Breeding method of superior oyster strains resistant to Vibrio spp. based on whole genome selection

Abstract

The present invention belongs to the field of animal disease-resistant breeding, and specifically relates to a method for breeding oysters with Vibrio resistance based on whole genome selection. In view of the problem that the existing whole genome selection technology is mostly used for vertebrates with specific immune functions, but the effect on invertebrates lacking specific immunity is not obvious, phenotypic evaluation and reference population construction are carried out in the summer when the disease breaks out, and individuals with the same genetic background, the same growth period and the same physiological stage as the reference population are selected as candidate parents. The optimal Bayes A model is used to estimate the genomic breeding value in the actual breeding process, thereby overcoming the problems that the existing genomic selection technology is directly applied to the breeding of oysters with Vibrio resistance, which cannot achieve the expected technical effect, and the problems such as reduced accuracy of genomic breeding value estimation, insufficient selection precision, and overall poor disease resistance of the selected oyster strains. This breeding scheme has higher selection accuracy for oysters.

Description

Technical Field

[0001] The invention belongs to the field of animal disease-resistant breeding, and particularly relates to a method for breeding an excellent Vibrio-resistant oyster strain based on whole genome selection. Background Art

[0002] Oysters, commonly known as oysters and sea oysters, are important economic marine shellfish in my country and occupy an important proportion in my country's marine aquaculture. In 2021, my country's total oyster production was 5.8191 million tons, accounting for 38.13% of the total marine aquaculture production, and is one of the important marine aquaculture economic species. Due to its high protein, high glycogen, low fat and diverse vitamins, oysters have been given the reputation of "milk in the sea" and are important edible shellfish. The long oyster is the main oyster variety cultivated in northern my country, and a lot of breeding and improvement work has been carried out in the fields of growth, shell color, nutritional composition, glycogen content, etc., and fruitful results have been achieved. However, the current work on disease-resistant breeding of oysters is still in urgent need of development.

[0003] In recent years, a complex disease known as "summer mortality syndrome" has been impacting the oyster industry. This disease, which is prevalent in the summer and is typically caused by viruses and pathogenic Vibrio bacteria, causes mass oyster deaths. Rising summer temperatures lead to the proliferation of pathogens. Combined with the environmental stress of high temperatures, adult oysters become weak after ovulation and spermatogenesis, compromising their immune systems and making them susceptible to microbial infection and death, resulting in severe economic losses. Furthermore, oysters lack a specific immune system and are farmed in open ocean environments, making it impossible to prevent or treat diseases through vaccine development or direct control of waterborne pathogens. Therefore, genetically improving oyster disease resistance is the only effective solution to this disease problem.

[0004] Traditional breeding methods, such as population selection and family selection, suffer from low selection accuracy and slow genetic progress, especially for traits with low heritability and difficult to measure, such as disease resistance and reproductive traits. Breeding disease-resistant oysters through traditional selective breeding is time-consuming and slow, so the introduction of advanced biological tools will be a key approach to breeding improved oysters. Whole-genome selection, which leverages genome-wide variation, has been applied to selective breeding in livestock, poultry, and plants. This technology estimates the Genomic Estimated Breeding Value (GEBV) of candidate individuals with only genotypes from a reference population with both phenotypes and genotypes, enabling selection without phenotypic measurements. Therefore, genomic selection is the optimal method for traits that are difficult to measure phenotypically, such as disease resistance and fillet yield.

[0005] However, at present, whole genome selection technology is rarely used in aquatic species. Chinese patent CN115287340 A discloses a method for breeding large yellow croaker strains resistant to visceral white spot disease based on whole genome selection, and Chinese patent CN106480189 B discloses a method for breeding disease-resistant fish strains based on whole genome selection. The method mainly includes the steps of reference population construction, resistance phenotype determination, genotype data acquisition, optimal genome selection model construction, breeding value evaluation, etc. However, since large yellow croaker, flounder, and semi-smooth tongue sole are all fish and vertebrates with specific immune systems, while oysters are invertebrates and do not have formed immune organs and tissues, but only primitive, non-specific defense systems, the direct application of the above methods to the breeding of oyster strains resistant to Vibrio cannot achieve the expected technical effect. Problems such as reduced accuracy of genome breeding value estimation, insufficient selection precision, and overall poor disease resistance of the selected oyster strains have occurred. Summary of the Invention

[0006] Based on the above reasons, the purpose of the present invention is to provide a method for breeding excellent oyster Vibrio-resistant strains based on whole genome selection. The oyster population screened by this method can significantly improve the oyster's resistance to Vibrio, accelerate the breeding of new disease-resistant oyster varieties, and ensure the healthy development of the oyster industry.

[0007] In order to achieve the above objectives, the technical solution of the present invention discloses a method for breeding superior oyster Vibrio-resistant strains based on whole genome selection, comprising the following steps:

[0008] Reference population construction: Select parents with different genetic backgrounds to construct a basic breeding population, and wait for the offspring to grow to one year old or above to serve as the reference population;

[0009] Resistance phenotype determination: Use a highly virulent strain of Vibrio alginolyticus as a model pathogen to conduct a challenge experiment and evaluate the resistance phenotype of the reference population;

[0010] Genotype data acquisition;

[0011] Construction of the optimal genomic selection model: Using cross-validation to obtain the Bayes A genomic selection model with the best prediction accuracy for breeding value estimation;

[0012] Evaluation of genomic breeding values ​​of candidate parents: Individuals from the same population, with the same parents, and cultured in the same sea area as the reference population were selected as candidate parents, and the genomic breeding values ​​of the candidate parents were estimated using the Bayes A model;

[0013] Resistance selection population breeding;

[0014] Select Effectiveness Assessment.

[0015] Furthermore, the reference population is selected from adult oysters in the disease outbreak season.

[0016] Furthermore, the reference population phenotype assessment is for healthy oysters, no OsHV-1 (OysterHerpesvirus type 1) virus is detected, and the Vibrio load is less than 50 CFU / g.

[0017] Furthermore, the challenge experiment steps are:

[0018] Bacterial culture: Vibrio alginolyticus Cg5 was cultured in 2216E culture medium. After 12 hours of incubation, the culture was centrifuged and the bacterial pellet was washed three times with sterile seawater and diluted to 5 × 10 8 CFU / mL, bacterial concentration was measured using a UV spectrophotometer;

[0019] Bacterial injection: Each oyster was injected with 0.1 mL of Vibrio suspension into the adductor muscle, with an injection dose of 5 × 10 7 After the injection, the oysters were cultured in seawater with a pH of 8.1, a dissolved oxygen of 8 mg / L, a temperature of 22°C, and a salinity of 30-32‰ for observation.

[0020] Furthermore, after the challenge experiment, the binary phenotype of survival status (survival status) of individuals after the challenge and the survival time (survival time) from the start of the challenge experiment to the death of oysters were used as measurement criteria to measure the resistance phenotype of the reference population to Vibrio alginolyticus.

[0021] Furthermore, the genotype acquisition step is: using genome sequencing technology or SNP genotyping chip to genotype the whole genome SNP of long oysters, obtaining the genotype data of the reference population, and performing genotype filling after filtering the minor allele frequency and genotype missing rate for the construction of the genomic selection model.

[0022] Furthermore, the genomic breeding value evaluation step of the candidate parent is as follows: oyster individuals of the candidate population are anesthetized by being immersed in a 50 g / L magnesium chloride solution, and after the oysters are opened, 50 mg of gill filaments are taken for DNA extraction and genotyping.

[0023] Furthermore, the resistance breeding group breeding step is: selecting the top 15% of individuals with the highest breeding values ​​as resistance parents, and the other individuals as control parents, and breeding the resistance breeding group and the control group separately.

[0024] Furthermore, the selection effectiveness evaluation includes the evaluation of Vibrio resistance in the larval stage and the evaluation of Vibrio resistance in the adult stage.

[0025] The beneficial effects of the present invention are:

[0026] 1. The present invention addresses the problem that existing whole-genome selection technology is mostly used for vertebrates with specific immune functions, but is not very effective for invertebrates that lack specific immune systems. Phenotypic evaluation and reference population construction are carried out in the summer when the disease breaks out, and individuals with the same genetic background, growth period, and physiological stage as the reference population are selected as candidate parents. The optimal Bayes A model is used to estimate the genomic breeding value in the actual breeding process. This overcomes the problem that the existing genomic selection technology cannot achieve the expected technical effect when directly applied to the breeding of superior Vibrio-resistant oyster strains, resulting in reduced genomic breeding value accuracy, insufficient selection precision, and overall poor disease resistance of the selected oyster strains. This breeding program has higher selection accuracy for oysters. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Attachment Figure 1 Flowchart of the implementation of genome-wide selection breeding for Vibrio resistance in long oysters

[0028] Attachment Figure 2 (A) The prediction accuracy of genomic breeding values ​​for survival status traits under different genomic selection models; (B) The prediction accuracy of genomic breeding values ​​for survival time traits under different genomic selection models;

[0029] Attachment Figure 3 The population structure characterized by principal component analysis for the reference and candidate populations

[0030] Attachment Figure 4 is the genomic breeding value of the resistance selection population in the candidate population and the genomic breeding value of the control population

[0031] Attachment Figure 5 (A) is the survival rate of each group in the larval stage after Vibrio infection, (B) is the survival curve of each group in the adult stage after Vibrio infection, (C) is the survival time of each group in the adult stage after Vibrio infection, and (D) is the survival rate of each group during the large-scale mortality outbreak in summer. DETAILED DESCRIPTION

[0032] The present invention will be explained below with reference to the following examples. It will be understood by those skilled in the art that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention.

[0033] The first embodiment of the present invention discloses a method for breeding superior oyster strains resistant to Vibrio spp. based on whole genome selection, comprising the following steps:

[0034] Reference population construction: Select parents with different genetic backgrounds to construct a basic breeding population, and wait for the offspring to grow to one year old or above to serve as the reference population;

[0035] Resistance phenotype determination: Use a highly virulent strain of Vibrio alginolyticus as a model pathogen to conduct a challenge experiment and evaluate the resistance phenotype of the reference population;

[0036] Genotype data acquisition;

[0037] Construction of the optimal genomic selection model: Using cross-validation to obtain the Bayes A genomic selection model with the best prediction accuracy for breeding value estimation;

[0038] Evaluation of genomic breeding values ​​of candidate parents: Individuals from the same population, with the same parents, and cultured in the same sea area as the reference population were selected as candidate parents, and the genomic breeding values ​​of the parents were estimated using the Bayes A model;

[0039] Resistance selection population breeding;

[0040] Select Effectiveness Assessment.

[0041] In this embodiment, parents with different genetic backgrounds are selected to construct the basic breeding population in order to ensure the genetic diversity of the breeding population. The present invention selects oysters with different genetic backgrounds from China, Japan, South Korea, and the United States.

[0042] In this embodiment, the offspring of the parents are grown to one year old or above before being used as the reference population to ensure that both the reference population and the candidate population are adults. In existing whole-genome selection breeding technologies for vertebrates, the reference population is generally juveniles and the candidate population is generally adults. This selection method has little effect on the accuracy of genomic breeding value prediction for species with specific immune systems. However, for oysters that lack adaptive systemic immunity, the immune system of their juveniles is not yet fully developed. In disease-resistant genomic selection breeding, the use of juveniles as the reference population has a greater impact on the accuracy of genomic breeding value prediction. Therefore, in this embodiment, adults in the same growth period are selected for both the reference population and the candidate population.

[0043] In this embodiment, the Bayes A model was selected as the optimal genome selection model based on cross-validation results in the construction of the optimal genome selection model. This was selected based on the differences in the immune systems of oysters and existing vertebrates such as fish. Due to the differences in the oyster immune system, those skilled in the art were not clear before implementation which model would have the highest accuracy for genome selection in oysters. Therefore, four genome selection models were constructed, specifically:

[0044] Screening from rrBLUP and three Bayesian methods (Bayes A, Bayes B, and Bayesian Lasso), they can be expressed as:

[0045] y=μ + Zu + e

[0046] Where y is the phenotypic value, μ is the population mean, Z is the SNP marker matrix (with values ​​of 0, 1, and 2), u is the marker effect value vector, and e is the residual. rrBLUP generates the effect value of each marker through maximum likelihood estimation, while the three Bayesian methods estimate the effect value of each SNP through Gibbs sampling. The product of the effect value u of each individual marker and the marker matrix is ​​the genomic breeding value of the individual. The entire dataset was randomly divided into a training set (90% of the data volume) and a test set (10% of the data volume) for cross-validation. The training set used phenotypic and genotypic data to estimate the effect value of each marker. The breeding value of the test set was calculated using the estimated effect value matrix of the training set and the marker matrix of the test set. The Pearson correlation coefficient between the test set phenotype and its estimated breeding value was used to measure the predictive accuracy of the model. Cross-validation results showed that the Bayes A model had higher predictive accuracy among the four models, with an accuracy of 0.1572 when estimating the binary trait of survival or failure and an accuracy of 0.1534 when estimating survival time (see Appendix). Figure 2 ); thus determining the Bayes A model as the best prediction model.

[0047] In this embodiment, in the evaluation of the genomic breeding values ​​of candidate parents, the purpose of selecting individuals from the same population as the reference population, with the same parents, and cultured in the same sea area as the candidate parents is to ensure that their growth stage and physiological state are consistent with the reference population, thereby improving the accuracy of the genomic breeding value estimation.

[0048] Population structure analysis showed that the reference population and the candidate population had the same population structure, indicating that they had the same population origin, thus ensuring the accuracy of breeding value evaluation (Appendix Figure 3 The breeding values ​​of the parents estimated by the genomic selection model ranged from 0.410 to 0.703, indicating that there were significant resistance differences among the candidate parents and that they could serve as potential indicators for measuring Vibrio resistance.

[0049] In a further embodiment, the reference population is selected from adult oysters during the disease outbreak season. In the prior art, the reference population is generally selected from juveniles, and the selected season is not restricted. This is because the species selected for whole genome selection in the prior art are generally vertebrates, whose immune systems are specific immune, and the difference between juveniles and adults is not obvious. However, oysters lack a specific immune system, so the selection of juveniles as a reference population is more different from that of adults. At the same time, different seasons and different growth periods have a greater impact on their immunity. Therefore, it is necessary to select the summer when the disease breaks out to perform resistance phenotype measurement, specifically:

[0050] After one year of aquaculture in the natural sea area, the oysters were transferred to the laboratory for two weeks of temporary culture to adapt to the environment before the outbreak of the disease in the summer. They were then challenged with Vibrio alginolyticus. Oysters were tested for Vibrio and viral pathogens using tissue homogenate plating and PCR reactions to ensure that the oysters used were healthy and disease-free oysters (OsHV-1 was not detected and the Vibrio load was less than 50 CFU / g). The Vibrio alginolyticus Cg5 strain was cultured in 2216E culture medium, incubated for 12 hours, and then centrifuged. The bacterial pellet was washed repeatedly three times with sterile seawater and diluted to 5×10 8 CFU / mL, and the bacterial concentration was measured using a UV spectrophotometer. Each oyster was injected with 0.1 mL of Vibrio suspension into the adductor muscle, and the injection dose for each oyster was 5×10 7 CFU. After the injection, 1,402 oysters were cultured in seawater with a pH of 8.1, a dissolved oxygen of 8 mg / L, a temperature of 22°C, and a salinity of 30-32‰ for observation. During the infection experiment, the seawater was changed every two days. During the experiment, the death and survival of oysters were recorded every two hours, and the dead oysters were immediately removed from the tank and the adductor muscles were preserved in DNA preservation solution. The challenge experiment lasted for a total of 12 days. At the end of the experiment, a total of 982 oysters died, with a cumulative mortality rate of 70.04%. The adductor muscles of individuals that survived the challenge experiment were sampled and preserved in DNA preservation solution.

[0051] In a further embodiment, the oysters used for the reference population phenotype assessment are healthy, have no OsHV-1 (Oyster Herpesvirus type 1) virus detected, and have a Vibrio load of less than 50 CFU / g. This step can improve screening accuracy.

[0052] In a further embodiment, the step of evaluating the genomic breeding value of the candidate parent is as follows: oyster individuals of the candidate population are anesthetized by being immersed in a 50 g / L magnesium chloride solution, and after opening their shells, 50 mg of gill filaments are taken for DNA extraction and genotyping.

[0053] In a further embodiment, the resistance breeding population breeding step is: selecting the top 15% of individuals with the highest breeding values ​​as resistance parents, and the other individuals as control parents, and breeding the resistance breeding population and the control population separately.

[0054] In a further embodiment, the selection effectiveness evaluation includes evaluation of Vibrio resistance in larval stage and evaluation of Vibrio resistance in adult stage.

[0055] The following will be described in conjunction with specific embodiments:

[0056] (1) Construction of a reference population of Vibrio alginolyticus in long oysters

[0057] 86 oysters with diverse genetic backgrounds (from China, Japan, South Korea, and the United States) were selected as parental stock to construct a breeding base. Gonadal development was observed microscopically. Oysters with mature gonads were dissected, and sperm and eggs were obtained for artificial insemination at a sperm-egg ratio of 5:1 to 10:1. A glass rod was used to mix the sperm and eggs, and the development of the fertilized eggs was observed microscopically every 10 minutes. After the second polar body released the fertilized eggs, they were placed in fresh water for incubation. After successful hatching, they were artificially fed with platyphylla and golden algae. When the larvae developed into eyed larvae, scallop shells were added as a substrate for attachment and metamorphosis. One week after all larvae had attached, the metamorphosed spat were transferred to outdoor nursery ponds for artificial rearing. Approximately 40 days later, when the shells of the spat reached 2-3 mm in height, they were transported to the sea area of ​​Rongcheng City, Shandong Province, for natural culture.

[0058] (2) Measurement of resistance phenotype of the long oyster Vibrio alginolyticus reference population

[0059] After one year of aquaculture in the natural sea area, the oysters were transferred to the laboratory for two weeks of temporary culture to adapt to the environment before the outbreak of the disease in the summer. They were then challenged with Vibrio alginolyticus. Oysters were tested for Vibrio and viral pathogens using tissue homogenate plating and PCR reactions to ensure that the oysters used were healthy and disease-free oysters (OsHV-1 was not detected and the Vibrio load was less than 50 CFU / g). The Vibrio alginolyticus Cg5 strain was cultured in 2216E culture medium, incubated for 12 hours, and then centrifuged. The bacterial pellet was washed repeatedly three times with sterile seawater and diluted to 5×10 8 CFU / mL, and the bacterial concentration was measured using a UV spectrophotometer. Each oyster was injected with 0.1 mL of Vibrio suspension into the adductor muscle, and the injection dose for each oyster was 5×10 7CFU. After the injection, 1,402 oysters were cultured in seawater with a pH of 8.1, a dissolved oxygen of 8 mg / L, a temperature of 22°C, and a salinity of 30-32‰ for observation. During the infection experiment, the seawater was changed every two days. During the experiment, the death and survival of oysters were recorded every two hours, and the dead oysters were immediately removed from the tank and the adductor muscles were preserved in DNA preservation solution. The challenge experiment lasted for a total of 12 days. At the end of the experiment, a total of 982 oysters died, with a cumulative mortality rate of 70.04%. The adductor muscles of individuals that survived the challenge experiment were sampled and preserved in DNA preservation solution.

[0060] (3) Genotype acquisition of the long oyster alginolytic Vibrio reference population

[0061] Using simplified genome sequencing, 295 Crassostrea gigas were genotyped, including 131 susceptible individuals that died first and 164 resistant individuals that survived. DNA from each individual was digested with the restriction endonucleases EcoRI and MspI, and the digested products were ligated to sequencing adapters using T4 DNA ligase. Sequencing libraries purified by magnetic bead sorting were then subjected to paired-end sequencing on the Illumina platform. Single-nucleotide polymorphisms (SNPs) were identified using the Stacks program, and SNPs with a minor allele frequency less than 0.05 and a missingness greater than 10% were filtered out. Genotypes for genomic selection analysis were imputed using Beagle and converted to numerical format using Plink. Genotyping using simplified genome sequencing yielded 48,000 high-quality SNP markers for subsequent analysis.

[0062] (4) Construction of genome selection model for Vibrio alginolyticus in long oysters

[0063] The accuracy of genomic breeding value prediction was estimated using the Bayes A model. The accuracy was 0.1572 when estimating the binary trait of survival or failure, and 0.1534 when estimating survival time ( Figure 2 ).

[0064] (5) Estimation of genomic breeding values ​​of candidate parents

[0065] The candidate population and the reference population come from the same population, have the same parents, and are cultured in the same sea area to ensure that their growth stage and physiological state are consistent with the reference population. The oyster individuals of the candidate population were anesthetized by soaking them in a 50g / L magnesium chloride solution. After the shells were opened, 50mg of gill filaments were taken for DNA extraction and genotyping. A total of 120 candidate parents were successfully genotyped, and genotype data were obtained. After filtering and filling the genotype data, the genomic breeding values ​​of the parents were estimated using the Bayes A model with the highest accuracy in cross-validation. Population structure analysis showed that the reference population and the candidate population had the same population structure, indicating that they had the same population origin, thereby ensuring the accuracy of the breeding value assessment ( Figure 3 The breeding values ​​of the parents estimated by the genomic selection model ranged from 0.410 to 0.703, indicating that there were significant resistance differences among the candidate parents and that they could serve as potential indicators for measuring Vibrio resistance.

[0066] (6) The top 15% of individuals with the highest breeding values ​​were selected as resistant parents, and the other individuals were selected as control parents to breed the resistant breeding population and the control population. The lowest genomic breeding value of the resistant parents was 0.592, and its average value was 0.641, which was significantly higher than the 0.511 of the control population ( Figure 4 Candidate parents with mature gonads are dissected to obtain sperm and eggs for artificial insemination. After successful hatching, they are artificially fed with flat algae and golden algae. When the larvae develop into eyed larvae, scallop shells are placed as a substrate for attachment and metamorphosis. After metamorphosis, when the shells of the juveniles reach 2-3 mm in height, they are transferred to natural sea areas for culture. The resistance breeding population and the control population are bred and reared using the same methods and maintained in the same waters to ensure a consistent environment.

[0067] (7) Selection effectiveness evaluation

[0068] To demonstrate the effectiveness of the selection, larvae and adult shellfish from the selected and control populations were challenged with Vibrio spp. and their survival rates were compared. Furthermore, the survival rates of the various populations during a summer mass mortality outbreak in a natural marine area were compared to verify the effectiveness of the selected populations in resisting Vibrio spp.

[0069] ① Assessment of Vibrio resistance in larval stage

[0070] Larvae that have developed to the shell top stage are transferred to 100 mL glass beakers. The larvae density is adjusted with sieve silk so that the number of larvae in each glass beaker is roughly the same. After one day of temporary rearing, the oyster larvae are subjected to an immersion infection experiment. Vibrio alginolyticus is added to the 100 mL beaker to make the concentration of Vibrio in the seawater 1×10 6CFU / mL, the experiment was carried out for a total of 7 days to evaluate the resistance of the larvae to Vibrio alginolyticus. During the experiment, the water temperature was maintained at 26 ± 1℃ and the salinity was maintained at 30 ± 1‰. At the end of the challenge experiment, 70 μL of neutral red was added to each beaker with a pipette and stirred evenly with a glass rod to stain each larva. After 3 hours, formaldehyde was added to fix it and placed under a 4×100 microscope to count the larval mortality. A total of 11,314 larvae were selected for the challenge experiment. After 7 days of the challenge experiment, the survival rate of the control group was 23.92%, and the survival rate of the resistant breeding group was 33.71%, a relative increase of 40.92% ( Figure 5 A).

[0071] ② Evaluation of Vibrio resistance in adult shellfish

[0072] The first-year-old oysters cultured in the natural sea were transferred to the laboratory and acclimated for two weeks before being challenged with Vibrio alginolyticus. After anesthesia, each oyster was injected with 0.1 mL of Vibrio suspension into the adductor muscle. The injection dose for each oyster was 5×10 7 CFU. After the injection, the oysters were cultured in seawater with a pH of 8.1, a dissolved oxygen of 8 mg / L, a temperature of 20°C, and a salinity of 30-32‰ for observation. The experiment lasted for a total of 12 days, and the number of oyster deaths per day in the breeding group and the control group, as well as the survival time during the challenge experiment, were counted. A total of 1,312 oysters were used for Vibrio challenge. After Vibrio challenge, the survival rate of the control group was 85.34%, the survival rate of the control group was 72.06%, and the survival rate of the resistant group increased by 18.42% compared with the control group ( Figure 5 B). In addition, the average survival time of the resistant breeding population was 10.8 days, an increase of 12.73% compared to the 9.58 days of the control population ( Figure 5 C).

[0073] ③ The control group and the breeding group were placed in the natural sea area of ​​Rongcheng City, Shandong Province for summering. The number of oysters surviving before and after summer was counted to evaluate the disease resistance of the breeding group when a large-scale death outbreak occurred in summer. The oysters were transported to the Rongcheng sea area of ​​Shandong, China for floating cage culture. There were 30 individuals in each layer of the cage. The survival of the long oyster Vibrio resistant breeding group and the control group was counted before and after summer. The survival rate of each group was calculated by counting the number of live and dead oysters in the cage. A total of 2,132 oysters were used for summer survival comparison. After summering, the survival rate of the resistant group was 68.33%, and the survival rate of the control group was 61.02%. The survival rate of the breeding group increased by 11.98% compared with the control group ( Figure 5 D).

[0074] The present invention establishes a breeding method for oyster disease resistance traits based on genomic selection technology. It is the first time that whole genome selection breeding technology has been used to carry out disease resistance selection breeding of oysters, and an excellent strain with resistance to vibriosis has been selected. Compared with traditional methods, this technology does not require the candidate parents to be attacked by viruses, which protects the offspring from vertical transmission of pathogens from the parents and the influence of pathogenic microorganisms during the seedling breeding process. In addition, this technology is suitable for disease resistance traits with low heritability, and selection is carried out based on genomic information, avoiding errors in phenotypic selection caused by environmental factors. A genomic selection reference population is constructed using adult oysters during the disease outbreak season, and individuals with the same population source and growth stage are selected as candidate parents for selection breeding to ensure the representativeness of the reference population's resistance phenotype, making it more practical.

[0075] This technology constructs a genomic selection model, which allows the breeding value of candidate populations to be directly estimated using genomic information without the need for phenotypic information. On the one hand, it protects candidate parents from the dangers of Vibrio infection during phenotypic testing, thereby avoiding vertical transmission of pathogens and ensuring biosecurity during the seedling breeding period. On the other hand, it allows direct selection based on the genomic breeding value estimated from genomic information, resulting in higher selection accuracy.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for breeding superior oyster strains resistant to Vibrio based on whole genome selection, characterized in that: The following steps are involved: Reference population construction: Select parents with different genetic backgrounds to construct a basic breeding population, and wait for the offspring to grow to one year old or above to serve as the reference population; Resistance phenotype determination: Use a highly virulent strain of Vibrio alginolyticus as a model pathogen to conduct a challenge experiment and evaluate the resistance phenotype of the reference population; Genotype data acquisition; Construction of the optimal genomic selection model: Using cross-validation to obtain the Bayes A genomic selection model with the best prediction accuracy for breeding value estimation; Evaluation of genomic breeding values ​​of candidate parents: Individuals from the same population, with the same parents, and cultured in the same sea area as the reference population were selected as candidate parents, and the genomic breeding values ​​of the candidate parents were estimated using the Bayes A model; Resistance selection population breeding; Select effectiveness assessment; Wherein, the reference population is selected from adult oysters in the disease outbreak season; The steps of the challenge experiment are as follows: Bacterial culture: Vibrio alginolyticus Cg5 was cultured in 2216E culture medium. After 12 hours of incubation, the culture was centrifuged and the bacterial pellet was washed three times with sterile seawater and diluted to 5 × 10 8 CFU / mL, bacterial concentration was measured using a UV spectrophotometer; Bacterial injection: Each oyster was injected with 0.1 mL of Vibrio suspension into the adductor muscle, with an injection dose of 5 × 10 7 CFU; After the injection, the oysters were cultured in seawater with a pH of 8.1, a dissolved oxygen of 8 mg / L, a temperature of 22°C, and a salinity of 30-32‰ for observation; After the challenge experiment, the binary phenotype of survival status of the individual after the challenge and the survival time from the start of the challenge experiment to the death of the oyster are used as the measurement criteria to measure the resistance phenotype of the reference population to Vibrio alginolyticus; The oysters used for the reference population phenotype assessment were healthy, had no OsHV-1 virus detected, and had a Vibrio load of less than 50 CFU / g; The genomic breeding value evaluation steps of the candidate parents are as follows: oyster individuals of the candidate population are immersed in a 50 g / L magnesium chloride solution for anesthesia, and after the oysters are opened, 50 mg of gill filaments are collected for DNA extraction and genotyping.

2. The breeding method according to claim 1, characterized in that: The genotype acquisition step comprises: performing genotyping of the whole genome SNPs of the long oyster using genome sequencing technology or SNP genotyping chip to obtain genotype data of the reference population, and performing genotype filling after filtering based on the minor allele frequency and genotype missing rate for the construction of the genomic selection model.

3. The breeding method according to claim 1, characterized in that: The resistance breeding population breeding step is: select the top 15% individuals with the highest breeding values ​​as resistance parents, and the other individuals as control parents, and breed the resistance breeding population and the control population separately.

4. The breeding method according to claim 1, characterized in that: The selection effectiveness evaluation includes the evaluation of Vibrio resistance in the larval stage and the evaluation of Vibrio resistance in the adult stage.

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