CN114214432A - Molecular marker for identifying melanin diffusion gene locus of chicken and application of molecular marker in assisted breeding - Google Patents
Molecular marker for identifying melanin diffusion gene locus of chicken and application of molecular marker in assisted breeding Download PDFInfo
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- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a molecular marker for identifying a melanin spread gene locus of a chicken and application thereof in assisted breeding, belonging to the field of molecular marker assisted breeding. The molecular marker is located at the 532bp downstream position of the starting codon of the MC1R gene (GenBank: KF379749.1), and G/A base mutation exists. The invention determines the gene type of the polymorphic locus related to the feather color by the feather color test and the detection of the parent and the F1 generation MC1R gene locus gene types, and establishes the MC1R gene homozygote strain by a molecular marker-assisted selection method so as to achieve the effects of purifying the MC1R gene of the pure-line chicken and improving the black feather rate of the commercial chicken, and the method can be used for large-scale seed selection and quickens the breeding process.
Description
Technical Field
The invention relates to the field of molecular marker assisted breeding, in particular to a molecular marker for identifying a melanin diffusion gene locus of a chicken and application thereof in assisted breeding.
Background
In poultry breeding, the feather color is always an important character for breeding and breeding high-quality chickens at home and abroad, and the relation between the feather color phenotype and the genotype is researched, so that products which accord with the preference of consumers can be better cultivated, and the market demand is met. Meanwhile, the feather color gene on the sex chromosome can be used for establishing a male and female identification system, so that the production efficiency is greatly improved. The melanocortin receptor 1(MC1R) gene is closely related to the black feather character of poultry, and MC1R is one of the melanocortin receptor family members, plays a key role in vivo pigment deposition and is identified as a main determinant of melanin synthesis and distribution.
The Yangtai small black chicken is a small-body type laying hen complete set system which is jointly cultivated by Yangzhou university and Jiangsu North agriculture and pasture science and technology limited company, and is a laying hen variety suitable for Jiangsu and peripheral markets thereof. The complete set has the appearance characteristics of black feather and green feet, and the production characteristics of producing light pink shell eggs, bright eggshells, good meat quality, low feed intake and the like, and also has the advantage of high culled chicken residual value and stronger competitive advantage. However, during the breeding of the cultivars, it was found by the combining ability measurement that: about 7.5 percent of the Yangtai small black chicken production performance optimal combined chicken flocks are non-black feather chickens, and the consistency of the variety characters is influenced to a certain extent. At present, no method for screening the black feather color gene exists.
Disclosure of Invention
The invention aims to provide a molecular marker for identifying a chicken melanin diffusion gene locus and application thereof in auxiliary breeding, so as to solve the problems in the prior art, obtain polymorphic loci related to feather color through genotype detection, determine the gene type, and establish an MC1R gene homozygous strain by using a molecular marker-assisted selection method, so as to achieve the effects of purifying pure line chicken MC1R gene homozygous rate and improving commercial chicken black feather rate, and can be used for large-scale seed selection and quicken the breeding process.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a molecular marker for identifying a melanin spread gene locus of a chicken, which is positioned at the 532bp downstream of an initiation codon of an MC1R gene (GenBank: KF379749.1) and has G/A base mutation.
The invention also provides a product for identifying the melanin spread gene locus of the chicken, which comprises the molecular marker.
Preferably, the product comprises a kit, a reagent or a biochip.
The invention also provides application of the molecular marker in chicken genetic breeding.
Preferably, the method is applied to auxiliary selection of the chicken black feather character.
The invention also provides a method for selecting the chicken black feather character by using the molecular marker in an auxiliary way, which comprises the following steps:
(1) obtaining chicken DNA to be detected, and amplifying to obtain a target gene by taking the obtained DNA as a template;
(2) sequencing the obtained amplification product, judging the genotype, and selecting a cock and a hen with polymorphic sites of AA genotypes according to the genotype to breed to obtain the black feather chicken.
Preferably, in step (1), the amplification primers are:
upstream primer (SEQ ID NO: 1): 5'-CATCCCCTCTGCCTCGTGAC-3', respectively;
downstream primer (SEQ ID NO: 2): 5'-ACCTACTACCGCAACAACGC-3' are provided.
Preferably, the amplification reaction system is: mu.L of 10. mu.M forward primer 0.5. mu.L, 10. mu.M reverse primer 0.5. mu.L, 2 XTaq mix 10. mu.L, water 8. mu.L, 200 ng/. mu.L DNA 1. mu.L.
Preferably, the PCR reaction procedure is pre-denaturation at 95 ℃ for 5 minutes; 30 seconds at 95 ℃, 30 seconds at 56 ℃, 50 seconds at 72 ℃ and 37 cycles; 72 ℃ for 10 minutes; and kept at 4 ℃ until electrophoresis.
The invention discloses the following technical effects:
the E locus is an important expanded locus for controlling feather and hair color of birds and mammals, directly influences the distribution of eumelanin and melanophil, and determines that G/A gene mutation exists at a 532bp position downstream of an initiation codon of MC1R gene through screening. The invention confirms the gene type of the polymorphic locus related to the feather color through the feather color test and the detection of the parent and the gene type of the F1 generation MC1R gene locus, and establishes the MC1R gene homozygote strain by utilizing the molecular marker-assisted selection, so as to realize the effects of purifying the homozygote of the MC1R gene of the pure chicken and improving the black feather rate of the commercial chicken, and further lay a foundation for large-scale breeding and accelerating the breeding process.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is an agarose gel electrophoresis of PCR amplification products;
FIG. 2 shows the results of parental cross tests with different genotypes; a is a sequencing peak picture corresponding to SNP locus homozygote genotype AA of male parent MC1R gene; b is a sequencing peak diagram corresponding to SNP locus heterozygote genotype AG of the male parent MC1R gene; c is a sequencing peak picture corresponding to SNP locus heterozygote genotype GG of female parent MC1R gene; d is a sequencing peak picture corresponding to the SNP site heterozygote genotype AG of the MC1R gene of the F1 generation; e is a sequencing peak picture corresponding to SNP locus heterozygote genotype GG of the MC1R gene of the F1 generation;
FIG. 3 is the peak pattern of MC1R locus of homozygote and heterozygote male parent and F1 generation and the correlation analysis chart of the feather color character thereof in the test of test cross.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
1. Test specimen
The test sample comprises 15 pure black feather cocks (bred by hybridization of black feather yuan chickens and royal chickens), 15 luo island red pure hens (from Beijing Zhongnong bang-like laying hen breeding Limited liability company) and all test animals provided by Jiangsu North agricultural and big farming and pasturing science and technology Limited.
2. Test method
(1) Cross test for feather color
The test selects 15 black feather cocks and 15 Luo island red hens, and is operated according to the conventional artificial insemination method according to the ratio of 1:1 for insemination, wherein the insemination is continuously performed on the first two days, hatching eggs are collected and numbered on the third day, the hatching eggs are collected for 14 days and are hatched in two batches, and the hatching eggs with the same number are filled into the same hatching bag when the eggs are landed. The feathering of the F1 chicks of each group was observed and recorded at the time of hatching (see Table 1), and blood was collected.
TABLE 1 parental and F1 generation gender and feather trait statistics
(2) DNA template
0.2mL of wing vein blood of the test parent cock and hen was collected, and blood DNA templates of the parent and F1 generation chicks were extracted using a blood DNA extraction kit.
(3) PCR polymerase chain reaction and product detection
The extracted DNA was used as a template in PCR and PCR amplification was carried out using the provided PCR kit, and the reaction system and reaction conditions are detailed in Table 2 and Table 3, respectively.
An amplification primer:
an upstream primer: 5'-CATCCCCTCTGCCTCGTGAC-3', respectively;
a downstream primer: 5'-ACCTACTACCGCAACAACGC-3' are provided.
TABLE 2 PCR reaction System for MC1R Gene
TABLE 3 PCR amplification procedure for the MC1R Gene
mu.L of the PCR amplification product was subjected to 1.5% agarose gel electrophoresis, and the product was sequenced as a single band of 692bp in size (see FIG. 1), whereby base information of 532bp downstream of the start codon of MC1R gene was obtained from different individuals. This site is the SNP site to be detected, and the base sequences on the left and right sides are as follows: GGTGAGCGTCAGCAACCTGGCC (G/A) AGACGCTCTTCATGCT.
3. The association analysis of the polymorphic site and the black feather character
The peak diagrams of the SNP sites of all parents and partial F1 generation chicks in the cross-testing test are shown in figure 2, and the sequencing peaks of the male parent corresponding to the base of the site are shown in figures 2A and 2B. FIG. 2A represents AA genotype peak (base of arrow is A), SNP site peak is A peak, which is homozygote; FIG. 2B represents the AG genotype (bases of red arrow are A and G), and the presence of both A and G peaks at this site indicates that the tested individuals are heterozygotes and the genotype is AG. The sequencing peak of the female parent is shown in figure 2C, the SNP site peak of the individual detected by the female parent is shown as G peak (the base of the arrow is G), and the individual SNP site peak is homozygote. The sequencing peak maps of the F1 generation are shown in FIGS. 2D and 2E, and FIG. 2D represents the AG genotype, which shows that the individuals of the F1 generation are heterozygotes and the genotype is AG; in addition, the F1 individuals also had a G peak, which was homozygote.
As can be seen from the sequencing peak maps of the parent and the F1 generation (see figure 3 in detail), when the target base sequence of the parent is A, and the peak map is a single peak (homozygote), the F1 generation of the parent has the black feather character; the target basic sequence of the parent is A/G, when the peak image is double peak (heterozygote), the F1 generation feather color of the parent has character separation phenomenon, namely black feather: golden feather is 1: 1. therefore, the MC1R gene polymorphism site has obvious association with the feather color character and can be used as a molecular marker-assisted breeding method. The method is simple and convenient, is simple to operate, is not limited by genetic background, has wide application range, and can identify the genotypes of different individuals at the site of the melanin spreading gene (E gene).
4. Test for verifying the polymorphic site and the Black feather Property
Collecting 578 parts of blood of pure black-feather cocks, detecting polymorphism of sites of the pure black-feather cocks, respectively selecting 30 feathers of individuals with AA and AG genotypes, respectively carrying out semen deposition on a group A cock (AA genotype) and a group B cock (AG genotype) and a 60 Luo island red hen (1: 1), continuously collecting hatching eggs, immediately hatching the eggs after 14 days, loading the hatching eggs of the same hen into the same hatching bag when the eggs are landed at the age of 19 embryo, and counting phenotype and gender of each group of chicks when the chicks are hatched.
TABLE 4 feather color and gender statistics of the offspring chicks of the different genotypes of the cock
From the feathering of the offspring of A, B groups, it is known that the offspring of AA genotype cock is black feather, while the offspring of AG genotype cock has separated feathers (black feather: golden feather: 1), and the MC1R gene polymorphism site is emphasized as the molecular marker of black feather character. In addition, research results show that the black feather of the black chicken is autosomal inheritance of a melanin diffusion gene (E gene), and has an epistatic effect on a honeysuckle feather gene(s) of the Luo island red chicken.
5. Collecting blood of pure black-feather chickens, detecting the genotype of the pure black-feather chickens, and selecting the cocks and hens with AA genotype as site polymorphism by combining the genotypes of the 578 parts of the pure black-feather chickens to breed a new strain.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
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<120> molecular marker for identifying melanin spread gene locus of chicken and application thereof in assisted breeding
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Claims (9)
1. A molecular marker for identifying a melanin spread gene locus of a chicken is characterized in that the molecular marker is positioned at the 532bp downstream of an initiation codon of an MC1R gene and has a G/A base mutation.
2. A product for identifying a melanin spreading gene locus of a chicken, comprising the molecular marker of claim 1.
3. The product of claim 2, wherein the product comprises a kit, a reagent, or a biochip.
4. Use of the molecular marker of claim 1 in genetic breeding of chicken.
5. The use as claimed in claim 4 for the assisted selection of the chicken black feather trait.
6. A method for selecting chicken black feather trait by using the molecular marker of claim 1, comprising the steps of:
(1) obtaining chicken DNA to be detected, and amplifying to obtain a target gene by taking the obtained DNA as a template;
(2) sequencing the obtained amplification product, judging the genotype, and selecting a cock and a hen with polymorphic sites of AA genotypes according to the genotype to breed to obtain the black feather chicken.
7. The method of claim 6, wherein in step (1), the amplification primers are:
an upstream primer: 5'-CATCCCCTCTGCCTCGTGAC-3', respectively;
a downstream primer: 5'-ACCTACTACCGCAACAACGC-3' are provided.
8. The method of claim 6, wherein the amplification reaction system is: mu.L of 10. mu.M forward primer 0.5. mu.L, 10. mu.M reverse primer 0.5. mu.L, 2 XTaq mix 10. mu.L, water 8. mu.L, 200 ng/. mu.L DNA 1. mu.L.
9. The method of claim 6, wherein the PCR reaction program is pre-denatured at 95 ℃ for 5 minutes; 30 seconds at 95 ℃, 30 seconds at 56 ℃, 50 seconds at 72 ℃ and 37 cycles; 72 ℃ for 10 minutes; and kept at 4 ℃ until electrophoresis.
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| CN116287287A (en) * | 2022-12-13 | 2023-06-23 | 华南农业大学 | Molecular detection method applied to small Bai Ji green foot character and application thereof |
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| CN110029175A (en) * | 2019-04-22 | 2019-07-19 | 广西大学 | A kind of breeding method using molecular labeling quickly breeding black silk plumage Gallus domesticlus brisson |
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| CHICKEN(GRCG6A): "rs314881228", 《ENSEMBL》 * |
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| CN116287287A (en) * | 2022-12-13 | 2023-06-23 | 华南农业大学 | Molecular detection method applied to small Bai Ji green foot character and application thereof |
| CN116287287B (en) * | 2022-12-13 | 2023-11-10 | 华南农业大学 | Molecular detection method applied to small Bai Ji green foot character and application thereof |
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