CN103563851A - Method for cultivating dominant-green-shin white feather broiler strain based on molecular assistant selection - Google Patents
Method for cultivating dominant-green-shin white feather broiler strain based on molecular assistant selection Download PDFInfo
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Abstract
The invention discloses a method for cultivating a dominant-green-shin white feather broiler strain based on molecular assistant selection and belongs to the technical field of molecular breeding. According to the method for cultivating the dominant-green-shin white feather broiler strain based on molecular assistant selection, the method of molecular assistant selection is adopted for appraisal of a genotype of a broiler dominant white feather locus, so that the genotype of the white feather locus is rapidly judged, the dominant-green-shin white feather broiler strain with the II genotype is produced, complexity of test cross selective breeding is avoided, a generation interval is shortened, the breeding progress is accelerated, and therefore the cultivation cost is reduced. Meat of the dominant-green-shin white feather broiler strain cultivated through the method is fine and tender, taste of the meat of the broiler strain is delicious, the production performance of the broiler strain is high, color feather residues do not exist on a carcass, and therefore the carcass is beautiful. In addition, the strain can serve as a male parent and be hybridized with a high-yield laying hen strain or a fast-growing large broiler strain in a matched mode so that high-yield or fast-growing large dominant-green-shin white feather broilers can be produced respectively.
Description
Technical field
The present invention is specifically related to the breeding method of the dominant white plumage broiler chicken strain of a kind of blue or green shin based on molecule assisted Selection, belongs to molecular breeding technical field.
Background technology
China's native chicken breed aboundresources, its fine and tender taste, meat flavour deliciousness, but in appearance coloured plumages such as Huang Yu, numb plumage that show as more.For cold freshly-slaughtered poultry market, coloured plumage broiler chicken Chang Yin butchers rear feather and is not easy de-clean and affects the attractive in appearance of carcass.Fast for external fast large-scale white plumage growth of meat chicken speed, Carcass Traits is good, but meat can not show a candle to the local chicken of China.Therefore, significant in the cultivation of dominant white plumage high quality meat chicken.
In traditional breeding method, usually adopt the method for test cross, according to the phenotypic character of filial generation chicken, reserve seed for planting.Dominant white homozygote individuality and the dominant white heterozygote by test cross, Breeding and expanding population, eliminated in Recessive Chicken colony are individual.And test cross is loaded down with trivial details, 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 the dominant white plumage broiler chicken strain of a kind of blue or green shin based on molecule assisted Selection.
In order to realize above object, the technical solution adopted in the present invention is:
A breeding method for the dominant white plumage broiler chicken strain of blue or green shin of molecule assisted Selection, comprises the following steps:
(1) utilize blue or green shin broiler chicken kind to hybridize as female parent as male parent and fast large-scale white plumage broiler chicken kind, produce F1 generation, the white plumage male and female of the blue or green shin of the F1 generation of selecting and remain chicken;
(2) F1 generation selfing obtains F2 generation, and the F2 that selects and remain is for the white plumage male and female of blue or green shin chicken;
(3) utilize the RFLP-PCR method of the artificial PvuII of introducing to measure F2 for the genotype of the white plumage male and female of blue or green shin chicken, the dominant Bai Yu site of selecting and remain is the genotypic individuality of II, eliminating dominant Bai Yu site is Ii and the genotypic individuality of ii, and obtaining dominant Bai Yu site is the genotypic male and female chicken of II;
(4) the II genotype individuality obtaining is carried out traversed by and expands numerous, according to family, select and individual choice method, improve speed of production and chicken meat quality, purify simultaneously and fix the appearance character of the white plumage of blue or green shin, after 3 seed selections more than generation, obtaining dominant Bai Yu site is the dominant white plumage broiler chicken strain of the genotypic blue or green shin of II.
In described step (1), blue or green shin broiler chicken kind is gu-shi chicken, langshan chicken, Huaibei fiber crops chicken, Huainan Spotted-brown Chickens, the black chicken in Ji, Dehua, Hei Ji, Wuhua, Mount Huang, Chongren Chicken, shouguang chicken, Lushi chicken, Jing Yangji, the large chicken of Yun county sun, Dong'an chicken, Land of Peach Blossoms chicken, precious jade chicken, the black chicken of camellia chicken and field etc.
In described step (1), fast large-scale white plumage broiler chicken kind is that Luo Si 308, section treasured, Ai Weiyin, Ha Bade, Chinese mugwort pull out increasingly (AA), hypo sieve, enlightening is high.
In described step (3), utilize the RFLP-PCR method of the artificial PvuII of introducing, comprise the following steps: the chicken complete genome DNA to be measured that comprises PMEL17 gene of take is template, take primer pair P as primer PCR amplification PMEL17 gene the tenth exon, obtain amplified fragments; Amplified fragments is carried out to Pvu II enzyme and cut, enzyme is cut to product and carry out agarose gel electrophoresis, according to agarose gel electrophoresis result, identify the genotype in the dominant Bai Yu of chicken site;
Described primer pair P is:
Upstream primer P-F:5 '-AGTGGCACCACGCTCACCGTGGGG
cAGCT-3 ' (as shown in SEQ ID No.1),
Downstream primer P-R:5 '-GAGGGGGTGAGTGCTGTGTTCTC-3 ' (as shown in SEQ ID No.2).
Described chicken complete genome DNA adopts the thick formulation of benzene phenol-chloroform or Proteinase K method to extract.
Described pcr amplification reaction system is: 2 * Taq PCR MasterMix, 6.25 μ L, ddH
2o 4.25 μ L, P-F 0.5 μ L, P-R 0.5 μ L, DNA profiling 1.0 μ L.
Described 2 * Taq PCR MasterMix contains Taq archaeal dna polymerase, dNTPs, magnesium chloride and PCR reaction buffer, is commercial goods, can be purchased from companies such as the precious biology in Sheng Gong, Dalian, Shanghai, hundred Tykes, Beijing, Beijing Kang Wei.
Described pcr amplification reaction program is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 20s, 30 circulations; 72 ℃ are extended 10min; 4 ℃ of preservations.
Described Pvu II endonuclease reaction system is: amplified fragments 10 μ L, Buffer 1.5 μ L, Pvu II 0.3 μ L, ddH
2o 3.2 μ L.
Described Pvu II endonuclease reaction condition is: 37 ℃, enzyme is cut and spent the night.
The mass concentration of described Ago-Gel is 2%.
The voltage of described agarose gel electrophoresis is 120V, and electrophoresis time is 60min.
The described method of identifying the dominant white plumage loci gene type of chicken is: II genotype shows as 160bp band; Ii genotype shows as 124bp and 160bp band; Ii genotype shows as 124bp.
Preferably, the dominant Bai Yu site that step (4) is obtained is the dominant white plumage broiler chicken strain of the genotypic blue or green shin of II as male parent and high-yield egg chicken kind or fast pork chicken breed corss combination, produces high yield type or the dominant white plumage broiler chicken of fast large-scale blue or green shin.
Beneficial effect of the present invention:
The present invention adopts the method for molecule assisted Selection to identify the genotype in the dominant Bai Yu of chicken site, can determine fast the genotype in this site, producer gene type is the dominant white plumage high quality meat chicken strain of the blue or green shin of II, can avoid the loaded down with trivial details of test cross seed selection, shorten the generation interval, accelerate breeding process, thereby reduce, cultivate cost.
The present invention is directed to the wild homozygote individuality of dominant white plumage and insert this feature at a 9bp of PMEL17 gene exon10 existence, design upstream primer is introduced Pvu II restriction enzyme site, and whether whether have insetion sequence is mutual corresponding relation with existing between restriction enzyme site.Wherein, primer pair P designs for the nucleotide sequence that does not contain the PMEL17 gene of 9bp insertion, introduce after Pvu II restriction enzyme site, pcr amplification product can be cut to 124bp and 27bp(27bp by Pvu II and not show during short electrophoresis very much) fragment (as shown in SEQ ID No.3); After amplification, there is not this restriction enzyme site of Pvu II in the gene order of inserting containing 9bp, thus its PCR product enzyme to cut rear fragment be that 160bp(is as shown in SEQ ID No.4).
Dominant white its fine and tender taste of plumage broiler chicken strain of blue or green shin that adopts the inventive method to cultivate, meat flavour is delicious, and productivity is high, does not have coloured plumage vestiges on carcass, makes carcass more attractive in appearance.In addition, layer breed or broiler chicken inter breed crossing are supporting, can produce respectively high yield type or the dominant white plumage broiler chicken of fast large-scale blue or green shin.
Accompanying drawing explanation
Fig. 1 be in the embodiment of the present invention 1 each sample at the restriction enzyme digestion and electrophoresis figure in the dominant Bai Yu of chicken site;
Note: numbering is described as follows: 5,8,9 is II genotype, and 3,6,7 is Ii genotype; 1,2,4 is ii genotype, and M is DNA marker.
Embodiment
Following embodiment is only described in further detail the present invention, but does not form any limitation of the invention.
The breeding method of the dominant white plumage broiler chicken strain of blue or green shin based on molecule assisted Selection in the present embodiment, comprises the following steps:
(1) utilize gu-shi chicken pure lines cock to hybridize as female parent as male parent and Ai Wei mattress hen, produce F1 generation chicken, the white plumage male and female of the blue or green shin of the F1 generation chicken of selecting and remain in a small amount of individual ，F1Bai plumage of coloured plumage colony that superseded hybridization occurs;
(2) F1 generation selfing obtains F2 generation, and the F2 that selects and remain is for the white plumage male and female of blue or green shin chicken;
(3) utilize the RFLP-PCR method of the artificial PvuII of introducing to measure F2 for the genotype of the white plumage male and female of blue or green shin chicken, specifically comprise the following steps:
1. the collection of sample
F2, for the blue or green shin white feather chicken wing venous blood collection of selecting and remain, is added to 1/3 anticoagulant, by Proteinase K method, extract blood DNA, DNA Sample storage is standby in 4 ℃ of Refrigerator stores;
2. design of primers
Take PMEL17 gene order (GenBank Accession No.AY636129) as template design primer is to P, as follows:
Upstream primer P-F:5 '-AGTGGCACCACGCTCACCGTGGGG
cAGCT-3 ',
Downstream primer P-R:5 '-GAGGGGGTGAGTGCTGTGTTCTC-3 ';
3. pcr amplification
Take primer pair P as primer, set up 12.5 μ L amplification systems: 2 * Taq PCR MasterMix(is purchased from Beijing hundred Imtech) 6.25 μ L, ddH
2o 4.25 μ L, P-F 0.5 μ L, P-R 0.5 μ L, DNA profiling 1.0 μ L;
Response procedures is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 20s, 30 circulations; 72 ℃ are extended 10min; 4 ℃ of preservations;
4. the enzyme of pcr amplification product is cut
The Pvu II enzyme of 15 μ L is cut system: amplified fragments 10 μ L, Buffer 1.5 μ L, Pvu II 0.3 μ L, ddH
2o 3.2 μ L; Endonuclease reaction condition: 37 ℃, enzyme is cut and spent the night;
5. detection and the result of pcr amplification product enzyme after cutting judged
The enzyme of getting after 8 μ L pcr amplifications is cut product, 2% agarose gel electrophoresis (electrophoretic voltage: 120V; Electrophoresis time: 60min) point sample, electrophoresis finishes rear employing gel imaging system and detects, and electrophoresis pattern is referring to Fig. 1;
According to the size of the band occurring in electrophoresis pattern, judge: when there is the fragment of 160bp, be dominant white plumage homozygote (II genotype, phenotype is white plumage); When there is the fragment of 124bp, be wild type homozygote (ii genotype, phenotype is coloured plumage or recessive white feather); When there is the fragment of 160bp, 124bp, be dominant white plumage heterozygote (Ii genotype, phenotype is white plumage);
(4) according to genotype result of determination, the dominant Bai Yu site of selecting and remain is the genotypic individuality of II, and eliminating dominant Bai Yu site is Ii and the genotypic individuality of ii, and obtaining dominant Bai Yu site is the genotypic male and female chicken of II;
(5) the II genotype individuality obtaining is carried out traversed by and expands numerous, according to family, select and individual choice method, improve speed of production and chicken meat quality, purify simultaneously and fix the appearance character of the white plumage of blue or green shin, after 3 seed selections more than generation, obtaining dominant Bai Yu site is the dominant white plumage broiler chicken strain of the genotypic blue or green shin of II;
(6) dominant Bai Yu site step (5) being obtained is the dominant white plumage broiler chicken strain of the genotypic blue or green shin of II as male parent and Bai Laihang, gu-shi chicken corss combination, obtains the dominant white plumage broiler chicken commercial generation of high yield type or fast large-scale blue or green shin.
Claims (10)
1. a breeding method for the dominant white plumage broiler chicken strain of the blue or green shin based on molecule assisted Selection, is characterized in that: comprise the following steps:
(1) utilize blue or green shin broiler chicken kind to hybridize as female parent as male parent and fast large-scale white plumage broiler chicken kind, produce F1 generation, the white plumage male and female of the blue or green shin of the F1 generation of selecting and remain chicken;
(2) F1 generation selfing obtains F2 generation, and the F2 that selects and remain is for the white plumage male and female of blue or green shin chicken;
(3) utilize the RFLP-PCR method of the artificial PvuII of introducing to measure F2 for the genotype of the white plumage male and female of blue or green shin chicken, the dominant Bai Yu site of selecting and remain is the genotypic individuality of II, eliminating dominant Bai Yu site is Ii and the genotypic individuality of ii, and obtaining dominant Bai Yu site is the genotypic male and female chicken of II;
(4) the II genotype individuality obtaining is carried out traversed by and expands numerous, according to family, select and individual choice method, improve speed of production and chicken meat quality, purify simultaneously and fix the appearance character of the white plumage of blue or green shin, after 3 seed selections more than generation, obtaining dominant Bai Yu site is the dominant white plumage broiler chicken strain of the genotypic blue or green shin of II.
2. the breeding method of the dominant white plumage broiler chicken strain of the blue or green shin based on molecule assisted Selection according to claim 1, is characterized in that: in described step (1) blue or green shin broiler chicken kind be in gu-shi chicken, langshan chicken, Huaibei fiber crops chicken, Huainan Spotted-brown Chickens, the black chicken in Ji, Dehua, Hei Ji, Wuhua, Mount Huang, Chongren Chicken, shouguang chicken, Lushi chicken, Jing Yangji, the large chicken of Yun county sun, Dong'an chicken, Land of Peach Blossoms chicken, precious jade chicken, the black chicken of camellia chicken and field any.
3. the breeding method of the dominant white plumage broiler chicken strain of the blue or green shin based on molecule assisted Selection according to claim 1, is characterized in that: in described step (1) fast large-scale white plumage broiler chicken kind be that Luo Si 308, section treasured, Ai Weiyin, Ha Bade, Chinese mugwort pull out increasingly, hypo Luo, enlightening senior middle school any.
4. the breeding method of the dominant white plumage broiler chicken strain of the blue or green shin based on molecule assisted Selection according to claim 1, it is characterized in that: the RFLP-PCR method of utilizing the artificial PvuII of introducing in described step (3), comprise the following steps: the chicken complete genome DNA to be measured that comprises PMEL17 gene of take is template, take primer pair P as primer PCR amplification PMEL17 gene the tenth exon, obtain amplified fragments; Amplified fragments is carried out to Pvu II enzyme and cut, enzyme is cut to product and carry out agarose gel electrophoresis, according to agarose gel electrophoresis result, identify the genotype in the dominant Bai Yu of chicken site;
Described primer pair P is:
Upstream primer P-F:5 '-AGTGGCACCACGCTCACCGTGGGG
cAGCT-3 ',
Downstream primer P-R:5 '-GAGGGGGTGAGTGCTGTGTTCTC-3 '.
5. the breeding method of the dominant white plumage broiler chicken strain of the blue or green shin based on molecule assisted Selection according to claim 4, is characterized in that: described pcr amplification reaction system is: 2 * Taq PCR MasterMix6.25 μ L, ddH
2o 4.25 μ L, P-F 0.5 μ L, P-R 0.5 μ L, DNA profiling 1.0 μ L.
6. the breeding method of the dominant white plumage broiler chicken strain of the blue or green shin based on molecule assisted Selection according to claim 4, is characterized in that: described pcr amplification reaction program is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 20s, 30 circulations; 72 ℃ are extended 10min; 4 ℃ of preservations.
7. the breeding method of the dominant white plumage broiler chicken strain of the blue or green shin based on molecule assisted Selection according to claim 4, is characterized in that: described Pvu II endonuclease reaction system is: amplified fragments 10 μ L, Buffer 1.5 μ L, Pvu II 0.3 μ L, ddH
2o 3.2 μ L; Pvu II endonuclease reaction condition is: 37 ℃, enzyme is cut and spent the night.
8. the breeding method of the dominant white plumage broiler chicken strain of the blue or green shin based on molecule assisted Selection according to claim 4, is characterized in that: the mass concentration of described Ago-Gel is 2%; The voltage of agarose gel electrophoresis is 120V, and electrophoresis time is 60min.
9. the breeding method of the dominant white plumage broiler chicken strain of the blue or green shin based on molecule assisted Selection according to claim 4, is characterized in that: the described method of identifying the dominant white plumage loci gene type of chicken is: II genotype shows as 160bp band; Ii genotype shows as 124bp and 160bp band; Ii genotype shows as 124bp.
10. the breeding method of the dominant white plumage broiler chicken strain of the blue or green shin based on molecule assisted Selection according to claim 1, it is characterized in that: the dominant Bai Yu site that step (4) is obtained is the dominant white plumage broiler chicken strain of the genotypic blue or green shin of II as male parent and high-yield egg chicken kind or fast pork chicken breed corss combination, produces high yield type or the dominant white plumage broiler chicken of fast large-scale blue or green shin.
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| CN104293905A (en) * | 2014-04-11 | 2015-01-21 | 河南农业大学 | Primer, kit and detection method for detecting chicken green shin character linkage SNP locus genotype |
| CN104823919A (en) * | 2015-05-18 | 2015-08-12 | 贵州省畜牧兽医研究所 | Cultivation method and application of recessive white feather new strain of Xiaoxiang chicken in southeast of Guizhou province |
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| CN109913464A (en) * | 2019-01-02 | 2019-06-21 | 湖南洪江嵩云禽业有限公司 | Pmel17 gene relevant to snowy peak black-bone chicken chest muscle melanin deposition and genetic marking method |
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| CN104293905A (en) * | 2014-04-11 | 2015-01-21 | 河南农业大学 | Primer, kit and detection method for detecting chicken green shin character linkage SNP locus genotype |
| CN103931557A (en) * | 2014-05-08 | 2014-07-23 | 周珠英 | Method for cultivating new variety crossbred chicken by using Daiyun black chicken as male parent |
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| CN106386683A (en) * | 2016-09-21 | 2017-02-15 | 佛山科学技术学院 | A cultivation method for a disease-resistant line of Qingyuan partridge chickens without endogenous avian leukosis ev 21 viruses |
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| CN109371032A (en) * | 2018-11-23 | 2019-02-22 | 湖南农业大学 | Snowy peak black-bone chicken chest muscle melanin deposition pmel17 gene and genetic marking method |
| CN109371032B (en) * | 2018-11-23 | 2021-10-22 | 湖南农业大学 | Xuefeng silky fowl pectoral muscle melanin deposition pmel17 gene and genetic marking method |
| CN109913464A (en) * | 2019-01-02 | 2019-06-21 | 湖南洪江嵩云禽业有限公司 | Pmel17 gene relevant to snowy peak black-bone chicken chest muscle melanin deposition and genetic marking method |
| CN109913464B (en) * | 2019-01-02 | 2021-10-22 | 湖南云飞凤农业有限公司 | Pmel17 gene related to melanin deposition of pectoralis muscles of Xuefeng silky fowl and genetic marking method |
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