A method for identifying antiviral traits of largemouth bass

Abstract

The present invention relates to a method for identifying the antiviral traits of largemouth bass, and belongs to the technical field of fish genetic breeding. The present invention provides a method for identifying the antiviral traits of largemouth bass, detecting the SNP site of the largemouth bass genome to be tested, the SNP site is located at the 38930790th nucleotide on chromosome 3 of the largemouth bass genome, and the resistance of samples with genotype CC to frog iris virus is significantly higher than that of samples with genotypes GG and GC; the GenBank accession number of the largemouth bass genome is: ASM1485139v1. The present invention determines the SNP site in the immunity and disease resistance of largemouth bass, provides relevant effective molecular markers for carrying out molecular breeding of largemouth bass resistant to frog iris virus, and provides a theoretical basis for analyzing the genetic mechanism of its disease resistance traits.

Description

Technical Field

[0001] The invention relates to the technical field of fish genetic breeding, and in particular to a method for identifying anti-virus traits of largemouth bass. Background Art

[0002] Largemouth bass (Micropterus salmoides), commonly known as California bass, belongs to the genus Micropterus, class Osteichthyes, subclass Actinopterygii, order Perciformes, family Cehtrachidae, and is a high-quality freshwater fish. With the expansion of largemouth bass farming, its disease problem has become more and more prominent. Largemouth bass ranavirus (LMBV) infection of largemouth bass causes frog iris virus disease, which can cause large-scale fish outbreaks and deaths, causing fish disasters.

[0003] Quarantine of largemouth bass seedlings and prevention and control of early infection are the main measures to control the outbreak of frog iridovirus disease. After dissection, the swim bladder can be seen to be swollen and covered with red gas glands, and sometimes there is yellow or brown waxy secretion in the swim bladder. At present, traditional Chinese medicine and vaccines against LMBV have been developed, but it is difficult to fundamentally prevent and control LMBV infection. At present, the molecular biological prevention and control methods of LMBV for largemouth bass mainly stay at the detection and diagnosis of LMBV infection.

[0004] Du Hongyao conducted a preliminary study on the function of TRIM23 in virus-host interaction of largemouth bass. By analyzing the differential gene TRIM23 in largemouth bass infected with ulcerative syndrome virus (LBUSV), it was shown that TRIM 23 affects viral replication by negatively regulating IFN response (Du Hongyao. Preliminary study on the function of TRIM23 in virus-host interaction of largemouth bass [D]. Anhui Agricultural University, 2023. DOI: 10.26919 / d.cnki.gannu.2023.000465.). Chinese patent CN117947179A discloses a molecular marker and screening method for largemouth bass to resist SNPRV (rhabdovirus), screened the molecular markers related to the anti-infection performance of largemouth bass, and used them to screen parents resistant to rhabdovirus. Therefore, there is an urgent need to strengthen the molecular biology research on largemouth bass resistance to LMBV, especially the relevant resistance molecular markers, in order to obtain resistant largemouth bass varieties and fundamentally prevent and control LMBV infection. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for identifying the antiviral traits of largemouth bass.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In the first aspect, the present invention provides a method for identifying the antiviral traits of largemouth bass, detecting the SNP site of the largemouth bass genome to be tested, wherein the SNP site is located at the 38930790th nucleotide on chromosome 3 of the largemouth bass genome, and the resistance of samples with genotype CC to frog iris virus is significantly higher than that of samples with genotypes GG and GC; the GenBank accession number of the largemouth bass genome is: ASM1485139v1.

[0008] The present invention can obtain the antiviral trait of largemouth black bass by detecting the genotype of the SNP site, and provides a method for identifying the antiviral trait of largemouth black bass.

[0009] Furthermore, the virus is frog iridescent virus.

[0010] Furthermore, the method comprises the following steps:

[0011] S1: Extract genomic DNA of the sample to be identified;

[0012] S2: Detect the genotype of the SNP site of the genomic DNA in step S1;

[0013] S3: Determine the antiviral properties of the identified sample based on the genotype described in step S2.

[0014] Furthermore, the genomic DNA of step S1 is used as a template, primers are designed according to the position information of the SNP site, a PCR reaction is performed to obtain a PCR amplification product, and the genotype of the SNP site is analyzed.

[0015] In a specific embodiment of the present invention, the nucleotide sequences of the primers are shown in SEQ ID NOs: 1-3.

[0016] In a second aspect, the present invention provides a method for breeding virus-resistant largemouth bass, wherein the method is used to detect the genotype of the SNP site of the largemouth bass, and the largemouth bass with the genotype CC is used as the parent, and the parents are bred to obtain a virus-resistant largemouth bass variety.

[0017] In a third aspect, the present invention provides a PCR reagent for detecting SNP sites in the genome of largemouth bass, wherein the PCR reagent comprises primers having nucleotide sequences as shown in SEQ ID NOs: 1-3.

[0018] In a fourth aspect, the present invention provides the use of the PCR reagent in identifying antiviral-related traits of largemouth bass, preparing a kit for identifying antiviral traits of largemouth bass, and / or in breeding antiviral largemouth bass.

[0019] In a fifth aspect, the present invention provides a kit for identifying the antiviral traits of largemouth bass, wherein the kit contains the PCR reagent.

[0020] In a specific embodiment of the present invention, the kit further contains Flu-Arms 2×PCR Mix and water.

[0021] The genomic DNA of the largemouth bass to be tested is used as a DNA template, and the primers with nucleotide sequences shown in SEQ ID NOs: 1 to 3 are used for PCR amplification to obtain an amplified product. The base at the 25 bp position of the amplified product is detected to determine the genotype of the SNP site of the largemouth bass to be tested, and the resistance of the largemouth bass to frog iris virus to the tested largemouth bass is judged.

[0022] The PCR amplification program was as follows: 95°C for 10 min; 95°C for 15 s, gradient annealing from 61 to 55°C for 60 s (-0.6°C / cycle, decreasing by 0.6°C each cycle), for 10 cycles; 95°C for 15 s, 55°C for 60 s, for a total of 30 cycles; 30°C for 30 s.

[0023] The PCR amplification system (total volume 10 μL) is: DNA template 5 ng ~ 250 ng (preferably 5 ng ~ 50 ng), Flu-Arms 2×PCR Mix 5 μL, upstream typing primer F1 (10 μM) 0.05 μL ~ 0.25 μL (preferably 0.1 μL), downstream typing primer F2 (10 μM) 0.05 μL ~ 0.25 μL (0.1 μL), downstream universal primer R (10 μM) 0.1 μL ~ 0.5 μL (preferably 0.3 μL), add water to 10 μL.

[0024] The nucleotide sequence of the amplified product of the primers shown in SEQ ID NOs: 1 to 3 is shown in SEQ ID NO: 4. When the SNP site is located at the 25 bp of the 5' end of the amplified product (i.e., g 38930790 of chromosome 3 of the reference largemouth bass genome of GenBank accession number: ASM1485139v1), the largemouth bass samples of CC genotype are significantly more resistant to frog iridescence virus than those of GG and GC genotypes, and survive longer after infection with frog iridescence virus.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention discloses a method for identifying the antiviral trait of largemouth bass, and designs a reagent for detecting SNP sites in the genome of largemouth bass, wherein the reagent includes primers with nucleotide sequences as shown in SEQ ID NOs: 1 to 3, and is used to detect the genotype of the SNP site g 38930790 of largemouth bass. Through association analysis, different genotypes of the site are significantly correlated with resistance to frog iridescence virus, wherein the resistance of largemouth bass samples with CC genotype to frog iridescence virus is significantly higher than that of GG and GC genotypes, and the survival time after infection with frog iridescence virus is long. The present invention determines the SNP sites in the immunity and disease resistance of largemouth bass, provides relevant effective molecular markers for carrying out molecular breeding of largemouth bass resistant to frog iridescence virus, and provides a theoretical basis for analyzing the genetic mechanism of its disease resistance traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are the symptoms of largemouth bass infected with frog iris virus. A is a healthy largemouth bass; B is a largemouth bass infected with frog iris virus.

[0028] Figure 2 is the distribution density of high-quality SNPs on 22 chromosomes.

[0029] Figure 3 This is a genome-wide association analysis diagram of largemouth bass anti-frog iridovirus. A is a Manhattan plot; B is a quantile-quantile (QQ) plot.

[0030] Figure 4 Fluorescence images of different genotypes of largemouth bass SNP site g 38930790C>G.

[0031] Figure 5 The effect of SNP polymorphism on resistance of largemouth bass to frog iridovirus.

[0032] Figure 6 This is a statistical chart of the survival time of different genotypes of largemouth bass SNP site g38930790C>G.

[0033] Figure 7 The survival rate of the offspring of largemouth bass with and without SNP locus selection after being infected with frog iris virus for 14 days. DETAILED DESCRIPTION

[0034] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0035] Example 1: Using frog iris virus to attack largemouth bass

[0036] 1. Experimental Methods

[0037] 1000 healthy largemouth bass were selected for testing. The species was "Youyu No. 3" and the body length was about 5.5 cm. The largemouth bass were injected intraperitoneally with largemouth bass frog iridovirus. 4 TCID 50 / tail, and obtained virus-injected largemouth bass.

[0038] The virus-injected largemouth bass were kept normally, the water temperature was controlled at 28℃, and they were observed once in the morning, noon and evening every day. The time of onset and death of the largemouth bass was recorded, and the tail fins of the dying largemouth bass were collected in time and preserved in anhydrous ethanol.

[0039] like Figure 1 A and Figure 1 As shown in B, the 3rd to 4th day after infection is the peak period of mortality for largemouth bass. The main symptoms of dead largemouth bass are circling, dragging feces, roaming, bleeding fins, enlargement of gill cover edge and spleen, etc.

[0040] Example 2 Largemouth bass genome resequencing and SNP site screening

[0041] The death and survival of the largemouth bass in Example 1 after virus injection were classified. The 50 largemouth bass ranked in the top 50 in terms of death time were selected from the dead individuals as the susceptible group. The 50 largemouth bass that were still alive and disease-free 14 days after virus injection were selected from the surviving individuals as the disease-resistant group. The tail fin samples of the largemouth bass in the above groups were collected and their genomic DNA (OD 260 / OD 280 =1.8-2.0, concentration>100ng / μL), and the genomic DNA of 100 largemouth bass to be tested was obtained.

[0042] The whole genome sequencer Hiseq X10 PE150 was used to sequence and quality control the genomic DNA of 100 largemouth bass to be tested. An average of 10,556,429,233 bp of high-quality data was obtained for each sample, with high-quality bases (Q value>30) accounting for 92.48% and an average GC content of 42.45%, and raw reads were obtained.

[0043] The largemouth bass genome (ASM1485139v1) released by NCBI was used as the reference genome. The raw reads obtained from sequencing each sample were quality-controlled and aligned, and the obtained variant sites were quality-controlled and filtered to obtain 2,064,240 high-quality SNPs.

[0044] The generalized linear model GLM (Q) was calculated using gemma software (v0.98.1), and association analysis was performed on the target trait in the population to locate candidate genes associated with the target trait. The GWAS analysis model is expressed as: y = Xα + Qβ + Kμ + e.

[0045] Among them, y is the phenotype vector, that is, the survival time of largemouth bass after being infected with largemouth bass frog iridovirus, X is the genotype matrix, α is the genotype effect vector, Q is the fixed effect matrix (which can be information such as population structure), β is the fixed effect vector, K is the random effect matrix, mainly referring to the kinship matrix, μ is the random effect vector, and e is the residual vector.

[0046] For each SNP site, we test whether α is 0 and the probability value of α being 0. p is used to measure the degree of association between the marker genotype and the phenotype. The smaller the p value, the smaller the probability of α being 0, and the more likely the marker is associated with the trait.

[0047] The distribution of high-quality SNPs on 22 chromosomes is as follows Figure 2 As shown, the SNP density map shows that the density is highest on chromosome 1 (chr 1) and lowest on chromosome 15 (chr 15) of the largemouth bass genome.

[0048] The susceptible population was assigned a value of 1 and the resistant population was assigned a value of 0 as the phenotypic trait, and an association analysis was performed with the genotypes obtained by resequencing. A total of 2348 SNP loci associated with the LMBV resistance trait were obtained under the threshold line of -log10 p=4.0 ( Figure 3 A). Among them, the most significant SNP is mainly located on chromosome 3 of the largemouth bass genome. Use R to draw a quantile-quantile (QQ) plot to visualize the association analysis results, such as Figure 3 As shown in B. The results show that the p-value of the significant SNP (-log10p=4) is greater than its expected value, indicating that the GLM model is reliable for the sample data.

[0049] Thus, one SNP site was obtained: g38930790C>G on chromosome 3 of the largemouth bass genome, with three genotypes: GG, GC and CC.

[0050] Example 3 Correlation analysis between the anti-frog iridovirus trait and the SNP locus genotype

[0051] The SNP loci obtained in Example 2 of the present invention were verified in another population containing 311 healthy largemouth bass.

[0052] 1. Design primers: Using SNP primer software, according to the location information of the SNP sites determined in Example 1, and referring to the chromosome gene sequence of the largemouth bass genome (GenBank accession number: ASM1485139v1), a specific primer combination for PCR amplification was designed. The sequence of each primer is shown in Table 1.

[0053] Table 1

[0054]

[0055] 2. According to the method of Example 1, 311 healthy largemouth bass were infected with largemouth bass frog iris virus, the onset and death time of the 311 experimental largemouth bass were recorded, the tail fins of the dying fish were collected in time, and the genomic DNA of the 311 largemouth bass was extracted and stored at -20°C for later use.

[0056] 3. Use the upstream typing primer F1, downstream typing primer F2 and downstream universal primer R in Table 1 to perform PCR amplification on the genomic DNA of 311 largemouth bass, detect the amplification product by the fluorescent dye method, and analyze and output the corresponding genotype according to the different fluorescent colors to obtain the genotype of the SNP site described in the present invention in different samples.

[0057] The PCR amplification program was as follows: 95°C for 10 min; 95°C for 15 s, gradient annealing from 61 to 55°C for 60 s (-0.6°C / cycle, decreasing by 0.6°C each cycle), for 10 cycles; 95°C for 15 s, 55°C for 60 s, for a total of 30 cycles; 30°C for 30 s.

[0058] The PCR amplification system was: 5 ng to 50 ng DNA template, 0.1 μL upstream typing primer F1 (10 μM), 0.1 μL downstream typing primer F2 (10 μM), 0.3 μL downstream universal primer R (10 μM) and 5 μL Flu-Arms2×PCR Mix, and water was added to make the total PCR reaction system 10 μL.

[0059] According to the genotyping results, the number of individuals with different genotypes at the SNP loci was counted, the genotype frequencies and gene allele frequencies at different SNP loci were calculated, the survival time was counted, and the correlation (p value) between the survival time of different genotypes corresponding to the infection with largemouth bass frog iridovirus was calculated.

[0060] 2. Experimental Results

[0061] like Figure 4 The figure shows the fluorescence images of different genotypes of the SNP sites in Example 2. The red color represents the GG genotype, the blue color represents the CC genotype, and the green color represents the GC genotype.

[0062] like Figure 5 and Figure 6 As shown, the SNP site of Example 2 is significantly correlated with the resistance of largemouth bass to frog iris virus. The resistance of largemouth bass to frog iris virus of CC genotype of SNP site of Example 2 is significantly higher than that of GG genotype (p<0.01), and significantly higher than that of GC genotype (p<0.05). The nucleotide sequence of the PCR amplification product is shown in SEQ ID NO: 4, and the 25 bp at the 5' end of the amplification product corresponds to the SNP site of Example 2.

[0063] Example 4 A kit for identifying the characteristics of largemouth bass anti-frog iridovirus

[0064] 1. Composition of the kit

[0065] Upstream typing primer F1 (SEQ ID NO: 1), downstream typing primer F2 (SEQ ID NO: 2), downstream universal primer R (SEQ ID NO: 3), Flu-Arms 2×PCR Mix and water.

[0066] 2. How to use the kit

[0067] PCR amplification was performed using the genomic DNA of the largemouth bass to be tested as a DNA template.

[0068] The PCR amplification program was as follows: 95°C for 10 min; 95°C for 15 s, gradient annealing from 61 to 55°C for 60 s (-0.6°C / cycle, decreasing by 0.6°C each cycle), for 10 cycles; 95°C for 15 s, 55°C for 60 s, for a total of 30 cycles; 30°C for 30 s.

[0069] The PCR amplification system was: 5 ng to 50 ng DNA template, 0.1 μL upstream typing primer F1 (10 μM), 0.1 μL downstream typing primer F2 (10 μM), 0.3 μL downstream universal primer R (10 μM) and 5 μL Flu-Arms2×PCR Mix, and water was added to make the total PCR reaction system 10 μL.

[0070] After PCR amplification, the amplified product is obtained, and the amplified product is analyzed to determine the genotype of the SNP site of the largemouth bass to be tested, and to judge the resistance of the largemouth bass to frog iridovirus.

[0071] 3. Interpretation of PCR test results

[0072] The nucleotide sequence of the amplified product is shown in SEQ ID NO: 4. When the SNP site is located at the 25bp at the 5' end of the amplified product (i.e., g 38930790 of chromosome 3 of the reference largemouth bass genome with GenBank accession number: ASM1485139v1), the resistance of largemouth bass samples with CC genotype to frog iris virus is significantly higher than that of GG and GC genotypes, and the survival time is long after infection with largemouth bass frog iris virus.

[0073] Example 5 A virus-resistant largemouth bass breeding method

[0074] The genotype of the SNP site of the largemouth bass was detected by using the kit of Example 4, and the largemouth bass with the genotype of CC was used as the parent. The parent was bred to obtain a largemouth bass variety resistant to frog iris virus.

[0075] Example 6: Validation of virus-resistant largemouth bass breeding method

[0076] 1. Experimental Methods

[0077] The method of Example 5 was used to obtain a largemouth bass with a genotype of CC at the SNP site, which was used as a parent. The parent was bred to obtain largemouth bass offspring resistant to largemouth bass frog iris virus, and 1000 offspring with a body length of 4 to 5 cm were selected as a breeding group.

[0078] The offspring of 1000 common largemouth black bass with a body length of 4 to 5 cm were selected as the unselected group.

[0079] Each group of largemouth seabass was challenged with poison in the same manner as in Example 1, and the survival rate of each group of largemouth seabass 14 days after the challenge was calculated.

[0080] 2. Experimental Results

[0081] like Figure 7 As shown, the survival rate of largemouth bass in the selected group was 35.2%, which was significantly higher than that in the non-selected group.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for identifying antiviral traits of largemouth bass, characterized in that: The genotype of the SNP site of the largemouth bass genome to be tested is detected, the SNP site is located at the 25bp of the nucleotide sequence shown in SEQ ID NO: 4, and the largemouth bass sample with the genotype CC of the SNP site has significantly higher resistance to frog iridescence virus than the samples with the genotypes GG and GC; the reference genome GenBank accession number of the largemouth bass is: ASM1485139v1, and the virus is frog iridescence virus.

2. The method according to claim 1, characterized in that The method comprises the following steps: S1: Extract genomic DNA of the sample to be identified; S2: Detect the genotype of the SNP site of the genomic DNA in step S1; S3: Determine the antiviral properties of the identified sample based on the genotype described in step S2.

3. The method according to claim 2, characterized in that Using the genomic DNA from step S1 as a template, primers are designed according to the position information of the SNP site, a PCR reaction is performed to obtain a PCR amplification product, and the genotype of the SNP site is analyzed.

4. The method according to claim 3, characterized in that The nucleotide sequences of the primers are shown in SEQ ID NOs: 1-3.

5. A method for breeding largemouth bass resistant to frog iris virus, characterized in that: The genotype of the SNP site of largemouth bass is detected by the method described in any one of claims 1 to 4, a largemouth bass with a genotype of CC is selected as a parent, and the parents are bred to obtain a largemouth bass variety resistant to frog iris virus.

6. Use of primers with nucleotide sequences as shown in SEQ ID NOs: 1 to 3 in identifying largemouth bass resistant to frog iridovirus.

7. Use of primers with nucleotide sequences as shown in SEQ ID NOs: 1 to 3 in preparing a kit for identifying largemouth bass resistant to frog iris virus.

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