CN103571963B - Primer, kit and detection method for detecting recessive white feather locus genotype of chicken - Google Patents
Primer, kit and detection method for detecting recessive white feather locus genotype of chicken Download PDFInfo
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- CN103571963B CN103571963B CN201310589394.4A CN201310589394A CN103571963B CN 103571963 B CN103571963 B CN 103571963B CN 201310589394 A CN201310589394 A CN 201310589394A CN 103571963 B CN103571963 B CN 103571963B
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
The invention discloses a primer, a kit and a detection method for detecting a recessive white feather locus genotype of a chicken, belonging to the technical field of biology. According to the invention, a specific primer P1 of an allelic gene c is designed according to the fact that 7.7kb insertion (ev1) exists in a fourth intron of a TYR gene, a specific primer P2 of the allelic gene c is designed according to the fact that 7.7kb insertion (ev1) does not exist in a fourth intron of a PMEL17 gene, a primer P3 is a common downstream primer of the primer P1 and the primer P2, a whole genome of a to-be-detected chicken containing the TYR gene is taken as a template, the primers P1, P2 and P3 are put in the same PCR (Polymerase Chain Reaction), an amplified fragment of the PCR is subjected to agarose gel electrophoresis after once PCR, and the recessive white feather locus genotype of the TYR gene of the chicken is identified according to the result of the agarose gel electrophoresis. The method greatly improves the genotype judging efficiency and accuracy, the method is simple, the genotype judging time is short, no sequencing or restriction enzyme is needed, no special instrument is required, the cost is low, and the method is easy to popularize.
Description
Technical field
The present invention is specifically related to a kind of primer for detecting chicken recessive white feather loci gene type, and comprises test kit and the detection method of this primer, belongs to biological technical field.
Background technology
Along with growth in the living standard, the consumption idea of people changes mass type into from the scalar type in past.The requirement of people to chicken meat is also more and more higher.The indigenous chicken of China has the good feature of meat, but its speed of growth is comparatively slow, and feed conversion rate is low.And though external fast large-scale broiler chicken fast growth, feed conversion rate are high, its meat is poor.For addressing this problem, large-scale for state's extra income broiler chicken and domestic local high quality meat chicken are hybridized, the meat that can either improve the large-scale broiler chicken of state's extra income also can accelerate the speed of growth and the production performance of local high quality meat chicken.But due to the impact of the long-term food habits of our people, the chicken of coloured plumages such as Chinese Consumer's preference jute plumage, black plumage, especially Shelter in South China Cities does not accept the white feather chicken of growth fast.In order to the phenotype of chicken meets the demand of Chinese Consumer's after making hybridization, just need the Recessive Chicken isozygotied, Recessive Chicken production performance is high, and can keep the plumage look of coloured plumage after hybridizing with China coloured plumage, is significant in China's high-quality a breed of chicken.
TYR enzyme gene (Tyrosine, TYR) plays very important effect in melanin synthesis process.Have and report: whether TYR gene intron 4 exists the insertion of 7.7kb, relevant to recessive white feather.
Summary of the invention
The object of this invention is to provide a kind of primer for detecting chicken recessive white feather loci gene type.
Meanwhile, the present invention also provides a kind of test kit for detecting chicken recessive white feather loci gene type.
Finally, the present invention also provides a kind of method detecting chicken recessive white feather loci gene type.
In order to realize above object, the technical solution adopted in the present invention is:
For detecting a primer for chicken recessive white feather loci gene type, comprise primer P1, P2 and P3,
Upstream primer P1:5 '-CCCAGTTCCCTTCAATTT-3 ' (as shown in SEQ ID No.1);
Upstream primer P2:5 '-TGGCCGACCACTATTCCCTA-3 ' (as shown in SEQ ID No.2);
Downstream primer P3:5 '-CTGATGGGCTTGCTTGAGGT-3 ' (as shown in SEQ ID No.3).
For detecting a test kit for chicken recessive white feather loci gene type, mainly comprise primer P1, P2 and P3,
Upstream primer P1:5 '-CCCAGTTCCCTTCAATTT-3 ';
Upstream primer P2:5 '-TGGCCGACCACTATTCCCTA-3 ';
Downstream primer P3:5 '-CTGATGGGCTTGCTTGAGGT-3 '.
Preferably, a kind of test kit for detecting chicken recessive white feather loci gene type, also comprises 2 × Taq PCR MasterMix, sterilizing ultrapure water, and comprises the DNA Marker of 600bp, 500bp, 400bp, 300bp, 200bp and 100bp.
Described 2 × Taq PCR MasterMix contains Taq archaeal dna polymerase, dNTPs, magnesium chloride and PCR reaction buffer, is commercial goods, can purchased from the company such as the raw work in Shanghai, Dalian precious biology, Tyke, Beijing hundred, Beijing Kang Wei.
Detect a method for chicken recessive white feather loci gene type, comprise the following steps:
(1) to comprise the chicken complete genome DNA to be measured of TYR gene for template, in same PCR reaction system, utilize primer P1-P3 pcr amplification chicken TYR gene C allelotrope, utilize primer P2-P3 pcr amplification chicken TYR gene c allelotrope, obtain amplified production;
Described primer P1, P2 and P3 are respectively:
Upstream primer P1:5 '-CCCAGTTCCCTTCAATTT-3 ',
Upstream primer P2:5 '-TGGCCGACCACTATTCCCTA-3 ',
Downstream primer P3:5 '-CTGATGGGCTTGCTTGAGGT-3 ';
(2) agarose gel electrophoresis is carried out to amplified production, according to the genotype in agarose gel electrophoresis result qualification chicken TYR gene recessive white feather site.
In described step (1), pcr amplification reaction system is: 2 × Taq PCR MasterMix 6.25 μ L, ddH
2o 4.25 μ L, P1 0.5 μ L, P2 0.5 μ L, P3 0.5 μ L, DNA profiling 0.5 μ L, totally 12.5 μ L.
In described step (1), pcr amplification reaction program is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations; 72 DEG C extend 10min; 4 DEG C of preservations.
In described step (2), the mass concentration of sepharose is 1%.
In described step (2), the voltage of agarose gel electrophoresis is 120V, and electrophoresis time is 30min.After electrophoresis terminates, with gel imaging system, sepharose photograph is detected, sentence type.
Identify in described step (2) that the method for chicken TYR gene recessive white feather loci gene type is: CC genotypic expression is 267bp band; Cc genotypic expression is 451bp and 267bp band; Cc genotypic expression is 451bp.
Beneficial effect of the present invention:
For detecting the primer of chicken recessive white feather loci gene type in the present invention, be there is 7.7kb insertion (ev1) for TYR gene intron 4 to design allelotrope c Auele Specific Primer P1; There is not 7.7kb insertion (ev1) for PMEL17 gene intron 4 and design allele C Auele Specific Primer P2; Primer P3 is the common downstream primer of primer P1 and P2.Such primer P1-P3 can only increase c allelotrope, and the clip size of amplification is 451bp; Primer P2-P3 can only increase C allelotrope, and the clip size of amplification is 267bp.Namely TYR gene recessive white feather loci gene type can be detected accurately, fast and easily by electrophoresis detection somatotype: CC genotypic expression is 267bp band after pcr amplification TYR gene; Cc genotypic expression is 451bp and 267bp band; Cc genotypic expression is 451bp.
The present invention is to comprise the chicken complete genome DNA to be measured of TYR gene for template, primer P1, P2 and P3 are placed in a PCR reaction, again agarose gel electrophoresis is carried out to pcr amplified fragment after a PCR reaction, according to the genotype in agarose gel electrophoresis result qualification chicken TYR gene recessive white feather site.Compared with prior art, the molecular biology method that the present invention sets up can improve genotypic judgement efficiency and accuracy greatly, and method is easy, and the somatotype time is short, and do not need order-checking and restriction enzyme, do not need special instrument, cost is low, easily popularizes.
The present invention demonstrates its accuracy by sentencing type to other chicken colonies, result shows that the method can diagnose the genotype of the TYR gene in recessive white feather site effectively, may be used for the molecular marker assisted selection in chicken recessive white feather site, thus for shortening breeding time, accelerating breeding process provides strong guarantee.
Accompanying drawing explanation
Fig. 1 is the techniqueflow schematic diagram of the embodiment of the present invention 1;
Fig. 2 is the PCR electrophorogram in each sample TYR gene recessive white feather site in embodiment 1,
Note: numbering is described as follows: 1,2,3,6,11,12 is CC genotype, and 7,8,9 is cc genotype, and 4,5,10 is Cc 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.
Embodiment 1
The techniqueflow schematic diagram of the present embodiment as shown in Figure 1, mainly comprises the following steps:
(1) collection of sample
Gather Agricultural University Of He'nan and plant chicken house Recessive Chicken strain 30, Chinese poultry resource gene pool Yangzhou Recessive Chicken 30, Lushi's layer of green-shell egg conservation factory Lushi layer of green-shell egg 30, wing venous blood collection, adds 1/3 antithrombotics, is stored in 4 DEG C of Refrigerator stores for subsequent use.
(2) extraction of genomic dna
Reference Sambrock et al(2002) method.
(3) amplimer
Primer P1, P2 and P3 is designed according to the TYR gene order (GenBank Accession NC_006088.3) that NCBI announces, as follows:
Upstream primer P1:5 '-CCCAGTTCCCTTCAATTT-3 ',
Upstream primer P2:5 '-TGGCCGACCACTATTCCCTA-3 ',
Downstream primer P3:5 '-CTGATGGGCTTGCTTGAGGT-3 '.
(4) pcr amplification
PCR reaction system adopts mixing application of sample method, the number that the quantity of the various components namely needed for each reaction system and the PCR needed for 1 secondary response react, calculate the total amount of various reactive component, join in 1 1.5mL centrifuge tube, brief centrifugation after abundant mixing, be dispensed into again in each 0.2mL Eppendorf PCR pipe, then add template DNA respectively, the more laggard performing PCR amplification of brief centrifugation.
PCR reaction system is: 2 × Taq PCR MasterMix(is purchased from Beijing hundred Imtech) 6.25 μ L, ddH
2o 4.25 μ L, P1 0.5 μ L, P2 0.5 μ L, P3 0.5 μ L, DNA profiling 0.5 μ L, totally 12.5 μ L.Response procedures is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations; 72 DEG C extend 10min; 4 DEG C of preservations.
(5) detection of pcr amplification product and result judge
Get 8 μ L pcr amplification products, the agarose gel electrophoresis (electrophoretic voltage: 120V of 1%; Electrophoresis time: 30min) point sample, gel imaging system detects, and electrophoretogram is see Fig. 2.
Size according to the band occurred in electrophoretogram judges: if only have the fragment of 267bp, be then recessive white feather homozygote; If there is the fragment of 267bp and 451bp, then it is recessive white feather heterozygote; If only have the fragment of 451bp, be then wild-type homozygote, result is as shown in table 1.
Table 1 detection validation crowd surveillance result cartogram
Claims (5)
1. detect a method for chicken recessive white feather loci gene type, it is characterized in that: comprise the following steps:
(1) to comprise the chicken complete genome DNA to be measured of TYR gene for template, in same PCR reaction system, utilize primer P1-P3 pcr amplification chicken TYR gene C allelotrope, utilize primer P2-P3 pcr amplification chicken TYR gene c allelotrope, obtain amplified production;
Described primer P1, P2 and P3 are respectively:
Upstream primer P1:5 '-CCCAGTTCCCTTCAATTT-3 ',
Upstream primer P2:5 '-TGGCCGACCACTATTCCCTA-3 ',
Downstream primer P3:5 '-CTGATGGGCTTGCTTGAGGT-3 ';
(2) agarose gel electrophoresis is carried out to amplified production, according to the genotype in agarose gel electrophoresis result qualification chicken TYR gene recessive white feather site;
Wherein, identify in described step (2) that the method for chicken TYR gene recessive white feather loci gene type is: CC genotypic expression is 267bp band; Cc genotypic expression is 451bp and 267bp band; Cc genotypic expression is 451bp.
2. the method for detection chicken recessive white feather loci gene type according to claim 1, is characterized in that: in described step (1), pcr amplification reaction system is: 2 × Taq PCR MasterMix 6.25 μ L, ddH
2o 4.25 μ L, P1 0.5 μ L, P2 0.5 μ L, P3 0.5 μ L, DNA profiling 0.5 μ L, totally 12.5 μ L.
3. the method for detection chicken recessive white feather loci gene type according to claim 1, is characterized in that: in described step (1), pcr amplification reaction program is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations; 72 DEG C extend 10min; 4 DEG C of preservations.
4. the method for detection chicken recessive white feather loci gene type according to claim 1, is characterized in that: in described step (2), the mass concentration of sepharose is 1%.
5. the method for detection chicken recessive white feather loci gene type according to claim 1, is characterized in that: in described step (2), the voltage of agarose gel electrophoresis is 120V, and electrophoresis time is 30min.
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106417155A (en) * | 2016-07-20 | 2017-02-22 | 华南农业大学 | Method for avoiding appearance of laying hen type phenotypic offspring caused by hybridization of white-feather Xuefeng black-bone chicken and jute-feather Xuefeng black bone chicken |
| CN108085400B (en) * | 2017-12-29 | 2019-09-10 | 中国农业大学 | The identification and special marker of the white red plumage reason mutated-genotype of Leghorn |
| CN109652558A (en) * | 2018-08-10 | 2019-04-19 | 贵州大学 | A kind of primer and its application for identifying incense burner pheasant recessive white feather genes type |
| CN108977550A (en) * | 2018-08-10 | 2018-12-11 | 贵州大学 | A kind of primer and its application for identifying incense burner pheasant recessive white feather and coloured plumage homozygosis |
| 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 |
| 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 |
| CN111909989A (en) * | 2020-08-12 | 2020-11-10 | 北京康普森农业科技有限公司 | Rapid genotyping detection method for recessive leucocyte of chicken |
| CN114959064A (en) * | 2022-06-17 | 2022-08-30 | 华南农业大学 | Molecular detection method for identifying recessive white feather genotype of chicken by anticoagulation whole blood method and application thereof |
| CN116790767B (en) * | 2023-07-21 | 2024-02-20 | 华南农业大学 | TYR gene molecular marker related to chicken skin blackness and application thereof |
| CN116926207A (en) * | 2023-08-07 | 2023-10-24 | 华南农业大学 | Primer, kit and application for detecting recessive white feather gene carrier of chicken based on LAMP |
Citations (1)
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| CN101696452A (en) * | 2009-10-19 | 2010-04-21 | 华中农业大学 | Method for identifying molecules of recessive white feather genes of chicken |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101696452A (en) * | 2009-10-19 | 2010-04-21 | 华中农业大学 | Method for identifying molecules of recessive white feather genes of chicken |
Non-Patent Citations (1)
| Title |
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| 多重PCR筛查高发群体缺失型α-地中海贫血基因的研究;区采莹等;《中国热带医学》;20021231;第2卷(第2期);129-131 * |
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