A method for creating a fertile alloheptaploid silver crucian carp female reproductive system resistant to herpesvirus

Abstract

The present invention belongs to the technical field of fish genetic breeding, and specifically relates to a method for creating a fertile alloheptaploid silver crucian carp gynogenetic line that is resistant to herpes simplex virus. First, a gynogenetic triploid silver crucian carp mother is bred with a sexually reproducing diploid amblycephala bream father to obtain offspring G1, and then individuals whose genomes have been integrated with a set of amblycephala bream sperm chromosome sets are screened from offspring G1, i.e., alloheptaploid silver crucian carp, which has fertile gynogenetic ability. Herpes simplex virus infection experiments have confirmed that the alloheptaploid silver crucian carp clone line has 100% resistance to herpes simplex virus. The breeding route provided by the present application can create a new silver crucian carp strain with significant resistance to herpes simplex virus and maintaining gynogenetic ability, which is an important way to cultivate new silver crucian carp varieties and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of fish genetic breeding, and in particular relates to a method for creating a fertile alloheptaploid silver crucian carp gynogenetic reproductive system that is resistant to herpesvirus. Background Art

[0002] As a classic and highly effective cell engineering breeding technique, polyploid breeding has been widely used in animal and plant breeding, including in the selection of farmed fish and shellfish. Allopolyploids, produced by the integration and doubling of the genomes of two different parents, often combine the advantages of hybridization and polyploidization, exhibiting superior growth and greater stress resistance than their diploid ancestors. Optimizing or developing new and efficient ploidy manipulation techniques, creating superior germplasm, and providing core breeding material for the development of new varieties are key areas of genetic breeding in aquatic animals.

[0003] Allogistic silver crucian carp ( C. gibelio ) is an important aquaculture species in my country. However, due to the high-density and single-culture farming model commonly used in the main breeding areas of silver crucian carp, the herpes simplex virus ( Ca The epidemic disease caused by HV) broke out in the main breeding areas of silver crucian carp and caused huge economic losses. Among them, the main breeding variety in the crucian carp breeding market, "Zhongke No. 3" or A + Pair Ca HV is extremely susceptible and has a mortality rate of 100%. Ca HV allopolyploids of gibel carp are an important demand of the crucian carp aquaculture industry.

[0004] The silver crucian carp has multiple reproductive modes such as parthenogenetic gynogenesis and sexual reproduction. When performing allospermia gynogenesis, the silver crucian carp also has the ability to incorporate chromosome sets and chromosome fragments of sperm from other fish into the egg nucleus for coordinated development. Taking advantage of this ability, the applicant team used white crucian carp, red crucian carp, etc. as parents to create a series of new polyploid silver crucian carp. However, the white crucian carp is amphidiploid (AABB), and the synthesized new polyploid silver crucian carp has a set of 50 chromosome sets (AB) derived from the father, forming a double tetraploid crucian carp with a genome composition of AAAABBBBB. This excessive ploidy will lead to an imbalance in the nucleus-cytoplasm ratio of the cell, bringing some negative effects, and will not show the advantages of polyploidy. For example, growth and herpes virus infection experiments on the obtained new polyploid silver crucian carp showed that it had no growth advantage compared with the maternal population and its resistance to herpes virus was not significantly improved. The applicant team's previous patent CN115486412B disclosed a method for obtaining a new double triploid clone line by backcrossing the red crucian carp with a new double tetraploid male fish that integrates the chromosome set of the red crucian carp. However, this method diluted the genome composition of the original mother "Zhongke No. 3" in the new polyploid silver crucian carp after two generations of integration and hybridization. Its original excellent traits, such as the silver-black body color of "Zhongke No. 3" that is very popular in the market, will be affected by the genome of the red crucian carp.

[0005] Therefore, breeding a new polyploid silver crucian carp germplasm with high resistance to crucian carp herpes virus, reduced negative effects, and better traits is still a technical problem that needs to be solved urgently. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a method for creating a fertile alloheptaploid gibel carp gynogenetic reproductive system resistant to herpesvirus.

[0007] The present invention adopts the following technical solutions:

[0008] A method for creating a fertile alloheptaploid silver crucian carp gynogenetic reproductive system resistant to crucian herpesvirus, comprising the following steps:

[0009] S1. The offspring G1 was obtained by breeding the gynogenetic triploid silver carp (AAABBB) with the sexual diploid bluebrike (CC).

[0010] S2. Individuals (AAABBBC) whose genomes incorporated a set of sperm chromosomes of Megalobrama amblycephala were screened from the progeny G1, namely, alloheptaploid gibel carp, which had fertile female nuclear reproduction ability.

[0011] Preferably, the gynogenetic triploid gibel crucian carp mother in step S1 is the allogeneic gibel crucian carp "Zhongke No. 3".

[0012] Preferably, the method further comprises an S3 propagation step, wherein the alloheptaploid gibel crucian carp is used as the female parent, and the sperm of the male parent Xingguo red carp is used to stimulate the eggs of the female parent to perform gynogenesis to obtain offspring G2, which is an alloheptaploid gibel crucian carp clone line.

[0013] Preferably, the eggs of female individuals in the alloheptaploid gibel crucian carp are dug out for inspection, and female fish with well-matured eggs are selected for breeding. Individuals with a fertilized egg hatching rate of more than 20% are alloheptaploid gibel crucian carp with fertile female nuclear reproductive ability.

[0014] Preferably, in step S1, the specific method of breeding is: in the crucian carp breeding season, the male semen is mixed with the semen: trypsin solution at a volume ratio of 1:10, and the semen is treated at 23°C for 15 minutes; the female eggs are mixed with the treated semen for fertilization, and the obtained fertilized eggs continue to hatch; the mass concentration of the trypsin solution is 1%.

[0015] Preferably, in step S1, the method for screening allogeneic heptaploid gibel crucian carp from the offspring G1 is as follows: clip the fin rays of the fry to take a trace amount of blood, add CyStain DNA 1Step solution and mix well, detect the DNA content by flow cytometry, use the maternal triploid gibel crucian carp and the paternal amblycephala bream as controls, and select individuals with DNA content between the triploid gibel crucian carp and the sum of the DNA content of triploid gibel crucian carp + amblycephala bream, which are the allogeneic heptaploid gibel crucian carp.

[0016] The beneficial effects of the present invention are:

[0017] Since the white crucian carp, red crucian carp and other amphidiploid crucian carp all belong to the genus Carassius auratus, the virus will replicate in their bodies after infection with the Carassius auratus herpes virus, and there are only differences in the survival rate after infection. Therefore, the heterologous neopolyploid crucian carp obtained using white crucian carp, red crucian carp, etc. are still susceptible. These amphidiploid crucian carp are not the best paternal parents for obtaining the new polyploid silver crucian carp with absolute disease resistance. The amblycephala bream belongs to the diploid cyprinid family that is distantly related to the silver crucian carp. It has important characteristics such as fast growth, high back and similar body color to the silver crucian carp. In this application, the inventor team proved for the first time that the amblycephala bream is not infected with the Carassius auratus herpes virus. Therefore, by Ca A set of non-infectious Ca The new polyploid of silver crucian carp obtained from the chromosome set of HV amblycephala can improve the susceptibility of the original mother to viruses and greatly improve its antiviral ability, thereby obtaining a new polyploid of silver crucian carp with absolute disease resistance advantage.

[0018] In the traditional hybrid breeding process, due to the reproductive isolation between the silver carp and the crucian carp, sexual reproduction is not possible to produce fertile hybrid offspring. However, this method utilizes the special reproductive characteristics of the silver carp to break through the reproductive isolation between the silver carp and the silver carp, and obtains fertile allogeneic heptaploid silver carp offspring. At the same time, the polyploid silver carp maintains its ability to reproduce with parthenogenetic female nuclei, which can permanently fix its disease resistance advantage. The method of this application not only obtains the crucian carp and bream hybrids that are difficult to obtain through traditional hybridization, but also breaks through the traditional belief that aneuploidy is infertile. Herpes virus infection experiments have confirmed that allogeneic heptaploid silver carp clones are infected Ca Only a trace amount of virus particles were detected in the body after HV, indicating that the virus replication in the body was significantly inhibited, so the disease would not occur and cause death, that is, it has 100% anti-herpes virus ability.

[0019] This application provides an important reference for parent selection in polyploid breeding, and species with more distant genetic relationships can be selected to improve more traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This application provides a route for creating the alloheptaploid gibel crucian carp clone line.

[0021] Figure 2 This is a histogram of the DNA content in the blood cells of the alloheptaploid gibel crucian carp and its parents in Example 1.

[0022] Figure 3 The genomic composition of the alloheptaploid gibel carp was identified by whole-genome in situ hybridization in Example 1, wherein the blue fluorescent signal is the chromosome stained by DAPI, the green signal is the hybridization signal of the maternal gibel carp genomic probe labeled with digoxigenin, and the red signal is the hybridization signal of the genomic probe of the amblycephala bream labeled with biotin.

[0023] Figure 4 This is the genome composition of the G2 generation of gibel crucian carp produced by whole genome in situ hybridization identification and screening in Example 2, and the red fluorescent signal is the chromosome of the amblycephala bream.

[0024] Figure 5 This is the genome composition of the G1 generation of gibel crucian carp that was not reproduced identified by whole genome in situ hybridization in Example 2. The red fluorescent signal is the chromosome of Megalobrama amblycephala.

[0025] Figure 6 The infection was caused by alloheptaploid silver carp, the female silver carp and the male amblycephala Ca Survival curve after HV.

[0026] Figure 7 The infection was caused by alloheptaploid silver carp, the female silver carp and the male amblycephala Ca Viral copy number after HV.

[0027] Figure 8 The infection was caused by alloheptaploid silver carp, the female silver carp and the male amblycephala Ca Pathological section of HV posterior renal tissue. DETAILED DESCRIPTION

[0028] To facilitate understanding, the technical solution of the present invention is described in more detail below with reference to specific embodiments.

[0029] Example 1

[0030] Creation of a fertile alloheptaploid gynogenetic clone of silver crucian carp

[0031] like Figure 1 As shown, a gynogenetic triploid gibel carp mother (AAABBB) was bred with a sexual diploid amblycephala bream father (CC) to obtain the offspring G1; individuals with a DNA content between that of the triploid gibel carp and the sum of the DNA contents of the triploid gibel carp and amblycephala bream, that is, individuals with a set of amblycephala bream sperm chromosome set incorporated into their genome (AAABBBC), were screened from the offspring G1, which were alloheptaploid gibel carp with fertile gynogenetic ability.

[0032] The specific operations are:

[0033] 1. Prepare 1% trypsin solution

[0034] Weigh 0.1 g of trypsin powder and add it to 10 mL of sperm preservation solution to make the working concentration 1%. Dissolve and mix evenly and set aside. Store at 4°C and use immediately after preparation.

[0035] 2. Treating the sperm of Megalobrama amblycephala with trypsin solution

[0036] During the breeding season, sexually mature male fish of the distantly related diploid species Megalobrama amblycephala (genome composition CC, 48 chromosomes) were selected and subjected to meiotic division to produce reduced sperm (C). 1 mL of semen was added to 10 mL of the above-mentioned trypsin solution and treated at 23°C for 15 min, with repeated inversion during the treatment to ensure complete treatment.

[0037] 3. The treated sperm is fertilized with the mature eggs of Zhongke No. 3

[0038] Gonadally mature albiculture crucian carp, "Zhongke No. 3," were selected and injected with oxytocin. The injected broodstock were then placed in a tank with running water at a constant temperature of 21°C. The effect lasts approximately 10 hours, so the oxytocin was injected in the evening, and spawning was carried out in the morning for the next experiment. After spawning, the mature eggs of the albiculture crucian carp, "Zhongke No. 3," were squeezed into a 10 cm dish. Then, dry insemination was performed with trypsin-treated Megalobrama amblycephala sperm for 15 minutes. The sperm and eggs were mixed using a dry feather and then spread onto white porcelain plates filled with aerated water at 23°C (approximately 2,000 to 4,000 fertilized eggs per plate).

[0039] 4. Fertilized egg hatching and selection

[0040] During the incubation period of the fertilized eggs in the white porcelain dish, change the water every 12 hours and add a small amount of methylene blue to fix any dead fertilized eggs. After 24 hours, remove any blue dead eggs fixed by the methylene blue from the white porcelain dish to prevent water contamination and affect the normal development of the fertilized eggs. Once the surviving fertilized eggs have hatched and formed membranes, transfer them to a fish tank and lightly oxygenate them.

[0041] 5. Ploidy detection and breeding of candidate offspring

[0042] When the above fry were cultured to six months of age, a small amount of blood (about 0.1 μL) was collected by clipping the fin rays. The blood was added to 200 μL of 4°C pre-cooled CyStain DNA 1Step (Partec CyStain DNA 1Step, Germany) solution, mixed, and placed on ice until the DNA content was detected by the CytoFLEX flow cytometer. The female triploid gibel carp and the male amblycephala bream were used as controls. Individuals with DNA contents between the triploid gibel carp and the sum of the triploid gibel carp and amblycephala bream DNA contents were selected, which were alloheptaploid gibel carp. (See References) Figure 2 .

[0043] In this example, a total of 15 alloheptaploid gibel carp (AAABBBC) incorporating a set of chromosome sets from Megalobrama amblycephala sperm were screened.

[0044] 6. Screening and reproduction of fertile offspring

[0045] In polyploid breeding, aneuploids are usually infertile due to their inability to undergo normal meiosis, for example, triploid seedless watermelons. In this application, identified alloheptaploid silver crucian carp were cultured to sexual maturity. After examining the gonadal development of the alloheptaploid silver crucian carp, mature individuals with oocytes developed to stage IV were selected for reproduction by injecting oxytocin. Their eggs were then fertilized with ultraviolet-inactivated Xingguo red carp sperm. The fertilized eggs hatched and the membranes emerged, thus obtaining a batch of alloheptaploid silver crucian carp gynogenetic clones.

[0046] In the present embodiment, after 15 tail individuals screened for are cultivated to sexual maturity, the female fish egg development situation is checked.Select healthy and strong body shape, no deformity and abdomen is soft, expand, the female fish parent with clear ovary outline adopts ovulator to dig eggs and check: ovulator is slowly inserted in the genital pore, then a little to the left or right, rotate several times and extract gently, can take out a small amount of ovum, then add a small amount of fixing transparent liquid, soak 2-3 minute back and observe the egg nucleus position.If the nuclear position eccentricity or polarization of all or most of ovum, then show that egg maturity is good; As white nucleus occupies central position, show that egg development is relatively poor; If most of ovum does not have white core to occur, then mostly are degenerate ovum or overmature, are not suitable for breeding. Subsequently, the qualified female fish were bred by injecting oxytocin, and their mature eggs were dry-fertilized with ultraviolet-inactivated Xingguo red carp sperm. After the fertilized eggs hatched from the membrane, the hatching rate was statistically >20%, and the parents were considered fertile. In this embodiment, one fertile alloheptaploid parent was finally screened, and a batch of gynogenetic clone offspring of this individual were obtained.

[0047] 7. Genetic identification of offspring of gynogenetic clones

[0048] In order to verify that all the offspring of the alloheptaploid silver carp were heptaploid, three offspring were randomly selected to prepare the metaphase chromosome division phase. Then, the genome of the female silver carp was labeled with digoxigenin and the genome of the male amblycephala was labeled with biotin by whole genome in situ hybridization. Figure 3 As shown, panel a represents an overlay of all signals, panel b shows the red signal from a biotin-labeled genomic probe for Megalobrama amblycephala, panel c shows the green signal from a digoxigenin-labeled genomic probe for Carassius auratus, and panel d shows the blue fluorescent signal from chromosomes stained with DAPI. It can be observed that the gynogenetic offspring of the alloheptaploid Carassius auratus is indeed a heptaploid, with a set of Megalobrama amblycephala sperm (24 chromosomes marked by the red signal) integrated into the Carassius auratus.

[0049] Example 2

[0050] Method for creating a fertile alloheptaploid gynogenetic clone of gibel crucian carp:

[0051] The methods for fertilization, hatching, cultivation, and ploidy detection of Megalobrama amblycephala and triploid Carassius auratus in this Example 2 are the same as those in Example 1 and will not be repeated here.

[0052] In this example, blood was collected from the obtained offspring G1 individuals for DNA content detection, and 13 silver crucian carps were screened out with DNA contents between the father, Megalobrama amblycephala (diploid), and the mother, Gibelio carp (triploid). They were selected as candidate alloheptaploids for further breeding experiments.

[0053] Ten silver carp were randomly selected from the 13 screened silver carp for breeding experiments. All ten silver carp were able to produce G2 generations through female reproduction. From the G2 generations produced by each silver carp, three offspring were randomly selected to prepare metaphase chromosome divisions, and then whole genome in situ hybridization experiments were performed. The offspring tested were all alloheptaploid silver carp. Figure 4 The results are for the test of 6 randomly selected G2 individuals. It can be confirmed that all 10 gibel crucian carp that produced the G2 generation are fertile alloheptaploid gibel crucian carp.

[0054] The remaining 3 unscreened individuals of Carassius auratus were also subjected to chromosome fluorescence in situ hybridization. Figure 5 It can be seen that the three silver crucian carp individuals are alloceptaploid silver crucian carp.

[0055] Based on the above experiments, it can be confirmed that the alloheptaploid traits and fertility traits in the G1 generation obtained in the breeding method of the present application are highly correlated, and all the alloheptaploids obtained can undergo gynogenesis.

[0056] Example 3

[0057] Ca HV artificial infection experiment

[0058] Prepare fresh Ca HV virus suspension was diluted with PBS buffer to 10 0 , 10 1 , 10 2 , 10 3 , 10 4 60 healthy silver crucian carp Zhongke 3 were randomly divided into 6 groups (10 in each group) and injected with the serially diluted virus filtrate. Each carp was injected with 50 μL of the virus filtrate dilution at the base of the pectoral fin. The control group was injected with the same amount of PBS buffer. Observation was performed daily, and the mortality rate was calculated. The median lethal dose was 10 2 dilution.

[0059] The viral copy number was calculated using fluorescence quantitative PCR. Ca The 637 bp helicase gene fragment of HV was used as the quantitative standard to construct a standard curve. The specific steps are as follows:

[0060] 1) Amplification Ca The HV 637 bp helicase fragment was purified and recovered using a gel recovery kit.

[0061] 2) The purified helicase fragment was inserted into the pMD®18-T plasmid to obtain pMD- Ca HV plasmid (3,329 bp).

[0062] 3) Calculate the copy number of the plasmid based on the relationship between the molar number, relative molecular mass and concentration. Ca The HV plasmid was diluted 10-fold as a standard and quantitatively analyzed by fluorescence quantitative PCR. The standard curve was constructed using SPSS software.

[0063] 4) Total DNA from the samples was extracted using a DNA extraction kit and used as a template for quantitative PCR analysis under the same conditions. The viral copy number of each sample was calculated based on the standard curve.

[0064] Finally, the half-lethal dose was calculated to be 50 μL Ca The HV diluted filtrate contained 3.214×10 7 , and this concentration was used as the injection concentration in subsequent experiments.

[0065] The allogeneic heptaploid silver crucian carp gynogenetic clones, the female parent triploid silver crucian carp "Zhongke No. 3", and the male parent Megalobrama amblycephala were temporarily cultured in a 32 cm × 24 cm × 36 cm aquarium in the laboratory. The water temperature was gradually transitioned to 23℃ (±1℃). The experimental fish were kept for two weeks to observe whether there were any abnormalities and to detect the absence of Ca HV virus. Feed the animals daily as normal during the temporary period, but withhold feeding for 24 h before and after virus injection. Inject 50 μL of the virus into the base of the pectoral fin. Ca The virus suspension after HV filtration (the number of virus particles was 3.214×10 7 Wild-type individuals served as the control group, and each fish was injected with an equal volume of PBS buffer. Three replicates were run for each infection group, each containing 30 fish. The culture water was maintained at 23°C (±1°C), continuously filtered, and replaced daily. When no fish died, one-third of the water was replaced daily. If fish died, one-half of the water was replaced daily, and dead fish were removed promptly to maintain water cleanliness.

[0066] After infection, the mortality of gibel crucian carp was recorded every day until 14 dpi (days post infection), and the survival rate of fish was analyzed. Figure 6 As shown, the results showed that the female parent "Zhongke No. 3" began to die on the 6th day and all died on the 8th day, with a final survival rate of 0%; while the paternal group of Megalobrama amblycephala and the allogeneic heptaploid silver crucian carp had a final survival rate of 100%, indicating that the allogeneic heptaploid silver crucian carp has a very high ability to resist crucian herpes virus after viral infection.

[0067] Furthermore, the viral copy numbers in the head and kidney tissues of the three fish were calculated, e.g. Figure 7 As shown, the parent "Zhongke No. 3" is A + Tied to CaAfter HV infection, the virus in the body increases exponentially, while almost no virus particles can be detected at different time points after infection in the amblycephalic bream, and trace amounts of virus particles can be detected in the alloheptaploids. At the same time, pathological sections of the head and kidney tissues were analyzed. Figure 8 120 h after virus infection, the pathological damage of the head and kidney tissues of the alloheptaploid group was significantly lower than that of the mother silver carp A. + Tie.

[0068] These results indicate that maternal A + Pair Ca Highly susceptible to HV, the male amblycephalic bream is not infected Ca HV, while the alloheptaploid clones are infected Ca However, HV has a high antiviral ability because its viral replication in the body is significantly inhibited and therefore it will not cause illness or death.

[0069] The above experiments prove that the breeding route provided in this application can create a new crucian carp strain with significant resistance to herpes virus and also maintains female reproductive ability, and is therefore an important way to cultivate new varieties of silver crucian carp.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for producing a fertile alloheptaploid gibel carp gynogenetic system resistant to herpesvirus, characterized in that: The following steps are involved: S1. The gynogenetic triploid silver crucian carp mother AAABBB was bred with the sexually reproducing diploid Megalobrama amblycephala father CC to obtain offspring G1, wherein the gynogenetic triploid silver crucian carp mother was the allogeneic silver crucian carp "Zhongke No. 3"; S2. From the progeny G1, the individual AAABBBC, whose genome has been integrated with a set of sperm chromosomes of Megalobrama amblycephala, was screened. The female individuals of the alloheptaploid gibel carp have fertile gynogenetic ability.

2. The method for producing a fertile alloheptaploid gibel carp gynogenetic reproductive system resistant to herpesvirus according to claim 1, characterized in that: The method also includes an S3 propagation step, in which a female individual of the alloheptaploid silver crucian carp is used as the mother parent, and the sperm of the father parent Xingguo red carp is used to stimulate the eggs of the mother parent to perform gynogenesis to obtain offspring G2, which is an alloheptaploid silver crucian carp clone system.

3. The method for creating a fertile alloheptaploid gibel carp gynogenetic reproductive system resistant to herpesvirus according to claim 2, characterized in that: The eggs of female individuals in the alloheptaploid gibel crucian carp were dug out for inspection, and females with well-matured eggs were selected for breeding. Individuals with a fertilized egg hatching rate greater than 20% were alloheptaploid gibel crucian carp with fertile female nuclear reproductive ability.

4. The method for creating a fertile alloheptaploid gibel carp gynogenetic reproductive system resistant to herpesvirus according to claim 1, characterized in that: In step S1, the specific breeding method is: during the crucian carp breeding season, the male parent's semen is mixed with a trypsin solution at a volume ratio of 1:10, and the semen is treated at 23° C. for 15 minutes; the female parent's eggs are mixed with the treated semen for fertilization, and the resulting fertilized eggs are continued to be hatched; the mass concentration of the trypsin solution is 1%.

5. The method for creating a fertile alloheptaploid gibel carp gynogenetic reproductive system resistant to herpesvirus according to claim 1, characterized in that: In the step S1, the method for screening allogeneic heptaploid gibel crucian carp from the offspring G1 is as follows: clip the fin rays of the fry to obtain a trace amount of blood, add CyStain DNA 1Step solution and mix evenly, detect the DNA content by flow cytometry, use the maternal triploid gibel crucian carp and the paternal amblycephala bream as controls, and select individuals with a DNA content between the triploid gibel crucian carp and the sum of the DNA contents of the triploid gibel crucian carp and the amblycephala bream, which are the allogeneic heptaploid gibel crucian carp.

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