CN102605064A - Multi-gene pyramiding breeding method for thoroughbred milk goats - Google Patents

Abstract

The invention discloses a multi-gene pyramiding breeding method for thoroughbred milk goats. Genome DNA (deoxyribonucleic acid) of a milk goat continuously giving birth to two or more lambs is used as a template, four pairs of primers respectively expand intron 2 and exon 10 of a prolactin receptor gene and an untranslation region (5'UTR) and exon 1 of luteotropin beta calcmeurin 5', the sizes of expanded products are judged by agarose gel electrophoresis, site mutation of the expanded products of the four pairs of primers are screened by DNA sequencing technology, then polyacrylamide gel electrophoresis is used for performing genetic typing and gene frequency analysis for SNPs (single nucleotide polymorphisms) of four sites of the prolactin receptor gene and the luteotropin beta calcmeurin, the relation of polymorphism of a multiple-birth milk goat individual (F1 generation) and the number of born lamps and the relation of different genetype combinations and the number of the born lamps are analyzed, parental generation (F0 generation) is reviewed, filial generation (F2 generation) is tracked, the relation of the genetype combination of an ewe individual and the number of the born lamps is detected, and contribution of different genetypes in terms of prolific trait formation is analyzed.

Description

A kind of selection of multi-gene pyramiding of fine breed milk goats

Technical field

The invention belongs to the molecular genetics field; Be specifically related to a kind of selection of multi-gene pyramiding of fine breed milk goats; The single-chain DNA base mutation polymorphum of seized individual hprl receptor (PRLR) gene of this method Using P CR-SSCP and dna sequencing technology for detection and lutropin beta subunit (LH β) gene; Relation through analyzing high yield milk goat individual (F1 generation) polymorphum and litter size is confirmed suitable molecule marker, analyzes different genotype combination of high yield milk goat individual (F1) and the relation between the litter size, and traces back parental generation (F0 generation); Follow the tracks of filial generation (F2 generation); Detect the individual genotype combination of ewe and the relation of litter size, the contribution rate of research different genotype in the polyembryony proterties forms selected the individual genotype combination of high yield then; Analyze the apolegamy mode that the high yield idiotype is combined to form, use the multiple gene polymerization breeding core group that these apolegamy modes form the many lambs of milk goat again.

Background technology

The seed selection of polyembryony proterties is one of important goal of milk goat breeding; The polyembryony proterties is the quantitative character by the minor-polygene decision; Heritability very low (being about about 0.23); The conventional breeding means are difficult to make great progress in than short duration, this to a great extent limit the development of China's milk goat aquaculture.Development along with the animal gene group; A large amount of dna molecular markers is by development and application; Animal genome map is drawn progressively perfect, and increasing functional gene with important economic implications is excavated in succession, and the research that is closely related with the goat reproductive trait also obtains remarkable progress.Gene pyramiding (Gene pyramiding) is through genetic engineering means, be dispersed in different varieties or the strain excellent genes through hybridize, backcross, means such as composite hybridization are aggregated in the same kind.Most of economic characters of livestock and poultry have additive effect, and genetic expression is accumulative action, promptly focus on a mimic gene in the kind more multilist reach abundant more.At present, both at home and abroad about the research of sheep molecular breeding mainly concentrate on sheep Markers for Detection and with the dependency aspect of proterties, still only stay in conceptively for gene pyramiding breeding technique great majority, its Study on Theory is relatively lagged behind.

(Single Nucleotide Polymorphisms SNP) just is meant in the genomic dna sequence polymorphum that the replacement owing to single Nucleotide (A/T/C/G) causes to SNP.SNPs can be two or more allelic polymorphic, and therefore, usually said SNPs comprises the variation of the insertion of base, disappearance, insertion/disappearance and Tumor-necrosis factor glycoproteins copy number.A SNP is illustrated in the variation that a Nucleotide is arranged on certain site of genome, is mainly caused by conversion of single base (replacing another kind of pyrimidine or a kind of purine is replaced another kind of purine with a kind of pyrimidine) and transversion (purine and pyrimidine exchange).SNPs with transversion form variation accounts for 2/3; Other several kinds of SNP are on similar level; Because CpG (nucleotide pair; After wherein G follows C closely in the DNA chain) cytosine(Cyt) of dinucletide is to be prone to the site of undergoing mutation in the genome most, wherein great majority are methylated, spontaneously deaminize and form thymus pyrimidine.In any known or unknown gene or near all possibly find quantity not wait SNPs, can be divided between gene coding region SNPs (cSNPs), gene periphery SNPs (pSNPs) and gene three types of SNPs (iSNPs) etc. according to their distribution position in genome.Generally speaking, cSNP is fewer, because the aberration rate in the coding region only occupies 1/5 of sequence on every side, but therefore its tool significance in the research of inherited disease and breeding receives much attention.According to the influence to inherited character, cSNPs can be divided into two kinds again: a kind of is synonym cSNPs, and promptly the change of encoding sequence does not influence the aminoacid sequence that it is translated due to the SNP, and mutating alkali yl is identical with " implication " of mutating alkali yl not; Another kind is non-synonym cSNPs, thereby the change that the i.e. change of base sequence will cause the change of coded amino acid to produce protein sequence possibly finally have influence on proteinic function.Therefore, concerning the nonsynonymous mutation of coding region SNPs, they possibly have direct material impact to gene function, especially for the coding region sudden change, more may cause coded albumen generation significant change, thereby influence the performance of its function.Insertion/the deletion mutantion of base on the gene order changes thereby will cause coded amino acid whose change to produce protein sequence equally, so that has influence on proteic biological function, thereby causes the phenotype to individuality to have material impact.Moreover, in population genetic research, these SNPs are also significant in the research of population genetic and organic evolution as genetic marker.

Because SNPs is two equipotential gene molecule markers; So; In theory in a diplont colony; SNPs is made up of 2,3 or 4 allelotrope, but in fact 3 or 4 allelic SNPs are very rare, so SNPs is called two equipotential gene molecule markers usually simply.At present, mainly adopt several kinds of different routes to find SNPs: i.e. determined dna sequence method, PCR-SSCP and dna sequencing combined techniques, AS-PCR method, primer extension and oligonucleotide ligation etc.In these SNP detection techniques, the 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; Certainly, utilize PCR-SSCP and dna sequencing combined techniques to detect SNP and can suitably reduce testing cost, still; The experimentation of PCR-SSCP is long, operates more loaded down with trivial detailsly, and has the false negative problem in the experimentation; So, also also nonideal SNP detection means; The AS-PCR method 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; And primer extension and oligonucleotide ligation technology for detection SNP site need detection platform such as plate reader, gene chip, micro-sphere array technology and mass spectrograph, and exploitativeness is not strong for general molecule laboratory.

(prolactin is a kind of prepituitary gland peptide hormone PRL) to prolactin antagonist, and the effect of the prolactin antagonist of having reported has kind more than 300, can they be divided into six big types: 1) breeding and lactation; 2) g and D; 3) E&M, 4) brain and behavior; 5) immunomodulatory; 6) electrolyte balance.(prolactinreceptor PRLR) regulates by hprl receptor in these effects.Oneself detects hprl receptor in the various tissues that comprise brain, uterus, blastodisc, ovary.Thought the organ that does not contain PRLR in the past, for example olifactory nerve epithelium, fetal adrenal cortex, stomach, intestines and tunica mucosa bronchiorum, kidney duct epithelium, choroid plexus, Tiroidina, liver pancreas epithelium also have the expression of PRLR.Hprl receptor belongs to the cytokine receptor family member, and this family member also comprises some interleukin, a colony-stimulating factor (G-CSF), rHuGM-CSF, LIF (LIF), erythropoietin (EPO), TSF, obese factor etc.Although these members are obviously irrelevant in heredity, they all contain the aminoacid sequence of high conservative in the cell foreign lands.In view of the multiple effect of hprl receptor, deep day by day to the research of hprl receptor both at home and abroad.

Metakentrin (LH) has very important effect for the prolificacy of keeping male and jenny, the main effect of depending on LH of Mammals ovulation.Have a lot of scholars all metakentrin being carried out darker exploration, and these researchs have all got into molecular biological field.For example, the complete understanding that synthesizes basic molecular mechanism with LH is needed the research in LH α and LH β promoter regulation zone.Concerning subunit gene, obviously be that many cis elements instruct cell specific expression, simultaneously sex steroid and GnRH to be reacted, these researchs comprise the subunit promotor from 3 species.Find that it is that it discharges with discontinuous impulse form that metakentrin has a tangible characteristic when studying widely in that the physiological function of lutropin is done, impel follicle maturity and ovulation at LH peak of preovulatory final stage formation.The data of horse Yulin and Zhang Shou etc. shows; Before the ovulation; The progesterone that under the effect of LH, produces can trigger the formation and the release of ovulation enzyme, and under the effect of LH, sophisticated ovarian follicle can be secreted prostaglandin(PG); The matter smooth muscle fibers shrank between the latter possibly make around the mature follicle, impelled follicular rupture and ovulation.In addition, in the cycle, LH secretion peak, ovulation in 20-24 after this hour appear in the several hrs after the climax of normally oestrusing at spontaneous estrus.The postponement hint LH secretion peak of ovulation and the non-coordinative role between the ovulation or LH secretion peak are not enough to stimulate induced ovulation.Secrete the growth and the maturation of peak energy termination granulosa cell simultaneously, help them to be divided into the corpus luteum granulosa cell, impel the secretion of progesterone to help gestation.Therefore, when the sheep spontaneous estrus, carry out the test-tube ectogenic LH that gives simultaneously.The appearance at LH peak in can acceleration bodies reaches the purpose that improves conception rate thereby impel corpus luteum after mature follicle ovulation and the ovulation to form.Therefore, lutropin is a kind of important gonad-stimulating hormone, has very important effect for keeping female and prolificacy buck.

For this reason; With outstanding polyembryony individuality is the research starting point; Trace back and follow the tracks of through the outstanding ewe of typical case is Id; Analyze different genotype and genotype and be combined in the contribution rate of polyembryony proterties in forming, explore the transfer law and the aggregation scheme thereof of excellent genes, be intended to assist the practice of multiple gene polymerization breeding that theoretical foundation and technical support are provided for the foundation and the molecule marker of multiparous new strain.

Summary of the invention

The objective of the invention is to, a kind of selection of multi-gene pyramiding of fine breed milk goats is provided.

In order to realize above-mentioned task, the present invention takes following technical solution:

A kind of selection of multi-gene pyramiding of fine breed milk goats is characterized in that, is template with continuous product 2 lambs and above milk goat genomic dna; Under the PCR condition; Utilize 4 couples of primer P1, P2, P3 and P4, increase respectively prolactin receptor gene and lutropin beta subunit gene, wherein:

Described primer P1 is following:

Upstream primer 1F:5 '-TGTCAGTAAGCGTCAGAGGGC-3 ';

Downstream primer 1R:5 '-GGCTGGTGGAAGGTCACTCTT-3 ';

Described primer P2 is following:

Upstream primer 2F:5 '-CTTACCACAACATTGCTGACG-3 ';

Downstream primer 2R:5 '-GTTTAGCAGAGAACAAGGGGG-3 ';

Described primer P3 is following:

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

Downstream primer 3R:5 '-CAAAGCCTGAGTCCAAC-3 ';

Described primer P4 is following:

Upstream primer 4F:5 '-CCTGAGGCACTGGCCTGTCC-3 ';

Downstream primer 4R:5 '-CACCATGCTGGGGCAGTAGCC-3 ';

Primer P1 amplification prolactin receptor gene intron 2, primer P2 amplification prolactin receptor gene exons 10 is used for examination goat luteotropin acceptor gene site mutation;

Primer P3 amplification lutropin beta subunit gene 5 ' non-translational region, primer P4 amplification lutropin beta subunit gene exons 1 is used for examination goat lutropin beta subunit gene site mutation;

According to agarose gel electrophoresis 4 pairs of primer extension products being carried out size judges; Adopt the examination of dna sequencing technology to prolactin receptor gene intron 2 and exons 10 then, and lutropin beta subunit gene 5 ' non-translational region and exons 1 have the sudden change of 4 bases;

Utilize polyacrylamide gel electrophoresis that the SNPs in these 4 sites is carried out gene type and gene frequency analysis again; Analyze the relation of individual polymorphum of high yield milk goat and litter size and the relation between different genotype combination and the litter size; And trace back parental generation, and follow the tracks of filial generation, detect the individual genotype combination of ewe and the relation of litter size; Analyze the contribution of different genotype in the polyembryony proterties forms; Select the individual genotype combination of high yield, analyze the apolegamy mode that the high yield idiotype is combined to form, use the multiple gene polymerization breeding core group that these apolegamy modes form the many lambs of milk goat.

The condition of described pcr amplification is:

12 μ L reaction systems: comprise 0.25U Taq archaeal dna polymerase, 2 * Buffer of 6 μ L, the goat genome DNA sample of 0.4 μ L, each 0.4 μ L of the upstream and downstream primer of each primer of 10pmol/ μ L and sterilization ultrapure water 4.7 μ L.

The PCR response procedures is following:

1) utilize the PCR response procedures of primer P1 to be: 95 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s, 68 ℃ of annealing 30s, 72 ℃ are extended 30s, so carry out 35 circulations, and last 72 ℃ are fully extended 10min, 4 ℃ of preservations.

2) utilize the PCR response procedures of primer P2 to be: 95 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 35 circulations altogether, and last 72 ℃ are fully extended 10min, 4 ℃ of preservations.

3) utilize the PCR response procedures of primer P3 to be: 95 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 35 circulations altogether, and last 72 ℃ are fully extended 10min, 4 ℃ of preservations.

4) utilize the PCR response procedures of primer P4 to be: 95 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 35 circulations altogether, and last 72 ℃ are fully extended 10min, 4 ℃ of preservations.

There is the sudden change of C → T in described prolactin receptor gene intron 2 at 114bp, and there is the sudden change of C → A in exons 10 at 319bp; There is the sudden change of C → A in 5 ' non-translational region of described lutropin beta subunit gene at 153bp, and there is the sudden change of T → C in exons 1 at 64bp.

Described gene type is the pcr amplification product that detects and analyze primer P1, P2, P3 and P4 with 10% polyacrylamide gel electrophoresis, and the result is following:

Primer P1 site: the base of homozygous GG in the 114bp position is C, the base of heterozygous GH in the 114bp position be C, the base of homozygous HH in the 114bp position is T;

Primer P2 site: the base of homozygous CC in the 319bp position is C, the base of heterozygous CD in the 319bp position be A, the base of homozygous DD in the 319bp position is A;

Primer P3 site: the base of homozygous QQ in the 153bp position is A, the base of heterozygous PQ in the 153bp position be A, the base of homozygous PP in the 153bp position is CC;

Primer P4 site: the base of homozygous MM in the 64bp position is C, the base of heterozygous LM in the 64bp position be C, the base of homozygous LL in the 64bp position is T.

The individual genotype of described high yield is combined as: GGCCPPLL (F1 generation with F2 generation), GGCCPQMM (F1 generation) and GGCDPPLL (F1 for) and GGCCQQLL (F0 for).

The contribution rate of described different genotype in the polyembryony proterties forms does; The genotypic assembly effect value of CC is genotypic higher by 14.12% than CD; The genotypic assembly effect value of MM is genotypic higher by 3.8% than LM; The genotypic assembly effect value of PP is genotypic higher by 15.67% than QQ, and the genotypic assembly effect value of LL is genotypic higher by 11.48% than LM; The genotypic assembly effect value of PQ is genotypic higher by 11.02% than QQ, and the genotypic assembly effect value of CD is genotypic higher by 10.69% than DD

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

4 SNPs of the functional gene PRLR intron relevant with goat yeaning traits 2 and exons 10 and LH β gene 5 ' UTR and exons 1 have been proposed; These 4 SNPs 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.

SNPs to these two genes has carried out gene type and gene frequency analysis; Not only analyzed F1 for the relation between different genotype and genotype combination and the lambing proterties; And trace back parental generation (F0 generation), follow the tracks of filial generation (F2 generation), detect the genotype combination of ewe individuality and the relation of litter size; The contribution rate of research different genotype in the polyembryony proterties forms; Select the individual genotype combination of high yield, analyze the apolegamy mode that the high yield idiotype is combined to form, use the multiple gene polymerization breeding core group that these apolegamy modes form the many lambs of milk goat.

Description of drawings

Fig. 1 is that goat PRLR gene utilizes primer P1 to carry out the pcr amplification electrophorogram, and among the figure, M representes Marker I fragment, and fragment length is from falling down to be followed successively by: 100bp, 200bp, 300bp, 400bp, 500bp, 600bp;

Fig. 2 is that goat PRLR gene utilizes primer P2 to carry out the pcr amplification electrophorogram, and among the figure, M representes: Marker I fragment, and fragment length is from falling down to be followed successively by: 100bp, 200bp, 300bp, 400bp, 500bp, 600bp;

Fig. 3 is that goat LH β gene utilizes primer P3 to carry out the pcr amplification electrophorogram, and among the figure, M representes Marker I fragment, and fragment length is from falling down to be followed successively by: 100bp, 200bp, 300bp, 400bp, 500bp, 600bp;

Fig. 4 is that goat LH β gene utilizes primer P4 to carry out the pcr amplification electrophorogram, and among the figure, M representes Marker I fragment, and fragment length is from falling down to be followed successively by: 100bp, 200bp, 300bp, 400bp, 500bp, 600bp;

Fig. 5 is that the SSCP that goat PRLR gene utilizes primer P1 to carry out pcr amplification product detects electrophorogram, among the figure, and 1,3 expression: GH genotype; 2 expressions: GG genotype; 4,5 and 6 expressions: HH genotype;

Fig. 6 is that the SSCP that goat PRLR gene utilizes primer P2 to carry out pcr amplification product detects electrophorogram, among the figure, and among the figure, 1,6 expression: CD genotype; 3,4 and 5 expressions: CC genotype; 2 expressions: DD genotype;

Fig. 7 is that the SSCP that goat LH β gene utilizes primer P3 to carry out pcr amplification product detects electrophorogram, among the figure, and 1,3 and 5 expressions: PP genotype; 2 and 4 expressions: PQ genotype; 6 expressions: QQ genotype;

Fig. 8 is that the SSCP that goat LH β gene utilizes primer P4 to carry out pcr amplification product detects electrophorogram, among the figure, and 1,2 and 4 expressions: LL genotype; 3 expressions: LM genotype; 5 expressions: MM genotype.

Below through extraction, detection and concentration analysis to continuous product 2 lambs and above milk goat genomic dna; And under the pcr amplification condition, utilize primer P1, P2, P3 and the PRLR intron 2 of P4 amplification and the polyacrylate hydrogel electrophoretic analysis embodiment of exons 10 and LH β gene 5 ' UTR and exons 1 amplified production to come the present invention is done further detailed description.

Embodiment

A, utilize primer P1 and P2 respectively to PRLR intron 2 and exons 10 and utilize primer P3 and P4 to the pcr amplification of LH β gene 5 ' UTR and exons 1 and the detection of polymorphum thereof

1, the collection of goat blood sample and processing

Get goat blood sample 5mL, add ACD (the Hydrocerol A 2.4g of 0.2 μ L; Trisodium citrate 6.6g; Glucose 7.35g; Be settled to 50mL, autoclaving) anti-freezing, put into ice chest after slowly putting upside down 3 times ,-80 ℃ of preservations are subsequent use.

Present embodiment adopts can the milk goat blood sample 265 parts to continuous product 2 lambs and above Xi Nongsa, and picking up from Qianyang, Shaanxi Province Sa can sheep sheep stud and biology ltd of green new millennium of Shaanxi.

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, and pH to 8.0 is transferred in sterilization, 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.

Add the SDS of mass concentration 10% in the dna solution of (10) 500 μ L, making its final concentration is 0.1%, adds Proteinase K to final concentration and reaches 50 μ g/mL.

About (11) 5 ℃ of insulation 10h.

(12) phenol, chloroform, primary isoamyl alcohol (25: 24: 1) mixture and chloroform are distinguished extracting once, phenol, chloroform, iso pentane alcohol mixture consumption and chloroform equal-volume.

(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 the washing with alcohol with mass concentration 70%, 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, and calculate dna content and OD at 260nm, 280nm place ₂₆₀/ OD ₂₈₀Ratio.Like OD ₂₆₀/ OD ₂₈₀Ratio contains more protein or phenol less than 1.6 in the interpret sample, then should carry out purifying; If ratio greater than 1.8, then should consider to remove the RNA purifying.

DNA concentration (μ g/mL)=50 * OD ₂₆₀Value * extension rate

(3) structure in kind DNA pond

After DNA detection finishes, take out certain amount and be diluted to 50ng/ μ L, then from Xi Nongsa can milk goat 265 individuals, concentration be to get 5 μ L the sample of DNA of 50ng/ μ L to mix and be built into the DNA pond.

4, pcr amplification primer design

The polymerase chain reaction primer is gone up the sequences Design (accession number: AF042358 and AF041257) of the sheep PRLR gene that GenBank provides according to NCBI; Primer P1 and primer P2 are with primer P1 amplification PRLR gene intron 2, with primer P2 amplification PRLR gene extron 10, examination goat PRLR gene P1 and P2 site mutation; According to sheep LH beta gene sequence (GenBank accession number: S54695) design primer P3 and P4, amplification lutropin beta subunit gene 5 ' non-translational region and exons 1, examination goat lutropin beta subunit gene site mutation;

Described primer P1 is following:

Upstream primer 1F:5 '-TGTCAGTAAGCGTCAGAGGGC-3 ';

Downstream primer 1R:5 '-GGCTGGTGGAAGGTCACTCTT-3 ';

Described primer P2 is following:

Upstream primer 2F:5 '-CTTACCACAACATTGCTGACG-3 ';

Downstream primer 2R:5 '-GTTTAGCAGAGAACAAGGGGG-3 ';

Described primer P3 is following:

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

Downstream primer 3R:5 '-CAAAGCCTGAGTCCAAC-3 ';

Described primer P4 is following:

Upstream primer 4F:5 '-CCTGAGGCACTGGCCTGTCC-3 ';

Downstream primer 4R:5 '-CACCATGCTGGGGCAGTAGCC-3 ';

5, pcr amplification

The DNA pond that agricultural Sa can milk goat colony respectively is a masterplate; Utilize primer P1 and P2 under the PCR condition, increase respectively goat PRLR intron 2 and exons 10; And utilize primer P3 and P4 increase respectively LH β gene 5 ' UTR and exons 1, pcr amplification condition and program are as follows:

The condition of pcr amplification is: 12 μ L reaction systems, comprise 0.25U Taq archaeal dna polymerase (sky, Beijing root Science and Technology Ltd.), and 6 μ L, 2 * Buffer (includes Mg ²⁺, dNTPs etc., the Mix of sky, Beijing root Science and Technology Ltd.), the goat genome DNA sample of 0.4 μ L, each 0.4 μ L of the upstream and downstream primer of each primer of 10pmol/ μ L and sterilization ultrapure water 4.7 μ L.

The PCR response procedures is following:

(1) the PCR response procedures of primer P1 is: 95 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 30s, 68 ℃ of annealing 30s, 72 ℃ are extended 30s, so carry out 35 circulations, and last 72 ℃ are fully extended 10min, 4 ℃ of preservations.

(2) the PCR response procedures of primer P2 is: 95 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, so carry out 35 circulations, and last 72 ℃ are fully extended 10min, 4 ℃ of preservations.

(3) the PCR response procedures of primer P3 is: 95 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 30s, so carry out 35 circulations, and last 72 ℃ are fully extended 10min, 4 ℃ of preservations.

(4) the PCR response procedures of primer P4 is: 95 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s, so carry out 35 circulations, and last 72 ℃ are fully extended 10min, 4 ℃ of preservations.

6, PCR product purification and order-checking

Pcr amplification carries out agarose gel electrophoresis after accomplishing, and electrophoresis result such as Fig. 1, Fig. 2, Fig. 3 and shown in Figure 4 can know the band of seeing 176bp, 203bp, 231bp and 195bp, the success of illustration purpose gene fragment amplification;

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, the centrifugal 1min of 12000r/min 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, the centrifugal 1min of 12000r/min 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, the centrifugal 1min of 12000r/min outwells waste liquid, and adsorption column is reentered in the collection tube.

(6) in adsorption column, add 500 μ L rinsing liquids, the centrifugal 1min of 12000r/min outwells waste liquid, and centrifugal adsorption column is put into collection tube, and the centrifugal 2min of 12000r/min 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 is placed 2min.The centrifugal 1min of 12000r/min collects dna solution.

(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 the above PCR purified product that is template with 2 goat colony DNA ponds and carry out two-way order-checking, peak figure analyzes to order-checking, and wherein in same site two different peaks being arranged is that single nucleotide mutation has taken place; PRLR gene intron 2 (P1) goes out to exist the sudden change of C → T at 114bp, and there is the sudden change of C → A in exons 10 (P2) 319bp; There is the sudden change of C → A in LH β gene 5 ' UTR (P3) at the 153bp place, there is the sudden change of T → C in exons 1 (P4) at the 64bp place.

B, utilize goat PRLR intron 2 and the exons 10 that primer P1 and P2 amplification obtains and utilize primer P3 and the polyacrylamide gel electrophoresis analysis of 4 SNPs of LH β gene 5 ' UTR that the P4 amplification obtains and exons 1

Utilize primer P1 and P2 can carry out pcr amplification by 265 parts of genomic dnas of milk goat to Xi Nongsa respectively, obtain 265 dna fragmentations that comprise goat PRLR intron 2 and exons 10; Utilize primer P3 and P4 can carry out pcr amplification by 265 parts of genomic dnas of milk goat to Xi Nongsa respectively again; Obtain 265 dna fragmentations that comprise goat LH β gene 5 ' UTR and exons 1; Utilize following method that the PRLR intron of each goat 2 and exons 10 and LH β gene 5 ' UTR and exons 1 are carried out gene type then, concrete grammar is following:

Get each 5 μ L of pcr amplification product of primer P1, P2, P3 and P4; Mix with the sex change damping fluid of 5 μ L (95% methane amide, the EDTA of 0.5mol/L, the blue or green FF of 0.025% YLENE and 0.025% tetrabromophenol sulfonphthalein) respectively, 95 ℃ of sex change 10min put into mixture of ice and water effect 10min then rapidly; Last point sample is in 10% non-denaturing polyacrylamide gel solution; Under 4 ℃ condition, 280V electrophoresis 10min, electrophoresis 4.5h under the 210V weighing apparatus press strip spare afterwards.After electrophoresis finished, in the silver nitrate solution of adding 0.1%, jog 10min on shaking table used the distilled water wash gel 2 times then, added colour developing liquid (NaOH of 5g, the Na of 0.1g ₂CO ₃, add 0.5mL formaldehyde, be settled to 250mL), jog 15min on shaking table uses the distilled water wash gel then, the preservation of taking pictures (Fig. 5, Fig. 6, Fig. 7 and Fig. 8).

The frequency statistics analysis in C, 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 (table 1 and table 2) of colony.

The association analysis of D, goat PRLR gene and LH β genetic effect

The litter size of production data: Xi Nongsa ability milk goat the 1st, 2,3 and 4 tires.

Association analysis sample: have the Xi Nongsa of complete lambing characteristics record can milk goat 265.

The association analysis model:

Utilize the dependency of factors such as SPSS (13.0) software analysis kind, age, field and gene locus and economic characters.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 t analysis, ANOVA or multivariate linear model analyzing gene type effect.

In data processing; Different according to the factor that influences the lambing proterties; Consider in the field-effect (Farm), variety effect (Breed), sire effect (S), sire effect (SD) between dam, age (Age), genotype effect (Genotype) and the relevant effect of doing mutually; Adopted following fixed model to analyze, simultaneously, accepted or rejected according to practical situation.Complete model is following:

y _ijkmnpq＝μ+Farm _i+Breed _j+S _p+SD _q+Age _k+Genotype _m+X _n+e _ijkmnpq

Wherein: y _Ijkmnpq: individual phenotype record; μ: population mean; Farm _i: an other effect; Breed _j: variety effect; S _p: the sire effect; SD _q: effect between dam in the sire; Age _k: age effect; Genotype _m: marker gene type effect; X _nMake effect mutually for various secondarys with more than the secondary, as: Farm * Breed, Farm * Age, Farm * Genotype, Breed * Age, Breed * Genotype, Age * Genotype, Breed * Age * Genogype etc.; e _Ijkmnpq: random error; Utilization SPSS (13.0) software is analyzed data, and uses the least square fitting linear model, and litter size between each genotype is carried out significance test of difference.Analytical results is shown in table 1 and table 2:

The association analysis of table 1: Xi Nongsa ability milk goat PRLR gene P1 and P2 different genotype and litter size

Different lowercase alphabet differentials different significantly (P＜0.05) in the same row, following table together.

The association analysis of table 2: Xi Nongsa ability milk goat LH β gene P3 and P4 different genotype and litter size

Statistical result showed adopts lambing proterties significant correlation, particularly GG and the individual litter size of CC genotype of primer P1 and P2 amplification goat PRLR gene intron 2 and exons 10 and Xi Nongsa ability milk goat to be significantly higher than other genotype individual (P＜0.05); Equally, adopt primer P3 and P4 amplification LH β gene 5 ' non-translational region (5 ' UTR) with exons 1 and Xi Nongsa can milk goat lambing proterties significant correlation, particularly PP and LL genotype individuality litter size be significantly higher than other genotype individualities (P＜0.05); Therefore, adopt primer P1, P2, P3 and P4 to detect the SNP site and can be used as the molecule marker that goat yeaning traits is selected.

The different genotype combination of E, PRLR and LH β gene is to the influence of milk goat litter size

According to pedigree information, trace back parental generation, follow the tracks of filial generation, utilization SPSS (13.0) software analysis, the relation of F1 generation, parental generation (F0 generation) and combination of filial generation genotype and litter size is shown in table 3, table 4 and table 5.

The relation of milk goat genotype combination of table 3:F1 Dai Xinongsa ability and litter size

The relation of milk goat genotype combination of table 4F0 Dai Xinongsa ability and litter size

The relation of milk goat genotype combination of table 5F2 Dai Xinongsa ability and litter size

Can know that from table 3, table 4 and table 5 the individual gene property of high yield is combined as GGCCPPLL (F1 generation with F2 generation), GGCCPQMM (F1 generation) and GGCDPPLL (F1 for) and GGCCQQLL (F0 for).The contribution rate of different genotype in the polyembryony proterties forms does; The genotypic assembly effect value of CC is genotypic higher by 14.12% than CD; The genotypic assembly effect value of MM is genotypic higher by 3.8% than LM; The genotypic assembly effect value of PP is genotypic higher by 15.67% than QQ, and the genotypic assembly effect value of LL is genotypic higher by 11.48% than LM; The genotypic assembly effect value of PQ is genotypic higher by 11.02% than QQ; The genotypic assembly effect value of CD is genotypic higher by 10.69% than DD; Therefore; Combination of gene property and the contribution rate of different genotype in the polyembryony proterties forms that can be individual according to high yield be analyzed the apolegamy mode that the high yield idiotype is combined to form, and use the multiple gene polymerization breeding core group that these apolegamy modes form the many lambs of milk goat.

Claims (6)

1. the selection of a multi-gene pyramiding of fine breed milk goats is characterized in that, is template with continuous product 2 lambs and above milk goat genomic dna; Under the PCR condition; Utilize 4 couples of primer P1, P2, P3 and P4, increase respectively prolactin receptor gene and lutropin beta subunit gene, wherein:

Described primer P1 is following:

Upstream primer 1F:5 '-TGTCAGTAAGCGTCAGAGGGC-3 ';

Downstream primer 1R:5 '-GGCTGGTGGAAGGTCACTCTT-3 ';

Described primer P2 is following:

Upstream primer 2F:5 '-CTTACCACAACATTGCTGACG-3 ';

Downstream primer 2R:5 '-GTTTAGCAGAGAACAAGGGGG-3 ';

Described primer P3 is following:

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

Downstream primer 3R:5 '-CAAAGCCTGAGTCCAAC-3 ';

Described primer P4 is following:

Upstream primer 4F:5 '-CCTGAGGCACTGGCCTGTCC-3 ';

Downstream primer 4R:5 '-CACCATGCTGGGGCAGTAGCC-3 ';

Primer P1 amplification prolactin receptor gene intron 2, primer P2 amplification prolactin receptor gene exons 10 is used for examination goat luteotropin acceptor gene site mutation;

Primer P3 amplification lutropin beta subunit gene 5 ' non-translational region, primer P4 amplification lutropin beta subunit gene exons 1 is used for examination goat lutropin beta subunit gene site mutation;

According to agarose gel electrophoresis 4 pairs of primer extension products being carried out size judges; Adopt the examination of dna sequencing technology to prolactin receptor gene intron 2 and exons 10 then, and lutropin beta subunit gene 5 ' non-translational region and exons 1 have the sudden change of 4 bases;

Utilize polyacrylamide gel electrophoresis that the SNPs in these 4 sites is carried out gene type and gene frequency analysis again; Analyze the relation of individual polymorphum of high yield milk goat and litter size and the relation between different genotype combination and the litter size; And trace back parental generation, and follow the tracks of filial generation, detect the individual genotype combination of ewe and the relation of litter size; Analyze the contribution of different genotype in the polyembryony proterties forms; Select the individual genotype combination of high yield, analyze the apolegamy mode that the high yield idiotype is combined to form, use the multiple gene polymerization breeding core group that these apolegamy modes form the many lambs of milk goat.

2. the method for claim 1, it is characterized in that: the condition of described pcr amplification is:

12 μ L reaction systems: comprise 0.25U Taq archaeal dna polymerase, 6 μ L, 2 * Buffer, the goat genome DNA sample of 0.4 μ L, each 0.4 μ L of the upstream and downstream primer of each primer of 10pmol/ μ L and sterilization ultrapure water 4.7 μ L;

The PCR response procedures is following:

1) utilize the PCR response procedures of primer P1 to be: 95 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s, 68 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 35 circulations altogether, and last 72 ℃ are fully extended 10min, 4 ℃ of preservations;

2) utilize the PCR response procedures of primer P2 to be: 95 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, so carry out 35 circulations, and last 72 ℃ are fully extended 10min, 4 ℃ of preservations;

3) utilize the PCR response procedures of primer P3 to be: 95 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 30s, so carry out 35 circulations, and last 72 ℃ are fully extended 10min, 4 ℃ of preservations;

4) utilize the PCR response procedures of primer P4 to be: 95 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s, so carry out 35 circulations, and last 72 ℃ are fully extended 10min, 4 ℃ of preservations.

3. the method for claim 1 is characterized in that, there is the sudden change of C → T in described prolactin receptor gene intron 2 at 114bp, and there is the sudden change of C → A in exons 10 at 319bp; There is the sudden change of C → A in 5 ' non-translational region of described lutropin beta subunit gene at 153bp, and there is the sudden change of T → C in exons 1 at 64bp.

4. the method for claim 1 is characterized in that, described gene type is the pcr amplification product that detects and analyze primer P1, P2, P3 and P4 with 10% polyacrylamide gel electrophoresis, and the result is following:

Primer P1 site: the base of homozygous GG in the 114bp position is C, the base of heterozygous GH in the 114bp position be C, the base of homozygous HH in the 114bp position is T;

Primer P2 site: the base of homozygous CC in the 319bp position is C, the base of heterozygous CD in the 319bp position be A, the base of homozygous DD in the 319bp position is A;

Primer P3 site: the base of homozygous QQ in the 153bp position is A, the base of heterozygous PQ in the 153bp position be A, the base of homozygous PP in the 153bp position is CC;

Primer P4 site: the base of homozygous MM in the 64bp position is C, the base of heterozygous LM in the 64bp position be C, the base of homozygous LL in the 64bp position is T.

5. the method for claim 1 is characterized in that, the individual genotype of described high yield is combined as:

F1 generation and F2 generation: GGCCPPLL;

F1 generation: GGCCPQMM and GGCDPPLL;

F0 generation: GGCCQQLL.

6. the method for claim 1 is characterized in that, the contribution rate of described different genotype in the polyembryony proterties forms is:

The genotypic assembly effect value of CC is genotypic higher by 14.12% than CD;

The genotypic assembly effect value of MM is genotypic higher by 3.8% than LM;

The genotypic assembly effect value of PP is genotypic higher by 15.67% than QQ;

The genotypic assembly effect value of LL is genotypic higher by 11.48% than LM;

The genotypic assembly effect value of PQ is genotypic higher by 11.02% than QQ,

The genotypic assembly effect value of CD is genotypic higher by 10.69% than DD.

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