NIPBL gene for judging chicken sex and application

Abstract

The invention belongs to the technical field of biomolecules, and particularly relates to an NIPBL gene for judging the sex of chickens and application of the NIPBL gene. The NIPBL gene has a segment with the difference of 606bp on chicken Z and W chromosomes, and is also a deletion segment on the W chromosome, the chicken sex can be quickly and effectively identified by detecting the sequence condition, the chicken with the deletion of the molecular marker sequence segment is a female individual, and the chicken without the deletion of the molecular marker sequence segment is a male individual; the invention further provides a specific primer, the chicken sex can be accurately judged through a PCR amplification technology and agarose gel electrophoresis, a strip is clear, the method is small in technical difficulty, easy and convenient to operate and high in accuracy, the sex of chicks or chickens difficult to distinguish in appearance can be effectively identified, the method has practical significance on identification of varieties small in male and female appearance difference, and the method is worthy of popularization and application. The method disclosed by the invention is strong in technical applicability and good in repeatability, can be used for accurately identifying the gender of the poultry, and has a wide application prospect.

Description

Technical Field

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

[0002] Since different types of poultry have obvious gender preferences during the production process, it is of great significance to develop simple and convenient sex identification technology. 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 narrow and limited to some specific breeds. Anal inversion identification must be performed at the age of 1 day after hatching, which is very irritating to the chicks, and 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] The sex chromosome system of most mammals is XX / XY type, XY heterogametic development forms males, and XX homogametic development forms females. In this type of animal, the Sry gene located on the Y chromosome can regulate the process of sex determination. Based on the different distribution of this gene on the X and Y chromosomes, there is a Chinese patent CN1664110A that discloses a method of using PCR technology to amplify the Sry gene sequence to identify the sex of mammals. Unlike mammals, the sex chromosome system of birds is ZZ / ZW type, ZW heterogametic development forms females, and ZZ homogametic development forms males, which is exactly opposite to the sex chromosome control sex development pattern of mammals. At present, the molecular method for identifying the sex of chickens is mainly based on the CHD1 gene sequence, which is distributed on both the Z and W chromosomes of birds, and the lengths of CHD1-Z and CHD1-W are different due to different evolutionary rates. The length of PCR amplification can be used to determine the sex of birds. Using the CHD1 gene, studies at home and abroad have explored molecular sex detection for rare poultry, including chickens, and further used this gene to identify the sex of pigeons, quails, parrots, etc. by improving specific primer sequences (Chinese patent CN101962677A). Therefore, the method of determining sex based on the difference in the length of the fragments distributed on different sex chromosomes of the same gene is feasible. Summary of the invention

[0004] In view of the above problems, the object of the present invention is to provide a NIPBL gene and application for determining the sex of chickens.

[0005] The technical contents of the present invention are as follows: 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 606 bp between the NIPBL gene on the chicken chromosome Z and the chromosome W, and the sequence is shown in SEQ ID NO.1; The individuals with or without the sequence fragment of SEQ ID NO.1 are female individuals, and the individuals without or without the sequence fragment of SEQ ID NO.1 are male individuals; The gene accession number (Gene ID) of the NIPBL gene on chromosome Z in the NCBI database is 427439, and the sequence range is: 11847125-12006549; The gene accession number of the NIPBL gene on the W chromosome in the NCBI database is 427025, and the sequence range is: 5323305-5482338.

[0006] 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 through gene sequence sequencing, the presence or absence of a sequence fragment of SEQ ID NO.1 indicates a female individual (ZW-type chromosome), and the absence of a sequence fragment of SEQ ID NO.1 indicates a male individual (ZZ-type chromosome).

[0007] The present invention also provides a gene fragment for determining the sex of a chicken, wherein the gene fragment is located on the NIPBL gene of the chicken chromosome, and its nucleotide sequence on the chicken Z chromosome is shown as SEQ ID NO.2, and its nucleotide sequence on the W chromosome is shown as SEQ ID NO.3.

[0008] 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.

[0009] The present invention also provides a primer pair for determining the molecular marker of the sex of chickens. 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.

[0010] The present invention also provides a method for determining the sex of a chicken, comprising the following steps: 1) Extract genomic DNA from the chicken individuals to be tested; 2) Using the genomic DNA to be tested as a template, perform PCR amplification using the above primer pairs; 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; The PCR reaction system: 2X PCR Taq Master Mix 12.5 μL, forward and reverse primers 0.5 μL each, DNA template 200 ng, ddH2O added to 25 μL; The PCR reaction procedure is as follows: 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.

[0011] The sequence length of the single band is 1009 bp, as shown in SEQ ID NO.2; The sequence lengths of the double bands are 1009 bp and 403 bp, respectively, as shown in SEQ ID NO.2 and SEQ ID NO.3.

[0012] 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; The detection product comprises the above-mentioned primer pair.

[0013] The beneficial effects of the present invention are as follows: The molecular marker for determining the sex of a chicken of the present invention is a segment of the NIPBL gene on the chicken chromosome Z and W with a difference of 606 bp, which is also a missing segment on the W chromosome. By detecting the sequence, the sex of the chicken can be quickly and effectively identified. The individual with the missing molecular marker sequence segment is a female individual, and the individual without the missing molecular marker sequence segment is a male individual. The present invention also provides specific primers. Through PCR amplification technology and agarose gel electrophoresis, the bands are clear and the sex of the chicken can be accurately determined. The method has low technical difficulty, simple operation and high accuracy, can effectively identify the sex of chicks or chickens that are difficult to distinguish in appearance, and has practical significance for the identification of varieties with small differences in male and female appearance. The technical applicability of the present invention is strong, the repeatability is good, the sex of poultry can be accurately identified, and the application prospect is broad. Through early embryo sex identification, the specialized breeding of poultry of different sexes is realized, the breeding group structure is optimized, and the breeding efficiency and economic benefits are improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a sequence comparison of the 606 bp deletion fragment of the NIPBL gene on chromosome Z and chromosome W; Figure 2 Figure 1 is the amplification result of CHD1 specific primers; Figure 3 This is a diagram showing the amplification results of the specific primers of the present invention. DETAILED DESCRIPTION

[0015] 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.

[0016] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0017] Example 1 Acquisition of NIPBL gene 1) Obtaining the sex chromosome sequence of the NIPBL gene The NIPBL genomic sequence on chicken chromosomes was searched from the NCBI database (https: / / www.ncbi.nlm.nih.gov). The gene accession number of the NIPBL gene on chromosome Z in the NCBI database was 427439, and the sequence range was 11847125-12006549; the gene accession number of the NIPBL gene on chromosome W in the NCBI database was 427025, and the sequence range was 5323305-5482338.

[0018] 2) Sequence alignment The sequences on chromosome W and Z of NIPBL obtained above were compared using DNAMAN software. Figure 1 As shown, the NIPBL gene fragment located on the Z chromosome has a nucleotide sequence as shown in SEQ ID NO.2, and the NIPBL gene fragment located on the W chromosome has a nucleotide sequence as shown in SEQ ID NO.3, wherein the W chromosome has a 606 bp deletion fragment compared with the Z chromosome, and its nucleotide sequence is shown in SEQ ID NO.1, which is used as a molecular marker to determine the sex of chickens.

[0019] 3) Primer design and synthesis 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; Primer F (SEQ ID NO. 4): 5′-GTATGCTGGTTTCATTCACGTAAG; Primer R (SEQ ID NO. 5): 5′-GCTTATGTCAACTGTAGTG.

[0020] Specific primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0021] Example 2 Validation of NIPBL gene for determining chicken sex 1. Sample Collection and DNA Extraction 1) Select chicken tissue and store it in a 1.5 mL centrifuge tube; 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; 3) Add 200 μL AL reagent to the sample, vortex at high speed for 10 seconds, and incubate at 70°C for 10 minutes; 3) Add 200 μL of anhydrous ethanol to the sample and vortex at high speed for 10 seconds; 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; 5) Discard the filtrate and put the column back into the collection tube; Add 500 μL of Buffer GW1 (diluted with ethanol) to the column and centrifuge at 10,000 × g for 1 min; 6) Discard the filtrate and put the column back into the collection tube; Add 650 μL of Buffer GW2 (diluted with ethanol) to the column and centrifuge at 10,000 × g for 1 min; 7) Discard the filtrate and put the column back into the collection tube; Centrifuge at 10,000 × g for 1 minute. 8) Place the column back into a new 1.5 mL centrifuge tube; 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; 9) Discard the DNA binding column, determine the concentration, and store at -20°C.

[0022] 2. PCR Amplification The target fragment was amplified by polymerase chain reaction (PCR) using the above-synthesized primer pair, and electrophoresis was performed using 1×TAE as a buffer and 1% agarose gel for about 25 minutes at a voltage of 135 V / min. The product was photographed and stored using a gel imaging system.

[0023] The PCR system is shown in Table 1, and the amplification reaction conditions are shown in Table 2.

[0024] Table 1 Components of PCR reaction

[0025] Table 2 PCR amplification program

[0026] The current molecular method for chicken sex identification is mainly based on the CHD1 gene sequence, using the published CHD1-specific primers (F: 5'-GTTACTGATTCGTCTACGAGA-3', R: 5'-TTGAAATGATCCAGTGCTTG-3') to amplify the chicken DNA. Figure 2 As shown, the hen amplified two bands, 450 bp and 600 bp respectively; the rooster amplified a single band, with a length of 600 bp.

[0027] Based on the above results, 5 DNA samples were selected from the individuals whose genders were determined to be female and male and mixed them separately. The mixed pool DNA of female and male was used as template, and PCR reaction was performed at different annealing temperatures using primers F and R of Example 1. At the same time, the result of CHD1 primer was used as positive control, and water was used as template as negative control. The results are as follows: Figure 3 As shown, two bands were detected in female chickens, and compared with the marker, the product lengths were consistent with the theoretical results of 403bp and 1009bp; one band was detected in male chickens, and the product length was consistent with the theoretical result of 1009bp; 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.

[0028] In addition, when the specific primers provided by the present invention are annealed at a temperature of 42°C, male chickens show a single clear band, while female chickens show obvious double bands, which is the best effect. Figure 3 The detection results of this study are consistent with the determination results of CHD1 gene.

[0029] In summary, a gene NIPBL that can be used to identify the sex of chickens and its application are obtained. By amplifying the genomic sequences on the W and Z chromosomes of NIPBL including the deleted fragment through 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). The presence of the deleted fragment is a female individual (ZW type chromosome), and the absence of the deleted fragment is a male individual (ZZ type chromosome).

Claims

1. A molecular marker for determining the sex of a chicken, characterized in that: The molecular marker is a nucleotide sequence with a length difference of 606 bp between the NIPBL genes on the chicken Z chromosome and the W chromosome, and its sequence is shown in SEQ ID NO.

1.

2. The molecular marker for determining the sex of a chicken according to claim 1, characterized in that: The gene accession number of the NIPBL gene on chromosome Z in the NCBI database is 427439, and the sequence range is: 11847125-12006549.

3. The molecular marker for determining the sex of a chicken according to claim 1, characterized in that: The gene accession number of the NIPBL gene on the W chromosome in the NCBI database is 427025, and the sequence range is: 5323305-5482338.

4. An application of the molecular marker for determining the sex of chickens as claimed in claim 1, characterized in that: The nucleotide sequence of the molecular marker is shown as SEQ ID NO.

1. By sequencing the gene sequence of chicken chromosomes, those with a missing sequence fragment of SEQ ID NO.1 are female individuals, and those without a missing sequence fragment of SEQ ID NO.1 are male individuals.

5. A gene fragment for determining the sex of a chicken, characterized in that: The gene fragment is located on the NIPBL gene of the chicken chromosome, and its nucleotide sequence on the chicken Z chromosome is shown as SEQ ID NO.2, and its nucleotide sequence on the chicken W chromosome is shown as SEQ ID NO.

3.

6. An application of the gene fragment for determining the sex of a chicken according to claim 5, characterized in that: By sequencing the gene sequence of chicken chromosomes, the individuals with the sequence containing only SEQ ID NO.2 are determined to be male individuals, and the individuals with the sequence containing both SEQ ID NO.2 and SEQ ID NO.3 are determined to be female individuals.

7. A primer pair for determining the sex of a chicken using a molecular marker, characterized in that: 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 gene fragment according to claim 5.

8. 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 and using the primer pair described in claim 7 to perform PCR amplification; 3) The PCR product was subjected to agarose gel electrophoresis, and the sex 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.

9. The method for determining the sex of a chicken according to claim 8, characterized in that: The sequence length of the single band is 1009 bp, as shown in SEQ ID NO.2; The sequence lengths of the double bands are 1009 bp and 403 bp, respectively, as shown in SEQ ID NO.2 and SEQ ID NO.

3.

10. A detection product for determining the sex of a chicken, characterized in that: The detection product includes a detection reagent or a test kit; The composition of the detection product includes the primer pair described in claim 7.

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