CN115058522A - Molecular marker for identifying broiler varieties containing white rocco blood sources and application - Google Patents
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Abstract
The invention discloses a molecular marker for identifying broiler breeds containing belokch blood margin and application thereof, wherein the molecular marker is an SNP (single nucleotide polymorphism) site located at 16131bp of a mitochondrial genome, and has A/G polymorphism, when the base at the position is A, the broiler breeds do not contain the belokch blood margin, and when the base at the position is G, the broiler breeds containing the belokch blood margin are obtained. The method is simple to operate, easy to operate in a laboratory, convenient for basic popularization and use and wide in market application prospect, and in addition, the detection kit developed based on the method can generate considerable economic benefits and good social values.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a molecular marker for identifying broiler varieties with white roche blood relationship and application thereof.
Background
Most local varieties in China are colored feathers, including yellow feather, jute feather, hemp feather, brown feather, red feather, black feather and the like, and are collectively called yellow-feather broilers. Yellow-feathered broilers are the unique local chicken species in China, the sale mode is mainly live chickens, and the annual output is about 45 hundred million. The yellow-feathered broilers are divided into 3 types of fast, medium and high-quality types according to different growth speeds. The development requirements of 'large-scale cultivation, centralized slaughtering, cold chain transportation and cold fresh marketing' of poultry industry in China are that slaughtering processing type varieties are pushed out in many times. Before being sold in the market, broilers of different grades can be distinguished through appearance such as feather color, and after slaughtering, the broilers are difficult to distinguish only by carcass appearance. The production efficiency of yellow-feather broilers in China is low generally. The white loxk is a female parent of fast large white feather broilers, integrates the functions of producing more eggs and increasing the growth speed, and greatly improves the production speed of filial generation. At present, most of fast yellow-feathered broiler varieties cultivated in China use white rook chickens as female parents. Therefore, many bad marketers in the market are good, and the hybrid broiler chickens are sold as pure local chickens, so that the market is disordered. The occurrence of these phenomena not only hinders the upgrade of the broiler industry structure, but also indirectly causes the lack of integrity in the broiler industry, so that the whole industry faces the trust crisis. Therefore, a fast, accurate and easy-to-operate technique for identifying chicken products with different grades is needed.
At present, two methods of morphological identification and molecular biological identification are mainly used for identifying the varieties of animals and plants. The morphological identification is easily affected by the environment and seasons, and has the advantages of long identification time, large workload, and inaccurate and scientific detection. The molecular identification technology can overcome the defects of morphological identification, has the advantages of rapidness and no environmental influence, and is gradually used for variety identification.
Early molecular marker identification methods were as follows: markers such as Random Amplified Polymorphism (RAPD), Restriction Fragment Length Polymorphism (RFLP), etc., have disadvantages such as unstable typing, excessive genotype, no discrimination, non-co-dominance, etc., and thus they are only studied and are not basically included in the variety identification standards. Through the development and practice of variety identification for decades, only a Single Sequence Repeat (SSR) labeling method and a Single Nucleotide Polymorphism (SNP) labeling method are reserved. Single Nucleotide Polymorphism (SNP) refers to a polymorphism in DNA sequence caused by a variation of a single nucleotide at the genomic level. As the SNP has the advantages of high density, good compatibility, high flux, high speed, low cost, high association degree with phenotypic traits and the like, the SNP is widely applied to variety DNA identity identification as a molecular marker of a class of crops and domestic animals. However, no research report for identifying the broiler varieties with the white rocco blood margin by using an SNP molecular marker method is found so far.
Disclosure of Invention
One of the purposes of the invention is to provide a molecular marker for identifying broiler varieties with white roche blood relationship aiming at the defects in the prior art.
Another object of the present invention is to provide a primer for detecting the above molecular marker.
The third object of the present invention is to provide the use of the above molecular marker.
The fourth object of the present invention is to provide a detection method using the above molecular marker.
The purpose of the invention is realized by the following technical scheme:
a molecular marker for identifying broiler varieties with alborok blood relationship is an SNP (Single nucleotide polymorphism) site located at 16131bp of a mitochondrial genome, and has A/G polymorphism, when the base at the position is A, the varieties do not contain alborok blood relationship, and when the base at the position is G, the varieties are broiler varieties with alborok blood relationship.
A method for identifying broiler varieties with white roch blood relationship comprises the steps of extracting chicken genome DNA, detecting a base sequence at 16131bp of a mitochondrial genome, and determining that the broiler varieties do not contain white roch blood relationship when the base at the position is A and the broiler varieties with white roch blood relationship when the base at the position is G.
The extraction of the chicken genomic DNA of the invention can be carried out by extracting the chicken genomic DNA to be identified in a conventional manner, for example, extracting the chicken genomic DNA from blood, chicken, feather, tissue or egg.
A method for identifying broiler varieties with white rocco blood margins is characterized in that when the base at 16131bp of a mitochondrial genome is G, a restriction enzyme cutting site of BanII endonuclease of GGGCT/C can be formed, and when the base is A, GGACTC cannot be restricted by the BanII endonuclease.
The invention also provides a primer pair of the molecular marker for identifying the broiler varieties of the white roche blood relationship, wherein the primer pair comprises the following components in parts by weight:
a forward primer: 5'-GCAGCCTCAGTCCTCATCC-3'
Reverse primer: 5'-ACTAGAGCTTGTGCGTGGG-3' are provided.
The invention also provides a molecular marker for identifying the broiler varieties with the alburn kindred, wherein the primer pair is used for amplifying the genome DNA of the chicken varieties to be identified, and then the amplified products are subjected to enzyme digestion by using the restriction enzyme BanII to obtain one 455bp amplified fragment which is the broiler varieties without the alburn kindred, and the broiler varieties with the alburn kindred of which the two amplified fragments are 346bp and 109bp are the broiler varieties with the alburn kindred.
The invention also provides a kit containing the primer pair and used for identifying the genotype of the broiler varieties with the white roche blood relationship.
The invention also provides application of the molecular marker or the primer pair or the kit in identification of white roche blood related broiler varieties.
The invention also provides application of the molecular marker or the primer pair or the kit in screening of white rocco related broiler varieties.
The invention also provides application of the molecular marker or the primer pair or the kit in screening broiler varieties without the white roche blood margin.
The invention also provides a method for identifying the broiler varieties with the albuck kindred, which comprises the steps of amplifying the genome DNA of the chickens to be identified through the primer pair, then carrying out enzyme digestion on the amplification products by using the restriction enzyme BanII to obtain one 455bp amplification fragment which is the broiler variety without the albuck kindred, and obtaining 346bp and 109bp amplification fragments which are the broiler varieties with the albuck kindred.
In a specific embodiment, the invention also provides a rapid identification method of the genotype of the broiler variety of the beloka kindred, which comprises the following steps:
1) extracting genome DNA in chicken to be detected;
2) carrying out PCR amplification reaction by using the DNA extracted in the step 1) as a template and using the primer pair disclosed by the invention;
3) the PCR reaction system in the step 2) is as follows: 2 XPCR Mix (Nanjing Nuojingzu biology, Ltd.) 25. mu.L, 10. mu. mol/L forward primer 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, ultrapure water 21. mu.L;
4) the PCR reaction procedure in the step 2) is as follows: 35 cycles at 95 deg.C for 5min, (95 deg.C for 30s, 60 deg.C for 30s, and 72 deg.C for 60s), and 72 deg.C for 10 min;
5) and (3) carrying out enzyme digestion on the amplified product by using restriction enzyme BanII, carrying out agarose gel electrophoresis detection on the enzyme digestion product, wherein the electrophoresis result shows that 2 bands of the broiler varieties are related to the white roc blood, and only 1 band of the broiler varieties is related to the broiler varieties which do not contain the white roc blood.
6) The enzyme digestion reaction system in the step 5) is as follows: the reaction system comprises 43 mu L of PCR product, 5 mu L of 10 Xbuffer solution and 2 mu L of BanII enzyme (5U), the samples are mixed evenly and then centrifuged, and the mixture is placed in a PCR instrument for incubation for 1h at 37 ℃.
Has the advantages that:
the method for identifying the broiler varieties of the Bairoche blood margin utilizes the difference of mitochondrial sequences, and has the advantage that the broiler varieties of the Bairoche blood margin can be identified quickly and accurately. In addition, the method is simple to operate, easy to operate in a laboratory, convenient for basic popularization and use and wide in market application prospect, and the detection kit developed based on the method can generate considerable economic benefits and good social values.
Drawings
FIG. 1: and (3) carrying out molecular identification on the PCR amplified fragment electrophoresis result of the broiler varieties with the white roche blood margin.
FIG. 2: sequencing peak pattern at base mutation position.
FIG. 3: agarose electrophoresis results after enzyme digestion of BanII.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1
Establishment of molecular detection method for broiler variety of 1 Bairoc kinder
1.1 primer design
The applicant carries out full-length sequencing on mitochondrial genomes of broiler variety groups with or without the Bailock blood margin, finds that the two groups have variation at a 16131bp position, and designs a primer to amplify the candidate site.
A forward primer: 5'-GCAGCCTCAGTCCTCATCC-3'
Reverse primer: 5'-ACTAGAGCTTGTGCGTGGG-3'
1.2PCR amplification
The PCR reaction system is as follows:
2 XPCR Mix (Nanjing NuoZan Bio Inc.) 25. mu.L, 10. mu. mol/L forward primer 1. mu.L each, 50-100. mu.g/ml template DNA 2. mu.L, 21. mu.L of ultrapure water.
The PCR reaction program is: 5min at 95 deg.C, 35 cycles (30 s at 95 deg.C, 30s at 60 deg.C, 60s at 72 deg.C) and 10min at 72 deg.C.
1.3 electrophoretic detection
The PCR product was detected by 1.3% agarose gel electrophoresis, and the results are shown in FIG. 1. The detection result shows that the PCR products are all specific 455bp fragments, the PCR products are sent to Shanghai biological Limited company for sequencing, the sequencing sequences are compared to screen variation sites, an (A-G) variation site is found at 148bp, a sequencing peak diagram is shown in figure 2, wherein the position of the variation site at 148bp of the amplified fragment on a chicken mitochondrial genome is 16131bp (the reference sequence is NC-007235), and only when the base of the site is G, the enzyme cutting site of the BanII endonuclease of GGGCT/C can be formed, and the site is a white rock-edge broiler variety; when the basic group is A, GGACTCC can not be cut by BanII endonuclease, and the broiler chicken variety does not contain white rocco. Namely, the broiler varieties of the Bairoc kindred are controlled by a single gene locus, and the broiler varieties of the Bairoc kindred can be distinguished by utilizing the mutation locus.
Because the mitochondrial genome sequence does not undergo homologous recombination, the gene type of heterozygote does not exist, the site base is AA homozygote without enzyme cutting site when the site base is A, and the site base is GG homozygote with enzyme cutting site when the site base is G. The PCR product is cut by BanII enzyme, the reaction system is 43 mu L of PCR product, 5 mu L of 10 Xbuffer solution and 2 mu L of BanII enzyme (5U), the samples are evenly mixed and centrifuged, and the mixture is placed in a PCR instrument for incubation for 1h at 37 ℃. After the reaction was completed, detection was performed by 2.0% agarose gel electrophoresis. The AA type electrophoresis detection has only one 455bp strip, the GG type is a homozygote type with a restriction enzyme cutting site, and the electrophoresis detection has 346bp and 109bp strips, and the results are shown in a figure 3 (wherein 1-8 are broiler varieties without white roc blood relationship, and 9-16 are broiler varieties with white roc blood relationship).
Example 2
This example performed a population expansion validation of white rocco-cut broiler breeds using the method and primers described in example 1. Broiler breeds without white roc blood relationship (white ear yellow chicken, camellia chicken, Henan fighting chicken, Gushi chicken, Langshan chicken, Xianju chicken and Beijing fatty chicken) come from the national local chicken breeder gene bank (Jiangsu), and each breed is 30; 3 white rocco kindred broiler breeds come from 3 large-scale breeding enterprises. 30 per variety. The GG type proportion of the molecular marker in the broiler varieties without the Bailock blood margin accounts for 80%, and the GG type proportion of the molecular marker in the broiler varieties without the Bailock blood margin accounts for AA types. Further verifies that the SNP locus can effectively and reliably identify the broiler varieties of the Bairocco blood margin (as shown in Table 1).
Table 1 shows the genotype distribution of the broiler varieties with white roche blood relationship detected by the molecular markers of the invention.
Claims (10)
1. The molecular marker is an SNP locus at 16131bp of a mitochondrial genome, and has A/G polymorphism, when the base at the position is A, the variety does not contain the white roche blood margin, and when the base at the position is G, the variety is the broiler variety of the white roche blood margin.
2. A method for identifying broiler varieties with white roch blood margin is characterized in that base sequences at 16131bp positions of a mitochondrial genome are detected for extracting chicken genome DNA, when bases at the positions are A, the broiler varieties without white roch blood margin are detected, and when bases at the positions are G, the broiler varieties with white roch blood margin are detected.
3. The method of claim 2, wherein extracting chicken genomic DNA is extracting genomic DNA from chicken blood, chicken meat, feathers, tissues, or eggs.
4. A molecular marker primer pair for identifying broiler varieties of white roche blood margin is disclosed, wherein:
a forward primer: 5'-GCAGCCTCAGTCCTCATCC-3'
Reverse primer: 5'-ACTAGAGCTTGTGCGTGGG-3' are provided.
5. A molecular marker for identifying broiler varieties with alblock kindred is characterized in that the genomic DNA of a chicken to be detected is amplified through the primer pair shown in claim 4, then the amplified product is subjected to enzyme digestion by using restriction enzyme BanII, one 455bp amplified fragment is obtained and is a broiler variety without alblock kindred, and 346bp and 109bp amplified fragments are obtained and are broiler varieties with alblock kindred.
6. A kit comprising the primer set according to claim 4.
7. Use of the molecular marker of claim 1, the primer pair of claim 4 or the kit of claim 6 for identifying white roche related broiler varieties.
8. Use of the molecular marker of claim 1, the primer pair of claim 4 or the kit of claim 6 for screening white roche-related broiler breeds or screening white roche-free broiler breeds.
9. A method for identifying broiler varieties with alburn kindred is characterized in that genomic DNA of chickens to be identified is amplified through a primer pair shown in claim 4, then an amplification product is subjected to enzyme digestion by using a restriction enzyme BanII, one 455bp amplification fragment which is a broiler variety without alburn kindred is obtained, and 346bp and 109bp amplification fragments which are broiler varieties with alburn kindred are obtained.
10. A method for screening broiler breeds with or without the white roch margin is characterized in that genomic DNA of chickens to be screened is amplified through a primer pair shown in claim 4, then an amplification product is subjected to enzyme digestion by using a restriction enzyme BanII, one 455bp amplification fragment which is a broiler breed without the white roch margin is obtained, and two 346bp and 109bp amplification fragments which are broiler breeds with the white roch margin are obtained.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116622859A (en) * | 2023-05-30 | 2023-08-22 | 江苏省家禽科学研究所 | Molecular biological identification method and application of pure-bred tea-flower chicken |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116622859A (en) * | 2023-05-30 | 2023-08-22 | 江苏省家禽科学研究所 | Molecular biological identification method and application of pure-bred tea-flower chicken |
| CN116622859B (en) * | 2023-05-30 | 2023-11-14 | 江苏省家禽科学研究所 | Molecular biological identification method and application of pure-bred tea-flower chicken |
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