Method for breeding poultry by heavy ion beam mutagenesis

Abstract

This invention discloses a method for heavy ion beam mutagenesis breeding of poultry. To address the challenges of efficient, rapid, and safe breeding in poultry, this method utilizes heavy ion beam irradiation of poultry progenitor cells (PGCs) to induce radiation mutagenesis, constructing a mutant cell library. The PGCs in this library are then isolated into single cells to form monoclonal populations. Targeted deep sequencing is used to screen for positive PGCs with gene mutations from the S2-after monoclonal population, and the mutated gene is preliminarily identified. Positive PGCs are injected via microinjection into recipient embryos after their own PGCs have been ablated, resulting in chimeras. Individuals developed from these chimeras undergo molecular screening and identification. M1 generation individuals containing the identified mutated gene are then used for breeding to obtain offspring strains that stably inherit the mutated gene. This method is efficient, rapid, safe, and avoids the ethical and regulatory risks associated with gene editing technologies, making it of significant application value for mutagenesis breeding in poultry.

Description

Technical Field

[0001] This invention belongs to the field of poultry genetics and breeding and biotechnology, specifically relating to a method for breeding poultry based on physicochemical mutagenesis. Background Technology

[0002] The poultry industry is an important part of global livestock farming, but outbreaks of infectious diseases (such as avian influenza, avian leukosis, and duck hepatitis) often cause huge economic losses. Furthermore, physiological inhibition caused by environmental factors such as heat stress, cold stress, and high-density herd stress also seriously restricts the healthy development of the poultry industry. Selecting and breeding superior poultry with strong disease resistance, stress tolerance, and good growth traits can significantly reduce disease risks and drug dependence, improve survival rates and production performance, and build a solid biosecurity barrier from the source.

[0003] Traditional breeding methods rely on individual-level selection, that is, first breeding individuals and then selecting the target trait through population selection. This process is usually time-consuming, costly, and has limited efficiency. Although gene editing technologies (such as CRISPR / Cas9) can directly manipulate embryos, they have high technical barriers, carry off-target risks, and face ethical and regulatory restrictions; more importantly, the current mainstream gene editing tools are relatively expensive to use.

[0004] Mutation breeding is a breeding technique that induces genetic variations in organisms through physical, chemical, or biological factors, and then selects superior mutants to cultivate new varieties. Tracing back to Muller's (1927) discovery that X-rays could induce gene mutations in fruit flies and Stadler's (1928) discovery of the mutagenic effects of X-rays on corn and barley, it was proven that artificial mutagenesis can create heritable variations. The core theoretical basis of mutation breeding is gene mutation; its essence is to use mutagenesis to interfere with the genetic material (DNA) of an organism, causing changes in its sequence. Compared with traditional breeding, mutation breeding has advantages such as simple operation, wide application range, and high mutation frequency.

[0005] Heavy ion beam mutagenesis is an advanced physical breeding technique that has attracted much attention in recent years. This technique induces genetic variation by irradiating biological samples with an accelerated heavy ion beam (such as carbon, oxygen, or neon ions). The core advantages of heavy ion beam mutagenesis are twofold: first, the high energy transfer linear density allows the heavy ion beam to cause localized cluster damage to DNA, thus exhibiting a higher relative biological effect; second, its unique energy deposition mode enables concentrated energy deposition at a specific depth within the sample. This method can induce a variety of beneficial variations, including unnatural mutations, thereby greatly expanding the genetic resources and selection range for biological breeding.

[0006] Currently, Chinese patent ZL201911223356.0 discloses a technology for obtaining target gene mutants of the M1 generation through targeted deep sequencing, thereby creating new rice germplasm with important utilization value, such as low cadmium accumulation, aroma, storage resistance, suitability for direct seeding, and low glycemic index. However, there are no reports on combining heavy ion beam mutagenesis technology with poultry breeding and selection, nor are there any mature, efficient, and standardized methods specifically for the rapid cultivation of new poultry germplasm.

[0007] Therefore, developing a poultry breeding technology that is efficient, fast, safe, and free from ethical regulations and usage risks such as gene editing technology has significant application value. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above and provide a poultry heavy ion beam mutagenesis breeding method. This method induces mutations in poultry PGCs through heavy ion beam radiation and uses the mutated PGCs to cultivate new poultry germplasm with resistance or superior traits. This method can significantly shorten the breeding cycle and improve breeding efficiency.

[0009] To solve the above-mentioned technical problems, the present invention proposes a method for heavy ion beam mutagenesis breeding of poultry, comprising the following steps:

[0010] S1: Radiation mutagenesis was induced by irradiating poultry PGCs with heavy ion beams to construct a mutant cell library. The radiation mutagenesis dose was a non-lethal mutagenesis dose. S2: Isolate PGCs from the mutant cell library into single cells to form monoclonal cells; S3: Targeted deep sequencing was used to screen positive PGCs with gene mutations from the single-clone population after S2, and the mutated genes were preliminarily identified. S4: The positive PGCs are injected into the recipient embryo after its own PGCs have been ablated by microinjection to obtain a chimera; the transplanted embryos are hatched to obtain gonadal chimera chicks (M1 generation). S5: Molecular screening and identification are performed on individuals obtained after chimera development. M1 generation individuals containing the mutated gene are identified and offspring strains that can inherit the mutated gene are obtained through breeding.

[0011] Primordial germ cells (PGCs), as precursors to sperm and eggs, carry a complete set of genetic information for an individual. Their unique advantage lies in their ability to be cultured and genetically manipulated in vitro, subsequently transplanted into recipient embryos, and ultimately migrate to the gonads to develop into functional gametes. Therefore, PGC technology allows for a more direct and efficient way to obtain genetically improved offspring, making it the mainstream reproductive engineering technology in current poultry breeding. However, there is currently no mature, efficient, and standardized method specifically designed for the rapid breeding of new poultry germplasm by combining heavy ion mutagenesis technology with poultry PGC systems. Therefore, the poultry high-efficiency breeding technology based on heavy ion beam irradiation mutagenesis developed in this invention has significant application value. This invention pioneers precise selection at the cellular level to cultivate target individuals; the "select-then-breed" approach significantly shortens the breeding cycle and improves the efficiency of target selection compared to the traditional "breed-then-select" approach. The technical solution of this invention is safe and controllable, making it more conducive to large-scale promotion and application.

[0012] In the above method, preferably, in step S1, the heavy ion beam is a carbon ion beam ( 12 C 6+ The carbon ion beam has an energy of 30-80 MeV / u; the radiation mutagenesis dose is 0.1-5 Gy, preferably 0.5-3 Gy, and the irradiation time is 30-60 s. During irradiation, poultry PGCs are supported on a pre-cooled culture medium at 4°C. This preferred radiation mutagenesis method can further induce mutations in poultry PGCs across the entire genome, creating new genetic variations and reducing metabolic damage during irradiation.

[0013] In the above method, preferably, after step S1, the obtained mutant cell library is first placed in M1 generation cell culture medium for recovery culture, then replaced with normal culture medium, and then step S2 is performed. The culture medium for the M1 generation cells contains antioxidants and stem cell factor (SCF).

[0014] Our research indicates that heavy ion beam irradiation of progenitor cells (PGCs) may induce cell cycle arrest and apoptosis. Irradiation also generates a large amount of reactive oxygen species (ROS), which disrupt the mitochondrial membrane potential and function of PGCs, leading to energy metabolism disorders and further exacerbating DNA damage and apoptosis. Therefore, to improve cell viability after irradiation and maintain their stemness and proliferative capacity, we added a convalescent culture period.

[0015] More preferably, in the above method, the antioxidant is at least one of N-acetylcysteine, β-mercaptoethanol, and vitamin C; the concentration of N-acetylcysteine ​​in the M1 generation cell culture medium is controlled at 1-2 mM, the concentration of β-mercaptoethanol in the M1 generation cell culture medium is controlled at 50-200 μM, the concentration of vitamin C in the M1 generation cell culture medium is controlled at 30-50 mM, and the amount of stem cell factor added is 40-60 ng / mL.

[0016] In the aforementioned method, step S2 can non-directionally screen for positive PGCs with gene mutations. However, for sample pools where interesting target mutation signals are found in mixed pool detection, DNA can be extracted from individual clones within that sample pool. This can be achieved by screening for single clones using Sanger sequencing after PCR amplification of the target gene, thereby improving the accuracy, directionality, and heritability of the mutation. Preferably, step S2 includes: preparing a single-cell suspension by dilution and cell sieving to reduce the concentration of the mutant cell library (M1 generation cell suspension); treating the cells in the mutant cell library with an antibody-magnetic bead complex encoding a selected target gene protein; passing the cells through a magnetic field sorting column; transferring the single cells after column separation to well plates for culture; and after cell expansion, harvesting some cells for cryopreservation and using others for DNA extraction. This preferred method allows for faster screening of the desired target gene mutation, further improving the applicability of the method in targeted breeding processes.

[0017] In the preferred embodiment of the above method, step S3, the targeted deep sequencing refers to the resequencing and screening of the entire M1 generation genome. This involves using high-throughput technology to perform high-depth sequencing on the target region of the target gene to detect low-frequency mutations in the population. The sequencing depth depends on the number of individuals in the pool and the lowest allele frequency of the target gene mutation. When the number of individuals in the pool is 50, the sequencing depth is ≥10000×. If a mutation is detected, proceed to step S4; if no mutation is detected, terminate the operation or repeat steps S1-S3 until a mutation of the target gene is detected, then proceed to step S4. Specifically, a portion of cells from M1 generation cell clones are typically collected to form a pool. Generally, 50 clones are selected to form one pool. After extracting DNA from the pooled cells, high-depth targeted sequencing, capture sequencing, PCR, or a combination thereof are performed on the target region. One of the functional hit criteria is the insertion or deletion of bases, frameshifting of coding regions, disruption of key structural domains, or mutations in splicing sites / promoter core elements. The target region includes the coding region and regulatory region of the target gene.

[0018] Preferably, the target genes in the above-described method include one or more of ANP32A, PTX3, Del-1, GDF-15, SPOP, chNHE1, Tva, PTPN6, PTPN11, Cbl-b, PDCD1, MSTN, Follicatin, FLRG, TfR, and SOCS1. To achieve an anti-avian influenza phenotype, this invention can target ANP32A, PTX3, Del-1, GDF-15, SPOP, etc., genes that have been proven to significantly affect the replication and infectivity of avian influenza viruses. To achieve an anti-avian leukosis phenotype, this invention can target multiple receptor genes such as chNHE1 and Tva; to achieve an anti-goose parvovirus phenotype, this invention targets the Tfr receptor gene to block viral invasion; to achieve an anti-duck viral hepatitis phenotype, this invention targets the SOCS1 receptor gene. Mutations in these genes can prevent the virus from entering cells, thereby producing resistance. To achieve a disease-resistant phenotype against multiple diseases, this invention targets negative regulatory genes of natural killer cells (NK cells), including but not limited to PTPN6, PTPN11, Cbl-b, and PDCD1, to relieve NK cell functional inhibition and thus enhance their antiviral immunity. To achieve a phenotype of efficient muscle development, this invention can target and regulate the MSTN gene and its propeptide segment, or intervene in its downstream negative regulatory factors such as Follicatin and FLRG, to block the Myostatin-Smad signaling pathway, thereby relieving its inhibitory effect on muscle growth and promoting skeletal muscle development and body fat optimization in poultry.

[0019] The preferred method described above, specifically the ablation of the recipient embryo's own PGCs, includes: using a combination of physical and chemical methods, irradiating with 3-5 Gy X-rays, and injecting an emulsified busulfan solution (preferably 50-75 μg / embryo) into the yolk to reduce the recipient embryo's own PGCs. This preferred combination of physical and chemical treatment ablates the recipient embryo's own PGCs, clearing its germinal crest niche, reducing toxicity, and increasing targeting, thereby maximizing the space for the migration and colonization of exogenous positive PGCs and improving the colonization efficiency of positive PGCs in the recipient embryo's gonads.

[0020] The above method, preferably, the specific operation of the microinjection includes: after the recipient embryo has been incubated for at least 2.5 days, positive PGCs are introduced into the blood of the recipient embryo through microinjection. The PGCs will use their inherent homing ability to migrate with the blood flow and colonize the developing gonadal ridge.

[0021] In the preferred embodiment of the above method, the molecular screening and identification in step S5 includes: after the individual of the chimera has reached sexual maturity, the genotype of its germ cells (sperm is collected from males, and early embryos are tested from females) is used to confirm whether it contains the M1 generation of the mutated gene. If it does not contain it, the operation is terminated; if it does contain it, it is confirmed as a positive germline chimera and subsequent reproductive operations are carried out.

[0022] The above method uses the selected M1 generation individuals carrying the mutated gene as core breeding material, and employs different breeding schemes for subsequent generations based on the sex of the individuals, aiming to obtain a new homozygous strain with stable heritable traits. Preferably, in step S5, the specific method for obtaining a offspring strain with stable heritable mutated genes through reproduction includes: (a) If the M1 generation individuals are of a single sex, the chimera of the M1 generation is mated with the wild type to produce offspring M2 generation. The M2 generation individuals are genotyped and heterozygous individuals carrying the mutated gene are screened out. The heterozygous individuals of the M2 generation are then cross-crossed to produce the M3 generation, from which homozygous individuals are screened out. (b) If the selected M1 generation individuals are of different sexes, the M1 generation chimeras are crossbred to produce the M2 generation, from which homozygous individuals are selected.

[0023] Compared with the prior art, the advantages of the present invention are as follows: 1. Revolutionary improvement in breeding efficiency and significant reduction in cycle: Traditional hybridization breeding requires multiple generations of repeated selection and takes several years; while this invention obtains mutant traits by directly inducing PGCs, which can be directly inherited by offspring, avoiding the cumbersome hybridization and backcrossing process, and is expected to shorten the breeding cycle of new varieties.

[0024] 2. Significantly optimized mutation frequency and spectrum: This invention boasts high mutagenesis efficiency and a rich variety of mutation types. The heavy ion beam exhibits a high relative biological effect, with a mutation frequency far exceeding that of conventional X-rays or chemical mutagens. This provides a larger mutant library for screening individuals with breakthrough superior traits (such as disease resistance and stress resistance).

[0025] 3. Creation of New Germplasm Resources: This invention makes it possible to create entirely new genetic variations and germplasm resources. Heavy ion irradiation can produce beneficial mutations that are rare or non-existent in nature, thereby cultivating new poultry germplasm, which is difficult to achieve using traditional breeding methods.

[0026] 4. Safety and Broad Application Prospects: This method is a physical mutagenesis method, avoiding the residues and environmental pollution problems associated with chemical mutagens. Compared to obtaining favorable mutations through long-term artificial selection or hybridization to screen for natural variations, it can significantly increase the mutation frequency and obtain more superior mutation types in a shorter time. Furthermore, the genetic modification mediated by PGCs results in breeding materials that do not contain exogenous transgenic components, making them more acceptable to the public and compliant with relevant regulations, thus possessing good potential for industrial application. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a flowchart of a poultry breeding method according to a specific embodiment of the present invention.

[0029] Figure 2 This is a schematic diagram of the ANP32A mutation screening results of the chicken targeted screening breeding method in Embodiment 1 of the present invention.

[0030] Figure 3 The diagram shows the effect of different doses of X-ray irradiation on embryonic development in the chicken directional breeding method of Embodiment 1 of the present invention. The diagram on the left, A, shows the embryonic development results after treatment with pure physical methods (X-rays with different irradiation doses), and the diagram on the right, B, shows the embryonic development results after treatment with chemical and physical methods.

[0031] Figure 4 This is a diagram of the individual rearing of the mutant chicken in Example 1 of the present invention.

[0032] Figure 5 These are comparative images of the lungs of mutant chickens infected with avian influenza virus in Embodiment 1 of the present invention. Image A on the left is an image of the lungs of wild-type chickens, and image B on the right is an image of the lungs of mutant chickens.

[0033] Figure 6 The figures shown are of the goose PGCs culture status and identification results in the directional breeding method of geese in Example 2 of the present invention. Figure A shows the growth status of goose PGCs, and Figure B shows the glycogen staining results of goose PGCs.

[0034] Figure 7 This is a picture of an individual raising of the mutant goose in Example 2 of the present invention.

[0035] Figure 8 This is a necropsy image of the intestines of a mutant goose infected with GFV in Example 2 of the present invention.

[0036] Figure 9 This is a necropsy image of the kidneys of a mutant goose infected with GFV in Example 2 of the present invention. Detailed Implementation

[0037] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0038] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0039] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0040] Given that there is no precedent for the heavy ion beam mutagenesis method for poultry PGCs of the present invention, the definition of animal generations in this specification is as follows, based on the biological characteristics of the technical system: M0 generation: refers to PGCs before undergoing heavy ion beam mutagenesis treatment; M1 generation: refers to the cell population obtained from M0 generation PGCs after mutagenesis treatment, and the animal individuals formed by the development of these cells; M2 generation: refers to offspring produced by M1 generation individuals through sexual reproduction; M3 generation: refers to the offspring produced by M2 generation individuals through sexual reproduction.

[0041] Example 1: This embodiment uses chicken PGCs as donor cells and selects the ANP32A gene as the target to obtain individuals resistant to avian influenza virus (AIV). Figure 1 As shown, the specific methods and steps include the following.

[0042] First, a suitable combination of mutagenic dosage parameters was selected, specifically 1.5 Gy and an irradiation time of 35 s. The process continued until the cell number reached 10... 6 Then, according to the above conditions, the heavy ion beam ( 12 C 6+ The poultry PGCs were irradiated with an energy of 80 MeV / u, and the culture medium was pre-cooled at 4°C during the irradiation process. Then, the obtained mutant cell library was placed in M1 generation cell culture medium for recovery culture. The M1 generation cell culture medium contained 1 mM N-acetylcysteine ​​and 40 ng / mL SCF.

[0043] After the convalescent culture period, the sorted M1 generation cells were serially diluted 10-fold and passed through a cell sieve to prepare single-cell suspensions. These suspensions were then incubated with an antibody-magnetic bead complex coated with ANP32A antibody. The treated cells were then passed through a magnetic separation column. Single cells were cultured in 96-well plates with normal medium. Once monoclonal colonies formed, portions of cells from each of the 50 monoclonal colonies were collected and pooled into 40 cell pools. DNA was extracted from these pooled cells using a kit for sequencing.

[0044] Targeted depth sequencing at a depth of 10000× was used to identify ANP32A mutations in multiple cell pools, with some mutations located in exon 4, causing key amino acid mutations (see [link]). Figure 2 This weakens the ANP32A protein's support for AIV polymerase function. Monoclonal screening yields monoclonal cells lacking ANP32A function, which are considered positive cells. Compared to cells that failed the magnetic separation column, the number of cells with the target gene mutation in the sorted sample is significantly higher than in the sample that failed the magnetic separation column, and false positives are more easily avoided.

[0045] Chicken hatching eggs were irradiated with X-rays (5 Gy, 0.5 Gy / min), followed by the injection of 50 μg of busulfan into the yolk to reduce the ablation of its own progeny genetic cells (PGCs) and form a recipient embryo (see [link to article]). Figure 3 (Figure B). But if only physical methods are used (see Figure B). Figure 3 (Figure A) This will cause delayed embryonic development and significantly affect breeding efficiency.

[0046] The aforementioned positive cells were microinjected into the bloodstream of recipient embryos (3500 positive cells per embryo), allowing them to colonize the germinal ridges of the recipient embryos and form chimeras. The transplanted embryos were then hatched to obtain chimeric chicks (M1 generation). Mutant individuals were identified by detecting the ANP32A genotype in the M1 generation germ cells.

[0047] Wild-type individuals are then hybridized with them, and the genotypes of the hybrid offspring are identified to obtain heterozygous individuals of different sexes carrying the ANP32A gene mutation (M2 generation). The M2 generation individuals are then intercrossed to obtain homozygous individuals (M3 generation).

[0048] After confirming the individual's ANP32A genotype using Sanger sequencing, an AIV challenge test was performed. Using 10 6 EID 50 The H5N1 subtype AIV strain was administered via nasal drops to wild-type and mutant chickens, and observed for 14 days. Mutant chickens exhibited good mental status, normal feed intake, and no respiratory or neurological symptoms (see...). Figure 4The current in vivo disease resistance test results are early preliminary results, and the trend of fewer than ten individuals in the test is observed. Preliminary findings indicate a disease-resistant phenotype; the chick images are of mutant individuals being raised. Viral load testing shows that the viral load in throat and cloacal swabs is significantly lower than that in the wild-type control group. Histopathological observation shows no obvious lesions in the lung tissue of mutant individuals (see...). Figure 5 Based on this experiment and combined with domestic and international research progress, the results show that the ANP32A mutant chicken prepared using this invention has complete resistance to avian influenza virus.

[0049] Example 2: This embodiment uses goose PGCs as donor cells and selects the goose transferrin receptor (TfR) gene as the target to obtain individuals resistant to goose parvovirus (GPV). The specific method steps are as follows.

[0050] First, achieve in vitro culture of goose PGCs (see...) Figure 6 , Figure 6 Figures A and B confirm that the isolated cells are PGCs. A suitable mutagenic dose combination of 1 Gy and an irradiation time of 30 s were used for heavy ion beam (…). 12 C 6+ The cells were irradiated with an energy of 80 MeV / u. The mutagenized cells were processed according to the corresponding steps in Example 1, but a total of 50 cell pools (50 single clones in each pool) were obtained. The DNA of the pooled cells was extracted using a kit for sequencing.

[0051] Targeted depth sequencing at a depth of 10000× revealed TfR gene mutations in some cell pools, with Indels in exons 4-6 causing substitutions of key amino acids in the TfR protein. Single-clone screening yielded positive cells with functional loss of virus-binding ability. These positive cells were then cultured in larger quantities for microinjection.

[0052] Recipient embryos were prepared according to the corresponding procedures in Example 1. The aforementioned positive cells were microinjected into the bloodstream of the recipient embryos (3000 cells per embryo), allowing them to colonize the recipient's goiter ridge and form chimeras. The transplanted embryos were hatched to obtain chimeric goslings (M1 generation). Mutant individuals were confirmed by detecting the TfR genotype of the M1 generation germ cells. The TfR genotype of the goslings was verified by PCR and sequencing, confirming the mutant individuals.

[0053] The selected M1 generation consisted of individuals of different sexes. These M1 individuals were crossbred to obtain homozygous individuals (M2 generation) carrying the TfR gene mutation. The TfR genotype was confirmed to be nonfunctional by Sanger sequencing.

[0054] After confirming the individuals were homozygous through sequencing, a challenge experiment with a virulent GPV strain was conducted. Using 10... 5 TCID 50 A highly virulent strain of GPV was administered orally to 3-day-old goslings, and the chicks were observed for 21 days. The mutant geese exhibited normal growth and development, active feeding, and no typical clinical symptoms such as diarrhea or paralysis (see...). Figure 7 Histopathological examination showed that the intestinal villi and kidney tissue structures were intact, without lymphocyte infiltration, necrosis or other lesions (see...). Figure 8 and Figure 9 The results showed that the TfR mutant geese prepared using this invention exhibited complete resistance to goose parvovirus.

Claims

1. A method for breeding poultry using heavy ion beam mutagenesis, characterized in that, Includes the following steps: S1: Radiation mutagenesis was induced by irradiating poultry PGCs with heavy ion beams to construct a mutant cell library. The radiation mutagenesis dose was a non-lethal mutagenesis dose. S2: Isolate PGCs from the mutant cell library into single cells to form monoclonal cells; S3: Targeted deep sequencing was used to screen positive PGCs with gene mutations from the single-clone population after S2, and the mutated genes were preliminarily identified. S4: The positive PGCs are injected into the recipient embryo after its own PGCs have been ablated by microinjection to obtain a chimera; S5: Molecular screening and identification are performed on individuals obtained after chimera development. M1 generation individuals containing the mutated gene are identified and offspring strains that can inherit the mutated gene are obtained through breeding.

2. The method according to claim 1, characterized in that, In step S1, the heavy ion beam is a carbon ion beam with an energy of 30-80 MeV / u; the radiation mutagenesis dose is 0.1-5 Gy, preferably 0.5-3 Gy, and the irradiation time is 30-60 s. During the irradiation process, poultry PGCs are carried on a pre-cooled culture medium at 4°C.

3. The method according to claim 1, characterized in that, After step S1, the obtained mutant cell library is first placed in M1 generation cell culture medium for recovery culture, then replaced with normal culture medium, and then step S2 is performed. The culture medium for the M1 generation cells contains antioxidants and stem cell factors.

4. The method according to claim 3, characterized in that, The antioxidant is at least one of N-acetylcysteine, β-mercaptoethanol, and vitamin C; the concentration of N-acetylcysteine ​​in the M1 generation cell culture medium is controlled at 1-2 mM, the concentration of β-mercaptoethanol in the M1 generation cell culture medium is controlled at 50-200 μM, the concentration of vitamin C in the M1 generation cell culture medium is controlled at 30-50 mM, and the amount of stem cell factor added is 40-60 ng / mL.

5. The method according to any one of claims 1 to 4, characterized in that, The specific operation method of step S2 includes: preparing a single-cell suspension by dilution and cell sieving, then treating the cells in the mutant cell library with an antibody-magnetic bead complex encoding a protein of a selected target gene, passing the cells through a magnetic field sorting column, transferring the single cells after passing through the column to a well plate for culture, and after the cells have expanded in culture, collecting some cells for cryopreservation and using the rest for DNA extraction.

6. The method according to claim 5, characterized in that, In step S3, the targeted deep sequencing refers to whole-genome resequencing of generation M1; when the number of individuals in the pool is 50, the sequencing depth is ≥10000×; if a mutation is detected, proceed to the subsequent step S4; if no mutation is detected, terminate the operation or repeat steps S1~S3 until a mutation of the target gene is detected and then proceed to step S4.

7. The method according to claim 5, characterized in that, The target genes include one or more of ANP32A, PTX3, Del-1, GDF-15, SPOP, chNHE1, Tva, PTPN6, PTPN11, Cbl-b, PDCD1, MSTN, Follicatin, FLRG, TfR, and SOCS1.

8. The method according to any one of claims 1 to 4, characterized in that, The specific method for ablating the recipient embryo's own PGCs includes: irradiating with 3-5 Gy X-rays and injecting the emulsified busulfan solution into the yolk.

9. The method according to any one of claims 1 to 4, characterized in that, The specific operation of the microinjection includes: at least 2.5 days after the recipient embryo is incubated, positive PGCs are introduced into the blood of the recipient embryo through microinjection. The PGCs will use their inherent homing ability to migrate with the blood flow and colonize the developing gonadal ridge.

10. The method according to any one of claims 1 to 4, characterized in that, The molecular screening and identification in step S5 includes: after the individual of the chimera has reached sexual maturity, the genotype of its germ cells is tested to confirm whether it contains the M1 generation of the mutated gene. If it does not contain it, the operation is terminated. If it does contain it, it is confirmed as a positive germline chimera and subsequent reproductive operations are carried out.

11. The method according to any one of claims 1 to 4, characterized in that, In step S5, the specific method for obtaining offspring strains capable of stably inheriting the mutated gene through breeding includes: (a) If the M1 generation individuals are of a single sex, the chimera of the M1 generation is mated with the wild type to produce offspring M2 generation. The M2 generation individuals are genotyped and heterozygous individuals carrying the mutated gene are screened out. The heterozygous individuals of the M2 generation are then cross-crossed to produce the M3 generation, from which homozygous individuals are screened out. (b) If the selected M1 generation individuals are of different sexes, the M1 generation chimeras are crossbred to produce the M2 generation, from which homozygous individuals are selected.

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