A molecular biological identification method and application of AB and CD roosters in a white-feathered broiler chicken breeding system

Abstract

The present invention discloses a molecular biological identification method and application of AB and CD roosters in a white-feathered broiler chicken matching system. The molecular marker used for identification is a SNP site located at 76216bp (Gallus_gallus‑4.0) of chromosome 16 of the chicken genome, which has a G / A polymorphism. When the base at this position is G, it is an AB rooster; when the base at this position is A, it is a CD rooster. The method of the present invention can quickly and accurately identify AB and CD roosters in a white-feathered broiler matching system, is convenient for grassroots promotion and use, and has broad market application prospects. In addition, the detection kit developed based on the method of the present invention can generate considerable economic benefits and good social value.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a molecular biology identification method and application of AB and CD roosters in a matching system of white-feathered broiler chickens. Background Art

[0002] At present, most broiler breeds at home and abroad are complementary lines, which make full use of the hybrid advantages between different lines to improve the performance of economic traits of offspring. my country's broilers are composed of three types: white-feathered broilers, yellow-feathered broilers and 817 broilers. White-feathered broilers adopt a four-line complementary breeding method ( Figure 1 ), the male lines A and B have faster growth rates but slightly worse reproductive performance; the female lines C and D have better reproductive performance but slightly worse growth rates. Yellow-feathered broilers are mostly three-line or two-line matching.

[0003] 817 broiler chickens are produced by crossbreeding white-feathered broiler parent generation sire roosters (AB roosters) with high-yielding brown-shelled egg-laying chickens ( Figure 2 ). The name 817 comes from the screening of hybrid combinations completed by the Poultry Research Institute of Shandong Academy of Agricultural Sciences on August 17, 1988. This model fully utilizes the advantages of "fast" white-feathered broilers and "high yield" of commercial brown-shelled laying hens. With the advantages of low seed production cost, high production efficiency and strong adaptability, it quickly occupied part of the yellow-feathered broiler market. At present, the annual output of 817 broilers is about 2.5 billion, which is an important part of my country's broiler category.

[0004] The speed of fresh broiler chickens going on the market will be further accelerated, and the advantages of 817 broiler chickens will be further highlighted. Foreign white-feathered broiler breeding companies believe that this breeding model infringes their intellectual property rights, and have tightened the supply of A and B series roosters, resulting in a shortage of AB series roosters. Some companies use parent-generation maternal roosters (CD series roosters) ( Figure 1 The offspring of C♂x D♀ are generally eliminated and replaced. The main performance indicators of 817 broilers produced with CD roosters, such as growth rate and feed conversion efficiency, are lower than those of 817 broilers produced with AB roosters, which has caused certain market confusion.

[0005] Therefore, there is an urgent need for a fast, accurate and easy-to-operate technology to identify AB and CD roosters in a white-feathered broiler system to ensure the orderly, healthy and stable development of my country's 817 broiler industry. Summary of the invention

[0006] One of the purposes of the present invention is to provide a molecular biological identification method for AB and CD roosters in a matching line of white-feathered broiler chickens in view of the shortcomings of the prior art.

[0007] A second object of the present invention is to provide primers for detecting the above-mentioned molecular markers.

[0008] The third object of the present invention is to provide the use of the above-mentioned molecular markers.

[0009] A fourth object of the present invention is to provide a detection method using the above-mentioned molecular markers.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] A molecular marker for identifying AB and CD roosters in a matching line of white-feathered broiler chickens. The molecular marker is a SNP site located at 76216bp (Gallus_gallus-4.0) of chromosome 16 of the chicken genome, which has a G / A polymorphism. When the base at this position is G, it is an AB rooster; when the base at this position is A, it is a CD rooster.

[0012] The present invention also provides a primer pair for identifying molecular markers of AB and CD roosters in a matching line of white-feathered broiler chickens, wherein:

[0013] Forward primer (SEQ ID NO. 1): 5'-CGGAGTCACTGACAGCAGAG-3'

[0014] Reverse primer (SEQ ID NO. 2): 5'-GCAGCATCTGATGGACACCT-3'.

[0015] The present invention also provides a kit containing the primer pair for identifying the genotypes of AB and CD roosters in a matching line of white-feathered broiler chickens.

[0016] The present invention also provides the use of the molecular markers or primers or kit pairs in identifying or breeding AB and CD roosters in a matching system of white-feathered broiler chickens.

[0017] The present invention also provides the use of the above molecular markers or primers or kit pairs in screening or auxiliary screening of AB roosters.

[0018] The present invention also provides the use of the above molecular markers or primers or kit pairs in screening or auxiliary screening of CD line roosters.

[0019] In a specific example, the application described in the present invention is specifically to extract the genomic DNA of the chicken breed to be tested, use the genomic DNA of the chicken breed to be tested as a template, perform PCR amplification on SEQ ID NO.1 / SEQ ID NO.2 through primers to obtain a PCR amplification product, and use Sanger sequencing to detect the genotype of the SNP site at the 206bp of the amplified sequence. When the base at this position is G, it is an AB rooster; when the base at this position is A, it is a CD rooster.

[0020] In a specific embodiment,

[0021] In the application of the present invention, the genomic DNA in the chicken to be tested can be extracted according to conventional methods in the art, and the sample used for extraction can be a commonly used sample, such as blood, chicken, feathers, tissue, oral swab, etc.

[0022] In a specific embodiment, in the application of the present invention,

[0023] The reaction system for PCR amplification was: 25 μL of 2×PCR Mix (Nanjing Novozyme Biotechnology Co., Ltd.), 1 μL of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

[0024] In a specific embodiment, in the application of the present invention, the reaction procedure of PCR amplification is: 95°C for 5 min, 35 cycles of (95°C for 30 s, 58°C for 30 s, 72°C for 60 s), and 72°C for 10 min.

[0025] The Sanger sequencing of the present invention can be performed according to conventional methods in the art. For example, after the reaction is completed, the PCR product is subjected to Sanger sequencing on an ABI3730 sequencer and sequence alignment is performed.

[0026] The AB and CD roosters in the white-feathered broiler matching system to be detected in the present invention can amplify a 388bp sequence (SEQ ID NO.3 or SEQ ID NO.4) by amplifying the sequence, and determine the genotype of the SNP site at the 206bp of the 388bp sequence. When the base at this position is G, it is an AB rooster (SEQ ID NO.3); when the base at this position is A, it is a CD rooster (SEQID NO.4).

[0027] Beneficial effects:

[0028] The present invention utilizes the difference of chicken genome sequence to identify AB and CD roosters in the matching line of white-feathered broiler chickens. The advantage is that it can quickly and accurately identify AB and CD roosters in the matching line of white-feathered broiler chickens, and crack down on unscrupulous merchants who sell inferior products as good ones. In addition, the method is simple to operate, easy to operate in the laboratory, and convenient for grassroots promotion and use, and has broad market application prospects. In addition, the detection kit developed based on the method of the present invention can generate considerable economic benefits and good social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Diagram of the four-line production model of white-feather broiler chickens.

[0030] Figure 2 817Broiler production model diagram.

[0031] Figure 3 Sequencing peak diagram of AB line rooster.

[0032] Figure 4 Sequencing peak diagram of CD line rooster. DETAILED DESCRIPTION

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present 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.

[0034] Example 1

[0035] Identification of 1AB and CD roosters

[0036] 1.1 Sample collection

[0037] Blood samples of AB and CD roosters in the white-feathered broiler breeding system were collected from a white-feathered broiler production enterprise in Henan and a white-feathered broiler production enterprise in Shandong. Thirty samples were collected from each enterprise and each line.

[0038] 1.2 PCR amplification

[0039] The primers designed by the present invention are used for PCR amplification.

[0040] Forward primer: 5′-CGGAGTCACTGACAGCAGAG-3′;

[0041] Reverse primer: 5′-GCAGCATCTGATGGACACCT-3′.

[0042] The PCR reaction system is:

[0043] 2×PCR Mix (Nanjing Novozyme Biotechnology Co., Ltd.) 25 μL, 10 μmol / L forward and reverse primers 1 μL each, 50-100 μg / ml template DNA 2 μL, ultrapure water 21 μL.

[0044] The PCR reaction program was: 95°C for 5 min, 35 cycles of (95°C for 30 s, 58°C for 30 s, 72°C for 30 s), and 72°C for 3 min.

[0045] 1.3 Sequencing

[0046] After the reaction, the PCR products were sequenced by Sanger sequencing on an ABI 3730 sequencer.

[0047] 1.4 SNP distribution statistics

[0048] The genotype of the SNP site at the 206th bp of the amplified 388bp sequence was determined. When the base at this position was G, it was an AB rooster; when the base at this position was A, it was a CD rooster. The statistical results are shown in Table 1.

[0049] Table 1 SNP distribution of AB and CD roosters in white broiler breeding lines from different sources

[0050]

[0051] Example 2

[0052] Verification of the generational transmission of AB and CD roosters in the 2-marker locus white-feathered broiler breeding line

[0053] 2.1 Sample collection

[0054] Blood samples of AB and CD line roosters in the white-feathered broiler breeding system were collected from a white-feathered broiler production enterprise in Henan Province. Thirty samples were collected from each line in each generation for three consecutive generations.

[0055] 2.2 PCR amplification

[0056] The primers designed by the present invention are used for PCR amplification.

[0057] Forward primer: 5′-CGGAGTCACTGACAGCAGAG-3′;

[0058] Reverse primer: 5′-GCAGCATCTGATGGACACCT-3′.

[0059] The PCR reaction system is:

[0060] 2×PCR Mix (Nanjing Novozyme Biotechnology Co., Ltd.) 25 μL, 10 μmol / L forward and reverse primers 1 μL each, 50-100 μg / ml template DNA 2 μL, ultrapure water 21 μL.

[0061] The PCR reaction program was: 95°C for 5 min, 35 cycles of (95°C for 30 s, 58°C for 30 s, 72°C for 30 s), and 72°C for 3 min.

[0062] 2.3 Sequencing

[0063] After the reaction, the PCR products were sequenced by Sanger sequencing on an ABI 3730 sequencer.

[0064] 2.4 SNP distribution statistics

[0065] The genotype of the SNP site at the 206th bp of the amplified 388bp sequence was determined. When the base at this position was G, it was an AB rooster; when the base at this position was A, it was a CD rooster. The statistical results are shown in Table 2.

[0066] Table 2 SNP distribution of AB and CD roosters in different generations of white broiler chickens

[0067]

[0068] The results showed that the locus could be stably inherited for three consecutive generations, further verifying the high efficiency of the present invention on AB and CD line roosters.

Claims

1. A molecular marker for identifying AB and CD roosters in a matching line of white-feathered broiler chickens, the molecular marker being a SNP site located at 76216 bp on chromosome 16 of a chicken genome, the genome version being Gallus_gallus-4.0, which has a G / A polymorphism, the molecular marker sequence being as shown in SEQ ID NO.3 or SEQ ID NO.4, and a SNP site being present at 206 bp of the sequence shown in SEQ ID NO.3 or SEQ ID NO.4, when the SNP site is G, it is an AB rooster; when the SNP site is A, it is a CD rooster.

2. A primer pair for identifying molecular markers of AB and CD roosters in a matching line of white-feathered broiler chickens, characterized in that The primer pair is shown as SEQ ID NO.1 and SEQ ID NO.

2.

3. A kit for identifying the genotypes of AB and CD roosters in a matching line of white-feathered broiler chickens, comprising the primer pair described in claim 2.

4. Use of the primer pair according to claim 2 or the kit according to claim 3 in identifying or breeding AB and CD roosters in a matching line of white-feathered broiler chickens.

5. The use according to claim 4, characterized in that: The samples for extracting genomic DNA from the chicken to be tested are blood, chicken, feathers, tissue or oral swab.

6. Use of the primer pair according to claim 2 or the kit according to claim 3 in screening or auxiliary screening of AB roosters.

7. The use according to claim 6, characterized in that: The samples for extracting genomic DNA from the chicken to be tested are blood, chicken, feathers, tissue or oral swab.

8. Use of the primer pair according to claim 2 or the kit according to claim 3 in screening or auxiliary screening of CD line roosters.

9. The use according to claim 8, characterized in that: The samples for extracting genomic DNA from the chicken to be tested are blood, chicken, feathers, tissue or oral swab.

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