Preparation method of all-female grass carp parent and application thereof

Abstract

This invention provides a method for preparing all-female grass carp broodstock and its application, belonging to the field of grass carp breeding and aquaculture technology. The preparation method includes the following steps: screening female donor grass carp; preparing female grass carp reproductive stem cells and cell transplantation recipient fish; transplanting the female grass carp reproductive stem cells into the cell transplantation recipient fish, selecting transplantation-positive individuals; raising the transplantation-positive individuals at a certain density; using grass carp genomic marker primers, grass carp male sex marker-specific primers, and cell transplantation recipient fish genomic marker primers to screen for semen samples that are positive for grass carp genomic markers, negative for grass carp male sex markers, and negative for cell transplantation recipient fish genomic markers. The fish providing these semen samples are the all-female grass carp broodstock. The preparation time for all-female grass carp broodstock using this method is less than one year, and the success rate is significantly higher than existing technologies.

Description

Technical Field

[0001] This invention belongs to the field of freshwater fish farming technology, and particularly relates to the field of grass carp breeding and farming technology, specifically to a method for preparing all-female grass carp broodstock and its application. Background Technology

[0002] Existing research has shown that all-female grass carp populations obtained through gynogenesis have advantages such as strong disease resistance, rapid growth, and high survival rate. Therefore, large-scale breeding of all-female grass carp populations will play a significant role in promoting the development of the grass carp aquaculture industry. This will improve the economic benefits of aquaculture while reducing the use of medications and protecting the aquatic ecological environment, thus yielding significant economic, ecological, and social benefits.

[0003] Currently, there are four main methods for the direct breeding of all-female fish: (1) artificial selection; (2) sex hormone-induced direct sex reversal; for example, Chinese patent CN108377936B discloses a method for sex reversal of mandarin fish by using a mixture of 17α-methyltestosterone hormone and feed, which is then fed to mandarin fish by bait fish before the differentiation of the gonads. Chinese patent application CN103798173A discloses that starting from the 5th day after the hatching of yellow catfish and snakehead fry, feed treated with 30ppm methyltestosterone and 50ppm flutamide or 30ppm estrone and 50ppm tamoxifen citrate is used to continuously feed the fish for 30 days to obtain offspring with a female or male ratio of more than 90%. (3) gynogenesis method; for example, CN115735855A discloses a method for breeding all-female mandarin fish through gynogenesis. The paper "Cytological Observation on Inhibiting First Cleavage in Grass Carp to Achieve Gynogenesis by Heat Shock Method" (DOI:10.3321 / j.issn:1000-3207.2003.02.010) discloses a technique for inhibiting first cleavage in grass carp to achieve gynogenesis by heat shock method. The success rate of obtaining all-female diploid grass carp embryos was 12.3%, and the success rate of obtaining all-female diploid larvae was only 1.57%. (4) Method combining gynogenesis and hormone sex reversal; for example, CN106489799A discloses a method for establishing a purebred all-female yellow catfish germplasm bank by combining artificial gynogenesis technology and hormone sex reversal technology from wild original strains of yellow catfish with excellent traits selected from the Yangtze River system.

[0004] Of the methods mentioned above, manual selection is cumbersome, labor-intensive, and cannot accurately distinguish the sex of fish fry, making it unsuitable for large-scale production of all-female fish. While direct sex hormone induction can produce batches of fish of a single sex, the resulting commercial fish may have hormone residues and cannot guarantee all-female reproduction. Gynogenesis technology also has the following problems: 1) long cultivation cycle and difficulty in sex identification of fry; 2) low frequency of diploid occurrence; 3) low survival rate of fry, with few surviving to sexual maturity. Therefore, it is necessary to develop a method for preparing all-female grass carp with a short cultivation cycle, high success rate, and suitability for large-scale aquaculture. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for preparing all-female grass carp broodstock. This method offers advantages such as short preparation time and high success rate.

[0006] Specifically, in order to achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing all-female grass carp broodstock includes the following steps:

[0008] S1. Select female grass carp as donors;

[0009] S2. Preparation of female grass carp reproductive stem cells and cell transplant recipient fish

[0010] S3. Transplant the female grass carp reproductive stem cells into the cell transplant recipient fish and select individuals with positive transplant results.

[0011] S4. The transplanted positive individuals are raised at a certain density;

[0012] S5. Using grass carp genome marker primers, grass carp male sex marker specific primers, and cell transplant recipient fish genome marker primers, semen samples that are positive for grass carp genome markers and negative for grass carp male sex markers and cell transplant recipient fish genome markers are screened out. The fish that provide the semen samples are the parents of all-female grass carp.

[0013] In a preferred embodiment, the cell transplant recipient fish is prepared from zebrafish.

[0014] In a preferred embodiment, the grass carp genome marker primers are as follows: forward primer gc_mst n_F: 5'-GTGGGTAATGGAGATATAACG-3'; reverse primer gc_mstn_R: 5'-GCTT TAGCAAGGCTGAGA-3'; the grass carp male sex marker specific primers are as follows: forward primer Sca971_32_446_F: 5'-ACCAATAGATGAATTATTTTTCAGGC-3'; reverse primer Sca971_32_446_R: 5'-ACATTGTTGTCTGTATGCTCTGAC-3'; the zebrafish genome marker primers are as follows: forward primer zf-nr5a1a-F: 5'-GATGAGAGGTGGAAGA AATAAG-3'; reverse primer zf-nr5a1a-R: 5'-GCTTCATACCTGCTCCT-3'.

[0015] In a preferred embodiment, step S1 includes the following steps: selecting grass carp juveniles 5-10cm long, cutting off the tail fin to extract DNA, performing PCR amplification and gel electrophoresis, and those without bands are genetically female grass carp, which are used as female donor grass carp.

[0016] In a further preferred embodiment, the PCR amplification primers are as follows:

[0017] Forward primer Sca971_32_446_F: 5'-ACCAATAGATGAATTATTTTTCAGGC-3'; Reverse primer Sca971_32_446_R: 5'-ACATTGTTGTCTGTATGCTCTGAC-3'.

[0018] In a preferred embodiment, the female grass carp reproductive stem cells mentioned in step S2 account for more than 30% of the total number of living cells.

[0019] In a preferred embodiment, step S2 includes the following steps: dissecting the female grass carp donor obtained in step S1, obtaining the gonads, and digesting them with tissue digestion solution to prepare a single-cell suspension; filtering, and centrifuging at discontinuous density gradients with 60%, 50%, 40%, 35%, 30%, and 20% Percoll cell separation solution to obtain cell layers of 30%–35% and 35%–40% concentration, which are then washed, stained with live cells, concentrated, and placed in Leibovitz's L-15 medium containing 3% fetal bovine serum to obtain a female grass carp reproductive stem cell suspension. The tissue digestion solution includes 2.5 mg / mL trypsin digest, 100 U / mL collagenase, and 0.5 mg / mL DNase.

[0020] In a preferred embodiment, step S3 includes the following steps: culturing the cell transplant recipient fish in a constant temperature incubator for 4 days, anesthetizing it, and then placing it on an agarose plate; transplanting a suspension containing the female grass carp reproductive stem cells into the genital ridge of the cell transplant recipient fish; placing the resulting transplanted fish in 0.3×Danieau's buffer and raising it at a constant temperature until day 5; selecting the transplanted fish whose genital ridge glows under a stereofluorescence microscope, which are the transplanted positive individuals. The success rate of female grass carp reproductive stem cell transplantation obtained according to this method is ≥75%.

[0021] In a further preferred embodiment, the recipient fish for cell transplantation is cultured in a 28.5°C incubator for 4 days, anesthetized for 5 minutes with 0.2 mg / mL MS-222, and then placed on a 2% agarose plate. A suspension containing the female grass carp reproductive stem cells is transplanted into the genital ridge of the recipient fish. The resulting transplanted fish are placed in 0.3×Danieau's buffer and kept at 28.5°C for 5 days. Under a stereofluorescence microscope, the transplanted fish exhibiting luminescence at the genital ridge are identified as positive transplant individuals. The success rate of female grass carp reproductive stem cell transplantation obtained using this method is >82%.

[0022] In a preferred embodiment, step S2 includes the following steps: injecting zebrafish fertilized eggs with a morpholino antisense oligonucleotide targeting DND1 mRNA during the I-cell stage to eliminate endogenous germ cells and obtain cell transplantation recipient fish; the sequence of the antisense oligonucleotide is: 5'-GCTGGGCATCCAT GTCTCCGACCAT-3'.

[0023] In a preferred embodiment, the density-based rearing method in step S4 is as follows: for transplant-positive individuals 1-7 days after transplantation, the rearing density is 50 individuals / L of water; for transplant-positive individuals 8-30 days after transplantation, the rearing density is 20 individuals / L of water; for transplant-positive individuals 31-60 days after transplantation, the rearing density is 10 individuals / L of water; for transplant-positive individuals more than 61 days after transplantation, the rearing density is 5 individuals / L of water; the water temperature is controlled at 25-27°C, and feeding is performed 3 times a day.

[0024] In a preferred embodiment, step S5 includes the following steps: anesthetizing the transplant-positive individuals 4 months after transplantation, aspirating secretions from the genital protuberance, extracting DNA from the DNA in a proteinase K solution, performing PCR amplification using the grass carp genome marker primers and the grass carp male sex marker-specific primers, performing gel electrophoresis on the amplification products, selecting transplant-positive individuals that are positive for grass carp genome markers and negative for grass carp male sex-specific markers and negative for cell transplant recipient fish genome markers, and extracting their semen. The grass carp genome marker primers are as follows: forward primer gc_mstn_F: 5'-GTGGGTAATGGAGATATAACG-3'; reverse primer gc_mstn_R: 5'-GCTTTAGCA AGGCTGAGA-3'; the grass carp male sex marker specific primers are as follows: forward primer Sca971_32_446_F: 5'-ACCAATAGATGAATTATTTTTCAGGC-3'; reverse primer Sca971_32_446_R: 5'-ACATTGTTGTCTGTATGCTCTGAC-3'; the cell transplant recipient fish is prepared from zebrafish, and the zebrafish genome marker primers are as follows: forward primer zf-nr5a1a-F: 5'-GAT GAGAGGTGGAAGAAATAAG-3'; reverse primer zf-nr5a1a-R: 5'-GCTTCATACC TGCTCCT-3'.

[0025] When the all-female grass carp broodstock prepared according to the method described above is used for hybridization with wild grass carp, all the resulting hybrid offspring are identified as female. Therefore, this all-female grass carp broodstock can be used to achieve large-scale breeding and / or farming of all-female grass carp.

[0026] Compared with the prior art, the present invention has the following beneficial effects: (1) The time required to prepare all-female grass carp broodstock in the present invention is less than one year, which is significantly shorter than the 5-6 years (the time for female grass carp to reach sexual maturity) in the prior art. (2) The success rate of preparing all-female grass carp broodstock in the present invention is greater than 10%, while the success rate in the prior art is less than 2% (1.57%), which is a significant improvement. (3) The all-female grass carp broodstock obtained by the preparation method of the present invention can realize the large-scale preparation of all-female grass carp, and also provides a new method for breeding all-female grass carp. Attached Figure Description

[0027] Figure 1 This is a graph showing the gel electrophoresis results of the female grass carp donors in Example 1.

[0028] Figure 2 These are stereofluorescence microscope comparison images taken during the screening of transplant-positive individuals in Example 1.

[0029] Figure 3 This is a gel electrophoresis identification result of screening all-female grass carp parents in Example 1;

[0030] Figure 4 The image shows the results of gel electrophoresis identification of the offspring obtained by hybridizing the all-female grass carp parent prepared in Example 1 with wild grass carp in Example 2. Detailed Implementation

[0031] The following description, in conjunction with embodiments, clearly and completely illustrates the technical solutions of the present invention, enabling those skilled in the art to fully understand the invention. Obviously, the described embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Any equivalent modifications or substitutions made by those skilled in the art to the following embodiments without creative effort are within the protection scope of the present invention.

[0032] The methods not described in detail in the following examples are all conventional methods well known to those skilled in the art. The main components of Danieau's buffer are as follows: NaCl 17.4 mM, KCl 0.21 mM, MgSO4 0.12 mM, Ca(NO3)2 0.18 mM, and HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) 1.5 mM. Percoll cell separation medium (catalog number: 17-0891-01) was purchased from General Electric (GE). Proteinase K (catalog number: ST537) was purchased from Beyotime Biotechnology. The main components of the tissue digestion solution are as follows: 2.5 mg / mL trypsin cell digestion solution (Beyotime, catalog number C0192), 100 U / mL type I collagenase (Beyotime, catalog number ST2294), and 0.5 mg / mL DNase I (Solepro, catalog number D8071). The components of the fish sperm preservation solution are: 0.137M NaCl, 5.4mM KCl, 0.25mM Na2HPO4, 0.44mM KH2PO4, 1.3mM CaCl2, 1.0mM MgSO4, and 4.2mM NaHCO3.

[0033] Example 1

[0034] This embodiment provides a method for preparing all-female grass carp broodstock, including the following steps:

[0035] 1. Screening and identification of female grass carp donors

[0036] Genetically female grass carp were selected using male sex markers: 5-10cm juvenile grass carp were selected, and DNA was extracted from the tail fin. PCR amplification was performed, and the amplified product was 446bp in size. The amplified product was then subjected to 2% agarose gel electrophoresis. The results are as follows: Figure 1 As shown. Figure 1 Grass carp without stripes are female, while those with stripes are male.

[0037] The PCR amplification primers are as follows (Reference: DOI:10.1038 / s41598-017-08476-y):

[0038] Forward primer Sca971_32_446_F: 5'-ACCAATAGATGAATTATTTTTCAGGC-3';

[0039] Reverse primer Sca971_32_446_R: 5'-ACATTGTTGTCTGTATGCTCTGAC-3'.

[0040] The PCR amplification reaction system is as follows: 10 μL of 2×PCR mix (Novazia, P222-01), 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 1 μL of DNA template (100 ng / μL), 7 μL of pure water, and a total volume of 20 μL.

[0041] The PCR amplification reaction conditions were as follows: 95℃ for 15 seconds, 56℃ for 15 seconds, 72℃ for 15 seconds, 30 cycles; 72℃ for 5 minutes.

[0042] 2. Preparation of female grass carp reproductive stem cells (reproductive stem cells account for ≥30%) and recipient fish for cell transplantation (recipient fish germ cell removal efficiency ≥95%).

[0043] 2.1 Preparation of Grass Carp Gonadal Single-Cell Suspension: Ten female grass carp juveniles (as described above) were dissected to obtain gonads. The gonads were diced as finely as possible in a petri dish using a scalpel, and 5 mL of tissue digestion solution was added. The tissue digestion solution containing the gonads was transferred to 5 mL centrifuge tubes and incubated in a 35°C water bath for 3 hours, with 1 mL pipette pipetting every 30 minutes, 20 times each time. After 3 hours, the cells were filtered through a 40 μm cell sieve (Shanghai Sangon Biotech, catalog number F613461-9001-BD). The filtered cells were used for the next step of centrifugation enrichment of grass carp germ cells.

[0044] 2.2 Centrifugation and Enrichment of Grass Carp Germ Cells: Percoll cell separation solutions with concentrations of 60%, 50%, 40%, 35%, 30%, and 20% were prepared using D-PBS buffer for cell culture. In a 15 mL centrifuge tube, 2 mL of each Percoll cell separation solution was slowly added sequentially along the tube wall using a pipette. Then, the gonadal cell suspension filtered in step 2.1 was slowly added along the tube wall using a pipette. The tubes were centrifuged at 800 g for 30 minutes at 20°C. After centrifugation, the centrifuge tubes were carefully removed, avoiding vigorous shaking. Each cell layer was carefully aspirated from top to bottom using a pipette. The 30%–35% and 35%–40% cell layers were combined into a 5 mL low-absorption centrifuge tube. Twice the volume of D-PBS buffer was added, and the mixture was thoroughly mixed. After centrifugation at 800g for 5 minutes, the supernatant was carefully discarded, and the precipitate was retained. 2 mL of D-PBS buffer was added again, and the cells were vortexed and centrifuged at 800g for 5 minutes. The washing steps were repeated once more. Finally, the cells were resuspended in 20 μL of Leibovitz's L-15 medium (Gibco, catalog number 11415-064) containing 3% fetal bovine serum to obtain a suspension of female grass carp donor reproductive stem cells.

[0045] 2.3 Detection of the proportion of reproductive stem cells: Take 5 μL of the obtained female grass carp donor reproductive stem cell suspension and drop it onto an adhesive glass slide (Shitai, catalog number 188105W), and let it stand for 2 minutes. Use an immunohistochemical pen (Beyotime, catalog number P0139-1pc) to draw a circle with a diameter of about 1 cm around the cell droplet. Perform the immunofluorescence experiment as follows: (1) Fix the sections with 200 μL of 4% PFA-PBS for 20 minutes at room temperature; (2) Wash the sections three times with 200 μL of PBST (containing 0.1% Triton X-100), each time for 5 minutes; (3) Wash the sections three times with 200 μL of 2% BSA-1% DMSO-0.1% Triton X 100-PBS buffer, each time for 6 minutes, and the last time for 1 hour; (4) Dilute the zebrafish Nanos2 polyclonal antibody (the antibody disclosed in CN113173988B) with 200 μL of 2% BSA-1% DMSO-0.1% Triton X 100-PBS buffer at a volume ratio of 1:200, add it to the cells, cover with a paraffin film, and incubate the sections at 4°C for 12 hours; (5) Wash the sections three times with 200 μL of 2% BSA-1% DMSO-0.1% Triton X-100-PBS buffer. (6) Wash three times with 100-PBS buffer for 6 min each time; (7) Dilute Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) (Beyotime, catalog number A0423) at a volume ratio of 1:500 with 200 μL 2% BSA-1% DMSO-0.1% TritonX 100-PBS buffer and incubate at room temperature for 2 hours; (8) Soak three times with 200 μL 2% BSA-1% DMSO-0.1% TritonX 100-PBS buffer for 5 min each time; (9) Wash three times with 200 μL 1% BSA-1% DMSO-0.1% TritonX PBS buffer for 5 min each time; DAPI staining solution (Beyotime, catalog number C1002) diluted with 100-PBS at a volume ratio of 1:1000, with a final DAPI concentration of 5 μg / mL, was stained at room temperature for 30 minutes; (9) the slide was covered with a 20 μL cover slip containing anti-quenching agent and stored at 4°C in the dark.

[0046] Immunofluorescence results were photographed under a fluorescence microscope with a 10x objective lens, and cell counting was performed using ImageJ image processing software. The number of DAPI-positive cells (total cells) and the number of zebrafish Nanos2 polyclonal antibody-positive cells (germinal stem cells) in a single field of view were calculated. The proportion of germinal stem cells was calculated using the following formula: Proportion of germinal stem cells = Number of zebrafish Nanos2 polyclonal antibody-positive cells (germinal stem cells) / Number of DAPI-positive cells (total cells). To effectively improve transplantation success rates, the proportion of germinal stem cells should be maintained at over 30%.

[0047] 2.4 Preparation of zebrafish receptors: Zebrafish fertilized eggs were injected with 1 nL of 50 μM morpholino anti-sense oligos targeting DND1 mRNA during the I-cell stage. The sequence was 5'-GCTGGGCATCCATGTCTCCGACCAT-3' (Reference: DOI:10.1016 / S0960-9822(03)00537-2) to eliminate endogenous germ cells. The proportion of recipient fish with completely eliminated germ cells obtained using this method was over 95%.

[0048] 3. Transplantation of female grass carp reproductive stem cells (success rate of reproductive cell transplantation ≥75%)

[0049] The zebrafish recipients, whose germ cells were completely removed as prepared in step 2.4, were cultured in a constant temperature incubator at 28.5℃ for 4 days. After anesthetizing with 0.2 mg / mL MS-222 (ethyl m-aminobenzoate methanesulfonate) for 5 minutes, they were placed on a 2% agarose plate. The female grass carp germ cell suspension was transplanted into the genital ridge of the recipient fish. 136 recipient fish that underwent germ cell transplantation were selected and placed in 0.3×Danieau's buffer, and cultured at 28.5℃ for 5 days. Glowing was observed at the genital ridge under a stereofluorescence microscope (e.g., luminescence at the genital ridge). Figure 2 As shown, Figure 2 (Only one set of comparisons is shown in the image) Transplant-positive individuals were raised, and a total of 112 transplant-positive individuals with luminous genital ridges were obtained.

[0050] 4. Breeding of transplanted positive individuals

[0051] Positive transplanted individuals should be housed according to the following density: 50 fish / L of water for fish 1-7 days post-transplantation; 20 fish / L of water for fish 8-30 days post-transplantation; 10 fish / L of water for fish 31-60 days post-transplantation; and 5 fish / L for fish older than 61 days post-transplantation. Maintain water temperature between 25-27°C and feed three times daily.

[0052] 5. Screening and identification of broodstock of all-female grass carp (100% accuracy in identifying broodstock of all-female grass carp).

[0053] Forty-seven fish, four months post-transplantation, were anesthetized with 0.2 mg / mL MS-222. 1-2 μL of secretion was aspirated from the genital protuberance of adult fish using a capillary tube and placed in a 20 μg / mL proteinase K solution for DNA extraction. The specific procedure was as follows: digestion in a 55℃ water bath for 1 hour, incubation at 95℃ for 5 minutes to inactivate proteinase K, centrifugation at 10000g for 5 minutes, and the supernatant was used as a PCR template. Using the extracted DNA as a template, PCR amplification was performed using grass carp genomic marker primers and grass carp male sex marker-specific primers (the PCR reaction system and amplification procedure were the same as in step 1, only the specific primers and DNA templates differed). The amplification products were subjected to 2% agarose gel electrophoresis. Simultaneously, the PCR amplification products of male grass carp genomic DNA, female grass carp genomic DNA, and zebrafish genomic DNA were used as controls. The DNA extraction method, PCR amplification system, and procedure were the same for all groups (only the specific DNA templates and amplification primers differed). The identification results are as follows: Figure 3 As shown, grass carp with positive genomic markers and negative male-specific grass carp markers and negative zebrafish genomic markers are the parents of all-female grass carp. A total of 6 all-female grass carp parents were obtained; the success rate of preparing all-female grass carp parents was 12.8%.

[0054] The grass carp genome marker primers are as follows:

[0055] gc_mstn_F:5'-GTGGGTAATGGAGATATAACG-3';

[0056] gc_mstn_R: 5'-GCTTTAGCAAGGCTGAGA-3';

[0057] The specific primers for male sex markers in grass carp are as follows:

[0058] Sca971_32_446_F:5'-ACCAATAGATGAATTATTTTTCAGGC-3';

[0059] Sca971_32_446_R: 5'-ACATTGTTGTCTGTATGCCTGAC-3';

[0060] The zebrafish genome marker primers are as follows:

[0061] zf-nr5a1a-F: 5'-GATGAGAGGTGGAAGAAATAAG-3';

[0062] zf-nr5a1a-R: 5'-GCTTCATACCTGCTCCT-3'.

[0063] Example 2

[0064] In this embodiment, sperm from the all-female grass carp parent prepared in Example 1 was used to artificially inseminate wild grass carp eggs to determine the sex of the hybrid offspring.

[0065] Obtaining wild-type grass carp eggs: In mid-May, wild-type grass carp eggs are obtained using artificial spawning induction. Obtaining whole female grass carp sperm: Prepare 2 mL of fish sperm preservation solution. Take 10 adult zebrafish carrying whole female grass carp sperm, anesthetize them for 5 minutes with 0.2 mg / mL MS-222, gently wipe the fluid near the cloaca with a cotton swab on a moist sponge, and use a 10 μL pipette to draw positive fish sperm into the fish sperm preservation solution. Dry artificial insemination: Take about 10,000 wild-type grass carp eggs and add them to 2 mL of positive fish sperm solution. Gently stir with a glass rod to mix the sperm and eggs evenly. Add 1 L of culture water at about 25℃, let stand for 5 minutes, and then place them in an incubation tank. Incubate with running water at about 25℃ and aeration. The fry hatch after 4-5 days.

[0066] Molecular identification was performed using PCR: Seven hatched juvenile grass carp were placed into ten 1.5 mL centrifuge tubes, and 50 μL of 20 μg / mL proteinase K solution was added to each tube to extract genomic DNA (the method was the same as step 5 in Example 1). The extracted DNA was used as a template for PCR amplification using grass carp genomic marker primers and grass carp male sex marker-specific primers. The amplification products were subjected to 2% agarose gel electrophoresis. Additionally, one male and one female grass carp were selected, and genomic DNA was extracted from each. The extracted DNA was used as a template for PCR amplification using grass carp genomic marker primers and grass carp male sex marker-specific primers. The amplification products were subjected to 2% agarose gel electrophoresis. The grass carp genomic marker primers, grass carp male sex marker-specific primers, PCR reaction conditions, and amplification procedures were the same as in step 5 of Example 1. The gel electrophoresis results are shown below. Figure 4 As shown, from Figure 4 As can be seen above, the offspring obtained by hybridizing the sperm of the all-female grass carp parent prepared in Example 1 with the eggs of wild grass carp only have the bands of grass carp genome molecular markers, and there are no bands of male grass carp sex-specific markers. This is consistent with the bands of female grass carp genome DNA, indicating that the offspring obtained by hybridization are all female grass carp.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Various modifications and variations can be made to the present invention by any person skilled in the art. Any simple equivalent changes and modifications made based on the scope of protection of the present invention and the content of the specification should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a full female parent grass carp, characterized in that, Includes the following steps: S1. Select 5-10cm long grass carp juveniles, cut off the tail fins to extract DNA, perform PCR amplification, and perform gel electrophoresis on the amplification products. Those without bands are genetically female grass carp and will be used as female donor grass carp. The PCR amplification primers are as follows: Forward primer Sca971_32_446_F: 5'-ACCAATAGATGAATTATTTTTCAGGC-3'; Reverse primer Sca971_32_446_R: 5'-ACATTGTTGTCTGTATGCTCTGAC-3'; S2. Preparation of female grass carp reproductive stem cells and cell transplant recipient fish; the female grass carp reproductive stem cells account for more than 30% of the total number of living cells; zebrafish fertilized eggs are injected with morpholino antisense oligonucleotides targeting DND1 mRNA at the I-cell stage to eliminate endogenous germ cells and obtain cell transplant recipient fish; the sequence of the antisense oligonucleotide is: 5'-GCTGGGCATCCATGTCTCCGACCAT-3'; S3. Transplant the female grass carp reproductive stem cells into the cell transplant recipient fish and select transplant positive individuals; including the following steps: place the cell transplant recipient fish in a constant temperature incubator for 4 days, anesthetize it, and place it on an agarose plate; transplant the suspension containing the female grass carp reproductive stem cells into the genital ridge of the cell transplant recipient fish; place the resulting transplanted fish in 0.3×Danieau's buffer and raise it at a constant temperature for 5 days, and select the transplanted fish with luminescence at the genital ridge under a stereofluorescence microscope, which are the transplant positive individuals; S4. The transplanted positive individuals are raised at a certain density; S5. Using grass carp genomic marker primers, grass carp male sex marker-specific primers, and cell transplant recipient fish genomic marker primers, semen samples that are positive for grass carp genomic markers, negative for grass carp male sex markers, and negative for cell transplant recipient fish genomic markers are screened. The fish providing the semen samples are the all-female grass carp parents. The grass carp genomic marker primers are as follows: Forward primer gc_mstn_F: 5'-GTGGGTAATGGAGATATAACG-3'; Reverse primer gc_mstn_R: 5'-GCTTTAGCAAGGCTGAGA-3'; The grass carp male sex marker-specific primers are as follows: Forward primer Sca971_32_446_F: 5'-ACCAATAGATGAATTATTTTTCAGGC-3'; Reverse primer Sca971_32_446_R: 5'-ACATTGTTGTCTGTATGCTCTGAC-3'; The cell transplant recipient fish was prepared from zebrafish, and the zebrafish genome marker primers are as follows: Forward primer zf-nr5a1a-F: 5'-GATGAGAGGTGGAAGAAATAAG-3'; Reverse primer zf-nr5a1a-R: 5'-GCTTCATACCTGCTCCT-3'.

2. The method for preparing all-female grass carp parents according to claim 1, characterized in that, Step S2 includes the following steps: dissecting the female grass carp donor obtained in step S1, obtaining the gonads, digesting them with tissue digestion solution to prepare a single-cell suspension; filtering, and centrifuging at discontinuous density gradients with 60%, 50%, 40%, 35%, 30%, and 20% Percoll cell separation solution to obtain cell layers of 30%–35% and 35%–40% concentration, washing, staining with live cells, and concentrating the cells, then placing them in Leibovitz's L-15 medium containing 3% fetal bovine serum to obtain a female grass carp reproductive stem cell suspension.

3. The method of claim 1, wherein the all-female grass carp parents are prepared by crossing the all-female grass carp parents of claim 1 with the all-male grass carp parents of claim 2. The density-based rearing method described in step S4 is as follows: For transplant-positive individuals 1-7 days after transplantation, the rearing density is 50 individuals / L of water; for transplant-positive individuals 8-30 days after transplantation, the rearing density is 20 individuals / L of water; for transplant-positive individuals 31-60 days after transplantation, the rearing density is 10 individuals / L of water; for transplant-positive individuals more than 61 days after transplantation, the rearing density is 5 individuals / L of water; the water temperature is controlled at 25-27℃, and they are fed 3 times a day.

4. The method of claim 1, wherein the all-female grass carp parents are prepared by crossing the all-female grass carp parents of claim 1 with the all-male grass carp parents of claim 2. Step S5 includes the following steps: anesthetizing the positive transplant individuals 4 months after transplantation, aspirating secretions from the genital protuberance, extracting DNA from the DNA in a proteinase K solution, performing PCR amplification using the grass carp genomic marker primers and the grass carp male sex marker-specific primers, performing gel electrophoresis on the amplification products, selecting the positive transplant individuals that are positive for the grass carp genomic marker and negative for the grass carp male sex-specific marker and negative for the cell transplant recipient fish genomic marker, and extracting their semen.

5. The method for preparing all-female grass carp broodstock according to any one of claims 1 to 4, or the application of the all-female grass carp broodstock prepared by the method according to any one of claims 1 to 4 in the breeding of all-female grass carp.

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