CN-122004156-A - Method for cultivating GCRV-resistant grass carp strain based on artificial gynogenesis

CN122004156ACN 122004156 ACN122004156 ACN 122004156ACN-122004156-A

Abstract

The invention relates to the technical field of fish disease-resistant breeding, in particular to a method for cultivating GCRV-resistant grass carp strain based on artificial gynogenesis. The method comprises the steps of (1) parent selection and cultivation, (2) artificial spawning, (3) gynogenesis induction, (4) resistance verification, namely, carrying out a grass carp reovirus challenge test on the gynogenesis grass carp fries to verify GCRV resistance, and (5) breeding and expanding propagation, namely, selecting screened gynogenesis grass carp resistant individuals, and using the selected gynogenesis grass carp resistant individuals as core parents after cultivation for subsequent breeding and expanding propagation of GCRV resistant grass carp populations. The GCRV-resistant grass carp strain cultivated by the method has the advantages of stable resistance, excellent growth and the like, and the corresponding cultivation method has the characteristics of short period, high efficiency, strong operability and the like, can effectively solve the problem of controlling bleeding diseases of grass carp, ensures the healthy and sustainable development of grass carp cultivation industry, and has great economic value and social significance.

Inventors

QIN QINBO
LIU SHAOJUN
BAI JINHAI
HUANG XU
WANG ZHONGQING
XU XIDAN
YU QIFANG
ZHANG KUN
JIANG YUCHEN

Assignees

湖南师范大学

Dates

Publication Date: 20260512
Application Date: 20260402

Claims (10)

1. A method for cultivating a GCRV-resistant grass carp strain based on artificial gynogenesis, comprising the steps of: (1) Selecting and culturing parents, namely selecting a male black carp population with high genetic diversity as a heterogeneous sperm donor, selecting a female grass carp population with high growth speed and sexual maturity as a female parent, and carrying out standardized culture on the parents; (2) Artificial induced spawning, namely injecting induced spawning hormone into the male black carp and the female grass carp respectively, inducing semen discharge and ovulation, and obtaining semen and ovum; (3) The gynogenesis induction comprises collecting semen of male black carp, diluting, inactivating by ultraviolet irradiation, mixing the inactivated semen with mature ovum of female grass carp to form fertilized ovum, cold shock treating fertilized ovum, and doubling chromosome induction to obtain gynogenesis grass carp fries; (4) Screening resistant individuals, namely screening out the gynogenesis grass carp fries to obtain the gynogenesis grass carp resistant individuals with GCRV resistance; (5) Selecting the screened gynogenesis grass carp resistant individuals, and culturing the gynogenesis grass carp resistant individuals to serve as core parents for subsequent breeding and propagation of GCRV resistant grass carp populations.
2. The method of claim 1, wherein the heterologous sperm donor in step (1) is selected from the group consisting of a male black carp from an aquatic specie stock farm or a natural quality water area, the genetic diversity of which is assessed by whole genome second generation sequencing at a depth of 30 x to 60 x, and a K-mer distribution frequency analysis to assess genome heterozygosity, the K-mer length being set to 17-21 bp, and the selected male black carp having a genome heterozygosity of not less than 0.80%.
3. The method according to claim 1, wherein the female parent in the step (1) is selected and cultivated specifically, the female grass carp selected is an individual with strong constitution, no injury and gonad development to IV stage, the female grass carp is subjected to intensified cultivation for 30 days before induced spawning, the water quality is kept fresh during cultivation, and the female grass carp is fed with high-protein compound feed.
4. The method according to claim 1, wherein the artificial induced spawning in the step (2) is operated under the conditions that the induced spawning water temperature is controlled to be 22-28 ℃, and the mixed oxytocin is injected into the female parent and the heterologous sperm donor respectively by adopting a single injection mode, wherein the mixed oxytocin dose injected into female grass carp is LRH-A10-15 mug/kg body weight + HCG 800-1000 IU/kg body weight + domperidone 5-8 mg/kg body weight, and the mixed oxytocin dose injected into male black carp is LRH-A3-5 mug/kg body weight + HCG 100-150 IU/kg body weight.
5. The method of claim 1, wherein the specific operation and parameters of semen dilution and ultraviolet inactivation treatment in the step (3) are that Hank's solution or isotonic solution is adopted to dilute semen with a dilution volume ratio of 4-10:1, the diluted semen is tiled to form a uniform thin layer, the diluted semen is irradiated and inactivated by an ultraviolet lamp for 25-35min, the sperm motility is observed in real time by an optical microscope during the inactivation process, the inactivation is stopped when 70% -80% of sperms lose the motility, fertilized eggs are formed, the parameters of the fertilized eggs cold shock treatment are that the inactivated sperms and mature eggs of grass carp are mixed, normal temperature water is added for fertilization, and after fertilization is completed, the fertilized eggs are placed under the condition of 4-6 ℃ for cold shock treatment, and the treatment duration is 12-20min.
6. The method of claim 1, wherein the screening of resistant individuals in step (4) comprises the following two protocols: Carrying out a grass carp reovirus challenge test on the gynogenesis grass carp fries, verifying and screening to obtain a gynogenesis grass carp resistant individual with GCRV resistance; And in the second scheme, genetic characteristic analysis is carried out on the gynogenesis grass carp fries, and GCRV-resistant gynogenesis grass carp resistant individuals are obtained through screening based on analysis results.
7. The method of claim 6, wherein the grass carp reovirus challenge test comprises the specific steps of soaking a GCRV-II strain for challenge when the grass carp is 5 months old, diluting the GCRV-II virus stock solution by 60-80 times, respectively soaking a group of the grass carp in the virus diluent and a common grass carp control group in the virus diluent for 10-20 min times, keeping the test water temperature at 28+/-1 ℃, and recording the death time and pathological characteristics of each group of individuals, wherein the survival rate of the grass carp is improved by 20% -30% compared with that of the control group, namely, the GCRV resistance of the grass carp is obviously enhanced.
8. The method of claim 6, wherein the genetic profile analysis step specifically comprises the steps of collecting tissue samples of gynogenesis grass carp and common grass carp respectively, performing whole genome resequencing, performing selection signal joint analysis by adopting three methods of FST, pi ratio and XP-CLR, screening to obtain candidate genes, performing KEGG function enrichment analysis on the candidate genes to obtain a remarkable enrichment pathway, screening out core candidate genes according to the signal intensity of the pathway, extracting non-synonymous mutation sites, splice sites and SNP sites of a promoter region in the core candidate genes as candidate molecular markers for resisting GCRV characteristics, and screening core individuals carrying target resistance genotypes from gynogenesis grass carp populations based on the candidate molecular markers.
9. The method of claim 8, wherein the step of screening core individuals carrying the target resistance genotype from the population of gynogenesis grass carp based on the candidate molecular markers is specifically carried out by designing allele-specific detection primers for candidate SNP loci on the chr3 chromosome, carrying out genotype detection on the individuals of gynogenesis grass carp to be detected by using KASP typing technology to obtain genotype information of each SNP locus, giving corresponding weight coefficients according to genetic effects of excellent allele resistance traits of each locus on GCRV, calculating comprehensive scores of molecular markers of the individuals of gynogenesis grass carp to be detected on all candidate SNP loci, sequencing the population of gynogenesis grass carp based on the comprehensive scores, and screening the individuals of gynogenesis grass carp with comprehensive scores higher than the population average or 20% -30% in front of the population as core breeding population of the anti-GCRV traits.
10. The method according to claim 1, wherein the core parent in the step (5) is cultivated under the conditions that the screened gynogenesis grass carp resistant individuals are selected, placed in a cultivation water body with standard water quality and stable environment, fed with complete compound feed, periodically monitored for growth conditions and health indexes, and cultivated to sexual maturity for subsequent breeding.

Description

Method for cultivating GCRV-resistant grass carp strain based on artificial gynogenesis Technical Field The invention relates to the technical field of fish disease-resistant breeding, in particular to a method for cultivating GCRV-resistant grass carp strain based on artificial gynogenesis. Background Grass carp (Ctenopharyngodon idella) is one of the most important freshwater farmed fishes in China, has the characteristics of fast growth, high feed conversion rate, good meat quality and the like, and is an important animal protein source in daily diet of residents. According to the data of Chinese fishery statistics annual book (2024), the grass carp yield in 2023 is 594.13 ten thousand tons, accounting for 17.4% of the total yield of freshwater aquaculture fishes. However, as the scale of cultivation increases and the degree of intensification increases, viral diseases have become a major bottleneck restricting the healthy development of grass carp farming. The grass carp hemorrhagic disease caused by grass carp reovirus (GRASS CARP Reovirus, GCRV) has the characteristics of acute disease, strong infectivity, high mortality and the like, has been classified as a second-class animal epidemic disease by the agricultural rural department, causes huge economic loss to grass carp farming every year, and seriously threatens industrial safety. GCRV is a double-stranded RNA virus, and can be classified into three genotypes of GCRV-I, GCRV-II and GCRV-III according to genome sequence and biological characteristics. The GCRV-II strain is the most main epidemic strain in grass carp culture areas in China, and particularly in grass carp of 1 age, a large-scale epidemic situation is prone to be exploded, the disease course is short after the disease is developed, and the prevention and control difficulty is high. Due to the pair GCRVThe mechanism of infection of II is not clear, and efficient prevention and control means for GCRV-II are lacking at present. The existing prevention and control measures mainly depend on conventional means such as vaccination, water disinfection, feed additives and the like, but the problems of short protection period, unstable effect, environmental pollution risk and the like exist in the methods, and the disease problem can not be fundamentally solved. In the aspect of disease resistance breeding, the traditional methods such as family selection, group selection and the like need multi-generation continuous breeding, have long period (usually about 20 years), low efficiency and are difficult to accurately locate and fix disease resistance genes. Although molecular marker assisted selective breeding has advanced to a certain extent, stable main disease resistance gene markers have not been identified so far because GCRV resistance belongs to a complex polygenic control character, and the breeding practice requirements cannot be met. Therefore, a new grass carp germplasm with stable GCRV resistance is created from the genetic level, so that' seed disease resistance is realized, and the grass carp hemorrhagic disease prevention and control difficult problem is solved, and the sustainable development of industry is ensured. The artificial gynogenesis technology is an important technical means in the field of fish genetic breeding, and the core principle is that a genetic inactivated heterologous sperm is utilized to activate oocyte to start development, and then chromosome doubling treatment is carried out to obtain diploid offspring mainly inheriting female parent genetic materials. The technology not only can quickly fix excellent characters and shorten the breeding period. More uniquely, genetically inactivated heterologous sperm lose complete fertilization capacity, but the trace genetic material fragments produced by the genetically inactivated heterologous sperm can still be integrated into the genome of the offspring of gynogenesis, thereby imparting new genetic characteristics to the offspring and achieving trait improvement, a phenomenon known as the "oligospermia effect". Early studies demonstrated that microsatellite markers of paternal origin could be detected in the inactivated heterologous sperm-induced gynogenesis grass carp and that these individuals exhibited significant differences in expression over the innate immune pathway. However, the prior art does not develop systematic research on the GCRV-II resistance of grass carp, on one hand, the prior art does not accurately screen heterogeneous sperm donors suitable for gynogenesis of grass carp, lacks evaluation of genetic diversity and disease resistance potential of the donors, and on the other hand, a complete technical system of heterosperm induction-resistance screening-genetic analysis-strain propagation is not established, so that the technology cannot be applied to production breeding in a falling to the ground. Therefore, based on the artificial gynogenesis technology, the heterogenic sperms with high genetic dive