CN103563849A - Breeding method for high-yield black-skin chicken strain - Google Patents
Breeding method for high-yield black-skin chicken strain Download PDFInfo
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Abstract
The invention discloses a breeding method for a high-yield black-skin chicken strain and belongs to the technical field of molecular breeding. The genotype of a black-skin site of a chicken is authenticated with a molecular marker-assisted selection method, so that the genotype of the site can be quickly determined, and the shortcomings of complicated testcross, long generation interval and the like in conventional breeding are overcome. The method disclosed by the invention is used for producing the high-yield black-skin chicken strain of which the genotype is FmFm, so that the generation interval is shortened, the breeding progress is accelerated, and the breeding cost is reduced; furthermore, the egg laying performance of the strain is high, and the nutritional value of eggs is high; the black-skin characters of culled hens are uniform, and carcasses are more attractive. Furthermore, the strain is used as a male parent hybridized with high-laying-rate chickens, so that black-skin chickens with higher production performance can be produced.
Description
Technical field
The present invention is specifically related to a kind of breeding method of high yield type crow skin chicken strain, belongs to molecular breeding technical field.
Background technology
Black-bone chicken is medicinal poultry well-known at home and abroad, within 1874, is admitted in the world for standard variety, and blackbone Chicken and black-bone chicken egg have special medicinal, nutrition and ornamental value.But the egg laying performance of black-bone chicken is low.And the egg laying performance of external high-yield egg chicken is very good.In order to improve its productivity, breeder often will import the blood relationship of high-yield egg chicken in the practices of breeding, and it is separated that this will make black skin proterties occur, and then the price of hen is eliminated in impact.In order to keep again eliminating the black skin proterties of hen when productivity is provided, it is extremely important that the judgement of black skin loci gene type just seems.In traditional breeding method, usually adopt the method for test cross, according to the phenotypic character of filial generation chicken, reserve seed for planting.The black skin heterozygote of eliminating in Wu Fu chicken colony by test cross, Breeding and expanding population is individual.But test cross process is comparatively loaded down with trivial details, and the cycle is long, and can be subject to the big or small impact of group expanding.
Summary of the invention
The breeding method that the object of this invention is to provide a kind of high yield type crow skin chicken strain.
In order to realize above object, the technical solution adopted in the present invention is:
A breeding method for high yield type crow skin chicken strain, comprises the following steps:
(1) utilize black skin chicken breed to hybridize as female parent as male parent and high-yield egg chicken kind, produce F1 generation chicken;
(2) F1 generation selfing obtains F2 generation, and the F2 that selects and remain is for black skin chicken;
(3) utilize quantitative fluorescent PCR to measure F2 for the relative copy number of black skin chicken EDN3 gene, determine that F2 is for the genotype of black skin chicken individuals, the black skin site of selecting and remain is the genotypic individuality of FmFm, and eliminating black skin site is the genotypic individuality of Fmfm, and obtaining black skin site is the genotypic male and female chicken of FmFm;
(4) the FmFm genotype individuality obtaining is carried out traversed by and expands numerous, according to family, select and individual choice method, improve egg production, purify simultaneously and fix black skin proterties, after 4 seed selections more than generation, obtaining black skin site is the genotypic high yield type crow of FmFm skin chicken strain.
Black skin chicken breed in described step (1) is for a silk plumage black-bone chicken, rivers and mountains black-bone chicken, Jinhu County's crow Salted chicken, Yugan black-bone chicken, Yun county sun black-bone chicken, snowy peak black-bone chicken, mountain region, Sichuan black-bone chicken, crow are covered black-bone chicken, Tengchong snow cock, Lueyang chicken (Heihe black-bone chicken), he stays black-bone chicken, Wuliang Shan Mountain black-bone chicken, Yanjin black-bone chicken etc.
High-yield egg chicken kind in described step (1) is that Bai Laihang, Luo Man, Hai Lan, Hai Saikesi, Ni Ke, bar cloth cock, Hei Kang, Te Jia, snow Buddhist, Yi Sha, No. 3, Hong, Jing Fen, Hebei, capital great Wu, agricultural university, new poplar are brown, new Yang Bai etc.
In described step (3), utilize quantitative fluorescent PCR to measure F2 for the relative copy number of black skin chicken EDN3 gene, determine that F2 is for the genotypic method of black skin chicken individuals, comprise the following steps: the chicken Whole Blood Genomic DNA to be measured that comprises EDN3 gene of take is template, the primer pair P of take carries out fluorescent quantitative PCR as primer, and the DNA sequence dna of amplified fragments is as shown in SEQ IDNo.3;
Described primer pair P is:
Upstream primer P-F:5 '-CTTGACTTTTGGGATTTACCT-3 ' (as shown in SEQ ID No.1),
Downstream primer P-R:5 '-ACTGGTGCAGTATTTTCTGTT-3 ' (as shown in SEQ ID No.2).
The total length of this extension increasing sequence is 134bp, is positioned on the EDN3 gene of chicken crow skin site, can detect chicken crow skin gene loci EDN3 gene and whether have copy number variation.
Described fluorescent quantitative PCR reaction system is: quantitative fluorescent PCR 2 * buffer solution 10 μ L, ddH
2o7 μ L, P-F0.5 μ L, P-R0.5 μ L, DNA profiling 2 μ L.
Described quantitative fluorescent PCR 2 * buffer solution mainly comprises 5U/ μ L Taq DNA Polymerase, 10 * Buffer, 25mM MgCl
2, 2mM dNTPs, SYBR Green I.
Described fluorescent quantitative PCR response procedures is: 95 ℃ of denaturation 2min; 40 circulations: 95 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 30s; 72 ℃ are extended 10min; 20 ℃ of preservations.
Concrete, in described step (3), utilize quantitative fluorescent PCR to measure F2 for the relative copy number of black skin chicken EDN3 gene, determine that F2 is for the genotypic method of black skin chicken individuals, further comprising the steps of: to utilize calibration curve to try to achieve the relative copy number of chicken EDN3 gene to be measured, according to its relative copy number, identify the genotype in chicken crow skin site.
The preparation method of described calibration curve is: using white skin chicken Whole Blood Genomic DNA as fluorescent quantitation standard items, concentration is respectively 80,40, and 20,10,5,2.5ng/ μ L, the primer pair P of take carries out fluorescent quantitative PCR as primer, the Ct value of above-mentioned six standard items of take is ordinate, the logarithm value of concentration of take is abscissa, and Criterion curve, tries to achieve calibration curve equation.
The described method of utilizing calibration curve to try to achieve the relative copy number of chicken EDN3 gene to be measured is: before fluorescent quantitative PCR, first the DNA of chicken to be measured is diluted to same concentration 10ng/ μ L, again by the Ct value substitution calibration curve equation of chicken to be measured, obtain its corresponding logarithm concentration, be converted into again concentration, be the relative copy number of this chicken EDN3 gene to be measured.
Described identifies that according to its relative copy number the genotypic method in chicken crow skin site is: when the relative copy number of chicken EDN3 gene to be measured is 2, this individuality is black skin homozygote (FmFm); When the relative copy number of chicken EDN3 gene to be measured is 1.5, this individuality is black skin heterozygote (Fmfm); When the relative copy number of chicken EDN3 gene to be measured is 1, this individuality is non-black skin homozygote (fmfm).
Preferably, the black skin site that step (4) is obtained is that the genotypic high yield type crow of FmFm skin chicken is supporting as male parent and high-yield egg chicken inter breed crossing, produces the blacker skin chicken of high productivity energy.
Beneficial effect of the present invention:
Adopt the high yield type crow skin chicken strain that method producer gene type of the present invention is FmFm, not only avoided the loaded down with trivial details of test cross seed selection, shortened the generation interval, accelerated breeding process, reduced cultivation cost; And this strain egg laying performance is good, egg nutrient is worth high; Eliminate hen crow skin proterties homogeneous, carcass is more attractive in appearance.In addition, this strain, as male parent and high-yield egg chicken corss combination, can be produced to the blacker skin chicken of high productivity energy.
The present invention adopts the method for molecule assisted Selection to identify chicken crow skin loci gene type, can determine fast the genotype in this site, shortens breeding time, improves breeding efficiency, has overcome the shortcomings such as in traditional conventional breeding, test cross is loaded down with trivial details, the generation interval is long.
Embodiment
Following embodiment is only described in further detail the present invention, but does not form any limitation of the invention.
Embodiment 1
The breeding method of high yield type crow skin chicken strain in the present embodiment, comprises the following steps:
(1) utilize silk plumage black-bone chicken pure lines cock as male parent and come aircraft carrier chicken to hybridize as female parent in vain, producing F1 generation chicken;
(2) F1 generation selfing obtains F2 generation, and the F2 that selects and remain is for black skin chicken;
(3) utilize quantitative fluorescent PCR to measure F2 for the relative copy number of black skin chicken EDN3 gene, determine that F2, for the genotype of black skin chicken individuals, specifically comprises the following steps:
1. the collection of sample
F2, for the black skin chicken wings venous blood collection of selecting and remain, is added to 1/3 anticoagulant, by Proteinase K method, extract blood DNA;
2. DNA sample concentration is adjusted
Utilize micro-spectrophotometer to carry out concentration determination the DNA of testing sample, according to surveyed concentration, each testing sample DNA concentration is added to TE and be diluted to 10ng/ μ L, after dilution, with micro-ultraviolet specrophotometer, measure, 4 ℃ of Refrigerator stores are standby;
3. the preparation of standard items
Getting at random the DNA sample of a white plumage broiler chicken, measure after concentration, be diluted to 80ng/ μ L, as standard items mother liquor, is 80,40,20,10,5,2.5ng/ μ L by mother liquor doubling dilution, as 6 standard items of production standard curve;
4. design of primers
The sequence of genotype identification primer pair P used is as follows:
Upstream primer P-F:5 '-CTTGACTTTTGGGATTTACCT-3 ',
Downstream primer P-R:5 '-ACTGGTGCAGTATTTTCTGTT-3 ';
5. qPCR amplification
Take primer pair P as amplimer, set up 20 μ L systems: quantitative fluorescent PCR 2 * buffer solution (comprises 5U/ μ L TaqDNA Polymerase, 10 * Buffer, 25mM MgCl
2, 2mM dNTPs, SYBR Green I) and 10 μ L, ddH
2o7 μ L, P-F0.5 μ L, P-R0.5 μ L, DNA profiling 2 μ L; Response procedures is: 95 ℃ of denaturation 2min; 95 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 30s, 40 circulations; 72 ℃ are extended 10min; 20 ℃ of preservations;
6. the judgement of the processing of qPCR amplification data and genotype result
Take 80,40,20,10,5, the Ct value of 6 standard items of 2.5ng/ μ L is ordinate, the logarithm of concentration of take is abscissa, Criterion curve is obtained the equation y=-0.285x+7.665 of calibration curve, R
2=0.9986 and the amplification efficiency 92.72% of this reaction;
Bring the Ct value of individuality to be measured into equation, obtain its corresponding logarithm concentration, then be converted into concentration, be the relative copy number of this individual EDN3 gene to be measured;
The relative copy number of the EDN3 gene of white skin individuality is made as to 1, and when the relative copy number of individuality to be measured is 2, the genotype in this site is FmFm; When the relative copy number of individuality to be measured is 1.5, the genotype in this site is Fmfm(because sample to be tested DNA concentration calibration and quantitative fluorescent PCR test all can exist certain experimental error, this error is controlled within 20%, does not affect the type of sentencing to black skin site);
(4) according to genotype result of determination, the black skin site of selecting and remain is the genotypic individuality of FmFm, and eliminating black skin site is the genotypic individuality of Fmfm;
(5) the FmFm genotype individuality obtaining is carried out traversed by and expands numerous, according to family, select and individual choice method, improve egg production, purify simultaneously and fix black skin proterties, after 4 seed selections more than generation, obtaining black skin site is the genotypic high yield type crow of FmFm skin chicken strain;
(6) using the black skin site obtaining, be that the genotypic high yield type crow of FmFm skin chicken is supporting as male parent and high-yield egg chicken inter breed crossing, produce the blacker skin chicken of high productivity energy.
Claims (10)
1. a breeding method for high yield type crow skin chicken strain, is characterized in that: comprise the following steps:
(1) utilize black skin chicken breed to hybridize as female parent as male parent and high-yield egg chicken kind, produce F1 generation chicken;
(2) F1 generation selfing obtains F2 generation, and the F2 that selects and remain is for black skin chicken;
(3) utilize quantitative fluorescent PCR to measure F2 for the relative copy number of black skin chicken EDN3 gene, determine that F2 is for the genotype of black skin chicken individuals, the black skin site of selecting and remain is the genotypic individuality of FmFm, and eliminating black skin site is the genotypic individuality of Fmfm;
(4) the FmFm genotype individuality obtaining is carried out traversed by and expands numerous, according to family, select and individual choice method, improve egg production, purify simultaneously and fix black skin proterties, after 4 seed selections more than generation, obtaining black skin site is the genotypic high yield type crow of FmFm skin chicken strain.
2. the breeding method of high yield type according to claim 1 crow skin chicken strain, is characterized in that: the black skin chicken breed in described step (1) is for a silk plumage black-bone chicken, rivers and mountains black-bone chicken, Jinhu County's crow Salted chicken, Yugan black-bone chicken, Yun county sun black-bone chicken, snowy peak black-bone chicken, mountain region, Sichuan black-bone chicken, crow are covered black-bone chicken, Tengchong snow cock, Lueyang chicken, he stays black-bone chicken, Wuliang Shan Mountain black-bone chicken or Yanjin black-bone chicken.
3. the breeding method of high yield type crow skin chicken strain according to claim 1, is characterized in that: the high-yield egg chicken kind in described step (1) is Bai Laihang, Luo Man, Hai Lan, Hai Saikesi, Ni Ke, bar cloth cock, Hei Kang, Te Jia, snow Buddhist, Yi Sha, No. 3, Hong, Jing Fen, Hebei, capital great Wu, agricultural university, new poplar is brown or new Yang Bai.
4. the breeding method of high yield type according to claim 1 crow skin chicken strain, it is characterized in that: in described step (3), utilize quantitative fluorescent PCR to measure F2 for the relative copy number of black skin chicken EDN3 gene, determine that F2 is for the genotypic method of black skin chicken individuals, comprise the following steps: the chicken Whole Blood Genomic DNA to be measured that comprises EDN3 gene of take is template, the primer pair P of take carries out fluorescent quantitative PCR as primer, and the DNA sequence dna of amplified fragments is as shown in SEQ ID No.3;
Described primer pair P is:
Upstream primer P-F:5 '-CTTGACTTTTGGGATTTACCT-3 ',
Downstream primer P-R:5 '-ACTGGTGCAGTATTTTCTGTT-3 '.
5. the breeding method of high yield type crow skin chicken strain according to claim 4, is characterized in that: described fluorescent quantitative PCR reaction system is: quantitative fluorescent PCR 2 * buffer solution 10 μ L, ddH
2o7 μ L, P-F0.5 μ L, P-R0.5 μ L, DNA profiling 2 μ L.
6. the breeding method of high yield type crow skin chicken strain according to claim 4, is characterized in that: described fluorescent quantitative PCR response procedures is: 95 ℃ of denaturation 2min; 40 circulations: 95 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 30s; 72 ℃ are extended 10min; 20 ℃ of preservations.
7. the breeding method of high yield type according to claim 4 crow skin chicken strain, it is characterized in that: in described step (3), utilize quantitative fluorescent PCR to measure F2 for the relative copy number of black skin chicken EDN3 gene, determine that F2 is for the genotypic method of black skin chicken individuals, further comprising the steps of: to utilize calibration curve to try to achieve the relative copy number of chicken EDN3 gene to be measured, according to its relative copy number, identify the genotype in chicken crow skin site.
8. the breeding method of high yield type according to claim 7 crow skin chicken strain, it is characterized in that: the described method of utilizing calibration curve to try to achieve the relative copy number of chicken EDN3 gene to be measured is: before fluorescent quantitative PCR, first the DNA of chicken to be measured is diluted to same concentration 10ng/ μ L, by in the Ct value substitution calibration curve equation of chicken to be measured, obtain its corresponding logarithm concentration, be converted into again concentration, be the relative copy number of this chicken EDN3 gene to be measured.
9. the breeding method of high yield type according to claim 7 crow skin chicken strain, it is characterized in that: described identifies that according to its relative copy number the genotypic method in chicken crow skin site is: when the relative copy number of chicken EDN3 gene to be measured is 2, this individuality is black skin homozygote; When the relative copy number of chicken EDN3 gene to be measured is 1.5, this individuality is black skin heterozygote.
10. the breeding method of high yield type according to claim 1 crow skin chicken strain, it is characterized in that: the black skin site that step (4) is obtained is that the genotypic high yield type crow of FmFm skin chicken is supporting as male parent and high-yield egg chicken inter breed crossing, produces the blacker skin chicken of high productivity energy.
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| CN104642265A (en) * | 2015-02-05 | 2015-05-27 | 河南农业大学 | Quick balanced breeding method of chicken breeds with high quality |
| CN106070044A (en) * | 2016-06-29 | 2016-11-09 | 来宾市忻城县润华牧业有限责任公司 | A kind of reduction is raised in cages kind by the breeding method of common chicken wild nature |
| CN106172195A (en) * | 2016-07-19 | 2016-12-07 | 贵州省普安县万象生态农业综合开发有限公司 | A kind of method improving Seedling mountain, Pu'an Gallus Domesticus laying rate |
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