Molecular biological identification method for fast large and small white feather broiler chickens and application

Abstract

The invention discloses a molecular biological identification method and application of fast large and small white feather broiler chickens, a molecular marker is obtained by amplifying genome DNA of a chicken to be detected by adopting sequences shown as SEQ ID NO.1 and SEQ ID NO.2, allele is detected by capillary electrophoresis, and the chicken containing 309 or 311 bp allele is the small white feather broiler chicken; the chicks which do not contain 309 and 311 bp alleles are fast and large white feather broiler chicks; the method utilizes allele difference to identify the fast large and small white feather broiler chickens, and has the advantage that the fast large and small white feather broiler chickens can be identified quickly and accurately. Moreover, the method is simple to operate, easy to operate in a laboratory, convenient to popularize and use in a basic level 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 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 large and small white-feathered broiler chicks. Background Technology

[0002] my country's broiler chicken industry comprises three types: fast-growing white-feathered broilers, small white-feathered broilers, and yellow-feathered broilers. White-feathered broilers have a fast growth rate and high feed conversion efficiency, reaching market age at 38-42 days and a weight of approximately 2.5-3.0 kg. Yellow-feathered broilers mainly refer to local Chinese chicken breeds and bred varieties containing local chicken bloodlines, reaching market age at 60-150 days and a weight of 1.0 kg-2.5 kg. Small white-feathered broilers, also known as 817 broilers, are generally bred by crossing fast-growing white-feathered broilers with high-yielding brown-shelled egg-laying chickens. They have the advantage of low breeding costs, reaching market age at 35-49 days and a weight of 1.1-2.0 kg.

[0003] Fast-growing broiler breeder chickens are larger, consume more feed, and lay relatively fewer eggs, typically around 180 eggs per production cycle, with a cost of about 2.5 yuan per chick. Small-sized broiler breeder chickens, derived from high-laying hens, are relatively smaller, lay more eggs, typically around 300 eggs per production cycle, with a cost of about 0.8 yuan per chick. Fast-growing and small-sized broiler chicks are similar in appearance and cannot be distinguished by looks. Some breeders, in pursuit of profit, mix small-sized broiler chicks with fast-growing chicks, causing market confusion.

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

[0005] One of the objectives of this invention is to address the shortcomings of existing technologies by providing a molecular biological identification method for large and small white-feathered broiler chicks.

[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 distinguishing between fast-growing and small-sized white-feathered broiler chicks, wherein the molecular marker is obtained by amplifying the genomic DNA of the chicken to be tested using the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and detecting alleles by capillary electrophoresis. Chicks containing 309 or 311 bp alleles are small-sized white-feathered broiler chicks; chicks without 309 and 311 bp alleles are fast-growing white-feathered broiler chicks.

[0011] This invention also provides a primer pair for identifying molecular markers in fast-growing and small-growing broiler chicks, 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 the genotypes of fast-growing and small-sized white-feathered broiler chicks.

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

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

[0017] The present invention also provides a method for identifying fast-growing and small-sized white-feathered broiler chicks. The method involves amplifying the genomic DNA of the white-feathered broiler to be tested using primer pairs SEQ ID NO.1 and SEQ ID NO.2, and detecting alleles using capillary electrophoresis. Chicks containing 309 or 311 bp alleles are small-sized white-feathered broiler chicks; chicks without 309 and 311 bp alleles are fast-growing white-feathered broiler chicks.

[0018] The present invention also provides a method for screening fast-growing and small-sized white-feathered broiler chicks. The method involves amplifying the genomic DNA of the fast-growing white-feathered broiler to be tested using primer pairs SEQ ID NO.1 and SEQ ID NO.2, and detecting alleles by capillary electrophoresis. Chicks containing 309 or 311 bp alleles are small-sized white-feathered broiler chicks; chicks without 309 and 311 bp alleles are fast-growing white-feathered broiler chicks.

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

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

[0021] 2) Using the DNA extracted in step 1) as a template, perform PCR amplification 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 309 or 311 bp allele were small white-feathered broiler chicks; those not containing the 309 and 311 bp alleles were fast-growing white-feathered broiler chicks.

[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 present invention can distinguish between fast-growing and small-sized white-feathered broiler chickens by whether the population contains the 309 / 311 bp allele.

[0027] Beneficial effects:

[0028] This invention utilizes allelic differences to distinguish between fast-growing and small-sized white-feathered broiler chicks. Its advantages lie in its ability to quickly and accurately differentiate between these two types of chicks. 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 Peak diagram of individuals containing a 309 bp allele;

[0030] Figure 2 Peak diagram of an individual containing a 311 bp allele. Detailed Implementation

[0031] 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.

[0032] Example 1

[0033] 1. Identification of large and small white-feathered broiler chicks

[0034] 1.1 Sample Collection

[0035] All experimental samples used in this study were provided by the National Poultry Production Performance Testing Station. The samples included six breeds of fast-growing white-feathered broilers (AA, Ross 308, Cobb 500, Shengze 901, Guangming 2, and Wod188) and four breeds of small white-feathered broilers (Yisheng 817, Yisheng 909, Fengyuan 817, and Wod168), with 30 individuals from each breed. Genomic DNA was extracted from these samples for subsequent experiments.

[0036] 1.2 PCR amplification

[0037] PCR amplification was performed using primers designed according to this invention.

[0038] Forward primer: 5'-AACTTGGTAAGGGAATTCCCT-3'; FAM fluorescent label added to the 5' end.

[0039] Reverse primer: 5'-GAATTAGTGCCCAGCTGTGCT-3'.

[0040] The PCR reaction system is as follows:

[0041] 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.

[0042] 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.

[0043] 1.3 Capillary electrophoresis detection

[0044] After the reaction, the PCR products were genotyped using an ABI 3730 analyzer. The peak diagrams of individuals containing the 309 or 311 bp alleles are shown below. Figure 1 or Figure 2 As shown.

[0045] 1.4 Allele Statistics

[0046] The allele distribution of different populations is shown in Table 1. According to Table 1, those containing the 309 or 311 bp allele are small white-feathered broilers, while those not containing the 309 or 311 bp allele are fast-growing white-feathered broilers.

[0047] Table 1. Allele distribution in fast-growing and small-sized white-feathered broiler chicks.

[0048]

[0049] Example 2

[0050] Two rapid tests to identify adulterated large-scale white-feathered broiler chickens

[0051] 2.1 Sample Collection

[0052] Feathers were collected from 100 fast-growing white-feathered broiler chicks that farmers reported were adulterated, and genomic DNA was extracted for subsequent experiments.

[0053] 2.2 PCR amplification

[0054] PCR amplification was performed using primers designed according to this invention.

[0055] Forward primer: 5'-AACTTGGTAAGGGAATTCCCT-3'; FAM fluorescent label added to the 5' end.

[0056] Reverse primer: 5'-GAATTAGTGCCCAGCTGTGCT-3'.

[0057] The PCR reaction system is as follows:

[0058] 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.

[0059] 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.

[0060] 2.3 Capillary electrophoresis detection

[0061] After the reaction was completed, the PCR products were genotyped using an ABI 3730 analyzer.

[0062] 2.4 Allele Statistics

[0063] Fifteen mice were found to contain 309 or 311 bp alleles, while 75 mice did not contain 309 or 311 bp alleles.

[0064] 2.5 Group rearing verification

[0065] Fifteen birds containing the 309 or 311 bp allele and 75 birds not containing the 309 or 311 bp allele were raised in separate groups. The 15 birds containing the 309 or 311 bp allele weighed around 1.5 kg at 6 weeks of age, while the other 75 birds weighed over 2.5 kg. They were confirmed to be 15 small white-feathered broiler chickens disguised as broiler chickens.

[0066] In summary, this molecular marker is specific between fast-growing and small-sized broiler chicks. This also demonstrates the high efficiency of this invention in identifying fast-growing and small-sized broiler chicks.

Claims

1. A molecular marker for distinguishing between fast-growing and small-sized white-feathered broiler chicks, characterized in that, The molecular markers were obtained by amplifying the genomic DNA of the chickens to be tested using the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and detecting alleles by capillary electrophoresis. Chickens containing 309 or 311 bp alleles were classified as small white-feathered broiler chicks; chickens without 309 and 311 bp alleles were classified as fast-growing white-feathered broiler chicks.

2. A primer pair for identifying fast-growing and small-growing white-feathered broiler chicks, said primer pair as shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. A kit containing the primer pair described in claim 2 for identifying fast-growing and small-sized white-feathered broiler chicks.

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 large and small white-feathered broiler chicks.

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

2. Alleles were detected by capillary electrophoresis. Chicks containing 309 or 311 bp alleles were classified as small broiler chicks; chicks without 309 and 311 bp alleles were classified as fast-growing broiler chicks.

6. A method for screening fast-growing white-feathered broiler chicks, characterized in that, Genomic DNA of broiler chicks to be tested was amplified using primer pairs SEQ ID NO.1 and SEQ ID NO.

2. Alleles were detected by capillary electrophoresis. Chicks without 309 and 311 bp alleles were classified as fast-growing broiler chicks; chicks with 309 or 311 bp alleles were classified as small-growing broiler chicks.

7. A method for identifying or screening large and small white-feathered broiler chicks, characterized in that, Includes the following steps: 1) Extract genomic DNA from the large and small white-feathered broiler chicks to be tested; 2) Using the DNA extracted in step 1) as a template, perform PCR amplification using the primer pair described in this invention; 3) After the reaction, the PCR products were genotyped using an ABI 3730 analyzer. Those containing the 309 or 311 bp allele were small white-feathered broiler chicks; those not containing the 309 and 311 bp alleles were fast-growing white-feathered broiler chicks.

8. The application according to claim 7, characterized in that, The PCR reaction system consisted of: 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 application according to claim 7, characterized in that, The PCR reaction program was as follows: 95℃ for 5 min, (95℃ for 30 s, 58℃ for 30 s, 72℃ for 60 s) for 35 cycles, and 72℃ for 10 min.

10. The application according to any one of claims 5 to 9, characterized in that, The chicken genomic DNA to be tested was obtained from the chicken's blood, meat, and feathers.

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