Method for cultivating GCRV-resistant grass carp strain based on artificial gynogenesis

Abstract

The invention relates to the technical field of fish disease-resistant breeding, in particular to a method for cultivating a GCRV (Growth CRV Virus)-resistant grass carp strain based on artificial gynogenesis. Comprising the following steps: (1) selection and cultivation of parents; (2) artificial spawning induction; (3) inducing gynogenesis; (4) resistance verification: carrying out a grass carp reovirus attack test on the gynogenetic grass carp fry to verify the GCRV resistance of the gynogenetic grass carp fry; and (5) breeding and expanding propagation: selecting the screened gynogenetic grass carp resistant individuals, cultivating the gynogenetic grass carp resistant individuals as core parents, and applying the core parents to subsequent breeding and expanding propagation of GCRV resistant grass carp groups. The GCRV-resistant grass carp strain cultivated by the method has the advantages of stable resistance, excellent growth and the like, the corresponding cultivation method has the characteristics of short period, high efficiency, strong operability and the like, the problem of grass carp hemorrhagic disease prevention and control can be effectively solved, the healthy and sustainable development of the grass carp breeding industry is guaranteed, and the method has great economic value and social significance.

Description

Technical Field

[0001] This invention relates to the field of fish disease-resistant breeding technology, and in particular to a method for cultivating GCRV-resistant grass carp strains based on artificial gynogenesis. Background Technology

[0002] grass carp( Ctenopharyngodon idella Grass carp is one of the most important freshwater farmed fish in my country, characterized by rapid growth, high feed conversion rate, and good meat quality, making it an important source of animal protein in the daily diet of residents. According to data from the *China Fisheries Statistical Yearbook (2024)*, my country's grass carp production reached 5.9413 million tons in 2023, accounting for 17.4% of the total freshwater farmed fish production. However, with the expansion of farming scale and the increase in intensification, viral diseases have become a major bottleneck restricting the healthy development of grass carp farming. Among them, grass carp hemorrhagic disease caused by Grass Carp Reovirus (GCRV) is characterized by rapid onset, high infectivity, and high mortality. It has been listed as a Class II animal disease by the Ministry of Agriculture and Rural Affairs, causing huge economic losses to the grass carp farming industry every year and seriously threatening the industry's security.

[0003] GCRV is a double-stranded RNA virus, and based on its genome sequence and biological characteristics, it can be divided into three genotypes: GCRV-I, GCRV-II, and GCRV-III. Among them, GCRV-II is currently the most prevalent strain in grass carp farming areas in my country, especially prone to large-scale outbreaks in one-year-old grass carp, characterized by a short disease course and high difficulty in control. Due to the lack of understanding of GCRV... The infection mechanism of GCRV-II remains unclear, and currently, there is a lack of highly effective control measures. Existing control measures mainly rely on conventional methods such as vaccination, water disinfection, and feed additives. However, these methods have problems such as short protection periods, unstable effects, and environmental pollution risks, and cannot fundamentally solve the disease problem. In terms of disease-resistant breeding, traditional methods such as family selection and population selection require multiple generations of continuous breeding, which is time-consuming (usually nearly 20 years), inefficient, and difficult to accurately locate and fix disease-resistant genes. Although molecular marker-assisted selection breeding has made some progress, because GCRV resistance is a complex trait controlled by multiple genes, no stable major disease-resistant gene markers have been identified to date, which cannot meet the needs of breeding practice. Therefore, creating new grass carp germplasm with stable GCRV resistance at the genetic level to achieve "disease resistance through breeding" is a key way to solve the problem of grass carp hemorrhagic disease control and ensure the sustainable development of the industry.

[0004] Artificial gynogenesis is an important technique in fish genetic breeding. Its core principle is to activate oocytes through genetically inactivated xenospermia, initiating development, followed by chromosome doubling to obtain diploid offspring that primarily inherit maternal genetic material. This technique not only rapidly fixes desirable traits and shortens the breeding cycle, but more uniquely, although genetically inactivated xenospermia loses its complete fertilization capacity, the trace genetic material it produces can still be integrated into the genome of gynogenic offspring, thus conferring new genetic characteristics and achieving trait improvement. This phenomenon is known as the "xenospermia effect." Previous studies have confirmed that paternal microsatellite markers can be detected in grass carp undergoing gynogenesis induced by inactivated xenospermia, and these individuals show significant differences in expression along innate immune pathways. However, existing technologies have not yet conducted systematic research on GCRV-II resistance in grass carp: on the one hand, existing technologies have not accurately screened heterologous sperm donors suitable for the development of gynogenesis in grass carp, and lack assessment of donor genetic diversity and disease resistance potential; on the other hand, a complete technical system of "heterosperm induction-resistance screening-genetic analysis-strain propagation" has not yet been established, which makes it impossible to apply the technology to production breeding.

[0005] Therefore, based on artificial gynogenesis technology, the introduction of heterologous sperm with high genetic diversity to induce grass carp to produce gynogenic offspring, combined with virus challenge tests to screen resistant individuals, has led to the cultivation of a stable GCRV-resistant grass carp strain, providing technical support for the genetic prevention and control of grass carp hemorrhagic disease. Summary of the Invention

[0006] This invention provides a method for cultivating GCRV-resistant grass carp strains based on artificial gynogenesis. Its purpose is to address the shortcomings of existing technologies, such as the lack of systematic research on introducing heterologous genetic material through artificial gynogenesis to enhance grass carp resistance to GCRV-II, and the absence of a technical system applicable to production breeding. This invention fills a gap in the technical field.

[0007] To achieve the above objectives, this invention provides a method for cultivating GCRV-resistant grass carp strains based on artificial gynogenesis, comprising the following steps: (1) Selection and breeding of parent stock: Select male black carp population with high genetic diversity as heterologous sperm donors, and select female grass carp population with fast growth rate and sexual maturity as maternal stock, and carry out standardized breeding of the parent stock; (2) Artificial spawning: Male black carp and female grass carp were injected with spawning hormones to induce sperm and egg release, and sperm and eggs were obtained; (3) Gynogenesis induction: Collect the semen of the male black carp, dilute it and inactivate it by ultraviolet irradiation; mix the inactivated sperm with the mature eggs of female grass carp to form fertilized eggs, subject the fertilized eggs to cold shock treatment to induce chromosome doubling, and obtain gynogenic grass carp seedlings; (4) Screening of resistant individuals: GCRV-resistant gynogenetic grass carp fry were screened from the gynogenetic grass carp fry. (5) Breeding and propagation: Selected female-developed grass carp resistant individuals are cultivated and used as core parents for subsequent breeding and propagation of GCRV resistant grass carp populations.

[0008] The selection of heterologous sperm is a crucial step in the successful development of artificial gynogenetic cells, directly affecting the efficiency of gynogenetic development and the potential for phenotypic improvement in offspring. The selection and cultivation of heterologous sperm donors must adhere to the principle of "prioritizing genetic diversity and matching disease resistance potential," and must simultaneously meet the following conditions: First, they must possess rich genetic diversity, providing a sufficient genetic variation basis for the heterosperm effect; second, they must possess strong disease resistance and stress resistance potential, and the resistance gene fragments contained in their genetic material can influence grass carp offspring through the heterosperm effect.

[0009] Black carp ( Cyprinus carpio *Culter alburnus* var. *quanzhounensis*, native to Guilin, Guangxi, is a variant of carp developed through long-term rice paddy domestication. Due to its long-term exposure to natural populations and low levels of artificial selection, *Culter alburnus* exhibits significantly higher genetic diversity than other carp varieties. It also possesses strong tolerance to low oxygen levels, stress resistance, and disease resistance, providing a richer genetic variation base for gynogenic offspring of grass carp, thus facilitating the selection and fixation of resistance-related desirable traits. Furthermore, previous experiments have confirmed that distant hybridization between *Culter alburnus* and grass carp fails to produce viable offspring. Therefore, surviving individuals obtained by inducing grass carp egg development using ultraviolet-inactivated *Culter alburnus* semen can be confirmed as gynogenic offspring, eliminating the need for additional genetic testing, simplifying the technical process, and improving breeding efficiency.

[0010] Preferably, the selection of heterologous sperm donors in step (1) is as follows: male black carp are sourced from aquatic breeding farms or natural high-quality waters, and their genetic diversity is assessed by whole-genome next-generation sequencing with a sequencing depth of 30× to 60×; the heterozygosity of the genome is assessed by K-mer distribution frequency analysis, with the K-mer length set to 17-21 bp, and the heterozygosity of the selected male black carp is not less than 0.80%. The heterozygosity of koi is usually 0.40% to 0.50%, and this application limits the heterozygosity of the male black carp to not less than 0.80% to ensure that the heterologous sperm has a higher level of genetic diversity. A higher heterozygosity means that its genome contains richer allelic variations and potential functional sites. This genetic information can still play a regulatory role in the early development stage of oocytes through the heterosperm effect after sperm inactivation, thereby affecting the expression of the maternal genome. Compared to using cyprinid fish with low genetic diversity as heterologous sperm donors, introducing high-heterozygous carp sperm can achieve stronger genetic effects and higher screening efficiency for resistance traits during gynogenesis, thereby significantly enhancing the breeding potential of GCRV-resistant grass carp strains.

[0011] Preferably, the selection and cultivation of the mother fish in step (1) specifically involves: selecting female grass carp that are healthy, free from injury or disease, and whose gonads have developed to stage IV; conducting intensive cultivation for 30 days before induced spawning, maintaining clean water quality during the cultivation period, and feeding them high-protein compound feed.

[0012] Preferably, the operating conditions and dosage for artificial spawning in step (2) are as follows: the spawning water temperature is controlled at 22-28℃, and a single injection method is used to inject mixed oxytocin into the mother and the heterologous sperm donor respectively; the mixed oxytocin dosage injected into female grass carp is: LRH-A 10-15μg / kg body weight + HCG 800-1000 IU / kg body weight + domperidone 5-8 mg / kg body weight; the mixed oxytocin dosage injected into male black carp is: LRH-A 3-5μg / kg body weight + HCG 100-150 IU / kg body weight.

[0013] Preferably, the specific operation and parameters of the semen dilution and ultraviolet inactivation treatment in step (3) are as follows: the semen is diluted with Hank's solution or isotonic solution at a volume ratio of 4-10:1; the diluted semen is spread into a uniform thin layer and irradiated with ultraviolet light for 25-35 minutes; during the inactivation process, sperm motility is observed in real time with an optical microscope at intervals of 60-90 seconds, and inactivation is stopped when 70%-80% of the sperm lose their motility.

[0014] Preferably, the parameters for the cold shock treatment of the fertilized eggs in step (3) are as follows: inactivated sperm are mixed with mature grass carp eggs and fertilized in room temperature water; after fertilization, the fertilized eggs are placed at 4-6℃ for cold shock treatment for 12-20 minutes.

[0015] Preferably, the screening of resistant individuals in step (4) includes the following two schemes: Option 1: Conduct grass carp reovirus challenge tests on the gynogenetic grass carp fry to verify and screen for GCRV-resistant gynogenetic grass carp individuals; Option 2: Perform genetic characteristic analysis on the gynogenetic grass carp fry, and screen for GCRV-resistant gynogenetic grass carp individuals based on the analysis results.

[0016] Preferably, the specific steps of the grass carp reovirus challenge test are as follows: when the gynogenetic grass carp are 5 months old, they are challenged by immersion in the GCRV-II virus strain; the GCRV-II virus stock solution is diluted 60-80 times, and the gynogenetic grass carp group and the ordinary grass carp control group are immersed in the diluted virus solution for 10-20 minutes respectively; the test water temperature is maintained at 28±1 ℃, and the mortality time and pathological characteristics of each group are recorded. The survival rate of the gynogenetic grass carp is 20%-30% higher than that of the control group, which indicates that its GCRV resistance is significantly enhanced.

[0017] Preferably, the genetic characteristic analysis steps specifically include: collecting tissue samples from gynogenetic grass carp and common grass carp for whole-genome resequencing; using FST, π ratio, and XP-CLR methods for combined selection signal analysis to screen for candidate genes; performing KEGG functional enrichment analysis on the candidate genes to obtain significantly enriched pathways; and screening core candidate genes based on the signal intensity of the pathways; extracting non-synonymous mutation sites, splicing sites, and SNP sites in the promoter region of the core candidate genes as candidate molecular markers for GCRV resistance; and screening core individuals carrying the target resistance genotype from the gynogenetic grass carp population based on the candidate molecular markers.

[0018] Preferably, the step of screening core individuals carrying the target resistance genotype from the gynogenetic grass carp population based on the candidate molecular markers specifically involves: designing allele-specific detection primers for candidate SNP loci on the chr3 chromosome; using KASP genotyping technology to perform genotyping on the gynogenetic grass carp individuals to be tested, obtaining genotype information for each SNP locus; assigning corresponding weight coefficients to the genetic effect of the superior GCRV resistance allele at each locus on the resistance trait, and calculating the comprehensive molecular marker score of the gynogenetic grass carp individuals to be tested at all candidate SNP loci; ranking the gynogenetic grass carp population according to the comprehensive score, and selecting gynogenetic grass carp individuals with a comprehensive score higher than the population average or ranking in the top 20%–30% of the population as the core breeding population for the GCRV resistance trait.

[0019] Preferably, the cultivation conditions for the core parent in step (5) are as follows: select the screened gynogenetic grass carp resistant individuals, place them in aquaculture water with qualified water quality and stable environment, feed them complete compound feed, monitor their growth status and health indicators regularly, and cultivate them to sexual maturity for subsequent breeding.

[0020] The above-described solution of the present invention has the following beneficial effects: (1) Cultivating stable resistant strains to achieve fundamental control: This invention uses male black carp as heterologous sperm donors, which, while inducing gynogenesis in grass carp, introduce resistance-related genetic or epigenetic variations that may originate from black carp into the maternal genome; at the same time, it is combined with challenge verification with GCRV-II type virus strain to accurately ensure that the strain's resistance meets the standards. Compared with traditional control methods such as vaccine immunization and chemical disinfection, this invention improves germplasm at the genetic level to achieve "disease resistance through breed", which not only ensures stable inheritance of resistance, but also avoids the drawbacks of traditional methods such as short protection period, fluctuating effect and water pollution.

[0021] (2) A systematic breeding system with strong operability: This invention provides a systematic solution, including: a) screening criteria for heterologous sperm donors based on genomic data; b) a sperm inactivation process that balances genetic inactivation with preservation of the "heterosperm effect"; c) a challenge phenotype verification process; d) analysis of the genetic basis of resistance at the whole genome level and marker mining; e) a method for constructing stable strains combined with molecular marker-assisted selection. This system is logically rigorous and highly operable, ensuring the availability, verifiability, and heritability of resistance traits.

[0022] (3) Shorten the breeding cycle and improve breeding efficiency: Traditional grass carp disease resistance breeding (such as family selection and population selection) requires 4-5 generations of continuous selection to fix the superior traits. However, this invention can quickly obtain offspring with high homozygosity by using artificial gynogenesis technology. Combined with molecular marker-assisted selection, it can quickly fix the superior traits of the maternal parent and the related genetic characteristics introduced by the heterosperm effect, shortening the breeding cycle to 2-3 generations. This effectively solves the core problem of traditional breeding that is "long cycle and slow results", and greatly improves breeding efficiency.

[0023] In summary, the GCRV-resistant grass carp strain developed in this invention has advantages such as stable resistance and excellent growth. The corresponding breeding method has the characteristics of short cycle, high efficiency and strong operability. It can effectively solve the problem of grass carp hemorrhagic disease prevention and control, ensure the healthy and sustainable development of grass carp farming, and has significant economic value and social significance. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 A flowchart of the method provided for this invention; Figure 2 Genome heterozygosity diagram of male black carp; Figure 3 This is a pathological feature diagram of common grass carp infected with GCRV-II. Figure 4 A pathological feature diagram of gynogenetic grass carp infected with GCRV-II; Figure 5 Survival curves of common grass carp and gynogenetic grass carp after infection with GCRV-II; Figure 6 A map of single nucleotide polymorphism sites identified through genetic structure analysis of gynogenetic grass carp and common grass carp populations; Figure 7 Cross-validation error analysis diagram for gynogenetic grass carp and ordinary grass carp populations; Figure 8 A diagram illustrating the ancestral composition of gynogenetic grass carp and common grass carp populations; Figure 9 Neighbor-joining tree analysis diagram of gynogenetic grass carp and common grass carp populations; Figure 10 The results are the combined screening results based on three selection signal analysis and detection methods; Figure 11A functional enrichment analysis diagram of gynogenetic grass carp and common grass carp populations. Detailed Implementation

[0026] To make the technical problems, solutions, and advantages of this invention clearer, a detailed description will be provided below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0027] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0028] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a locking connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0029] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] Example 1 A method for cultivating GCRV-resistant grass carp strains based on artificial gynogenesis, the flowchart of which is shown below. Figure 1 As shown, it includes the following steps: (1) Male black carp were selected from Hunan Yuelushan Aquatic Breeding Technology Co., Ltd. Cyprinus carpio Thirty var. Quanzhounensis were used as heterologous sperm donors. They were 3 years old, weighed 1.5-2.0 kg, were in good health, and free from disease.

[0031] Genome heterozygosity was assessed using whole-genome next-generation sequencing (NGS) at a sequencing depth of 30× to 60×. The procedure involved harvesting tail fin tissue samples from male carp, extracting DNA using a plant and animal genomic DNA extraction kit (Takara, Beijing) according to the manufacturer's instructions, and performing paired-end sequencing using the DNBseq platform. The raw short-read sequencing data were analyzed for quality control using the FastP software (v0.20.0). K-mer analysis was performed using GCE software (v1.0.0), with a K-mer length set to 17. K-mer frequency distribution was statistically analyzed, and genome heterozygosity was calculated.

[0032] Sequencing results ( Figure 2 The results showed that the heterozygosity of the male black carp was 1.06%, which is higher than that of conventional koi varieties, meeting the requirements of this invention for genetic diversity of heterologous sperm donors.

[0033] Female grass carp were selected from aquaculture populations in the Yangtze River basin. Ctenopharyngodon idella 50 tails were selected as the female parent, aged 4 years, weighing 5-6 kg, and required to be healthy, free from injury or disease, with enlarged abdomen and gonads developed to stage IV.

[0034] A 30-day intensive rearing period is conducted before spawning induction. Rearing conditions are: water temperature 22-28℃, dissolved oxygen ≥5.0mg / L, ammonia nitrogen ≤0.2mg / L, and nitrite ≤0.1mg / L. The fish are fed a formulated feed with 40% crude protein content twice daily, at a rate of 2.5%-3.0% of their body weight.

[0035] (2) When the water temperature stabilizes at 25℃, artificial labor induction is performed on the parent plants. A single injection method is used, and the oxytocin is prepared as a mixed solution and used immediately after preparation.

[0036] The injection dosage for female grass carp is LRH-A 212 μg / kg body weight + HCG 900 IU / kg body weight + domperidone 6 mg / kg body weight, dissolved in 0.7% physiological saline, with each grass carp receiving approximately 5 mL of the solution. The injection site is the inner side of the base of the pectoral fin, with the needle inserted at a 45° angle to the fish body to a depth of approximately 0.5-1 cm.

[0037] The injection dosage for male black carp is: LRH-A 4μg / kg body weight + HCG 130 IU / kg body weight, with a total injection volume of 2mL per black carp. The injection site and method are the same as for female grass carp.

[0038] Ten hours after injection, the male fish's sperm release was checked. When milky white semen flowed out when the male fish's abdomen was gently pressed, artificial semen collection was performed, and the semen was collected using a dry centrifuge tube. Twelve hours after injection, the female fish's ovulation was checked. When the female fish exhibited chasing behavior, abdominal swelling, and eggs flowing out when gently pressed, artificial egg collection was performed promptly.

[0039] (3) Dilute carp semen with Hank's solution pre-cooled to 4°C at a volume ratio of 5:1. Spread 8 mL of the diluted semen evenly in a pre-cooled glass culture dish with a diameter of 20 cm, with a liquid layer thickness of about 2 mm. Place the culture dish on a circular shaker (SanLiang, SL-O180-S) with an ice plate on it, and set the rotation speed to 110 r / min. Irradiate vertically for 28 min using two 16W ultraviolet lamps (Philips TUV 16W), with the lamps 20 cm above the liquid surface. During irradiation, take a small amount of semen every 60 seconds and observe sperm motility under an optical microscope. When 75% of the sperm are observed to have lost their motility (manifested as the sperm ceasing to wag or only the tail twitching slightly), the inactivation is terminated, and an inactivated sperm suspension is obtained.

[0040] Take 100 mL of mature grass carp eggs and place them in a dry fertilization basin. Add 5 mL of inactivated sperm suspension and mix well. Then add 100 mL of aerated water at 25°C and gently stir for fertilization for 2.5 minutes. Immediately after fertilization, transfer the fertilized eggs to 4°C water for cold shock treatment for 15 minutes to inhibit the expulsion of the second polar body and induce chromosome doubling.

[0041] After cold shock treatment, the fertilized eggs were transferred to a flowing water hatching tank at 25±0.5℃ with a dissolved oxygen level of 6.0-7.0 mg / L. Fertilization occurred 25 hours later, yielding approximately 4500 newly hatched fry. These were then fertilized in a 1000 m² rearing tank to cultivate live food. After 15 days, artificial formulated feed was introduced, three times daily. After 30 days of rearing, 2300 female grass carp fry (2-3 cm in length) were obtained, representing a survival rate of 51.1%.

[0042] (4) When the gynogenetic grass carp fry reached 5 months of age, 40 healthy individuals of uniform size (body length 11-13cm, weight 35-45g) were selected as the experimental group, and 40 common grass carp of the same size were selected as the control group. Both the experimental and control groups were placed in a recirculating aquaculture system for 14 days to acclimatize. The culture conditions were: water temperature 28±1℃, pH 7.0-7.5, dissolved oxygen ≥6mg / L. During the acclimatization period, the fish were fed formulated feed twice daily at a rate of 2% of their body weight.

[0043] GCRV-II virus strain was used for challenge experiments. GCRV-II virus stock solution, frozen at -80℃, was thawed in a 28℃ water bath for 2 hours and diluted 80-fold to prepare a working virus solution. The immersion infection method was used for challenge. The experimental and control groups were immersed in the working virus solution for 10 minutes to ensure full exposure to the virus. After immersion, the fish were immediately returned to their original rearing tanks. The fish were observed for 14 consecutive days after challenge, and the time of death, number of deaths, and pathological characteristics of the grass carp in each group were recorded. Results ( Figure 3 , 4 5) The results showed that in the control group, ordinary grass carp began to die on the 5th day after infection, with a cumulative mortality rate of 72.5% and a final survival rate of 27.5%. The dead individuals exhibited typical GCRV infection symptoms, such as petechiae on the body surface, gill swelling and congestion, and intestinal congestion. In the experimental group, gynogenetic grass carp had a cumulative mortality rate of 47.5% and a final survival rate of 52.5%. The pathological symptoms of the dead individuals were significantly milder than those in the control group, mainly manifested as mild gill congestion and a small number of petechiae on the body surface, with milder lesions in the internal organs.

[0044] Statistical analysis showed that the survival rate of the experimental group was significantly higher than that of the control group (P<0.05), proving that gynogenetic grass carp have strong resistance to GCRV-II.

[0045] Example 2: Based on Example 1, blood samples were collected from 40 gynogenetic grass carp (GGC) and common grass carp (CY) for whole-genome resequencing. Sequencing library construction and sequencing were performed by the Tianjin Institute of Biotechnology, China, on the Illumina platform. The Burrows-Wheeler alignment tool (v.0.7.8) was used, and the obtained paired-end sequencing reads were aligned to the grass carp T2T reference genome using the MEM algorithm (parameters: mem -t 4 -k 32 -M). Variance detection and hard filtering were performed using SAMtools mpileup and GATK software. Further quality control based on genetic parameters was performed using VCFtools (v.0.1.15) software to ensure data reliability. To reduce the impact of linkage disequilibrium, independent molecular markers were screened using PLINK software (v1.9). Neighbor-joining trees were constructed using treebest software to infer population phylogenetic relationships. The population genetic structure was analyzed using ADMIXTURE software. To mine genomic selection features, FST, π ratio, and XP-CLR analyses were combined. Three methods were integrated to achieve comprehensive detection of selection signals. Overlapping signals with all three indicators ranking in the top 5% were extracted to identify candidate selected genomic regions. Candidate regions were annotated using ANNOVAR software, and GO functional annotation and KEGG pathway enrichment analysis of candidate genes were performed using the clusterProfiler package.

[0046] Table 1. Candidate SNP sites associated with GCRV resistance obtained in this invention.

[0047] The results showed that, through variant annotation, a total of 8,661,553 single nucleotide polymorphism (SNP) sites were identified in the genome of the gynogenetic grass carp. Figure 6 The transformation / transversion ratio (TS / TV) was 1.51, indicating that the population genome structure was normalized and that nonsynonymous mutations occurred more frequently than synonymous mutations.

[0048] Genetic analysis was conducted on a gynogenetic grass carp population and a general grass carp population based on high-quality SNP data to clarify the kinship and genetic differences between the populations. ① Cross-validation error analysis results showed that ( Figure 7 K=2 is the optimal genetic grouping model; ancestral component analysis shows that ( Figure 8 When K=2, the CY population is dominated by green ancestral components, while the GGC population shows a significant increase in the proportion of blue ancestral components, clearly demonstrating the differences in genetic background between the populations. ② Neighbor-joining tree analysis results ( Figure 9 The results clearly show the phylogenetic relationship between the two groups, which are far apart in genetic space, consistent with the ancestral component analysis and PCA results.

[0049] Based on the joint screening results of three selection signal analysis and detection methods ( Figure 10 This invention identified 5,084 significant SNP sites in gynogenetic grass carp and common grass carp populations, corresponding to 1,403 candidate genes after annotation. Functional enrichment analysis showed that ( Figure 11 These genes were significantly enriched in the calcium signaling pathway (P<0.05). The identification of selection signals within the calcium signaling pathway further supports the view that the immune capacity of gynogenic grass carp has undergone enhanced evolution. Calcium signaling plays a central role in immune cell activation and antiviral response regulation, and this function is highly conserved in various vertebrates. This invention further screened the core candidate gene with the strongest selection signal within the calcium signaling pathway based on the intensity of the selection signal, and extracted non-synonymous mutation sites, splicing sites, and SNP sites in the promoter region of this gene as candidate molecular markers for GCRV resistance. Finally, 77 candidate SNP sites associated with GCRV resistance were identified, as shown in Table 1.

[0050] (6) For the 77 candidate SNP loci located on the chr3 chromosome of grass carp and significantly associated with the GCRV resistance trait, identified in this invention, conserved flanking sequences of each SNP locus were extracted with reference to the grass carp T2T reference genome sequence. Based on these sequences, allele-specific upstream primers and universal downstream primers for KASP genotyping were designed. Caudal fin samples were randomly collected from 300 gynogenetic grass carp individuals to be tested. Genomic DNA was extracted using a commercial genomic DNA extraction kit (Takara, Beijing). After NanoDrop quality control, genotyping of the target SNP loci was performed using KASP technology. Samples and loci with a genotyping success rate of less than 95% were removed to obtain a high-quality genotype dataset of the individuals to be tested. Weighting weights were set based on the allele effect values ​​(β values) of each SNP locus in resistant individuals, and a weighted disease resistance genetic score was calculated for each individual to be tested. Individuals in the population were ranked according to the comprehensive score, and individuals with the comprehensive score in the top 30% of the population were selected as the core breeding population for the GCRV resistance trait.

[0051] (7) Select gynogenetic grass carp resistant individuals screened through genetic characteristic analysis and transfer them to aquaculture ponds for cultivation. The water pH should be 7.5-8.5, ammonia nitrogen ≤0.2 mg / L, nitrite ≤0.05 mg / L, and water temperature should be naturally adjusted according to the season. Feed them a complete formulated feed (32% crude protein, 8% crude fat, 5% crude fiber) twice daily at a rate of 3%-5% of their body weight, adjusted according to feeding behavior and water temperature. After cultivation, surviving grass carp can be used as core broodstock for subsequent breeding and large-scale propagation of GCRV-resistant grass carp populations.

[0052] This embodiment successfully bred a highly resistant grass carp core parent by combining artificial gynogenesis technology with GCRV resistance assessment. Its GCRV challenge survival rate is significantly higher than that of ordinary grass carp, providing reliable technical support for the industrialization of GCRV resistant grass carp strains.

Claims

1. A method for cultivating GCRV-resistant grass carp strains based on artificial gynogenesis, characterized in that, Includes the following steps: (1) Selection and breeding of parent stock: Select male black carp population with high genetic diversity as heterologous sperm donors, and select female grass carp population with fast growth rate and sexual maturity as maternal stock, and carry out standardized breeding of the parent stock; (2) Artificial spawning: Male black carp and female grass carp were injected with spawning hormones to induce sperm and egg release, and sperm and eggs were obtained; (3) Gynogenesis induction: Collect the semen of the male black carp, dilute it and inactivate it by ultraviolet irradiation; mix the inactivated sperm with the mature eggs of female grass carp to form fertilized eggs, subject the fertilized eggs to cold shock treatment to induce chromosome doubling, and obtain gynogenic grass carp seedlings; (4) Screening of resistant individuals: GCRV-resistant gynogenetic grass carp fry were screened from the gynogenetic grass carp fry. (5) Breeding and propagation: Selected female-developed grass carp resistant individuals are cultivated and used as core parents for subsequent breeding and propagation of GCRV resistant grass carp populations.

2. The method as described in claim 1, characterized in that, The selection of heterologous sperm donors in step (1) is as follows: male black carp are sourced from aquatic breeding farms or natural high-quality waters, and their genetic diversity is assessed by whole-genome next-generation sequencing with a sequencing depth of 30× to 60×; the genome heterozygosity is assessed by K-mer distribution frequency analysis with a K-mer length of 17-21 bp, and the genome heterozygosity of the selected male black carp is not less than 0.80%.

3. The method as described in claim 1, characterized in that, The selection and cultivation of the mother fish in step (1) are as follows: the selected female grass carp are healthy, free from injury or disease, and have developed their gonads to stage IV; before induced spawning, they are subjected to intensive cultivation for 30 days, during which the water quality is kept fresh and they are fed high-protein compound feed.

4. The method as described in claim 1, characterized in that, The operating conditions and dosage for artificial spawning in step (2) are as follows: the spawning water temperature is controlled at 22-28℃, and a single injection method is used to inject mixed oxytocin into the mother and the heterologous sperm donor respectively; the mixed oxytocin dosage injected into female grass carp is: LRH-A 10-15μg / kg body weight + HCG 800-1000 IU / kg body weight + domperidone 5-8 mg / kg body weight; the mixed oxytocin dosage injected into male black carp is: LRH-A 3-5μg / kg body weight + HCG 100-150 IU / kg body weight.

5. The method as described in claim 1, characterized in that, The specific operation and parameters of the semen dilution and ultraviolet inactivation treatment in step (3) are as follows: the semen is diluted with Hank's solution or isotonic solution at a volume ratio of 4-10:1; the diluted semen is spread into a uniform thin layer and irradiated with ultraviolet light for 25-35 minutes; during the inactivation process, sperm motility is observed in real time with an optical microscope at intervals of 60-90 seconds, and inactivation is stopped when 70%-80% of the sperm lose their motility; the parameters for the formation of fertilized eggs and the cold shock treatment of the fertilized eggs are as follows: the inactivated sperm is mixed with mature grass carp eggs and fertilized with room temperature water; after fertilization, the fertilized eggs are placed at 4-6℃ for cold shock treatment for 12-20 minutes.

6. The method as described in claim 1, characterized in that, Step (4) involves screening for resistant individuals using the following two methods: Option 1: Conduct grass carp reovirus challenge tests on the gynogenetic grass carp fry to verify and screen for GCRV-resistant gynogenetic grass carp individuals; Option 2: Perform genetic characteristic analysis on the gynogenetic grass carp fry, and screen for GCRV-resistant gynogenetic grass carp individuals based on the analysis results.

7. The method as described in claim 6, characterized in that, The specific steps of the grass carp reovirus challenge experiment were as follows: when gynogenetic grass carp were 5 months old, they were challenged by immersion in GCRV-II virus strain; the GCRV-II virus stock solution was diluted 60-80 times, and the gynogenetic grass carp group and the ordinary grass carp control group were immersed in the diluted virus solution for 10-20 minutes respectively; the test water temperature was maintained at 28±1 ℃, and the mortality time and pathological characteristics of each group were recorded. The survival rate of the gynogenetic grass carp was 20%-30% higher than that of the control group, which indicated that its GCRV resistance was significantly enhanced.

8. The method as described in claim 6, characterized in that, The genetic characteristic analysis steps specifically include: collecting tissue samples from gynogenetic grass carp and common grass carp for whole-genome resequencing; using FST, π ratio, and XP-CLR methods for combined selection signal analysis to screen for candidate genes; performing KEGG functional enrichment analysis on the candidate genes to obtain significantly enriched pathways; and screening core candidate genes based on the signal intensity of the pathways; extracting non-synonymous mutation sites, splicing sites, and SNP sites in the promoter region of the core candidate genes as candidate molecular markers for GCRV resistance; and screening core individuals carrying the target resistance genotype from the gynogenetic grass carp population based on the candidate molecular markers.

9. The method as described in claim 8, characterized in that, The specific steps for screening core individuals carrying the target resistance genotype from the gynogenetic grass carp population based on the candidate molecular markers are as follows: designing allele-specific detection primers for candidate SNP loci on the chr3 chromosome, using KASP genotyping technology to perform genotyping on the gynogenetic grass carp individuals to be tested, and obtaining genotype information for each SNP locus; Based on the genetic effect of the superior GCRV resistance allele at each locus on the resistant trait, a corresponding weight coefficient was assigned, and the molecular marker comprehensive score of the gynogenetic grass carp individual to be tested at all candidate SNP loci was calculated. The gynogenetic grass carp population was ranked according to the comprehensive score, and gynogenetic grass carp individuals with a comprehensive score higher than the population average or in the top 20% to 30% of the population were selected as the core breeding population for the GCRV resistance trait.

10. The method as described in claim 1, characterized in that, The cultivation conditions for the core parent in step (5) are as follows: select the screened gynogenetic grass carp resistant individuals, place them in aquaculture water with qualified water quality and stable environment, feed them complete compound feed, monitor their growth status and health indicators regularly, and cultivate them to sexual maturity for subsequent breeding.

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