Method for breeding animals based on heavy ion beam mutagenesis

Abstract

The application belongs to the field of animal mutagenic breeding, and specifically discloses a method for breeding animals based on heavy ion beam mutagenesis, which comprises the following steps: irradiating cells or tissues of target animals with a non-lethal mutagenic dose of heavy ion beam to induce gene mutation and obtain a mutant cell library; performing preliminary judgment and screening on the mutant cell library to obtain a candidate cell library carrying gene mutation; culturing the candidate cell library to obtain individuals carrying gene mutation; and breeding the individuals carrying gene mutation to obtain offspring lines capable of inheriting gene mutation. The application effectively reduces the frequency of non-target mutation while ensuring mutagenic efficiency, improves the success rate of obtaining mutant animals, and greatly reduces the breeding cycle and cost.

Description

Technical Field

[0001] This invention relates to the field of animal breeding, and more specifically to a method for breeding animals based on heavy ion beam mutagenesis. Background Technology

[0002] Traditional animal genetic improvement relies heavily on natural mutations and conventional hybridization, a process that often requires several generations of breeding, is time-consuming, and inefficient. While advancements in molecular biology have led to various molecular improvement approaches in recent years, their industrialization remains constrained by technological barriers, potential off-target risks, and regulatory hurdles. Therefore, it is necessary to explore alternative breeding pathways to improve breeding efficiency, shorten breeding cycles, reduce overall costs, and accelerate industrialization.

[0003] In the plant field, physicochemical mutagenesis breeding has been validated through long-term practice. Mutagenesis can generate genetic diversity far exceeding the frequency of natural variation in a relatively short period, significantly expanding the available germplasm resources. Since the 20th century, X-rays, gamma rays, and various chemical mutagens have been widely used in crops such as rice, wheat, barley, and soybeans, resulting in the development of numerous new materials and varieties with important agronomic traits, promoting variety renewal and industrial upgrading. It should be noted that traditional mutagenesis also has significant drawbacks, mainly manifested in the randomness of mutation sites and the fact that contemporary individuals are often chimeric, making direct selection difficult. Homozygous mutants are usually only screened after the first generation of segregation, leading to low accuracy and efficiency in obtaining innovative germplasm. Nevertheless, physicochemical mutagenesis, because it does not rely on transposition or the introduction of exogenous fragments and can obtain stable genetic variations in a wide range of species, still holds an irreplaceable fundamental position and application value.

[0004] Traditional physiochemical mutagenesis faces significant challenges in the engineering application of animal systems, primarily in three aspects. First, conventional mutagenesis methods generate a broad, non-targeted mutational load across the entire genome, easily accumulating adverse alterations to key genes, leading to abnormal embryonic development or even death, significantly reducing the success rate of obtaining fertile individuals and achieving genetic fixation. Second, animals cannot be subjected to large-scale irradiation and sowing like crop seeds. Mutagenesis and treatment of fertilized eggs or early embryos are technically more complex, making it difficult to establish a large-scale process. Simultaneously, the high cost of animal population rearing and phenotypic identification hinders high-throughput reverse genetic screening. Third, hybridization breeding requires obtaining consistent mutations at the same gene locus across multiple breeds and strains to ensure a stable mutational background in the hybrid offspring at the biallelic level. Traditional mutagenesis and screening methods struggle to simultaneously ensure consistency across multiple strains, accurate localization, and traceability. While it's conceivable to obtain mutant cells first and then obtain individuals through somatic cell nuclear transfer, directly using mutagenized mixed-pool cells to prepare cloned animals in the absence of an efficient screening and retrospective system would result in extremely high human and material resource investment and low success rates, failing to meet the needs of breeding applications.

[0005] Among physical mutagenesis methods, heavy ion beam mutagenesis has significant advantages due to its concentrated energy deposition distribution, controllable trajectory, and relatively unique biological effects. Compared with conventional radiation or chemical mutagenesis, heavy ions can produce a rich variety of mutation types and facilitate the technological control of mutation scale and distribution through optimization of dosimetry and linear energy transfer parameters. Within a reasonable parameter window, heavy ion mutagenesis is expected to maintain mutagenesis efficiency and phenotypic plasticity while reducing the accumulation of genome-wide irrelevant variations, lowering the risk of cellular dysfunction and embryonic lethality caused by non-target effects, and providing more favorable conditions for the engineering, replicability, and transformability of animal mutagenesis breeding. However, current heavy ion mutagenesis in animals still lacks a mature engineering system. Key systematic dosimetry and linear energy transfer parameters have not yet been established, the efficient coupling process between mutagenesis and molecular screening is not yet perfect, and the closed-loop connection with somatic cell nuclear transfer needs to be standardized. These factors limit its potential for large-scale application in animal husbandry, medical models, and cross-species genetic research.

[0006] Furthermore, traditional heavy ion mutagenesis methods, due to their high-energy irradiation, often lead to reduced cell viability, slower growth rates, and even mass cell death, posing significant challenges to subsequent screening and the acquisition of mutants. Generally, it is difficult to obtain sufficient target mutant cells through heavy ion irradiation, which limits its application in animal breeding.

[0007] Therefore, it is particularly urgent to establish a complete system of heavy ion-based animal mutagenesis technology. 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 to provide a method for breeding animals based on heavy ion beam mutagenesis.

[0009] A method for breeding animals based on heavy ion beam mutagenesis includes the following steps:

[0010] S1 Controllable Mutation Introduction: Gene mutations are induced by irradiating the cells or tissues of the target animal with a non-lethal dose of heavy ion beam to obtain a mutant cell library. S2 Screening and Identification: The mutant cell library is preliminarily screened to obtain candidate cell libraries carrying gene mutations; S3 Cultivating Mutant Individuals: Cultivating the candidate cell bank to obtain individuals carrying gene mutations; S4 strain establishment: The individuals carrying the gene mutation from step S3 are bred to obtain offspring strains that can inherit the gene mutation.

[0011] In the above method, preferably, the cells or tissues of the target animal include somatic cells, primordial germ cells (PGCs), or fertilized eggs in the single-cell stage of the target animal.

[0012] More preferably, the target animal is a fish, the cell or tissue of the target animal is a fertilized egg in the single-cell stage, and the gene mutations targeted by the screening and identification include one or more mutations in the bmp6, sp7, runx2b, lepr, and mstn genes.

[0013] Heavy ion beam irradiation mutagenesis is a physical mutagenesis technique developed in recent years. Utilizing the high linear energy transfer and strong biological effects of heavy ions, it can induce diverse DNA variations at relatively low doses. Compared to traditional chemical or radiation mutagenesis, it features high mutation efficiency, controllable damage, and good genetic stability. However, existing technologies are primarily used in agriculture, enabling direct detection of a target gene region in the M1 generation and identification of individuals with target gene mutations. This allows for targeted or non-targeted screening of specific genes and direct entry into the breeding process without waiting for phenotypic manifestation. Applying this technology to fish presents numerous challenges, the core challenge being how to efficiently and controllably generate valuable mutations. The present invention combines heavy ion beam mutagenesis with M1 generation screening and identification technology. By irradiating single-celled fertilized eggs with heavy ion beams and incorporating molecular screening process design, when using rapid homozygous reproduction pathways such as parthenogenesis or germ cell transplantation, the fish breeding cycle can be shortened from several years to 1-2 generations, significantly reducing costs and improving the stability and efficiency of gene mutation acquisition.

[0014] In the preferred embodiment of the above-mentioned breeding method, in step S2, the fertilized eggs are oriented and fixed before irradiation mutagenesis, so that their animal pole faces the direction of the heavy ion beam. This ensures that the heavy ion beam is incident along the polarity direction of the embryo, avoiding the scattering and non-uniform deposition of irradiation energy in a high-water-content environment under unoriented irradiation conditions. This reduces mutagenesis drift caused by scattering and non-uniform deposition in high-water-content embryos. This step ensures that mutation events preferentially occur in the core nuclear region rather than being absorbed by the surrounding cytoplasm, improving mutagenesis efficiency and the probability of mutations entering the germline, thus solving the key problem of "uncontrollable energy deposition leading to mutagenesis instability".

[0015] We also found that, due to the strong oxygen metabolism dependence of early fish embryos, heavy ion mutagenesis can induce a localized peak in reactive oxygen species (ROS) and rapid consumption of dissolved oxygen. Without proper incubation buffer regulation, this can lead to a significant increase in early embryonic death or malformation rates, creating a contradictory relationship of "increased mutation rate – drastically decreased survival rate." Therefore, simply reducing or increasing the dosage in fish systems may not yield a stable, selectable mutant population. For this reason, in the aforementioned breeding method, preferably, in step S2, a high-oxygen incubation solution containing buffering components is prepared as the carrier medium for single-cell fertilized eggs, with a dissolved oxygen concentration of 6.5–8.0 mg / L, to maintain homeostasis of embryonic oxygen metabolism during irradiation, prevent early death due to oxidative stress, and further avoid early embryonic death caused by excessive oxidative damage. More preferably, the hyperoxia incubation solution is prepared by the following steps: using E3 culture medium or HBSS basal solution as the base medium, adding 0.5%~2.0% trehalose (preferably about 1.0%) and 0.5~1 mM NaHCO3 by mass, and slowly aerating at 4°C for 30~60 min to raise the dissolved oxygen to 6.5~8.0 mg / L, then sealing and raising the water temperature to the embryo incubation temperature. This measure enables the present invention to obtain a higher survival rate of mutant populations at lower doses, overcoming the problem of uncontrolled oxygen free radicals leading to death in fish mutagenesis.

[0016] More preferably, the target animal is a fish, and the reproduction is one or a combination of the following three pathways: a) Cross the M1 generation individuals containing the gene mutation with wild-type individuals, and introduce the "directed backcross + linkage molecular marker" monitoring method. Perform one or more directed backcrosses between the M1 generation individuals carrying the gene mutation and the corresponding wild-type individuals, and use linkage molecular markers near the gene locus to selectively track and genotype the M2 and subsequent populations. Select and breed in the M2 generation and subsequent generations to obtain homozygous mutant lines with clear genetic background and stable trait expression. b) Using parthenogenesis, the gametes of the M1 generation individuals containing the gene mutation were induced to directly develop into homozygous diploid M2 generation; c) The germ cells of the M1 generation individuals containing the gene mutation are transplanted into the recipient fish that has undergone germ cell removal treatment, and the recipient fish is used to generate donor-derived gametes.

[0017] Ensuring uniform and efficient mutagenesis reaching fish reproductive cells, and precisely balancing lethal and mutagenic doses, are further contributions of this invention. Through a synergistic design of "controlled mutagenesis time window + fixed energy incident direction + ROS buffered incubation medium," this technology achieves high mutation density while maintaining a hatching survival rate of ≥92%, realizing the coexistence of low dose, high mutation rate, and high survival rate, and enabling the identification of heritable mutations in the M1 generation. By combining the above-mentioned process parameters, this invention not only achieves high-frequency genetic variation at lower doses but also enables rapid identification and precise screening of mutagenic effects, thereby fundamentally shortening the breeding cycle, reducing costs, and improving efficiency.

[0018] Further preferably, the target animal is a bird, and the cells or tissues of the target animal are PGCs of the target animal. The gene mutations targeted for screening and identification preferably include one or more mutations among ANP32A, PTX3, Del-1, GDF-15, SPOP, chNHE1, Tva, PTPN6, PTPN11, Cbl-b, PDCD1, MSTN, Follicatin, FLRG, TfR, and SOCS1. Primordial germ cells (PGCs), as precursors to sperm and eggs, carry the complete genetic information of a poultry 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 can more directly and efficiently obtain genetically improved offspring, making it the mainstream reproductive engineering technology in current poultry breed 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 PGCs. Therefore, the high-efficiency poultry breeding technology based on heavy ion beam irradiation mutagenesis of PGCs developed in this invention has significant application value. This invention pioneers precise selection at the cellular level to breed target individuals; the "select-then-breed" approach significantly shortens the breeding cycle and improves the efficiency of selecting breeding targets 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.

[0019] More preferably, when the target animal is a bird, 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).

[0020] 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.

[0021] More preferably, when the target animal is poultry, 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.

[0022] Further preferably, when the target animal is a bird, the mutagenized PGCs are screened for the target gene. Specifically, if a sample pool containing a target gene mutation signal is found during pooled detection, DNA is extracted from each clone in that sample pool. Single clones can be obtained by screening using digital PCR, KASP typing, or PCR amplification followed by Sanger sequencing of the target gene. If no mutation is found, the aforementioned operation is repeated by replacing the sample pool. Preferably, the specific method for separating PGCs from the mutant cell library into single cells further includes: preparing a single-cell suspension by dilution and cell sieving to reduce the concentration of the mutant cell library (M1 generation cell suspension). Single-cell clones can be obtained using immunomagnetic bead sorting, flow cytometry, single-cell sorting, or limiting dilution methods, depending on the expression location of the target gene-encoded protein and the detection method. For target genes unsuitable for cell surface antibody sorting, molecular typing screening can be performed directly after single-clone culture. This preferred method allows for faster screening of the desired target gene mutation, further improving the applicability of the method in targeted breeding processes.

[0023] More preferably, when the target animal is a mammal, the cells or tissues of the target animal are somatic cells of the target animal, and the target gene includes one or more of CD163, ANPEP, and LDLR. This technical solution establishes a three-stage process using somatic cells as a carrier. First, amplifiable somatic cells are irradiated with heavy ions, followed by high-throughput molecular screening, and genotyping and retrospection are completed at the monoclonal cell level. Finally, positive clones are used as donors for somatic cell nuclear transfer or equivalent embryo reconstruction to achieve individualized fixation and propagation of gene mutations. This process combines the diversity of mutagenesis with the precision of screening, which is beneficial for efficiently obtaining gene mutations in a multi-species context and significantly reduces the cost of large-scale population mutagenesis and phenotypic screening at the animal level, thus improving screening efficiency. After the above method is completed, the obtained mammalian individuals can be molecularly confirmed and the target trait can be evaluated. If it is confirmed that they carry a functional mutation generated by heavy ion mutagenesis in the target gene and exhibit the predetermined target trait, then the mammalian individual or its offspring will have greater application value.

[0024] More preferably, when the target animal is a mammal, after in vitro mutagenesis using the heavy ion beam irradiation, the mutant cell library is constructed by adding a combination of α-linolenic acid (ALA) and basic fibroblast growth factor (bFGF) to the culture medium of the mutagenized somatic cells. The mutant cell library constructed using this preferred scheme significantly improves the growth status (cell viability) of cells after heavy ion mutagenesis. More preferably, the concentration of α-linolenic acid in the culture medium is controlled at 5-20 μM, and the concentration of basic fibroblast growth factor is controlled at 10-30 ng / mL. By proposing the combination of α-linolenic acid (ALA) and growth factors in the culture medium, the growth status of irradiated cells is improved, the viability of irradiated cells is increased, and thus the efficiency of obtaining monoclonal mutant cells is improved. This scheme promotes cell repair and proliferation by regulating cell membrane fluidity and intracellular lipid metabolism, thereby improving the survival and growth rate of cells after heavy ion mutagenesis. Through this optimized treatment, we can obtain more mutant cells while reducing cell death, providing sufficient cell resources for subsequent somatic cell nuclear transfer and the preparation of genetically modified animals.

[0025] In the above method, preferably, the heavy ion beam can be a carbon ion beam, a nitrogen ion beam, etc., but the heavy ion beam is preferably a carbon ion beam (such as ¹²C). 6+The energy ranges from 30 to 80 MeV / u, with a dose range of 0.1 to 5 Gy, preferably 0.5 to 3 Gy. The irradiation time can be 30 to 60 s. Optimized heavy ion beam irradiation parameters ensure sufficient mutation generation while maintaining high cell viability. Correlation curves between dose, viability, and mutational burden were established experimentally to determine the mutagenesis window for different species and cell types. Compared to conventional radiation, heavy ion mutagenesis results in more concentrated energy deposition and a relatively controllable distribution of induced mutation sites. It can significantly increase the proportion of functional mutations while reducing genome-wide background variation, avoiding the cell or embryonic lethality problem caused by multi-site mutations commonly found in traditional mutagenesis.

[0026] Preferably, the preliminary screening described above involves molecular typing screening targeting a specific site / gene / pathway, including one or a combination of whole-genome resequencing, whole-exome sequencing, targeted deep sequencing, capture sequencing, digital PCR, KASP typing, and Sanger sequencing. Further, targeted deep sequencing refers to high-coverage sequencing of the target gene, target site, or target pathway-related region. When the pool contains at least 50 individuals, the average coverage depth is not less than 5000×; when the pool contains at least 100 individuals, the average coverage depth is not less than 10000×; when the pool contains at least 500 individuals, the average coverage depth is 30000~50000×. If a gene mutation is detected, proceed to step S3; if no gene mutation is detected, terminate the operation or repeat steps S1~S2 until a mutation in the target gene is detected, then proceed to step S3.

[0027] The process after step S3 and before step S4 further includes molecular screening and identification. The molecular screening and identification includes: after the individual carrying the gene mutation reaches sexual maturity, the genotype of its germ cells is detected to confirm whether it contains the gene mutation in the M1 generation individual. If it does not contain the gene mutation, the operation is terminated or the mutagenesis and screening are repeated until a target gene with the mutation is identified and the process proceeds to step S4. If the gene is found, it is confirmed as a positive germline chimera and the subsequent reproductive operation in step S4 is carried out.

[0028] Introducing heavy ion beam mutagenesis combined with M1 generation high-depth targeted sequencing technology into animal breeding can not only obtain high-frequency genetic variations at lower doses, but also, when combined with high-throughput sequencing technology, enable rapid identification and precise screening of mutagenic effects, thereby fundamentally shortening the breeding cycle, reducing costs and improving efficiency.

[0029] The above method, preferably, includes one of the following cultivation methods in step S3: incubation, feeding, somatic cell nuclear transfer to reconstruct embryonic development, and injection into a recipient embryo after its own PGCs have been ablated.

[0030] More preferably, when the target animal is a bird, the specific method for ablating 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 genital ridge niche, reducing toxicity and increasing targeting, thereby maximizing the space advantage for the migration and colonization of exogenous positive PGCs and improving the colonization efficiency of positive PGCs in the recipient embryo's gonads. The specific operation of the microinjection includes: after the recipient embryo has been incubated for at least 2.5 days, introducing positive PGCs into the recipient embryo's bloodstream via microinjection. The PGCs will utilize their inherent homing ability to migrate with the bloodstream and colonize the developing genital ridge.

[0031] More preferably, when the target animal is a mammal, the reconstructed embryo includes somatic cell nuclear transfer, embryo aggregation, and equivalent nucleocytoplasmic reprogramming steps. Further, the reconstructed embryo development using somatic cell nuclear transfer specifically refers to using the positive somatic cell clone as a donor to perform somatic cell nuclear transfer to reconstruct the embryo, and cultivating an individual containing a gene mutation. Further, the positive somatic cell clone is obtained from a candidate cell bank using a single-clone establishment and backtracking strategy; the single-clone establishment employs limiting dilution, unlimited dilution, or flow cytometry sorting; the backtracking strategy employs a barcode-backtracking strategy or a batch traceability strategy of "cryopreservation-extraction-bank construction".

[0032] Compared with the prior art, the advantages of the present invention are as follows: 1. The method and technical system of this invention optimizes heavy ion irradiation conditions for animal breeding and establishes reproducible mutagenesis conditions; it combines mutagenesis with high-throughput molecular screening to achieve efficient acquisition, screening, and identification of animal mutants; and it ensures the stable preservation and traceability of positive clones by constructing a monoclonal and retrospective mechanism. Through the systematic integration of the above elements, the application potential of heavy ion mutagenesis in animal breeding is released, forming an independent innovation platform that can be promoted and transformed.

[0033] 2. The heavy ion mutagenesis method used in this invention has the characteristics of concentrated energy deposition and high controllability, which can effectively reduce the frequency of non-target mutations and reduce the risk of embryo lethality while ensuring mutagenesis efficiency.

[0034] 3. Combining heavy ion irradiation of somatic cells with high-throughput monoclonal screening and somatic cell nuclear transfer technology significantly improves the success rate of obtaining mutant animals and greatly reduces the breeding cycle and cost.

[0035] 4. The system of the present invention can perform mutagenesis screening on the same target gene or target pathway in multiple varieties and lines, thereby obtaining mutant materials with similar functions or consistent trait directions with greater probability and convenience, thus providing a unified source of mutation for hybridization breeding and ensuring the stability of hybrid offspring at the biallelic level.

[0036] 5. The method of this invention is applicable to the breeding of disease resistance, production performance and disease models in multiple species such as fish, poultry and mammals. It has significant advantages such as high precision, short cycle, low cost and cross-species scalability, providing an innovative technical approach and industrialization foundation for building an independent and controllable new animal mutation breeding system.

[0037] In summary, this invention provides an animal breeding and model preparation platform that does not rely on specific exogenous nuclease digestion. By constructing an integrated technical system based on heavy ion mutagenesis, molecular screening, and somatic cell cloning, it achieves the efficient acquisition of gene-mutated individuals (even individuals with specific target gene mutations). This invention breaks through the application bottleneck of traditional physicochemical mutagenesis in animals, establishing a systematic engineering process from mutagenesis parameter standardization to screening verification and embryo reconstruction, which can be widely applied to different species and different breeding objectives; it provides an innovative approach and technical support for constructing a new animal breeding technology system. Attached Figure Description

[0038] 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.

[0039] Figure 1 This is a flowchart of the fish heavy ion mutagenesis breeding method in a specific embodiment of the present invention.

[0040] Figure 2 This is the local sequence alignment result of the insertion mutation site detected in the bmp6 gene of paddy carp in Example 1 of this invention. The mutation is located near site 6344761 on chromosome B24. The reference sequence (Ref) is AAG, the variant sequence (Alt) is AAGAG, the upper row is the wild-type (WT) reference sequence, and the lower row is the corresponding sequence of the mutagenized sample (MUT). "+" indicates that an insertion mutation has occurred at this site in the mutant.

[0041] Figure 3These are comparative photographs of the growth phenotypes of lepr mutant individuals and wild-type paddy field carp obtained in Example 2 of this invention. Figures A, B, and C show wild-type paddy field carp individuals that have not undergone heavy ion beam mutagenesis treatment, while Figures D, E, and F show candidate individuals that have undergone heavy ion beam mutagenesis and showed lepr-related mutation sites detected in the M1 generation molecular screening.

[0042] Figure 4 The electrophoresis results are those of PCR amplification of DNA samples from different sources using carp species-specific primers in Example 3 of this invention.

[0043] Figure 5 This is a schematic diagram of the fertilized egg and early cleavage morphology formed after the sperm produced by the recipient fertilizes a normal egg in Embodiment 3 of the present invention.

[0044] Figure 6 This is a flowchart of a poultry breeding method according to a specific embodiment of the present invention.

[0045] Figure 7 This is a schematic diagram of the ANP32A mutation screening results of the chicken targeted screening breeding method in Example 4 of the present invention.

[0046] Figure 8 The figure shows the effect of different doses of X-ray irradiation on embryonic development in the chicken targeted breeding method of Example 4 of the present invention. Figure A shows the embryonic development results after treatment with pure physical methods (X-rays with different irradiation doses), and Figure B shows the embryonic development results after treatment with chemical and physical methods.

[0047] Figure 9 This is a diagram showing the individual rearing of the mutant chicken in Example 4 of the present invention.

[0048] Figure 10 These are comparative images of the lungs of mutant chickens infected with avian influenza virus in Example 4 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.

[0049] Figure 11 This is a flowchart of the preparation of heavy ion-mediated target gene mutation cloned pigs in Example 5 of the present invention.

[0050] Figure 12 The results of a comparative experiment on the effect of adding ALA / bFGF to the culture medium in Example 5 of this invention on improving the activity of irradiated cells are presented.

[0051] Figure 13 This is a preferred flowchart of the mutation breeding method in a specific embodiment of the present invention. Detailed Implementation

[0052] 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.

[0053] 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.

[0054] 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.

[0055] The terms used in this invention are defined as follows: M0 generation: refers to biological material before mutagenesis treatment; for fish: single-celled fertilized eggs; for birds: primordial germ cells (PGCs); for mammals: somatic cells (such as fibroblasts). M1 generation: refers to materials obtained after heavy ion beam mutagenesis of M0 generation biological materials. At the cellular level: the mutagenized cell population (mutant cell bank); at the individual level: animal individuals directly developed and cultured from the mutagenized cells; M1 generation individuals may be chimeras, that is, they contain both mutant cells and wild-type cells in their bodies.

[0056] M2 generation: refers to the first generation offspring produced by M1 generation individuals through sexual reproduction (self-fertilization or hybridization). The genotype of M2 generation individuals may be heterozygous or homozygous.

[0057] M3 generation: refers to the offspring produced by M2 generation individuals through sexual reproduction. Homozygous individuals can be obtained in the M3 generation by interbreeding M2 generation heterozygotes.

[0058] PGCs: Primordial Germ Cells are the precursor cells of sperm and eggs, used as mutagenic material in avian breeding.

[0059] Heavy ion beam: refers to heavy ions (such as carbon ions¹²C) accelerated by an accelerator. 6+ Beams containing nitrogen ions, etc., have high linear energy transfer (LET) characteristics and can be used to induce gene mutations in biological materials.

[0060] Non-lethal mutagenic dose: refers to an irradiation dose that can induce gene mutations without causing mass cell or embryo death. In this invention, the non-lethal mutagenic dose is typically 0.1~5 Gy, preferably 0.5~3 Gy, to maintain cell or embryo survival rate above 50%.

[0061] Functional mutations: These are mutations that lead to changes in gene function, including frameshift mutations, nonsense mutations, key domain disruptions, splicing site variations, etc., which are distinct from synonymous mutations or non-coding region mutations.

[0062] like Figure 13 As shown, a preferred method of the present invention for breeding animals based on heavy ion beam mutagenesis includes the following steps: S1 Controlled Mutation Introduction: Gene mutations are induced by irradiating the cells or tissues of the target animal with a non-lethal mutagenic dose of heavy ion beam to obtain a mutant cell library; the cells or tissues of the target animal include somatic cells, primordial germ cells or fertilized eggs in the single-cell stage of the target animal. S2 Screening and Identification: The mutant cell library is preliminarily screened to obtain candidate cell libraries carrying gene mutations; S3 Cultivating Mutant Individuals: Cultivating the candidate cell bank to obtain individuals carrying gene mutations; After the individuals carrying the gene mutation reach sexual maturity, molecular screening is used to identify the genotype of their germ cells to confirm whether they contain M1 generation individuals with gene mutation. If they do not contain it, the operation is terminated or mutagenesis and screening are repeated until the target gene with mutation is identified and the process proceeds to step S4. If it is found, it is confirmed to be a positive germline chimera and the subsequent breeding operation in step S4 is carried out. Molecular screening involves pooling the DNA of M1 generation individuals and performing high-depth targeted sequencing on one or more selected gene regions to identify the presence of mutations in those genes. The depth of high-depth targeted sequencing is set according to the pool size: when the pool contains at least 50 individuals, the average coverage depth is no less than 5000×; when the pool contains at least 100 individuals, the average coverage depth is no less than 10000×; and when the pool contains at least 500 individuals, the average coverage depth is no less than 30000×. Molecular screening and identification can first utilize bioinformatics analysis to discover mutations, and then verify the M1 generation individuals carrying one or more specific gene mutations obtained after molecular screening and identification using at least one of PCR (dPCR), competitive allele-specific PCR (KASP), and Sanger sequencing to confirm their mutations and individual genotypes, thus obtaining M1 generation individuals containing specific gene mutations. The preferred verification steps include: first, using digital PCR (dPCR) technology to verify candidate mutation signals in the pooled DNA to confirm their actual existence; then, for the verified mutation sites, using competitive allele-specific PCR (KASP) genotyping to perform high-throughput screening of all individuals corresponding to the pooled DNA to quickly identify one or more candidate individuals carrying specific gene mutations; finally, using Sanger sequencing to perform a final verification of the mutation sites of the target gene in the screened candidate individuals to accurately confirm their mutation type and genotype. S4 strain establishment: Individuals carrying gene mutations from step S3 are bred (using conventional hybridization, parthenogenesis, or germ cell transplantation, etc.) to obtain offspring strains that can inherit gene mutations. These offspring strains can then be used for industrial applications.

[0063] (a) Mutation breeding of fish One such Figure 1 The fish heavy ion mutagenesis breeding method of the present invention includes the following steps: S1: Parental preparation and fertilized egg acquisition: Using standard artificial breeding techniques for the target fish species, healthy fertilized eggs in the single-cell stage were obtained as M0 generation mutagenesis material; when collecting fertilized eggs, microscopic confirmation was used to confirm that they were in the stage before the first cleavage to ensure that they were in a single-cell state. S2: Heavy Ion Beam Irradiation Mutagenesis: During the single-cell stage of the fertilized eggs, to improve the controllability of energy deposition in the target area (zygotic nucleus / blastodisc), the fertilized eggs are oriented and fixed before irradiation mutagenesis, so that their animal pole faces the direction of the heavy ion beam, allowing the heavy ion beam to be incident along the direction of embryonic polarity; then, the single-cell stage fertilized eggs are subjected to irradiation mutagenesis treatment using a heavy ion beam; the heavy ion beam includes, but is not limited to, a carbon ion beam, but is preferably a carbon ion beam, and the energy of the carbon ion beam is preferably 80 MeV / u; the dose of heavy ion beam mutagenesis is a non-lethal dose that can induce mutations and ensure embryonic survival, preferably 0.5~3 Gy, more preferably 0.5~1 Gy; the LET of the heavy ion beam is 30~50 keV / μm; at the same time, in order to balance the rapid burst of local free radicals (ROS) induced by heavy ion irradiation and the high dependence of early embryonic metabolism on oxygen, a high-oxygen incubation solution containing buffer components (dissolved oxygen preferably 6.5~8.0) is prepared in this specific embodiment. The high-oxygen incubation solution (mg / L) is used as the carrier mother solution for single-cell stage fertilized eggs to maintain the homeostasis of embryonic oxygen metabolism during irradiation; the high-oxygen incubation solution is preferably prepared by the following steps: using E3 culture medium or HBSS basal solution as the base medium, adding 1% trehalose and 0.5~1 mM NaHCO3 by mass fraction, and slowly aerating at 4℃ for 30~60 min to raise the dissolved oxygen to 6.5~8.0 mg / L, then sealing and raising the water temperature to the embryo incubation temperature; S3: Embryo Hatching and Population Building: The fertilized eggs from step S2 are hatched and raised under suitable hatching conditions for the fish species to obtain the M1 generation population.

[0064] S4: Molecular Screening and Identification: Molecular screening and identification is performed on a portion of the M1 generation individuals to identify M1 individuals carrying one or more selected gene mutations. Molecular screening and identification involves pooling the DNA of the M1 generation individuals and performing high-depth targeted sequencing on one or more selected gene regions to identify the presence of mutations in those genes. The depth of high-depth targeted sequencing is set according to the pool size: when the pool contains at least 50 individuals, the average coverage depth is not less than 5000×; when the pool contains at least 100 individuals, the average coverage depth is not less than 10000×; and when the pool contains at least 500 individuals, the average coverage depth is not less than 30000×. After molecular screening and identification, at least one of PCR (dPCR), competitive allele-specific PCR (KASP), and Sanger sequencing can be used to verify the M1 generation individuals carrying one or more specific gene mutations obtained after molecular screening and identification. After confirming the mutation and individual genotype, M1 generation individuals containing specific gene mutations are obtained. The preferred verification steps include: First, using digital PCR (dPCR) technology to verify candidate mutation signals in the pooled DNA to confirm their actual existence; then, for the verified mutation sites, using competitive allele-specific PCR (KASP) genotyping to perform high-throughput screening of all individuals corresponding to the pooled DNA to quickly identify one or more candidate individuals carrying specific gene mutations; finally, for the screened candidate individuals, using Sanger sequencing to perform a final verification of the mutation sites of the target gene to accurately confirm their mutation type and genotype. S5: Later breeding and cultivation: For the M1 generation individuals with specific gene mutations identified in step S4, offspring strains that can stably inherit the gene mutation are obtained through breeding.

[0065] Example 1: Screening and breeding of bmp6 gene-related mutation sites in paddy field carp A heavy ion beam mutagenesis breeding method for a rice paddy carp strain without intermuscular spines includes the following steps: S1: Parent preparation and fertilized egg acquisition: Select healthy paddy field carp as parents and obtain fertilized eggs through standard artificial breeding techniques; confirm under a microscope that they are in the single-cell stage before the first cleavage begins.

[0066] S2: Heavy ion beam irradiation mutagenesis: Before irradiation, preparation was carried out by positioning the fertilized eggs with their animal poles facing the direction of the heavy ion beam and fixing them in an agarose mold. The eggs were then treated with a modified HBSS incubation medium containing 1% trehalose and 0.7 mM NaHCO3 at a dissolved oxygen concentration of 7.0 mg / L to optimize the irradiation environment. Then, during the single-cell stage, a carbon ion beam (¹²C) of 80 MeV / u was used. 6+ Irradiation was performed at a dose of 1.0-5.0 Gy (more preferably 1 Gy in this embodiment); the LET of the heavy ion beam was 30-50 keV / μm (more preferably 50 keV / μm in this embodiment); after irradiation, the fertilized eggs were quickly transferred to standard incubation conditions.

[0067] S3: Embryo Hatching and M1 Generation Population Construction: Under suitable hatching conditions for this fish species, the irradiated fertilized eggs were hatched and conventionally fed to obtain an M1 generation variant population of approximately 3,000 fish.

[0068] S4: Molecular Identification and Screening of Generation M1: Genomic DNA was extracted from the Generation M1 population, and pooled sequencing was constructed at a scale of 100 tails / pool. In this embodiment, after proportional conversion, the sequencing depth of the pooled sequencing at 100 tails / pool was set to ≥10,000× to ensure that chimeric mutations with a minimum allele frequency of 0.1% could be detected. A targeted capture panel was constructed for the major gene bmp6 of the intermuscular spur, and high-depth targeted sequencing was performed. Based on meeting the minimum depth threshold (≥10,000×), the average sequencing depth of each pool was further increased to ≥30,000× in this embodiment. The sequencing results shown in Table 1 show that a stable mutagenic mutation signal was detected in the region where the bone formation-related gene bmp6 is located. An insertion mutation was detected at a specific site on chromosome B24, with the reference sequence being AAG and the mutation form being AAGAG. This mutation was supported by multiple independent sequencing reads in the pooled sequencing, and its mutation ratio was significantly higher than the background sequencing error level, indicating that this mutation is a real mutagenic genetic variation. For the bmp6-related mutation sites, digital PCR (dPCR), allele-specific PCR, or competitive allele-specific PCR (KASP) typing techniques are further used to verify the candidate mutation signals in the pool and to screen individuals by typing, thereby identifying M1 generation candidate individuals carrying the bmp6 mutation sites.

[0069] S5: Subsequent breeding and strain establishment of the target mutant: Using the identified mutant individuals as core breeding materials, the M2 generation segregating population is obtained through conventional self-pollination. Based on linkage molecular markers at the mutation site and its adjacent regions, genotyping and typing screening are performed on all M2 generation individuals, and individuals exhibiting homozygous mutations at the target site are selected.

[0070] Table 1: Sequencing information of bmp6 gene-related mutations

[0071] The above sequencing results are derived from high-depth targeted sequencing analysis of DNA pooled samples from M1 generation individuals. The total sequencing depth is the cumulative depth of all covering reads within the target region. This mutation signal was stably supported in the sequencing data, and its mutation rate was significantly higher than the background sequencing error level, indicating that this mutation is a real mutagenetic variation. The reference sequence corresponding to chromosome B24 is NC_056620.1 (see [link to relevant documentation]). Figure 2 ).

[0072] Figure 2Sequencing results showed that, compared with the wild type, the mutagenized sample of Example 1 exhibited a base insertion (AAG→AAGAG) near locus 6344761 on chromosome B24. In the alignment, heavy ion beam mutagenesis marked with "+" successfully introduced a stable insertional genetic variation into the bmp6 gene region, providing molecular evidence for subsequent screening of bone formation and intermuscular spur-related traits based on bmp6. Table 2 shows the comparison results of fertilized egg hatching survival rate, mutation detection rate, and malformation rate under different treatment conditions.

[0073] Table 2: Effects of different treatment conditions on fertilized egg hatching survival rate and mutation detection efficiency

[0074] Table 2 shows a comparative analysis of different LET, dosage, and incubation conditions. Low dosage (1 Gy) and high LET (50 keV / μm) can maintain a high hatching survival rate while improving mutation detection rate and controlling the deformity rate. This trend is further enhanced when combined with ROS-buffered incubation. Under high dosage conditions, although the mutation detection rate is improved, the hatching survival rate decreases significantly and the deformity rate increases. The results of the above comparative experiments indicate that within the lower dosage range, by selecting a higher LET and optimizing incubation conditions, a better balance can be achieved between deformity rate, survival rate, and mutation detection.

[0075] Example 2: Obtaining lepr gene-related mutation sites and combining them with growth trait screening This embodiment provides a method for obtaining lepr gene-related mutation sites in paddy field carp using heavy ion beam mutagenesis combined with M1 generation molecular screening, and using it for screening populations with different growth traits, to illustrate the applicability of the present invention in screening for growth-related gene mutations and trait grouping, specifically including the following steps.

[0076] S1: Parental preparation and fertilized egg acquisition: Following the standard artificial breeding procedure for paddy field carp, sexually mature and healthy male and female parents were selected for artificial spawning and insemination to obtain healthy fertilized eggs in the single-cell stage as M0 generation mutagenesis material. After collection, the fertilized eggs were confirmed under a microscope to be in a single-cell state before the onset of the first cleavage.

[0077] S2: Heavy Ion Beam Irradiation Mutagenesis: Single-celled fertilized eggs were oriented and fixed so that their animal poles faced the direction of the heavy ion beam. Subsequently, the fertilized eggs were subjected to irradiation mutagenesis using a carbon ion beam. The carbon ion beam energy was 80 MeV / u, and the irradiation dose was controlled within the non-lethal range of 0.5~1 Gy. A hyperoxia incubation solution containing buffering components was used as a carrier stock solution during irradiation to maintain metabolic homeostasis of the embryos. The preparation process of the hyperoxia incubation solution in this embodiment was the same as in Example 1.

[0078] S3: Embryo Hatching and Population Building: Irradiated fertilized eggs are hatched and raised under suitable conditions to obtain an M1 generation population.

[0079] S4: Molecular Screening and Identification: DNA was extracted from a portion of the M1 generation individuals and mixed into pools for high-depth targeted sequencing analysis of the target gene region. Sequencing results showed a mutation signal detected in the lepr gene-related region. This mutation had stable sequencing support depth in the pooled sequencing, and its mutation frequency was higher than the background sequencing error level. For the aforementioned lepr-related mutation sites, digital PCR (dPCR) or allele-specific molecular detection methods were further used to verify candidate mutation signals in the pooled sequencing. Combined with competitive allele-specific PCR (KASP) genotyping technology, individuals in the pooled sequencing were screened to identify M1 generation candidate individuals carrying the aforementioned lepr-related mutation sites.

[0080] S5: Later-stage breeding and phenotypic grouping: M1 generation individuals carrying lepr-related mutation sites were fed and bred, and their growth indicators such as body length and weight were measured under the same or similar breeding conditions. The population was analyzed based on the body length-weight relationship model, and the traits of the mutated population were compared with those of conventional paddy field carp. The results are shown in Table 3.

[0081] Table 3: Comparison of traits between the LEPR mutant rice paddy carp population obtained by mutagenesis and the conventionally farmed population.

[0082] Table 4 summarizes the representative mutation sites detected in the lepr gene region by high-depth targeted sequencing in the M1 generation of rice paddy carp in this embodiment. The listed mutations include deletion and insertion variants, distributed at different chromosomal locations. The table provides the chromosome number, genomic coordinates, reference sequence (Ref), mutation type (Variant), mutation type, total sequencing depth, number of sequencing reads supporting the mutation, and corresponding mutation frequency for each mutation site. All listed mutations were supported by multiple independent reads in pooled sequencing, and their mutation frequencies were significantly higher than the background sequencing error level, indicating that these mutations are real mutagenic genetic variations and can serve as candidate mutation sites for subsequent molecular screening and trait association analysis.

[0083] Table 4: Results of Detection of Lepr Gene-Related Mutation Sites

[0084] The comparison photos of the growth phenotypes of the lepr mutant individuals and wild-type paddy field carp obtained in this embodiment are as follows: Figure 3 As shown in the figure, A, B, and C are wild-type paddy field carp individuals that have not undergone heavy ion beam mutagenesis, while D, E, and F are candidate individuals that have undergone heavy ion beam mutagenesis and showed lepr-related mutation sites in the M1 generation molecular screening. All individuals were raised under the same culture conditions and sampled at the same time point. The figure shows that, compared to wild-type individuals (AC), lepr mutant individuals (DF) exhibited significant differences in body length and weight. This phenotypic difference is consistent with the molecular screening results based on lepr gene mutations described in Example 2, indicating that the mutagenesis and screening method can obtain candidate individuals with identifiable differences in growth traits at the population level.

[0085] Example 3: Shortening the generation cycle using germ cell transplantation technology A method for reproductive propagation of paddy field carp carrying target gene mutations is provided to illustrate the applicability and scalability of the present invention under different reproductive pathways.

[0086] Steps S1 to S4 in this embodiment are the same as in Embodiment 1 (or the corresponding steps in Embodiment 2 can be used). Through the above steps, M1 generation candidate individuals carrying target gene mutations (including but not limited to lepr, bmp6, or other functional gene-related mutations) can be obtained. Subsequent steps can be implemented as follows.

[0087] S5 (Recipient Preparation and Cell Transplantation): To accelerate the genetic transmission of the target mutation, a germ cell transplantation pathway is employed. Testicular tissue from M1 generation candidate individuals carrying the target gene mutation is enzymatically digested to isolate and obtain donor germ cell stem cells. Simultaneously, antisense oligonucleotides (MOs) are injected during the embryonic stage of goldfish with short sexual maturation cycles to prepare degametized recipients. The donor germ cell stem cells are then transplanted into the body cavity of 2-day-old recipient goldfish larvae. S6 (Gamete Acquisition and Identification): The recipient fish are raised to sexual maturity (this process is much shorter than that of paddy field carp), and their semen is collected and identified by species-specific molecular markers to confirm that the obtained semen comes from the donor paddy field carp; In this embodiment, three individuals are randomly selected from the recipient goldfish that have completed germ cell transplantation and survived to sexual maturity as test subjects, and are respectively denoted as recipient 1, recipient 2 and recipient 3. S7 (Rapid Propagation and Background Isolation): Using the mutant sperm obtained through this "surrogate reproduction" method, artificial insemination can be performed with the eggs of normal female paddy field carp, resulting in an M2 generation population carrying the target gene mutation in a significantly shorter time. The mutant sperm is selected from any recipient goldfish individual identified as a donor source in Example 3; the eggs are derived from normal female paddy field carp parents.

[0088] The method in this embodiment combines our targeted screening optimization, which ensures that the obtained gametes not only possess the characteristics of the donor species but also carry the target mutations locked by heavy ion mutagenesis and molecular screening by detecting molecular markers related to the formation or growth of intermuscular spines (such as the bmp6 and lepr gene mutation sites in Examples 1 and 2). This approach not only significantly shortens the time to obtain the next generation but also, by using goldfish as a host, isolates to some extent the physiological burden of heavy ion mutagenesis on the donor carp and the accompanying cumulative effects of mutations.

[0089] Figure 4 The electrophoresis results are those of PCR amplification of DNA samples from different sources using carp and goldfish species-specific primers (see Table 5 below, cited from Franěk et al., 2019) in Example 3. Recipient 1, Recipient 2, and Recipient 3 are three goldfish individuals that underwent germ cell transplantation and reached sexual maturity in Example 3. Lane 1: Goldfish genomic DNA (gDNA); Lane 2: Carp genomic DNA (gDNA); Lane 3: Wild carp sperm DNA; Lanes 4-6: Sperm DNA produced by Recipient 1, Recipient 2, and Recipient 3. Figure 4 The results showed that specific amplified bands consistent with the carp genome and wild carp sperm could be detected in the sperm DNA produced by the recipient, indicating that the sperm produced by the recipient originated from the reproductive cells of the donor carp.

[0090] Table 5: Goldfish and carp-specific primers used to confirm that the recipient transplanted cells originated from the donor (see Roman Franěk, Marinovi Z, Luji J, et al. Cryopreservation and transplantation of common carp spermatogonia[J]. PLoS One, 2019, 14(4):e0205481.doi.org / 10.1371 / journal.pone.0205481)

[0091] Figure 5 This diagram illustrates the fertilized egg and early cleavage morphology formed after the donor-derived mutant sperm produced by the recipient goldfish in Example 3 was fertilized with the eggs of a normal female paddy field carp. The sperm was derived from... Figure 4 Any recipient goldfish individual identified as a donor source. Figure 5 As can be seen from the schematic diagram of the fertilized egg and early cleavage morphology formed after the sperm produced by the recipient fertilizes a normal egg, the sperm is shown to have normal fertilization ability.

[0092] As can be seen from the above examples of fish mutation breeding, this invention strictly limits mutation to the single-cell stage of fertilized eggs, allowing the resulting mutations to be simultaneously distributed into cell lineages, including primordial germ cells (PGCs), during subsequent cleavage. Therefore, the heritability of the mutation is established at the moment of its occurrence. Based on this, this invention proposes combining heavy ion beam mutagenesis with M1 generation targeted deep sequencing, enabling direct molecular-level identification and locking of heritable mutant individuals already in the germ cell lineage during the mutagenesis generation, without waiting for subsequent genotype isolation and verification. This invention pioneers the application of the core technology combination of "heavy ion mutagenesis + M1 generation targeted deep sequencing screening" in fish screening breeding. For the first time in fish breeding, it achieves early and precise molecular-level identification of target traits controlled by major genes in the mutagenesis generation (M1 generation). Compared with traditional methods, this invention shortens the breeding cycle from several generations to as short as 1-2 generations, significantly improving screening efficiency and providing an efficient, precise, and universally applicable technical path for rapidly creating new germplasm and new varieties of various fish.

[0093] Furthermore, the directional irradiation design of this invention effectively overcomes the adverse effects of high water content, large yolk volume, and easy scattering of energy deposition in fish fertilized eggs. This allows induced mutations to be synchronously distributed into cell lineages, including primordial germ cells (PGCs), during early cleavage, laying a structural foundation for obtaining heritable mutations. This invention introduces a high-oxygen incubation solution containing buffering components as the mother solution for fertilized eggs during heavy ion beam irradiation in the single-cell stage. By increasing and stabilizing dissolved oxygen levels, it effectively balances the contradiction between the rapid burst of local free radicals (ROS) induced by heavy ion irradiation and the high-oxygen metabolic demands of early embryonic development. This measure significantly improves the survival rate and developmental stability of fertilized eggs within the non-lethal mutagenic dose range, making heavy ion mutagenesis operable and reproducible in fish.

[0094] Finally, in terms of rapidly creating new germplasm and new varieties of fish, this invention, through heavy ion beam mutagenesis combined with subsequent molecular screening and selection breeding processes, can obtain fish populations with rapid growth characteristics. Under the same or similar culture conditions, joint analysis of the body length and weight of the obtained populations revealed that the populations can be stably distinguished into fast-growing and slow-growing groups in terms of growth phenotype. Specifically, the fast-growing group significantly outperformed the unmutated or conventionally cultured groups in key growth indicators such as body length and weight, and exhibited a higher growth index b value in the body length-weight relationship model, displaying positive allometric growth characteristics, indicating that its weight gain rate was significantly enhanced relative to body length gain (see detailed embodiments). These results demonstrate that the new fish strains obtained by this invention have formed stably distinguishable phenotypic differences from existing varieties in terms of growth efficiency and growth trajectory, and can serve as an effective basis for identifying rapid growth functional traits.

[0095] (II) Mutation breeding of poultry Example 4: 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 6 As shown, the specific methods and steps include the following.

[0096] 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.

[0097] 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.

[0098] 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 7 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.

[0099] 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 8 (Figure B). But if only physical methods are used (see Figure B). Figure 8 (Figure A) This will cause delayed embryonic development and significantly affect breeding efficiency.

[0100] 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.

[0101] 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).

[0102] 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 9The 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 10 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.

[0103] As can be seen from the above examples, traditional hybridization breeding requires multiple generations of repeated selection, taking several years. In contrast, this invention, through direct mutagenesis of PGCs, yields mutant traits that can be directly inherited by offspring, avoiding the cumbersome hybridization and backcrossing processes, and potentially shortening the breeding cycle for new varieties. This invention provides the possibility of creating entirely new genetic variations and germplasm resources. Heavy ion irradiation can produce beneficial mutations that are rare or nonexistent in nature, thereby cultivating new poultry germplasm, which is difficult to achieve with traditional breeding methods. Furthermore, the method of this invention is a physical mutagenesis, without the residues of chemical mutagens or environmental pollution problems. Compared with obtaining beneficial 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. In addition, the breeding materials obtained through PGC-mediated genetic modification 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.

[0104] (III) Mutagenesis breeding of mammals Example 5: Mutagenesis screening and obtaining CD163 gene mutant cells using porcine fetal fibroblasts (PEFs) as an example This embodiment uses PEFs as donor cells to systematically demonstrate the specific operation procedures and methods of using a heavy ion beam mutagenesis platform for pig breeding (see [link]). Figure 11 ).

[0105] First, fetal pig tissues were collected from gestational pigs at 30-40 days of gestation. Primary PEFs were prepared by digestion with collagenase and trypsin and then amplified in DMEM medium containing 10%-20% FBS until a fusion rate of 70%-80% was achieved.

[0106] Heavy ion beam irradiation was performed using a 50 Me V / u carbon ion beam (¹²C) provided by the Lanzhou Heavy Ion Research Facility. 6+Cells were irradiated in a synchrotron radiation device and randomly divided into 0 Gy control group, 0.5 Gy, 1 Gy, 2 Gy, and 3 Gy mutagenesis treatment groups. Cell viability was recorded at 24 h and 72 h after irradiation, and the effect of irradiation on cell viability was further evaluated by colony formation assay.

[0107] To improve cell growth after heavy ion beam irradiation, this invention proposes adding a combination of ALA and bFGF to the culture medium. By optimizing the cell culture environment, regulating cell membrane fluidity and lipid metabolism, and enhancing cell repair and proliferation capabilities, the growth of cells after heavy ion beam irradiation is improved. In specific implementation, porcine fetal fibroblasts (PEFs) were selected as experimental subjects, and different concentrations of ALA (5 μM, 10 μM, 20 μM) and bFGF (10 ng / mL, 20 ng / mL, 30 ng / mL) were used for irradiation with a 2 Gy heavy ion beam and then cultured. Experimental results showed that the combination of ALA and bFGF significantly improved the activity of cells irradiated by heavy ion beam, with a particularly preferred concentration of 10 μM ALA and 10 ng / mL bFGF (see [link to relevant documentation]). Figure 12 The red arrow indicates the addition of the combination: ALA 10 μM + bFGF 10 ng / mL, which increased the viability of irradiated cells by 58% compared to the control group (see [reference]). Figure 12 This optimization scheme provides a stable foundation for obtaining mutant cells in the future.

[0108] Mutagenized cells were seeded in culture plates at a density of approximately 100–500 cells / well. After confluence, a portion of the cells were cryopreserved, while the remainder were used for genomic DNA extraction. The library design covered exons and regulatory regions of candidate genes or key pathways. High-throughput sequencing (e.g., Illumina platform) was used to analyze mutation frequency and type at a sequencing depth of 50,000×. Functional hit criteria included: frameshift mutations, deletion of key domains, altered splicing signals, or disruption of promoter core elements. Samples meeting these criteria proceeded to single-clone isolation and subsequent validation.

[0109] Single-cell colony establishment was performed using flow cytometry for single-cell sorting and distribution into 96-well plates for culture. The colonies were expanded to approximately 10-1. 5 After cell division, each cell was genotyped, and positive clones were stored in a liquid nitrogen cryopreservation bank, with barcodes simultaneously established for traceability.

[0110] This embodiment is illustrated by... Figure 11 The process shown can systematically obtain diverse mutant clones of genes without relying on nuclease editing, and realize a closed-loop operation from mutagenesis to screening, laying the foundation for subsequent mammalian breeding or model construction.

[0111] To verify the feasibility of the mutagenesis scheme in this embodiment, the CD163 gene was selected as the target, with the aim of obtaining pig somatic cells with this gene mutation, so as to provide nuclear transfer donor cells for the subsequent creation of PRRSV-resistant cloned pigs.

[0112] First, porcine PEFs were irradiated with a 1 Gy heavy ion beam, and a sequencing library targeting exons 1-7 of CD163 was constructed. Results showed that among the 62 mixed-cell samples tested, two cell lines had small-fragment deletion mutations, one with a 5-base deletion and the other with a 16-base deletion, both leading to frameshift mutations and premature termination of the CD163 gene (see Table 6 below). These two functionally deficient CD163 monoclonal cells were obtained through monoclonal screening.

[0113] Table 6: Sequencing results of mixed-pool cells targeting CD163

[0114] Subsequently, using positive clones as donors, reconstructed embryos were prepared using somatic cell nuclear transfer technology. Enucleated oocytes were fused with donor cells, followed by electrofusion and chemical activation, and cultured in vitro to the blastocyst stage before being transplanted into recipient sows in estrus at the same time. The transplantation success rate was approximately 40%, ultimately yielding healthy piglets. Molecular confirmation was performed after the cloned pigs were born, including PCR and Sanger sequencing to verify the CD163 genotype, and immunohistochemistry to detect CD163 protein expression levels. The cloned pigs' genotype will be identical to that of the nuclear donor, and the CD163 protein will prematurely terminate translation due to a frameshift mutation. According to existing literature (Xu et al. eLife, 2020; Yang et al. Antiviral Research, 2018), these CD163-inactivated pigs will exhibit significant PRRSV resistance.

Claims

1. A method for breeding animals based on heavy ion beam mutagenesis, characterized in that, Includes the following steps: S1 Controllable Mutation Introduction: Gene mutations are induced by irradiating the cells or tissues of the target animal with a non-lethal dose of heavy ion beam to obtain a mutant cell library. S2 Screening and Identification: The mutant cell library is preliminarily screened to obtain candidate cell libraries carrying gene mutations; S3 Cultivating Mutant Individuals: Cultivating the candidate cell bank to obtain individuals carrying gene mutations; S4 strain establishment: The individuals carrying the gene mutation from step S3 are bred to obtain offspring strains that can inherit the gene mutation.

2. The method according to claim 1, characterized in that, The cells or tissues of the target animal include somatic cells, primordial germ cells, or fertilized eggs in the single-cell stage.

3. The method according to claim 2, characterized in that, The target animal is a fish, and the cells or tissues of the target animal are fertilized eggs in the single-cell stage. The gene mutations targeted by the screening and identification include one or more mutations in the bmp6, sp7, runx2b, lepr, and mstn genes.

4. The method according to claim 3, characterized in that, Before heavy ion beam irradiation, the fertilized eggs are oriented and fixed so that their animal pole faces the direction of the heavy ion beam, and the heavy ion beam is incident along the direction of embryonic polarity. A high-oxygen incubation solution containing buffer components is prepared as the carrier mother solution for single-cell stage fertilized eggs, and the dissolved oxygen concentration is 6.5~8.0 mg / L.

5. The method according to claim 4, characterized in that, The hyperoxic incubation solution is prepared by the following steps: using E3 culture medium or HBSS base medium as the base medium, adding 0.5%~2.0% trehalose and 0.5~1mM NaHCO3 by mass, and slowly aerating at 4℃ for 30~60 min to raise the dissolved oxygen to 6.5~8.0 mg / L, then sealing and raising the water temperature to the embryo incubation temperature.

6. The method according to claim 3, characterized in that, The reproduction occurs through one or a combination of the following three pathways: a) Cross the M1 generation individuals containing the gene mutation with wild-type individuals, and introduce the "directed backcross + linkage molecular marker" monitoring method. Perform one or more directed backcrosses between the M1 generation individuals carrying the gene mutation and the corresponding wild-type individuals, and use linkage molecular markers near the gene locus to selectively track and genotype the M2 and subsequent populations. Select and breed in the M2 generation and subsequent generations to obtain homozygous mutant lines with clear genetic background and stable trait expression. b) Using parthenogenesis, the gametes of the M1 generation individuals containing the gene mutation were induced to directly develop into homozygous diploid M2 generation; c) The germ cells of the M1 generation individuals containing the gene mutation are transplanted into the recipient fish that has undergone germ cell removal treatment, and the recipient fish is used to generate donor-derived gametes.

7. The method according to claim 2, characterized in that, The target animal is a bird, and the cells or tissues of the target animal are PGCs of the target animal. The gene mutations targeted for screening and identification include one or more mutations among 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 claim 7, characterized in that, The obtained mutant cell library was first placed in M1 generation cell culture medium for recovery period culture, then replaced with normal culture medium, and then the PGCs in the mutant cell library were separated into single cells to form monoclonal cells before proceeding to step S2. The culture medium for the M1 generation cells contains antioxidants and stem cell factors. 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.

9. The method according to claim 8, characterized in that, The specific operation method for separating PGCs from mutant cell libraries into single cells also includes: preparing single-cell suspensions by dilution and cell sieving, and then obtaining single-cell clones by immunomagnetic bead sorting, flow cytometry sorting, single-cell sorting or limiting dilution method according to the expression location and detection method of the target gene encoded protein; for target genes that are not suitable for cell surface antibody sorting, molecular typing screening can be performed directly after single-clone culture.

10. The method according to claim 2, characterized in that, The target animal is a mammal, the cells or tissues of the target animal are somatic cells of the target animal, and the target gene includes one or more of CD163, ANPEP, and LDLR.

11. The method according to claim 10, characterized in that, After in vitro irradiation mutagenesis using the heavy ion beam, a combination of α-linolenic acid and basic fibroblast growth factor is added to the culture medium of the mutagenized somatic cells; the concentration of α-linolenic acid in the culture medium is controlled at 5~20 μM, and the concentration of basic fibroblast growth factor is controlled at 10~30 ng / mL.

12. The method according to any one of claims 1 to 11, characterized in that, The heavy ion beam is a carbon ion beam with an energy of 30-80 MeV / u; the irradiation mutagenesis dose is 0.1-5 Gy, preferably 0.5-3 Gy, and the irradiation time is 30-60 s.

13. The method according to any one of claims 1 to 11, characterized in that, The preliminary screening is a molecular typing screening targeting a specific site / gene / pathway, including one or a combination of whole-genome resequencing, whole-exome sequencing, targeted deep sequencing, capture sequencing, digital PCR, KASP typing, and Sanger sequencing.

14. The method according to claim 13, characterized in that, The targeted deep sequencing refers to high-coverage sequencing of target genes, target sites, or target pathway-related regions. When the pool contains at least 50 individuals, the average coverage depth is not less than 5000×; when the pool contains at least 100 individuals, the average coverage depth is not less than 10000×; when the pool contains at least 500 individuals, the average coverage depth is 30000~50000×. If a gene mutation is detected, proceed to the next step S3; if no gene mutation is detected, terminate the operation or repeat steps S1~S2 until a mutation of the target gene is detected, then proceed to step S3. The process after step S3 and before step S4 further includes molecular screening and identification. The molecular screening and identification includes: after the individual carrying the gene mutation reaches sexual maturity, the genotype of its germ cells is detected to confirm whether it contains the M1 generation individual with the gene mutation. If it does not contain it, the operation is terminated or the mutagenesis and screening are repeated until the target gene with the mutation is identified and the process proceeds to step S4. If it contains it, it is confirmed as a positive germline chimera and the subsequent reproductive operation in step S4 is carried out.

15. The method according to any one of claims 1 to 11, characterized in that, The cultivation method in step S3 includes one of the following: incubation, feeding, somatic cell nuclear transfer to reconstruct embryonic development, and injection into the recipient embryonic development after its own PGCs have been ablated.

16. The method according to claim 15, characterized in that, The target animal is a mammal; the reconstructed embryo includes somatic cell nuclear transfer, embryo aggregation, and equivalent nucleocytoplasmic reprogramming steps. The somatic cell nuclear transfer reconstructing embryonic development specifically refers to using the positive somatic cell clone as a donor to perform somatic cell nuclear transfer reconstructing embryos and cultivating individuals containing gene mutations. The positive somatic cell clones are obtained from the candidate cell bank using a single-clone establishment and backtracking strategy. The establishment of the single clone adopts the limiting dilution method, the infinite dilution method, or the flow sorting method, and the traceability strategy adopts the barcode-traceability strategy or the "freeze-extraction-library construction" batch traceability strategy.

17. The method according to claim 15, characterized in that, The target animal is a bird, and the embryos are developed and cultivated using the recipient embryos after the PGCs injected into them are ablated. Specifically, the positive PGCs are injected into the recipient embryos after the PGCs are ablated by microinjection to obtain chimeras. The specific method for ablating the recipient embryo's own PGCs includes: irradiating with 3-5 Gy of X-rays and injecting the emulsified busulfan solution into the yolk; 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.

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