CN101705288A - Mononucleotide polymorphism of milk goat MFG-E8 genes and detection method thereof - Google Patents

Abstract

The invention discloses a mononucleotide polymorphism of milk goat MFG-E8 genes and a detection method thereof. The invention takes the whole genome DNA of the milk goat to be detected containing the MFG-E8 genes as the template, takes the primer coupled P (comprising P1, R1 and newP) as the primer, and takes the PCR amplified milk goat MFG-E8 as the genes; the restriction enzyme EcoRV is utilized to digest PCR amplifying products, and then polyacrylamide gel electrophoresis is carried out on the amplified segments after enzyme digestion; the mononucleotide polymorphism of the 12892nt of the milk goat MFG-E8 genes is identified according to the results of polyacrylamide gel electrophoresis. As the MFG-E8 gene function relates to development of breast, butter fat, total solids and other milk elements, the detection method provided by the invention lays a foundation for the establishment of the relation between the SNP and the milk component of the MFG-E8 genes, so as to be used for the marker-assisted selection of the properties of Chinese milk goat and the rapid establishment of milk goat species group with excellent genetic resources.

Description

The single nucleotide polymorphism of milk goat MFG-E 8 genes 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 a kind of single nucleotide polymorphism of milk goat MFG-E 8 genes 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 ResornanceEnerg 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.

MFG-E8 is to be found first in Ruzhong and Mammals epithelial cell as a kind of glycoprotein, is the another kind of major protein composition of milk fat ball film.MFG-E8 glycoprotein is the contact molecule between apoptotic cell and the active phagocytic cell, and in the mammary gland deterioration process, MFG-E8 is at the identification apoptotic cell, and the epithelial cell aspect that the activation phagocytic cell is engulfed apoptosis plays an important role.The disappearance of MFG-E8 will postpone the removing of the mammary epithelial cell and the dairy fats ball of apoptosis, the growth and the differentiation of infringement mammary gland, and causing simultaneously fails is obstructed and the generation of mammitis.This albumen impels the interaction of smart ovum in fertilization process.In addition, MFG-E8 plays a role in the maturation of pig lactadherin and in the capacitation and discovers.MFG-E8 also is a kind of important milk-content that can stop the invasion of enteron aisle cause of disease and infect.

At present, the research for the MFG-E8 gene focuses mostly at mouse and human immunology.Also be more common in mouse both at home and abroad with human about the research of animal MFG-E8 gene genetic variation, do not see the report of milk goat MFG-E 8 genes heritable variation or SNP research.The research scarcity in present Chinese milk goat MFG-E 8 genes heritable variation field, the related research of the functional study of this gene locus and heritable variation thereof and economic characters (as: height, body length, milk yield, suckling becomes to grade proterties) is still blank.

Summary of the invention

The problem that the present invention solves is to provide a kind of single nucleotide polymorphism and detection method thereof of milk goat MFG-E 8 genes, utilize the RFLP-PCR method single nucleotide polymorphism that sudden change may cause the proteins encoded expression level to change at the synonym on its gene locus to detect, eliminate in advance and eliminate the individuality that carries bad gene, accelerate to have the foundation of high-quality economic characters milk goat population.

The present invention is achieved through the following technical solutions:

The method for quick of milk goat MFG-E 8 genes single nucleotide polymorphism and application thereof, its gene mononucleotide polymorphism comprises:

At milk goat MFG-E 8 genes 12892bp is the nucleotide polymorphisms of C or T.

The detection method of the single nucleotide polymorphism of above-mentioned milk goat MFG-E 8 genes is:

With the milk goat complete genome DNA to be measured that comprises the MFG-E8 gene is template, is primer with primer to P, the pcr amplification milk goat MFG-E 8 genes; After restriction enzyme EcoRV digestion pcr amplification product, the amplified fragments after again enzyme being cut carries out polyacrylamide gel electrophoresis; Identify the single nucleotide polymorphism of milk goat MFG-E 8 genes 12892bp according to the polyacrylamide gel electrophoresis result;

Described primer to P is:

Upstream primer: cctactacgc acgactggat at 22;

Downstream primer: acaaggctca aagaaactcc 20.

Described pcr amplification reaction program is:

94 ℃ of pre-sex change 4min; 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 20s, 30～35 circulations; 72 ℃ are extended 10min.

Described polyacrylamide gel electrophoresis is that mass concentration is 10% polyacrylamide gel electrophoresis.

Describedly identify that according to the polyacrylamide gel electrophoresis result single nucleotide polymorphism of the 12892nd of milk goat MFG-E 8 genes is: the TT genotype shows as the 195bp band; The TC genotype shows as 195bp, 175bp and 20bp band; The CC genotype shows as 175bp and 20bp band; Wherein, single nucleotide polymorphism CC genotype is the synonym sudden change.

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

The invention discloses the nucleotide polymorphisms of the functional gene MFG-E8 relevant with the milk goat lactation, this nucleotide polymorphisms can be as a molecular genetic marker; The synonym sudden change that comprises in this polymorphism may cause the proteins encoded expression level to change, and eliminates the individuality that carries bad gene in advance, can accelerate to have the foundation of high-quality economic characters milk goat population.

The present invention utilizes the RFLP-PCR method may produce the single nucleotide polymorphism that the proteins encoded expression level changes to the synonym on milk goat MFG-E 8 genes 12892bp sudden change and detects, when 12892bp sports C by T, the codon AAT of original coding Asp undergos mutation and is AAC, cause influencing the efficient of translation at translation process owing to carrying this amino acid whose tRNA abundance difference, thereby influence this proteic expression amount at the mRNA that transcribes.

Milk goat MFG-E 8 genes method for detecting single nucleotide polymorphism provided by the invention, sport the transition mutations of C by T at 12892bp, introduce base mispairing by the 3 ' end that design of primers is artificial at downstream primer, when becoming C by T, form restriction enzyme EcoRV recognition site behind the pcr amplification MFG-E8 gene in the mispairing position, and suddenly change when not taking place, can not form the EcoRV recognition site behind the pcr amplification MFG-E8 gene; Can detect the MFG-E8 gene mononucleotide polymorphism accurately, fast and easily by the electrophoresis detection somatotype: not comprising the 195bp band is CC genotype individuality; Not comprising the 175bp band is TT genotype individuality; Comprising 195bp and 175bp band simultaneously is TC genotype individuality; And then to the gene frequency change monitoring of the MFG-E8 gene SNP of population.

The present invention has carried out gene type and gene frequency analysis to the EcoRV site of MFG-E8 gene, and and each of Saanen goat lactation amount in tire year between carried out the proterties association analysis; The result shows: it is not remarkable that the EcoRV site influence difference to the milk yield (first tire, second tire, triplet and the 4th tire) of three individual chi proterties (the long and chest measurement of height, body) and four parity, but milk fat content and the total solid of Xi Nongsa energy milk goat had remarkably influenced; The Nucleotide polymorphic site of milk-content MFG-E8 gene can become the mark of molecular genetic assistant breeding.

Description of drawings

Fig. 1 is the agarose electrophoresis detected result figure of milk goat MFG-E 8 genes pcr amplification after product;

Fig. 2 cuts the rear electrophoresis result for the 195bp PCR product that milk goat MFG-E 8 genes comprises the mutational site through the EcoRV enzyme;

Fig. 3 is the different genotype sequencer map of milk goat MFG-E 8 genes SNP.

Embodiment

The present invention utilizes the RFLP-PCR method may produce the single nucleotide polymorphism that the proteins encoded expression level changes to milk goat MFG-E 8 genes 12892bp synonym sudden change and detects, below in conjunction with the present invention is described in further detail, the explanation of the invention is not limited.

A, milk goat MFG-E 8 genes contain the 7th exon region PCR primer design

The present invention is reference according to the sequence with the isogenic animal ox of milk goat, and specifically the sequence with the Angus (NC_007319) that NCBI was announced is reference; Utilize Primer 5.0 designs to increase and comprise the PCR primer of milk goat MFG-E 8 genes the 7th exon, its primer sequence is as follows:

Upstream primer: tcttttccct tcactgcctc 20;

Downstream primer: acaaggctca aagaaactcc 20;

With above-mentioned primer to the milk goat genome amplification, the gene fragment of the 397bp that comprises milk goat MFG-E 8 genes the 7th exons 1 2671bp～13063bp can increase, increase the segmental electrophoresis detection in back as shown in Figure 1, and wherein, swimming lane 1～6 is for detecting fragment, swimming lane M is Marker (100bp, 200bp, 300bp, 400bp, 500bp, 600bp); Sequencing result is shown in SEQ ID NO.1, to the fragment of amplification check order identify after, find that working as 12892bp (is the 930th in MFG-E8 gene C DS district, be positioned at the 225th of sequence table) T when sporting C, amino acid code of 310bp of causing encoding sports AAC by AAT, thereby forms same sense mutation;

Because (SNP site) no nature restriction enzyme site at 12892bp place can not be checked by direct enzyme cutting, and if manually design the mispairing of PCR primer at the 12891bp place, be T by the A mispairing; When 12892bp sports C by T, the 18bp～23bp the sequence of the primer amplification MFG-E8 gene product of design is GATATC, formed the restriction enzyme site of restriction enzyme EcoRV, when when 12892bp does not suddenly change, the 18bp～23bp the sequence of the primer amplification MFG-E8 gene product of design is GATATT, and restriction enzyme EcoRV can not discern; So just can detect this site SNP polymorphism; Therefore designing pcr amplification primer P is:

Upstream primer: cctactacgc acgactggat at 22;

Downstream primer: acaaggctca aagaaactcc 20;

Wherein, the base mismatch of introducing is the T of downstream primer 22bp; The fragment of the 195bp of 12869bp～13063bp that the corresponding gene fragment that increases of primer P is the MFG-E8 gene when with restriction enzyme EcoRV the gene fragment enzyme of the corresponding amplification of primer P being cut digestion, can not be cut open and maybe can be cut into 2 sections.

B, carry out the MFG-E8 gene fragment of pcr amplification milk goat to be measured with primer P

1, milk goat sample collection and extracting genome DNA

1) collection of milk goat sample

The present invention specifically with 688 ewes of the population of 2 place of china milk goat kinds as detected object, specifically gathering sample and see Table 1: Xi Nongsa can milk goat (244), Central Shanxi Plain milk goat (424);

The collection of table 1 milk goat sample

2) separation of blood sample genomic dna, extraction, purifying

1) freezing blood sample (being mainly hemocyte) room temperature is thawed, and shifts 1.0mL to 2.0mL Eppendorf centrifuge tube, adds equal-volume PBS liquid, abundant mixing, 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;

3) add Proteinase K to 3 μ L (20mg/mL) and mixing, 55 ℃ are spent the night to clarification, and defecator not can add 1 μ L Proteinase K mixing and continue digestion until 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 chloroform, primary isoamyl alcohol mixed solution (24: 1) 500 μ L, abundant mixing 20min, 4 ℃, the centrifugal 10min of 12000r/min changes supernatant liquor in another 1.5mL centrifuge tube over to;

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

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

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

10) dried DNA is dissolved in the TE liquid 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.

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

12) 5 ℃ are incubated about 10h;

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

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

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

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

3) pcr amplification

The PCR reaction system adopts mixes the application of sample method, promptly according to the number of the required PCR reaction of the quantity of the required various components of each reaction system and 1 secondary response, calculate the total amount of various reactive components, join in 1 1.5mL centrifuge tube, fully instantaneous centrifugal behind the mixing, divide again to install in each 0.2mLEppendorf PCR pipe, add template DNA then, instantaneous more centrifugal laggard performing PCR amplification;

The PCR reaction system sees Table 2:

Table 2PCR reaction system

The PCR response procedures:

94 ℃ of pre-sex change 4min;

72 ℃ are extended 10min;

Genomic dna to 688 samples of 2 milk goat kinds carries out pcr amplification, obtains to comprise in the milk goat MFG-E 8 genes of 688 individualities the dna fragmentation of the 195bp in this SNP site.

C, EcoRV enzyme are cut the MFG-E8 gene fragment of digestion pcr amplification

1, EcoRV endonuclease reaction digestion system (25～30 μ L): 10～15 μ L PCR products, 10 * damping fluid (containing BSA), 2.5～3.0 μ L, EcoRV (10U/ μ L) is 1.0～1.5 μ L, 11.5～16.5 μ L sterilization pure water (H ₂O);

2, enzyme is cut digestion condition: digest 5～10h in 37 ℃ of constant incubators;

Polyacrylamide gel electrophoresis analysis behind d, the EcoRV digestion PCR product

1) polyacrylamide gel (PAGE) of making 10%, 200V voltage electrophoresis 45min behind the last sample, 0.01%AgNO ₃, the 2%NaOH colour developing.

2) treat that the different dna fragmentation of molecular weight separates when clear, in BIO-RAD Gel Doc 2000 gel imaging system imagings;

3) according to polyacrylamide gel electrophoresis interpretation of result SNP polymorphism:

Analyze with the photograph of BIO-RAD Gel Doc 2000 gel imaging systems, judge the polymorphism of SNP:

When the 12892bp of MFG-E8 gene sports C by T, owing to introduced base mispairing, the 18bp of the MFG-E8 gene product of pcr amplification～23bp sequence is GATATC, cut the amplified fragments enzyme at GAT^ATC restriction enzyme EcoRV identification back, cut the upstream primer fragment of 20bp, amplified fragments is cut to 2 sections; And the 12892bp of MFG-E8 gene does not undergo mutation, and restriction enzyme EcoRV can not discern the restriction enzyme site that base mispairing is introduced, and amplified fragments is not cut open;

Because milk goat is 2 times of bodies, so the polyacrylamide gel electrophoresis result of the polymorphism of the SNP of the 12892bp of the genomic MFG-E8 gene of milk goat is:

The TT genotype shows as 195bp one band; The TC genotype shows as 195bp, 170bp and 20bp three bands; The CC genotype shows as 175bp and 20bp two bands; Because 20bp is less, exceed the imaging visual field so analyze the swimming position at polyacrylamide gel electrophoresis, but still can differentiate TT genotype, TC genotype and CC genotype accurately by 195bp and 175bp band: not comprising the 195bp band is CC genotype individuality; Not comprising the 175bp band is TT genotype individuality; Comprising 195bp and 170bp band simultaneously is TC genotype individuality;

As shown in Figure 2, wherein, swimming lane 1 does not comprise the 175bp band, it is a TT genotype individuality, and swimming lane 2 does not comprise the 195bp band, is CC genotype individuality, comprise 209bp and 70bp band, swimming lane 3, swimming lane 4 comprise 195bp and 175bp band simultaneously, be heterozygote TC genotype individuality, swimming lane M is Marker I (600bp, 500bp, 400bp, 300bp, 200bp, 100bp).

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 the sequencer map of individual its 12892bp of heterozygote TC genotype that comprises 175bp and 195bp band is expressed as T or C really, shown in Fig. 3 a, the 4th peak is two peaks from left to right, and CC genotype, TT genotype are respectively C, T, shown in Fig. 3 b, c.

The frequency statistics analysis in e, milk goat MFG-E 8 genes SNP site

1) gene and genotype frequency

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 _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.

T gene frequency rangeability in different milk goat kind MFG-E8 gene SNPs is 54.1%～57.0%, and C gene frequency rangeability is between 43.0%～45.9%, and is as shown in table 3.The gene frequency in this SNP site is greater than 1.0%, meets molecule marker in the improvement of breed greater than 5.0%～10% requirement, possesses the population genetic diversity feature.

The 12892nd SNP gene frequency distribution table of table 3 milk goat MFG-E 8 genes

The association analysis of f, genetic effect and proterties

Genotype data: EcoRV (TT, TC and CC)

Production data: three individual footages are according to (height, body length and chest measurement), data of giving milk of four parity (first tire, second tire, triplet and the 4th tire) and milk-content data.

The association analysis sample: have the west farming of body chi record and the Sa can milk goat totally 668, have the Sa of first tire to the, four tire lactations record can milk goat 170,74 of the Sa energy milk goats of milk-content record are arranged, all milk goats be ewe.

The association analysis model:

Utilize the dependency of SPSS (13.0) 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 least-square analysis that data are proofreaied and correct again; According to data characteristics, utilize the effect of multivariate linear model analyzing gene type to proterties.

Genotype to the effect model of body chi proterties is:

Yij＝μ+Ai+Gj+eij

Wherein: y _IjBe individual phenotype record; μ is a population mean; Ai is the fixed effect at age; Gj is genotypic fixed effect; Eij is a random error.

Genotype to the effect model of milk character is:

Yij＝μ+YSi+Gj+(GYS)ij+eij.

Wherein: Yij is individual phenotype record; μ is a population mean; Ysi is the seasonal effect of lambing; (GYS) ij is various main effect secondarys and makes effect more than the secondary mutually; Eij is a random error.

Utilization SPSS (16.0) software is analyzed data, and uses the least square fitting linear model, to carrying out significance test of difference with the milk yield index between each genotype.

The result shows: in the discernible SNP of EcoRV site, three kinds of genotype are to the lactation amount influence of body chi and first tire to the, four tires all not significantly (P＞0.05), and are specifically as shown in table 4; Three kinds of genotype influence significant difference (P＜0.05) to milk fat content and total solid, and specifically as shown in table 5: the CC type is better than the CT type, and the CT type is better than the TT type.Illustrate that the EcoRV site can become a molecular genetic marker that improves the milk goat milk yield.

The SNP site of table 4.EcoRV identification and the association analysis of body chi and first tire to the, four tires.

The SNP site of table 5.EcoRV identification and the association analysis of milk-content.

Annotate: have same letter and represent difference not remarkable (P＞0.05), alphabetical different table differential different significantly (P＜0.05).

The nucleotides sequence tabulation

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

＜120〉single nucleotide polymorphism of milk goat MFG-E 8 genes and detection method thereof

<210>1

<211>397

<212>DNA

＜213〉milk goat (Capra hircus)

<220>

<221>y

<222>(225)

<400>1

tcttttccct?tcactgcctc?gctctcacgc?ccccacgcgt?aggctgtgga?tcccagatct 60

gggtgacagc?cccccacccc?cacctccttg?ttggcaggat?gcactgaacc?cctaggcctg 120

aaggataata?ccatccccaa?caagcagatc?acagcctcca?gctactacaa?aacctggggc 180

ctgagtgcct?ttagctggtt?tccctactac?gcacgactgg?ataaytgggg?caagttcaac 240

gcctggaccg?cccagaccaa?cagtgcctct?gagtggctgc?aggtgagtca?ggctcctctg 300

gggatgcagg?gttggggttg?ggtggggctg?tgggaatcta?tgaccctggc?ccaaccctgg 360

cctctgcttg?ccaagaggga?gtttctttga?gccttgt 397

Claims (5)

1. the single nucleotide polymorphism of a milk goat MFG-E 8 genes is characterized in that, its gene mononucleotide polymorphism comprises:

At milk goat MFG-E 8 genes 12892bp is the nucleotide polymorphisms of C or T.

2. the detection method of the described milk goat MFG-E 8 genes single nucleotide polymorphism of claim 1 is characterized in that, is template with the milk goat complete genome DNA to be measured that comprises the MFG-E8 gene, is primer with primer to P, the pcr amplification milk goat MFG-E 8 genes; After restriction enzyme EcoRV digestion pcr amplification product, the amplified fragments after again enzyme being cut carries out polyacrylamide gel electrophoresis; Identify the single nucleotide polymorphism of milk goat MFG-E 8 genes 12892nt according to the polyacrylamide gel electrophoresis result;

Described primer to P is:

Upstream primer: cctactacgc acgactggat at 22;

Downstream primer: acaaggctca aagaaactcc 20.

3. the detection method of milk goat MFG-E 8 genes single nucleotide polymorphism as claimed in claim 2 is characterized in that, described pcr amplification reaction program is:

94 ℃ of pre-sex change 4min; 94 ℃ of sex change 30s, ℃ 56 ℃ of annealing 30s, 72 ℃ are extended 20s, 30～35 circulations; 72 ℃ are extended 10min.

4. the detection method of milk goat MFG-E 8 genes single nucleotide polymorphism as claimed in claim 2 is characterized in that, described polyacrylamide gel electrophoresis is 10% polyacrylamide gel electrophoresis.

5. the detection method of milk goat MFG-E 8 genes single nucleotide polymorphism as claimed in claim 2, it is characterized in that identify that according to the polyacrylamide gel electrophoresis result single nucleotide polymorphism of milk goat MFG-E 8 genes 12892nt is: the TT genotype shows as 195bp one band; The TC genotype shows as 195bp, 175bp and 20bp three bands; The CC genotype shows as 175bp and 20bp two bands; Wherein, single nucleotide polymorphism CC genotype is the synonym sudden change.

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