Different small white broiler chicken molecular biology identification method and application

Abstract

A molecular biology identification method and application of different small white-feathered broilers, the molecular marker is that the genomic DNA of the small white-feathered broiler is amplified by using the sequence shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the alleles are detected by capillary electrophoresis, the small white-feathered broiler containing 205 bp alleles is F type small white chicken, and the small white-feathered broiler not containing 205 bp alleles is M type small white chicken; the present application identifies different small white-feathered broilers by using the difference of the alleles, and the advantage lies in that the F type and M type small white chicken can be quickly and accurately identified. Moreover, the method is simple in operation, easy for laboratory operation, convenient for grass-roots promotion and use, has a wide market application prospect, in addition, the detection kit developed based on the method of the present application can produce considerable economic benefits and good social value.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and in particular relates to a molecular biological identification method and its application for different small white-feathered broiler chickens. Background Technology

[0002] In my country, broiler chickens are mainly divided into three categories: white-feathered broilers (also known as fast-growing white-feathered broilers), yellow-feathered broilers, and small white-feathered broilers. Among them, small white-feathered broilers are bred through crossbreeding of parent roosters (white-feathered broilers) with high-yielding brown-shelled laying hens. This model effectively combines the dual advantages of the "rapid" growth of white-feathered broilers and the "high" egg production of commercial brown-shelled laying hens. It features low breeding costs, high production efficiency, strong adaptability, and suitability for slaughtering and processing, thus rapidly expanding its market share and capturing a portion of the market originally belonging to yellow-feathered broilers.

[0003] Currently, the annual output of small white-feathered broiler chickens is approximately 2.5 billion, making them an indispensable and important category in my country's broiler industry. Driven by consumption upgrades and improved cold chain logistics, the market entry of chilled broiler chickens has accelerated significantly. Among them, small white-feathered broiler chickens, with their higher slaughtering and processing efficiency, better end-user adaptability, and cost-effectiveness, will see their market advantages become increasingly prominent. The surge in market demand has led to a shortage of parent stock roosters. Some companies are using female parent stock roosters as a substitute, producing small white-feathered broiler chickens (hereinafter referred to as M-type small white chickens, such as...) produced from these female roosters. Figure 2 As shown, the growth rate and feed conversion efficiency of F-type broiler chickens produced by breeding roosters are lower than those of small white-feathered broilers (hereinafter referred to as F-type broilers). Figure 1 As shown in the figure, this has caused some market chaos.

[0004] Therefore, there is an urgent need for a fast, accurate, and easy-to-operate technology to identify different small white-feathered broiler chickens, so as to ensure the orderly, healthy, and stable development of my country's small white-feathered broiler chicken industry. Summary of the Invention

[0005] One of the objectives of this invention is to provide a molecular biological identification method for different small white-feathered broiler chickens, addressing the shortcomings of existing technologies.

[0006] A second objective of this invention is to provide primers for detecting the aforementioned molecular markers.

[0007] A third objective of this invention is to provide the use of the aforementioned molecular markers.

[0008] The fourth objective of this invention is to provide a detection method utilizing the aforementioned molecular markers.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A molecular marker for identifying different small white-feathered broiler chickens, wherein the molecular marker is obtained by amplifying the genomic DNA of small white-feathered broiler chickens using the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and detecting alleles by capillary electrophoresis; chickens containing a 205 bp allele are classified as type F small white-feathered chickens; chickens without a 205 bp allele are classified as type M small white-feathered chickens.

[0011] This invention also provides a primer pair for identifying molecular markers in different small white-feathered broiler chickens, wherein:

[0012] Forward primer (SEQ ID NO.1): 5'-AACTTGGTAAGGGAATTCCCT-3'

[0013] Reverse primer (SEQ ID NO.2): 5'-GAATTAGTGCCCAGCTGTGCT-3'.

[0014] The present invention also provides a kit containing the above-mentioned primer pairs for identifying different genotypes of small white-feathered broiler chickens.

[0015] The present invention also provides the application of the above-mentioned molecular markers or primers or kits in the identification of different small white-feathered broiler chickens.

[0016] The present invention also provides the application of the above-mentioned molecular markers or primers or kits in screening small white-feathered broiler chickens.

[0017] The present invention also provides a method for identifying F-type and M-type broiler chickens. The genomic DNA of the broiler chicken to be tested is amplified using primer pairs SEQ ID NO.1 and SEQ ID NO.2, and alleles are detected by capillary electrophoresis. Chickens containing 205 bp alleles are classified as F-type broiler chickens; chickens not containing 205 bp alleles are classified as M-type broiler chickens.

[0018] The present invention also provides a method for screening F-type and M-type broiler chickens. The genomic DNA of the broiler chicken to be tested is amplified by primer pairs SEQ ID NO.1 and SEQ ID NO.2, and alleles are detected by capillary electrophoresis. Chickens containing 205 bp alleles are classified as F-type broiler chickens; chickens not containing 205 bp alleles are classified as M-type broiler chickens.

[0019] In one specific embodiment, the present invention also provides a more specific method for rapid identification or screening of small white-feathered broiler chickens, comprising the following steps:

[0020] 1) Extract genomic DNA from the small white-feathered broiler chickens to be tested;

[0021] 2) Using the DNA extracted from small white-feathered broiler chickens in step 1) as a template, PCR amplification was performed using the primer pair described in this invention;

[0022] 3) After the reaction, the PCR products were genotyped using an ABI 3730 analyzer. Those containing the 205 bp allele were classified as F-type white chickens; those not containing the 205 bp allele were classified as M-type white chickens.

[0023] The PCR reaction system in step 2) of this invention is as follows: 25 μL of 2×PCR Mix (Nanjing Novizan Biotechnology Co., Ltd.), 1 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

[0024] The PCR reaction program in step 2) of this invention is as follows: 95℃ for 5 min, (95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s) for 35 cycles, and 72℃ for 10 min.

[0025] The extraction of genomic DNA from the chicken breed to be tested in this invention can be carried out using common methods. The extraction site can be a location commonly used in the field, such as, but not limited to, chicken blood, chicken meat, feathers, etc.

[0026] The small white-feathered broiler chickens to be tested in this invention can be distinguished by whether the population contains a 205 bp allele.

[0027] Beneficial effects:

[0028] This invention utilizes allele differences to identify different small white-feathered broiler chickens. Its advantages include the ability to quickly and accurately distinguish between F-type and M-type small white chickens. Furthermore, the method is simple to operate, easy to perform in laboratories, and readily applicable at the grassroots level, possessing broad market application prospects. In addition, the detection kit developed based on this invention can generate considerable economic benefits and significant social value. Attached Figure Description

[0029] Figure 1 Production model diagram of type F white chickens.

[0030] Figure 2 M-shaped white chicken production model diagram.

[0031] Figure 3 Peak diagram of 205 bp alleles. Detailed Implementation

[0032] The following examples are used to illustrate the present 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 all commercially available products.

[0033] Example 1

[0034] Identification of different small white-feathered broiler chickens

[0035] 1.1 Sample Collection

[0036] Blood samples from F-type and M-type white chickens were collected from small-scale broiler chicken production enterprises in Henan, Shandong, and Anhui provinces. Thirty samples of each type of F-type and M-type white chicken were collected from each enterprise. Genomic DNA was extracted from these samples for subsequent experiments.

[0037] 1.2 PCR amplification

[0038] PCR amplification was performed using primers designed according to this invention.

[0039] Forward primer: 5'-AACTTGGTAAGGGAATTCCCT-3'; FAM fluorescent label added to the 5' end.

[0040] Reverse primer: 5'-GAATTAGTGCCCAGCTGTGCT-3'.

[0041] The PCR reaction system is as follows:

[0042] 2×PCR Mix (Nanjing Novizan Biotechnology Co., Ltd.) 25 μL, 1 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

[0043] The PCR reaction program was as follows: 95℃ for 5 min, (95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s) for 35 cycles, and 72℃ for 10 min.

[0044] 1.3 Capillary electrophoresis detection

[0045] After the reaction, the PCR products were genotyped using an ABI 3730 analyzer. The peak diagram of individuals containing the 205 bp allele is shown below. Figure 3 As shown.

[0046] 1.4 Allele Statistics

[0047] The allele distribution of different groups is shown in Table 1. According to Table 1, those containing the 205 bp allele are all F-type white chickens, and those not containing the 205 bp allele are all M-type white chickens.

[0048] Table 1. Allele distribution of different small white-feathered broiler chickens

[0049]

[0050] Example 2

[0051] 2. Genotyping of F-type and M-type white chicks as parents

[0052] 2.1 Sample Collection

[0053] For the identified F-type and M-type white chickens, blood was collected from 30 paternal and 30 maternal chickens, and genomic DNA was extracted.

[0054] 2.2 PCR amplification

[0055] PCR amplification was performed using primers designed according to this invention.

[0056] Forward primer: 5'-AACTTGGTAAGGGAATTCCCT-3'; FAM fluorescent label added to the 5' end.

[0057] Reverse primer: 5'-GAATTAGTGCCCAGCTGTGCT-3'.

[0058] The PCR reaction system is as follows:

[0059] 2×PCR Mix (Nanjing Novizan Biotechnology Co., Ltd.) 25 μL, 1 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

[0060] The PCR reaction program was as follows: 95℃ for 5 min, (95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s) for 35 cycles, and 72℃ for 10 min.

[0061] 2.3 Capillary electrophoresis detection

[0062] After the reaction, the PCR products were genotyped using an ABI 3730 analyzer. The peak diagram of individuals containing the 205 bp allele is shown below. Figure 3 As shown.

[0063] 2.4 Allele Statistics

[0064] The allele distribution of different groups is shown in Table 2. According to Table 2, only the F-type white chicken father contains the 205 bp allele and passes it on to the next generation of F-type white chickens; neither the M-type white chicken father nor mother contains the 205 bp allele; and the offspring M-type white chickens will not contain the 205 bp allele.

[0065] Table 2. Genotype distribution of different small white-feathered broiler chicken parents (paternal and maternal).

[0066]

[0067] The above results further verify the high efficiency of the present invention on different small white-feathered broiler chickens.

Claims

1. A molecular marker for distinguishing between F-type and M-type small white-feathered broiler chickens, characterized in that, The molecular markers were used to amplify the genomic DNA of small white-feathered broiler chickens using the sequences shown in SEQ ID NO.1 and SEQ ID NO.

2. Alleles were detected by capillary electrophoresis. Chickens containing a 205 bp allele were classified as F-type small white chickens, while those without a 205 bp allele were classified as M-type small white chickens.

2. A primer pair for identifying molecular markers in F-type and M-type small white-feathered broiler chickens, said primer pair being shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. A kit containing the primer pair described in claim 2 for identifying F-type and M-type miniature white-feathered broiler genotypes.

4. The application of the molecular marker of claim 1, the primer pair of claim 2, or the kit of claim 3 in the identification or screening of F-type and M-type small white-feathered broiler chickens.

5. A method for distinguishing between F-type and M-type white chicks, characterized in that, Genomic DNA of the small white-feathered broiler chickens to be tested was amplified using primer pairs SEQ ID NO.1 and SEQ ID NO.

2. Alleles were detected by capillary electrophoresis. Chickens containing a 205 bp allele were classified as type F small white chickens; those not containing a 205 bp allele were classified as type M small white chickens.

6. A method for screening F-type and M-type white penises, characterized in that, Genomic DNA of the small white-feathered broiler chickens to be tested was amplified using primer pairs SEQ ID NO.1 and SEQ ID NO.

2. Alleles were detected by capillary electrophoresis. Chickens containing a 205 bp allele were classified as type F small white chickens; those not containing a 205 bp allele were classified as type M small white chickens.

7. A method for identifying or screening F-type and M-type small white-feathered broiler chickens, characterized in that, Includes the following steps: 1) Extract genomic DNA from the small white-feathered broiler chickens to be tested; 2) Using the DNA extracted from the small white-feathered broiler chickens in step 1) as a template, PCR amplification was performed using the primer pair shown in SEQ ID NO.1 and SEQ ID NO.2; 3) After the reaction, the PCR products were genotyped using an ABI 3730 analyzer. Those containing the 205 bp allele were classified as F-type white chickens; those not containing the 205 bp allele were classified as M-type white chickens.

8. The identification or screening method according to claim 7, characterized in that, The PCR reaction system in step 2) is as follows: 25 μL of 2×PCR Mix, 1 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

9. The identification or screening method according to claim 7, characterized in that, The PCR reaction program in step 2) is as follows: 95℃ for 5 min, (95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s) for 35 cycles, and 72℃ for 10 min.

10. The identification or screening method according to any one of claims 5 to 9, characterized in that, The genomic DNA was extracted from blood, chicken meat, and feathers.

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