A kit for evaluating the resistance of largemouth bass to frog iridovirus

Abstract

The present invention relates to a kit for evaluating the resistance of largemouth black bass to frog iridescence virus, and belongs to the technical field of aquaculture. The present invention provides a kit for evaluating the resistance of largemouth black bass to frog iridescence virus, and the kit contains a primer combination with a nucleotide sequence as shown in SEQ ID NO: 1-3. The present invention screens the SNP site related to the resistance of largemouth black bass to frog iridescence virus through whole genome association analysis, which is located at g 38943374 on chromosome 3 of the largemouth black bass genome, is an A / C allele mutation, and has genotypes of CC, AA and AC. The resistance of samples with genotype CC to frog iridescence virus is significantly higher than that of samples with genotypes of AA and AC, and PCR primers and kits for detecting SNP sites are designed to identify and evaluate the germplasm of largemouth black bass, provide a research and development basis for disease-resistant breeding of largemouth black bass, promote the genetic breeding process of largemouth black bass, and improve the economic benefits of breeding.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to a kit for evaluating the resistance of largemouth bass to frog iridovirus. Background Art

[0002] Ranavirus is a complex icosahedral double-stranded DNA virus that can infect amphibians, reptiles, fish and other animals, and cause serious diseases or even death. Largemouth bass (Micropterus salmoides) is native to the Mississippi River Basin in North America. It is a eurythermal fish with the characteristics of fast growth, few diseases, delicious meat, strong adaptability, easy fishing and short breeding cycle. Largemouth bass ranavirus (LMBV) that infects largemouth bass was first isolated from largemouth bass in Lake Vail, Florida, USA in 1991. Largemouth bass infected with Ranavirus are difficult to distinguish clinically. In foreign countries, the main symptoms are that the fish lose balance, tend to float near the water surface, and may have an enlarged or red swim bladder; in China, the main symptoms are surface ulcers and muscle necrosis; this may be affected by changes in the environment and feed.

[0003] Genome wide association study (GWAS) is a process of detecting genetic variation (marker) polymorphisms of multiple individuals across the entire genome to obtain genotypes, and then performing statistical analysis on the genotypes and observable traits, i.e., phenotypes, at the population level. The genetic variation (marker) that is most likely to affect the trait is screened out based on statistics or significant p-values, and then the linkage disequilibrium relationship between markers and functional genes is used to explore genes related to trait variation.

[0004] At present, researchers have screened out a series of key disease-resistant SNP molecular markers through GWAS for use in aquaculture. Chinese patent CN114617085A discloses a method for breeding a disease-resistant family of saddle grouper. The method uses GWAS technology to find molecular markers significantly associated with saddle grouper resistance to nervous necrosis virus, screens out disease-resistant parents based on significantly associated markers, and then combines family selection to breed disease-resistant offspring with disease-resistant parents, and finally obtains a strain of saddle grouper resistance to nervous necrosis virus, especially resistance to red-spotted grouper nervous necrosis virus. Chinese patent CN112501312A discloses a reagent for detecting a combination of SNP molecular markers related to grouper resistance to nervous necrosis virus. SNP sites are screened through GWAS technology, and the genotype of the SNP site is associated with the phenotypic traits of the sample. The key mutation sites that can affect the phenotypic traits are screened out through statistical analysis. By detecting the SNP genotype of the red-spotted grouper, it can be effectively determined whether it has the resistance to nervous necrosis virus. However, there is still a lack of SNP sites and related detection technologies and products for frog iridovirus in largemouth bass. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a kit for evaluating the resistance of largemouth bass to frog iridovirus.

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

[0007] In a first aspect, the present invention provides a kit for evaluating the resistance of largemouth bass to frog iridovirus, characterized in that the kit contains a primer combination with nucleotide sequences as shown in SEQ ID NOs: 1-3.

[0008] The kit of the invention evaluates the trait of the largemouth bass against frog iridovirus by detecting the genotype of the SNP site in the largemouth bass genome.

[0009] In a specific embodiment of the present invention, the kit further contains Flu-Arms 2×PCR Mix.

[0010] In a specific embodiment of the present invention, the kit further contains water.

[0011] In a second aspect, the present invention provides the use of the kit in evaluating the resistance of largemouth bass to frog iridovirus.

[0012] In a third aspect, the present invention provides a method for evaluating the resistance of largemouth bass to frog iridescence virus, detecting a SNP site in the largemouth bass genome, wherein the SNP site is located at g 38943374 on chromosome 3 of the largemouth bass genome, is an A / C allele mutation, and has genotypes of CC, AA and AC. The resistance of samples with genotype CC to frog iridescence virus is significantly higher than that of samples with genotypes AA and AC.

[0013] The GenBank accession number of the largemouth bass genome is: ASM1485139v1.

[0014] Further, the kit is used for detection.

[0015] 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 to perform PCR amplification to obtain amplified products, and the amplified products are detected 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 iris virus to the tested largemouth bass.

[0016] The PCR amplification system (total volume 10 μL) can be: DNA template 5 ng ~ 250 ng, Flu-Arms2×PCR Mix 5.0 μL, upstream typing primer (10 μM) 0.05 μL ~ 0.25 μL, downstream typing primer (10 μM) 0.05 μL ~ 0.25 μL, downstream universal primer (10 μM) 0.1 μL ~ 0.5 μL, supplemented with ddH2O to 10 μL.

[0017] In a specific embodiment of the present invention, the PCR amplification system with a total volume of 10 μL can be: 5 ng to 50 ng DNA template, 5 μL Flu-Arms 2×PCR Mix, 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), supplemented with ddH2O to a total PCR amplification system volume of 10 μL.

[0018] 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.

[0019] The nucleotide sequence of the amplification product of the primers shown in SEQ ID NOs: 1 to 3 is shown in SEQ ID NO: 4.

[0020] When the SNP site was located at the 25bp of the 5' end of the amplified product (i.e., g 38943374 on chromosome 3 of the reference largemouth bass genome with GenBank accession number: ASM1485139v1; g 38943374 on chromosome 3), largemouth bass samples with CC genotype were significantly more resistant to frog iridovirus than those with AA and AC genotypes, and survived longer after infection with frog iridovirus.

[0021] In a fourth aspect, the present invention provides a method for assisting the breeding of largemouth bass with high resistance to frog iris virus, characterized in that a sample with a CC genotype at the SNP site is obtained by the method as a parent, and the parent is cultivated to obtain a largemouth bass with high resistance to frog iris virus.

[0022] In a fifth aspect, the present invention provides the use of a reagent for detecting SNP sites in the genome of a largemouth bass in evaluating the largemouth bass's resistance to frog iridovirus and / or in preparing a kit for evaluating the largemouth bass's resistance to frog iridovirus;

[0023] The SNP site is located at g 38943374 on chromosome 3 of the largemouth bass genome, is an A / C allele mutation, and the genotypes are CC, AA and AC. The GenBank accession number of the largemouth bass genome is: ASM1485139v1.

[0024] In a sixth aspect, the present invention provides the use of a reagent for detecting SNP sites in the genome of a largemouth bass in the preparation of a kit for evaluating the largemouth bass's resistance to frog iris virus;

[0025] The SNP site is located at g 38943374 on chromosome 3 of the largemouth bass genome, is an A / C allele mutation, and the genotypes are CC, AA and AC. The GenBank accession number of the largemouth bass genome is: ASM1485139v1.

[0026] In a seventh aspect, the present invention provides the use of a reagent for detecting SNP sites in the genome of a largemouth bass in assisting the breeding of largemouth bass with high resistance to frog iris virus;

[0027] The SNP site is located at g 38943374 on chromosome 3 of the largemouth bass genome, is an A / C allele mutation, and the genotypes are CC, AA and AC. The GenBank accession number of the largemouth bass genome is: ASM1485139v1.

[0028] Furthermore, the reagent is a primer, and the nucleotide sequence of the primer is shown in SEQ ID NO: 1-3.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention screens out a SNP site related to the frog iris virus resistance trait of largemouth black bass through whole genome association analysis, which is located at g38943374 on chromosome 3 of the largemouth black bass genome, is an A / C allele mutation, and has genotypes of CC, AA and AC. The resistance of samples with genotype CC to frog iris virus is significantly higher than that of samples with genotypes AA and AC, and PCR primers and a kit for detecting the SNP site are designed to identify the germplasm of largemouth black bass, provide a research and development basis for disease-resistant breeding of largemouth black bass, and the like, which is conducive to promoting the genetic breeding process of largemouth black bass and improving the economic benefits of largemouth black bass farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 These are the symptoms of largemouth bass infected with frog iris virus (LMBV). A is a healthy largemouth bass; B is a largemouth bass infected with LMBV.

[0032] Figure 2 Distribution density of SNPs on largemouth bass chromosomes after filtering for quality control.

[0033] Figure 3 This is the genome-wide association analysis of largemouth bass resistance to LMBV. A is a Manhattan plot; B is a quantile-quantile (QQ) plot.

[0034] Figure 4 This is the PCR fluorescence image of different genotypes of largemouth bass g 38943374C>A locus. Red indicates AA genotype, blue indicates CC genotype, and green indicates AC genotype.

[0035] Figure 5 This is a statistical chart of the survival time of different genotypes of largemouth bass g 38943374C>A site.

[0036] Figure 6 This is the survival rate of the offspring of largemouth bass selected for the g 38943374C>A site after being infected with frog iris virus. DETAILED DESCRIPTION

[0037] 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.

[0038] Example 1 A kit for evaluating the resistance of largemouth bass to frog iridovirus

[0039] 1. Composition of each reagent in the kit

[0040] (1) Primer set: upstream typing primer, downstream typing primer and downstream universal primer.

[0041] (2) Flu-Arms 2×PCR Mix and water.

[0042] In the primer set, the nucleotide sequences of the upstream typing primer, the downstream typing primer and the downstream universal primer are shown in Table 1.

[0043] Table 1 Nucleotide sequences of upstream typing primers, downstream typing primers and downstream universal primers

[0044]

[0045] 2. How to use the kit

[0046] (1) extracting DNA from largemouth bass samples to be tested;

[0047] (2) Using the DNA extracted in step (1) as a template, a PCR reaction is performed to obtain a PCR reaction product.

[0048] The total volume was 10 μL. The PCR reaction system was shown in Table 2, and the PCR reaction conditions were shown in Table 3.

[0049] Table 2 PCR reaction system

[0050] Reagents content Upstream typing primer (10 μM) 0.1μL Downstream typing primer (10μM) 0.1μL Downstream universal primer (10 μM) 0.3μL Flu-Arms 2×PCR Mix 5μL DNA template 5ng~50ng water The total PCR reaction volume is 10 μL

[0051] Table 3 PCR reaction conditions

[0052]

[0053]

[0054] (3) The reaction products were detected by fluorescent dye method. According to the different fluorescent colors, the corresponding genotypes were analyzed and output, and the resistance of largemouth bass samples to frog iridovirus was evaluated.

[0055] 3. Interpretation of test kit results

[0056] The nucleotide sequence of the amplified product is shown in SEQ ID NO: 4. The SNP site is located at the 25bp at the 5' end of the nucleotide sequence of the amplified product (i.e., g38943374 of chromosome 3 of the reference largemouth bass genome with GenBank accession number: ASM1485139v1). Largemouth bass samples with CC genotype have significantly higher resistance to frog iris virus than those with AA and AC genotypes, and have a longer survival time after being infected with largemouth bass frog iris virus.

[0057] Example 2: Using the Rana iridovirus to attack the Rana iridodovirus

[0058] 1. Experimental Methods

[0059] 1000 healthy “Youyu No. 3” (body length about 5.5 cm) were intraperitoneally injected with largemouth bass frog iris virus. 4 TCID 50 / , get the poison-killing largemouth bass.

[0060] The poisoned largemouth bass were raised normally, the water temperature was maintained at 28℃, the symptoms of the largemouth bass were observed once in the morning, noon and evening every day, the onset and death time were recorded, and the tail fins of the dying largemouth bass were collected and stored in anhydrous ethanol for later use.

[0061] 2. Experimental Results

[0062] The peak of mortality of largemouth bass occurred on the 3rd to 4th day after infection. The main symptoms of dead largemouth bass were bleeding on the edge of the gill cover and fin rays and enlarged spleen. They also showed activities such as dragging stool, roaming and spinning ( Figure 1 A and Figure 1 B) The experimental fish were basically stable 7 days after the challenge, and the mortality rate of largemouth bass during the whole experiment was 78.8%.

[0063] Example 3 Largemouth bass genome resequencing

[0064] 1. Experimental Methods

[0065] 1. The caudal fin samples of the top 50 black bass that died in the order of time after being infected with the iridescent virus of the black bass frog in Example 1 were used as susceptible population samples; the caudal fin samples of the 50 black bass that survived the infection with the iridescent virus of the black bass frog in Example 1 for 14 days were used as disease-resistant population samples, and the genomic DNA of each sample was extracted to detect its purity and concentration. The concentration of the genomic DNA of the extracted sample was greater than 100 ng / μL, and the OD260 / OD280 was 1.8-2.0, and the subsequent whole genome resequencing could be performed.

[0066] 2. (1) Use an enzyme to randomly break the genomic DNA in step 1 into short DNA fragments, perform blunt-end repair, and obtain blunt-end repaired DNA fragments.

[0067] (2) Add dA tails to both ends of the blunt-end repaired DNA fragment and connect them to sequencing adapters to obtain a DNA fragment connected to the adapter.

[0068] (3) Purify the DNA fragments connected to the adapter using AMPure XP magnetic beads to obtain magnetic bead-purified DNA fragments.

[0069] (4) Perform PCR amplification on the 300-400 bp DNA fragments in the DNA fragments purified by magnetic beads to obtain a library to be sequenced.

[0070] (5) After purification and library testing, the sequencing library was sequenced using the whole genome sequencer Hiseq X10 PE150 with a sequencing depth of 10×. After preliminary quality control, 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.

[0071] 3. Using the largemouth bass genome published on NCBI (GenBank accession number: ASM1485139v1) as the reference genome, the raw reads obtained in step 2 were quality controlled to obtain clean reads, and then the clean reads were compared with the reference genome for short sequences to obtain the variant sites of each sample.

[0072] Plink2 software was used to perform quality control filtering on the variant sites, removing non-binary sites, sites with second allele frequencies less than 0.05, sites with deletion rates greater than 0.5, and sites with heterozygous ratios greater than 0.8, and a total of 2,902,562 SNP sites were obtained. Further SNP quality control filtering was performed to obtain 2,064,240 high-quality SNP stable sites.

[0073] 2. Experimental Results

[0074] The SNP density plot shows that the density is highest on chromosome 1 (chr 1) and lowest on chromosome 15 (chr 15) of the largemouth bass genome. Figure 2 ).

[0075] Example 4: Conducting a genome wide association study (GWAS) and screening for disease resistance SNP sites

[0076] 1. Experimental Methods

[0077] For the SNP sites screened in Example 3, 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 related to the target trait. The GWAS analysis model is expressed as: y = Xα + Qβ + Kμ + e.

[0078] 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.

[0079] 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.

[0080] 2. Experimental Results

[0081] 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 SNPs are mainly located on chromosome 3 of the largemouth bass genome. Quantile-quantile (QQ) plots were drawn using R to visualize the association analysis results ( Figure 3 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.

[0082] Thus, one SNP site was obtained: g38943374C>A on chromosome 3 of the largemouth bass genome, with three genotypes: CC, AC and AA.

[0083] Example 5 Correlation analysis between genotype and resistance to frog iris virus

[0084] 1. Experimental Methods

[0085] The correlation between the SNP site of Example 4 of the present invention and the resistance to frog iridovirus of largemouth bass was verified in another group containing 311 largemouth bass.

[0086] 1. Referring to the chromosome gene sequence of the largemouth bass genome (GenBank accession number: ASM1485139v1), according to the position information of the SNP site determined in Example 4, the PCR amplification specific primer combination was designed using SNP primer software. The primer sequences are shown in Table 1 of Example 1.

[0087] 2. 311 healthy “Youyu No. 3” (body length about 5.5 cm) were intraperitoneally injected with largemouth bass frog iris virus at an absolute lethal dose of 10 4.5 TCID 50 / , get the poison-attacking largemouth bass.

[0088] The poisoned largemouth bass were kept normally, the water temperature was maintained at 28℃, the symptoms of the largemouth bass were observed once in the morning, noon and evening every day, the onset and death time were recorded, the tail fins of the dying largemouth bass were collected, the genomic DNA of the tail fins was extracted, and stored at -20℃ for later use.

[0089] The specific primers in Table 1 were used to perform PCR amplification reaction on the genomic DNA of the caudal fin of 311 largemouth bass to obtain amplified products, which were detected by the fluorescent dye method. According to different fluorescent colors, the corresponding genotypes were analyzed and output to obtain the genotypes of the SNP loci in different samples. According to the genotyping results, the number of individuals with different genotypes at each locus was counted, and the genotype frequency, allele frequency, survival time (h) ± standard deviation and the difference (p value) between each genotype at different SNP loci were calculated.

[0090] The PCR reaction system and reaction conditions were the same as those in Example 1.

[0091] 2. Experimental Results

[0092] like Figure 4 Shown is the PCR fluorescence graph of different genotypes of largemouth bass g 38943374C>A site.

[0093] As shown in Table 4 and Figure 5 As shown, the SNP site was significantly associated with the resistance of largemouth bass to frog iridovirus, and the resistance of largemouth bass to frog iridovirus of the CC genotype of SNP was significantly higher than that of the AA and AC genotypes (p<0.01).

[0094] Table 4 Effect of SNP polymorphism on resistance of largemouth bass to frog iridovirus

[0095] genotype Number of individuals Genotype frequency Allele frequency Survival time (h) ± standard deviation p-value CC 83 0.2399 C：0.4950 92.82±6.0144 CC-AC: 0.0067 AC 154 0.5101 A：0.5050 69.0384±5.2728 AC-AA: 0.9507 AA 74 0.25 69.4056±5.5344 CC-AA: 0.0073

[0096] Example 6 A method for assisting the breeding of largemouth bass with high resistance to frog iris virus

[0097] 1. Experimental Methods

[0098] The genotype of the SNP site of the largemouth bass was detected using the kit of Example 1. The 25bp at the 5' end of the nucleotide sequence of the amplified product showed that the genotype of the SNP site was CC. The largemouth bass was used as the parent. The parent was cultivated to obtain offspring, that is, a largemouth bass variety with high resistance to largemouth bass frog iris virus.

[0099] Example 7 Verification of the resistance of largemouth bass to frog iris virus after assisted breeding

[0100] The method of Example 6 was used to carry out auxiliary breeding of largemouth bass, and from the obtained offspring largemouth bass, 1,000 offspring largemouth bass with a body length of 4 to 5 cm were selected as the breeding group. The offspring of common largemouth bass with the same body length and number were selected as the non-breeding group.

[0101] The breeding group and the non-breeding group were infected with the largemouth bass frog iridovirus according to the method of Example 2, and the survival rate of each group was recorded 14 days after the largemouth bass frog iridovirus infection.

[0102] 2. Experimental Results

[0103] The offspring of largemouth bass assisted by the method of Example 6 had a survival rate of 35.90% 14 days after infection with largemouth bass frog iris virus, which was significantly higher than that of ordinary largemouth bass offspring.

[0104] 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 kit for evaluating the resistance of largemouth bass to frog iridovirus, characterized in that: The kit contains a primer combination with nucleotide sequences as shown in SEQ ID NOs: 1-3.

2. The kit according to claim 1, characterized in that The kit also contains Flu-Arms 2×PCRMix.

3. The kit according to claim 1, characterized in that The kit also contains water.

4. Use of the kit according to any one of claims 1 to 3 in evaluating the resistance of largemouth bass to frog iridovirus.

5. A method for evaluating the resistance of largemouth bass to frog iridovirus, characterized in that: The SNP site in the genome of largemouth bass was detected. The SNP site was located at g 38943374 on chromosome 3 of the largemouth bass genome, which was an A / C allele mutation. The genotypes were CC, AA and AC. The resistance of samples with genotype CC to frog iridovirus was significantly higher than that of samples with genotypes AA and AC. The GenBank version number of the largemouth bass genome is: ASM1485139v1.

6. The method according to claim 5, characterized in that The detection is performed using the kit according to any one of claims 1 to 3.

7. A method for assisting the breeding of largemouth bass with high resistance to frog iris virus, characterized in that: The sample with CC genotype at the SNP site is obtained by the method described in claim 5 or 6 as a parent, and the parent is cultured to obtain a largemouth bass with high resistance to frog iris virus.

8. Use of a reagent for detecting SNP sites in the genome of largemouth bass in evaluating the resistance of largemouth bass to frog iridovirus and / or in preparing a kit for evaluating the resistance of largemouth bass to frog iridovirus; The SNP site is located at g 38943374 on chromosome 3 of the largemouth bass genome, is an A / C allele mutation, and the genotypes are CC, AA and AC. The GenBank version number of the largemouth bass genome is: ASM1485139v1; samples with genotype CC are significantly more resistant to frog iris virus than samples with genotypes AA and AC.

9. Application of reagents for detecting SNP sites in the genome of largemouth bass in assisting the breeding of largemouth bass with high resistance to frog iris virus; The SNP site is located at g 38943374 on chromosome 3 of the largemouth bass genome, is an A / C allele mutation, and the genotypes are CC, AA and AC. The GenBank version number of the largemouth bass genome is: ASM1485139v1; samples with genotype CC are significantly more resistant to frog iris virus than samples with genotypes AA and AC.

10. The use according to claim 8 or 9, characterized in that: The reagent is a primer, and the nucleotide sequence of the primer is shown in SEQ ID NO: 1-3.

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