Method for creating sterile heterologous heptaploid crucian carp with anti-herpes virus ability

Abstract

The present application belongs to the technical field of fish genetic breeding, and particularly relates to a method for creating sterile allohexaploid crucian carp with herpes virus resistance. First, double-triploid crucian carp females are bred with double-diploid crucian carp males, and the ploidy of the obtained offspring G1 is detected. Double-triploid crucian carp females with double-tetraploid offspring G1 are selected as the required female parent, and the females are controlled to lay eggs again. The obtained eggs are fertilized with double-diploid crucian carp sperm inactivated by ultraviolet rays, and double-triploid maintenance line offspring G2 is obtained after hatching. The female offspring G2 is used as the female parent, and diploid Topmouth gudgeon is used as the male parent to breed offspring G3, which is the sterile allohexaploid crucian carp with herpes virus resistance. The breeding route provided in the present application can create a new crucian carp strain with significant herpes virus resistance and sterility, and has a broad application prospect.

Description

Technical Field

[0001] This invention belongs to the field of fish genetic breeding technology, specifically relating to a method for creating a sterile allogeneic heptaploid crucian carp with resistance to herpesvirus. Technical Background

[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 are produced by the integration and doubling of the genomes of two different parent species. They often combine the advantages of hybridization and polyploidization, exhibiting better growth advantages and stronger stress resistance than their diploid counterparts. Optimizing or developing new and efficient polyploid manipulation techniques to create superior germplasm and provide core breeding materials for new varieties is one of the important directions in aquatic animal genetic breeding.

[0003] Crucian carp belong to the genus *Carassius* in the family Cyprinidae. This genus is a complex polyploid system, mainly including the amphiploid crucian carp (*Carassius auratus*) (AABB), which has 100 chromosomes and reproduces sexually, and the amphiploid silver crucian carp (*C. gibelio*) (AAABBB), which has more than 150 chromosomes and reproduces through parthenogenesis. Crucian carp are also an important aquaculture fish. However, they are commonly infected with crucian carp herpesvirus (CaHV), which causes acute gill hemorrhage, causing extremely serious damage to the industry. Acute gill hemorrhage has a very rapid onset, with infected individuals typically dying within a week. Currently, there is no effective treatment, and prevention remains the primary approach in aquaculture, with limited success.

[0004] The Culter alburnus, belonging to the genus Culter in the subfamily Culterinae of the family Cyprinidae, is a diploid (CC) fish with 48 chromosomes that reproduces sexually. Previous studies have shown that hybridization of double diploid crucian carp eggs (AB) with diploid Culter alburnus sperm (C) results in incompatibility due to the distant phylogenetic relationship between the two fish species, leading to the failure of the resulting hybrid offspring (ABC). The applicant team previously attempted to fertilize Culter alburnus sperm with double triploid silver crucian carp eggs (AAABBBB), but because silver crucian carp reproduce through parthenogenesis, the sperm does not fuse with the egg nucleus. The Culter alburnus sperm only stimulates egg activation to initiate embryonic development, resulting in offspring that are still silver crucian carp (AAABBBB), not a hybrid.

[0005] Breeding allopolyploids that integrate the genomes of crucian carp and topmouth culter may be an effective way to create multi-trait improved varieties. However, there are currently no successful cases of integrating the two genomes to form normal offspring. 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 sterile allogeneic heptaploid crucian carp with anti-herpesvirus ability.

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

[0008] A method for creating a sterile, allogeneic heptaploid crucian carp with anti-herpesvirus ability includes the following steps:

[0009] S1. Screening of maternal parent: Breeding double triploid female crucian carp with double diploid male crucian carp to obtain offspring G1. Detect the ploidy of offspring G1. If all offspring G1 are double tetraploid, select the corresponding double triploid female crucian carp as the required maternal parent.

[0010] S2. Creation of maintenance line: The maternal parent selected in step S1 lays eggs again, and the resulting eggs are fertilized with ultraviolet-inactivated double diploid crucian carp sperm. After hatching, the double triploid maintenance line offspring G2 is obtained.

[0011] S3. Creation of allogeneic heptaploid crucian carp: Using the female fish in offspring G2 as the maternal parent and the diploid Culter alburnus as the paternal parent, breeding was carried out to obtain offspring G3, which is a sterile allogeneic heptaploid crucian carp with the ability to resist herpesvirus.

[0012] Preferably, in step S1, the female parent of the ditriploid silver carp in gynogenesis is the heterozygous silver carp "Zhongke No. 3".

[0013] Preferably, in steps S1 and S3, the reproduction process involves dry insemination to obtain fertilized eggs, which are then hatched.

[0014] Preferably, in step S1, 50 tails are randomly selected from the offspring G1 to test their ploidy. If all 50 selected tails are ditetraploid, then all offspring G1 are ditetraploid.

[0015] Preferably, in step S1, the method for detecting the ploidy of offspring G1 is as follows: a small amount of blood is taken by cutting off the fin rays of the fish fry, and CyStain DNA1Step solution is added and mixed well. Then, the DNA content of the blood cells of each individual fish is detected by flow cytometry.

[0016] Preferably, in step S2, the ultraviolet inactivation operation is as follows: take double diploid crucian carp semen, dilute the semen with sperm preservation solution at a volume ratio of 1:50, spread the diluted semen in a petri dish, place the petri dish on an ice box, and irradiate it at 20cm under a 15W ultraviolet lamp for 6-8 minutes to obtain ultraviolet-inactivated sperm.

[0017] Compared with the prior art, the advancements of this application are as follows:

[0018] Traditional methods involve directly hybridizing sexually reproduced diploid crucian carp (AABB) with diploid culter fish (CC) or bighead carp (CC), and the offspring (ABC) are all lethal. In their previous research, the inventors' team created a new double triploid population by integrating the genomes of gynogenetic and sexually reproducing crucian carp. They discovered that most of the females in this population regained their ability to reproduce through asexual gynogenesis, while a few females acquired a unique reproductive method (see Lu M, Zhang QC, Zhu ZY, et al. An efficient approach to synthesize sterile allopolyploids through the combined reproduction mode of ameiotic oogenesis and sperm-egg fusion in the polyploidCarassius complex[J]. Science Bulletin, 2023, 68(10):1038-1050. and Lu Meng, Zhou Li, Gui Jianfang, The discovery of the "non-meiotic fusion" reproductive method and its significance in breeding practice[J]. Science Bulletin, 2024, 69(19):2704-2706.). This new double triploid female not only reproduces through asexual gynogenesis... Silver carp The inventors inherited the ability to produce unmeiotic eggs through non-meiosis and also the ability to fuse egg and sperm nuclei to form zygotes, a unique reproductive method they named "non-meiotic fusion" reproduction. The triploid eggs (AAABBB) produced by diploid crucian carp with this characteristic have a higher ploidy than the double haploid eggs (AB) produced by diploid crucian carp. These eggs have higher compatibility with distant paternal genomes, thus this characteristic can be used to create allopolyploid populations. However, current reports related to "non-meiotic fusion" reproduction focus more on fertility control in offspring.

[0019] 1) In this application, based on previous research, the inventors have for the first time successfully integrated the genomes of crucian carp and Culter alburnus, creating an alloheptaploid crucian carp (AAABBBBC), which has overcome the previous problem of incompatibility between distant hybridization of crucian carp and Culter alburnus.

[0020] 2) The inventors discovered for the first time that the Culter alburnus is not infected with crucian carp herpesvirus. Crucian carp herpesvirus infection experiments showed that the Culter alburnus not only possesses extremely strong resistance to crucian carp herpesvirus, but can also inherit this resistance to the created allogeneic heptaploid Culter alburnus, confirming that the Culter alburnus is the key paternal parent for creating a new antiviral crucian carp strain. The allogeneic heptaploid Culter alburnus created in this application exhibits a 96.97% higher resistance to herpesvirus compared to "Zhongke No. 3," and is almost completely unaffected by the virus.

[0021] 3) The allogeneic heptaploid crucian carp created in this application is sterile, and even if it escapes during the breeding process, it will not cause potential ecological risks. At the same time, since its maternal "maintenance line" and paternal "Culter alburnus" are both fertile, effective control over fertility can be achieved in seed production, which is conducive to protecting the intellectual property rights of the aquatic seed industry. Attached Figure Description

[0022] Figure 1 A roadmap for the creation of a sterile allogeneic heptaploid crucian carp with anti-herpesvirus capabilities, provided for this application.

[0023] Figure 2 The images show the morphological features of the allogeneic heptaploid crucian carp and the allogeneic heptaploid bighead carp created in this application.

[0024] Figure 3 To distinguish the chromosomes derived from the maternal crucian carp and the paternal culter or bighead carp in the metaphase of lymphocytes of allogeneic heptaploid crucian carp and allogeneic heptaploid bighead carp using whole-genome in situ hybridization.

[0025] Figure 4 The results are experimental findings on the anti-Carp Herpesvirus CaHV in heterologous heptaploid crucian carp.

[0026] Figure 5 The morphology and section of the gonads of the allogeneic heptaploid crucian carp during the breeding season. Detailed Implementation

[0027] To facilitate understanding, the technical solution of the present invention will be described in more detail below with reference to specific embodiments.

[0028] like Figure 1 As shown, a method for creating a sterile allogeneic heptaploid crucian carp with anti-herpesvirus ability includes the following three stages:

[0029] Phase 1: Screening for triploid crucian carp maternal parents with "non-meiotic fusion" reproductive lines

[0030] A double triploid female crucian carp was bred with a double diploid male crucian carp to obtain offspring G1. The ploidy of offspring G1 was tested. If offspring G1 was double tetraploid, the corresponding double triploid female crucian carp was selected as the desired maternal parent. Specifically, during the breeding season, a group of sexually mature female parents were selected from the double triploid crucian carp population. After artificial spawning, eggs (AAABBB) were collected from each parent and fertilized with sperm (AB) from double diploid crucian carp. The fertilized eggs were hatched separately into fry according to the maternal parent, resulting in offspring G1. The ploidy of 50 fry was randomly tested from each group. If the ploidy of all fry was double tetraploid (AAAABBBB), the double triploid maternal parent corresponding to this batch of fry was determined to have "non-meiotic fusion" reproductive capacity. If the ploidy of all fry was double triploid (AAABBB), the double triploid maternal parent corresponding to this batch of fry was determined to have gynogenetic reproductive capacity. Select a triploid female fish with "non-meiotic fusion" reproductive capacity as the desired maternal parent.

[0031] The specific steps for this stage are as follows:

[0032] S1. Selection of male and female parent fish: During the breeding season, select female triploid crucian carp with swollen and soft abdomens. If the eggs released are plump and dispersed when the vent is gently squeezed, the female fish is selected as the sexually mature parent fish. Similarly, if a large amount of thick, milky-white semen flows out when the vent of the male diploid crucian carp is gently squeezed, the male fish is selected as the sexually mature parent fish.

[0033] S2. Artificial dry fertilization: The female diploid crucian carp selected in S1 were artificially induced to produce mature eggs. The eggs were mixed with the semen of the male diploid crucian carp in a plastic dish and then poured into a porcelain dish with water for fertilization. During the process, the eggs were stirred with a feather to make them evenly adhere to the bottom of the porcelain dish.

[0034] S3. Ploidy detection of offspring fry: Fertilized eggs were hatched into fry and raised for 1 month until they reached a body length of 2.5 cm. 50 fish were randomly selected from each group, and 0.1-0.5 μL of blood was collected from the tail. CyStain DNA 1Step (source: Partec, Germany) solution was added and quickly mixed. The blood cells were rapidly fixed and stained using this solution. The DNA content of the blood cells of each individual fish was then measured by flow cytometry.

[0035] In this phase, following the steps outlined above, a total of 13 sexually mature double triploid female crucian carp and 1 sexually mature double diploid male crucian carp were selected for breeding experiments. The offspring of 11 females were found to be double triploid after ploidy testing, while the offspring of the other 2 females were found to be double tetraploid, representing 15.4% of the females who underwent non-meiotic fusion reproduction.

[0036] Phase Two: Creating a "maintenance line" of "non-meiotic fusion" reproductive triploids

[0037] By controlling water temperature, light, and nutrition, the double triploid crucian carp selected in the first stage of "non-meiotic fusion" reproduction were made to spawn again. Their eggs were then fertilized with ultraviolet-inactivated double diploid crucian carp sperm, and artificial gynogenesis was carried out to expand the population, creating the "non-meiotic fusion" double triploid "maintenance line" G2.

[0038] The specific steps for this stage are as follows:

[0039] S1. Controlling female parent fish to spawn again: The triploid female crucian carp selected in the first stage who underwent "non-meiotic fusion" reproduction were cultured in a rearing system with a water temperature of 20-23℃ and sufficient light. They were fed high-protein crucian carp feed twice daily, along with one feeding of natural animal protein such as bloodworms. The feeding cycle was 15-20 days. If the eggs released from the female's vent were plump and dispersed when gently squeezed, the female was considered capable of spawning again.

[0040] S2. Ultraviolet inactivation treatment of double diploid crucian carp semen: Take double diploid crucian carp semen and dilute it with sperm preservation solution at a volume ratio of 1:50. Add 4ml of diluted semen to a plastic culture dish with a diameter of 10cm. Shake the dish and spread it evenly at the bottom. Then place a horizontal shaker under a 15W ultraviolet lamp and place the culture dish on the shaker with an ice box in the middle to maintain a low temperature. At the same time, keep the semen surface 20cm directly below the lamp tube and irradiate the semen for 6-8 minutes to obtain ultraviolet-inactivated sperm.

[0041] S3. Creating a double triploid "maintenance line" that reproduces without meiosis: Artificial spawning was induced in the double triploid female crucian carp cultured in S1 that reproduced without meiosis. The eggs were mixed with the sperm treated in S2 in a plastic dish and then poured into a water-filled porcelain dish for fertilization. After hatching, the resulting fertilized eggs were used to obtain the double triploid "maintenance line" fry G2 that reproduced without meiosis.

[0042] Based on the first phase of the experiment, one female was selected from two diploid crucian carp that underwent non-meiotic fusion reproduction. This female was then fertilized with UV-inactivated diploid crucian carp sperm to obtain approximately 2,000 artificial gynogenetic "maintenance lines" G2. The offspring of these diploid "maintenance lines" still possess the ability to reproduce without meiosis.

[0043] Phase 3: Creating a sterile alloheptaploid population

[0044] Using the "maintenance line" of a new double triploid broiler produced without meiosis as the maternal parent and the diploid Culter alburnus as the paternal parent, an allopolyploid was created. The specific operation was as follows:

[0045] The G2 individuals obtained in the second stage were cultured until sexual maturity. After artificial induction of spawning in the sexually mature individuals, their eggs were fertilized with sperm from the Chinese crucian carp using the dry insemination method. The resulting fertilized eggs were then incubated to produce offspring G3, thus obtaining allogeneic heptaploid Chinese crucian carp.

[0046] In this stage, 20 individuals were randomly selected from the artificial gynogenetic "maintenance line" G2 prepared in the second stage, and their eggs were fertilized with the sperm of 3 Culter alburnus. The average fertilization rate and embryo hatching rate of fertilization with Culter alburnus sperm were 90.47% ± 1.79% and 97.23% ± 0.68%, respectively.

[0047] Comparative Example

[0048] Selecting distantly related diploid bighead carp as the male parent, and following the steps of the third stage above, using the new double triploid "maintenance line" of "non-meiotic fusion" as the female parent, dry insemination was performed between its eggs and bighead carp sperm. The resulting fertilized eggs were hatched, and the offspring G3 were hatched to obtain allogeneic heptaploid bighead carp.

[0049] Twenty individuals were randomly selected from the artificial gynogenetic "maintenance line" G2 prepared in the second stage, and their eggs were fertilized with the sperm of three bighead carp. The average fertilization rate and embryo hatching rate of fertilization with bighead carp sperm were 85.62% ± 2.46% and 97.49% ± 0.19%, respectively.

[0050] See Figure 2 The allogeneic heptaploid crucian carp and the allogeneic heptaploid bighead carp basically maintain the overall shape of crucian carp, with only the body color and mouth shape being affected by the paternal parent.

[0051] experiment

[0052] 1. Chromosome analysis

[0053] Chromosomes derived from the maternal crucian carp and paternal topmouth culter / bighead carp were distinguished in the metaphase of lymphocytes from G3 generation alloheptaploid crucian carp and alloheptaploid bighead carp using whole-genome in situ hybridization. Results are shown in [link to results]. Figure 3 .from Figure 3 As shown in Figure A, the total number of chromosomes in the alloheptaploid *Culter alburnus* is approximately 174. The thin arrows in the figure indicate chromosomes from the maternal doubly triploid *Culter alburnus*, which exhibit strong green fluorescence only at the centromere, totaling approximately 150 chromosomes. The thick arrows indicate chromosomes from the paternal diploid *Culter chinensis*, which are uniformly covered with red fluorescence from head to tail, totaling 24 chromosomes. This result confirms that the chromosome set (AAABBBC) of the alloheptaploid *Culter alburnus* consists of the complete chromosome set from the maternal doubly triploid *Culter alburnus* (AAABBB) and one chromosome set from the paternal *Culter chinensis* (C). Similarly, from... Figure 3As shown in Figure B, the chromosomes derived from the maternal crucian carp and paternal bighead carp are present in the metaphase of the lymphocytes of the alloheptaploid bighead carp. This indicates that the experiment successfully created alloheptaploid crucian carp and alloheptaploid bighead carp.

[0054] 2. Crucian carp herpesvirus infection experiment

[0055] Artificial infection experiments with CaHV herpesvirus were conducted on the obtained G3 generation allogeneic heptaploid crucian carp and allogeneic heptaploid bighead carp to verify their antiviral properties. The experimental procedure is as follows:

[0056] S1. Preparation of experimental fish to be challenged: Allogeneic heptaploid crucian carp, allogeneic heptaploid bighead carp and control group allogeneic silver crucian carp "Zhongke No. 3", double triploid "maintenance line", and fry of Culter alburnus and bighead carp were raised to a size of about 20g under the same environment. After being brought back to the laboratory, the culture water temperature was gradually transitioned to 23℃ (±1℃) and the fish were raised for two weeks and observed to confirm that there were no abnormalities in the experimental fish.

[0057] S2. Intraperitoneal injection of virus: The four groups of experimental fish were randomly divided into three parallel groups, totaling 18 groups of fish, with approximately 20-30 fish in each group, and were injected simultaneously. The volume of CaHV filtrate injected was 0.1 ml per 10 g body weight, and the viral load in the filtrate was 2.9 × 10⁻⁶. 8 One herpesvirus / mL, injected into the abdominal cavity from the base of the pectoral fin.

[0058] S3. Evaluation of disease resistance of each clonal line: After virus injection, the culture water temperature was maintained at 23℃ (±1℃), and the water was changed daily to keep the water clean. Dead individuals were removed promptly. The survival rate of the 18 groups of fish after intraperitoneal injection was recorded daily, with an infection period of 21 days. The average survival rate of the three parallel groups corresponding to each experimental group was calculated.

[0059] The results are as follows Figure 4 As shown. Figure 4 Figure A shows the experimental results of the allogeneic heptaploid crucian carp against crucian carp herpesvirus CaHV created in this application. In the control group, the allogeneic silver crucian carp "Zhongke No. 3" and the double triploid "maintenance line" all died within 9 days after being injected with the virus (the average survival rate was 0%). The Culter alburnus did not die after being injected with the virus, while the survival rate of the allogeneic heptaploid crucian carp after being injected with the virus was as high as 96.97%, indicating that the integration of the Culter alburnus genome significantly improved the disease resistance of the allogeneic heptaploid crucian carp. Figure 4 Figure B shows the experimental results of the alloheptaploid bighead carp strain created in this application resisting crucian carp herpesvirus (CaHV). In the control group, bighead carp did not die after being injected with the virus, while all alloheptaploid bighead carp died after injection, indicating that bighead carp are not infected with crucian carp herpesvirus. However, the integration of the bighead carp genome cannot enhance the disease resistance of the alloheptaploid bighead carp strain. In other words, the bighead carp's resistance to herpesvirus cannot be inherited by the alloheptaploid bighead carp strain, making it highly susceptible to herpesvirus infection. Therefore, the Culter alburnus is the key paternal parent for creating a new antiviral crucian carp strain.

[0060] 3. Fertility testing experiment

[0061] like Figure 5 As shown, during the breeding season, the gonads of 100 allogeneic heptaploid crucian carp G3 were dissected and histologically examined. The results showed that none of the tested individuals had any mature eggs in their ovaries or mature sperm in their testes, confirming that allogeneic heptaploid crucian carp are infertile.

[0062] The above experiments demonstrate that the breeding route provided in this application can create new crucian carp strains that are sterile and have significant resistance to crucian carp herpesvirus, thus making it an important approach for breeding new crucian carp varieties.

[0063] 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 foregoing 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 within the protection scope of the present invention.

Claims

1. A method for creating a sterile allogeneic heptaploid crucian carp with anti-herpesvirus ability, characterized in that, Includes the following steps: S1. Screening of maternal parent: Breeding double triploid female crucian carp with double diploid male crucian carp to obtain offspring G1. Detect the ploidy of offspring G1. If all offspring G1 are double tetraploid, select the corresponding double triploid female crucian carp as the required maternal parent. S2. Creation of maintenance line: The maternal parent selected in step S1 lays eggs again, and the resulting eggs are fertilized with ultraviolet-inactivated double diploid crucian carp sperm. After hatching, the double triploid maintenance line offspring G2 is obtained. S3. Creation of allogeneic heptaploid crucian carp: Using the female fish in offspring G2 as the maternal parent and the diploid Culter alburnus as the paternal parent, breeding was carried out to obtain offspring G3, which is a sterile allogeneic heptaploid crucian carp with the ability to resist herpesvirus.

2. The method for creating a sterile allogeneic heptaploid crucian carp with anti-herpesvirus ability as described in claim 1, characterized in that, In step S1, the female parent of the ditriploid silver carp in gynogenesis is the heterozygous silver carp "Zhongke No. 3".

3. The method for creating a sterile allogeneic heptaploid crucian carp with anti-herpesvirus ability as described in claim 1, characterized in that, In steps S1 and S3, fertilization is carried out using dry insemination to obtain fertilized eggs, which are then hatched.

4. The method for creating a sterile allogeneic heptaploid crucian carp with anti-herpesvirus ability as described in claim 1, characterized in that, In step S1, 50 tails are randomly selected from the offspring G1 to test their ploidy. If all 50 tails selected are ditetraploid, then all offspring G1 are ditetraploid.

5. The method for creating a sterile allogeneic heptaploid crucian carp with anti-herpesvirus ability as described in claim 1, characterized in that, In step S1, the method for detecting the ploidy of offspring G1 is as follows: a small amount of blood is taken by cutting off the fin rays of the fish fry, and CyStain DNA 1Step solution is added and mixed well. Then, the DNA content of blood cells of each individual fish is detected by flow cytometry.

6. The method for creating a sterile allogeneic heptaploid crucian carp with anti-herpesvirus ability as described in claim 1, characterized in that, In step S2, the ultraviolet inactivation operation is as follows: take double diploid crucian carp semen, dilute the semen with sperm preservation solution at a volume ratio of 1:50, spread the diluted semen in a petri dish, place the petri dish on an ice box, and irradiate it at 20cm under a 15W ultraviolet lamp for 6-8 minutes to obtain ultraviolet-inactivated sperm.

Images