CN114736969A - Method for selecting anti-nervous necrosis virus individual of epinephelus lanceolatus - Google Patents

Method for selecting anti-nervous necrosis virus individual of epinephelus lanceolatus Download PDF

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CN114736969A
CN114736969A CN202210196158.5A CN202210196158A CN114736969A CN 114736969 A CN114736969 A CN 114736969A CN 202210196158 A CN202210196158 A CN 202210196158A CN 114736969 A CN114736969 A CN 114736969A
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necrosis virus
nervous necrosis
epinephelus lanceolatus
snp
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王庆
秦启伟
杨敏
段旭琢
梁凯珊
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Guangdong Provincial Laboratory Of Lingnan Modern Agricultural Science And Technology
South China Agricultural University
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South China Agricultural University
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Abstract

The invention discloses a method for selecting an anti-nervous necrosis virus individual of epinephelus lanceolatus, which comprises the following steps: (1) obtaining SNP locus information of the epinephelus lanceolatus; (2) analyzing the disease-resistant weight value of the SNP marker and selecting the SNP marker; (3) obtaining candidate Epinephelus lanceolatus individual SNP locus information and selecting an individual with anti-nervous necrosis virus capability; (4) and (5) checking the selection accuracy of the disease-resistant individuals. The method screens the SNP marker with the maximum weight of resisting the nervous necrosis virus from the SNP markers of the epinephelus lanceolatus, so as to establish a method capable of selecting an individual resisting the nervous necrosis virus.

Description

Method for selecting anti-nervous necrosis virus individual of epinephelus lanceolatus
Technical Field
The invention belongs to the technical field of grouper breeding, and particularly relates to a selection method of an anti-nervous necrosis virus individual of grouper saddletree.
Background
Epinephelus lanceolatus, also known as Epinephelus lanceolatus or Epinephelus longata, belongs to the order Perciformes (Perciformes), the family Serranidae (Serranidae), and the genus Epinephelus (Epinephelus). The fish with the largest body type is also called as "Banwang". The epinephelus lanceolatus is large in size, rich in nutrition, delicious in meat quality and high in growth speed, and is a favorite seafood fish. Epinephelus lanceolatus has become a main object for mariculture in southern China, such as Guangdong, Hainan, Fujian, Guangxi and the like, and has great economic value.
At present, with the increasing of the breeding amount, diseases and insect pests in the breeding process of grouper become more and more obvious, in particular to Viral nervous necrosis disease (VNN), the pathogen of which is Nervous Necrosis Virus (NNV). The virus belongs to the genus of beta nodavirus, is a type of parvovirus, and can be divided into 4 genotypes: parafin nervous necrosis virus genotype (striedjack NNV, SJNNV), takifugu rubripes nervous necrosis virus genotype (Tiger filler NNV, TPNNV), verasper variegates nervous necrosis virus genotype (Barfin junder NNV, BFNNV) and Red spotted rocky nervous necrosis virus genotype (RGNNV). The RGNNV is the most harmful to the groupers, the groupers can be infected with the virus basically in the seedling stage, the nervous system and retina of the groupers are mainly damaged, the larval fish and the young fish are easy to be infected by the virus, and the infection death rate is over 90 percent and even reaches 100 percent. At present, no effective vaccine exists, the virus starts to attack at the juvenile fish stage, and the vaccine is difficult to inject.
Therefore, development of breeding of disease-resistant varieties is an effective way for coping with the virus and improving the yield of the groupers. However, in the breeding process, the breeding period of the epinephelus lanceolatus is found to be long, the first generation of the epinephelus lanceolatus needs 6 to 8 years to be generally bred, and the traditional breeding mode is not practical. Therefore, the breeding by combining the genetic information of the epinephelus lanceolatus per se can accelerate the breeding process, in addition, the disease-resistant character can not be observed through phenotype, particularly, the disease-resistant parent can not be selected for filial generation breeding, so that the breeding difficulty is further increased, and therefore, a method which is accurate and can predict the disease resistance of the epinephelus lanceolatus through the genetic information such as SNP markers is urgently needed.
Disclosure of Invention
The invention aims to provide a method for selecting an individual with the anti-nervous necrosis virus of epinephelus lanceolatus, which is established by screening the SNP marker with the maximum anti-nervous necrosis virus weight from the SNP markers of the epinephelus lanceolatus.
The above object of the present invention can be achieved by the following technical solutions: a method for selecting an individual of Epinephelus lanceolatus for resisting nervous necrosis virus comprises the following steps:
(1) obtaining SNP locus information of epinephelus lanceolatus: selecting healthy epinephelus lanceolatus fries, attacking the epinephelus lanceolatus fries with red spot nervous necrosis virus (RGNNV), collecting fin rays or muscles of dead fries for storage, setting a susceptible group and an anti-susceptible group, and performing whole genome re-sequencing and GWAS analysis to obtain SNP site information of the epinephelus lanceolatus;
(2) analyzing the disease resistance weight value of the SNP marker and selecting the SNP marker: taking the resistance and the susceptibility as phenotypes, calculating the weight of SNP loci by using CropGBM software, mining SNP loci related to the phenotypes, screening to obtain 722 SNP markers with the highest weight values, and selecting the SNP markers for the disease resistance individuals of the Epinephelus lanceolatus;
(3) obtaining candidate Epinephelus lanceolatus individual SNP locus information and selecting anti-nervous necrosis virus individuals: collecting fin rays of candidate group fishes, extracting fin ray genome DNA, performing whole genome re-sequencing, calculating the anti-disease breeding value of the candidate Epinephelus lanceolatus by using 722 SNP markers obtained in the step (2) and adopting a GAPIT3 method, and selecting individuals with the breeding value larger than 28 as anti-nervous necrosis virus individuals and individuals with the anti-nervous necrosis virus capacity smaller than 28 as anti-nervous necrosis virus individuals;
(4) checking the selection accuracy of disease-resistant individuals: when the candidate population is a fry with the length within 10cm, performing virus attack on the fish individuals with the breeding values of more than 28 and less than 28 obtained by calculation, respectively counting the mortality, and when the disease resistance of the individuals with the breeding values of more than 28 is strong and the disease resistance of the individuals with the breeding values of less than 28 is poor, selecting the nervous necrosis virus resistant individuals of the epinephelus lanceolatus by using the breeding values in the step (3) accurately; and (3) when the candidate population is a parent fish population, performing successful toxicity on filial generations propagated by parents with the breeding value of more than 28 and less than 28, and when the disease resistance of the filial generations propagated by the parents with the breeding value of more than 28 is stronger than that of the filial generations propagated by the parents with the breeding value of less than 28, accurately selecting the anti-nervous necrosis virus individual of the epinephelus lanceolatus parent by adopting the breeding value in the step (3).
In the method for selecting the anti-nervous necrosis virus individual of the epinephelus lanceolatus, the method comprises the following steps:
preferably, the healthy epinephelus lanceolatus fries obtained in the step (1) are uniform in size, 4-6 cm in length and 600-800 in number.
Preferably, the erythromelasma necrosis virus (RGNNV) in the step (1) is injected intraperitoneally, and the titer of the injected erythromelasma necrosis virus (RGNNV) is 107～108TCID50/m。
Preferably, the Epinephelus akaara nervous necrosis virus (RGNNV) in step (1) is isolated from Epinephelus akaara and obtained by laboratory cultivation using conventional methods, which can be referred to, but not limited to, the following references: separation and identification of perco carp source carp spring viremia virus and pathological observation thereof, Zhengli peace and the like, and Chinese aquatic science 2019.5; or the separation and partial characteristic study of the fish viral nervous necrosis virus, jiang jun, the university of chinese agriculture major academic thesis, 2008.
Preferably, the fin rays or muscles of the dead fish fries collected in the step (1) are stored, the observation is required to be carried out once every 2-3 hours, the death time point is recorded, the fin rays or muscles of the dead and old fish are cut and stored in alcohol with the volume percentage of 95%, and then the fish are transferred to a refrigerator at the temperature of-80 ℃ for long-term storage.
Preferably, 100-200 fish died first are selected as susceptible groups in the step (1), 100-200 fish without death are selected as resistant groups in the step (1), and if the number of fish without death is less than 100-200 fish, 100-200 fish of the rest live fish and fish died last are selected as resistant groups.
Preferably, when GWAS (whole genome association analysis) analysis is used in step (1), whole genome association analysis (GWAS) analysis is performed using the epinephelus lanceolatus genome as a reference genome in combination with the resistance and susceptibility to genes, wherein the reference genome is the epinephelus lanceolatus genome and is derived from: ASM528154v1https:// www.ncbi.nlm.nih.gov/genome/? term ASM528154v 1.
Preferably, in the step (2), the resistance and the susceptibility are taken as phenotypes, CropGBM (crop Genomic Breeding machine) software is used, and a lighting bm algorithm after a large amount of optimization is performed on a Gradient Boosting Decision Tree (GBDT) by using a calculation method, so that the weight of the SNP sites is calculated to excavate the SNP sites related to the phenotypes.
Preferably, the process of calculating the weight of the SNP sites in the step (2) to search the SNP sites related to the phenotype is as follows: calling plink, preprocessing genotype data according to the deletion rate and the heterozygosity, generating ped files, then preprocessing phenotype data, training a model and screening SNP sites, and finally screening to obtain 722 SNP markers with the highest weight values for selecting the disease-resistant individuals of the Epinephelus lanceolatus.
Preferably, an animal tissue DNA extraction kit is used for extracting the fin-ray genome DNA in the step (3), and the extracted DNA requires a single picking band when being subjected to gel electrophoresis.
Preferably, the breeding resistance value of the candidate Epinephelus lanceolatus is calculated in step (3) by a GAPIT3 method, wherein the GAPIT3 method is Genomic Association and differentiation Integrated Tool.
Preferably, the number of the candidate fish in the step (4) is more than 100.
Preferably, the toxic materials are attacked in the step (4) by using the akabane nervous necrosis virus (RGNNV), the akabane nervous necrosis virus (RGNNV) is injected into the abdominal cavity, and the titer of the injected akabane nervous necrosis virus (RGNNV) is 107～108TCID50/mL。
Compared with the prior art, the invention has the following advantages:
(1) the invention screens the SNP marker with the maximum weight of resisting the nervous necrosis virus from the SNP markers of the epinephelus lanceolatus, thereby establishing a method for screening the individual of the epinephelus lanceolatus resisting the nervous necrosis virus.
(2) The method has higher accuracy and higher use value, and the comprehensive judgment accuracy is higher;
(3) the method for selecting the anti-nervous necrosis virus individuals of the epinephelus lanceolatus provided by the invention solves the problem that the disease resistance of the epinephelus lanceolatus cannot be judged from the appearance;
(4) the method has the advantages of high efficiency, accuracy, small damage to parent fishes and the like;
(5) the method has obvious economic benefit and social benefit in the breeding aspect of the groupers.
Detailed Description
The present invention is further specifically described below by way of examples, but the embodiments of the present invention are not limited thereto.
Example 1
The method for selecting the anti-nervous necrosis virus individual of the epinephelus lanceolatus provided by the embodiment comprises the following steps of:
(1) acquisition of SNP locus information of epinephelus lanceolatus
Selecting healthy epinephelus lanceolatus fries (the healthy epinephelus lanceolatus fries are uniform in size, 4-6 cm in length and 600 tail in number), separating the epinephelus lanceolatus fries from the epinephelus lanceolatus by using RGNNV (RGNNV), and obtaining the epinephelus lanceolatus by adopting a conventional method for laboratory cultivation, wherein the cultivation method can refer to but not limited to the following documents, namely separation and identification of a perco carp source carp spring viremia virus and pathological observation thereof, Zheng Liping and the like, Chinese aquatic science, 2019.5, or research on separation and partial characteristics of the fish viral nervous necrosis virus, Jianfang Jun, Huazhong agriculture university Master thesis, 2008) is used for counteracting the virus, fin strips or muscles of dead fries are collected every day for preservation, and the death time point is recorded;
collecting the fin rays or muscles of dead fish fries for storage, observing every 2 hours, recording the death time point, shearing the fin rays and muscles of the fish which die and just die, storing in 95% (volume percentage) of alcohol, and then transferring to a refrigerator at-80 ℃ for long-term storage;
selecting fishes which die first as susceptible groups and live down after attacking as 100 tails of anti-susceptible groups, and performing whole genome re-sequencing and GWAS analysis to obtain SNP locus information of the epinephelus lanceolatus;
in GWAS (Whole genome association analysis), when the genome of Epinephelus lanceolatus is taken as a reference genome and combined with the resistance and susceptibility state to carry out the whole genome association analysis (GWAS), the reference genome is the genome of Epinephelus lanceolatus and is derived from the following sources:
ASM528154v1 https://www.ncbi.nlm.nih.gov/genome/？term＝ASM528154vl。
(2) analyzing the disease resistance weight value of the SNP marker and selecting the SNP marker: taking the resistance and the susceptibility as phenotypes, utilizing Crop Genomic Breeding machine (CropGBM) software, using a calculation method to carry out a lightgbm algorithm after a large amount of optimization on a Gradient Boosting Decision Tree (GBDT), calculating the weight of SNP sites, and mining the SNP sites related to the phenotypes for predicting the disease resistance of the epinephelus lanceolatus;
the process of calculating the weight of the SNP locus and mining the SNP locus related to the phenotype is as follows: calling plink, preprocessing genotype data according to deletion rate and heterozygosity, generating ped files, then preprocessing phenotype data, training a model and screening SNP sites, and finally screening to obtain 722 SNP sites with the highest weight values (shown in table 1) for predicting the disease resistance of the epinephelus lanceolatus.
TABLE 122 SNP loci with the highest weight values
Figure BDA0003525123440000051
Figure BDA0003525123440000061
Figure BDA0003525123440000071
Figure BDA0003525123440000081
Figure BDA0003525123440000091
Figure BDA0003525123440000101
Figure BDA0003525123440000111
Figure BDA0003525123440000121
Figure BDA0003525123440000131
Figure BDA0003525123440000141
Figure BDA0003525123440000151
Figure BDA0003525123440000161
Figure BDA0003525123440000171
Figure BDA0003525123440000181
Figure BDA0003525123440000191
Figure BDA0003525123440000201
Figure BDA0003525123440000211
Figure BDA0003525123440000221
(3) Obtaining candidate Epinephelus lanceolatus individual SNP locus information and selecting anti-nervous necrosis virus individuals: establishment of candidate population and whole genome re-sequencing: collecting fin rays of candidate group fishes (each fish is marked), extracting fin ray Genome DNA, performing whole Genome re-sequencing, processing and analyzing SNP locus data, performing Genome selection calculation under the condition of lacking a phenotype value, calculating individual breeding values by adopting 722 pieces of SNP locus information (table 1) screened in the step (2), calculating by using a Genome Association and Prediction Integrated Tool (GAPIT3) method, generating and outputting the breeding values of each fish after the calculation is finished, judging the fish as a disease-resistant individual when the breeding value is more than 28 according to the calculation, and judging the fish as the fish with poor disease resistance when the breeding value is less than 28;
the extraction of the DNA of the fin-ray genome adopts an animal tissue DNA extraction kit, and the extracted DNA requires a single picking band when gel electrophoresis is carried out.
(4) And (3) checking the selection accuracy of disease-resistant individuals: and (3) attacking the virus of individuals with the breeding value more than 28 and less than 28 obtained by calculation, respectively counting the death rate, and when the death rate is counted, if the disease resistance of the individuals with the breeding value more than 28 is strong (the death rate is low) and the disease resistance of the individuals with the breeding value less than 28 is poor (the death rate is high), judging that the breeding value can accurately select the individuals with high nervous necrosis virus resistance of the epinephelus lanceolatus.
During actual test, when the candidate population is a fry with the length within 10cm, performing virus attack on the fish individuals with the breeding values of more than 28 and less than 28 obtained by calculation, respectively counting the mortality, and when the disease resistance of the individuals with the breeding values of more than 28 is strong and the disease resistance of the individuals with the breeding values of less than 28 is poor, accurately selecting the nervous necrosis virus resisting individual of the Epinephelus lanceolatus by using the breeding values in the step (3); and (3) when the candidate population is a parent fish population, performing successful toxicity on filial generations propagated by parents with the breeding value of more than 28 and less than 28, and when the disease resistance of the filial generations propagated by the parents with the breeding value of more than 28 is stronger than that of the filial generations propagated by the parents with the breeding value of less than 28, accurately selecting the anti-nervous necrosis virus individual of the epinephelus lanceolatus parent by adopting the breeding value in the step (3).
Wherein the virus titer is 10 during virus attack7TCID50And (3) for viruses with the virus concentration of more than one mL, preferably performing intraperitoneal injection in a virus counteracting mode, wherein the size of the fish is about 4-5 cm during virus counteracting.
Example 2
To verify the accuracy of the authentication method in example 1, this example was verified using the following test:
(1) selecting 110 epinephelus lanceolatus seedlings of 3.15 +/-0.23 cm cultured by Hainan morning sea water product Limited in No. 6-20 in 2020, judging the disease resistance of the seedlings from the appearance, cutting a small number of tail fins, feeding the seedlings by using an independent feeding box, and marking the boxes;
(2) extracting DNA of each fish and performing re-sequencing to obtain SNP locus information of each fish;
(3) performing re-sequencing while attacking poison of each fish, and performing intraperitoneal injection of 30 μ L of the product with concentration of 2.46 × 107TCID50mL virus, record the marker number on dead fish box;
(4) calculating a breeding value according to the re-sequencing result by using Genome Association and Prediction Integrated Tool (GAPIT3), wherein the number of fish strips with the breeding value more than 28 is 11, and the number of fish strips with the breeding value less than 28 is 99 (Table 2);
TABLE 2 Breeding value and disease resistance of epinephelus lanceolatus fry
Figure BDA0003525123440000231
Figure BDA0003525123440000241
Figure BDA0003525123440000251
Figure BDA0003525123440000261
(5) Corresponding to the mark number of 11 fishes, the death rate of 3 fishes with the breeding value of more than 28 is calculated, the death rate is 27.27 percent, and the death rate of 89 fishes with the breeding value of less than 28 is 89.90 percent. The method provided by the invention is shown to have higher accuracy in determining the disease resistance of the groupers.
Example 3
To verify the accuracy of the authentication method in example 1, this example was also verified using the following test:
(1) 200 parent fishes of epinephelus lanceolatus, which are 123.15 +/-24.56 kg and are bred by Hainan morning sea water product Limited companies, are selected in No. 16 of 2020, at the moment, the disease resistance of the fries cannot be judged from the appearance, after fin tail fins are cut, electronic marks are marked on muscles of the back of each fish, and the mark corresponding to each fish is recorded.
(2) Extracting DNA of each fish and performing re-sequencing to obtain SNP locus information of each fish.
(4) The breeding values were calculated from the re-sequencing results using a Genome Association and Prediction Integrated Tool (GAPIT3), with 32 fish strips with a breeding value greater than 28 and 168 small fish strips 28 (Table 3).
(5) Breeding 32 disease-resistant parents and 168 disease-resistant parents corresponding to the marks, and obtaining 5.67 ten thousand tails of filial generations generated by the disease-resistant parents and 16.34 ten thousand tails of filial generations generated by the disease-resistant parents by adopting an artificial insemination mode. Is raised toAbout 4cm, randomly selecting 500 fishes per group for counteracting toxic substance, and performing intraperitoneal injection to obtain 30 μ L extract with concentration of 2.46 × 107TCID50Per mL of virus, mortality was recorded. The results indicated that the disease-resistant parent was judged to produce a progeny mortality rate of 60.80% and the disease-non-resistant parent was judged to produce a progeny mortality rate of 92.20% (table 3). The method has the advantage that the accuracy rate of judging the disease resistance of the parent of the grouper is high.
TABLE 3 parent fish breeding value and offspring disease resistance analysis of Epinephelus lanceolatus
Disease resistance judgment of parents Number of parents Average breeding value The number of offspring (Wan tailed) Sampling number of attacking toxin (tail) Mortality of sampled offspring
Disease-resistant parent 32 31.8647±2.1246 5.67 500 60.80％
Disease-resistant parent 168 18.9423±5.4798 16.34 500 92.20％
In conclusion, the result of observing the sample shows that the method has higher accuracy and higher use value, and the comprehensive judgment accuracy is higher. The method for identifying the nervous necrosis virus resisting individual of the epinephelus lanceolatus solves the problem that the disease resistance of the epinephelus lanceolatus cannot be predicted, and has the advantages of high efficiency, accuracy, small damage to parent fishes and the like. The technology has obvious economic benefit and social benefit in the breeding aspect of the groupers.
The present invention is illustrated by the following examples, which are not intended to limit the scope of the invention. Other insubstantial modifications and adaptations of the present invention can be made without departing from the scope of the present invention.

Claims (10)

1. A method for selecting an anti-nervous necrosis virus individual of epinephelus lanceolatus is characterized by comprising the following steps:
(1) obtaining SNP locus information of epinephelus lanceolatus: selecting healthy epinephelus lanceolatus fries, attacking the epinephelus lanceolatus fries with red spot nervous necrosis virus (RGNNV), collecting fin rays or muscles of dead fries for storage, setting a susceptible group and an anti-susceptible group, and performing whole genome re-sequencing and GWAS analysis to obtain SNP site information of the epinephelus lanceolatus;
(2) analyzing the disease resistance weight value of the SNP marker and selecting the SNP marker: taking the resistance and the susceptibility as phenotypes, calculating the weight of SNP loci by using CropGBM software, mining SNP loci related to the phenotypes, screening to obtain 722 SNP markers with the highest weight values, and selecting the SNP markers for the disease resistance individuals of the Epinephelus lanceolatus;
(3) obtaining candidate Epinephelus lanceolatus individual SNP locus information and selecting anti-nervous necrosis virus individuals: collecting fin rays of candidate group fishes, extracting fin ray genome DNA, performing whole genome re-sequencing, calculating the anti-disease breeding value of the candidate Epinephelus lanceolatus by using 722 SNP markers obtained in the step (2) and adopting a GAPIT3 method, and selecting individuals with the breeding value larger than 28 as anti-nervous necrosis virus individuals and individuals with the anti-nervous necrosis virus capacity smaller than 28 as anti-nervous necrosis virus individuals;
(4) checking the selection accuracy of disease-resistant individuals: when the candidate population is a fry with the length within 10cm, performing virus attack on the fish individuals with the breeding values of more than 28 and less than 28 obtained by calculation, respectively counting the mortality, and when the disease resistance of the individuals with the breeding values of more than 28 is strong and the disease resistance of the individuals with the breeding values of less than 28 is poor, selecting the nervous necrosis virus resistant individuals of the epinephelus lanceolatus by using the breeding values in the step (3) accurately; and (3) when the candidate population is a parent fish population, performing successful toxicity on filial generations propagated by parents with the breeding value of more than 28 and less than 28, and when the disease resistance of the filial generations propagated by the parents with the breeding value of more than 28 is stronger than that of the filial generations propagated by the parents with the breeding value of less than 28, accurately selecting the anti-nervous necrosis virus individual of the epinephelus lanceolatus parent by adopting the breeding value in the step (3).
2. The method for selecting an individual Epinephelus lanceolatus resistant to the nervous necrosis virus according to claim 1, wherein: the healthy epinephelus lanceolatus fries in the step (1) are uniform in size, 4-6 cm in length and 600-800 in number.
3. The method for selecting an individual Epinephelus lanceolatus resistant to nervous necrosis virus according to claim 1, wherein: the akabane nervous necrosis virus (RGNNV) in the step (1) adopts intraperitoneal injection, and the titer of the injected akabane nervous necrosis virus (RGNNV) is 107～108TCID50/mL。
4. The method for selecting an individual Epinephelus lanceolatus resistant to the nervous necrosis virus according to claim 1, wherein: collecting and storing the fin rays or muscles of the dead fish fries in the step (1), observing the fin rays or muscles every 2-3 hours, recording the death time point, shearing the fin rays or muscles of the dead and old fish, storing the fin rays or muscles in alcohol with the volume percentage of 95%, and then transferring the fin rays or muscles to a refrigerator at the temperature of-80 ℃ for long-term storage.
5. The method for selecting an individual Epinephelus lanceolatus resistant to nervous necrosis virus according to claim 1, wherein: selecting 100-200 fish died first as susceptible groups in the step (1); and (2) selecting 100-200 fish which do not die in the step (1) as an anti-infection group, and if the number of the fish which do not die is less than 100-200, selecting 100-200 fish which are the total of the remaining live fish and the fish which die finally as the anti-infection group.
6. The method for selecting an individual Epinephelus lanceolatus resistant to the nervous necrosis virus according to claim 1, wherein: in the step (2), the resistance and the susceptibility are used as phenotypes, CropGBM (crop Genomic Breeding machine) software is used, a lightgbm algorithm after a large amount of optimization is carried out on a Gradient Boosting Decision Tree (GBDT) by using a calculation method, and the weight of the SNP sites is calculated to mine the SNP sites related to the phenotypes.
7. The method for selecting an individual Epinephelus lanceolatus resistant to the nervous necrosis virus according to claim 1, wherein: the process of calculating the weight of the SNP locus in the step (2) and mining the SNP locus related to the phenotype is as follows: calling plink, preprocessing genotype data according to the deletion rate and the heterozygosity, generating ped files, then preprocessing phenotype data, training a model and screening SNP sites, and finally screening to obtain 722 SNP markers with the highest weight values for selecting the disease-resistant individuals of the Epinephelus lanceolatus.
8. The method for selecting an individual Epinephelus lanceolatus resistant to the nervous necrosis virus according to claim 1, wherein: and (4) extracting the DNA of the fin-ray genome in the step (3) by using an animal tissue DNA extraction kit.
9. The method for selecting an individual Epinephelus lanceolatus resistant to the nervous necrosis virus according to claim 1, wherein: and (4) when the toxicity is attacked in the step (4), the number of the candidate group fishes is more than 100.
10. The method for selecting an individual Epinephelus lanceolatus resistant to nervous necrosis virus according to claim 1, wherein: adopting red-spotted grouper nervous necrosis virus (RGNNV) during the virus attack in the step (4), wherein the red-spotted grouper nervous necrosis virus (RGNNV) is injected into the abdominal cavity, and the titer of the injected red-spotted grouper nervous necrosis virus (RGNNV) is 107～108TCID50/mL。
CN202210196158.5A 2022-02-28 2022-02-28 Method for selecting anti-nervous necrosis virus individual of epinephelus lanceolatus Pending CN114736969A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115992265A (en) * 2023-03-22 2023-04-21 中山大学 Grouper whole genome liquid phase chip and application thereof
CN116516028A (en) * 2023-06-27 2023-08-01 中国海洋大学三亚海洋研究院 SNP locus related to anti-nervous necrosis virus character of leopard gill-acanthus japonicus and application thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115992265A (en) * 2023-03-22 2023-04-21 中山大学 Grouper whole genome liquid phase chip and application thereof
CN115992265B (en) * 2023-03-22 2023-07-14 中山大学 Grouper whole genome liquid phase chip and application thereof
CN116516028A (en) * 2023-06-27 2023-08-01 中国海洋大学三亚海洋研究院 SNP locus related to anti-nervous necrosis virus character of leopard gill-acanthus japonicus and application thereof
CN116516028B (en) * 2023-06-27 2023-09-15 中国海洋大学三亚海洋研究院 SNP locus related to anti-nervous necrosis virus character of leopard gill-acanthus japonicus and application thereof

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