A structural variation molecular marker related to gape character and its application in the breeding of self-alternate male and female mating line of wulong goose

Abstract

The application discloses a structural variation molecular marker related to a goose eye defect trait and application of the structural variation molecular marker in Wulong goose self-differentiated male and female mating line breeding. The application provides application of a substance for detecting whether CA is inserted between 272-273 of SEQ ID NO. 1 on a Z chromosome in a goose genome in any one of the following or in preparation of a product with any one of the following functions: A1) establishing goose self-differentiated male and female mating line breeding; A2) detecting or assisting in detecting goose offspring genders; A3) breeding homozygous eye defect geese; A4) breeding homozygous normal eye geese; A5) identifying or assisting in identifying genotypes of to-be-tested geese; A6) identifying or assisting in identifying eyelid traits of to-be-tested geese; the application selects the Wulong goose eye defect trait by determining the genotype of the to-be-tested goose, can save production cost and accelerate genetic progress, better serves the Wulong goose self-differentiated male and female mating line breeding, and has great economic application value and scientific research value.

Description

Technical Field

[0001] This invention belongs to the field of animal genetics and breeding technology, and relates to a structural variation molecular marker related to the eye-opening trait of geese and its application in the breeding of sex-separated mating lines for Wulong geese. Background Technology

[0002] In the brooding and rearing of commercial meat geese, male and female chicks typically need to be raised separately due to differences in their growth rates and environmental and nutritional requirements. This reduces feeding costs and improves economic efficiency. There are generally two methods for sexing goslings: one is to observe the reproductive organ structure through manual vent turning, and the other is to use sex-linked inheritance of physical characteristics such as feather color. Currently, sexing of goslings is still done through vent turning, which not only increases labor costs in the production process but also causes significant stress to the goslings. Furthermore, vent turning exposes the genitals, increasing the risk of exposure to pathogens and disease infection. Therefore, there is an urgent need to develop economical, efficient, and hygienic techniques for sexing goslings.

[0003] Studies have shown that the eye-opening trait in Wulong geese is a Z-linked recessive trait, and this trait is fully penetrating at hatching, making it easily observable with the naked eye and suitable for breeding sex-separation lines. Breeding sex-separation lines first requires establishing a genotype-purified eye-opening paternal parent and a normal-eyed maternal parent. Using traditional breeding methods, this process typically requires several generations. However, if the genes controlling the eye-opening trait are known, molecular marker-assisted breeding technology can achieve the desired breeding goals in just one generation of selection.

[0004] Molecular marker-assisted breeding (MMR) is a newly developed breeding technique that has been widely applied to the cultivation of new varieties of plants and animals. Genome-wide association studies (GWAS) can identify molecular markers closely associated with target traits. Using these markers to select for the target population can enable the target trait to achieve genotypic homozygosity within one generation, thereby improving breeding efficiency and reducing production costs. Summary of the Invention

[0005] The technical problem solved by this invention is how to establish a breeding system for differentiating male and female geese using new loci, which is used to select for the eye-opening trait in geese, thereby saving production costs and accelerating genetic progress.

[0006] To address the aforementioned technical problems, the first aspect of this invention provides the use of a substance for detecting the presence of the insertion sequence CA between SEQ ID NO. 1 segments 272-273 on chromosome Z in a goose genome, in any of the following applications or in the preparation of a product having any of the following functions:

[0007] A1) Establish breeding systems for male and female geese;

[0008] A2) Detection or auxiliary detection of the sex of goose offspring;

[0009] A3) Breeding homozygous open-eyed geese;

[0010] A4) Select and breed homozygous normal-eyed geese;

[0011] A5) Identify or assist in identifying the genotype of the goose to be tested;

[0012] A6) To identify or assist in the identification of the eyelid characteristics of the goose to be tested;

[0013] The goose's genotype is Z. M Z M Z M W, Z N Z N Z N W or Z M Z N ;

[0014] The Z M Z M In the genome of the male goose with the genotype to be tested, there is a 2bp insertion sequence CA between positions 272-273 on both Z chromosomes of SEQ ID NO. 1;

[0015] The Z M The W genotype indicates that there is a 2bp insertion sequence CA between positions 272-273 of chromosome Z in the genome of the female goose being tested;

[0016] The Z N Z N The genome of the male goose with the genotype to be tested did not contain the 2bp insertion sequence CA between positions 272 and 273 of SEQ ID NO. 1 on either of the two Z chromosomes;

[0017] The Z N The W genotype indicates that there is no 2bp insertion sequence CA between positions 272-273 on chromosome Z of the test goose in the genome of the female goose with the W genotype.

[0018] The Z M Z N The genotype of the male goose being tested is characterized by a 2bp insertion sequence CA between positions 272-273 of SEQ ID NO.1 on one Z chromosome and a absence of the 2bp insertion sequence CA between positions 272-273 of SEQ ID NO.1 on the other Z chromosome.

[0019] In the applications described above, the substances include the following:

[0020] B1) Primer pair;

[0021] The primer pair consists of a single-stranded DNA molecule shown in SEQ ID No. 2 and a single-stranded DNA molecule shown in SEQ ID No. 3;

[0022] B2) Reagents or kits containing the primer pairs.

[0023] A second aspect of the present invention provides a method for identifying or assisting in the identification of eyelid traits in geese to be tested, comprising the following steps: identifying the genotype of the geese to be tested;

[0024] If the genotype of the male goose being tested is Z M Z M Or the genotype of the female goose to be tested is Z. M If W, then the male or female goose being tested has the open-eye phenotype;

[0025] If the genotype of the male goose being tested is Z N Z N or Z M Z N Or the genotype of the female goose to be tested is Z. N If W, then the male or female goose being tested has a normal ocular phenotype;

[0026] The goose's genotype is Z. M Z M Z M W, Z N W and Z M Z N ;

[0027] The Z M Z M In the genome of the male goose with the genotype to be tested, there is a 2bp insertion sequence CA between positions 272-273 on both Z chromosomes of SEQ ID NO. 1;

[0028] The Z M The W genotype indicates that there is a 2bp insertion sequence CA between positions 272-273 of chromosome Z in the genome of the female goose being tested;

[0029] The Z N Z N The genome of the male goose with the genotype to be tested did not contain the 2bp insertion sequence CA between positions 272 and 273 of SEQ ID NO. 1 on either of the two Z chromosomes;

[0030] The Z N The W genotype indicates that there is no 2bp insertion sequence CA between positions 272-273 on chromosome Z of the test goose in the genome of the female goose with the W genotype.

[0031] The Z M Z N The genotype of the male goose being tested is characterized by a 2bp insertion sequence CA between positions 272-273 of SEQ ID NO.1 on one Z chromosome and a absence of the 2bp insertion sequence CA between positions 272-273 of SEQ ID NO.1 on the other Z chromosome.

[0032] The method described above for identifying the genotype of the goose to be tested includes the following steps: detecting whether a CA is inserted between chromosome 272 and 273 of SEQ ID NO.1 on the Z chromosome of the goose to be tested, and determining the genotype as follows:

[0033] If a CA is inserted between chromosomes 272-273 of SEQ ID NO.1 on both Z chromosomes of the goose to be tested, then the genotype of the male goose to be tested is Z. M Z M The genotype of the female goose being tested is Z. M W;

[0034] If there is no CA insertion between chromosomes 272-273 of SEQ ID NO.1 on either of the two Z chromosomes in the genome of the goose to be tested, then the genotype of the male goose to be tested is Z. N Z N The genotype of the female goose being tested is Z. N W;

[0035] If, in the genome of the goose to be tested, there is no CA insertion between SEQ ID NO. 1 segments 272-273 on one Z chromosome, and a CA insertion is found between SEQ ID NO. 1 segments 272-273 on the other Z chromosome, then the goose to be tested is a male goose, and its genotype is Z. M Z N .

[0036] The method described above, specifically the detection of whether a CA is inserted between chromosome 272 and 273 of SEQ ID NO.1 on the Z chromosome of the goose genome to be tested, includes the following steps:

[0037] Using the genomic DNA of the goose to be tested as a template, the primer pair described in the first aspect is used for amplification, and the resulting sequencing product is sent for sequencing. Based on the sequencing results, it is determined whether CA is inserted between SEQ ID NO.1 272-273 in the genome of the goose to be tested.

[0038] Thirdly, the present invention provides a method for identifying the genotype of a goose to be tested, as described in the second aspect.

[0039] Fourthly, this invention provides a method for breeding homozygous bright-eyed geese, specifically for breeding geese with the genotype Z mentioned in the second aspect.M Z M Male geese or those with genotype Z M W's mother goose.

[0040] Fifthly, the present invention provides a method for breeding homozygous normal-eyed geese, specifically for breeding geese with the genotype Z in the second aspect. N Z N Male geese or those with genotype Z N W's mother goose.

[0041] In the above text, the homozygous swan with open eyes refers to an individual or a group.

[0042] In the above text, the homozygous normal-eyed geese refer to individuals or groups.

[0043] Sixthly, the present invention provides a breeding method for establishing a sex-matching system for geese, comprising the following steps:

[0044] 1) In the second aspect, the genotype is Z M Z M Male geese and genotype Z N W's female geese are bred to produce offspring;

[0045] 2) Detect the eyelid morphology of offspring. Offspring with open-eyed phenotype are female geese or candidates, and offspring with normal eye phenotype are male geese or candidates, thus realizing the establishment of a breeding system for distinguishing male and female geese.

[0046] In a seventh aspect, the present invention provides a method for detecting or assisting in the detection of the sex of goose offspring, comprising the following steps:

[0047] 1) The genotype Z in the second aspect M Z M Male geese and genotype Z N W's female geese mate and produce offspring;

[0048] 2) Detect the eyelid morphology of the offspring. Offspring with open-eyed phenotype are female geese or candidates, and offspring with normal eye phenotype are male geese or candidates.

[0049] The goose mentioned above can be the Wulong Goose.

[0050] The inventors of this invention, through whole-genome sequencing of a large number of goose samples, mined molecular markers related to eyelid blepharoplasty (or non-eyelid blepharoplasty) in geese. Genome-wide association study (GWAS) analysis revealed a 2bp insertion sequence between chromosome 272-273 of SEQ ID NO. 1 on the goose genome. This mutation affects the eyelid blepharoplasty / non-eyelid trait in Wulong geese. Based on this molecular marker, screening for the eyelid blepharoplasty / non-eyelid trait in geese can be achieved. The genotype for the eyelid blepharoplasty trait is Z. M Z M ZM W, the genotype for the non-eyelid trait is Z. N Z N Z N W or Z M Z N .

[0051] The beneficial effects of this invention are as follows:

[0052] 1. It can achieve rapid purification of eyelid characteristics.

[0053] 2. It can help geese distinguish between male and female.

[0054] This invention selects for the eye-opening trait in Wulong geese by determining the genotype of the goose to be tested. This can save production costs and accelerate genetic progress, better serving the breeding of Wulong geese for sex-selective mating lines. It has great economic and scientific research value. Attached Figure Description

[0055] Figure 1 The graph shows the results of the whole-genome association analysis of the eye-opening trait in geese, where: the horizontal axis represents the chromosome number of the goose; and the vertical axis represents the -logP value.

[0056] Figure 2 Diagram illustrating the production model of the Wulong Goose's sex-separated breeding system.

[0057] Figure 3 The image shows the sequencing results of geese with different genotypes. Detailed Implementation

[0058] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0060] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0061] Example 1: Determination of molecular markers associated with the eye-opening trait in geese and their application in identifying the eye-opening trait or genotype in geese.

[0062] I. Determination of Molecular Markers Related to the Eye-Opening Trait in Geese

[0063] Experimental materials: 60 adult Wulong geese, of which 30 had open eyes (15 males and 15 females) and 30 had normal eyes (15 males and 15 females).

[0064] 1. Observation Record of the Eye-Opening Trait in Geese

[0065] Observe whether there is a notch in the upper eyelid and take a photo for record. Those with notches are called "eye gaps".

[0066] 2. Identification of molecular markers

[0067] 1) Blood sample collection: Collect venous blood from the goose wing to be tested using EDTA anticoagulant blood collection tubes and store at -20℃ for later use.

[0068] 2) Whole blood genomic DNA extraction: Refer to the instructions for the blood genomic DNA extraction kit (Tiangen Biotech Co., Ltd., DP319) for specific operating procedures.

[0069] 3) Genotyping: Genomic DNA was collected from each goose to be tested. Genome resequencing was performed using a DNBSEQ-T7 gene sequencer. The sequencing depth for each individual was 10×, the sequencing strategy was PE-150bp, the insert length was 350bp, and the data volume for each individual was no less than 10G. After quality control, the data were used to perform sequence alignment and genotype extraction using two bioinformatics software programs, BWA and GATK.

[0070] 3. Genome-wide association analysis of the eye-opening trait in geese

[0071] Genome-wide association analysis of the eye-opening phenotype and genotype in geese was performed using Emmax software. The most significant SNPs were subjected to Haploview linkage analysis, which revealed a 2bp insertion sequence CA present only in individuals with the eye-opening phenotype. This 2bp insertion sequence CA is located between chromosome 47280796 and 47280797 on chromosome Z (NC_089912.1), or between positions 272 and 273 of SEQ ID No. 1. The nucleotide sequence of this insertion fragment is SEQ ID No. 4, and the 2bp insertion sequence is located at positions 273-374 of SEQ ID No. 4. Individuals with the normal eye phenotype did not have the 2bp insertion sequence CA, and the nucleotide sequence of the fragment without the 2bp insertion sequence CA is SEQ ID No. 1.

[0072] SEQ ID No. 4 differs from SEQ ID No. 1 only in the insertion of CA.

[0073] Analysis revealed that the inserted sequence could fully explain the eye-blind trait of the Wulong goose, such as... Figure 1As shown, these are molecular markers for identifying structural variations.

[0074] The genotype of the goose being tested is determined by whether a 2bp CA is inserted between SEQ ID No. 1 and SEQ ID No. 4 in all Z chromosomes of the goose genome, or by whether all Z chromosomes of the goose genome contain the fragment shown in SEQ ID No. 1 or SEQ ID No. 4. M Z M Z M W, Z N Z N Z N W or Z M Z N ZZ represents a female goose, and ZW represents a male goose.

[0075] The Z M Z M In the genome of the male goose with the genotype to be tested, there is a 2bp insertion sequence CA between positions 272-273 on both Z chromosomes of SEQ ID NO. 1;

[0076] The Z M The W genotype indicates that there is a 2bp insertion sequence CA between positions 272-273 of chromosome Z in the genome of the female goose being tested;

[0077] The Z N Z N The genome of the male goose with the genotype to be tested did not contain the 2bp insertion sequence CA between positions 272 and 273 of SEQ ID NO. 1 on either of the two Z chromosomes;

[0078] The Z N The W genotype indicates that there is no 2bp insertion sequence CA between positions 272-273 on chromosome Z of the test goose in the genome of the female goose with the W genotype.

[0079] The Z M Z N The genotype of the male goose being tested is characterized by a 2bp insertion sequence CA between positions 272-273 of SEQ ID NO.1 on one Z chromosome and a absence of the 2bp insertion sequence CA between positions 272-273 of SEQ ID NO.1 on the other Z chromosome.

[0080] According to statistics related to the eye-opening trait in geese, Z M Z M Genotypes of male geese (Z chromosome homozygous) and Z M The female geese of the W chromosome (homozygous for the Z chromosome) all exhibited the open-eye phenotype, while the Z chromosome... N Z NGenotype of male goose (Z chromosome homozygous), Z N Female geese with the W genotype (homozygous for Z chromosome) or Z M Z N All male geese with the Z chromosome heterozygote genotype have normal eyes.

[0081] II. Establishment of a method for determining genotype by detecting molecular markers of structural variations and design of specific primers

[0082] 1. Primer design and synthesis

[0083] Based on the whole-genome sequencing results mentioned above, primers for identifying molecular markers of structural variation in geese were designed as follows:

[0084] Upstream primer: 5'-TTAACAGAAAACCTTGGTTGAAG-3' (SEQ ID No. 2);

[0085] Downstream primer: 5'-CGCACAATTAATTTTCCAAGGC-3' (SEQ ID No. 3).

[0086] 2. Establishment of a method for determining genotype by detecting molecular markers of structural variations.

[0087] 1) Extract genomic DNA from the venous blood of the goose wing to be tested;

[0088] 2) Using the genomic DNA above as a template, perform PCR amplification with upstream and downstream primers to obtain PCR amplification products.

[0089] The above PCR amplification products were sent for sequencing (e.g.) Figure 3 The results are as follows:

[0090] If none of the above PCR amplification products contain the fragment shown in SEQ ID No. 4 but do contain the fragment shown in SEQ ID No. 1, it indicates that there is no 2bp insertion sequence CA between positions 272-273 of SEQ ID No. 1 on the two Z chromosomes. Therefore, the genotype of the male goose being tested is Z. N Z N Genotype: The genotype of the female goose being tested is Z. N The W genotype indicates that both the male and female geese being tested have normal ocular surface types.

[0091] If all the PCR amplification products above contain the fragment shown in SEQ ID No. 4 but not the fragment shown in SEQ ID No. 1, it indicates that there is a 2bp insertion sequence CA between positions 272-273 of SEQ ID No. 1 on both Z chromosomes. Therefore, the genotype of the male goose being tested is Z. M Z M Genotype, the genotype Z of the female goose to be tested.M The W genotype indicates that both male and female geese tested exhibit the open-eye phenotype.

[0092] If the PCR amplification product exhibits a heterozygous peak, containing both the fragment shown in SEQ ID No. 1 and the fragment shown in SEQ ID No. 4, it indicates that there is a 2bp insertion sequence CA between positions 272-273 of SEQ ID No. 1 on one Z chromosome, and no 2bp insertion sequence CA between positions 272-273 of SEQ ID No. 1 on the other Z chromosome. Therefore, the goose being tested is a male goose with a genotype of Z. M Z N Genotype, with normal ocular phenotype.

[0093] Example 2: Application of structural variation molecular markers in identifying the sex of Wulong geese

[0094] For the Wulong goose population, two base groups were established based on genotype: one with bulging eyes and the other with normal eyes. The sex of the offspring population was then determined based on the bulging eye phenotype.

[0095] Experimental materials: A flock of 1000 Wulong geese bred at the Qingdao Agricultural University Waterfowl Breeding Base.

[0096] I. Establish groups for individuals with wide-set eyes and those with normal eye types.

[0097] 1. Determining genotype by detecting structural variation molecular markers

[0098] 1) Blood sample collection: Collect venous blood from the goose wing to be tested using EDTA anticoagulant blood collection tubes and store at -20℃ for later use.

[0099] 2) Whole blood genomic DNA extraction: Genomic DNA was extracted from all blood samples using conventional methods in this field.

[0100] 3) Genotype determination:

[0101] Using the genomic DNA of each goose as a template, the genotype of each individual was determined using the primers and methods designed in Example 1, Part 2.

[0102] If none of the above PCR amplification products contain the fragment shown in SEQ ID No. 4 but do contain the fragment shown in SEQ ID No. 1, it indicates that there is no 2bp insertion sequence CA between positions 272-273 of SEQ ID No. 1 on either chromosome of chromosome Z. Therefore, the genotype of the male goose being tested is Z. N Z N Genotype: The genotype of the female goose being tested is Z. N The W genotype indicates that both the male and female geese being tested have normal ocular surface types.

[0103] If all the PCR amplification products above contain the fragment shown in SEQ ID No. 4 but not the fragment shown in SEQ ID No. 1, it indicates that there is a 2bp insertion sequence CA between positions 272-273 of SEQ ID No. 1 on both chromosomes of chromosome Z. Therefore, the genotype of the male goose being tested is Z. M Z M Genotype, the genotype Z of the female goose to be tested. M The W genotype indicates that both male and female geese tested exhibit the open-eye phenotype.

[0104] If the PCR amplification product exhibits a heterozygous peak, containing both the fragment shown in SEQ ID No. 1 and the fragment shown in SEQ ID No. 4, it indicates that there is a 2bp insertion sequence CA between positions 272-273 of SEQ ID No. 1 on one homologous chromosome of chromosome Z, and there is no 2bp insertion sequence CA between positions 272-273 of SEQ ID No. 1 on the other homologous chromosome of chromosome Z. Therefore, the goose being tested is a male goose with the genotype Z. M Z N Genotype, with normal ocular phenotype.

[0105] 2. Establish homozygous iridomorphic group and homozygous normal eye type group.

[0106] 1) Establish a homozygous open-eye population:

[0107] The above genotype 1 is Z. M Z M The male goose and Z M The female geese were retained to form a group with open eyes (about 200 geese were selected, with a male-to-female ratio of 1:4).

[0108] 2) Establish a homozygous normal eye type group:

[0109] The genotype described above is Z. N Z N The male goose and Z N The female geese of W were retained to form a normal eye-shaped group (about 200 geese were selected, with a male-to-female ratio of 1:4).

[0110] 3. Verify the genetic stability of traits and genotypes

[0111] The genotypes and phenotypes of the offspring from these two populations were determined using the primers and methods designed in Example 1, Part 2.

[0112] The results showed that this trait could be stably inherited without segregation.

[0113] II. Early sex determination of geese based on the eye-opening phenotype

[0114] according to Figure 2The diagram illustrates the construction of a sex-separated breeding system for the Five Dragons Goose, as detailed below:

[0115] Twenty-five male geese (genotype Z) from the aforementioned open-eyed population were selected. M Z M Type Z), and 100 female geese of the normal eye type group (genotype Z). N The offspring were mated (W-type) and their eyelids were observed and their sex determined.

[0116] Two hundred one-day-old offspring were randomly selected. Phenotypic observation revealed that 96 individuals had the open-eyed phenotype and were identified as female geese; 104 individuals had the normal eye phenotype and were identified as male geese.

[0117] The sex of these individuals was verified by cloacal examination. Of the 96 geese with the open-eyed phenotype, all were female; of the 104 geese with the normal eye phenotype, 102 were male and 2 were female.

[0118] Therefore, the system established using this invention achieves a 100% accuracy rate in identifying female geese and a 98.1% accuracy rate in identifying the sex of male geese.

[0119] The genotypes of these offspring were determined using the primers and methods designed in Example 1, Part 2. The genotypes of the offspring were Z. M W, Z M Z N Based on the relationship between genotype and ocular phenotype mentioned above, Z M Type W is the female goose with the trait of open-eyedness, Z... M Z N The male goose had a normal eye. The female goose with the blepharoptosis had the genotype Z. M The W type, with a normal-eyed male goose genotype of Z. M Z N The method of sex determination was consistent with that of the present invention when other existing methods were used to test offspring.

[0120] This demonstrates that the method system for early sex identification of geese based on the eye-opening phenotype established in this invention is stable and reliable.

[0121] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for identifying or assisting in the identification of eyelid traits in a Wulong goose, comprising the following steps: identifying the genotype of the goose to be tested; if the genotype of the male goose to be tested is Z... M Z M Or the genotype of the female goose to be tested is Z. M If the genotype is W, then the male or female goose being tested has the open-eye phenotype; if the genotype of the male goose being tested is Z... N Z N or Z M Z N Or the genotype of the female goose to be tested is Z. N If W, then the male or female goose being tested has a normal ocular phenotype; The eyelid condition is described as either open-eyed or normal. The goose's genotype is Z. M Z M Z M W, Z N Z N Z N W and Z M Z N ; The Z M Z M In the genome of the male goose with the genotype to be tested, there is a 2bp insertion sequence CA between positions 272-273 on both Z chromosomes of SEQ ID NO. 1; The Z M The W genotype indicates that there is a 2bp insertion sequence CA between positions 272-273 of chromosome Z in the genome of the female goose being tested; The Z N Z N The genome of the male goose with the genotype to be tested did not contain the 2bp insertion sequence CA between positions 272 and 273 of SEQ ID NO. 1 on either of the two Z chromosomes; The Z N The W genotype indicates that there is no 2bp insertion sequence CA between positions 272-273 on chromosome Z of the test goose in the genome of the female goose with the W genotype. The Z M Z N The genotype of the male goose being tested is characterized by a 2bp insertion sequence CA between positions 272-273 of SEQ ID NO.1 on one Z chromosome and a absence of the 2bp insertion sequence CA between positions 272-273 of SEQ ID NO.1 on the other Z chromosome.

2. The method according to claim 1, characterized in that: The method for identifying the genotype of the goose to be tested includes the following steps: detecting whether a CA is inserted between chromosome 272 and 273 of SEQ ID NO.1 on the Z chromosome of the goose to be tested, and determining the genotype as follows: If an insertion sequence CA exists between chromosome 272-273 of SEQ ID NO.1 on the Z chromosome of the goose genome to be tested, then the genotype of the male goose to be tested is Z. M Z M The genotype of the female goose to be tested is Z. M W; If there is no CA insertion between chromosome 272 and 273 of SEQ ID NO.1 on the Z chromosome of the goose genome to be tested, then the genotype of the male goose to be tested is Z. N Z N The genotype of the female goose to be tested is Z. N W; If one Z chromosome in the genome of the goose to be tested has no CA insertion between SEQ ID NO. 1 and sequence 272-273, while another Z chromosome has a CA insertion between SEQ ID NO. 1 and sequence 272-273, then the goose to be tested is a male goose, and its genotype is Z. M Z N .

3. The method according to claim 2, characterized in that: The detection of whether the CA sequence is inserted between SEQ ID NO. 1 and SEQ ID NO. 272-273 on the Z chromosome of the goose genome to be tested includes the following steps: Using the genomic DNA of the goose to be tested as a template, the primer pair was used for amplification, and the sequencing product was sent for sequencing. Based on the sequencing results, it was determined whether the CA sequence was inserted between SEQ ID NO.1 272-273 on the Z chromosome of the goose to be tested. The primer pair consists of a single-stranded DNA molecule shown in SEQ ID No. 2 and a single-stranded DNA molecule shown in SEQ ID No.

3.

4. A method for breeding homozygous bright-eyed geese from the Wulong geese, comprising selecting geese with the genotype Z as described in any one of claims 1-3. M Z M Male geese or those with genotype Z M W's mother goose.

5. A method for breeding homozygous normal-eyed geese from Wulong geese, comprising selecting geese with the genotype Z in any one of claims 1-3. N Z N Male geese or those with genotype Z N W's mother goose.

6. A breeding method for establishing a sex-selective mating system for Wulong geese, comprising the following steps: 1) The genotype of any one of claims 1-3 is Z M Z M Male geese and genotype Z N W's female geese are bred to produce offspring; 2) Detect the eyelid morphology of offspring. Offspring with open-eyed phenotype are female geese or candidates, and offspring with normal eye phenotype are male geese or candidates, thus realizing the establishment of a breeding system for distinguishing male and female geese.

7. A method for detecting or assisting in the detection of the sex of offspring of Wulong geese, comprising the following steps: 1) The genotype of any one of claims 1-3 is Z M Z M Male geese and genotype Z N W's female geese mate and produce offspring; 2) Detect the eyelid morphology of the offspring. Offspring with open-eyed phenotype are female geese or candidates, and offspring with normal eye phenotype are male geese or candidates.

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