Breeding method for visceral sarcoidosis resistant strain of cynoglossus semilaevis based on whole genome selection

Abstract

The invention belongs to the technical field of molecular breeding of aquatic animals, and particularly relates to a breeding method of a cynoglossus semilaevis visceral sarcoidosis resistant strain based on whole genome selection. The method specifically comprises the following steps: selecting cynoglossus semilaevis parents with different genetic backgrounds, constructing a basic breeding group, and performing disease-resistant phenotype evaluation; selecting candidate group individuals having the same genetic background as the reference group, and estimating the genome breeding value of the candidate group by using a GBLUP model; screening genome breeding values from the candidate groups for sorting, selecting individuals at the front stage of sorting as disease-resistant parents, breeding a disease-resistant breeding group and a control group, and performing visceral sarcoidosis infection verification on the disease-resistant breeding group and the control group. Results show that the disease resistance of the disease-resistant breeding group is obviously superior to that of a control group. The method provided by the invention improves the accuracy of genome breeding value estimation, shortens the breeding period, has high application value and popularization prospect, and provides reference for disease-resistant breeding of other aquatic products.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular breeding of aquatic animals, and particularly relates to a method for breeding a strain of Semilaevis semilaevis resistant to visceral sarcoidosis based on whole genome selection. Background Art

[0002] Cynoglossus semilaevis Cynoglossus semilaevis ) is an important marine economic aquaculture fish in northern and southeastern my country. In recent years, marine mycobacterium ( Mycobacterium marinum ) causes huge economic losses to the farming industry of Semi-smooth Tongue Sole. The disease mainly attacks the internal organs of fish, causing nodules to form, and ultimately leading to organ dysfunction and high mortality. The prevalence of visceral sarcoidosis not only reduces the output and economic benefits of aquaculture, but also increases the cost of management and treatment. At present, the prevention and control of visceral sarcoidosis mainly relies on antibiotic treatment and environmental control. However, marine mycobacteria have strong drug resistance and environmental adaptability, which makes traditional prevention and control methods limited in effect. Faced with these challenges, finding more effective prevention and control measures has become an urgent task in the field of aquaculture.

[0003] Traditional breeding methods, such as group selection and family selection, have shortcomings such as low selection accuracy and slow genetic progress, especially when targeting low heritability and difficult-to-quantify traits (such as disease resistance traits, growth traits, etc.). In the process of breeding half-smooth tongue sole for visceral sarcoidosis, traditional breeding methods are inefficient due to long cycles, slow progress, and difficulty in screening individuals with excellent disease-resistant genotypes. Therefore, it is particularly important to use whole genome selection technology. Based on the variation information within the genome, this technology can estimate the genomic breeding value (GEBV) of candidate individuals through the phenotypic and genotypic data of the reference population without directly measuring the phenotypic value. This makes whole genome selection an ideal breeding method, especially when dealing with difficult-to-measure traits. Whole genome selection technology can comprehensively analyze the genome information of half-smooth tongue sole, evaluate the disease resistance potential of individuals without directly measuring the phenotype, accelerate the breeding process of disease resistance traits, and improve breeding efficiency and accuracy, thereby providing strong support for the breeding of excellent varieties of half-smooth tongue sole resistant to visceral sarcoidosis. Therefore, the introduction of whole genome selection technology is a necessary way to break through the existing breeding bottleneck and achieve efficient disease-resistant breeding. Summary of the invention

[0004] The present invention aims to provide a method for breeding an excellent strain of Semilaevis semilaevis resistant to visceral sarcoidosis based on whole genome selection.

[0005] To achieve the above purpose, the present invention adopts the following technical solution: A method for breeding a strain of Cynoglossus semilaevis resistant to visceral sarcoidosis based on whole genome selection: 1) Construction of a reference population of Cynoglossus semilaevis against visceral sarcoidosis Select half-smooth tongue sole parents with different genetic backgrounds to construct a basic breeding population, and wait for their offspring to grow to more than six months old as a reference population; 2) Determination of disease resistance phenotype of reference population of Cynoglossus semilaevis against visceral sarcoidosis Using marine mycobacterium strains as model pathogens, infection experiments were conducted on the reference populations to evaluate the disease resistance phenotype of the reference populations; 3) Obtaining the genotype of the reference population of Cynoglossus semilaevis for resistance to visceral sarcoidosis Whole genome resequencing was used to perform genotyping on the reference population, and the genotype data of the reference population was obtained, which was then used to construct the genomic selection model after filtering for minor allele frequency and genotype missing rate; 4) Analysis of genomic breeding value of candidate populations Individuals from the same population as the reference population, with similar genetic backgrounds and kept together were selected as candidate populations, and the GBLUP model was used to analyze the genomic breeding values ​​of the candidate populations; 5) Cultivation of anti-visceral sarcoidosis population Through the above-mentioned genomic breeding value analysis and ranking, individuals in the candidate group with high breeding values ​​analyzed by the GBLUP model are selected as disease-resistant parents for disease-resistant breeding, and the resulting offspring can achieve disease resistance.

[0006] In step 2), healthy individuals were selected for phenotypic evaluation of the reference population, and the Vibrio load was less than 20 CFU / g. The individual injection concentration was 5×10 6 CFU / mL of the model pathogen strain was prepared and then cultured to obtain the disease-resistant phenotype of the reference population.

[0007] The injected half-laevis semilaevis was cultured in seawater with a pH value of 8.0, a dissolved oxygen of 8.5 mg / mL, a temperature of 26° C., and a salinity of 30-32 for observation.

[0008] After the infection experiment is completed, the binary state of individual survival or death after the infection is completed and the survival time from the start of the infection experiment to the death of the half-smooth tongue sole are used as measurement standards to measure the disease resistance phenotype of the reference group to marine mycobacteria.

[0009] The genotype acquisition was performed using PLINK v1.90b4 software to process and filter the genotype data. The specific filtering criteria included: first, using --geno 0.01 to filter out SNPs with a missing rate of more than 1%; second, using --hwe10 -6SNPs with Hardy-Weinberg equilibrium test P values ​​less than 0.000001 were screened out. At the same time, --maf 0.05 was used to retain SNPs with minor allele frequencies greater than 5% to ensure data diversity. --mind 0.1 filtered out samples with missing rates exceeding 10%, and the final data output format was set to VCF.

[0010] The steps for obtaining the genomic breeding values ​​of the candidate population in step 4) are as follows.

[0011] (1) Genotype data acquisition: PLINK v1.90b4 software was used to perform genotyping on the candidate population and obtain its genotype data.

[0012] (2) Phenotypic data integration: Combined with the known phenotypic data of the reference population, the relationship between genotype and phenotype is established through the mixed model equation (MME) for subsequent calculations.

[0013] (3) Constructing the gene relationship matrix: Based on the genotype data of the candidate population and the reference population, calculate the standardized gene relationship matrix (G matrix). The calculation formula of the G matrix is ​​as follows:

[0014] Where X is the genotype matrix of the individual, is the allele frequency.

[0015] (4) Calculation of genomic breeding value: The genomic breeding value is calculated using the GBLUP association model. The calculation formula is as follows:

[0016] Among them, G is the G matrix of the candidate group and the reference group, is the inverse matrix of the reference group G matrix, is the phenotypic value of the reference population.

[0017] The candidate population is healthy half-smooth tongue sole parents that come from the same population as the reference population, have a similar genetic background, and are kept together and can be used for subsequent breeding.

[0018] The beneficial effects of the present invention are: The present invention overcomes the problems of low selection accuracy and slow breeding progress in the traditional breeding methods for half-smooth tongue sole resistance to visceral sarcoidosis by introducing whole genome selection technology, and has significant beneficial effects. This technology uses genotype information to accurately predict the genetic potential of disease-resistant traits, greatly improving the accuracy and efficiency of breeding. By evaluating the disease resistance of individuals through genomic breeding values ​​(GEBV), it is possible to screen out excellent individuals at an early stage, accelerate the breeding process, and shorten the breeding cycle. At the same time, the present invention supports the efficient management of large-scale breeding populations, improves the genetic potential of the population and the success rate of disease-resistant variety breeding, and reduces breeding costs. In summary, the present invention provides a scientific, efficient, and accurate solution for the breeding of half-smooth tongue sole resistance to visceral sarcoidosis, accelerates the breeding process of new varieties, and has important application value and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A flowchart for implementing genome selection breeding of Semilaevis semilaevis for resistance to visceral sarcoidosis provided in an embodiment of the present invention.

[0020] Figure 2 This is a distribution diagram of the death time of individuals who died during the measurement of disease resistance traits of a reference group during the specific implementation of the present invention.

[0021] Figure 3 Principal component analysis of population structure for the reference and candidate groups.

[0022] Figure 4 It is the genomic breeding value of the disease-resistant breeding population and the control population in the candidate population.

[0023] Figure 5 It is the verification result of marine mycobacterium infection of the disease-resistant breeding population in the specific implementation process of the present invention; wherein, the horizontal axis represents the death time, and the vertical axis represents the survival probability. DETAILED DESCRIPTION

[0024] The scheme of the present invention is explained below in conjunction with the embodiments. Those skilled in the art will understand that the following embodiments are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention.

[0025] The present invention significantly improves the breeding efficiency and accuracy of disease-resistant traits through accurate genomic breeding value estimation and efficient genomic selection model. The specific steps include: selecting half-smooth tongue sole parents with different genetic backgrounds, constructing a basic breeding population, and evaluating disease-resistant phenotypes; determining GBLUP as the model with the best prediction accuracy through 5-fold cross-validation; selecting individuals from a candidate population with the same genetic background as the reference population, and estimating the genomic breeding value of the candidate population using the GBLUP model; screening the genomic breeding value ranking from the candidate population, selecting the top ranked individuals as disease-resistant parents, breeding disease-resistant breeding populations and control populations, and verifying their visceral sarcoidosis infection ( Figure 1 ). The results showed that the disease resistance of the disease-resistant breeding population was significantly better than that of the control population. The method of the present invention improves the accuracy of genomic breeding value estimation, shortens the breeding cycle, has high application value and promotion prospects, and provides a reference for disease-resistant breeding of other aquatic species.

[0026] The half-smooth tongue sole population obtained by the method of the present invention can significantly improve the resistance to visceral sarcoidosis, accelerate the breeding of new half-smooth tongue sole varieties, and ensure the healthy development of the half-smooth tongue sole breeding industry.

[0027] In the following implementation manner, the reference populations with different genetic backgrounds are selected from Cynoglossus semilaevis with different genetic backgrounds selected from the Bohai Sea, the Yellow Sea, and artificially bred populations.

[0028] Example 1

[0029] When breeding different species based on the whole genome, it is necessary to build a prediction model suitable for their genetic background and target traits. Different models may have different applicability in different species and traits due to different calculation assumptions, parameter settings and sensitivity to genomic variation information. Therefore, choosing a suitable model is crucial to improving the accuracy and prediction effect of genomic breeding values ​​(GEBV).

[0030] In the context of the genome of Cynoglossus semilaevis, which model has the highest accuracy needs to be determined experimentally. To this end, this embodiment uses a cross-validation method for evaluation. The specific method includes cross-validation and screening from GBLUP and four Bayes methods (BayesA, BayesB, BayesCpi and BayesL), which can be expressed as: .

[0031] Where b represents the vector of fixed effects (including the first three principal components, as well as the weight, length and width of the fish), g represents the vector of additive effects, e represents the vector of residual effects, and X and Z are association matrices used to connect fixed effects and additive genetic values. GBLUP generates the effect value of each marker by maximum likelihood estimation, while the three Bayesian methods estimate the effect value of each SNP by Gibbs sampling. The product of the effect value b of each individual marker and the marker matrix is ​​the genomic breeding value of the individual. The entire data set was randomly divided into a training set (75% of the data) and a test set (25% of the data) by random sampling 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 prediction accuracy of the model was measured by the Pearson correlation coefficient between the phenotype of the test set and its estimated breeding value.

[0032] Table 1 The genomic prediction accuracy of half-smooth tongue sole's resistance to visceral sarcoidosis under different genomic selection models

[0033] From the above, we can see that the cross-validation results show that the GBLUP model has a higher prediction accuracy among the five models. The accuracy of estimating the binary trait of survival or not is 0.4925 ± 0.0274, and the accuracy of estimating the survival time is 0.5477 ± 0.0389 (Table 1); thus, the GBLUP model is determined to be the best prediction model.

[0034] Example 2

[0035] (1) Construction of a reference population for Cynoglossus semilaevis against visceral sarcoidosis Half-smooth tongue soles with different genetic backgrounds were selected as parents (179) to construct the basic breeding population. The gonadal development was determined by artificial touching, and half-smooth tongue soles with mature gonadal development were selected for artificial insemination. The obtained sperm and eggs were mixed in a sperm-egg ratio of 10:1, and stirred evenly with a glass rod. The development of the fertilized eggs was observed under a microscope every 10 minutes. After the fertilized eggs expelled the second polar body, they were transported to the Yellow Sea Aquatic Products Co., Ltd. in Haiyang City, Shandong Province for hatching. After successful hatching, they were artificially fed with plankton (such as Artemia larvae or rotifers) suitable for the nutritional needs of half-smooth tongue sole fry according to conventional breeding techniques. When the fry developed to the mouth-slit stage, they were gradually transitioned to feeding fine-grained compound feed to ensure that their nutritional needs were met. When the fry developed to the point where they could eat independently, they were transferred to the nursery pond for breeding management. About 40 days later, when the body length of the young fish reaches 2 to 3 cm, appropriate stocking density and environmental management techniques are combined to ensure that the growth and development of the farmed individuals reach an ideal level, which serves as a reference group.

[0036] (2) Determination of the disease resistance phenotype of the reference population of Cynoglossus semilaevis against visceral sarcoidosis When the half-smooth tongue sole of the reference group grew to 6 months old, the reference groups with different genetic backgrounds were transferred to the infection workshop for five days to adapt to the environment, and then the marine mycobacterium infection experiment was carried out. First, the half-smooth tongue sole reference group was tested for pathogens using tissue homogenate plate count method and PCR reaction to ensure that the reference group was not infected by pathogens and was in a healthy state before the experiment.

[0037] Secondly, the marine mycobacterium (strain number: QDU-CS2 model strain preserved by Qingdao University) was cultured in Middlebrook 7H10 liquid medium, and the culture method was shaking culture, and the shaking parameters were 28°C and 180rpm. After 5 days of culture, centrifugation was performed, and the bacterial precipitate was repeatedly washed 3 times with sterile seawater and diluted to 5×10 6 CFU / mL, and the bacterial concentration was measured using a UV spectrophotometer.

[0038] Finally, each half-smooth tongue sole of the healthy reference group was injected intraperitoneally with 100 μL of marine mycobacterium suspension at a concentration of 5×10 6 CFU / mL; after the injection, the half-smooth tongue sole was cultured in seawater with a pH value of 8.0, a dissolved oxygen of 10 mg / mL, a temperature of 26°C, and a salinity of 30-32‰ for observation. During the infection experiment, the seawater was changed twice a day and the survival and death of the half-smooth tongue sole was recorded. The dead half-smooth tongue sole was immediately removed and the tail fin tissue was quickly frozen with liquid nitrogen and stored in a -80°C refrigerator. The infection experiment lasted for 20 days. At the end of the experiment, a total of 682 half-smooth tongue soles died and 275 half-smooth tongue soles survived, with a survival rate of 28.73%. The results are shown in Figure 2 .

[0039] (3) Genotype acquisition of a reference population of Cynoglossus semilaevis for resistance to visceral sarcoidosis 320 half-smooth tongue soles from the above reference population (the 320 were the first 160 susceptible individuals that died after infection and the last 160 resistant individuals that survived) were selected for genotyping using whole genome resequencing. DNA was extracted from the 320 materials, and the second-generation resequencing Illumina library was constructed after extraction. The library was sequenced using the PE150 strategy, and the sequencing volume of each sample was 5 Gb. The original sequence was filtered by FastQC (v 0.12.1) and all clean reads were aligned with the half-smooth tongue sole reference gene group (NCBI number: GCF_000523025.1). GATK (v 3.8) was used to filter SNPs for each sample, and then PLINK v1.90b4 software was used with --geno 0.01 to filter out SNPs with a missing rate of more than 1%; secondly, --hwe 10 -6 SNPs with Hardy-Weinberg equilibrium test P values ​​less than 0.000001 were screened out. At the same time, --maf0.05 was used to retain SNPs with minor allele frequencies greater than 5% to ensure data diversity. --mind 0.1 filtered out samples with a missing rate of more than 10%. Finally, the data output format was set to VCF to obtain high-quality SNPs. That is, after genotyping using whole-genome resequencing, a total of 3.44 million high-quality SNPs were obtained for subsequent analysis.

[0040] (4) Estimation of genomic breeding values ​​of candidate populations The candidate population and the reference population come from the same population, have the same parents, and are raised in the same area. The purpose of keeping the individuals together as candidate parents is to ensure that their growth stage and physiological state are consistent with the reference population to improve the accuracy of genomic breeding value estimation.

[0041] The tail fins of the semi-laevis of the candidate population confirmed above were cut and DNA was extracted for genotyping. A total of 172 candidate parents were resequenced for whole genomes, and their genotype data were obtained according to the same filtering parameters as the reference population. After quality control of the genotype data, the genomic breeding value of the candidate population was estimated using the GBLUP model predicted to be the most accurate in cross-validation. In the GEBV estimation process, the GBLUP model assumes that all SNPs have the same additive genetic effect on the target trait, and uses the genomic kinship matrix (G-matrix) instead of the traditional phenotypic kinship matrix (A-matrix) to calculate the genetic similarity between individuals. Specifically, the BLUP method was used to estimate the SNP effect, and the GEBV value of each candidate parent was calculated in combination with the individual genotype information. The specific calculation formula is as follows.

[0042] The calculation formula of G matrix is:

[0043] Among them, X is the genotype matrix of the individual, is the allele frequency.

[0044] The GEBV calculation formula is as follows:

[0045] Among them, G is the G matrix of the candidate group and the reference group, is the inverse matrix of the reference group G matrix, is the phenotypic value of the reference population.

[0046] PCA population structure analysis was performed on the filtered genotype data. The results showed that the reference population and the candidate population had similar population structure characteristics, indicating that they had the same population origin, thus ensuring the accuracy of the genomic breeding value assessment (see Figure 3 The breeding values ​​of the parents estimated by the genomic selection model ranged from 0.18 to 0.46, indicating that there were significant differences in disease resistance between the candidate parents and that they could serve as potential indicators for measuring visceral sarcoidosis resistance (see Figure 4 ).

[0047] (5) Disease resistance breeding population After estimating and ranking the candidate population through the above steps, select the top 20% of the individuals in the candidate population in terms of genomic breeding values ​​as disease-resistant parents, and the other individuals as control parents. Then, the disease-resistant breeding population and the control breeding population are bred separately according to the existing breeding method.

[0048] After breeding, the breeding value calculated from the parental genomes of the disease-resistant breeding population was as low as 0.347 and as high as 0.425 on average, which was significantly higher than 0.243 of the control parent.

[0049] In the summer when the gonads of half-smooth tongue sole mature, the gonad development is determined by artificial touching, and half-smooth tongue sole with mature gonads are selected for artificial insemination. The obtained sperm and eggs are mixed in a sperm-egg ratio of 10:1, and stirred evenly with a glass rod. The development of the fertilized eggs is observed under a microscope every 10 minutes. After the fertilized eggs expel the second polar body, they are transported to the Yellow Sea Aquatic Products Co., Ltd. in Haiyang City, Shandong Province for hatching. After successful hatching, they are artificially fed with plankton (such as Artemia larvae or rotifers) that are suitable for the nutritional needs of half-smooth tongue sole fry. When the fry develop to the stage of mouth slit, they are gradually transitioned to feeding fine-grained compound feed to ensure that their nutritional needs are met. When the fry develop to the point where they can feed independently, they are transferred to the nursery pond for breeding management. About 40 days later, when the body length of the fry reaches 2~3 cm, appropriate breeding density and environmental management technology are combined to ensure that the growth and development of the cultured individuals reach an ideal level.

[0050] (6) Select effectiveness evaluation In the season when visceral sarcoidosis breaks out (mid-October every year), 300 six-month-old fry from the insect-resistant first generation (i.e., bred from the parents of the disease-resistant breeding population) and the control group were selected for marine mycobacterium infection experiments. The infection experiment process was consistent with the infection process for determining the disease resistance traits of the reference population.

[0051] Survival analysis of the infection results revealed significant differences in disease resistance between the resistant first generation and the control group. Specifically, at 11 days, the peak of the disease incidence, the survival rate of the disease-resistant breeding group was 61.2%, while that of the control group was 40%. At 20 days, the end of the infection experiment, the survival rate of the disease-resistant breeding group was 40.2%, while that of the control group was 21.4%. Figure 5 ).

[0052] The present invention establishes a breeding method for the visceral sarcoidosis resistance trait of half-smooth tongue sole based on genomic selection technology, and for the first time uses whole genome selection breeding technology to carry out the visceral sarcoidosis resistance trait selection breeding of half-smooth tongue sole, and selects excellent strains with visceral sarcoidosis resistance. Compared with traditional methods, this technology does not require the candidate parents to be subjected to toxicity tests, avoids the risk of pathogen transmission to parents and offspring, and effectively ensures the health of seedlings during breeding. In addition, this technology is particularly suitable for disease-resistant traits with low heritability. By carrying out breeding based on genomic information, the interference of environmental factors on the results of phenotypic breeding is avoided. A genome selection reference population is constructed using half-smooth tongue sole, and individuals with the same population source and growth stage are selected as candidate parents for selection breeding to ensure the representativeness of the resistance phenotype of the reference population, making it more valuable for practical application.

[0053] Finally, it should be noted that the above-described embodiments are only intended to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the specific contents of each embodiment have been described in detail, it should be understood by those skilled in the art that these technical solutions may be appropriately modified or some or all of the technical features may be replaced with equivalent features. These adjustments or replacements do not change the essence of the technical solutions and do not exceed the technical scope covered by the embodiments of the present invention.

Claims

1. A method for breeding a strain of Cynoglossus semilaevis resistant to visceral sarcoidosis based on whole genome selection, characterized in that: 1) Construction of a reference population of Cynoglossus semilaevis against visceral sarcoidosis Select half-smooth tongue sole parents with different genetic backgrounds to construct a basic breeding population, and wait for their offspring to grow to more than six months old as a reference population; 2) Determination of disease resistance phenotype of reference population of Cynoglossus semilaevis against visceral sarcoidosis Using marine mycobacterium strains as model pathogens, infection experiments were conducted on the reference populations to evaluate the disease resistance phenotype of the reference populations; 3) Genotype acquisition of reference population of Cynoglossus semilaevis for resistance to visceral sarcoidosis Whole genome resequencing was used to perform genotyping on the reference population to obtain genotype data of the reference population, which was then used to construct a genomic selection model after filtering for minor allele frequency and genotype missing rate; 4) Analysis of genomic breeding value of candidate populations Individuals from the same population as the reference population, with similar genetic backgrounds and kept together were selected as candidate populations, and the GBLUP model was used to analyze the genomic breeding values ​​of the candidate populations; 5) Cultivation of anti-visceral sarcoidosis population Through the above-mentioned genomic breeding value analysis and ranking, individuals in the candidate group with high breeding values ​​analyzed by the GBLUP model are selected as disease-resistant parents for disease-resistant breeding, and the resulting offspring can achieve disease resistance.

2. The method for breeding a strain of Cynoglossus semilaevis resistant to visceral sarcoidosis based on whole genome selection according to claim 1, characterized in that: In step 2), healthy individuals were selected for phenotypic evaluation of the reference population, and the Vibrio load was less than 20 CFU / g. The individual injection concentration was 5×10 6 CFU / mL of the model pathogen strain was prepared and then cultured to obtain the disease-resistant phenotype of the reference population.

3. The method for breeding a strain of Cynoglossus semilaevis resistant to visceral sarcoidosis based on whole genome selection according to claim 2, characterized in that: The injected half-laevis were cultured in seawater with a pH of 8.0, a dissolved oxygen of 8.5 mg / mL, a temperature of 26°C, and a salinity of 30-32 for observation.

4. The method for breeding a strain of Cynoglossus semilaevis resistant to visceral sarcoidosis based on whole genome selection according to claim 1 or 2, characterized in that: After the infection experiment is completed, the binary state of individual survival or death after the infection and the survival time from the start of the infection experiment to the death of the half-smooth tongue sole are used as measurement standards to measure the disease resistance phenotype of the reference population to marine mycobacterium.

5. The method for breeding a strain of Cynoglossus semilaevis resistant to visceral sarcoidosis based on whole genome selection according to claim 1, characterized in that: The genotype acquisition was performed using PLINK v1.90b4 software to process and filter the genotype data. The specific filtering criteria included: first, using --geno 0.01 to filter out SNPs with a missing rate of more than 1%; second, using --hwe 10 -6 SNPs with Hardy-Weinberg equilibrium test P values ​​less than 0.000001 were screened out. At the same time, --maf 0.05 was used to retain SNPs with minor allele frequencies greater than 5% to ensure data diversity. --mind 0.1 filtered out samples with missing rates exceeding 10%, and the final data output format was set to VCF.

6. The method for breeding a strain of Cynoglossus semilaevis resistant to visceral sarcoidosis based on whole genome selection according to claim 1, characterized in that: Step 4) The genomic breeding value of the candidate population is to obtain the genotype information of the candidate population using the filtering criteria in step 3), and store it in VCF format, and calculate the G matrix of the reference population and the candidate population according to the GBLUP model: And then calculate the genomic breeding value according to the formula: 。 7. The method for breeding a strain of Cynoglossus semilaevis resistant to visceral sarcoidosis based on whole genome selection according to claim 1 or 6, characterized in that: The candidate population is healthy half-smooth tongue sole parents that come from the same population as the reference population, have a similar genetic background, and are kept together and can be used for subsequent breeding.

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