A SMAD7 gene for determining the sex of chickens and its application

Abstract

The present invention belongs to the technical field of biomolecules, and specifically relates to a SMAD7 gene and application for determining the sex of chickens. Through molecular markers and designing new PCR primers, the migration distance difference of the two bands amplified by the ZW chromosome is 299bp, which is 150bp higher than the migration distance difference of the traditional sex identification gene CHD1 amplification system. The migration distance difference of the two target bands in the present invention is larger and easier to judge; and the fragments amplified by the designed PCR primers are smaller, only 517bp and 816p, and the PCR amplification time is significantly reduced, reducing the time for sex identification; and the present invention can directly amplify and agarose gel electrophoresis by extracting chicken tissue, and the sex of the chicken can be quickly determined according to the electrophoresis result, and the time of DNA extraction can be reduced. The duration can be controlled within 1 hour, the operation is simple, the identification result is accurate, it is convenient for grassroots promotion and use, and has a broad market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomolecules, and in particular relates to a SMAD7 gene for determining the sex of a chicken and an application thereof. Background Art

[0002] In laying hen production, about 7 billion one-day-old male chicks are eliminated every year due to gender reasons, which leads to huge economic losses. In broiler production, sex-separated feeding helps to build a more accurate production management system, thereby improving overall production performance. Therefore, given that different types of poultry show significant gender preferences in production, establishing an efficient and simple sex identification technology is of great significance to improving poultry production performance.

[0003] At present, sex identification in poultry production mainly relies on sex-linked genetic characteristics such as feather color and feather speed, but this method has a limited scope of application and is only applicable to certain specific breeds. The traditional anal inversion identification method needs to be performed on the first day after the chicks hatch, which is difficult to operate, labor-intensive, and not very accurate. In addition, this method also requires a lot of manpower and material resources, which significantly increases production costs. Therefore, the development of a simple, convenient and low-cost chicken sex identification method has become a problem that needs to be solved urgently in the current poultry industry. Summary of the invention

[0004] In view of the above problems, the object of the present invention is to provide a SMAD7 gene and 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 a chicken, wherein the molecular marker is a nucleotide sequence with a length difference of 68 bp between the SMAD7 gene on the chicken chromosome Z and the chromosome W, and the sequence is shown in SEQ ID NO.1, which is a sequence missing from the chromosome Z compared to the chromosome W;

[0007] The gene accession number of the SMAD7 gene on chromosome Z in the NCBI database is 429683, and the sequence range is: 1571674-1599896;

[0008] The gene accession number of the SMAD7 gene on the W chromosome in the NCBI database is 395544, and the sequence range is: 4307664-4334160.

[0009] The present invention also provides an application of a molecular marker for determining the sex of a chicken, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and by sequencing the gene sequence of a chicken chromosome, a chromosome having only one chromosome lacking the sequence fragment of SEQ ID NO.1 is a ZW-type chromosome, indicating a female individual, and a chromosome having both chromosomes lacking the sequence fragment of SEQ ID NO.1 is a ZZ-type chromosome, indicating a male individual.

[0010] The present invention also provides a gene fragment for determining the sex of a chicken, wherein the gene fragment is located on the SMAD7 gene of a chicken chromosome, and the nucleotide sequence of the gene fragment on the chicken Z chromosome is shown in SEQ ID NO.2; and the nucleotide sequence on the W chromosome is shown in SEQ ID NO.3.

[0011] 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.

[0012] The present invention also provides a PCR primer pair for determining the sex of a chicken. The forward primer and the reverse primer of the PCR 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.

[0013] The present invention also provides a method for determining the sex of a chicken, comprising the following steps:

[0014] 1) Extract genomic DNA from the chicken individuals to be tested;

[0015] 2) Using the genomic DNA to be tested as a template, perform PCR amplification using the above primer pairs;

[0016] 3) The PCR product was subjected to agarose gel electrophoresis, and the gender was determined based on the band pattern. If there was a single band, the individual was determined to be male, and if there were double bands, the individual was determined to be female;

[0017] The PCR reaction system: KOD mix 5 μL, forward primer 0.3 μL, reverse primer 0.3 μL, tissue template 1 μL, ddH2O added to 10 μL;

[0018] The PCR reaction procedure was as follows: pre-denaturation at 98°C for 3 min, denaturation at 98°C for 10 sec, annealing at 55°C for 5 sec, extension at 68°C for 2 sec, 34 cycles, and final extension at 68°C for 3 min.

[0019] The sequence length of the single band is 517 bp, as shown in SEQ ID NO.2;

[0020] The sequence lengths of the double bands are 517 bp and 816 bp, respectively, as shown in SEQ ID NO.2 and SEQ ID NO.3.

[0021] 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;

[0022] The detection product comprises the above-mentioned primer pair.

[0023] The beneficial effects of the present invention are as follows:

[0024] The present invention provides a molecular marker for determining the sex of a chicken, which is a segment of SMAD7 gene on a chicken Z chromosome and a chicken W chromosome with a length difference of 68 bp. By detecting the sequence, a female individual is identified as having only one chromosome with a missing segment, and a male individual is identified as having a missing segment on both chromosomes. The sex of the chicken can be identified quickly and effectively. The present invention also designs new PCR primers and amplification systems, and the migration distance difference of two bands amplified from the ZW chromosomes is 299 bp, which is 150 bp compared with the migration distance difference of the traditional sex identification gene CHD1 amplification system. The migration distance difference of the two target bands in the present invention is larger, and it is easier to determine in actual operation. In addition, the fragments amplified by the designed PCR primers are smaller, which are only 517 bp and 816 bp. bp, the PCR amplification time is significantly reduced, and the time for gender identification is reduced; and the present invention can directly perform amplification and agarose gel electrophoresis by extracting chicken tissue, and the gender of the chicken can be quickly determined according to the electrophoresis result, and the gender of chicks or chickens that are difficult to distinguish in appearance can be effectively identified, which is of practical significance for the identification of breeds with small differences in male and female appearance, and by reducing the DNA extraction time, the time can be controlled within 1 hour, the operation is simple, and the identification result is accurate. The technical applicability of the present invention is strong, the repeatability is good, the gender of poultry can be accurately identified, and it has broad application prospects. Through early embryo gender identification, specialized breeding of poultry of different genders can be achieved, the breeding group structure is optimized, the breeding efficiency and economic benefits are improved, and it is convenient for grassroots promotion and use, and has broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a sequence comparison diagram of the SMAD7 gene on chromosome Z and chromosome W;

[0026] Figure 2 This is a diagram showing the amplification results of the specific primers of the present invention. DETAILED DESCRIPTION

[0027] 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 forms of modifications to the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0028] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0029] Example 1

[0030] Acquisition of SMAD7 gene

[0031] 1) Obtaining the sex chromosome sequence of the SMAD7 gene

[0032] The SMAD7 genomic sequence on chicken chromosomes was searched from the NCBI database (https: / / www.ncbi.nlm.nih.gov). The gene accession number of the SMAD7 gene on the Z chromosome in the NCBI database was 429683, and the sequence range was 1571674-1599896; the gene accession number of the SMAD7 gene on the W chromosome in the NCBI database was 395544, and the sequence range was 4307664-4334160.

[0033] 2) Sequence alignment

[0034] The sequences on chromosome W and chromosome Z of SMAD7 obtained above were compared using DNAMAN software. Figure 1 As shown, the SMAD7 gene fragment located on the Z chromosome has a nucleotide sequence as shown in SEQ ID NO.2, and the SMAD7 gene fragment located on the W chromosome has a nucleotide sequence as shown in SEQ ID NO.3, wherein the Z chromosome has multiple missing fragments compared to the W chromosome, totaling 299bp, and a fragment with a length of 68bp is used as a molecular marker to determine the sex of chickens, and its nucleotide sequence is shown in SEQ ID NO.1.

[0035] 3) Primer design and synthesis

[0036] Specific primers were designed at the W and Z chromosome consensus sequences upstream and downstream of the above-mentioned deletion sequence. The primer positions are as follows: Figure 1 As shown;

[0037] Primer F (SEQ ID NO. 4): 5′-GCTTCATCTGGTCTCAAC-3′;

[0038] Primer R (SEQ ID NO. 5): 5′-CTCCTGATTAGGCCTTTA-3′.

[0039] Specific primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0040] Example 2

[0041] Verification of SMAD7 gene for determining chicken sex

[0042] 1. Sample Collection and DNA Extraction

[0043] 1) Select chicken tissue (embryo, not limited to embryo) and store it in a 1.5 mL centrifuge tube;

[0044] 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;

[0045] 3) Add 200 μL AL reagent to the sample, vortex at high speed for 10 seconds, and incubate at 70°C for 10 minutes;

[0046] 3) Add 200 μL of anhydrous ethanol to the sample and vortex at high speed for 10 seconds;

[0047] 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 stand for 1 minute, and then centrifuge at 10,000 × g for 1 minute;

[0048] 5) Discard the filtrate and put the column back into the collection tube;

[0049] Add 500 μL of Buffer GW1 (diluted with ethanol) to the column and centrifuge at 10,000 × g for 1 min;

[0050] 6) Discard the filtrate and put the column back into the collection tube;

[0051] Add 650 μL of Buffer GW2 (diluted with ethanol) to the column and centrifuge at 10,000 × g for 1 min;

[0052] 7) Discard the filtrate and put the column back into the collection tube;

[0053] Centrifuge at 10,000 × g for 1 minute.

[0054] 8) Place the column back into a new 1.5 mL centrifuge tube;

[0055] Add 30 μL of 70°C DNA elution buffer to the center of the column membrane, let stand for 3 minutes, and centrifuge at 10,000 × g for 1 minute;

[0056] 9) Discard the DNA binding column, determine the concentration, and store at -20°C.

[0057] 2. PCR Amplification

[0058] Five DNA samples were selected from the individuals determined to be female and male and mixed in a pool. The mixed pool DNA of female and male was used as a template, and the primers F and R of Example 1 were used at different annealing temperatures to perform PCR reaction to amplify the target fragment. 1×TAE was used as a buffer and 1% agarose gel was used for electrophoresis for about 37 minutes and 20 minutes. The product was photographed and saved using a gel imaging system.

[0059] The PCR system is shown in Table 1 , and KOD enzyme (OD OneTM PCR Master Mix–Blue, KMM-201) was used for amplification. The amplification reaction conditions are shown in Table 2 .

[0060] Table 1 Components of PCR reaction

[0061]

[0062] Table 2 PCR amplification program

[0063]

[0064] The results of CHD1 primers were used as positive control, and water was used as template for negative control. Figure 2 As shown, two bands were detected in female chickens, and compared with the marker, the product lengths were consistent with the theoretical results of 517bp and 816bp; one band was detected in male chickens, and the product length was consistent with the theoretical result of 517bp; 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.

[0065] The traditional molecular method for identifying the sex of chickens is mainly based on the CHD1 gene sequence. The ZZ-type amplification product of a rooster is 600bp, and the ZW-type amplification product of a hen is 450bp and 600bp respectively. The migration distance difference is 150bp and it takes about 2.5 hours.

[0066] Compared with the traditional method, the Z chromosome amplification band amplified by the present invention is short, and the W chromosome amplification band is long, which is different from the current amplification principle of poultry sex identification genes; the migration distance difference between the two bands amplified by the ZW chromosomes in the present invention is 299bp, and the migration distance difference between the two target bands is larger, which is easier to judge in the actual operation process, and the time consumption can be controlled within 1 hour.

[0067] In summary, a gene SMAD7 and its application that can be used to identify the sex of chickens are obtained. According to the chicken SMAD7 gene, a new sex identification primer is designed. The results show that the Z chromosome amplification length is 517 bp, the W chromosome amplification length is 816 bp, the Z chromosome amplification band is short, and the W chromosome amplification band is long; the rooster's sex chromosome combination is ZZ type, the amplified fragment length is 1 517bp band, the hen's sex chromosome combination is ZW type, and the amplified fragment length is 2 bands, respectively, Z sequence 517bp and W sequence 816bp. The PCR system adopted by the present invention directly amplifies and agarose gel electrophoresis is performed on chicken tissues, and the sex of the chicken can be quickly determined according to the electrophoresis results, which reduces the DNA extraction time, and the identification result is accurate, which is convenient for grassroots promotion and use, and has broad market application prospects.

Claims

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

2. The method for determining the sex of a chicken according to claim 1, characterized in that: The sequence length of the single band is 517 bp, as shown in SEQ ID NO.2; The sequence lengths of the double bands are 517 bp and 816 bp, respectively, as shown in SEQ ID NO.2 and SEQ ID NO.3.

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