A method for simultaneously breeding natural gynogenetic mylopharyngodon piceus and hybrid offspring of cyprinus carpio and carassius auratus

Abstract

The present application belongs to the field of fish breeding, and discloses a method for simultaneously breeding natural gynogenesis of Procyprinus and hybrid offspring of Cyprinus carpio, comprising the following steps: taking Procyprinus as the female parent and taking red crucian carp as the male parent to perform special pond breeding, after artificial induction of parturition, performing artificial insemination after the parent fish is in estrus, then performing flow water hatching on the fertilized eggs, after the fry is hatched, performing feeding, and then performing detection and screening on the fed fish, thereby obtaining the natural gynogenesis of Procyprinus and the hybrid offspring of Cyprinus carpio. The present application changes the activity of the ovum and sperm of the parent fish by performing distant hybridization of Procyprinus and red crucian carp and combining with specific types and dosages of parturition inducers, and thereby obtains the natural gynogenesis of Procyprinus and the hybrid offspring of Cyprinus carpio which are both sexually fertile.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fish breeding, and particularly relates to a method for simultaneously breeding natural gynogenesis of Ctenopharyngodon idellus and hybrid offspring of Ctenopharyngodon idellus and Carassius auratus. BACKGROUND

[0002] Distant hybridization refers to hybridization between organisms with genetic relationship in species, genus or even more distant relationship. It can transfer the genome of one species to another species through hybridization, so that the offspring appears changes at the chromosome level. Such changes are much greater than those obtained by close hybridization. Distant hybridization can also produce gynogenetic offspring, so as to obtain natural gynogenetic offspring. How to form new natural gynogenetic fish by using existing technology is a problem to be solved by those skilled in the art.

[0003] Ctenopharyngodon idellus is one of the most widely cultured freshwater fish in the world, and has a long history of cultivation in China. Ctenopharyngodon idellus has the characteristics of fast growth, varied diet and strong disease resistance. Ctenopharyngodon idellus is a variety of Ctenopharyngodon idellus, also known as Ctenopharyngodon idellus, belonging to Cypriniformes, Cyprinidae and Cyprininae. Ctenopharyngodon idellus is short and stout, with a small head, thin skin and tender meat, no muddy smell, body color purple red, fine leaf scale, and part of the internal organs can be seen due to the thin abdominal skin. It grows fast and has delicious and meaty meat with small spines, so it is favored by many breeders. However, due to long-term self-crossing and close inbreeding, its quality is deteriorating, and the most intuitive feeling is that the growth rate and resistance are declining. The natural gynogenetic offspring shows advantages in meat quality, disease resistance, stress resistance and growth rate, so forming gynogenetic offspring with both sexes and fertility through distant hybridization provides an important germplasm resource for genetic breeding. Exploring the distant hybridization between Ctenopharyngodon idellus and Carassius auratus is expected to obtain new gynogenetic offspring with both sexes and fertility, which has important significance in production practice and genetic breeding. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the deficiencies and defects mentioned in the above background technology, and to provide a method for simultaneously breeding natural gynogenesis of Ctenopharyngodon idellus and hybrid offspring of Ctenopharyngodon idellus and Carassius auratus.

[0005] To solve the above technical problems, the technical solution provided by the present application is:

[0006] A method for simultaneously breeding natural gynogenesis of Ctenopharyngodon idellus and hybrid offspring of Ctenopharyngodon idellus and Carassius auratus, characterized in that it comprises the following steps: taking Ctenopharyngodon idellus as the female parent and Carassius auratus as the male parent, performing special pond breeding, after artificial induction of labor, when the parent fish is in estrus, performing artificial insemination, then performing flow water hatching on the fertilized eggs, after the fry is hatched, performing feeding, then detecting and screening the fed fish, to obtain the natural gynogenesis of Ctenopharyngodon idellus and the hybrid offspring of Ctenopharyngodon idellus and Carassius auratus.

[0007] The method, preferably, the special pool breeding comprises the following steps: 2 3 months before the breeding season, selecting female grass carp and male red crucian carp with good body shape, no disease and injury, and obvious sexual maturity as parents for special pool breeding, 1 month before the breeding season, feeding with high-quality food, and performing water flow stimulation every day to promote the maturity of gonads.

[0008] Preferably, the artificial induction of spawning comprises the following steps: in the breeding season, artificially injecting the female parent fish with a mixed induction agent of luteinizing hormone-releasing hormone analogue and chorionic gonadotropin, the dose of the luteinizing hormone-releasing hormone analogue is 7.5 10 μg / kg, and the dose of the chorionic gonadotropin is 100 150 IU / kg, first injecting the female parent fish, then 3-5 hours later, selecting the male parent fish with white semen squeezed out by gently squeezing the abdomen, and artificially injecting the male parent fish with the mixed induction agent of luteinizing hormone-releasing hormone analogue and chorionic gonadotropin, the injection dose of the male parent fish is half of that of the female parent fish, and the injection is performed by using the abdominal cavity one-needle injection method at the base of the pectoral fin without scales.

[0009] Preferably, the artificial induction of spawning is performed in the breeding season and when the water temperature is stable at more than 22℃.

[0010] Preferably, after the injection of the mixed induction agent is completed, the female and male parent fish are put into the spawning pool at a quantity ratio of 1:3-4.

[0011] Preferably, the artificial insemination comprises the following steps: after the parent fish is in estrus, the parent fish is salvaged by using a net, the female grass carp with suitable spawning quantity is selected, the abdomen is gently squeezed, and the dark green eggs are squeezed into a clean porcelain basin, meanwhile, the red crucian carp with large sperm is selected, the abdomen is gently squeezed, the white semen is squeezed into the porcelain basin, and then the porcelain basin is quickly mixed and poured into the prepared brown sheet for insemination.

[0012] Preferably, the fish fry feeding comprises the following steps: after the fish fry is completely hatched, the fish fry is cultured in the hatching pool for 2 3 days, and then is transferred to the pond prepared in advance after the lumbar point appears; after the fish fry is put into the pond for 2 days, the soybean milk is sprayed, and the spraying is required to be uniform and fine.

[0013] Compared with the prior art, the present application has the following beneficial effects:

[0014] 1. The breeding method of the present application changes the activity of the eggs and sperm of the parents by performing distant cross breeding of the grass carp and the red crucian carp, and then the natural female gynogenesis grass carp and the carp crucian cross offspring with both sexes are obtained. In the control experiment in which the white crucian carp is used as the male parent, only the carp crucian cross offspring is produced.

[0015] 2、The present application is first obtained natural gynogenesis offspring by crossing when the chromosome number of both parents is 100, and the natural gynogenesis offspring and the crucian carp hybrid offspring are both hermaphroditic and fertile, and the DNA of the male sperm is consistent with the DNA content of the blood, so that the natural gynogenesis offspring and the crucian carp hybrid offspring will obtain polyploid offspring by selfing, and it is also very rare that the crucian carp hybrid offspring of the F2 generation can obtain polyploid offspring, so that the present application provides a very good foundation for improving the germplasm resources of grass carp.

[0016] 3、The breeding method of the present application has high fertilization rate, high hatching rate and high production efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0018] Figure 1 It is a schematic diagram of grass carp x red crucian carp cross, wherein A: grass carp (Common carp, 2n=100); B: red crucian carp (2n=100); C: natural gynogenesis grass carp (2nL); D: crucian carp hybrid offspring (2nJ);

[0019] Figure 2 It is the average DNA content of two kinds of offspring and parents, wherein A: the average DNA content of grass carp (1:99.76); B: the average DNA content of red crucian carp (1:97.50); C: the average DNA content of natural gynogenesis grass carp (1:103.27); D: the average DNA content of crucian carp hybrid offspring (1:106.31);

[0020] Figure 3 It is the chromosome number of two kinds of offspring and parents, wherein A: grass carp chromosome; B: red crucian carp chromosome; C: natural gynogenesis grass carp chromosome; D: crucian carp hybrid offspring chromosome;

[0021] Figure 4 It is the HE staining result of ovary and testis sex gland tissue of natural gynogenesis grass carp, wherein A: 12-month-old natural gynogenesis grass carp ovary; B: 12-month-old natural gynogenesis grass carp testis; C: 18-month-old natural gynogenesis grass carp ovary, the oocytes in the ovary develop into II, III and IV stages; D: 18-month-old natural gynogenesis grass carp testis;

[0022] Figure 5HE staining results of ovary and testis of the hybrid of common carp and crucian carp, wherein A: ovary of the hybrid of common carp and crucian carp at 12 months old; B: testis of the hybrid of common carp and crucian carp at 12 months old; C: ovary of the hybrid of common carp and crucian carp at 18 months old; D: testis of the hybrid of common carp and crucian carp at 18 months old;

[0023] Figure 6 Fluorescence in situ hybridization results of two kinds of offspring and parents, wherein A: grass carp; B: red crucian carp; C: naturally gynogenetic grass carp; D: hybrid of common carp and crucian carp;

[0024] Figure 7 Microsatellite results of naturally gynogenetic grass carp and parents, wherein 1-8 from left to right are microsatellite results of the female parent grass carp, 9-19 are microsatellite results of the male parent red crucian carp, and 20-29 are microsatellite results of the offspring naturally gynogenetic grass carp, and the black identification line in the figure is an insertion fragment specific to the male parent red crucian carp;

[0025] Figure 8 Agarose electrophoresis gel map of grass carp, red crucian carp, naturally gynogenetic grass carp and hybrid of common carp and crucian carp;

[0026] Figure 9 Sequence alignment results of 5S rDNA of two kinds of offspring and parents;

[0027] Figure 10 Average DNA content of sperm of naturally gynogenetic grass carp and hybrid of common carp and crucian carp, wherein A: DNA content of sperm of naturally gynogenetic grass carp; B: DNA content of sperm of the hybrid of common carp and crucian carp;

[0028] Figure 11 Self-offspring of naturally gynogenetic grass carp and hybrid of common carp and crucian carp, wherein A: self-offspring of naturally gynogenetic grass carp; B: self-offspring of the hybrid of common carp and crucian carp. DETAILED DESCRIPTION

[0029] In order to facilitate the understanding of the present application, the following will combine the description of the drawings and the preferred embodiments to make a more comprehensive and detailed description of the present application, but the protection scope of the present application is not limited to the following specific embodiments.

[0030] Unless otherwise defined, all the professional terms used in the following have the same meaning as generally understood by those skilled in the art. The professional terms used in the present application are only for the purpose of describing the specific embodiments and are not intended to limit the protection scope of the present application.

[0031] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by existing methods.

[0032] Embodiment:

[0033] I. Method for simultaneously breeding natural gynogenesis of Ctenopharyngodon idella and hybrid offspring of Ctenopharyngodon idella and Carassius auratus

[0034] 1. In the breeding season, the female parent fish of Ctenopharyngodon idella is artificially induced to oviposition by injecting a mixed labor-inducing agent of luteinizing hormone-releasing hormone analogue and chorionic gonadotropin, wherein the dosage of the luteinizing hormone-releasing hormone analogue is 10 μg / kg, and the dosage of the chorionic gonadotropin is 100 IU / kg; the male parent fish is artificially induced to oviposition by injecting the mixed labor-inducing agent of luteinizing hormone-releasing hormone analogue and chorionic gonadotropin 3 hours later, wherein the dosage of the mixed labor-inducing agent is half of that of the female parent fish, and the injection is performed by intraperitoneal injection at the base of the pectoral fin without scales; after the injection, the parent fish is placed in an oviposition pool, and then artificial insemination is performed after the parent fish is in estrus, and then the fertilized eggs are hatched in a flow water hatching pool; the fertilization rate and the hatching rate of 1000 randomly selected fertilized eggs are counted, and the fertilization rate is 93.03%, and the hatching rate is 87.13%. After the fry is hatched, the fry is bred, and then the bred fry is detected and screened, so that the natural gynogenesis of Ctenopharyngodon idella and the hybrid offspring of Ctenopharyngodon idella and Carassius auratus are obtained.

[0035] The natural gynogenesis of Ctenopharyngodon idella and the hybrid offspring of Ctenopharyngodon idella and Carassius auratus have the appearance as shown in Figure 1 The morphological characteristics (countable and quantifiable traits) of the natural gynogenesis of Ctenopharyngodon idella and the hybrid offspring of Ctenopharyngodon idella and Carassius auratus are compared, and the obtained data are processed by SPSS software, and the results are shown in Tables 1 and 2. The morphological characteristics of the natural gynogenesis of Ctenopharyngodon idella are closer to those of the female parent Ctenopharyngodon idella, and the morphological characteristics of the hybrid offspring of Ctenopharyngodon idella and Carassius auratus are more between those of the female parent and the male parent, which shows the hybrid characteristics.

[0036] Table 1 Comparison of countable traits of parent and offspring

[0037]

[0038] Note: The capital Roman numerals represent hard fin rays, and the Arabic numerals represent soft fin rays.

[0039] Note: The upper superscripts a, b, c, and d represent different significant differences, and the same superscript has no significant difference.

[0040] Table 2 Comparison of quantifiable traits of parent and offspring

[0041]

[0042] Note: The upper superscripts a, b, c, and d represent different significant differences, and the same superscript has no significant difference.

[0043] II. Method for detecting ploidy of distant hybrid offspring

[0044] 1. For the ploidy of hybrid offspring, this method will use flow cytometry to detect the DNA content. Among them, the parents and offspring are selected 10 tails of experimental fish for experiment. The specific method is as follows:

[0045] 1) The captured parents and hybrid offspring were carefully classified and labeled in the tank to facilitate subsequent experimental operation and data recording. This step is crucial to ensure the accuracy and repeatability of the experiment.

[0046] 2) Prepare sterile syringes and anticoagulants, and each syringe is pre-filled with 0.2 mL of anticoagulant. The use of anticoagulants is to prevent blood clotting and ensure that the blood sample remains liquid during the experiment, facilitating subsequent analysis.

[0047] 3) When drawing blood samples, we collect from the fish's tail vein to minimize damage to the fish. After collection, the blood sample is properly stored in an ice box to maintain its freshness and activity.

[0048] 4) Perform nuclear staining, prepare sterile EP tubes, and add 0.3 mL of DAPI staining solution and 1 mL of 8% physiological saline. DAPI is a fluorescent dye that specifically binds to DNA, allowing us to observe the morphology of the cell nucleus under a microscope.

[0049] 5) Use a 1 μL pipette to gradually add the drawn blood to the EP tube containing the DAPI staining solution until the liquid color turns slightly red. This step is to ensure that the blood sample is fully mixed with the staining solution.

[0050] 6) After sample preparation, place them in the dark for 10-15 min to allow the DAPI dye to fully penetrate the cell nucleus. After the light avoidance treatment is complete, use a 20 μm pore size filter to filter the sample to remove unbound dye and cell debris, then dilute it for detection on the flow cytometer, as shown in Figure 2 .

[0051] 2. In this method, 10 parent and offspring hybrid fish with a body weight of more than 50 g were selected and labeled for each experimental fish to ensure the accuracy of the experiment. Before the experiment, prepare frozen slides, sterilize 1.5 mL EP tubes, sterilize glass culture dishes, sterilize 15 mL EP tubes, sterilize surgical scissors, sterilize pipettes, physiological saline (4 g / 500 mL), KCL (2.8 g / mL), methanol, glacial acetic acid, Giemsa staining solution, Na2HPO4 (0.7098 g / 500 mL), NaH2PO4 (0.78005 g / mL), and other reagents.

[0052] 1) At 8:30 PM on the first day, inject PHA (PHA is phytohemagglutinin, the dosage of PHA is calculated by weight * dose / concentration, the dose of PHA is 10 μg / g, the calculation result is 0.0025 * weight) with a concentration of 4 mg / mL into the experimental fish at a 45-degree angle towards the tail at the chest fin; on the second day at 8:30 AM, inject the same concentration of PHA (the dose is 15 μg / g, the calculation result is 0.00375 * weight) into the same location of the experimental fish; 3 hours later, inject the same concentration of PHA (the dose is 6 μg / g, the calculation result is 0.0015 * weight) into the same location of the experimental fish, and inject colchicine (the dose of colchicine is 4 μg / g, the calculation result is 0.00075 * weight) with a concentration of 2.5 mg / ml into the same location on the other side of the experimental fish. After the third injection, place the experimental fish in the tank for 1 hour before the experiment.

[0053] 2) First, the selected experimental fish is subjected to bloodletting treatment. After completing the bloodletting, the fish is carefully cut along the direction of the cloaca to remove the kidney as completely as possible from the back. The removed kidney is immediately thoroughly washed with physiological saline to remove possible residual blood and other impurities. After washing, the kidney is gently placed in a pre-prepared sterile culture dish.

[0054] 3) An appropriate amount of physiological saline is added to the culture dish to facilitate the processing of the kidney tissue. Then, the culture dish is tilted to about 45 degrees to facilitate operation. Using surgical scissors, the kidney tissue is carefully cut into small pieces until it is completely dispersed into a uniform aqueous solution. This step is crucial to ensure that the kidney cells are fully dispersed and the subsequent experiment proceeds smoothly.

[0055] 4) The kidney tissue treated into an aqueous solution is transferred to a 15 mL EP tube. To dilute and uniformly mix the kidney cell suspension, we add physiological saline to the EP tube until the total volume reaches 4 mL. Then, using a pipette, the suspension is vigorously blown for about 200 times to promote cell dispersion. After blowing, we continue to add physiological saline until the total volume of the EP tube reaches 12 mL, and perform another 200 times of blowing to ensure the uniformity of the cell suspension and reduce cell aggregation.

[0056] 5) After completing the blowing step, we let the EP tube stand for 10 min to allow the solid particles in the cell suspension to settle. Then, we carefully transfer the supernatant in the upper layer to a new 15 mL EP tube, and then supplement physiological saline to the EP tube to make the total volume reach 11 mL. Use a centrifuge to centrifuge the EP tube at a speed of 1500 revolutions per minute (rpm) for 5 min. After centrifugation, carefully pour off the supernatant and retain the precipitate at the bottom of the tube.

[0057] 6) Add 4 mL KCL, gently blow with a pipette to mix, then add to 10 mL, stand for 60 min, every 10 min during the interval, suck the bottom of the precipitate and discard. After standing, centrifuge the test tube in the centrifuge at 1500 rpm for 5 min, discard the supernatant after centrifugation. After the removal of the precipitate is completed, 4 mL KCL solution is added to the EP tube, which is a solution for cell lysis and chromosome preparation. We use a pipette to gently blow to ensure that the KCL solution is fully mixed with the precipitate. Then, continue to add physiological saline to make the total volume reach 10 mL to dilute the suspension and promote the further separation of cell components. The EP tube is left to stand for 60 min. During this process, the bottom precipitate is carefully sucked and discarded every 10 min, and after the standing time is over, the EP tube is placed in the centrifuge again at a speed of 1500 rpm for 5 min. After centrifugation is completed, the supernatant is poured off, and only the precipitate at the bottom of the tube is retained.

[0058] 7) After the completion of the precipitation, 2 mL of Carnoy's fixative (methanol: glacial acetic acid = 1:3) is added to the EP tube, and the EP tube is gently blown to ensure that the fixative is fully mixed with the precipitate. To further ensure the fixing effect, continue to add fixative to 6 mL, and then let the mixture stand for 15 min. After fixing, the EP tube is centrifuged in the centrifuge at 1500 rpm for 5 min. After centrifugation, the supernatant is removed, and this fixing and centrifugation step is repeated three times. After the three fixing and centrifugation processes are completed, an additional 2 mL of fixative is added to the EP tube, and then the EP tube is sealed. In order to maintain the stability of the cell nucleus sample, the sealed EP tube is stored in a refrigerator at 4°C.

[0059] 8) Take out a frozen slide that has been stored at -20°C for 24 h, and use a pipette to add the fixative stored at 4°C to the frozen slide from the vertical top, and gently pass the slide over the outer flame of the alcohol lamp a few times.

[0060] 9) Stain the toasted slide with staining solution (5 ml Na2HPO4+ 5 ml NaH2PO4+ 20 drops of Giemsa staining solution) for 45 min. After staining, wash the slide under running water to remove the staining agent, and the running water is gentle during this period. After natural air drying, observe and take pictures under an electron microscope, and the results are shown in Figure 3 , which shows that the chromosome number of the hybrid offspring of natural gynogenesis of grass carp and crucian carp is 100.

[0061] III. Fertility detection method of distant hybrid offspring

[0062] 1、To gain a deeper understanding of the reproductive characteristics of distant hybrid offspring, this method uses gonadal paraffin section technology for detailed analysis. Before the experiment begins, a series of experimental materials and reagents need to be prepared, including sterile EP tubes, sterile surgical scissors, alcohol solutions of different concentrations (from 70% to 100%), Bouin's solution, xylene, wax cups (ratio of yellow wax to white wax is 1:3), tweezers, blades, glass slides, and hematoxylin and eosin staining solutions. The specific experimental steps are as follows:

[0063] 1) The experimental fish need to be handled carefully. After being captured from the breeding environment, the fish are immediately disinfected, and then carefully dissected to remove the gonadal tissue. The removed gonads are immediately placed in a pre-prepared sterile EP tube containing Bouin's solution for tissue fixation. Bouin's solution is a commonly used fixative that can effectively preserve tissue structure. The fixation process usually lasts 1-3 days, during which the Bouin's solution is replaced once to ensure that the tissue is fully fixed. After fixation is complete, the gonadal tissue is transferred to a 70% alcohol solution for storage.

[0064] 2) The next day after fixation, at 8:00 AM, begin the tissue dehydration process by placing the fixed tissue in 70%, 80%, 90%, 95%, and 100% alcohol solutions, respectively, for 1 hour each.

[0065] 3) After completing the dehydration step of the tissue, it needs to be transferred to a solution mixed from xylene and 100% alcohol at a ratio of 1:1 for further processing. The tissue needs to be soaked in this mixed solution for about 45 minutes to ensure full penetration. After the soaking process is complete, the tissue is removed from the mixed solution and transferred to a pure xylene solution for infiltration. The purpose of this step is to remove the residual alcohol in the tissue and gradually make the tissue transparent, which is convenient for subsequent embedding and sectioning. The infiltration time should be controlled within 15 minutes, and it may need to be repeated several times until the tissue is observed to be transparent.

[0066] 4) Before the experiment starts, preheat the oven to 60°C one hour in advance to ensure stable temperature. At the same time, place the wax cup and tweezers in the oven to melt the paraffin into a liquid state during preheating. This step is crucial for subsequent tissue embedding, as liquid paraffin can better penetrate and fix the tissue structure. After the paraffin is melted, carefully place the tissue into the liquid paraffin and continue the infiltration process in the 60°C oven for about 2 hours. This process helps the paraffin fully penetrate the tissue, ensuring the stability of the tissue during embedding. After infiltration is complete, use the preheated tweezers to transfer the tissue to a mold filled with liquid paraffin, and place it at room temperature to allow the paraffin to solidify. After the paraffin is completely solidified, carefully remove the formed paraffin block from the mold.

[0067] 5) Before performing tissue sectioning, first finely trim the paraffin block into a regular cuboid shape using a pre-prepared blade. This step is to ensure the quality and consistency of the sections. The trimmed wax block is then placed on the microtome and secured with a clamp for precise sectioning. Next, adjust the microtome's sectioning thickness to 5 μm, ensuring the integrity and clarity of the sections during the process. The cut paraffin sections are gently placed in warm water at 42 ℃ to allow them to naturally expand, preventing them from curling or damaging. Subsequently, take the previously prepared glass slides and evenly drop a drop of glycerol onto the surface of the slides. The glycerol helps the sections adhere better to the glass slides. Use the slide to gently cover the sections from above, ensuring they spread evenly on the slide. Finally, place the slide with the sections into a 42 ℃ slide warmer for a two-day drying process. This step helps further fix and dehydrate the sections, preparing them for subsequent staining and microscopic observation.

[0068] 6) After drying, the slides are placed in xylene for deparaffinization, typically for 6 hours to ensure effective deparaffinization. After deparaffinization, staining is performed.

[0069] 7) After completing the deparaffinization process of the tissue sections, the sections on the slides need to be rehydrated for subsequent staining. First, immerse the slides in different concentrations of alcohol solutions in order, starting from 100%, then 95%, 90%, 80%, and 70%, with each concentration soaking for 5 minutes. This step helps gradually remove paraffin residues from the sections while preventing them from drying out. After rehydration, immerse the slides in hematoxylin staining solution for 45 minutes. Hematoxylin staining allows the cell nuclei to appear dark blue, making them clearly visible under a microscope. After staining, we carefully wash the slides with pure water to remove excess dye. Next, briefly immerse the slides in a 0.5% hydrochloric acid solution for 2 seconds, which helps enhance the contrast of cell structures. Then, wash the slides again with pure water and immerse them in a 0.2% NaOH solution for 10 seconds to neutralize the acidic environment. After washing with pure water, the slides need to be re-immersed in alcohol solutions in order from 70% to 95%, with each concentration also soaking for 5 minutes. Finally, immerse the slides in eosin staining solution for staining, with a staining time of about 1 minute. Eosin staining is mainly used to stain the cytoplasm, making it appear red under a microscope. After staining, the slides are first immersed in 100% alcohol for 5 minutes to remove excess eosin dye, and then transferred to xylene for another 5 minutes to further remove residual staining agents.

[0070] 8) First, take out the xylene-treated slides and prepare the glycerol jelly as the mounting medium. Glycerol jelly has good refractive index and transparency, which is suitable for mounting. During the mounting process, special attention should be paid to avoid air bubbles under the coverslip. Air bubbles may interfere with the observation under the microscope and affect the clarity of the cell structure. After the mounting is completed, the slides are placed in the fume hood for air drying. The air drying process helps to solidify the glycerol jelly and ensures the stability of the mounting. After air drying is completed, the slides are ready for observation under the microscope, and the results are shown in Figure 4 As shown in Table 1, the hybrid offspring of natural gynogenesis of C. idella and C. carpio were both male and female fertile.

[0071] IV. Genetic identification method of distant hybrid offspring

[0072] 1. In this method, the chromosome fixation solution prepared in the ploidy detection experiment is used, and after normal dropping of the slide, it is placed in a 70°C oven for drying. At the same time, the water bath is preheated (70°C, 80°C). The probe used in this chapter is a specific probe 263 for Carassius auratus constructed using BAC library. The specific operation steps are as follows:

[0073] 1) When performing fluorescence in situ hybridization (FISH) experiment, we first need to prepare the probe hybridization solution. This includes mixing purified probe (5 μL), deionized formamide (DDM, 4 μL), 50% dextran sulfate (DS, 3 μL), and 20× sodium chloride citrate buffer (SSC, 2 μL). Next, the slide containing chromosomes is preheated in a 70°C oven for 1-2 h to facilitate subsequent hybridization.

[0074] 2) The slide is soaked in 2×SSC solution for 30 min to reduce background signal. Then, the slide is dehydrated by 70% and 100% alcohol gradient, each for 5 min, and then briefly placed in 70% 2×SSC / DDM solution for 2 min to further prepare for hybridization.

[0075] 3) After drying at room temperature, denatured hybridization solution is added to the slide to cover the chromosomes, and then the slide is mounted. The mounted slide is placed in a wet box and incubated at 37°C overnight to promote the binding of the probe to the target DNA.

[0076] 4) After incubation, the slide is washed in 2×SSC / DDM solution at 43°C for 5 min, and then sequentially washed in 2×SSC and 1×SSC for 5 min each. After that, the slide is dried at room temperature, 8 μL of fluorescein isothiocyanate (FITC) is added, and incubated in the dark for 20-30 min.

[0077] 5) The washing step is performed again, the glass slides are washed in the washing solution at 43 °C for 15 min, and then dried at room temperature. Finally, 6 μL of DAPI and a fluorescence quencher are added to enhance nuclear staining and reduce the fluorescence background. After completing the above steps, the slides can be observed under a fluorescence microscope to evaluate the hybridization effect and the specific binding of the probe. The results of the red crucian carp experiment show 100 signal points, and the hybridization results of the natural gynogenetic grass carp are also signal-free, which is consistent with the results of the female parent grass carp. In the experiment of the carp crucian carp hybrid offspring, 50 signal points are shown, which is exactly half of the number of signals of the father red crucian carp, and the results are shown in Figure 5

[0078] 2. Microsatellite primers are used to amplify specific DNA fragments to reveal the genetic background of natural gynogenetic grass carp. Microsatellite markers are powerful tools for studying genetic diversity and parentage due to their widespread distribution and high polymorphism in the genome. By PCR amplifying these microsatellite loci, natural gynogenetic grass carp can be compared with the parents. The specific experimental method is as follows:

[0079] 1) First, wash and dry a set of glass slides, then assemble them into a rubber shell. Then, carefully inject 5-10 mL of high-temperature dissolved 10% agarose into the gap between the glass slides and the rubber box to ensure no leakage. After solidification on the glass substrate, connect the glass substrate to the electrophoresis stand, and pay attention to the position of the positive and negative electrodes. Adjust the positioning knob to align them.

[0080] 2) Polyacrylamide gel preparation: Prepare 40 mL of 8% polyacrylamide gel in a 50 mL centrifuge tube. Add 800 μL of 10% APS solution and 20 μL of TEMED, and mix quickly. Use a 5 mL syringe to inject the mixture between the two glass plates, ensuring no air bubbles. Insert a 1.0 mm or 1.5 mm comb, and let it stand for about 30 min until the gel is formed.

[0081] 3) After the gel is formed, carefully remove the comb and adjust the sample well. Pour in 1% buffer to ensure that the buffer covers the well on one side and the electrode on the other side. Add 2.5 μL of sample in sequence. After the sample is applied, connect the power supply and set the voltage to 300 V. Electrophoresis for about 2.5-3 h until the indicator band moves to the bottom of the gel.

[0082] ​4) Silver nitrate development, when the electrophoresis is completed, turn off the power supply and unplug it from the power source. Pour the buffer into a container for recycling. Prepare 3 containers, 10 g C2H4O2 solution, 0.5 g AgNO3 is dissolved in 500 mL pure water, 9 g NaOH and 0.25 g Na2CO3 are dissolved in 500 mL pure water, and 2.5 mL CH2O is added to make a developing solution. Place the glass sheet in the C2H4O2 solution, gently remove the glass sheet, and then dye it with AgNO3 solution for 10 min. After dyeing, rinse quickly with water, and then add the developing solution for 10 min. After rinsing, rinse and absorb with water, record and analyze the results, and find that the red crucian carp has a unique DNA insert fragment. This insert fragment appears in the red crucian carp sample and the natural gynogenesis of the grass carp sample, but is not detected in the female grass carp.

[0083] 3. For genetic analysis experiments, 10 parent fish and hybrid offspring fish were selected. First, the tail fins of the fish were thoroughly cleaned with a 70% alcohol solution to remove any possible microorganisms and impurities. After cleaning, the tail fins were cut off using autoclaved scissors. The cut tail fins were immediately placed in pre-labeled EP tubes. To store these samples for a long time, they were stored in a refrigerator at -20°C. The specific experimental method is as follows:

[0084] 1) DNA extraction was performed using the OMG (Omega Tissue DNA Kit) kit. The stored tail fin samples were removed and cut into pieces using sterilized scissors and placed in sterilized EP tubes. 200 μL TL Buffer and 25 μL OB Protease Solution were added. After addition, water bath at 55°C for 5 min.

[0085] 2) After the water bath step is completed, 220 μL of BL buffer is added to the sample, and the EP tube is gently shaken to ensure thorough mixing. After mixing, the water bath temperature is adjusted to 70°C, and the sample is placed back in the water bath for further processing for 10 min.

[0086] 3) After the water bath treatment is completed, 220 μL of 100% alcohol is added to the sample, and after thorough shaking, the sample is transferred to the adsorption column using a pipette, and centrifuged in a centrifuge for 1 min.

[0087] 4) After centrifugation, the supernatant is carefully removed and a new collection tube is replaced. Next, 500 μL of HBC Buffer is added, and centrifuged for 30 s, then the supernatant is discarded. Add 700 μL of DNA Wash Buffer, centrifuge for 30 s, then discard the supernatant. This step needs to be repeated twice to ensure the purification of DNA.

[0088] 5) After the above washing steps, a new collection tube is changed and centrifugation is performed for 1 min. After the end of centrifugation, the cap is opened, and the tube is placed at room temperature for 5 min to allow the DNA to precipitate at the bottom of the tube. After the completion of precipitation, 35-50 μL of Elution Buffer preheated to 70 °C is added, and centrifugation is performed again for 2 min. In this way, the pure DNA is successfully extracted from the sample, which can be stored in an environment of -20 °C for subsequent experiments.

[0089] 6) The DNA is extracted from the selected fish samples, and 5s-specific primers are selected for PCR amplification. After PCR amplification, in order to further purify the target DNA fragment, the obtained amplification product is gel recovered for subsequent cloning and sequencing. The recovered and purified DNA fragment is then connected to a suitable vector and transformed into host cells for the screening and amplification of single clones. After screening, Sanger sequencing is performed by Shenguo Bioengineering (Shanghai) Co., Ltd., and the sequencing results are analyzed in detail using Jaiview software (version 2.11.3.2) for 5S rDNA sequencing results. The outcross offspring and the maternal Procyprinus do not differ in this coding region, which indicates that the genetic information of the offspring in this region mainly comes from the maternal; and some base changes are shown in the NTS region, which indicates that the offspring has certain genetic variation on 5S rDNA, as shown in Figure 8 .

[0090] V. Outcross offspring self-crossing and passing down

[0091] 1. The natural gynogenetic Procyprinus is used as the paternal and maternal parent, and the DNA content of the sperm is detected before the breeding period, and the results are shown in Figure 9 . The average DNA content of the sperm is consistent with the DNA content of the blood. During the breeding season, the mother parent fish with swollen, soft, and elastic abdomen is artificially induced by injecting a mixed labor-inducing agent of luteinizing hormone-releasing hormone analog and chorionic gonadotropin. The dose of the luteinizing hormone-releasing hormone analog is 10 μg / kg, and the dose of the chorionic gonadotropin is 100 IU / kg. The father parent fish is selected by gently squeezing the abdomen to squeeze out white sperm 3 h after the mother parent fish is injected, and a mixed labor-inducing agent of luteinizing hormone-releasing hormone analog and chorionic gonadotropin is artificially induced by injection. The injection dose is half of that of the mother parent fish, and the injection is performed by abdominal cavity injection at the base of the pectoral fin without scales. After the injection is completed, the parent fish is placed in an egg laying pool, and artificial insemination is performed after the parent fish is in estrus. Then, the fertilized eggs are hatched in a flow water hatching pool. The fertilization rate and the hatching rate are statistically analyzed by randomly selecting 300 fertilized eggs, and the fertilization rate reaches 73.9%, and the hatching rate is 63.5%. The appearance of the offspring is photographed under a microscope as Figure 10As shown.

[0092] 2. The DNA content of the sperm of the hybrid offspring of common carp and crucian carp was detected before the breeding period, and the results are shown in Table 1. Figure 9 As shown, the average DNA content of the sperm was consistent with the DNA content of the blood. The unfertilized eggs were observed under a microscope, and the results are shown in Table 2. Figure 10 During the breeding season, the female parent fish were artificially induced to ovulate by injecting a mixture of luteinizing hormone-releasing hormone analog and chorionic gonadotropin, the dose of the luteinizing hormone-releasing hormone analog was 10 μg / kg, and the dose of the chorionic gonadotropin was 100 IU / kg. The male parent fish were artificially induced to ovulate by injecting a mixture of luteinizing hormone-releasing hormone analog and chorionic gonadotropin 3 hours after the female parent fish, the injection dose was half of that of the female parent fish, and the injection was performed at the base of the pectoral fin without scales. After the injection, the parent fish were placed in an oviposition pool, and artificial insemination was performed after the parent fish showed estrus. The fertilized eggs were then hatched in a flow water hatching pool. The fertilization rate and hatching rate of 300 randomly selected fertilized eggs were statistically analyzed, and the fertilization rate reached 77.4%, and the hatching rate was 67.3%. The appearance of the offspring under a microscope is shown in Table 3. Figure 10 As shown.

Claims

1. A method for simultaneously breeding natural gynogenetic Mylopharyngodon piceus and hybrid offspring of Cyprinus carpio, characterized in that, The method comprises the following steps: taking Ctenopharyngodon idella as female parent and Carassius auratus as male parent, carrying out special pool feeding, carrying out artificial induction after artificial spawning, carrying out artificial insemination after the parent fish is in estrus, carrying out flow water hatching on the fertilized eggs, carrying out feeding after the fry is hatched, and carrying out detection and screening on the fed fish, so that the natural gynogenesis Ctenopharyngodon idella and the Carassius auratus hybrid offspring are obtained.

2. The method of claim 1, wherein, The special pool breeding includes the following steps: 2 3 months before the breeding season, selecting female grass carp and male red carp with good body shape, no disease and injury, and obvious sexual maturity characteristics as parents for special pool breeding. 1 month before the breeding season, the parents are fed with high-quality food, and the water flow is stimulated every day to promote the maturation of gonads.

3. The method of claim 1, wherein, The artificial induction includes the following steps: in the breeding season, the female parent fish with abdominal distension, softness and elasticity is artificially injected with mixed induction agent of luteinizing hormone releasing hormone analogue and chorionic gonadotropin, the dose of the luteinizing hormone releasing hormone analogue is 7.5 10 μg / kg, and the dose of the chorionic gonadotropin is 100 150 IU / kg; the female parent fish is injected first, and 3-5 hours later, the male parent fish with white semen squeezed out by light abdominal squeezing is selected and artificially injected with mixed induction agent of luteinizing hormone releasing hormone analogue and chorionic gonadotropin, the injection dose is half of that of the female parent fish, and the injection is performed by abdominal cavity one-needle injection method at the base of the pectoral fin without scales.

4. The method of claim 3, wherein, The artificial induction is carried out in the breeding season and when the water temperature is stable at 22 DEG C or above.

5. The method of claim 3, wherein, After the injection of the mixed induction agent is completed, the female and male parent fish are put into the spawning pool at a quantity ratio of 1:3-4.

6. The method of claim 1, wherein, The artificial insemination comprises the following steps: after the parent fish is in estrus, the parent fish is salvaged by using a net, the female Ctenopharyngodon idella with smooth spawning and suitable spawning quantity is selected, the abdomen is gently squeezed, the dark green eggs are squeezed into a clean porcelain basin, the male Carassius auratus with large sperm is selected, the abdomen is gently squeezed, the white sperm is squeezed into the porcelain basin, the feather is used to quickly mix, and then the mixture is quickly poured into a prepared brown sheet for insemination.

7. The method according to any one of claims 1 to 6, characterized in that, The feeding of the fry includes the following steps: after the fry is completely hatched, the fry is cultured in a hatching pool for 2 3 days, and then is transferred to a previously fattened pond after the appearance of the lumbar point; and after the fry is released into the pond for 2 days, the soybean milk is sprayed.

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