CN103602745B - Primers, kit and detection method for detecting gene type of dominant white feather site of chicken PMEL17 gene - Google Patents
Primers, kit and detection method for detecting gene type of dominant white feather site of chicken PMEL17 gene Download PDFInfo
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Abstract
The invention discloses primers, a kit and a detection method for detecting a gene type of a dominant white feather site of chicken PMEL17 gene and belongs to the technical field of biology. According to the detection method, a specific primer P1-R of allelic gene I is designed, aiming at a situation that a 9bp insert (-CTGGGCACC-) exists in a 10th exon of the PMEL17 gene; a specific primer P2-F of allelic gene i is designed, aiming at the situation that no 9bp insert exists in the 10th exon of the PMEL17 gene; the specific primer pair P1 of the allelic gene I is used for amplifying an allelic gene I of the PMEL17 gene; the specific primer pair P2 of the allelic gene i is used for amplifying an allelic gene i of the PMEL17 gene; after a PCR (Polymerase Chain Reaction), agarose gel electrophoresis is carried out; the gene type of the dominant white feather site of the PMEL17 gene is detected accurately, rapidly and conveniently according to the result of the agarose gel electrophoresis. Compared with the prior art, a molecular biological method established in the invention can greatly improve the judging efficiency and accuracy of the gene type; and the method is simple, easy to operate and low in cost.
Description
Technical field
The present invention is specifically related to a kind of primer for detection of the white plumage loci gene type of chicken PMEL17 gene dominant, and the test kit that comprises this primer and detection method, belongs to biological technical field.
Background technology
White feather chicken can be divided into the dominant white plumage that isozygotys, the dominant white plumage of heterozygosis and recessive white feather three types genotype.We need to select the dominant white feather genes type that isozygotys in the breeding of dominant white feather chicken, carry out satisfied breeding demand.Therefore utilize molecular biosciences means to diagnose certain individuality in the genotype in dominant Bai Yu site, this is extremely important and necessary concerning our breeding.
Dominant white feather genes seat is one of most important locus affecting the formation of chicken plumage look, and it is synthetic that the dominant allele I on it can hinder feather melanochrome, thereby make to carry this genotypic individual whole body feather, presents white.Confirmed at present the dominant white feather genes locus coding of chicken PMEL17 albumen (Premelanosome protein17, PMEL17): PMEL17 genes encoding premelanosome protein 17, it is melanocyte differential protein, is playing the part of important role in melanocytic differentiation and ripening process.Dominant white allelotrope causes because PMEL17 gene exon10 9bp inserts.Be that dominant allele I is that PMEL17 gene exon10 9bp inserts genotype, Recessive alleles i is that PMEL17 gene exon10 inserts genotype without 9bp.For the gene fragment of poor 9bp only, after conventional pcr amplification, agarose gel electrophoresis is difficult to somatotype.In recent years, people have been developed many for seeking the method for molecular genetic marker, and modal have single-strand conformation polymorphism technology (SSCP), PCR-RFLP and direct Sequencing technology etc., but SSCP complex operation, length consuming time, result easily causes erroneous judgement; It is a certain specific restriction enzyme site that common PCR-RFLP method requires pleomorphism site to be measured, range of application limitation; And direct Sequencing technical costs is higher.
Summary of the invention
The object of this invention is to provide a kind of primer for detection of the white plumage loci gene type of chicken PMEL17 gene dominant.
Meanwhile, the present invention also provides a kind of test kit for detection of the white plumage loci gene type of chicken PMEL17 gene dominant.
Finally, the present invention also provides a kind of method that detects the white plumage loci gene type of chicken PMEL17 gene dominant.
In order to realize above object, the technical solution adopted in the present invention is:
A primer for detection of the white plumage loci gene type of chicken PMEL17 gene dominant, comprises primer pair P1, P2,
Described primer pair P1 is:
Upstream primer, P1-F:5 '-CCGGCCTCTACTGCCTCAACGTCTCGT-3 ' (as shown in SEQ ID No.1);
Downstream primer, P1-R:5 '-GTGCCCAGAGCTTCGGCGATGAGCGGTG-3 ' (as shown in SEQ ID No.2);
Described primer pair P2 is:
Upstream primer, P2-F:5 '-CCACGCTCACCGTGGGGCTGCTGCTCAT-3 ' (as shown in SEQ ID No.3);
Downstream primer, P2-R:5 '-GAGGGGGTGAGTGCTGTGTTCTC-3 ' (as shown in SEQ ID No.4).
A test kit for detection of the white plumage loci gene type of chicken PMEL17 gene dominant, mainly comprises primer pair P1, P2,
Described primer pair P1 is:
Upstream primer, P1-F:5 '-CCGGCCTCTACTGCCTCAACGTCTCGT-3 ';
Downstream primer, P1-R:5 '-GTGCCCAGAGCTTCGGCGATGAGCGGTG-3 ';
Described primer pair P2 is:
Upstream primer, P2-F:5 '-CCACGCTCACCGTGGGGCTGCTGCTCAT-3 ';
Downstream primer, P2-R:5 '-GAGGGGGTGAGTGCTGTGTTCTC-3 '.
Preferably, a kind of test kit for detection of the white plumage loci gene type of chicken PMEL17 gene dominant, also comprises 2 * TaqPCR MasterMix, sterilizing ultrapure water, and the DNA Marker that comprises 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 be purchased from companies such as the raw work in Shanghai, the precious biology in Dalian, hundred Tykes, Beijing, Beijing Kang Wei.
A method that detects the white plumage loci gene type of chicken PMEL17 gene dominant, comprises the following steps:
(1) take the chicken complete genome DNA to be measured that comprises PMEL17 gene is template, take primer pair P1 as primer PCR amplification chicken PMEL17 gene I allelotrope, take primer pair P2 as primer PCR amplification chicken PMEL17 gene i allelotrope;
(2) amplified fragments of step (1) is carried out to agarose gel electrophoresis, according to agarose gel electrophoresis result, identify the genotype in chicken PMEL17 gene dominant Bai Yu site.
In described step (1), pcr amplification reaction system is: 2 * Taq PCR MasterMix12.5 μ L, ddH
2o 9.5 μ L, P1-F 0.4 μ L, P1-R 0.6 μ L, P2-F 0.6 μ L, P2-R 0.4 μ L, DNA profiling 1.0 μ L.
In described step (1), pcr amplification reaction program is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 64 ℃ of annealing 30s, 72 ℃ are extended 15s, 35 circulations; 72 ℃ are extended 10min.
In described step (2), the mass concentration of sepharose is 1.5%.
In described step (2), the voltage of agarose gel electrophoresis is 120V, and electrophoresis time is 40min.
The method of identifying the white plumage loci gene type of chicken PMEL17 gene dominant in described step (2) is: II genotype shows as 322bp and 224bp band; Ii genotype shows as 322bp, 224bp and 144bp band; Ii genotype shows as 322bp and 144bp band.
Beneficial effect of the present invention:
In the present invention, for detection of the primer of the white plumage loci gene type of chicken PMEL17 gene dominant, be to insert (CTGGGCACC-) design allelotrope I Auele Specific Primer P1-R for PMEL17 gene exon10 9bp; For PMEL17 gene exon10, without 9bp, insert design allelotrope i Auele Specific Primer P2-F.Primer P1-R and the P1-F I allelotrope that can only increase like this, the clip size of amplification is 224bp; Primer P2-R and the P2-F i allelotrope that can only increase, the clip size of amplification is 144bp.In addition, because the combination of upstream primer P1-F and downstream primer P2-R also can be increased, the clip size of amplification is 322bp, the impact that this amplification is not inserted by 9bp, and each individuality all can increase.
It is primer to P1 that I allele-specific primers is take in the present invention, pcr amplification chicken PMEL17 gene I allelotrope, the i allele-specific primers of take is primer to P2, pcr amplification chicken PMEL17 gene i allelotrope, then primer pair P1 and P2 are placed in a PCR reaction, after a PCR reaction, again pcr amplified fragment is carried out to agarose gel electrophoresis, according to agarose gel electrophoresis result, can detect accurately, fast and easily the white plumage loci gene type of PMEL17 gene dominant: II genotype shows as 322bp and 224bp band; Ii genotype shows as 322bp, 224bp and 144bp band; Ii genotype shows as 322bp and 144bp band.
The present invention adopts agarose gel electrophoresis direct-detection PCR product, compared with prior art: the molecular biology method that the present invention sets up has improved genotypic judgement efficiency and accuracy greatly, and method is easy, the somatotype time is short, do not need order-checking and restriction enzyme, do not need special instrument, cost is low, easily popularizes.
The present invention is by type sequence verification have been sentenced to by other chicken colonies its accuracy.Research shows that the method can diagnose the genotype in dominant Bai Yu site effectively, can be for the molecular marker assisted selection in the dominant Bai Yu site of chicken, and then set up the dominant white feather chicken population of isozygotying.The present invention has set up the authentication method of the dominant Bai Yu of a kind of chicken site molecule marker, can determine fast the genotype in dominant Bai Yu site, thereby shortens breeding time, accelerates breeding process.
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 PMEL17 gene dominant Bai Yu site in embodiment 1,
Note: swimming lane is distinguished the electrophoresis banding pattern of representation DNA Marker I and genotype ii, ii, Ii, II, II, ii, Ii, ii and Ii from left to right;
Fig. 3 is the different genotype sequencer map of PMEL17 gene in embodiment 1,
Note: Fig. 3 a is PMEL17 gene II genotype sequencer map (the 9bp base for inserting in square frame), and Fig. 3 b is PMEL17 gene ii genotype sequencer map.
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 24 of 400 of chicken house Recessive Chicken strains, 400 of Xichuan Gallus Domesticuss, 24 of dominant white feather chicken strains and plumage Gallus Domesticuss, 30 of Chinese poultry resource gene pool Yangzhou Recessive Chickens, 24 of the white Leghorns in testing station, Zhengzhou and Luo Man brown shell layer CD are 24, wing venous blood collection, add 1/3 antithrombotics, be stored in 4 ℃ of Refrigerator stores standby.
(2) extraction of genomic dna
Reference Sambrock et al(2002) method.
(3) design of primers
PMEL17 gene order (GenBank Accession No.AY636129) the design primer of announcing according to NCBI.
Primer pair P1 is:
Upstream primer, P1-F:5 '-CCGGCCTCTACTGCCTCAACGTCTCGT-3 ';
Downstream primer, P1-R:5 '-GTGCCCAGAGCTTCGGCGATGAGCGGTG-3 ';
Primer pair P2 is:
Upstream primer, P2-F:5 '-CCACGCTCACCGTGGGGCTGCTGCTCAT-3 ';
Downstream primer, P2-R:5 '-GAGGGGGTGAGTGCTGTGTTCTC-3 '.
(4) pcr amplification
PCR reaction system adopts mixes application of sample method, according to the number of the quantity of the required various components of each reaction system and the required PCR of 1 secondary response reaction, calculate the total amount of various reactive components, join in 1 1.5mL centrifuge tube, fully mix rear instantaneous centrifugal, divide again and install in each 0.2mL Eppendorf PCR pipe, then add respectively template DNA, more instantaneous centrifugal laggard performing PCR amplification; PCR reaction system is in Table 1.Response procedures is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 64 ℃ of annealing 30s, 72 ℃ are extended 20s, 38 circulations; 72 ℃ are extended 5min; 4 ℃ of preservations.Wherein, 2 * Taq PCR MasterMix is purchased from Beijing hundred Imtech.
Table 1PCR reaction system
| Sterilizing ultrapure water (H 2O） | 9.5μL |
| 2×Taq?PCR?MasterMix | 12.5μL |
| Primer P1-F(10pmol/L) | 0.4μL |
| Primer P1-R(10pmol/L) | 0.6μL |
| Primer P2-F(10pmol/L) | 0.6μL |
| Primer P2-R(10pmol/L) | 0.4μL |
| The DNA(50ng/ μ L of chicken to be detected) | 1.0μL |
| Cumulative volume | 25.0μL |
(5) detection of pcr amplification product and result are judged
Get 10 μ L pcr amplification products, 1.5% agarose gel electrophoresis (electrophoretic voltage: 120V; Electrophoresis time: 40min) point sample, gel imaging system detects, and electrophoretogram is referring to Fig. 2.According to the size of the band occurring in electrophoretogram, judge: if there is the fragment of 322bp and 224bp, be dominant white plumage homozygote (phenotype is white plumage); If there is the fragment of 322bp and 144bp, it is dominant white plumage heterozygote (phenotype is white plumage); If there is 322bp, the fragment of 224bp and 144bp, is wild-type homozygote (phenotype is coloured plumage or recessive white feather), and result is as shown in table 2.
Table 2 detection validation crowd surveillance result cartogram
(6) to the different genotype of PMEL17 gene, (II, ii) check order, sequencer map as shown in Figure 3.Fig. 3 a is PMEL17 gene II genotype sequencer map (the 9bp base for inserting in square frame), and Fig. 3 b is PMEL17 gene ii genotype sequencer map.
(7) selection of individuality to be checked is with superseded
According to the analytical results of table 2, eliminate Ii individual, cultivate the II individuality of selecting and remain of the dominant white feather chicken colony of isozygotying.The dominant white plumage individuality that will isozygoty reserve seed for planting expand numerous, by colony of isozygotying of formation, for breeding.
Claims (8)
1. for detection of a test kit for the white plumage loci gene type of chicken PMEL17 gene dominant, it is characterized in that: mainly comprise primer pair P1, P2,
Described primer pair P1 is:
Upstream primer, P1-F:5 '-CCGGCCTCTACTGCCTCAACGTCTCGT-3 ';
Downstream primer, P1-R:5 '-GTGCCCAGAGCTTCGGCGATGAGCGGTG-3 ';
Described primer pair P2 is:
Upstream primer, P2-F:5 '-CCACGCTCACCGTGGGGCTGCTGCTCAT-3 ';
Downstream primer, P2-R:5 '-GAGGGGGTGAGTGCTGTGTTCTC-3 '.
2. the test kit for detection of the white plumage loci gene type of chicken PMEL17 gene dominant according to claim 1, it is characterized in that: also comprise 2 * Taq PCR MasterMix, sterilizing ultrapure water, and the DNA Marker that comprises 600bp, 500bp, 400bp, 300bp, 200bp and 100bp.
3. a method that detects the white plumage loci gene type of chicken PMEL17 gene dominant, is characterized in that: comprise the following steps:
(1) take the chicken complete genome DNA to be measured that comprises PMEL17 gene is template, take primer pair P1 as primer PCR amplification chicken PMEL17 gene I allelotrope, take primer pair P2 claimed in claim 1 as primer PCR amplification chicken PMEL17 gene i allelotrope;
Described primer pair P1 is:
Upstream primer, P1-F:5 '-CCGGCCTCTACTGCCTCAACGTCTCGT-3 ',
Downstream primer, P1-R:5 '-GTGCCCAGAGCTTCGGCGATGAGCGGTG-3 ';
Described primer pair P2 is:
Upstream primer, P2-F:5 '-CCACGCTCACCGTGGGGCTGCTGCTCAT-3 ',
Downstream primer, P2-R:5 '-GAGGGGGTGAGTGCTGTGTTCTC-3 ';
(2) amplified fragments of step (1) is carried out to agarose gel electrophoresis, according to agarose gel electrophoresis result, identify the genotype in chicken PMEL17 gene dominant Bai Yu site.
4. the method for the white plumage loci gene type of detection chicken PMEL17 gene dominant according to claim 3, is characterized in that: in described step (1), pcr amplification reaction system is: 2 * Taq PCR MasterMix12.5 μ L, ddH
2o9.5 μ L, P1-F0.4 μ L, P1-R0.6 μ L, P2-F0.6 μ L, P2-R0.4 μ L, DNA profiling 1.0 μ L.
5. the method for the white plumage loci gene type of detection chicken PMEL17 gene dominant according to claim 3, is characterized in that: in described step (1), pcr amplification reaction program is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 64 ℃ of annealing 30s, 72 ℃ are extended 15s, 35 circulations; 72 ℃ are extended 10min.
6. the method for the white plumage loci gene type of detection chicken PMEL17 gene dominant according to claim 3, is characterized in that: in described step (2), the mass concentration of sepharose is 1.5%.
7. the method for the white plumage loci gene type of detection chicken PMEL17 gene dominant according to claim 3, is characterized in that: in described step (2), the voltage of agarose gel electrophoresis is 120V, and electrophoresis time is 40min.
8. the method for the white plumage loci gene type of detection chicken PMEL17 gene dominant according to claim 3, is characterized in that: the method for identifying the white plumage loci gene type of chicken PMEL17 gene dominant in described step (2) is: II genotype shows as 322bp and 224bp band; Ii genotype shows as 322bp, 224bp and 144bp band; Ii genotype shows as 322bp and 144bp band.
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| CN106011232B (en) * | 2016-05-12 | 2019-10-11 | 上海市农业科学院 | A method of selection chicken anti-adversity |
| 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 |
| 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 |
| CN111893193A (en) * | 2020-08-12 | 2020-11-06 | 北京康普森农业科技有限公司 | Rapid genotyping detection method for dominant white chicken |
| CN113913535A (en) * | 2021-11-30 | 2022-01-11 | 江西农业大学 | Causal gene for identifying blue peacock white feather character and application thereof |
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