: MspI identification

Production data: parity (first tire, second tire and triplet) and year milk yield

The association analysis model:

Utilize the dependency of SPSS (16.0) software analysis gene locus, male animal, lambing season, age and parity factor and milk character.Earlier data are described analysis, determine whether to exist outlier, utilize the least square analysis that data are proofreaied and correct again; According to data characteristics, utilize multivariate linear model analyzing gene type effect.Model is as follows:

y _ijklmn＝μ+Genotype _i+S _j+LS _k+LN _l+Age _m+Xn+e _ijklmn

Wherein: y _IjklmBe individual phenotype record; LN _lThe parity effect; S _jBe the sire effect; LS _k: lambing season effect; Age _mBe age effect; Xn is various secondarys and makes effect more than the secondary mutually, as: Age * Genotype, S _j* Genotype etc.; e _IjklmnBe random error; Utilization SPSS (16.0) software is analyzed data, and uses the least square fitting linear model, and milk yield index between each genotype is carried out significance test of difference.

The result shows (seeing Table 4): for discernible the 7307th the SNP site of MspI, the CG genotype is a preponderant genotype, has its triplet annual lactation amount of the genotypic milk goat of CG and all is higher than CC, GG genotype, but do not reach conspicuous level (P＞0.05).

Variance analysis between table 4MspI polymorphic site and each tire lactation amount of Saanen goat

The nucleotides sequence tabulation

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

＜120〉single nucleotide polymorphism of GHRHR genes in dairy goat and detection method thereof

<210>1

<211>426

<212>DNA

＜213〉milk goat (Capra hircus)

<220>

<221>s

<222>(231)

<400>1

acgccaccct?ctttcaccag?gagaacatgg?accactgcag?cttctccact?gtaacagtcg?????60

tgggtggggg?tgctggcgtg?ggcagggagg?ttggattaga?gatgtcagcc?tgtgcagtcc????120

agtgggctga?ccccggggct?ctggctttgc?caaggacaga?gcgggaaagc?acccctcccc????180

cttcccgccc?ctccttgggg?tcaagtccta?aatcctcttg?cgcccagccc?sgtcattccc????240

ttgctccact?ctctgctcca?tattctgtat?tctggtttca?ttccccatcc?cagagcccaa????300

cctagagcac?atttcactcc?gctcatgctt?tccatctcac?acttcctctg?ggctcggtct????360

gtgctgggtg?tgggtgtcag?gggctggaca?aagccaggtc?ctttcttcaa?gaagcaccca????420

ggatga???????????????????????????????????????????????????????????????426

Claims (5)

1. the single nucleotide polymorphism of a GHRHR genes in dairy goat is characterized in that, its gene mononucleotide polymorphism comprises:

The 231st at the GHRHR gene order table of the milk goat shown in SEQ ID NO.1 is the nucleotide polymorphisms of C or G.

2. the detection method of the single nucleotide polymorphism of the described GHRHR genes in dairy goat of claim 1 is characterized in that, may further comprise the steps:

With the milk goat complete genome DNA to be measured that comprises the GHRHR gene is template, is primer with primer to P, the pcr amplification GHRHR genes in dairy goat; After restriction enzyme MspI digestion pcr amplification product, the amplified fragments after again enzyme being cut carries out agarose gel electrophoresis; Identify the 231st bit base polymorphism of the milk goat GHRH gene order table shown in the SEQ ID NO.1 according to electrophoresis result;

Described primer to P is:

Upstream primer P1:acgccaccct ctttcaccc 19;

Downstream primer P2:catcctgggt gcttcttgaa g 21.

3. the detection method of the single nucleotide polymorphism of GHRHR genes in dairy goat as claimed in claim 2 is characterized in that, described pcr amplification reaction program is: 95 ℃ of pre-sex change 4～5min; 94 ℃ of sex change 30s, 57.0 ℃ of renaturation 35s, 72 ℃ are extended 35s, 30～35 circulations; 72 ℃ are extended 10min.

4. the detection method of the single nucleotide polymorphism of GHRHR genes in dairy goat as claimed in claim 2 is characterized in that, described agarose gel electrophoresis is that mass concentration is 2% agarose gel electrophoresis.

5. the detection method of the single nucleotide polymorphism of GHRHR genes in dairy goat as claimed in claim 2, it is characterized in that identify that according to the agarose gel electrophoresis result single nucleotide polymorphism of the 7307th of GHRHR genes in dairy goat is: the CC genotype shows as 291bp and 134bp band; The CG genotype shows as 21bp, 195bp, 134bp and 96bp band; The GG genotype shows as 195bp, 134bp and 96bp band.

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