Reagents for detecting SNP molecular marker combinations associated with grouper's resistance to nerve necrosis virus trait

Abstract

This application is a divisional application of patent application No. 202011520740X, filed on December 21, 2020, entitled "Reagent for Detecting SNP Molecular Marker Combinations Related to Grouper's Resistance to Neuronecrosis Virus." This invention discloses a reagent for detecting SNP molecular marker combinations related to the grouper's resistance to neuronecrosis virus. This invention identifies five SNP loci associated with the resistance to neuronecrosis virus in red-spotted grouper. When the genotype of SNP locus 1 is GG, SNP locus 2 is AC, SNP locus 3 is CC, SNP locus 4 is TA, and SNP locus 5 is TT, the mortality probability of red-spotted grouper infected with neuronecrosis virus is significantly lower than that of individuals with other genotypes. By detecting the above SNPs in red-spotted grouper, it is possible to effectively determine whether they possess the neuronecrosis virus resistance trait.

Description

[0001] This application is a divisional application of the invention patent application filed on December 21, 2020, with application number 202011520740 X, entitled "Reagent for detecting SNP molecular marker combinations related to anti-neural necrosis virus traits in grouper". Technical Field

[0002] This invention relates to applications in [the field of research]. Specifically, it relates to reagents for detecting combinations of SNP molecular markers associated with the anti-neural necrosis virus trait in grouper. Background Technology

[0003] The red-spotted grouper (Epinephelus akaara), commonly known as the red-spotted grouper, belongs to the order Perciformes, family Serranidae, and genus Epinephelus. It is mainly distributed in the western North Pacific. Characterized by its delicious flesh and high nutritional value, it is an important grouper species farmed in the coastal areas of South China.

[0004] Red-spotted grouper nervous necrosis virus (RGNNV) is very harmful to both adult and juvenile grouper, with a mortality rate of up to 100% in severe cases. It often causes huge economic losses to grouper farming and seriously threatens the healthy development of the grouper farming industry. However, there is currently no effective treatment for the disease caused by this virus, and prevention and monitoring are the main methods of treatment.

[0005] Chinese patent CN201610255794.5 discloses a method for detecting grouper nerve necrosis virus infection using Sandwich ELASA based on nucleic acid aptamers; CN201610465941.1 discloses a colloidal gold immunochromatographic test strip for detecting antibodies against seven-banded grouper nerve necrosis virus and its application; CN201910873715.0 discloses a colloidal gold test strip for detecting grouper nerve necrosis virus and its preparation and detection method; CN200910039534.4 discloses a primer set, detection method, and rapid diagnostic kit for detecting red-spotted grouper nerve necrosis virus; CN03114369.5 discloses a gene diagnostic kit and detection method for grouper viral nerve necrosis virus, but lacks a method for detecting grouper nerve necrosis virus resistance.

[0006] Genome-wide association study (GWAS) is a strategy that involves extensively screening a large number of single nucleotide polymorphism (SNP) molecular markers across the entire genome of multiple individuals in a sample, obtaining genotypes, and then performing association analysis between these SNP genotypes and the phenotypic traits of the sample. Statistical analysis is then used to identify key mutation sites that can influence these phenotypic traits. With the development of genomics research and sequencing technology, researchers have used this technique to discover and identify a large number of genetic variations associated with phenotypic traits. In recent years, this method has been widely used in the screening and identification of genes associated with important economic traits in aquatic animals. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and to provide a reagent for detecting SNP molecular marker combinations related to the anti-neural necrosis virus trait in grouper.

[0008] The first objective of this invention is to provide a reagent for detecting any one or more molecular markers in a combination of SNP molecular markers associated with the anti-neural necrosis virus trait in grouper.

[0009] A second objective of this invention is to provide primers for detecting the SNP site 1.

[0010] A third objective of this invention is to provide primers for detecting the SNP site 2.

[0011] A fourth objective of this invention is to provide primers for detecting the SNP site 3.

[0012] A fifth objective of this invention is to provide primers for detecting the SNP site 4.

[0013] A sixth objective of this invention is to provide primers for detecting the SNP site 5.

[0014] A seventh object of the present invention is to provide the use of the reagents, or any one or more of the primers, in the preparation of a grouper nerve necrosis virus resistance detection kit.

[0015] The seventh objective of this invention is to provide a grouper nerve necrosis virus resistance detection kit.

[0016] An eighth object of the present invention is to provide the use of one or more of the reagents, primers, or kits in grouper nerve necrosis virus resistance.

[0017] To achieve the above objectives, the present invention is implemented through the following solution:

[0018] This invention claims protection for a reagent for detecting any one or more molecular markers in a combination of SNP molecular markers associated with the anti-neural necrosis virus trait in grouper, said reagent being used to detect the genotype of SNP site 1, SNP site 2, SNP site 3, SNP site 4 and / or SNP site 5.

[0019] The SNP site 1 is located at the 301 bp starting from the 5' end of the nucleotide sequence shown in SEQ ID NO.1. When the sample genotype is GG, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0020] The SNP site 2 is located at the 300th bp from the 5' end of the nucleotide sequence shown in SEQ ID NO.2. When the sample genotype is AC, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0021] The SNP site 3 is located at the 301 bp starting from the 5' end of the nucleotide sequence shown in SEQ ID NO.3. When the sample genotype is CC, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0022] The SNP site 4 is located at the 300th bp from the 5' end of the nucleotide sequence shown in SEQ ID NO.4. When the sample genotype is TA, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0023] The SNP site 5 is located at the 301 bp starting from the 5' end of the nucleotide sequence shown in SEQ ID NO.5. When the sample genotype is TT, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0024] Preferably, the grouper shown is the red-spotted grouper.

[0025] Preferably, the reagent is a primer.

[0026] This invention also claims protection for the following primers:

[0027] The primers for detecting SNP site 1 have nucleotide sequences shown in SEQ ID NO. 6-7.

[0028] The primers for detecting SNP site 2 have nucleotide sequences shown in SEQ ID NO. 8-9.

[0029] The primers for detecting SNP site 3 have nucleotide sequences shown in SEQ ID NO. 10-11.

[0030] The primers for detecting SNP site 4 have nucleotide sequences shown in SEQ ID NO. 12-13.

[0031] The primers for detecting SNP site 5 have nucleotide sequences shown in SEQ ID NO. 14-15.

[0032] Furthermore, the present invention claims protection for the use of the reagent, or any one or more of the primers, in the preparation of a grouper nerve necrosis virus resistance detection kit.

[0033] The present invention also claims a grouper nerve necrosis virus resistance detection kit, comprising the aforementioned reagent.

[0034] Preferably, it includes any one or more primers for detecting SNP sites 1 to 5.

[0035] More preferably, primers for detecting SNP sites 1 to 5 are included.

[0036] More preferably, it includes primers with acid sequences as shown in SEQ ID NO. 6-15.

[0037] Most preferably, a grouper nerve necrosis virus resistance detection kit includes primers with nucleotide sequences as shown in SEQ ID NO. 6-15, and PCR reagents.

[0038] The usage method includes the following steps: The usage method of the kit is as follows:

[0039] (1) Extract genomic DNA from the red-spotted grouper to be tested;

[0040] (2) Using the DNA obtained in step (1) as a template, and using the nucleotide sequences shown in SEQ ID NO. 6~7, 8~9, 10~11, 12~13 and 14~15 as primers, PCR amplification was performed to obtain PCR amplification products;

[0041] (3) Sequencing the PCR amplification products obtained in step (2) to determine the genotypes of SNP sites 1-5 of the red-spotted grouper to be tested;

[0042] (4) Determine whether the red-spotted grouper to be tested is resistant to nerve necrosis virus based on the genotypes of SNP loci 1-5 determined in step (3):

[0043] SNP site 1 is located at the 35th bp at the 5' end of the amplification product of the primers shown in SEQ ID NO. 6-7. When the sample genotype is GG, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0044] SNP site 2 is located at the 265th bp at the 5' end of the amplification product of the primers shown in SEQ ID NO. 8-9. When the sample genotype is AC, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0045] SNP site 3 is located at the 181 bp at the 5' end of the amplification product of the primers shown in SEQ ID NO. 10-11. When the sample genotype is CC, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0046] SNP site 4 is located at the 69th bp at the 5' end of the amplification product of the primers shown in SEQ ID NO. 12-13. When the sample genotype is TA, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0047] SNP site 5 is located at the 297th bp at the 5' end of the amplification product of the primers shown in SEQ ID NO. 14-15. When the sample genotype is TT, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0048] Therefore, this invention also claims protection for the use of one or more of the reagents, primers, or kits in grouper nerve necrosis virus resistance.

[0049] In this invention, genome extraction is not particularly limited. It can be performed using the traditional phenol-chloroform method or a kit. In a specific embodiment of this invention, a kit is used for extraction. The kit is easy to operate, fast, and produces high-quality DNA.

[0050] In addition, the method for detecting the individual genotype of the red-spotted grouper in this invention is not particularly limited. Techniques such as time-of-flight mass spectrometry, sequencing, microarrays, single-strand conformation polymorphism polymerase chain reaction, and restriction fragment length polymorphism polymerase chain reaction can all be used for SNP detection.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] This invention identifies five SNP loci associated with resistance to neuronecrosis virus (NSV) in red-spotted grouper. When the genotype of SNP locus 1 is GG, SNP locus 2 is AC, SNP locus 3 is CC, SNP locus 4 is TA, and SNP locus 5 is TT, the mortality rate of red-spotted grouper infected with NSV is significantly lower than that of individuals with other genotypes. Therefore, detecting these SNPs in red-spotted grouper can effectively determine whether they possess NSV resistance. Thus, the SNP markers of this invention are closely related to the resistance of red-spotted grouper to NSV. Selecting individuals with resistant genotypes during parent breeding will improve the offspring's resistance to NSV. Using the markers of this invention for assisted breeding can accelerate the development of superior disease-resistant red-spotted grouper varieties.

[0053] The primer pairs at the aforementioned SNP loci can effectively detect the genotype of the red-spotted grouper. The results can determine whether the grouper is resistant to neuronecrosis virus (NSV) and provide a reference for parent selection. Therefore, the SNP locus primers of this invention can detect the genotype of red-spotted grouper, determine whether an individual is resistant to NSV, and can be effectively used for marker-assisted selection breeding of red-spotted grouper, accelerating the development of disease-resistant superior varieties.

[0054] The present invention also provides a kit for detecting the SNPs markers, which can effectively detect the resistance and susceptibility traits of red-spotted grouper to nerve necrosis virus and can be used for molecular marker-assisted breeding of red-spotted grouper. Attached Figure Description

[0055] Figure 1 This is an electrophoretic detection image of PCR amplification using primers for SNP site 1.

[0056] Figure 2 This is an electrophoretic detection image of PCR amplification using primers for SNP site 2.

[0057] Figure 3 This is an electrophoretic detection image of PCR amplification using primers for SNP site 3.

[0058] Figure 4 This is an electrophoretic detection image of PCR amplification using primers for SNP site 4.

[0059] Figure 5 Electrophoretic detection image of PCR amplification using primers for SNP site 5. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0061] Example 1: Screening of SNPs in the PPAR-δ gene of the red-spotted grouper

[0062] 1. Experimental Methods

[0063] (1) Source of the Red-spotted Grouper sample

[0064] The red-spotted grouper samples were taken from the same batch of artificially bred 2-month-old red-spotted grouper from the Xiaodeng Island Grouper Farm in Xiamen. 300 healthy fry weighing about 50g each were randomly selected.

[0065] Red-spotted grouper was injected intraperitoneally with a median lethal concentration (1 × 10⁻⁶). 7 The individuals who died within 3 days or had typical symptoms were considered susceptible to the disease, while those who survived for 10 days without obvious symptoms were considered resistant.

[0066] Fifty resistant individuals (resistant group) and 50 susceptible individuals (susceptible group) were randomly selected, their fins were cut off and preserved in anhydrous ethanol for genomic DNA extraction.

[0067] (2) Genomic DNA extraction from grouper samples

[0068] Genomic DNA extraction was performed using the Trelief™ Animal Genomic DNA Kit (TsingKe), following the instructions:

[0069] 1) Prepare tissue samples: Take an appropriate amount of grouper fin tissue into a 1.5 mL centrifuge tube and cut the fins into small pieces with sterilized scissors;

[0070] 2) Activate the silica membrane: Place the Spin Column in a Collection Tube, add 250 µL of Buffer BL, and centrifuge at 12,000 g for 1 min;

[0071] 3) Sample digestion: Take 20 µL of Proteinase K into a new 1.5 mL centrifuge tube, add 200 µL of ddH2O diluted tissue fragments, vortex for 10 s, then add 200 µL of Buffer gA1, vortex for 10 s, and incubate at 56℃ for 1–3 h or overnight, shaking 3–5 times during the process;

[0072] 4) After incubation, add 200 µL of anhydrous ethanol and vortex to mix.

[0073] 5) Transfer the entire solution obtained in step 4) into a Spin Column, centrifuge at 12,000 g for 1 min, and discard the filtrate;

[0074] 6) Add 500 µL of Buffer PW, centrifuge at 12,000 g for 30 s, and discard the filtrate;

[0075] 7) Repeat step 6) once;

[0076] 8) Add 500 µL Wash Buffer, centrifuge at 12,000 g for 30 s, and discard the filtrate;

[0077] 9) Spin-dry the column at 12,000 g for 2 min, discard the filtrate, place the Spin Column into a new 1.5 mL centrifuge tube, and let it air dry for 1 min.

[0078] 10) Add 50-100 µL of TE Buffer preheated to 65°C to the center of the adsorption membrane, let it stand at room temperature for 2 min, and centrifuge at 12,000 g for 2 min.

[0079] 11) Add the obtained solution back into the Spin Column and centrifuge at 12,000 g for 2 min;

[0080] 12) Take 2 µL of DNA for electrophoresis detection, and 1 μL for DNA concentration determination. Store at -20℃.

[0081] (3) Acquisition of SNPs related to the resistance trait of nerve necrosis virus in red-spotted grouper

[0082] Genomic DNA samples from 50 randomly selected red-spotted grouper fish from both the resistant and susceptible groups were sent to Guangzhou Yunsuo Biotechnology Co., Ltd. for simplified genome sequencing, and genome-wide association analysis of the resistance / susceptibility trait was performed.

[0083] 2. Experimental Results

[0084] The five most significantly associated sites with the highest -log10(P) values ​​were identified as the SNPs associated with the resistance trait of nerve necrosis virus in red-spotted grouper.

[0085] (1) The SNP site 1 is located at the 301 bp starting from the 5' end of the nucleotide sequence shown in SEQ ID NO.1. It is a G / C allele mutation with a genotype of GG or GC.

[0086] (2) SNP site 2 is located at the 300bp position from the 5' end of the nucleotide sequence shown in SEQ ID NO.2. It is an A / C allele mutation with a genotype of AA or AC.

[0087] (3) SNP site 3 is located at the 301 bp starting from the 5' end of the nucleotide sequence shown in SEQ ID NO.3. It is a T / C allele mutation with a genotype of CC or TC.

[0088] (4) SNP site 4 is located at the 300bp position from the 5' end of the nucleotide sequence shown in SEQ ID NO.4. It is a T / A allele mutation with a genotype of TT or TA.

[0089] (5) SNP site 5 is located at the 301 bp starting from the 5' end of the nucleotide sequence shown in SEQ ID NO.5. It is a T / G allele mutation with a genotype of TT or TG.

[0090] Example 2: PCR-sequencing verification analysis of SNPs associated with nerve necrosis virus resistance in red-spotted grouper.

[0091] 1. Experimental Methods

[0092] 1) Primer design

[0093] Based on the location information of the five SNP sites obtained in Example 1, the upstream and downstream sequences of these SNP sites were retrieved from the grouper genome database, and primers for the SNP sites were designed based on this sequence information. The primer information is as follows:

[0094] SNP-1:

[0095] Upstream primer F: 5'-CCTGCTCACTGGACCCTCAC-3' (SEQ ID NO.6);

[0096] Downstream primer R: 5'-CAGTCCCAAGCCACGAGAATA-3' (SEQ ID NO.7).

[0097] SNP-2:

[0098] Upstream primer F: 5'-CTGCCCGTCTGGGAAACTCT-3' (SEQ ID NO.8);

[0099] Downstream primer R: 5'-AGGAAATCGGCTCTGGTGTT-3' (SEQ ID NO.9).

[0100] SNP-3:

[0101] Upstream primer F: 5'-CACTTCCCTGCTGTCCTTTG-3' (SEQ ID NO.10);

[0102] Downstream primer R: 5'-CATCCACCCAGTGCTGAGAC-3' (SEQ ID NO.11).

[0103] SNP-4:

[0104] Upstream primer F: 5'-GGATGTTGAAAGCCGAGCCT-3' (SEQ ID NO.12);

[0105] Downstream primer R: 5'-AAACTGAAATCTTCTGCGATG-3' (SEQ ID NO.13).

[0106] SNP-5:

[0107] Upstream primer F: 5'-GACCTTTCCTTTAATTTCCCTT-3' (SEQ ID NO.14);

[0108] Downstream primer R: 5'-CTGTGGAGATTCAGGCGGTA-3' (SEQ ID NO.15).

[0109] 2) PCR amplification

[0110] Using the genomic DNA of the population sample from Example 1 as a template, PCR amplification was performed using the primers for the SNP sites described above. The PCR reaction system is as follows:

[0111] The PCR reaction system is as follows:

[0112]

[0113] The PCR reaction conditions are as follows:

[0114] 94℃ for 4 min; 94℃ for 10 s, 55℃ for 30 s, 72℃ for 2 min, 35 cycles; 72℃ for 10 min.

[0115] 3) SNP genotyping analysis of sequencing results

[0116] The PCR products were sent to TsingKe for sequencing to obtain the genotypes of each SNP locus. A correlation analysis was then performed between the genotyping results and resistance to neuronecrosis virus (NSV). The correlation analysis was conducted using the chi-square test in SPSS 17.0 statistical software.

[0117] II. Experimental Results

[0118] The results of the analysis of differences in alleles and genotypes of SNP loci between the two groups are shown in Table 1. P<0.05 indicates significant differences.

[0119] Table 1: Statistical analysis of genotype and allele frequencies of 5 SNP loci in the resistant and susceptible groups.

[0120]

[0121] As shown in Table 1, the genotype and allele frequencies of the above five SNP loci differed significantly between the resistant and susceptible groups (P<0.05). The genotypes significantly associated with resistance at these five SNP loci were: SNP 1: GG, SNP 2: AC, SNP 3: CC, SNP 4: TA, and SNP 5: TT; the genotypes significantly associated with susceptibility were: SNP 1: GC, SNP 2: AA, SNP 3: TC, SNP 4: TT, and SNP 5: TG. Therefore, the SNP markers of this invention can be used for breeding disease resistance in red-spotted grouper.

[0122] Example 3: A method for detecting the resistance of red-spotted grouper to nerve necrosis virus.

[0123] The resistance of the red-spotted grouper to nerve necrosis virus was determined by detecting five SNP sites as described in Example 2 on the sample.

[0124] Includes the following steps:

[0125] (1) Extract genomic DNA from the red-spotted grouper to be tested;

[0126] (2) Using the DNA obtained in step (1) as a template, and using the nucleotide sequences shown in SEQ ID NO. 6~7, 8~9, 10~11, 12~13 and 14~15 as primers, PCR amplification was performed to obtain PCR amplification products;

[0127] (3) Sequencing the PCR amplification products obtained in step (2) to determine the genotypes of SNP sites 1-5 of the red-spotted grouper to be tested;

[0128] (4) Determine whether the red-spotted grouper to be tested is resistant to nerve necrosis virus based on the genotypes of SNP loci 1-5 determined in step (3):

[0129] SNP site 1 is located at the 35th bp at the 5' end of the amplification product of the primers shown in SEQ ID NO. 6-7. When the sample genotype is GG, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0130] SNP site 2 is located at the 265th bp at the 5' end of the amplification product of the primers shown in SEQ ID NO. 8-9. When the sample genotype is AC, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0131] SNP site 3 is located at the 181 bp at the 5' end of the amplification product of the primers shown in SEQ ID NO. 10-11. When the sample genotype is CC, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0132] SNP site 4 is located at the 69th bp at the 5' end of the amplification product of the primers shown in SEQ ID NO. 12-13. When the sample genotype is TA, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0133] SNP site 5 is located at the 297th bp at the 5' end of the amplification product of the primers shown in SEQ ID NO. 14-15. When the sample genotype is TT, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

[0134] Example 4: A kit for detecting resistance to nerve necrosis virus in red-spotted grouper.

[0135] 1. Composition

[0136] Nucleotide sequences as shown in SEQ ID NO. 6–15 primers, and PCR reagents.

[0137] 2. Instructions for use

[0138] Same as Example 3.

[0139] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of primers for detecting the SNP locus 2 genotype associated with resistance to nerve necrosis virus in red-spotted grouper in the preparation of a red-spotted grouper nerve necrosis virus resistance detection kit, characterized in that, The primers are used to detect the genotype of SNP site 2; SNP site 2 is located at the 300 bp starting from the 5' end of the nucleotide sequence shown in SEQ ID NO.

2. When the sample genotype is AC, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

2. The application according to claim 1, characterized in that, The nucleotide sequences of the primers are shown in SEQ ID NO. 8-9.

3. The application of primers for detecting the SNP locus 2 genotype associated with resistance to neuronecrosis virus in red-spotted grouper in the breeding of red-spotted grouper with resistance to neuronecrosis virus, characterized in that, The primers are used to detect the genotype of SNP site 2; SNP site 2 is located at the 300 bp starting from the 5' end of the nucleotide sequence shown in SEQ ID NO.

2. When the sample genotype is AC, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

4. The application according to claim 3, characterized in that, The nucleotide sequences of the primers are shown in SEQ ID NO. 8-9.

5. The application of a red-spotted grouper nerve necrosis virus resistance detection kit in the breeding of red-spotted groupers with nerve necrosis virus resistance, characterized in that, The kit includes primers for detecting the genotype of SNP site 2, which is located at the 300 bp from the 5' end of the nucleotide sequence shown in SEQ ID NO.

2. When the sample genotype is AC, the probability of death after infection with neuronecrosis virus is significantly lower than that of individuals with other genotypes.

6. The application according to claim 5, characterized in that, The nucleotide sequences of the primers are shown in SEQ ID NO. 8-9.

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