CN102477427B - Special primer for assistant identification of green shell laying hens and application thereof - Google Patents
Special primer for assistant identification of green shell laying hens and application thereof Download PDFInfo
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Abstract
The invention discloses a special primer for the assistant identification of blue shell laying hens and an application thereof. The special primer provided in the invention is a primer pair composed of DNA represented by a sequence 1 in a sequence table and DNA represented by a sequence 2 in the sequence table. The application is carried out according to a method comprising the following steps: 1, carrying out PCR amplification on genomic DNA of a sample to be tested with a specific primer pair; 2, digesting the obtained PCR amplification product with MscI; and 3, carrying out agarose gel electrophoresis on the PCR amplification product and the obtained enzymatic digestion product, and determining the gene type of an individual according to the size of enzymatic digestion strips. The special primer of the invention, which provides a new technical means for breeding the blue shell laying hens, is helpful for accelerating the breeding development and saving the breeding cost, lays a foundation for accelerating the molecular breeding of the purity of the Dongxiang blue shell laying hens, provides conveniences for the breeding of the blue shell laying hens, and performs large effects on the breeding of the blue shell laying hens.
Description
Technical field
The present invention relates to a kind of primer special and application thereof of assisting identification of hens laying green-shelled eggs.
Background technology
Layer of green-shell egg is one of good laying hen of China, and the green-egg-shelled that produces is paid much attention to and pays close attention to by domestic and international expert because of its unique eggshell color and outstanding Egg Quality, and the attraction human consumer falls over each other in eagerness to buy.China's existing layer of green-shell egg pure lines are by some local variety seed selections, due to colony's wide material sources and also the seed selection time short, so with respect to the strains such as Bai Laihang through the height seed selection, layer of green-shell egg strain homozygosity is not high.According to the conventional breeding method, the seed selection that improves green shell purity is exactly eliminate to produce powder shell offspring's kind chicken and hybridization assays to select heterozygote when pure lines are reserved seed for planting, and once measures in theory and just can tell different genotype.But owing to being subject to cock phenotype can't directly measure and the reserve seed for planting constraint of the conditions such as rear algebraically is limited of laying eggs, need many generations continuously seed selection just can reach breeding objective.
Bartlett et al. (Bartlett J R, Jones C P and Smith E J.Linkage analysis of endogenous viral elementl, blue eggshell, and pea comb loci in chickens (J) .The Journal of Heredity, 1996,87 (1): 67-70) oocyan gene (O) of chicken is positioned on No. 1 the short arm of a chromosome, it and the endogenous virus factor (ev1) and pea comb site (P) are chain.(the Yang Ning such as Yang Ning, Zhao Rui, Li Xianyao, Xu Guiyun, Yang Changsuo, Yan Huaxiang. the research of chicken green-egg-shelled gene molecule marker. poultry research latest developments---the tenth national poultry academic discussion collection of thesis once, 2003.37-39) find that by cross experiment the green shell proterties of Chinese blue-shelled egg layer is also the dominant character by Dominant gene.Blue-shelled egg layer all carries oocyan gene, can be divided into homozygous (OO) and heterozygous (Oo).Homozygous blue-shelled egg layer and non-layer of green-shell egg (as the Bai Laihang laying hen) hybridization, F1 is for all producing green-egg-shelled.Heterozygous blue-shelled egg layer and non-layer of green-shell egg (as the Bai Laihang laying hen) hybridization, F1 is in generation, and part is produced green-egg-shelled, and part is produced powder shell egg.
The layer of green-shell egg egg productivity is few, pure lines laying period 160 left and right of on average laying eggs, thus generally carry out with it grading up with white Leghorn, with the raising egg productivity.Separation phenomenon generally all appears in filial generation, most of green-egg-shelled that produces, and some produces powder shell egg.May be because layer of green-shell egg derives from local variety, brown shell egg product kind greatly in China's local variety, so also might be mixed with the gene of brown shell egg in layer of green-shell egg, and brown shell layer and white shell egg chicken (being Bai Laihang greatly) filial generation produces powder shell egg, so layer of green-shell egg and Bai Laihang filial generation have the appearance (Zhao Rui of powder shell egg, Liu Zhenzhen, Xu Guiyun, Yang Ning. utilize PCR-SSCP to analyze the SNP mark of chicken green-egg-shelled gene. Journal of Agricultural Biotechnology .2006,14 (5): 673-676).
The genetic development of research oocyan gene, the homozygosity that improves the layer of green-shell egg strain is necessary.Utilize molecular marker assisted selection to carry out molecular breeding and not only can directly select, can also select in early days, greatly accelerate genetic progress.
Summary of the invention
The purpose of this invention is to provide primer special and the application thereof of assisting identification of hens laying green-shelled eggs.
Primer special provided by the invention is the primer pair that shown in the sequence 2 of DNA shown in the sequence 1 of sequence table and sequence table, DNA forms.
Described primer pair can be used for assisting identification of hens laying green-shelled eggs, also can be further used for the assisting identification of hens laying green-shelled eggs pure lines.
The present invention also protects a kind of method of assisting identification of hens laying green-shelled eggs, comprises the steps: that genomic dna take chicken to be measured as template, carries out pcr amplification with described primer pair, cuts pcr amplification product with restriction enzyme MscI enzyme; If pcr amplification product only has a kind of, and can not cut by being limited property restriction endonuclease MscI enzyme, chicken to be measured is candidate's layer of green-shell egg; If pcr amplification product has two kinds, and a kind of can not cutting by being limited property restriction endonuclease MscI enzyme wherein, another kind can be cut by being limited property restriction endonuclease MscI enzyme, and chicken to be measured is candidate's layer of green-shell egg; If pcr amplification product only has a kind of, and can cut by being limited property restriction endonuclease MscI enzyme, chicken to be measured is candidate's non-layer of green-shell egg.
The present invention also protects the method for a kind of assisting identification of hens laying green-shelled eggs pure lines, comprises the steps: that genomic dna take chicken to be measured as template, carries out pcr amplification with described primer pair, cuts pcr amplification product with restriction enzyme MscI enzyme; If pcr amplification product only has a kind of, and can not cut by being limited property restriction endonuclease MscI enzyme, chicken to be measured is candidate's layer of green-shell egg pure lines; If pcr amplification product has two kinds, and a kind of can not cutting by being limited property restriction endonuclease MscI enzyme wherein, another kind can be cut by being limited property restriction endonuclease MscI enzyme, and chicken to be measured is candidate's layer of green-shell egg heterozygote; If pcr amplification product only has a kind of, and can cut by being limited property restriction endonuclease MscI enzyme, chicken to be measured is candidate's non-layer of green-shell egg.
The enzyme of restriction enzyme MscI is cut sequence (wherein arrow indication position be restriction enzyme site):
5’-TGG↓CCA-3’
3’-ACC↑GGT-5’。
Described chicken to be measured can be Bai Laihang laying hen or blue-shelled egg layer.
The reaction system of described pcr amplification is: the reaction system of described pcr amplification is: genomic dna 50-100ng contains Mg
2+10 * pcr amplification damping fluid, 1.5 μ l, the dNTPs mixed solution 0.8 μ l of 4mmol/L, the 0.3 μ l of DNA (upstream primer) shown in the sequence 1 of the sequence table of 10 μ mol/L, the 0.3 μ l of DNA (downstream primer) shown in the sequence 2 of the sequence table of 10 μ mol/L, Taq archaeal dna polymerase 0.5U replenishes ddH
2O to 15 μ l.The reaction conditions of described pcr amplification is: 95 ℃ of sex change 5min; 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 30 circulations; 72 ℃ are extended 10min.
The reaction system that described enzyme is cut is: pcr amplification product 3.5 μ l, and 10 * enzyme cutting buffering liquid, 1 μ l, restriction enzyme MscI4U replenishes ddH
2O to 10 μ l.The reaction conditions that described enzyme is cut is: 37 ℃ of 2-4 hour.
Described primer pair can be used for being prepared as follows (A) or test kit (B):
(A) assistant identification is identified the test kit of layer of green-shell egg;
(B) test kit of assisting identification of hens laying green-shelled eggs pure lines.
The present invention also protects following (A) or the test kit (B) that contains described primer pair:
(A) assistant identification is identified the test kit of layer of green-shell egg;
(B) test kit of assisting identification of hens laying green-shelled eggs pure lines.
Described primer pair can be used for a breed of chicken, is to identify that with aforesaid method the candidate's obtain layer of green-shell egg pure lines carry out breeding.
Method of the present invention comprises the steps: with Auele Specific Primer, the genomic dna to sample to be tested to be carried out pcr amplification, then the MscI enzyme is cut pcr amplification product, at last pcr amplification product and enzyme are cut product and carry out agarose gel electrophoresis, can differentiate individual genotype according to enzyme slitting band size.Method advantage of the present invention is as follows: assorted band does not appear in electrophoresis, and it is very accurate that genotype is sentenced type; Consuming time shorter, only need approximately 4～6 hours; Testing cost is very low, and the testing cost of every chicken only needs 1.5 yuan of left and right; Simple to operate, and can realize that automatization, mass-producing detect; Can detect the genotype of layer of green-shell egg before laying eggs, realize early stage selection, and can differentiate whether cock carries Recessive alleles.Can develop detection kit according to the inventive method.When method of the present invention is carried out breeding, only need the individuality with Recessive alleles in the parent is rejected, can make commodity egg all produce green-egg-shelled, for the producer brings good benefit.The present invention provides new technique means for the layer of green-shell egg breeding work, help to accelerate Advances in Breeding, save the breeding cost, for the molecular breeding that accelerates the pure rate of blue-shelled egg layer is laid a good foundation, for the breeding work of layer of green-shell egg is provided convenience, will play a great role in the green-egg-shelled a breed of chicken.
Description of drawings
Fig. 1 is in embodiment 2, the electrophorogram of the first sample.
Fig. 2 is in embodiment 2, the electrophorogram of the second sample.
Fig. 3 is in embodiment 2, the electrophorogram of the third sample.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment if no special instructions, is ordinary method.Test materials used in following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.% in following embodiment if no special instructions, is the quality percentage composition.Synthetic and the examining order of primer is completed by giving birth to work Bioisystech Co., Ltd.
The foundation of the discovery of embodiment 1, polymorphic site and pira-PCR-RFLP detection method
One, the discovery of the expression amount difference of O gene in different laying hens and the design of primer pair
Utilize that quantitative fluorescent PCR is homozygous to layer of green-shell egg, the expression amount of layer of green-shell egg heterozygous and non-layer of green-shell egg interstitial part of uterine tube O gene analyzes, result shows, this gene expression amount in the layer of green-shell egg pure lines is high, expression amount is extremely low in non-layer of green-shell egg does not even express, and the expression amount in the layer of green-shell egg heterozygous is isozygotying between layer of green-shell egg and non-layer of green-shell egg.
As follows according to O gene design primer:
F (upstream primer): 5 '-ACTATATCTCCCCTTAAACTA-3 ' (sequence 1 of sequence table);
R (downstream primer): 5 '-CAAAATCGTCAAGATAAGAGATG
GC-3 ' (sequence 2 of sequence table).
Two, the discovery of polymorphic site
With blue-shelled egg layer heterozygous and the hybridization of Bai Laihang laying hen, obtain F1 generation.
1, the egg-laying deseription analysis in F1 generation
By hybridizing with the Bai Laihang laying hen, the F1 in determining step two is homozygous or heterozygous for individuality respectively.If F2 is layer of green-shell egg (proterties does not namely occur separates) for individuality, this F1 is pure lines for individuality.If F2 also has non-layer of green-shell egg for existing layer of green-shell egg in individuality, this F1 is heterozygous for individuality.
2, the foundation of the discovery of polymorphic site and pira-PCR-RFLP detection method
Extract respectively the genomic dna in F1 generation (or Bai Laihang laying hen) as template, carry out pcr amplification with the primer pair (F and R) that designs.
Cut the PCR product with restriction enzyme MscI enzyme, pcr amplification product and enzyme thereof are cut product carry out respectively agarose gel electrophoresis.
The pcr amplification product of all samples is all at identical position display one band, approximately 174bp; The enzyme in all homozygous F1 generation is cut product and is all shown a band, and the position is identical with the position of pcr amplification product, approximately 174bp; The enzyme of all Bai Laihang laying hens is cut product and is all shown a band, compares with the band of pcr amplification product, and enzyme is cut the position of product band away from point sample hole summary, approximately 150bp; The enzyme in all heterozygous F1 generation is cut product and is all shown two bands, and the position with pcr amplification product is identical, and the position that another enzyme with the Bai Laihang laying hen is cut product is identical.
The analysis reason is as follows: big or small same or similar (the approximately 174bp) of the pcr amplification product in Bai Laihang laying hen and homozygous F1 generation, but concrete Nucleotide there are differences (may be the SNP site), have the MscI enzyme in the pcr amplification product of Bai Laihang laying hen and cut recognition site, do not have the MscI enzyme in the pcr amplification product in homozygous F1 generation and cut recognition site; The pcr amplification product in homozygous F1 generation can not be cut by the MscI enzyme, so that enzyme is cut the position of product electrophoresis showed is identical with the PCR product; The pcr amplification product of Bai Laihang laying hen can be cut by the MscI enzyme, so that enzyme is cut the position of product electrophoresis showed is different from the PCR product, should also produce the approximately small segment of 24bp, and just this small segment does not show under this deposition condition; In heterozygous F1 generation, have Bai Laihang laying hen and two kinds of homozygous DNA simultaneously, so banding pattern is the stack of Bai Laihang laying hen and homozygous banding pattern, shows simultaneously the band identical with the pcr amplification product position and cut the identical band in product position with the enzyme of Bai Laihang laying hen.
Take the genomic dna of chicken to be measured as template, adopt that described primer pair carries out pcr amplification, enzyme is cut and electrophoresis, the banding pattern and the green shell proterties of the blue-shelled egg layer close association that produce, can be used as the molecule marker of green shell character screening, thereby can effectively reject recessive non-oocyan gene, improve the green shell rate of blue-shelled egg layer colony.
The application of embodiment 2, pira-PCR-RFLP detection method
Experiment material: 33 blue-shelled egg layers and 12 Bai Laihang laying hens; The country poultry is measured the center; Reference: Zhao Rui, Liu Zhenzhen, Xu Guiyun and Yang Ning. (2006). utilize PCR-SSCP to analyze the SNP mark of chicken green-egg-shelled gene. Journal of Agricultural Biotechnology 14 (5): 673-676..
One, individuality is carried out genotyping
Respectively each individuality is carried out genotyping, method is as follows:
1, extract genomic dna.
2, the genomic dna that extracts take step 1 is as template, and (F and R) carries out pcr amplification with primer pair, obtains the PCR product.
F (upstream primer): 5 '-ACTATATCTCCCCTTAAACTA-3 ' (sequence 1 of sequence table);
R (downstream primer): 5 '-CAAAATCGTCAAGATAAGAGATG
GC-3 ' (sequence 2 of sequence table).
PCR reaction system (15 μ l): genomic dna 50-100ng, 10 * pcr amplification damping fluid (contains Mg
2+) 1.5 μ l, dNTPs (4mmol/l) mixed solution 0.8 μ l, each 0.3 μ l of upstream and downstream primer (10 μ mol/l), Taq archaeal dna polymerase 0.5U uses ddH
2O postreaction system to 15 μ l.
PCR reaction conditions: 95 ℃ of sex change 5min; Cycling program is: 95 ℃ of sex change 30s, and 55 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 30 circulations; Last 72 ℃ are extended 10min.
3, enzyme is cut
Get the PCR product of step 2,37 ℃ of enzymes were cut 2-4 hour.
Endonuclease reaction system: with 3.5 μ l PCR products, 10 * enzyme cutting buffering liquid, 1 μ l, 4U restriction enzyme MscI mixing, replenish ddH
2O to 10 μ l.
4, electrophoresis
The enzyme of the pcr amplification product of step 2 and step 3 is cut product carry out respectively 3% sepharose (voltage 8V/cm, time 30min).Use the gel imaging system observations.
Cut the electrophoresis result of product according to the enzyme of pcr amplification product and step 3, all detected individualities can be divided into 3 kinds:
The first: pcr amplification product and enzyme are cut product and are respectively shown a band, and enzyme is cut product less than pcr amplification product; Pcr amplification product is 174bp approximately, and enzyme is cut approximately 150bp of product;
The second: pcr amplification product and enzyme are cut product and are respectively shown a band, and the position is identical; Equal about 174bp;
The third: pcr amplification product shows a band, and enzyme is cut product and shown two bands; Pcr amplification product is 174bp approximately, and enzyme is cut product and is respectively approximately 174bp and approximately 150bp.
The electrophorogram of the first is seen Fig. 1.In Fig. 1, the 1:PCR amplified production; 2: enzyme is cut product; M:Marker; The electrophorogram of the second is seen Fig. 2.In Fig. 2, the 1:PCR amplified production; 2: enzyme is cut product; M:Marker; The third electrophorogram is seen Fig. 3.In Fig. 3, the 1:PCR amplified production; 2: enzyme is cut product; M:Marker.
12 Bai Laihang laying hens are the first; In 33 blue-shelled egg layers, 23 is the second, and 10 is the third.
Two, egg-laying deseription analysis
Blue-shelled egg layer is hybridized with the Bai Laihang laying hen respectively, determine that respectively individuality is homozygous (pure lines) or heterozygous.If F1 is layer of green-shell egg (proterties does not namely occur separates) for individuality, this blue-shelled egg layer is pure lines.If F1 also has non-layer of green-shell egg for existing layer of green-shell egg in individuality, this blue-shelled egg layer is heterozygous.Result shows, shows that the blue-shelled egg layer of the second banding pattern is pure lines; The blue-shelled egg layer that shows the third banding pattern is heterozygous.
The application of embodiment 3, pira-PCR-RFLP detection method
Experiment material: commercial generation blue-shelled egg layer (heterozygous) is newly raised in Shanghai; China Agricultural University's poultry resources base blue-shelled egg layer (pure lines) (reference: Zhao Rui, Liu Zhenzhen, Xu Guiyun and Yang Ning. (2006). utilize PCR-SSCP to analyze the SNP mark of chicken green-egg-shelled gene. Journal of Agricultural Biotechnology 14 (5): 673-676..)。
Heterozygous and homozygous judging criterion are as follows: with blue-shelled egg layer respectively with the hybridization of Bai Laihang laying hen, determine that respectively individuality is homozygous (pure lines) or heterozygous.If F1 is layer of green-shell egg (proterties does not namely occur separates) for individuality, this blue-shelled egg layer is pure lines.If F1 also has non-layer of green-shell egg for existing layer of green-shell egg in individuality, this blue-shelled egg layer is heterozygous.
All individualities are carried out genotyping, and method the results are shown in Table 1 with embodiment 2.
No. 1 distribution of the short arm of a chromosome G 67334934T in several chicken groups of table 1 chicken
Primer pair of the present invention and method identify that the accuracy of the layer of green-shell egg that isozygotys has all reached 100%.This primer of primer pair of the present invention and method is used in rejects the individuality that contains G in layer of green-shell egg colony, especially more significant to the selection of cock.
Claims (11)
1. the primer pair that forms of DNA shown in the sequence 2 of DNA and sequence table shown in the sequence 1 of sequence table.
2. the application of the described primer pair of claim 1 in assisting identification of hens laying green-shelled eggs.
3. the application of the described primer pair of claim 1 in the assisting identification of hens laying green-shelled eggs pure lines.
4. the method for an assisting identification of hens laying green-shelled eggs comprises the steps: that genomic dna take chicken to be measured as template, carries out pcr amplification with the described primer pair of claim 1, cuts pcr amplification product with restriction enzyme MscI enzyme; If pcr amplification product only has a kind of, and can not cut by being limited property restriction endonuclease MscI enzyme, chicken to be measured is candidate's layer of green-shell egg; If pcr amplification product has two kinds, and a kind of can not cutting by being limited property restriction endonuclease MscI enzyme wherein, another kind can be cut by being limited property restriction endonuclease MscI enzyme, and chicken to be measured is candidate's layer of green-shell egg; If pcr amplification product only has a kind of, and can cut by being limited property restriction endonuclease MscI enzyme, chicken to be measured is candidate's non-layer of green-shell egg; Described chicken to be measured is Bai Laihang laying hen or blue-shelled egg layer.
5. the method for assisting identification of hens laying green-shelled eggs pure lines comprises the steps: that genomic dna take chicken to be measured as template, carries out pcr amplification with the described primer pair of claim 1, cuts pcr amplification product with restriction enzyme MscI enzyme; If pcr amplification product only has a kind of, and can not cut by being limited property restriction endonuclease MscI enzyme, chicken to be measured is candidate's layer of green-shell egg pure lines; If pcr amplification product has two kinds, and a kind of can not cutting by being limited property restriction endonuclease Msc I enzyme wherein, another kind can be cut by being limited property restriction endonuclease MscI enzyme, and chicken to be measured is candidate's layer of green-shell egg heterozygote; If pcr amplification product only has a kind of, and can cut by being limited property restriction endonuclease MscI enzyme, chicken to be measured is candidate's non-layer of green-shell egg; Described chicken to be measured is Bai Laihang laying hen or blue-shelled egg layer.
6. method as described in claim 4 or 5, it is characterized in that: the reaction system of described pcr amplification is: genomic dna 50-100ng contains Mg
2+10 * pcr amplification damping fluid, 1.5 μ l, the dNTPs mixed solution 0.8 μ l of 4mmol/L, DNA0.3 μ l shown in the sequence 1 of the sequence table of 10 μ mol/L, DNA0.3 μ l shown in the sequence 2 of the sequence table of 10 μ mol/L, Taq archaeal dna polymerase 0.5U replenishes ddH
2O to 15 μ l; The reaction conditions of described pcr amplification is: 95 ℃ of sex change 5min; 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 30 circulations; 72 ℃ are extended 10min; The reaction system that described enzyme is cut is: pcr amplification product 3.5 μ l, and 10 * enzyme cutting buffering liquid, 1 μ l, restriction enzyme MscI4U replenishes ddH
2O to 10 μ l; The reaction conditions that described enzyme is cut is: 37 ℃ of 2-4 hour.
7. the application of the described primer pair of claim 1 in the test kit of preparation assisting identification of hens laying green-shelled eggs.
8. the application of the described primer pair of claim 1 in the test kit of preparation assisting identification of hens laying green-shelled eggs pure lines.
9. the test kit that contains the assisting identification of hens laying green-shelled eggs of the described primer pair of claim 1.
10. the test kit that contains the assisting identification of hens laying green-shelled eggs pure lines of the described primer pair of claim 1.
11. the application of the described primer pair of claim 1 in a breed of chicken is to carry out breeding with candidate's layer of green-shell egg pure lines; Described candidate's layer of green-shell egg pure lines adopt the described method of claim 5 to identify and obtain.
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| CN103583470B (en) * | 2013-11-20 | 2015-09-09 | 河南农业大学 | A kind of breeding method of the blue or green shank and recessive white feather chicken strain based on molecule assisted Selection |
| CN104830843A (en) * | 2014-06-03 | 2015-08-12 | 四川农业大学 | Primer composition for discriminating green-shelled-egg chicken genotype and use thereof |
| CN104642264B (en) * | 2015-02-05 | 2017-02-15 | 河南农业大学 | Fast balance breeding method for native hen varieties |
| CN108315444A (en) * | 2018-04-19 | 2018-07-24 | 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） | A kind of multiple PCR primer and discrimination method differentiating layer of green-shell egg genotype |
| CN109456962B (en) * | 2018-12-21 | 2022-03-04 | 河北农业大学 | Method and kit for extracting DNA from eggshell |
| CN116516022A (en) * | 2023-05-30 | 2023-08-01 | 江苏省家禽科学研究所 | Molecular biological identification method and application of pure-bred Dongxiang green-shell layer chicken |
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