Grass carp excellent family fast passage method

Abstract

This invention provides a method for rapid propagation of superior grass carp families, belonging to the field of aquaculture and fish genetic breeding technology. The method includes: screening for disease-resistant superior families from established grass carp families; isolating and enriching reproductive stem cells from these superior families; transplanting the grass carp family reproductive stem cells into the abdominal cavity or genital ridge region of recipient fish with short sexual maturity cycles; cultivating the recipient fish to sexual maturity, producing sperm, and then fertilizing the sperm with grass carp eggs to obtain F1 generation offspring; performing molecular identification on the offspring to confirm that their genetic material originates from the donor family. This invention can shorten the propagation cycle of superior grass carp families from 4-5 years to approximately 1.5 years, achieving precise and efficient transfer of superior traits, and providing an effective technical means to shorten the grass carp breeding cycle and cultivate superior grass carp varieties.

Description

Technical Field

[0001] This invention relates to the fields of aquaculture and fish genetic breeding technology, and in particular to a method for rapid propagation of superior grass carp families. Background Technology

[0002] Grass carp (Ctenopharyngodon Idella), belonging to the class Osteichthyes, family Cyprinidae, subfamily Leuciscinae, and genus Ctenopharyngodon, is one of the core economic fish species in my country's freshwater aquaculture. Due to its delicious flesh, rich nutrition, and low farming costs, it boasts the largest farming scale among freshwater fish and occupies an irreplaceable position in the aquaculture industry. With the large-scale and intensive development of the aquaculture industry, grass carp farming faces problems such as frequent disease outbreaks and low efficiency in selecting superior varieties. Therefore, the targeted selection and fixation of superior traits such as disease resistance and rapid growth have become crucial for improving the efficiency of grass carp farming and ensuring the healthy development of the industry.

[0003] Traditional grass carp breeding relies on natural sexual maturation and reproduction, but the natural sexual maturation cycle of grass carp is as long as 4-5 years, resulting in an extremely long breeding cycle: it usually takes about 20 years to breed four generations of grass carp, and it takes more than 22 years to cultivate a new grass carp variety with stable and excellent traits. This situation seriously restricts the rapid aggregation of excellent traits in grass carp and the speed of variety improvement, making it difficult to meet the urgent needs of the aquaculture industry for disease-resistant and high-yielding grass carp varieties. Therefore, developing an efficient and controllable rapid propagation breeding technology for superior grass carp families to shorten the sexual maturation and breeding cycle has become an important research direction in the field of grass carp genetic breeding.

[0004] In recent years, reproductive stem cell transplantation technology has provided a novel approach to fish genetic breeding. The core principle of this technology is to transplant reproductive stem cells from a donor fish into a recipient fish, utilizing the microenvironment of the recipient's gonads to induce the proliferation and differentiation of the donor stem cells, ultimately producing functional gametes carrying the donor's genetic information. This has enabled cross-species reproductive stem cell transplantation and functional gamete production. However, current research remains at the stage of verifying the technical principles and preserving fish genetic resources under extreme conditions. A standardized breeding application system has not yet been established, and there are no reports of applying this technology to the rapid propagation and passage of grass carp reovirus (GCRV) resistant families. Therefore, it cannot solve the industry's pain points of long breeding cycles and low efficiency in transferring superior traits in grass carp.

[0005] Based on this, in response to the technical deficiencies and industry demands of traditional grass carp breeding, it is urgent to develop a rapid propagation method specifically for superior grass carp families resistant to grass carp reovirus (GCRV) by relying on reproductive stem cell transplantation technology. This method is of great significance for achieving accurate and rapid transmission of superior genetic background in grass carp and shortening the breeding cycle. Summary of the Invention

[0006] In view of this, the present invention provides a method for rapid propagation of superior grass carp families, overcoming the shortcomings of existing grass carp breeding techniques, such as long cycles and low efficiency. This method provides a rapid propagation method for grass carp families based on reproductive stem cell transplantation. This method can transfer the superior traits of a target superior grass carp family (GCRV-resistant grass carp family) to the next generation within a few months, thereby shortening the cycle of completing a generation in traditional breeding from several years to approximately 1.5 years, greatly accelerating the breeding process.

[0007] This method involves separating reproductive cells from superior grass carp families with long sexual maturation cycles and transplanting them into recipient fish with short sexual maturation cycles. This allows for the rapid generation of sperm from the superior grass carp families in a short period of time, enabling the rapid propagation of superior grass carp families and significantly shortening the propagation time. This method has significant application value.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] A method for rapid propagation of superior grass carp pedigrees includes the following steps:

[0010] (1) Screening of disease-resistant and fast-growing superior strains of grass carp

[0011] More than 120 grass carp families were constructed, and at 6 months of age, they were administered GCRV via intraperitoneal injection (dosage: 10). 7 Infect juvenile fish with RNA copies / 100μL, injection volume 20 μL, challenge duration 150 h, and screen out disease-resistant families with high survival rates.

[0012] (2) Obtaining germline stem cells from grass carp donors

[0013] Gonadal tissue was obtained from superior grass carp families that were screened and verified and then bred to 11 months of age (body length 25-30 cm). The tissue was then dissociated into a single-cell suspension by enzymatic digestion and the reproductive stem cells were enriched by density gradient centrifugation.

[0014] (3) Preparation of recipient fish: Collect wild-type AB strain zebrafish fertilized eggs, microinject antisense oligonucleotides of the dnd1 gene during the single-cell stage to knock out their endogenous primordial germ cells, and obtain recipient juveniles with germ cell deficiency after culturing for 4-5 days.

[0015] (4) Transplantation of reproductive stem cells

[0016] The donor grass carp reproductive stem cells enriched in step (2) were transplanted into the gonadal region of the recipient juvenile fish using microinjection technology.

[0017] (5) Recipient fish culture and artificial insemination

[0018] After transplantation, the recipient fish were cultured under suitable conditions for about 3 months until sexual maturity. Sperm from the selected recipient fish was collected and artificially inseminated with common grass carp eggs to obtain F1 generation offspring. (6) Offspring acquisition and identification

[0019] Embryos obtained after fertilization develop into larvae, i.e., F1 generation. Through morphological observation and molecular identification (such as species identification using grass carp species-specific DNA markers and pedigree analysis through simplified genome sequencing), it is confirmed that the F1 generation individuals are grass carp whose genetic material is entirely derived from the donor pedigree, and positive recipients are screened out.

[0020] By adopting the above technical solution, the present invention has the following beneficial effects:

[0021] (1) Significantly shorten the cycle of sexual maturity and breeding of grass carp families

[0022] This invention uses zebrafish, whose sexual maturity cycle is only about 3 months, as the recipient. Leveraging the rapid sexual maturation of the recipient fish, the transplanted grass carp reproductive stem cells rapidly proliferate and differentiate into functional gametes under the regulation of the recipient's gonadal microenvironment, significantly shortening the generation cycle. This invention completes GCRV challenge screening and confirms superior families at 6 months of age. The donor grass carp are then further cultured until 11 months of age for gonadal extraction and reproductive stem cell transplantation. This highly efficient coupling with the recipient fish's approximately 3-month sexual maturity cycle allows for the completion of a full genetic generation of grass carp within 1.5 years, shortening the traditional 4-5 year breeding cycle by more than 70%, greatly accelerating the fixation of superior traits in grass carp.

[0023] (2) Realize the industrialization of technical principles into breeding platforms and solve the problem of application implementation.

[0024] Existing research on cross-species reproductive stem cell transplantation mainly focuses on verifying technical principles and preserving genetic resources in extreme scenarios, and has not yet formed an industrialized breeding application system. The core innovation of this invention lies in the first-ever creative development of this technology into a standardized and reproducible breeding operation platform, specifically adapted for the rapid passage and propagation of superior traits in live grass carp families. This promotes the technology from laboratory principles to practical applications in aquaculture breeding, solving the "last mile" problem of industrialization and providing a new, efficient, and routine technical tool for the field of aquaculture breeding.

[0025] (3) To achieve accurate and faithful transmission of superior traits

[0026] This invention directly uses grass carp families with resistance to grass carp reovirus (GCRV) as donors, whose reproductive stem cells carry all the superior genetic information of the donor family. Through transplantation and gamete formation, the superior genetic background of the donor can be completely and faithfully transmitted to the F1 generation, effectively avoiding the problems of trait segregation and genetic information dilution that are prone to occur in traditional hybridization breeding, and greatly improving the accuracy and efficiency of targeted breeding such as disease resistance in grass carp.

[0027] (4) The standardized operating system ensures a high breeding success rate.

[0028] This invention establishes an operational system based on a mature zebrafish experimental model and validated reproductive stem cell transplantation technology. The overall method steps are clear and the process is controllable. The success rate of reproductive stem cell transplantation and the rate of obtaining functional gametes are both stably guaranteed, which can meet the needs of large-scale and standardized breeding applications.

[0029] (5) Highly compatible with cutting-edge biotechnology and adapted to the development trend of precision breeding.

[0030] The method of this invention can be seamlessly integrated with cutting-edge breeding technologies such as whole-genome selection and molecular marker-assisted selection to form a synergistic breeding system: firstly, genomic selection can be carried out on grass carp families through genomic breeding value calculation to accurately screen out superior individuals, and then a large number of offspring carrying superior traits can be quickly obtained by relying on this invention. It is an ideal technical platform for the future precision breeding of grass carp. Attached Figure Description

[0031] Figure 1 This is a flowchart of the method for rapid propagation of superior grass carp families according to the present invention.

[0032] Figure 2 Electrophoresis images for species identification of F1 offspring, in which Figure 2 In the diagram, A represents the PCR amplification results of genomic DNA from fry produced by recipient sperm using grass carp-specific primers (gc_F1 / R2). Lanes (1-6) contain fry samples produced by sperm from different zebrafish recipient individuals. Lanes (1-6) show a 290bp specific band consistent with the grass carp positive control (+). (M) represents the DNA marker, (-) represents the donor zebrafish sample, and B represents the blank control. Figure 2 In the diagram, B represents the PCR amplification results of genomic DNA from fish fry produced by recipient sperm using zebrafish-specific primers (nr5a1a). Lanes (1-6) represent fish fry samples produced by sperm from different zebrafish recipient individuals. Lanes (1-6) show no bands. Lane (+) represents zebrafish recipients, which produced a specific band of 428 bp. Lane (M) represents the DNA marker. Lane (-) represents wild grass carp samples. Lane B represents the blank control.

[0033] Figure 3These are photos of grass carp F1 generation fry obtained through artificial insemination, whose genetic material originates from the donor family. A is 5 days old, and B is 10 days old.

[0034] Figure 4 This is a principal component analysis (PCA) diagram of family genetic structure based on genome-wide SNP markers. Detailed Implementation

[0035] In the following examples, if the specific technical requirements or conditions for the reagents or instruments used are not specified, they were tested under standard experimental conditions. If no reagent company instructions are explicitly provided, the conditions recommended in those instructions were followed. All reagents or instruments whose manufacturers are not specified are commercially available, standard products.

[0036] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] I. Donor Screening

[0039] Grass carp from different families aged 6 months were challenged with grass carp reovirus (GCRV) at a dose of 10. 7 RNA copies / 100 μL, 20 μL / tail. Survival rate was calculated 150 h after injection. Superior families resistant to grass carp reovirus (GCRV) were selected. The survival rate of family 14 was 76.00%, and that of family 6 was 64.81% (see patent application number 202511208945.7). Families 14 and 6 were selected as donors. In this embodiment, when the grass carp were further cultured to 11 months of age, healthy individuals with a body length of 25-30 cm were selected from the verified superior families. Under aseptic conditions, they were dissected, and approximately 20 mg of testis or ovarian tissue was collected.

[0040] II. Isolation and Enrichment of Reproductive Stem Cells

[0041] Gonadal tissue was digested at 33°C for 2 hours using a mixed enzyme solution containing 0.25% trypsin, 300 U / mL collagenase, and 0.05% DNase I to prepare a single-cell suspension. Using Percoll density gradient media, separation solutions with concentrations of 30% and 40% were prepared. After centrifugation at 800g for 30 min, high-purity grass carp reproductive stem cells were separated and collected at the 30% and 40% concentration interface. The cells were labeled with CM-Dil fluorescent dye for later use.

[0042] III. Receptor Preparation

[0043] Wild-type AB strain zebrafish fertilized eggs were collected, and the antisense oligonucleotide 5′-GCTGGGCATCCATGTCTCCGACCAT-3′ (SEQ ID NO.1) of the dnd1 gene was microinjected during the single-cell stage to knock out their endogenous primordial germ cells. After culturing for 4-5 days, recipient larvae with germ cell deficiency were obtained.

[0044] IV. Stem Cell Transplantation

[0045] Approximately 30-50 labeled donor grass carp reproductive stem cells were microinjected into the abdominal cavity of recipient juvenile fish lacking reproductive cells, near the genital ridge. The juvenile fish were then cultured normally after transplantation.

[0046] V. Receptor Culture and Artificial Insemination

[0047] The recipient fish were reared at 28.5℃ for approximately 3 months until sexual maturity. Sperm was collected by gently pressing the abdomen, and mature grass carp eggs were retrieved. Dry artificial insemination was performed at a sperm-to-egg ratio of 1:1000. The fertilized eggs were incubated at a water temperature of 22-25℃ to obtain the F1 generation offspring.

[0048] VI. Species identification and pedigree identification of offspring

[0049] 1. Species identification: To confirm the origin of the genetic material, grass carp-specific DNA sequences were used for identification, as shown in SEQ ID NO.2:

[0050]

[0051] Amplification was performed using primers for detecting the above fragment. The primer sequences are as follows:

[0052] Forward primer gc_F1: 5'-ATTATGTCACTGCGGAGGC-3' (SEQ ID NO.3);

[0053] Reverse primer gc_R1: 5'-TGGAGGATGACAGCAGACCT-3' (SEQ ID NO.4).

[0054] The PCR amplification reaction system is as follows: 2×PCR mix (12.5μL), forward primer (10μM) 1μL, reverse primer (10μM) 1μL, DNA template (100ng / μL) 1μL, pure water 9.5μL, and the total volume is 25μL.

[0055] The PCR amplification conditions were as follows: 98℃ for 30s; 98℃ for 10s, 60℃ for 10s, 72℃ for 10s, 30 cycles; 72℃ for 5min.

[0056] Meanwhile, zebrafish-specific gene primers were used as a negative control for verification. The primer sequences are as follows:

[0057] Forward primer zf-nr5a1a-F: 5'-GATGAGAGGTGGAAGAAATAAG-3' (SEQ ID NO.5);

[0058] Reverse primer zf-nr5a1a-R: 5'-GCTTCATACCTGCTCCT-3' (SEQ ID NO.6).

[0059] Fin rays or muscle tissue from F1 generation larvae were collected, and whole-genome DNA was extracted. PCR was performed using primers gc-F1 / R1 on the genomes of wild grass carp, recipient zebrafish, and larvae obtained from transplanted recipient semen. Figure 2 The results showed that both wild grass carp and recipient sperm-derived fry could amplify the target band, while the zebrafish genome showed no band at all.

[0060] At the same time, such as Figure 2 As shown in B, all offspring samples showed no bands when amplified using zebrafish-specific primers, proving that the fertilization process excluded genetic interference from the host recipient fish, and the resulting offspring were purebred grass carp. By comparing the above amplification results, positive recipient individuals were selected for propagation.

[0061] 2. Patriarchal Identification: To confirm that the F1 generation offspring accurately inherited the genetic material of the donor family, this embodiment employed high-throughput sequencing technology for pedigree analysis. Representative individuals (5 offspring from each of families 6 and 14) were randomly selected from the F1 population obtained using this method, and their genomic DNA was extracted and resequencing. The sequencing data was then combined with genomic variation data from a grass carp pedigree germplasm resource bank constructed in the laboratory (containing adult fish populations from families 6 and 14, as well as 718 samples from other background families), and a joint principal component analysis (PCA) was performed. The results are as follows: Figure 4 As shown.

[0062] Figure 4 Cluster analysis showed that the method of this invention achieves efficient transmission of superior pedigrees. In the figure, pedigree 14 (red) and pedigree 6 (blue) are represented, with gray background dots representing the mixed background of the remaining 19 pedigrees. Triangles represent family representatives, circles represent the F1 offspring obtained through rapid transmission using the method of this invention, and shaded areas represent the 95% confidence ellipse. The results show that the F1 offspring (circles) obtained by this method are closely distributed around their respective family representatives (triangles) and fall entirely within the 95% confidence ellipse. This confirms at the whole-genome level that the offspring and the donor pedigree belong to the same genetic lineage, with no significant genetic drift or exogenous contamination. This demonstrates that this technology can stably transmit the genetic characteristics of a specific pedigree to offspring with high fidelity.

[0063] Through the above steps, this invention successfully transmitted the genetic background of grass carp resistant to GCRV to the F1 generation within approximately 1.5 years, completing the rapid propagation of this superior family, shortening the breeding cycle by more than 70% compared to traditional methods. This demonstrates the remarkable effectiveness of this method in rapidly fixing and propagating complex stress resistance traits, and it can be used for the simultaneous and rapid propagation and selection of multiple superior families, possessing enormous potential for industrial application.

[0064] As can be seen from the above embodiments, the present invention provides a method for rapid propagation of superior grass carp families. This method enables the precise and rapid transmission of superior genetic background in grass carp, shortens the breeding cycle, and provides efficient technical support for the breeding of superior grass carp breeds.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for rapid propagation of superior grass carp families, characterized in that, Includes the following steps: (1) Screening for disease-resistant superior strains of grass carp; (2) Obtaining reproductive stem cells from superior grass carp families: Gonadal tissue was obtained from selected disease-resistant grass carp families, and reproductive stem cells were isolated and enriched. (3) Preparation of recipient fish: Wild-type AB strain zebrafish fertilized eggs were collected and microinjected during the single-cell stage. dnd1 Antisense oligonucleotides of genes, knocking out their endogenous primordial germ cells; (4) Grass carp reproductive stem cell transplantation: The donor grass carp reproductive stem cells obtained in step (2) are transplanted into the recipient juvenile fish that lacks reproductive cells. (5) Recipient fish cultivation and artificial insemination: The recipient fish after transplantation are cultivated until sexual maturity, and artificial insemination is performed using the sperm produced by the recipient fish and the eggs of ordinary grass carp to obtain F1 generation offspring; (6) Progeny molecular identification: The F1 generation progeny is identified using species-specific molecular markers to confirm that its genetic material comes from the grass carp donor family, thereby retrospectively screening for positive recipients. The species-specific molecular marker is a grass carp-specific DNA sequence, the nucleotide sequence of which is shown in SEQ ID NO.

2.

2. The method according to claim 1, characterized in that, The target grass carp pedigree is a pedigree that is resistant to grass carp reovirus.

3. The method according to claim 1, characterized in that, The recipient fish is a zebrafish.

4. The method according to claim 3, characterized in that, The nucleotide sequence of the antisense oligonucleotide is shown in SEQ ID NO.

1.

5. The method according to claim 1, characterized in that, In step (4), the transplantation site is the abdominal cavity or genital ridge region of the recipient fish.

6. The method according to claim 1, characterized in that, In step (6), the molecular identification includes the following two steps: First, the species of the F1 generation offspring is identified by using grass carp-specific molecular markers, and the 290bp target band is amplified by PCR to confirm that the fish fry originated from the donor grass carp; Second, the offspring fish fry are genotyped by simplified genome sequencing technology, and the kinship is analyzed with a reference population containing the donor family lineage to confirm the genetic origin of the offspring.

7. The method according to claim 6, characterized in that, The nucleotide sequences of the primers used to amplify the species-specific molecular markers of grass carp are shown in SEQ ID NO.3 and SEQ ID NO.

4.

8. The application of the method according to any one of claims 1 to 7 in shortening the grass carp breeding cycle and / or producing high-yield, disease-resistant grass carp families.

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