CN101705289B - Single nucleotide polymorphism of GHRHR genes in dairy goat and detection method thereof - Google Patents
Single nucleotide polymorphism of GHRHR genes in dairy goat and detection method thereof Download PDFInfo
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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
Technical field
The invention belongs to the molecular genetics field, relate to SNP (SNP) with the functional gene of milk goat as molecular genetic marker, particularly the SNP of GHRHR genes in dairy goat and detection method thereof.
Background technology
Goat milk has very high nutritive value, does not contain sensibiligen, and nutritive ingredient is balanced, is prone to digest and assimilate, and forms similar with 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, VITAMINs 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 receives 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 applied 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; Through 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; More accurately estimate the breeding value of 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, through 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 SNP.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 SNP at 1% o'clock.
At present, mainly adopt several kinds of diverse ways to find SNPs:DNA sequencing method, PCR-SSCP and dna sequencing combined techniques, AS-PCR method, PCR-RFLP method, TaqMam technology and molecular beacon (Molecular Beacons) etc.In these SNP detection techniques, (1) determined dna sequence method is a SNP detection method the most accurately, 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 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 has boundless prospect, still as a kind of novel SNP detection method in the Application Areas in future; This method need design special primer; And can only be directed against the special genes site, simultaneously, also have the probability of flase drop in the testing process; Therefore, the characteristics that do not have widespread usage at present; (4) the TaqMam technology all is that (Fluorescence Resornance Energ Transfer FRET) sets up on the basis, utilizes fluorescent mark and instrument to reach testing goal in fluorescence energy transfer with molecular beacon (Molecular Beacons).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, after finding the SNP site, introduces restriction enzyme and cuts, and 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 expensive, 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, thus the activity of bringing out the PKA 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 through combining regulation and control tethelin (GH) with GHRH and synthetic, it is grown the factor as one and in the hypophysis somatotroph is bred, plays specific action 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 SNP and the detection method thereof of GHRHR genes in dairy goat; Utilize the polymorphum 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 realizes through following technical scheme:
A kind of SNP 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 SNP of above-mentioned GHRHR genes in dairy goat is:
Milk goat complete genome DNA to be measured to comprise the GHRHR gene is a 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 polymorphum 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 preparatory 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 polymorphum 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 milk goat lactation property; This nucleotide polymorphisms can be as a molecular genetic marker; Utilize the phenotype information of marker site information and quantitative character; More accurately estimate the breeding value of animal individual, improve efficiency of selection, accelerate the breeding progress.
SNP polymorphum to above-mentioned GHRHR gene the invention also discloses its detection method, through designing specific PCR primer amplification fragment, can enough RFLP methods simple, fast, cost is low, detect the polymorphum 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 polymorphum 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 order-checking, seek the mononucleotide polymorphic of this amplified fragments the PCR product purification; Mononucleotide polymorphic to finding carries out the proterties correlation analysis; And its detection method is provided; Make the nucleotide polymorphisms of GHRHR gene become a kind of can be fast, the convenient molecular genetic marker that detects, for accelerating to set up milk goat population foundation is provided with high-quality economic characters.
The clone of a, GHRHR genes in dairy goat partial dna sequence and the detection of polymorphum 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 subsequent use.
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 ability 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, 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.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 subsequent use.
(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 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 in another 1.5mL centrifuge tube over to.
(6) add the NaAc damping fluid of 0.1 times of volume and the ice-cold absolute ethyl 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 deposition 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 until DNA fully, 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 absolute ethyl 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.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 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 following 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 designed primer P is carried out pcr amplification, and PCR total reaction system is 25 μ L, sees table 1; PCR total reaction program is seen 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 |
| TV | 25.0 |
Table 2PCR response procedures
6, PCR product purification and order-checking
Pcr amplification carries out agarose gel electrophoresis after accomplishing, and electrophoresis result is as shown in Figure 1, can know the band of seeing 425bp, illustration purpose gene clone success; 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; According to the operation of test kit specification sheets, concrete steps are following:
(1) at first in adsorption column, add 500 μ L balance liquid BL, 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) in blob of viscose, add equal-volume solution PC, 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) will go up a step gained solution and add 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) in adsorption column, add 700 μ L rinsing liquids, centrifugal 1 minute of 12000rpm outwells waste liquid, and adsorption column is reentered in the collection tube.
(6) in adsorption column, add 500 μ L rinsing liquids, 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.
Serve marine life Engineering Co., Ltd to above two kind DNA ponds PCR purified product that is template and 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; C, two kinds of detected results of 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 polymorphum 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 through 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 said as table 1 and table 2 respectively, and 1.0% agarose gel electrophoretogram of pcr amplification product is as shown in Figure 2, can see the band of 425bp 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, with the analysis of taking a picture of Kodak DC 120 gel imaging analysis systems, and declares type, writes 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 gene types, be respectively CC, CG, GG, figure is as shown in Figure 3 as a result for the gel that its PCR-RFLP detects:
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 polymorphum.
(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, and 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 polymorphum
1, the diagnosis in colony's polymorphum
Utilize above-mentioned SNP pleiomorphism detecting method to 268 parts of DNA samples of Sa ability milk goat, 440 parts of DNA samples of Central Shanxi Plain milk goat, carry out the evaluation of SNP polymorphum respectively; Add up its SNP site frequency and with the association 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 is seen table 3.
The 7307th SNP gene frequency distribution table of table 3 GHRHR genes in dairy goat
Can find out from table 3: the C gene frequency of Sa ability 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 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, utilize multivariate linear model analyzing gene type effect.Model is following:
y
ijklmn＝μ+Genotype
i+S
j+LS
k+LN
l+Age
m+Xn+ei
jklmn
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 level of signification (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>SNP 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 (3)
1. the detection method of the SNP of a GHRHR genes in dairy goat is characterized in that, may further comprise the steps:
Milk goat complete genome DNA to be measured to comprise the GHRHR gene is a 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 polymorphum of the milk goat GHRH gene order table shown in the SEQ ID NO.1 according to electrophoresis result;
The SNP of identifying the 7307th of GHRHR genes in dairy goat according to the agarose gel electrophoresis result 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;
Described primer to P is:
Upstream primer P1:acgccaccct ctttcaccc 19;
Downstream primer P2:catcctgggt gcttcttgaa g 21.
2. the detection method of the SNP of GHRHR genes in dairy goat as claimed in claim 1 is characterized in that, described pcr amplification reaction program is: 95 ℃ of preparatory 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.
3. the detection method of the SNP of GHRHR genes in dairy goat as claimed in claim 1 is characterized in that, described agarose gel electrophoresis is that mass concentration is 2% agarose gel electrophoresis.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102190033A CN101705289B (en) | 2009-11-17 | 2009-11-17 | Single nucleotide polymorphism of GHRHR genes in dairy goat and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102190033A CN101705289B (en) | 2009-11-17 | 2009-11-17 | Single nucleotide polymorphism of GHRHR genes in dairy goat and detection method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
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| CN101705289B true CN101705289B (en) | 2012-06-27 |
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| CN110923332B (en) * | 2019-12-06 | 2022-07-05 | 江苏省农业科学院 | Molecular marker influencing early growth of sheep, detection method and application thereof |
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| CN101063169A (en) * | 2007-06-04 | 2007-10-31 | 西北农林科技大学 | PCR-RFLP method for detecting goat prolactin gene single nucleotide polymorphism |
| WO2009055805A2 (en) * | 2007-10-25 | 2009-04-30 | Newsham Genetics, Lc | Genetic markers and methods for improving swine genetics |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101063169A (en) * | 2007-06-04 | 2007-10-31 | 西北农林科技大学 | PCR-RFLP method for detecting goat prolactin gene single nucleotide polymorphism |
| WO2009055805A2 (en) * | 2007-10-25 | 2009-04-30 | Newsham Genetics, Lc | Genetic markers and methods for improving swine genetics |
Non-Patent Citations (6)
| Title |
|---|
| CUNFANG ZHANG et al..Association of polymorphisms of the GHRHR gene with growth traits in cattle.《Arch. Tierz., Dummerstorf》.2008,第51卷300-301. * |
| E. E. Connor et al..Mapping of the bovine growth hormone-releasing hormone receptor (GHRH-R) gene to chromosome 4 by linkage analysis using a novel PCR-RFLP.《J. Anim. Sci.》.1999,第77卷793-794. * |
| 张存芳.黄牛PTHRP、GHRH和GHRHR基因SNP及其与生长性状关联分析.《中国优秀硕士学位论文全文数据库 农业科技辑》.2008,第2008年卷(第11期),D050-72. * |
| 张海军等.山羊生长激素释放激素(GHRH)和抑制素βA(INHβA)基因多态性.《农业生物技术学报》.2007,第15卷(第5期),767-771. * |
| 胡沈荣等.5 个山羊品种GH 基因第Ⅱ、Ⅴ外显子的多态性及其应用.《西北农林科技大学学报(自然科学版)》.2007,第35卷(第12期),11-15,20. * |
| 陈宏等.山羊重要经济性状的分子标记研究.《中国草食动物》.2008,148-151. * |
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