CN115992264A - Chicken yellow and white skin color molecular identification primer group and method thereof - Google Patents
Chicken yellow and white skin color molecular identification primer group and method thereof Download PDFInfo
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The invention discloses a chicken yellow and white skin color molecular identification primer group and a method thereof. Belonging to the technical field of animal breeding. Comprising the following steps: quality control internal index primers and white skin specific primers, and methods of identification are provided. The method for identifying the yellow and white skin color of the chicken by using the DNA blood sample of the yellow and white skin color chicken establishes the molecular marker, so that the identification of the yellow and white skin color of the chicken is not limited by time, and the chicken can be accurately identified when required. PCR amplification is carried out by using the primers by using different types of chicken individual DNA templates, and when only one 258bp band can be amplified, the individual is judged to be a yellow skin individual; when two bands of 258bp and 482bp in size, respectively, were amplified, the individual was judged to be white skin.
Description
Technical Field
The invention relates to the technical field of animal breeding, in particular to a chicken yellow and white skin color molecular identification primer group and a method thereof.
Background
With the large-scale development of chicken raising industry and the requirement of biological safety prevention and control of chicken farm epidemic diseases, "concentrated slaughter and fresh market" become trend. Especially in the southern areas of China, "iced chicken" is becoming the mainstream of the market. In both areas, there is a habit of eating white chickens, and consumers are required to purchase yellow skin broilers (Sanhuang chickens). However, the phenotype of the skin color of the broiler chickens is not easy to accurately judge by naked eyes, and the difficulty is brought to broiler breeders and breeders. In recent years, with the deep research of chicken genome and molecular biology, the technology for screening the skin color of broiler chickens by using a molecular detection technology is mature; the positioned chicken yellow-white skin control genes can be utilized to screen white skin breeding hens in a breeding core group through a molecular method and eliminate the white skin breeding hens, so that the yellow skin alleles of the breeding hens are fixed (namely, the gene frequency reaches 100%), the yellow skin of commercial chicken individuals is ensured, and the market demand is met.
The color of chicken skin is mainly related to the deposition of carotenoids on the skin epidermis, yellow skin chicken is mainly the result of pigmentation of lutein etc. in feed such as corn, while white skin is mainly due to carotenoid degradation by enzymes decomposing pigments. Chicken epidermis pigmentation to form yellow or white skin is determined by alleles at autosomal W sites; there are two alleles at this site, where the W gene determines white skin and is dominant to the W gene (determines yellow skin). Previous studies utilized linkage analysis and IBD methods to map the W locus to a locus encoding BCO 2. The W gene encodes a functional BCO2 enzyme that can decompose carotenoids into colorless carotenoids to make the skin color of chickens appear white, whereas the W gene encodes a defective BCO2 enzyme that cannot effectively decompose carotenoids, so chickens appear as yellow skin. Studies have found that chickens with white skin at 6150201bp of chromosome 24 of chickens inserted four bases of CCAA. Provides possibility for accurately identifying the chicken yellow and white skin phenotype by using a molecular marking method.
Therefore, providing a primer set for identifying chicken yellow and white skin color molecules and a method thereof are a problem to be solved by the person skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a chicken yellow-white skin color molecular identification primer set and a method thereof. The method can accurately identify the skin color of the chicken without being limited by time and individual difference, thereby reducing error of macroscopic identification of skin color phenotype and reducing investment of time and cost.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a breeding hen yellow-white skin color molecular identification primer set comprising: the nucleotide sequences of the quality control inner index primer and the white skin specific primer are as follows:
quality control internal index primer:
f:5'-TTCCTTCATTTCAAGCGTTTCTG-3', as shown in SEQ ID No. 1;
r1 is 5'-CATTTTCCTGGAGTGCCTTTGT-3', and is shown as SEQ ID No. 2;
white skin-specific primers:
f:5'-TTCCTTCATTTCAAGCGTTTCTG-3', as shown in SEQ ID No. 1;
r2 and 5'-TGGGTTGAGGGTTGGTTGG-3' are shown in SEQ ID No. 3.
The invention also provides a detection kit based on the primer group.
The invention also provides an identification method based on the primer group or the kit, which comprises the following steps:
(1) Extracting genome DNA in a chicken blood sample to be identified;
(2) And (3) performing double PCR amplification, detecting PCR amplification products by electrophoresis, and identifying the skin color of the chicken to be detected according to the existence and the size of the amplified DNA fragment.
Preferably: PCR amplification system: mix 5. Mu. L, F1 1. Mu. L, R1 1.5. Mu. L, R2 2.0.5. Mu. L, dd-H2O 2. Mu. L, DNA 1. Mu.L; the amplification procedure was 95℃for 5min; 15s at 94 ℃,15 s at 56 ℃ and 30s×35 cycles at 72 ℃; extending at 72℃for 5min and at 15℃for 1min.
Preferably: criteria for identification: when only one 258bp band can be amplified, judging that the individual is a yellow skin individual; when two bands of 258bp and 482bp in size, respectively, were amplified, the individual was judged to be white skin.
The invention also provides application of the primer group, the kit or the identification method of any one of the primer group and the kit in the breeding industry.
Compared with the prior art, the invention discloses a molecular identification primer group for chicken yellow and white skin color and a method thereof, and the obtained technical effects are that the method for identifying chicken yellow and white skin color by using DNA blood samples of chicken with yellow and white skin color to establish molecular markers is used for identifying chicken yellow and white skin color, so that the identification of chicken yellow and white skin color is not limited by time, and can be accurately identified when needed.
PCR amplification is carried out by using the primers by using different types of chicken individual DNA templates, and when only one 258bp band can be amplified, the individual is judged to be a yellow skin individual; when two bands of 258bp and 482bp in size are amplified, the individual is judged to be white skin; if the amplified band is not obtained, the template DNA is bad in quality, and the DNA should be re-extracted for amplification.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic flow chart provided by the invention.
FIG. 2 is a diagram showing the positions of yellow-white skin color genes on chromosome 24 and the related sequences thereof.
FIG. 3 is a schematic diagram showing PCR electrophoresis of yellow-white skin color of each sample provided by the present invention: lane 1 in the figure is marker DNA molecular weight marker (100 bp-2000 bp ladder); 2. lanes 3 and 4 have 258bp and 482bp bands, which are white skin; 5. 6 has only 258bp band, which is yellow skin; lane 7 is a blank control.
Fig. 4 is an electrophoresis chart (part) for verifying accuracy of the identification method of yellow-white skin color molecules provided by the invention: lane M in the figure: DNA molecular weight marker (100 bp-2000 bp ladder); 32 to 36, 38 to 52, 54 to 57 and 60 are white skin; 63. 66-73, 75-78 are yellow skin.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The embodiment of the invention discloses a chicken yellow and white skin color molecular identification primer group and a method thereof. The flow of the embodiment shows opinion fig. 1.
Example 1
Establishment of chicken yellow and white skin color molecular method
1. Chicken blood sample DNA extraction
DNA in chicken blood samples of known yellow-white skin color was extracted first, 200. Mu.L of buffer was added, and then the DNA concentration was determined, which was diluted to 100 ng/. Mu.L with TE buffer.
2. Primer design
The optimal sequence was selected as a Primer based on the differences between the nucleotide sequence of the W gene found between the 24 th chromosome yellow-white skin color locus sequence of chicken chromosome 6149739 ～ 6150221bp (download address: https:// www.ncbi.nlm.nih.gov/gene/419792, see FIG. 2) and the yellow-white skin color gene published by NCBI, using the universal Primer design software Primer3Plus (https:// www.bioinformatics.nl/cgi-bin/Primer3Plus/prim 3Plus. Cgi). The primer is synthesized by Beijing qingke biotechnology Co.
Wherein: the primer sequences used for PCR amplification of the three primer system were as follows:
(1) Quality control inner index primers capable of being amplified from yellow and white skin colors:
f:5'-TTCCTTCATTTCAAGCGTTTCTG-3', as shown in SEQ ID No. 1;
r1 is 5'-CATTTTCCTGGAGTGCCTTTGT-3', and is shown as SEQ ID No. 2;
(2) Specific primers that only white skin can amplify:
f:5'-TTCCTTCATTTCAAGCGTTTCTG-3', as shown in SEQ ID No. 1;
r2 and 5'-TGGGTTGAGGGTTGGTTGG-3' are shown in SEQ ID No. 3.
3. Three primer system PCR amplification
The PCR reaction was 10. Mu.L (see Table 1, unit of PCR reaction:. Mu.L), and amplification was performed by a universal PCR apparatus.
The amplification procedure was: 95℃for 5min, (94℃for 15s,56℃for 15s,72℃for 30 s). Times.35 cycles, 72℃for 5min,15℃for 1min.
TABLE 1
| Name of the name | Volume of |
| mix | 5μL |
| F1 | 1μL |
| R1 | 0.5μL |
| R2 | 0.5μL |
| dd-H 2 O | 2μL |
| DNA | 1μL |
The PCR amplification products were electrophoretically detected in a 1.5% agarose gel and photographed using a BIO-RAD imaging system.
4. Yellow-white skin tone identification
Judging yellow-white skin color according to electrophoresis bands in a map (see figure 3), applying the primers, performing PCR amplification by using a three-primer system by using different types of individual chicken DNA templates, and judging that the individual is white skin when two bands with the sizes of 258bp (TTCCTTCATTTCAAGCGTTTCTGGGTCCACCTTGTACATGCAGTTGTTCTCACCAGTGACATAGTAGTCTCCTTTATACACAACGTAGCTCACATTGGCATTGTCACTTGGCTCTGAAACAAAACTCAAGCTCTGGTTAAGCTTACATGACATCATATCAAGCTAGACATGTTTAAAGAAATAAACCCATTTGCTGTGACGTTAATCCCCCTTGATTAGAAGTATCTCACTAATCTACAAAGGCACTCCAGGAAAATG shown as SEQ ID No. 4) and 482bp (TTCCTTCATTTCAAGCGTTTCTGGGTCCACCTTGTACATGCAGTTGTTCTCACCAGTGACATAGTAGTCTCCTTTATACACAACGTAGCTCACATTGGCATTGTCACTTGGCTCTGAAACAAAACTCAAGCTCTGGTTAAGCTTACATGACATCATATCAAGCTAGACATGTTTAAAGAAATAAACCCATTTGCTGTGACGTTAATCCCCCTTGATTAGAAGTATCTCACTAATCTACAAAGGCACTCCAGGAAAATGTCTGGACAAGTGAGGACAAAAGGACAAGAACTGGTACCTGCTGCATTTCCTCAGTTCACTGCTAGATCCCAACTACCTACTGGTGCTCTGTAGCTTCTTGGTACTAAGAAAGAAGACTTCTCTAGCCAAGGGAGTGCTTTATGTAGCTCAACAGCTATTAAATTTGGACATTCAAGCCTTGTCAACATTGTCAGTTGATCCAGAGCCAACCAACCCTCAACCCA shown as SEQ ID No. 5) are amplified; if the amplified band is not obtained, the template DNA is bad in quality, and the DNA should be re-extracted for amplification.
Example 2
Reliability verification method for chicken yellow and white skin color molecular identification
1. Collection of verification material
Blood samples 120 were collected from LiangFeng farm-grazing Limited liability company, guangxi Zhuang, autonomous region, nanning, wherein specific skin colors and sexes are shown in Table 2 (sample gender, skin color Table Unit: only).
TABLE 2
2 chicken yellow and white skin color molecular identification method for identifying skin color
Skin tone identification was performed as per the chicken yellow-white skin tone molecular identification method established in example 1.
3 results of molecular identification of chicken yellow-white skin color (partial results are shown in FIG. 4), wherein 60 white skin and 60 yellow skin are identified, and compared with the results of phenotypic identification of chicken yellow-white skin color, the accuracy is 100%
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. A breeding hen yellow-white skin color molecular identification primer set, comprising: the nucleotide sequences of the quality control inner index primer and the white skin specific primer are as follows:
quality control internal index primer:
f:5'-TTCCTTCATTTCAAGCGTTTCTG-3', as shown in SEQ ID No. 1;
r1 is 5'-CATTTTCCTGGAGTGCCTTTGT-3', and is shown as SEQ ID No. 2;
white skin-specific primers:
f:5'-TTCCTTCATTTCAAGCGTTTCTG-3', as shown in SEQ ID No. 1;
r2 and 5'-TGGGTTGAGGGTTGGTTGG-3' are shown in SEQ ID No. 3.
2. A detection kit based on the primer set of claim 1.
3. An identification method based on the primer set of claim 1 or the kit of claim 2, characterized by comprising the steps of:
(1) Extracting genome DNA in a chicken blood sample to be identified;
(2) And (3) performing double PCR amplification, detecting PCR amplification products by electrophoresis, and identifying the skin color of the chicken to be detected according to the existence and the size of the amplified DNA fragment.
4. The assay of claim 3, wherein the PCR amplification system: mix 5. Mu. L, F11. Mu. L, R10.5. Mu. L, R20.5.5. Mu. L, dd-H 2 O2 mu L, DNA mu L; the amplification procedure was 95℃for 5min; the cycle was 15s at 94℃for 15s, 15s at 56℃for 30 s.times.35 at 72℃for 5min and 1min at 72 ℃.
5. The authentication method of claim 4, wherein the criteria for authentication: when only one 258bp band can be amplified, judging that the individual is a yellow skin individual; when two bands of 258bp and 482bp in size, respectively, were amplified, the individual was judged to be white skin.
6. Use of the primer set of claim 1, or the kit of claim 2, or the identification method of any one of claims 3 to 5 in the aquaculture.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5650135A (en) * | 1994-07-01 | 1997-07-22 | The Board Of Trustees Of The Leland Stanford Junior University | Non-invasive localization of a light-emitting conjugate in a mammal |
| CN101619354A (en) * | 2009-07-02 | 2010-01-06 | 华中农业大学 | Method for identifying fast and slow feather molecules of chickens |
| CN101967480A (en) * | 2010-11-04 | 2011-02-09 | 广东智威农业科技股份有限公司 | Molecular marker relevant to chicken skin color and authentication method and application thereof |
| CN103834642A (en) * | 2014-02-14 | 2014-06-04 | 华南农业大学 | Chicken skin color related molecular marker and its application |
| KR20150050499A (en) * | 2013-10-30 | 2015-05-08 | 충남대학교산학협력단 | A method for discriminating of shank color of korean native chicken |
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|---|---|---|---|---|
| US5650135A (en) * | 1994-07-01 | 1997-07-22 | The Board Of Trustees Of The Leland Stanford Junior University | Non-invasive localization of a light-emitting conjugate in a mammal |
| CN101619354A (en) * | 2009-07-02 | 2010-01-06 | 华中农业大学 | Method for identifying fast and slow feather molecules of chickens |
| CN101967480A (en) * | 2010-11-04 | 2011-02-09 | 广东智威农业科技股份有限公司 | Molecular marker relevant to chicken skin color and authentication method and application thereof |
| KR20150050499A (en) * | 2013-10-30 | 2015-05-08 | 충남대학교산학협력단 | A method for discriminating of shank color of korean native chicken |
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| Title |
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