Method for breeding fish by heavy ion beam mutagenesis

Abstract

This invention discloses a heavy ion mutagenesis breeding method for fish, belonging to the field of fish breeding technology. To address the technical problem of how to efficiently apply mutagenesis breeding technology in fish genetic improvement, this method first breeds the target fish species to obtain fertilized eggs in the single-cell stage; then, it uses a heavy ion beam to induce mutagenesis in the single-cell stage fertilized eggs, hatches and raises the mutagenized eggs to obtain an M1 generation population; next, it performs molecular screening to identify M1 generation individuals carrying one or more gene mutations; finally, it uses breeding methods to obtain offspring strains that can stably inherit the stated gene mutations. 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 pathway for rapidly creating new germplasm and strains of various fish species.

Description

Technical Field

[0001] This invention belongs to the field of fish breeding technology, and in particular relates to a fish heavy ion beam mutagenesis breeding method based on physical mutagenesis. Background Technology

[0002] Currently, fish breeding still mainly relies on traditional selection breeding, hybridization breeding, molecular marker-assisted selection, and whole-genome selection techniques. For example, Chinese patent application CN120052307A discloses a "breeding method and system for a new hybrid strain of mandarin fish and tilapia," which utilizes a systematic combination of artificial hybridization, molecular marker-assisted selection, and strain fixation techniques to significantly improve the success rate and efficiency of mandarin fish and tilapia hybridization breeding. However, these existing methods have certain limitations. For example, traditional methods are time-consuming and somewhat arbitrary; molecular marker-assisted selection heavily relies on known candidate genes and marker information, making it difficult to handle complex traits controlled by multiple genes; and whole-genome selection requires the construction of large-scale reference populations and the accumulation of long-term, costly phenotypic data, resulting in high technical barriers and costs.

[0003] Radiation, as a ubiquitous physical factor in nature, is a crucial foundation for the formation of biological genetic diversity. Through long-term evolution, it continuously drives genomic variation by inducing DNA damage and repair processes. In breeding practice, physical mutagenesis technology is based on these biological principles, using artificially controlled radiation to directionally amplify the sources of genetic variation. Recent advancements in physical mutagenesis technology utilize the high linear energy density (LET) and strong relative biological effect (RBE) of heavy ion beams to induce diverse DNA variations at relatively low doses. Compared to traditional chemical or radiation-induced mutagenesis, it offers higher mutation efficiency and better genetic stability. This mutagenesis technology, combined with targeted deep sequencing, can directly detect target gene regions in the M1 generation, identifying individuals with target gene mutations. This enables targeted screening of specific genes and direct entry into the targeted breeding process, eliminating the need to wait for phenotypic manifestation. However, the aforementioned existing technologies are currently mainly applied to the breeding of new plant varieties such as rice, and have shown effects such as shortened breeding cycles and improved screening efficiency.

[0004] However, plant seeds are dense, homogeneous, static dry matter systems, resulting in relatively uniform energy deposition during irradiation. In animal breeding, especially fish breeding, the aforementioned techniques have not yet been reported to be systematically applied. In animal mutation breeding, particularly in fish genetic improvement research, chemical and radiation-induced mutations have been used in exploratory breeding practices, but these have primarily focused on random improvement at the trait level, and a targeted genetic improvement system guided by specific target genes has not yet been established. Traditional chemical mutagenesis (such as ENU) and gamma-ray mutagenesis typically introduce a large number of random mutations across the genome, with mutation sites distributed non-selectively in both somatic and germline lineages. Due to significant cell lineage differentiation in early fish embryonic development, the current generation (M1 generation) of mutated individuals generally exhibits a high degree of chimerism. To confirm the heritability of mutations, current fish mutation breeding typically still relies on indirect verification through phenotypic segregation in M2 and even higher generations, leading to a significantly prolonged breeding cycle.

[0005] Furthermore, the superposition of random mutations at multiple sites can easily damage multiple genes or regulatory regions simultaneously, often leading to physiological defects, developmental abnormalities, or decreased reproductive capacity. This makes it difficult to strike a balance between "mutation acquisition efficiency" and "individual survival rate" in terms of mutagenesis dosage, further limiting the application of this technology in fish genetic improvement. In contrast, in plant breeding, especially in crops such as rice, heavy ion beam mutagenesis has been proven to induce relatively controllable genetic variations at lower doses and can be combined with M1 generation molecular detection methods to achieve early screening of target gene mutations, thereby significantly shortening the breeding cycle. However, due to the fundamental differences between fish embryonic structure, germ cell formation, and developmental kinetics and those of plants, the aforementioned effective mutagenesis and screening systems in plants cannot be directly transplanted into fish mutagenesis breeding.

[0006] Moreover, the application of mutation breeding in fish still faces significant bottlenecks. Fish fertilized eggs are highly water-rich, rapidly cleaving systems with a distinct "animal-plant pole" polar structure. The yolk is large and can partially shield incident particles. Without control over the spatial orientation of the fertilized egg, the energy transfer of heavy ion beams in fish embryos is prone to scattering and uneven sedimentation, resulting in mutations being concentrated in non-nuclear regions or local cell clusters, making it difficult to effectively enter the primordial germline. This hinders "mutation-induced heritable selection in the next generation." This is fundamentally different from the stable energy deposition distribution in plants, where mutations can naturally enter offspring. Therefore, the established M1 generation molecular screening system in plants cannot be directly applied to fish mutation breeding.

[0007] Chinese patent application CN120188747A discloses a "method for breeding spotted sea bass using ARTP mutagenesis technology." This patent application utilizes ARTP mutagenesis technology to study the mutagenic effects of different mutagenesis parameters on spotted sea bass fertilized eggs, screen and optimize mutagenesis conditions, and establish an ARTP mutagenesis technology system for spotted sea bass to improve mutation efficiency and screen for superior varieties. However, from the perspective of fish breeding, the principle of ARTP mutagenesis technology is fundamentally different from that of heavy ion beams. On the one hand, ARTP mainly uses a large amount of reactive oxygen species and nitrogenous substances (RONS) generated during plasma discharge to act on biological cells or tissues. Its effective depth of action is limited, mainly inducing point mutations such as single base substitutions, with a low proportion of double-strand DNA breaks and loss-of-function mutations, making it difficult to stably obtain mutation types with clear functional effects on target genes. On the other hand, its mutagenic effect is highly sensitive to species, developmental stage, and parameter settings. In the absence of a supporting high-throughput molecular screening system, it is difficult to systematically identify and accurately target low-frequency chimeric mutations in the M1 generation. Furthermore, different fish species, and even different developmental stages of the same fish, exhibit vastly different sensitivities to mutagen. The mechanism of action of ARTP devices is relatively complex, and optimizing parameters (e.g., gas, power, time) relies heavily on experience, making precise control of mutagenesis a major challenge. The target detection limit of existing fish mutagenesis systems is typically ≥1-5% allele frequency, making it difficult to identify the low-frequency chimeric mutations prevalent in the M1 generation. Moreover, fish mutagenesis is often considered to result in high mutation rates leading to high lethality, making it difficult to strike a balance between viability and selectability in the mutagenesis dosage.

[0008] These problems severely restrict the efficient application of mutation breeding technology in fish genetic improvement. Summary of the Invention

[0009] 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 fish heavy ion beam mutagenesis breeding.

[0010] To solve the above-mentioned technical problems, the present invention proposes a method for fish heavy ion beam mutagenesis breeding, comprising the following steps:

[0011] S1: Breed the target fish species to obtain fertilized eggs in the single-cell stage; S2: Heavy ion beam mutagenesis is performed on fertilized eggs in the single-cell stage using heavy ion beams, wherein the dose of heavy ion beam mutagenesis is a non-lethal mutagenesis dose. S3: Hatch and raise the fertilized eggs from step S2 to obtain the M1 generation population; S4: Perform molecular screening on the M1 generation individuals to identify M1 generation individuals carrying one or more gene mutations; S5: For the M1 generation individuals with gene mutations identified in step S4, obtain offspring strains that can inherit the gene mutations through breeding methods.

[0012] Heavy ion beam mutagenesis is a physical mutagenesis technique developed in recent years. Utilizing the high linear energy transfer density (LET) and strong relative biological effect (RBE) of heavy ions, it can induce diverse DNA mutations at relatively low doses. Compared to traditional chemical or radiation mutagenesis, it features high mutation efficiency, controllable damage, and good genetic stability. At the same absorbed dose, the higher the LET value of radiation, the stronger the biological effect: the average LET of gamma rays is approximately 0.2 keV / µm, and that of X-rays is approximately 2.0-5.0 keV / µm. 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. However, applying this technique to animals, especially fish, presents numerous challenges, the core of which lies in how to efficiently and controllably generate valuable mutations. The above-mentioned technical solution of the present invention combines heavy ion beam mutagenesis with M1 generation molecular screening and identification technology. By irradiating fertilized eggs in the single-cell stage with heavy ion beams and combining them with 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, which can significantly reduce costs and improve the stability and efficiency of gene mutation acquisition.

[0013] In the aforementioned fish heavy ion beam mutagenesis breeding method, preferably, in step S2, the fertilized eggs are oriented and fixed before heavy ion beam mutagenesis, so that their animal pole faces the direction of the heavy ion beam current. This allows the heavy ion beam to be 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 reproductive system, thus solving the key problem of "uncontrollable energy deposition leading to mutagenesis instability".

[0014] 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. To address this, in the aforementioned fish heavy ion beam mutagenesis breeding method, preferably, in step S2, a high-oxygen incubation solution containing buffering components is prepared as the carrier stock solution 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. By constructing an external microenvironment regulation system for fertilized eggs, including dissolved oxygen concentration control, pH buffering, and osmotic protection, the physiological stability of fertilized eggs during irradiation is maintained, the conditions for the formation and transformation of irradiation-induced damage are altered, early lethality due to oxidative stress is prevented, and early embryonic death due to excessive oxidative damage is avoided.

[0015] More preferably, the hyperoxia incubation solution is prepared by the following steps: using E3 culture medium or HBSS basal medium as the base medium, 0.5%~2.0% (preferably about 1.0%) of trehalose and 0.5~1 mM NaHCO3 are added, and the solution is slowly aerated at 4°C for 30~60 min to raise the dissolved oxygen to 6.5~8.0 mg / L. The solution is then sealed and the water temperature is raised to the embryo incubation temperature. This measure further regulates the external microenvironment control system. Through the synergistic effect of trehalose and bicarbonate buffering system, combined with low-temperature pretreatment and dissolved oxygen regulation, the fertilized eggs maintain a stable osmotic pressure and pH environment during irradiation. This allows the 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] In the above-described fish heavy ion beam mutagenesis breeding method, preferably, in step S2, the heavy ion beam can be various heavy ions, including but not limited to carbon ion beams, oxygen ion beams, or nitrogen ion beams; wherein, preferably, a carbon ion beam (e.g., ¹²C) is used. 6 + The energy of the carbon ion beam is preferably 80 MeV / u.

[0017] In the above-described fish heavy ion beam mutagenesis breeding method, preferably, in step S2, the dose of heavy ion beam mutagenesis is 0.5~3 Gy, more preferably 0.5~1 Gy; the LET of the heavy ion beam is preferably 30~50 keV / μm. During heavy ion beam treatment, the formation of DNA damage depends not only on the total dose but also on the spatial distribution of energy deposition. LET, as a key parameter characterizing the energy deposition intensity per unit path length, determines the degree of energy concentration at the microscale, thereby affecting the structural characteristics of DNA damage. If LET is too low, energy deposition is relatively dispersed, and the resulting damage is mainly easily repairable; if LET is too high, local energy deposition is too concentrated, easily forming complex damage and increasing the accumulation of non-specific damage. This invention controls LET within the range of 30~50 keV / μm, combined with a stable external microenvironment regulation system, making the DNA damage formation process more stable and more suitable for subsequent screening and mutagenesis.

[0018] In the aforementioned fish heavy ion beam mutagenesis breeding method, preferably, step S4, the molecular screening and identification includes: mixing the DNA of the M1 generation individuals, performing high-depth targeted sequencing on a selected target gene region to identify whether a mutation of the target gene exists; if a mutation exists, step S4 is stopped and the process proceeds to step S5; if no mutation exists, another target gene region is selected for high-depth targeted sequencing until a target gene with a mutation is identified and the process proceeds to step S5. The target gene region generally refers to a gene region related to the target trait, and can be a target region targeting one or more major genes related to the target trait. The major genes related to the target trait are generally one or more major genes controlling the target trait. Introducing heavy ion beam mutagenesis combined with M1 generation high-depth targeted sequencing technology into fish breeding not only allows for the acquisition of high-frequency genetic variations at lower doses, but also enables rapid identification and precise screening of mutagenic effects when combined with high-throughput sequencing technology, thereby fundamentally shortening the breeding cycle, reducing costs, and improving efficiency.

[0019] In the aforementioned fish heavy ion beam mutagenesis breeding method, preferably, in step S4, the depth of the 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 30000~50000×, to ensure stable detection of mutations with allele frequencies as low as 0.1%. After initially identifying signals carrying beneficial mutations of the target gene (such as frameshift mutations, key domain disruptions, etc.) in the pool through bioinformatics analysis, a multi-step verification process can be used to precisely locate the mutated individuals.

[0020] In the above-mentioned fish heavy ion beam mutagenesis breeding method, preferably, after step S4 and before step S5, at least one of PCR (dPCR), competitive allele-specific PCR (KASP), and Sanger sequencing is used to verify the M1 generation individuals carrying one or more gene mutations obtained after molecular screening and identification, and after confirming their mutations and individual genotypes, an M1 generation individual containing a certain gene mutation is obtained.

[0021] A more preferred verification step includes: first, using digital PCR (dPCR) technology to verify candidate mutation signals in the pooled DNA to confirm their existence; then, for the verified mutation sites, using competitive allele-specific PCR (KASP) genotyping technology to perform high-throughput screening on all individuals corresponding to the pool, quickly identifying one or more candidate individuals carrying gene mutations; finally, for the screened candidate individuals, using Sanger sequencing to perform a final verification of the gene mutation site to accurately confirm their genotype. This process, employing a hierarchical convergent screening system combining pooled targeted deep sequencing with digital PCR and KASP genotyping, can stably detect mutation signals with an allele frequency as low as 0.05–0.14%, compared to the detection threshold of ≥1–5% commonly found in existing fish mutagenesis systems, representing a sensitivity improvement of approximately 10–80 times. This technology overcomes the long-standing problem of "low-frequency and difficult detection of chimeric mutations" in the field of fish mutagenesis, making direct screening of the M1 generation a feasible and reproducible technical strategy.

[0022] In the above-mentioned fish heavy ion beam mutagenesis breeding method, preferably, in step S5, the breeding method 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 select and breed in the M2 generation and subsequent generations; given that heavy ion mutagenesis may simultaneously generate background variations unrelated to the target mutation, this invention introduces a "directed backcross + linkage marker" monitoring strategy in path a, performing one or more directed backcrosses between the M1 individuals carrying the gene mutation and the corresponding wild-type individuals, so that non-target background variations are gradually diluted with each backcross generation; and selectively tracking and genotyping the M2 and subsequent populations with linkage molecular markers near the target site to ensure that the target mutation is maintained while background variations are gradually eliminated; this path can obtain homozygous mutant lines with clear genetic background and stable phenotypic expression within 2 to 4 generations; b) When a gene mutation results in a loss of function, frameshift, or key amino acid substitution that has a clear trait effect and the individual phenotype is normal, parthenogenesis (e.g., gynogenesis) should be preferred to induce the gametes of the M1 generation individuals containing the gene mutation to directly develop into homozygous diploid M2 generation. c) To address the problems of delayed gonadal development, decreased fertility, and accumulation of non-target background mutations in M1 individuals that may be caused by heavy ion mutagenesis, this invention can use M1 generation individuals carrying specific gene mutations as germ cell donors and transplant their germ cell stem cells into closely related degametized host individuals with short sexual maturation cycles, so that the host can produce gametes derived from the donor, shortening the time to obtain the next generation of gametes, and reducing the cumulative effect of mutations associated with heavy ion mutagenesis through host background isolation. That is, the germ cells of the M1 generation individuals containing the gene mutation are transplanted into recipient fish that have undergone degametization treatment, and the recipient fish is used to produce gametes derived from the donor.

[0023] More preferably, the parthenogenesis method specifically includes: activating the eggs of female fish in the M1 generation by inactivating heterologous sperm with ultraviolet light, and inhibiting the expulsion of the second polar body through cold shock treatment to induce diploid gynogenesis. After obtaining homozygous or quasi-homozygous M2 generation within one generation, to balance the establishment speed and genetic background stability, a meiosis II phase blocking method is preferred, and a mild backcross can be performed in the M2 or M3 generation to further reduce the burden of non-target background variation.

[0024] Further preferably, path c specifically includes: microinjecting antisense oligonucleotides (MO) of sterility-related genes into the single-cell stage of fertilized eggs of recipient fish to perform germ cell removal treatment; transplanting the germ cell suspension of the M1 generation individuals containing specific gene mutations into the gonadal region of the body cavity of 2-7 day old recipient fish; raising the recipient fish until sexual maturity and collecting the donor-derived gametes produced by them.

[0025] The above-described fish heavy ion beam mutagenesis breeding method is applicable to the genetic improvement of various fish species and multiple economic traits. More preferably, the target gene includes one or more of the bmp6, sp7, and runx2b genes.

[0026] Compared with the prior art, the advantages of the present invention are as follows: 1. This invention strictly limits mutagenesis to the single-cell stage of the fertilized egg, ensuring that the resulting mutations are 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 germline during the mutagenesis phase, 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 selection 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 phase (M1 generation). Compared to 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 pathway for rapidly creating new germplasm and new varieties of various fish.

[0027] 2. This invention strictly limits the mutagenesis process to the single-cell stage of the fertilized egg and spatially orients and fixes the fertilized egg before irradiation, aligning the animal pole with the direction of the heavy ion beam, thereby significantly improving the controllability of heavy ion beam energy deposition in the zygote nucleus / blastodisc region. 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, enabling the induced mutations to be synchronously distributed into cell lineages, including primordial germ cells (PGCs), in the early cleavage stage, laying a structural foundation for obtaining heritable mutations.

[0028] 3. 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 embryos. 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.

[0029] 4. In the rapid creation of 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. Among them, the fast-growing group is significantly higher than the unmutated or conventionally cultured groups in key growth indicators such as body length and weight, and exhibits a higher growth index b value in the body length-weight relationship model, showing positive allometric growth characteristics, indicating that its weight gain rate is significantly enhanced relative to body length gain (see detailed implementation). 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.

[0030] 5. In terms of rapidly creating new germplasm and strains of various fish species, the preferred technical solution of this invention can obtain individuals in fish with mutations in key genes related to skeletal development and intermuscular spine formation, thereby providing a molecular basis for targeted regulation of intermuscular spine-related traits. For example, in a preferred embodiment, targeted deep sequencing and molecular typing analysis of induced contemporary individuals detected mutations in bone formation-related genes such as bmp6. Existing studies have shown that bmp6 plays an important regulatory role in fish skeletal formation and intermuscular spine development. The aforementioned mutations provide clear molecular targets and technical pathways for subsequently obtaining phenotypes with reduced or absent intermuscular spines through genetic screening and breeding. Therefore, the method of this invention not only enables targeted intervention in intermuscular spine-related developmental pathways at the molecular level, but also lays a genetic foundation for the subsequent construction of new fish strains with reduced or absent intermuscular spines. Attached Figure Description

[0031] 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.

[0032] Figure 1 This is a flowchart of the fish heavy ion beam mutation breeding method in a specific embodiment of the present invention.

[0033] Figure 2This 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 the site has undergone an insertion mutation in the mutant.

[0034] Figure 3 These 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.

[0035] 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.

[0036] 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. 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 are no precedents in the field of fish physical mutation breeding pioneered by this invention, we have referenced the mature and logically clear generation nomenclature method in plant mutation breeding and redefined it in combination with the biological characteristics of this technical system: M0 generation: refers to fertilized eggs before undergoing mutagenesis treatment.

[0041] M1 generation: refers to fish individuals that are directly developed (cultivated) from M0 generation fertilized eggs after undergoing mutagenesis treatment.

[0042] M2 generation: refers to offspring produced by M1 generation individuals through sexual reproduction.

[0043] M3 generation: refers to the offspring produced by M2 generation individuals through sexual reproduction.

[0044] One such Figure 1 The fish heavy ion beam 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 this fish species to obtain the M1 generation population; 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 methods. Reproduction can be achieved through one or a combination of the following three methods: a) Hybrid breeding pathway: M1 generation individuals carrying a specific gene mutation are crossed with wild-type individuals, and selection is carried out in the M2 generation and subsequent generations. Given that heavy ion mutagenesis may simultaneously generate background variations unrelated to the mutation, a "directed backcross + linkage marker" monitoring strategy can be preferentially introduced in pathway a. The M1 individuals carrying the specific gene mutation are backcrossed with wild-type individuals once or several times in a directed manner, so that non-target background variations are gradually diluted with each backcross generation. Linked molecular markers near the target site are used to selectively track and genotype the M2 and subsequent populations to ensure that the gene mutation is maintained while background variations are gradually eliminated. This pathway can obtain homozygous mutant lines with clear genetic background and stable phenotypic expression within 2 to 4 generations. b) Rapid homozygous pathway: When a specific gene mutation results in a loss of function, frameshift, or key amino acid substitution, which has a clear phenotypic effect and the individual phenotype is normal, parthenogenesis (preferably gynogenesis, so that gametes carrying the mutation directly develop into diploid offspring) is used to induce the gametes of the M1 generation individuals containing the specific gene mutation to directly develop into homozygous diploid M2 generation. c) Germ cell transplantation pathway (“surrogate motherhood”): M1 generation individuals carrying specific gene mutations can be used as germ cell donors, and their germ cell stem cells can be transplanted into closely related degametized host individuals with short sexual maturation cycles, so that the host can produce gametes from the donor, shortening the time to obtain the next generation of gametes, and reducing the cumulative effect of heavy ion mutagenesis through host background isolation. That is, the germ cells of the M1 generation individuals containing specific gene mutations are transplanted into recipient fish that have undergone degametization treatment, and the recipient fish is used to produce gametes from the donor.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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 were 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.

[0049] 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.

[0050] Table 1: Sequencing information of bmp6 gene-related mutations

[0051] 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 ).

[0052] Figure 2Sequencing results showed that, compared with the wild type, the mutagenized sample of Example 1 had a base insertion (AAG→AAGAG) near the 6344761 site 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.

[0053] Table 2 shows the comparison results of fertilized egg hatching survival rate, mutation detection rate, and malformation rate under different treatment conditions.

[0054] Table 2: Effects of different treatment conditions on fertilized egg hatching survival rate and mutation detection efficiency

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] S3: Embryo Hatching and Population Building: Irradiated fertilized eggs are hatched and raised under suitable conditions to obtain an M1 generation population.

[0060] 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.

[0061] 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.

[0062] Table 3: Comparison of traits between the LEPR mutant rice paddy carp population obtained by mutagenesis and the conventionally farmed population.

[0063] 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.

[0064] Table 4: Results of Detection of Lepr Gene-Related Mutation Sites

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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)

[0072] 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.

Claims

1. A method for fish breeding using heavy ion beam mutagenesis, characterized in that, Includes the following steps: S1: Breed the target fish species to obtain fertilized eggs in the single-cell stage; S2: Heavy ion beam mutagenesis is performed on fertilized eggs in the single-cell stage using heavy ion beams, wherein the dose of heavy ion beam mutagenesis is a non-lethal mutagenesis dose. S3: Hatch and raise the fertilized eggs from step S2 to obtain the M1 generation population; S4: Perform molecular screening on the M1 generation individuals to identify M1 individuals carrying one or more gene mutations; S5: For the M1 generation individuals with gene mutations identified in step S4, obtain offspring strains that can inherit the gene mutations through breeding methods.

2. The fish heavy ion beam mutagenesis breeding method according to claim 1, characterized in that, In step S2, before heavy ion beam mutagenesis, the fertilized egg is first oriented and fixed so that its animal pole faces the direction of the heavy ion beam, so that the heavy ion beam is incident along the direction of embryonic polarity.

3. The fish heavy ion beam mutagenesis breeding method according to claim 1, characterized in that, In step S2, 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.

4. The fish heavy ion beam mutagenesis breeding method according to claim 3, 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 0.5~1 mM 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.

5. The fish heavy ion beam mutagenesis breeding method according to any one of claims 1 to 4, characterized in that, In step S2, the heavy ion beam is a carbon ion beam with an energy of 80 MeV / µm; the dose of heavy ion beam mutagenesis is 0.5~3 Gy, preferably 0.5~1 Gy; and the LET is 30~50 keV / µm.

6. The fish heavy ion beam mutagenesis breeding method according to any one of claims 1 to 4, characterized in that, In step S4, the molecular screening and identification includes: mixing the DNA of the M1 generation individual population, performing high-depth targeted sequencing on a selected target gene region, and identifying whether there is a mutation in the target gene; if there is, stop step S4 and proceed to step S5; if there is no mutation, select another target gene region and perform high-depth targeted sequencing until a target gene with a mutation is identified and proceed to step S5.

7. The fish heavy ion beam mutagenesis breeding method according to claim 6, characterized in that, In step S4, the depth of the high-depth targeted sequencing is set according to the size of the pool. 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×.

8. The fish heavy ion beam mutagenesis breeding method according to any one of claims 1 to 4, characterized in that, After step S4 and before step S5, the M1 generation individuals carrying one or more gene mutations obtained after molecular screening are re-verified using at least one of PCR, competitive allele-specific PCR, and Sanger sequencing. After confirming the mutations and individual genotypes, M1 generation individuals containing gene mutations are obtained.

9. The fish heavy ion beam mutagenesis breeding method according to claim 8, characterized in that, The specific steps of the review include: First, digital PCR technology was used to verify the candidate mutation signals in the mixed pool DNA to confirm their existence. Then, for the verified mutation sites, competitive allele-specific PCR typing was used to perform high-throughput screening of all individuals corresponding to the mixed pool to quickly identify one or more candidate individuals carrying gene mutations. Finally, Sanger sequencing was used to perform a final verification of the mutation sites of the gene in the selected candidate individuals.

10. The fish heavy ion beam mutagenesis breeding method according to any one of claims 1 to 4, characterized in that, In step S5, the reproductive method 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.

11. The fish heavy ion beam mutagenesis breeding method according to claim 10, characterized in that, The specific parthenogenesis method includes: using ultraviolet light to inactivate heterologous sperm to activate the eggs of female fish in the M1 generation, and using cold shock treatment to inhibit the expulsion of the second polar body and induce diploid gynogenesis; after obtaining homozygous or quasi-homozygous M2 generation, using the meiosis II phase block method, and performing mild backcrossing in the M2 or M3 generation. The specific path c includes: microinjecting antisense oligonucleotides of sterility-related genes into the single-cell stage of fertilized eggs of recipient fish to remove germ cells; transplanting the germ cell suspension of the M1 generation individuals containing the gene mutation into the gonadal region of the body cavity of 2-7 day old recipient fish; raising the recipient fish until sexual maturity and collecting the donor-derived gametes produced by them.

12. The fish heavy ion beam mutagenesis breeding method according to claim 6, characterized in that, The target genes include one or more of the following genes: bmp6, sp7, runx2b, lepr, and mstn.

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