A primer set for determining chicken gender by using chicken sex chromosome homologous fragment length difference, a PCR gender identification method and application thereof

Abstract

The application belongs to the technical field of biomolecules, and particularly relates to a primer set for determining the gender of a chicken by using the length difference of homologous fragments of chicken sex chromosomes, a PCR gender identification method and application. By directly comparing the sequences of Z and W chromosomes of a chicken, a stable length difference site is found in the Z / W homologous region. In the Z chromosome, the region forms a 459 bp amplification fragment, and in the W chromosome, a 104 bp deletion exists, forming a 355 bp amplification fragment. After designing a pair of PCR primers based on the difference, a 459 bp single band is produced by amplification of a male chicken sample, and 459 bp and 355 bp double bands are produced by amplification of a female chicken sample, so that the gender of the chicken is identified. The method is simple to operate, intuitive to interpret and low in cost, and can be used for rapid gender identification of chicken embryos, chicks, adult chickens and chicken tissue samples.

Description

Technical Field

[0001] This invention belongs to the technical field of biomolecules, specifically relating to a primer set, PCR sex determination method, and application for determining the sex of chickens by utilizing the difference in length of homologous segments of chicken sex chromosomes. Background Technology

[0002] The genetic sex of chickens is determined by the composition of sex chromosomes; males are ZZ type and females are ZW type. Chicken sex determination has important applications in chicken embryo research, chick grouping, breeder selection, experimental animal grouping, production performance testing, and genetic breeding research.

[0003] Currently, chicken sex determination mainly includes two methods: phenotypic identification and molecular identification. Phenotypic identification methods include vent examination, feather speed identification, and feather color identification. Among these, vent examination relies heavily on the operator's experience, is labor-intensive, and carries the risk of misjudgment; feather speed identification and feather color identification are limited by breed, genetic background, and breeding system, and are not applicable to all chicken breeds or experimental materials.

[0004] Molecular sex determination methods based on PCR amplification are widely used in poultry sex determination due to their high accuracy, flexible sample types, and relatively simple operation. Existing PCR detection methods mostly rely on the length difference of the Z / W fragments of the CHD1 gene, W chromosome-specific sequences, or other sex chromosome-related markers for detection. However, existing methods still have certain limitations. For example, some methods amplify long fragments, some rely on W chromosome-specific amplification and lack endogenous amplification controls, and some methods are affected by the detection platform, amplification efficiency, or interpretation method.

[0005] Therefore, it is necessary to develop a new molecular marker and PCR detection method for chicken sex identification, which can simultaneously amplify homologous fragments of the Z and W chromosomes in the same reaction system using a pair of primers, and then visually distinguish between male and female individuals by ordinary agarose gel electrophoresis. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a primer set, PCR sex determination method, and application for determining the sex of chickens by utilizing the difference in length of homologous segments of chicken sex chromosomes.

[0007] The technical content of this invention is as follows: This invention provides a molecular marker for sex identification in chickens, characterized in that the sequence of the molecular marker is as shown in SEQ ID NO.1, and it is located on the chicken Z chromosome at positions 71043928-71044031 corresponding to accession number NC_052572.1; The molecular marker is present on the chicken Z chromosome and absent on the chicken W chromosome, and is used for chicken sex identification.

[0008] The present invention also provides a PCR primer set for sex determination of chickens, wherein the nucleotide sequences of the forward primer and the reverse primer of the PCR primer set are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively; The PCR primer set was used to amplify homologous nucleic acid fragments on chicken Z and W chromosomes; The nucleotide sequence of the homologous nucleic acid fragment corresponding to the chicken Z chromosome obtained after amplification is shown in SEQ ID NO.4. It is 459 bp in length and is located at positions 71043897-71044355 on the chicken Z chromosome corresponding to accession number NC_052572.1. The nucleotide sequence of the homologous nucleic acid fragment corresponding to the chicken W chromosome is shown in SEQ ID NO.5. It is 355 bp in length and is located at position 7896015–7896369 on the chicken W chromosome, corresponding to accession number NC_052571.1.

[0009] This invention provides a PCR sex determination method for chickens, comprising the following steps: 1) Extract genomic DNA from the chicken samples to be tested; The chicken samples to be tested were derived from blood, feather follicles, feather marrow, muscle tissue, chicken embryo tissue, oral swabs, allantoic membrane, allantoic fluid, or other samples containing chicken genomic DNA. 2) Using the genomic DNA to be tested as a template, PCR amplification was performed using the above-mentioned PCR primer set; The PCR amplification reaction system consisted of: 12.5 μL of 2×PCR Master Mix, 0.5–1.0 μL of forward primer, 0.5–1.0 μL of reverse primer, 1 μL of tissue template, and ddH2O added to a final volume of 25 μL. The PCR amplification reaction program is as follows: 98℃ pre-denaturation for 1-3 min, 98℃ denaturation for 10-30 s, 60℃ annealing for 5-30 s, 68℃ extension for 5-30 s, 35 cycles, and 68℃ final extension for 2-5 min. 3) The PCR products were subjected to agarose gel electrophoresis. The sex was determined based on the band pattern. When there was a single band, the individual was determined to be male. When there were two bands, the individual was determined to be female. The sequence length of a single band is 459 bp, as shown in SEQ ID NO.4; The sequence lengths of the two bands are 459 bp and 355 bp, respectively, as shown in SEQ ID NO.4 and SEQ ID NO.5.

[0010] The present invention also provides a kit for sex determination of chickens, the kit comprising the above-mentioned PCR primer set; It also includes PCR reaction solution, DNA polymerase, dNTPs, buffer, and Mg. 2+ One or more of the following: positive control, negative control, and DNA molecular weight standard; The DNA polymerase is a thermostable DNA polymerase, a high-fidelity DNA polymerase, or a hot-start DNA polymerase.

[0011] The present invention also provides the application of the above-mentioned PCR primer set or kit in chicken embryo sex identification, chick sex identification, adult chicken sex identification, chicken genetic breeding, chicken embryo experimental grouping, breeding chicken selection or production group management.

[0012] The beneficial effects of this invention are as follows: The PCR primer set and method for chicken sex determination of this invention directly compares the sequences of the chicken Z chromosome and W chromosome, and finds a stable length difference site in the Z / W homologous region. This region forms a 459 bp amplified fragment on the Z chromosome, and a 355 bp amplified fragment on the W chromosome due to a 104 bp deletion. After designing a pair of PCR primers based on this difference, male chicken samples amplify a single 459 bp band, and female chicken samples amplify a double band of 459 bp and 355 bp, thereby realizing chicken sex determination. The method of this invention is simple to operate, intuitive to interpret, and low in cost, and can be used for rapid sex determination of chicken embryos, chicks, adult chickens, and chicken tissue samples. Attached Figure Description

[0013] Figure 1 A schematic diagram showing the deletion of 104 bp in the chicken Z / W homologous amplification region; Figure 2 This is a graph showing the PCR amplification results. Detailed Implementation

[0014] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0015] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0016] Example 1 Z / W chromosome homologous region screening The genome sequences of chicken Z and W chromosomes were obtained (reference genome: GRCg7b (GCF_016699485.2)). The accession numbers for Z and W chromosomes were NC_052572.1 and NC_052571.1, respectively. Homology alignment was performed on the Z and W chromosome sequences. The alignment analysis revealed a target sequence on the Z chromosome, which has a homologous segment on the W chromosome.

[0017] Further comparison revealed that, for example Figure 1 Sequence alignment revealed a 104 bp deletion on chromosome W relative to chromosome Z at alignment positions 31-134. This deletion is located on chromosome Z at positions 71043928-71044031, corresponding to accession number NC_052572.1, and its nucleotide sequence is shown in SEQ ID NO.1. This deletion resulted in the same primer pair amplifying only the 459 bp fragment from Z in rooster ZZ, and simultaneously amplifying both the 459 bp fragment from Z and the 355 bp fragment from W in hen ZW.

[0018] This 104 bp deletion difference constitutes a Z / W chromosome homologous molecular marker that can be used for sex determination in chickens.

[0019] Example 2 Primer design and synthesis Specific primers were designed at the common sequence positions of the W and Z chromosomes upstream and downstream of the above-mentioned 104 bp deletion sequence, with nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0020] The primer set amplified only the 459 bp fragment from Z in the rooster ZZ individual, and simultaneously amplified the 459 bp fragment from Z and the 355 bp fragment from W in the hen ZW individual.

[0021] The 459 bp fragment originating from Z is located on the chicken Z chromosome at positions 71043897-71044355, corresponding to accession number NC_052572.1. Its nucleotide sequence is shown in SEQ ID NO.4. The W-origin 355 bp fragment is located on the chicken Z chromosome at physical positions 7896015-7896369 corresponding to accession number NC_052571.1, and its nucleotide sequence is shown in SEQ ID NO.5.

[0022] Example 3 A PCR sex determination method for chickens 1) Collect chicken samples to be tested as PCR amplification templates. a) Select chicken tissue (embryo, but not limited to embryo) and preserve it in a 1.5 mL centrifuge tube; b) Add 200 μL of ATL and 20 μL of Proteinase K to the sample and incubate at 55°C with shaking for 1 hour; c) Add 200 μL of AL reagent to the sample, vortex at high speed for 10 seconds, and incubate at 70°C for 10 minutes. d) Add 200 μL of anhydrous ethanol to the sample and vortex at high speed for 10 seconds; e) Place Hipure DNA Mini Column I into 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 10000×g for 1 minute; f) Discard the filtrate and reassemble the column into the collection tube; Add 500 μL of Buffer GW1 (diluted with ethanol) to the column and centrifuge at 10000×g for 1 minute; g) Discard the filtrate and reassemble the column into the collection tube; Add 650 μL of Buffer GW2 (diluted with ethanol) to the column and centrifuge at 10000×g for 1 minute; h) Discard the filtrate and reassemble the column into the collection tube; Centrifuge at 10000×g for 1 minute; i) Reassemble the column into a new 1.5 mL centrifuge tube; Add 30 μL of DNA elution buffer at 70°C to the center of the membrane on the column, let stand for 3 minutes, and then centrifuge at 10000×g for 1 minute. j) Discard the DNA binding column, determine the concentration, and store at -20°C.

[0023] Chicken samples to be tested can also be blood, feather follicles, feather marrow, muscle tissue, chicken embryo tissue, oral swabs, or other materials containing chicken genomic DNA.

[0024] 2) PCR amplification was performed using the primer set from Example 2. Five DNA samples were selected from each of the sex-identified female and male individuals and pooled separately. Using the mixed DNA from the female and male pools as templates, PCR reactions were performed using primers F and R from Example 2 at different annealing temperatures to amplify the target fragments. PCR amplification reaction system: 12.5 μL of 2×PCR Master Mix, 0.5 μL of forward primer, 0.5 μL of reverse primer, 1 μL of tissue template, and ddH2O added to 25 μL; PCR amplification reaction program: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 60℃ annealing for 5 s, 68℃ extension for 5 s, 35 cycles, 68℃ final extension for 3 min. 3) Collect the PCR amplification products and detect them by agarose gel electrophoresis. Electrophoresis was performed using 1×TAE as buffer and 1% agarose gel.

[0025] Electrophoresis results are judged according to the following criteria: If a sample shows only a 459 bp band, the individual from which the sample originated is determined to be a male chicken. If a sample shows both 459 bp and 355 bp bands, the individual from which the sample is taken is determined to be a female chicken. If the target band is not found or severe nonspecific amplification occurs, the test is considered invalid.

[0026] According to Figure 2 The PCR electrophoresis results showed that the male sample showed a single band of 459 bp, while the female sample showed two bands of 459 bp and 355 bp, which is consistent with the sex determination method established in this invention.

[0027] As can be seen, this invention utilizes a pair of primers to simultaneously amplify homologous fragments of the Z and W chromosomes in the same reaction system, and can visually distinguish between male and female individuals by ordinary agarose gel electrophoresis, thereby achieving chicken sex identification.

Claims

1. A PCR sex determination method for chickens, characterized in that, Includes the following steps: 1) Extract genomic DNA from the chicken samples to be tested; 2) Using the genomic DNA to be tested as a template, PCR amplification was performed using a set of PCR primers; The nucleotide sequences of the forward and reverse primers of the PCR primer set are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively; 3) The PCR products were subjected to agarose gel electrophoresis. The sex was determined based on the band pattern. When there was a single band, the individual was determined to be male. When there were two bands, the individual was determined to be female. The sequence of the single band is shown in SEQ ID NO.4; The sequences of the two bands are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively.

2. The application of the PCR sex determination method for chickens as described in claim 1 in chicken embryo sex determination, chick sex determination, adult chicken sex determination, or breeding stock sex selection.

3. The application of a PCR primer set for chicken sex identification in chicken embryo sex identification, chick sex identification, adult chicken sex identification, or breeding stock sex selection, characterized in that, The nucleotide sequences of the forward and reverse primers of the PCR primer set are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively; The PCR primer set was used to amplify a molecular marker on the chicken Z chromosome. The sequence of the molecular marker is shown in SEQ ID NO.1, and it is located on the chicken Z chromosome at positions 71043928-71044031, corresponding to accession number NC_052572.

1. The application method includes: extracting genomic DNA from the chicken sample to be tested, performing PCR amplification using the PCR primer set, and performing agarose gel electrophoresis on the PCR product. When there is a single band, the individual to be tested is determined to be male; when there are two bands, the individual to be tested is determined to be female. The sequence of the single band is shown in SEQ ID NO.4; The sequences of the two bands are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively.

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