A SPIN1 gene for determining chicken sex and its application

Abstract

The present invention belongs to the technical field of biomolecules, and specifically relates to a SPIN1 gene for determining the sex of chickens and its application. The SPIN1 gene contains a fragment on the chicken's Z and W chromosomes, which differs by 330 bp. This fragment is a missing fragment on the Z chromosome. By detecting the sequence, the sex of chickens can be quickly and effectively identified. Individuals missing this molecular marker sequence fragment are female, while those not missing this molecular marker sequence fragment are male. The present invention also provides specific primers. Through PCR amplification technology and agarose gel electrophoresis, clear bands can be obtained to accurately determine the sex of chickens. This method has low technical difficulty, simple operation, and high accuracy. It can effectively identify the sex of chicks or chickens that are difficult to distinguish in appearance. It has practical significance for identifying breeds with small differences in male and female appearance. The technology of the present invention has strong applicability and good repeatability, can accurately identify the sex of poultry, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomolecules, and in particular relates to a SPIN1 gene for determining the sex of chickens and its application. Background Art

[0002] In recent years, the poultry industry has developed rapidly. While the number of poultry is growing, it is necessary to take into account issues such as quality development. Since different breeds of poultry have obvious gender preferences during the production process, the development of simple and convenient sex identification technology is of great significance. At present, in poultry production, sex identification is mainly carried out through sex-linked phenotypes such as feather color and feather speed, but its application range is relatively narrow and limited to certain breeds. Anal prolapse identification must be performed on the first day of hatching, which is very irritating to the chicks. In addition, due to the difficulty of identification, high labor intensity, and low accuracy, it often requires a lot of manpower and material resources. Therefore, the development of a simple, convenient, and low-cost method for chicken sex identification has broad application prospects.

[0003] Most mammals have an XX / XY sex chromosome system, with XY heterogametes forming males and XX homogametes forming females. In these animals, the Sry gene, located on the Y chromosome, regulates the sex determination process. Based on the differential distribution of this gene on the X and Y chromosomes, patent CN200410014222.5 discloses a method for identifying the sex of mammals by amplifying the Sry gene sequence using PCR technology. Unlike mammals, birds have a ZZ / ZW sex chromosome system, with ZW heterogametes forming females and ZZ homogametes forming males, a pattern that is the opposite of the sex chromosome-controlled sex development pattern in mammals. Current molecular methods for sexing chickens are primarily based on the CHD1 gene sequence. This gene is found on both the Z and W chromosomes of birds. Due to different rates of evolution, CHD1-Z and CHD1-W differ in length. The length of the PCR amplified sequence can be used to determine the sex of birds. Using the CHD1 gene, researchers at home and abroad have explored molecular sex determination for rare poultry species, including chickens. By improving specific primer sequences, this gene has been further used to identify the sex of pigeons, quail, and parrots (CN201010281972.4). Therefore, sex determination based on the length differences in the fragments of the same gene distributed on different sex chromosomes is feasible. Summary of the Invention

[0004] In view of the above problems, the present invention aims to provide a SPIN1 gene and its application for determining the sex of chickens.

[0005] The technical contents of the present invention are as follows:

[0006] The present invention provides a molecular marker for determining the sex of chickens. The molecular marker is a nucleotide sequence with a length difference of 330 bp between the SPIN1 genes on the chicken chromosome Z and the W chromosome, and the sequence is shown in SEQ ID NO.1.

[0007] Individuals with a missing sequence fragment of SEQ ID NO.1 are female individuals, and individuals without a missing sequence fragment of SEQ ID NO.1 are male individuals;

[0008] The gene accession number (Gene ID) of the SPIN1 gene on chromosome Z in the NCBI database is 395344, and the sequence range is: 43790850-43866043;

[0009] The gene accession number of the SPIN1 gene on the W chromosome in the NCBI database is 374014, and the sequence range is: 6500543-6669172.

[0010] The present invention also provides an application of a molecular marker for determining the sex of a chicken. The nucleotide sequence of the molecular marker is shown as SEQ ID NO.1. Through gene sequence sequencing, if one chromosome has a deletion of the sequence fragment of SEQ ID NO.1, it is a ZW type chromosome, which is a female individual; if both chromosomes have a deletion of the sequence fragment of SEQ ID NO.1, it is a ZZ type chromosome, which is a male individual.

[0011] The present invention also provides a gene fragment for determining the sex of a chicken. The gene fragment is located on the SPIN1 gene of a chicken chromosome. Its nucleotide sequence on the chicken Z chromosome is shown in SEQ ID NO.2, and its nucleotide sequence on the chicken W chromosome is shown in SEQ ID NO.3.

[0012] The present invention also provides an application of a gene fragment for determining the sex of a chicken. By sequencing the gene sequence, a chicken with a sequence containing only SEQ ID NO.2 is determined to be a male individual, and a chicken with a sequence containing both SEQ ID NO.2 and SEQ ID NO.3 is determined to be a female individual.

[0013] The present invention also provides a primer pair for determining the molecular marker of chicken sex. The forward primer and the reverse primer of the primer pair have nucleotide sequences as shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, and are used to amplify the above gene fragment.

[0014] The present invention also provides a method for determining the sex of a chicken, comprising the following steps:

[0015] 1) Extract genomic DNA from the chicken to be tested;

[0016] 2) Using the genomic DNA to be tested as a template, perform PCR amplification using the above primer pairs;

[0017] 3) Run the PCR product through agarose gel electrophoresis and determine the sex of the individual based on the band pattern. If a single band is observed, the individual is considered male; if a double band is observed, the individual is considered female.

[0018] The PCR reaction system was as follows: 12.5 μL of 2X PCR Taq Master Mix, 0.5 μL of each forward and reverse primers, 200 ng of DNA template, and ddH2O added to 25 μL;

[0019] The PCR reaction procedure:

[0020] Pre-denaturation at 94°C for 30 seconds, denaturation at 98°C for 10 seconds, annealing at 46-62°C for 30 seconds, extension at 72°C for 1 minute, 30 cycles, and final extension at 72°C for 2 minutes.

[0021] The sequence length of the single band is 88 bp, as shown in SEQ ID NO. 2;

[0022] The sequence lengths of the double bands are 88 bp and 418 bp, respectively, as shown in SEQ ID NO. 2 and SEQ ID NO. 3.

[0023] The present invention also provides a detection product for determining the sex of a chicken, the detection product comprising a detection reagent or a kit;

[0024] The detection product comprises the above-mentioned primer pair.

[0025] The beneficial effects of the present invention are as follows:

[0026] The molecular marker for determining the sex of a chicken of the present invention is a segment of the SPIN1 gene on the chicken's Z and W chromosomes, which differs by 330 bp and is also a deleted segment on the Z chromosome. By detecting the sequence, the sex of the chicken can be quickly and effectively identified. Individuals with the missing molecular marker sequence segment are female, while individuals without the missing molecular marker sequence segment are male. The present invention also provides specific primers. Through PCR amplification technology and agarose gel electrophoresis, clear bands can be obtained to accurately determine the sex of the chicken. The method has low technical difficulty, simple operation, and high accuracy. It can effectively identify the sex of chicks or chickens that are difficult to distinguish in appearance, and has practical significance for identifying breeds with small differences in male and female appearance. The technology of the present invention has strong applicability and good repeatability, can accurately identify the sex of poultry, and has broad application prospects. Through early embryo sex identification, specialized breeding of poultry of different sexes can be achieved, the breeding group structure is optimized, and breeding efficiency and economic benefits are improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a sequence comparison of the 330 bp deletion fragment of the SPIN1 gene on chromosomes Z and W;

[0028] Figure 2 Figure 1 is the amplification result of CHD1 specific primers;

[0029] Figure 3 This is a diagram showing the amplification results of the specific primers of the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0031] Unless otherwise specified, all raw materials and reagents in the present invention are raw materials and reagents from conventional markets.

[0032] Example 1

[0033] Acquisition of the SPIN1 gene

[0034] 1) Obtaining the sex chromosome sequence of the SPIN1 gene

[0035] The SPIN1 genomic sequence on chicken chromosomes was searched from the NCBI database (https: / / www.ncbi.nlm.nih.gov). The SPIN1 gene on chromosome Z in the NCBI database has a gene accession number of 395344 and a sequence range of 43790850-43866043; the SPIN1 gene on chromosome W in the NCBI database has a gene accession number of 374014 and a sequence range of 6500543-6669172.

[0036] 2) Sequence alignment

[0037] The sequences of SPIN1 on chromosomes W and Z were aligned using DNAMAN software. Figure 1 The nucleotide sequence of the SPIN1 gene fragment located on the Z chromosome is shown in SEQ ID NO. 2, and the nucleotide sequence of the SPIN1 gene fragment located on the W chromosome is shown in SEQ ID NO. 3. Compared with the W chromosome, the Z chromosome has a 330 bp deletion fragment, the nucleotide sequence of which is shown in SEQ ID NO. 1. These fragments are used as molecular markers to determine the sex of chickens.

[0038] 3) Primer design and synthesis

[0039] Specific primers were designed at the common sequence positions of chromosomes W and Z, respectively, upstream and downstream of the deletion sequence. The primer positions were as follows: Figure 1 As shown;

[0040] Primer F (SEQ ID NO. 4): 5′-GAATCAGCAGGGTGGTCTTAACTATG-3′;

[0041] Primer R (SEQ ID NO. 5): 5′-CAATTATTGGCTGCCTACTCTGC-3′.

[0042] Specific primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0043] Example 2

[0044] Validation of the SPIN1 gene for chicken sex determination

[0045] 1. Sample Collection and DNA Extraction

[0046] 1) Select chicken tissue and store it in a 1.5 mL centrifuge tube;

[0047] 2) Add 200 μL of ATL and 20 μL of Proteinase K to the sample and incubate at 55°C with shaking for 1 hour;

[0048] 3) Add 200 μL of AL reagent to the sample, vortex at high speed for 10 seconds, and incubate at 70°C for 10 minutes;

[0049] 3) Add 200 μL of anhydrous ethanol to the sample and vortex at high speed for 10 seconds;

[0050] 4) Place Hipure DNA Mini Column I in a 2 mL collection tube, add 750 μL of the mixture (including the precipitate) to the column, let it stand for 1 minute, and then centrifuge at 10,000 × g for 1 minute;

[0051] 5) Discard the filtrate and put the column back into the collection tube;

[0052] Add 500 μL of Buffer GW1 (diluted with ethanol) to the column and centrifuge at 10,000 × g for 1 min;

[0053] 6) Discard the filtrate and put the column back into the collection tube;

[0054] Add 650 μL of Buffer GW2 (diluted with ethanol) to the column and centrifuge at 10,000 × g for 1 minute;

[0055] 7) Discard the filtrate and put the column back into the collection tube;

[0056] Centrifuge at 10,000 × g for 1 minute.

[0057] 8) Place the column back into a new 1.5 mL centrifuge tube;

[0058] Add 30 μL of 70°C DNA elution buffer to the center of the column membrane, let it stand for 3 minutes, and then centrifuge at 10,000 × g for 1 minute;

[0059] 9) Discard the DNA binding column, determine the concentration, and store at -20°C.

[0060] 2. PCR Amplification

[0061] Amplify the target fragment by polymerase chain reaction (PCR) using the primer pairs synthesized above. Perform electrophoresis on a 1% agarose gel using 1× TAE as buffer for approximately 25 minutes at 135 V / min. Photograph the product using a gel imaging system and store it.

[0062] The PCR system is shown in Table 1, and the amplification reaction conditions are shown in Table 2.

[0063] Table 1 PCR reaction components

[0064]

[0065] Table 2 PCR amplification program

[0066]

[0067] The current molecular method for sexing chickens is mainly based on the CHD1 gene sequence. The chicken DNA was amplified using the published CHD1-specific primers (F: 5'-GTTACTGATTCGTCTACGAGA-3', R: 5'-TTGAAATGATCCAGTGCTTG-3'). Figure 2 As shown, double bands of 450 bp and 600 bp were amplified from the hen, while a single band of 600 bp was amplified from the rooster.

[0068] Based on the above results, 5 DNA samples were selected from each of the individuals identified as female and male and mixed separately. PCR reactions were performed using the mixed DNA of female and male as templates, using primers F and R from Example 1 at different annealing temperatures. At the same time, the results of the CHD1 primers were used as a positive control, and water was used as a negative control. The results are as follows: Figure 3As shown, two bands were detected in female chickens, and compared with the marker, the product lengths were consistent with the theoretical results of 418 bp and 88 bp; one band was detected in male chickens, and the product length was consistent with the theoretical result of 88 bp; when the DNA sample was replaced with water, no specific bands were detected, indicating that the primer pair synthesized by the present invention can clearly distinguish between female and male chickens.

[0069] In addition, when the specific primers provided by the present invention are annealed at a temperature of 62°C, male chickens show a single clear band, while female chickens show double bands, which is the best effect. Figure 3 The above test results are consistent with the determination results of CHD1 gene.

[0070] In summary, a gene SPIN1 that can be used to identify the sex of chickens and its application were obtained. By amplifying the genomic sequences on the W and Z chromosomes of SPIN1, including the deleted fragment, using the designed PCR primer pair, it can be determined that the deleted fragment can be used as a molecular marker for determining the sex of chickens (SEQ ID NO.1 sequence). Individuals with the deleted fragment are female (ZW-type chromosomes), and individuals without the deleted fragment are male (ZZ-type chromosomes).

Claims

1. A method for determining the sex of a chicken, characterized in that: The steps include: 1) Extract genomic DNA from the chicken to be tested; 2) Using the genomic DNA to be tested as a template, perform PCR amplification using primer pairs; The nucleotide sequences of the forward primer and the reverse primer of the primer pair are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively; 3) Run the PCR product through agarose gel electrophoresis and determine the sex of the individual based on the band pattern. If a single band is observed, the individual is considered male; if a double band is observed, the individual is considered female. The sequence of the single band is shown in SEQ ID NO.2; The sequences of the double bands are shown as SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

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