CN103583470A - Molecular-assisted-selection cultivation method for green-shin recessive-white-feather chicken line - Google Patents
Molecular-assisted-selection cultivation method for green-shin recessive-white-feather chicken line Download PDFInfo
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Abstract
The invention discloses a molecular-assisted-selection cultivation method for a green-shin recessive-white-feather chicken line, and belongs to the technical field of molecular breeding. The molecular-assisted-selection cultivation method is adopted for authenticating the genotypes of dominant white feather sites of chickens, the genotypes of the dominant white feather sites can be rapidly determined, the green-shin recessive-white-feather chicken line with the genotypes being ii is produced, troubles of test cross breeding can be avoided, a generation interval is shortened, a breeding process is accelerated, and cultivation cost is reduced. The green-shin recessive-white-feather chicken line cultivated in the molecular-assisted-selection cultivation method has tender and delicious meat, is high in production performance, can be used as commercial chickens to be put into production directly, and can also be applied by being matched with green-shin jute-feather chickens.
Description
Technical field
The present invention is specifically related to a kind of breeding method of the blue or green shank and recessive white feather chicken strain based on molecule assisted Selection, belongs to molecular breeding technical field.
Background technology
Along with growth in the living standard, people's consumption idea changes mass type into from the scalar type in past.People are also more and more higher to the requirement of chicken meat.The local chicken of China has the advantages that meat is good, but its growth rate is slower, and feed conversion rate is low.Though and external fast large-scale broiler chicken fast growth, feed conversion rate are high, its meat is poor.For addressing this problem the large-scale broiler chicken of ，Jiang state extra income and domestic local high quality meat chicken is hybridized, the meat that can either improve the large-scale broiler chicken of state's extra income also can be accelerated growth rate and the productivity of local high quality meat chicken.But due to the impact of the long-term eating habit of China people, the white feather chicken of Fast Growth is not accepted in chicken, the especially Shelter in South China Cities of coloured plumages such as Chinese Consumer's preference jute plumage, black plumage.The demand that meets Chinese Consumer's in order to make to hybridize the phenotype of rear chicken, the Recessive Chicken that just need to isozygoty, the plumage look that can keep coloured plumage after the coloured plumage hybridization of Recessive Chicken productivity Gao，Qie Yu China, for being significant in China's high-quality a breed of chicken.
Summary of the invention
The breeding method that the object of this invention is to provide a kind of blue or green shank and recessive white feather chicken strain 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 blue or green shank and recessive white feather chicken strain of molecule assisted Selection, comprises the following steps:
(1) utilize blue or green shank and recessive white feather chicken as male parent, white Leghorn is hybridized as female parent, produces F1 generation;
(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 white plumage male and female of the genotypic blue or green shin of ii chicken;
(4) getting dominant Bai Yu site is the selfing of the white plumage male and female of the genotypic blue or green shin of ii chicken, utilize the method mensuration self progeny's of step (3) genotype, repeat above-mentioned steps to genetic stability, obtaining dominant Bai Yu site is the genotypic blue or green shank and recessive white feather chicken strain of ii.
In described step (1), blue or green shank and recessive white feather chicken is the blue or green shin white feather chicken after blue or green shin jute plumage local chicken sudden change, and through the artificial RFLP-PCR method of introducing PvuII, measuring dominant Bai Yu site is ii genotype.
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 that the genotypic blue or green shank and recessive white feather chicken strain of ii and the inter breed crossing of blue or green shin yellow pockmarked feather chicken are supporting, produces coloured plumage broiler chicken.
Described blue or green shin yellow pockmarked feather chicken kind is gu-shi chicken, the Ji, Huaibei, limit fiber crops chicken, Huainan Spotted-brown Chickens, Wuhua chicken, Chongren Chicken, Lang Yaji, Lushi chicken, Land of Peach Blossoms chicken, precious jade chicken, camellia chicken etc.
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 blue or green shank and recessive white feather chicken strain 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).
Blue or green its fine and tender taste of shank and recessive white feather chicken strain that adopts the inventive method to cultivate, meat flavour is delicious, and productivity is high, both can be used as commercial chicken and has directly put into production, and can also carry out supporting application with blue or green shin yellow pockmarked feather chicken.
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 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 blue or green shank and recessive white feather chicken strain based on molecule assisted Selection in the present embodiment, comprises the following steps:
(1) utilize blue or green shank and recessive white feather chicken in gu-shi chicken colony to make male parent, white Leghorn is hybridized as female parent, produces F1 generation;
(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 stripe size 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) getting dominant Bai Yu site is the selfing of the white plumage male and female of the genotypic blue or green shin of ii chicken, utilize the method mensuration self progeny's of step (3) genotype, repeat above-mentioned steps to genetic stability, obtaining dominant Bai Yu site is the genotypic blue or green shank and recessive white feather chicken strain of ii;
(6) dominant Bai Yu site step (5) being obtained is that the genotypic blue or green shank and recessive white feather chicken strain of ii is supporting as male parent and the inter breed crossing of blue or green shin yellow pockmarked feather chicken, produces coloured plumage broiler chicken.
Claims (10)
1. a breeding method for the blue or green shank and recessive white feather chicken strain based on molecule assisted Selection, is characterized in that: comprise the following steps:
(1) utilize blue or green shank and recessive white feather chicken as male parent, white Leghorn is hybridized as female parent, produces F1 generation;
(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 white plumage male and female of the genotypic blue or green shin of ii chicken;
(4) getting dominant Bai Yu site is the selfing of the white plumage male and female of the genotypic blue or green shin of ii chicken, utilize the method mensuration self progeny's of step (3) genotype, repeat above-mentioned steps to genetic stability, obtaining dominant Bai Yu site is the genotypic blue or green shank and recessive white feather chicken strain of ii.
2. the breeding method of the blue or green shank and recessive white feather chicken strain based on molecule assisted Selection according to claim 1, is characterized in that: in described step (1), blue or green shank and recessive white feather chicken is the blue or green shin white feather chicken after blue or green shin jute plumage local chicken sudden change.
3. the breeding method of the blue or green shank and recessive white feather chicken strain 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 '.
4. the breeding method of the blue or green shank and recessive white feather chicken strain based on molecule assisted Selection according to claim 3, is characterized in that: 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.
5. the breeding method of the blue or green shank and recessive white feather chicken strain based on molecule assisted Selection according to claim 3, 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.
6. the breeding method of the blue or green shank and recessive white feather chicken strain based on molecule assisted Selection according to claim 3, 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.
7. the breeding method of the blue or green shank and recessive white feather chicken strain based on molecule assisted Selection according to claim 3, 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.
8. the breeding method of the blue or green shank and recessive white feather chicken strain based on molecule assisted Selection according to claim 3, 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.
9. the breeding method of the blue or green shank and recessive white feather chicken strain 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 that the genotypic blue or green shank and recessive white feather chicken strain of ii and the inter breed crossing of blue or green shin yellow pockmarked feather chicken are supporting, produces coloured plumage broiler chicken.
10. the breeding method of the blue or green shank and recessive white feather chicken strain based on molecule assisted Selection according to claim 9, is characterized in that: described blue or green shin yellow pockmarked feather chicken kind is gu-shi chicken, the Ji, Huaibei, limit fiber crops chicken, Huainan Spotted-brown Chickens, Wuhua chicken, Chongren Chicken, Lang Yaji, Lushi chicken, Land of Peach Blossoms chicken, precious jade chicken or camellia chicken.
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| CN107099587A (en) * | 2017-04-27 | 2017-08-29 | 河南农业大学 | The method of the UTR variations of one kind detection chicken CDS2 genes 3 ' |
| 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 |
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