CN112430673A - Reagent for detecting SNP locus genotype related to resistance of grouper nervous necrosis virus - Google Patents
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Abstract
The invention discloses a reagent for detecting SNP locus genotype related to resistance of nervous necrosis viruses of grouper, which comprises a reagent body and a reagent body. The invention discloses an SNP locus related to the resistance of nervous necrosis virus, and the death probability of Epinephelus akaara with the genotype of CT infected with nervous necrosis virus is obviously lower than that of individuals with the genotype of CC or TT. The genotype of the SNP of the epinephelus akaara can be detected, and whether the epinephelus akaara has the anti-infection property of the nervous necrosis virus can be effectively determined. The SNP marker is closely related to the resistance of the epinephelus akaara to the nervous necrosis virus, and the selection of individuals with CT genotypes in parent breeding is favorable for improving the resistance of progeny to nervous necrosis diseases.
Description
Technical Field
The invention relates to the technical field of molecular biology and genetic breeding, in particular to a reagent for detecting SNP locus genotype related to resistance of a grouper nervous necrosis virus.
Background
Epinephelus akaara is commonly known as erythema, and Epinephelus is mainly distributed in the west part of the North Pacific ocean, and is produced in the Taiwan area of the Zhoushan island from the south of the Zhoushan island to the south of the south China's Taiwan strait. The grouper has the characteristics of delicious meat quality and high nutritional value, and is an important grouper breeding variety in coastal areas of south China.
The red-spotted grouper nervous necrobiosis virus (RGNNV) has great harm to adult and juvenile groupers of groupers, the death rate can reach 100% in serious cases, huge economic loss is often caused to grouper cultivation, the healthy development of the grouper cultivation industry is seriously threatened, however, no effective treatment means is available for diseases caused by the virus at present, and mainly prevention and control monitoring are performed.
Chinese patent CN201610255794.5 discloses a method for detecting nervous necrosis virus infection of grouper based on Sandwich ELASA of aptamer; CN201610465941.1 discloses a colloidal gold immunochromatographic assay test paper for detecting a Epinephelus septemfasciatus nervous necrosis virus antibody and application thereof; CN201910873715.0 discloses a colloidal gold test strip for detecting the nervous necrosis virus of grouper and a preparation and detection method thereof; CN200910039534.4 discloses a primer group, a detection method and a rapid diagnostic kit for detecting the red-spotted grouper nervous necrosis virus; CN03114369.5 discloses a kit for diagnosing viral nervous necrosis virus genes of grouper and a detection method, but a method for detecting resistance of the viral nervous necrosis virus of the grouper is lacked.
PPAR-delta (Peroxisome proliferator-activated receptor delta) is one of the members of the Peroxisome proliferator-activated receptor family PPARs (Peroxisome proliferator-activated receptors), a class of transcription factors that can regulate various biological processes of the body, such as lipid metabolism, cell survival, cancer cell invasion, angiogenesis, oncogenic signaling pathway, immune response, etc., by inhibiting or activating the expression of a target gene on a Peroxisome proliferator-responsive element (PPRE) bound to a promoter region upstream of the target gene. In recent years, the regulation of PPARs in immune response has been receiving more and more attention from researchers.
In recent years, with the development of modern biotechnology such as cell biology, molecular biology and the like, the DNA molecular marker technology is gradually applied to the breeding research work of new disease-resistant varieties, and the huge application potential is shown, and the development and application of disease-resistant molecular markers can greatly improve the effectiveness and the predictability of breeding. The SNP marker is a biallelic genetic variation, compared with other molecular markers, the detection is more automatic, the statistics are more convenient, and the SNP marker is the most ideal genetic marker.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a reagent for detecting the SNP locus genotype related to the resistance of the grouper nervous necrosis virus.
The first purpose of the invention is to provide a reagent for detecting SNP locus genotype related to resistance of the grouper nervous necrosis virus.
The second purpose of the invention is to provide the application of the reagent in the preparation of a kit for detecting the resistance of the nervous necrosis virus of the grouper.
The third purpose of the invention is to provide a kit for detecting the resistance of the nervous necrosis virus of the grouper.
The fourth purpose of the invention is to provide the application of the reagent or the kit for detecting the resistance of the nervous necrosis virus of the grouper in the molecular breeding of the grouper, which is characterized in that the grouper with the resistance of the nervous necrosis virus of the grouper is bred.
In order to achieve the purpose, the invention is realized by the following scheme:
the invention discloses an SNP locus related to the resistance of a nervous necrosis virus, the SNP locus is positioned at the 520 th basic group at the 5' end of a nucleotide sequence shown in SEQ ID NO.1, and the death probability of Epinephelus akaara with the genotype of CT infected with the nervous necrosis virus is obviously lower than that of individuals with the genotypes of CC or TT.
Therefore, the invention claims a reagent for detecting the SNP locus genotype related to the resistance of the grouper nervous necrosis virus, the reagent is used for detecting the genotype of the SNP locus, the SNP locus is positioned at the 520 th base at the 5' end of the nucleotide sequence shown in SEQ ID NO.1, and the death probability of the grouper with the SNP locus genotype of CT after being infected with the nervous necrosis virus is obviously lower than that of an individual with the genotype of CC or TT.
Preferably, the grouper is grouper akaara.
Preferably, the reagent is a primer with a nucleotide sequence shown in SEQ ID NO. 2-3.
SEQ ID NO.2：5’-gtgggaggaatgctgatgtg-3’；
SEQ ID NO.3：5-attcttcttcaccgtggaggc-3’。
The invention also claims application of the reagent in preparation of a kit for detecting the resistance of the nervous necrosis virus of the grouper.
The invention further claims a kit for detecting the resistance of the nervous necrosis virus of the grouper, which contains the reagent.
Preferably, the reagent is a primer with a nucleotide sequence shown in SEQ ID NO. 2-3.
The using method of the kit comprises the following steps:
(1) extracting the genome DNA of the epinephelus akaara to be detected;
(2) performing PCR amplification by using the DNA obtained in the step (1) as a template and using nucleotide sequences shown in SEQ ID NO. 2-3 as primers to obtain a PCR amplification product;
(3) sequencing the PCR amplification product obtained in the step (2) to determine the genotype of the SNP marker of the epinephelus akaara to be detected;
(4) determining whether the epinephelus akaara to be detected is susceptible to the nervous necrosis virus according to the genotype of the SNP marker determined in the step (3):
that is, the 520 th base C or T of the nucleotide sequence (584 bp) shown in SEQ ID NO.1 from the 5' end is obviously related to the resistance traits of the red spotted grouper nervous necrosis virus, the genotype of the SNP marker of the sample red spotted grouper is CT, the sample is more resistant to the nervous necrosis virus, namely, the death probability of the red spotted grouper infected with the nervous necrosis virus with the genotype of the SNP site CT is obviously lower than that of other genotype individuals.
In the using method of the kit for detecting the resistance of the nervous necrosis virus of the pikes, the genome extraction is not particularly limited, the traditional phenol chloroform method can be adopted for extraction, and the kit can also be adopted for extraction.
The individual genotype detection method of the epinephelus akaara is not particularly limited, and the SnapShot, flight time mass spectrum, sequencing, chip, single-strand conformation polymorphism polymerase chain reaction, restriction fragment length polymorphism polymerase chain reaction and other technologies can be used for SNP detection. The SnapShot method for detecting the SNP locus has the advantages of high accuracy, strong flexibility, large flux and short detection period, so that the embodiment of the invention adopts the SnapShot method for detecting the SNP marker.
The invention further claims application of the reagent and the kit for detecting the resistance of the nervous necrosis viruses of the grouper in molecular breeding of the grouper, and breeding the grouper with the resistance of the nervous necrosis viruses of the grouper.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses an SNP locus related to the resistance of nervous necrosis virus, and the death probability of Epinephelus akaara with the genotype of CT infected with nervous necrosis virus is obviously lower than that of individuals with the genotype of CC or TT. The genotype of the SNP of the epinephelus akaara can be detected, and whether the epinephelus akaara has the anti-infection property of the nervous necrosis virus can be effectively determined.
The SNP marker is closely related to the resistance of the epinephelus akaara to the nervous necrosis virus, and the selection of individuals with CT genotypes in parent breeding is favorable for improving the resistance of progeny to nervous necrosis diseases.
The invention also provides a pair of primers for detecting the SNP locus, the genotype of the epinephelus akaara to be detected can be effectively detected by utilizing the primer pair of the SNP locus, and the result can judge whether the epinephelus akaara to be detected has the anti-induction property to the nervous necrosis virus on one hand and provide reference for parent selection on the other hand. Therefore, the SNP locus primer can be used for detecting the genotype of the Epinephelus akaara, judging whether the individual has the resistance to the nervous necrosis virus, and can be effectively used for molecular marker-assisted selective breeding of the Epinephelus akaara and accelerating the breeding process of disease-resistant excellent varieties of the Epinephelus akaara.
The invention also provides a kit for detecting the SNP marker, which can effectively detect the anti-infection property of the epinephelus akaara to the nervous necrosis virus and is used for the molecular marker assisted breeding of the epinephelus akaara.
Drawings
FIG. 1 is an electrophoretic detection map of PCR amplification using SNP site primers.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
EXAMPLE 1 screening of SNPs of PPAR-delta Gene of Epinephelus akaara
1. Experimental methods
(1) Epinephelus akaara sample source
The sample epinephelus akaara to be detected is taken from 2-month-old artificially propagated epinephelus akaara fish of the same batch in a small hill path island of mansion gate, and 300 healthy fish fries with the weight of about 50 g/tail are randomly selected.
By intraperitoneal injectionInjecting into Epinephelus akaara with semi-lethal concentration (1 × 10)7TCID50mL), the dead individuals and the individuals with typical disease symptoms are susceptible individuals within 3 days, and the individuals which survive after 10 days and have no obvious disease symptoms are resistant individuals.
Randomly selecting 50 resistant individuals (resistant group) and 50 susceptible individuals (susceptible group), shearing fin rays, and storing in absolute ethyl alcohol for genome DNA extraction.
(2) Grouper sample genome DNA extraction
Genomic DNA extraction Using TrelieftAmal Genomic DNA Kit (TsingKe) Kit, the following procedure was followed according to the instructions:
1) preparing a tissue sample: taking a proper amount of grouper fin ray tissues in a 1.5mL centrifuge tube, and shearing the fin ray into pieces by using sterilized scissors;
2) activating the silica gel membrane: placing Spin Column in Collection Tube, adding 250 μ L Buffer BL, and centrifuging at 12,000g for 1 min;
3) sample digestion: add 20. mu.L of Proteinase K to a new 1.5mL centrifuge tube and add 200. mu.L of ddH2Carrying out vortex oscillation on the tissue fragments subjected to O dilution for 10s, then adding 200 mu L of Buffer gA1, carrying out vortex oscillation for 10s, and incubating for 1-3 h or overnight at 56 ℃, wherein the oscillation is carried out for 3-5 times;
4) after the incubation is finished, adding 200 mu L of absolute ethyl alcohol, and uniformly mixing by vortex oscillation;
5) transferring all the solution obtained in the step 4) into Spin Column, centrifuging for 1min at 12,000g, and removing the filtrate;
6) adding 500 mu L of Buffer PW, centrifuging for 30s at 12,000g, and discarding the filtrate;
7) repeating the step 6) once;
8) adding 500 mu L of Wash Buffer, centrifuging for 30s at 12,000g, and discarding the filtrate;
9) throwing for 2min at 12,000g, discarding the filtrate, putting Spin Column into a new 1.5mL centrifuge tube, opening the cover and airing for 1 min;
10) adding 50-100 mu L of TE Buffer preheated to 65 ℃ in advance to the center of the adsorption film, standing at room temperature for 2min, and centrifuging at 12,000g for 2 min;
11) adding the obtained solution into Spin Column again, and centrifuging at 12,000g for 2 min;
12) 2. mu.L of DNA was subjected to electrophoresis, and 1. mu.L was used for determination of DNA concentration, and stored at-20 ℃.
(3) Obtaining of DNA sequence of PPAR-delta gene of Epinephelus akaara
Taking the DNA of the red spot grouper fin-shaped strip extracted in the previous step as a template, designing a Primer by using Primer 5.0 software according to the DNA sequence of the PPAR-delta gene in the epinephelus coioides genomic database as a reference, and finally obtaining the genomic DNA sequence of the PPAR-delta gene of the red spot grouper through PCR amplification, product sequencing, sequence splicing and comparison.
(4) Screening of SNPs of PPAR-delta gene of Epinephelus akaara
The screening of the SNPs sites of the PPAR-delta gene of the epinephelus akaara is carried out by a direct sequencing method. Designing primers according to the obtained PPAR-delta genomic DNA sequences, carrying out PCR amplification by taking the genomic DNA of 5 samples of each of the anti-susceptible group and the susceptible group as a template, sending the obtained PCR product to TsingKe company for sequencing, observing a peak diagram of a sequencing result, carrying out multiple comparison analysis on the sequencing result, and predicting the SNPs and the genotypes corresponding to the SNPs.
The PCR reaction system is as follows:
the PCR reaction conditions were as follows:
4min at 94 ℃; at 94 ℃ for 10s, at 55 ℃ for 30s, at 72 ℃ for 2min, for 35 cycles; 10min at 72 ℃.
2. Results of the experiment
8 candidate SNP sites are obtained by primary screening.
Example 2 confirmation of SNPs of PPAR-delta Gene of Epinephelus akaara
1. Experimental methods
According to 8 candidate PPAR-delta gene SNP loci and sequences nearby, which are obtained in example 1, 50 susceptible samples and 50 resistant samples are randomly selected as templates, the SnapShot method is adopted to carry out genotyping on the SNPs obtained in example 1, and correlation analysis is carried out on the genotyping result and the nervous necrosis virus resistance.
The SnapShot method detection was performed by TsingKe, and the amplification primer information and the single-base extension primer information are shown in tables 1 and 2, respectively. Correlation analysis against nervous necrosis virus genotyping of 8 SNPs and correlation analysis with nervous necrosis virus resistance was performed using the chi-square test in SPSS 17.0 statistical software (P <0.05 indicates significant difference).
Table 1: snapshot method for detecting SNP locus peripheral primer information
Table 2: snapshot method for detecting single base extension primer information of SNP locus
2. Results of the experiment
The results of the analysis of the differences between the two groups for alleles and genotypes are shown in Table 3. Through the association analysis of SPSS software, 1 SNP locus (SNP5) related to the resistance of the nervous necrosis virus is obtained, and the SNP5 locus is located at the 520bp position from the 5' end of the nucleotide sequence (full length 584bp) shown in SEQ ID NO. 1.
Table 3: statistical analysis of SNPs of grouper PPAR-delta gene in anti-susceptible group and susceptible group
As can be seen from Table 1, at this locus, the frequencies of genotypes CC, CT and TT in the disease-resistant population were 37.5%, 60.4% and 2.1%, and the frequencies of genotypes CC, CT and TT in the susceptible population were 57.1%, 38.8% and 2.1%, and the statistical analysis result showed that the allele frequencies at this locus were significantly different in the susceptible and resistant populations (P < 0.05). Further proves that the 520 th base C or T of the nucleotide sequence (the full length 584bp) shown in SEQ ID NO.1 from the 5' end is obviously related to the red spotted grouper nervous necrosis virus resistance trait, and is an SNP marker related to red spotted grouper diseases.
The method comprises the steps of carrying out SNP marker detection on the Epinephelus akaara to be detected, and determining the anti-infection performance of the Epinephelus akaara to be detected on the nervous necrosis virus. The method comprises the following steps:
(1) extracting the genome DNA of the epinephelus akaara to be detected;
(2) performing PCR amplification by using the DNA obtained in the step (1) as a template and using nucleotide sequences shown in SEQ ID NO. 2-3 as primers to obtain a PCR amplification product,
SEQ ID NO.2：5’-gtgggaggaatgctgatgtg-3’。
SEQ ID NO.3：5’-attcttcttcaccgtggaggc-3’。；
(3) sequencing the PCR amplification product obtained in the step (2) to determine the genotype of the SNP marker of the epinephelus akaara to be detected;
(4) determining whether the epinephelus akaara to be detected is susceptible to the nervous necrosis virus according to the genotype of the SNP marker determined in the step (3):
that is, the 520 th base C or T of the nucleotide sequence (584 bp) shown in SEQ ID NO.1 from the 5' end is obviously related to the resistance traits of the red spotted grouper nervous necrosis virus, the genotype of the SNP marker of the sample red spotted grouper is CT, the sample is more resistant to the nervous necrosis virus, namely, the death probability of the red spotted grouper infected with the nervous necrosis virus with the genotype of the SNP site CT is obviously lower than that of other genotype individuals.
Example 4 kit for detecting disease resistance of Epinephelus akaara to nervous necrosis virus
A, make up
Primers with nucleotide sequences shown as SEQ ID NO. 2-3 and PCR amplification reagents;
wherein, SEQ ID NO. 2: 5'-gtgggaggaatgctgatgtg-3', respectively;
SEQ ID NO.3：5’-attcttcttcaccgtggaggc-3’。
second, use method
The same as in example 3.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
<110> southern China university of agriculture
<120> a reagent for detecting SNP locus genotype associated with resistance to nervous necrosis virus of grouper
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 584
<212> DNA
<213> Epinephelus akaara
<400> 1
gtgggaggaa tgctgatgtg aaactgcagc gaggcgagtg atgagtcaac ctgtcttttt 60
catcagataa taatgagata aagtcacatg tacataagtt ttactcaaat ggcaaaatga 120
ttggtttttg gctgcagaat actgaataaa atgttcaaaa taatctgatg accgaagact 180
taaatgtcaa cagagtaaaa ctaaactaaa aatagttttc tttaatgcag ataaaactaa 240
aatgtcagac tttgagtcaa ccaaaacgtg actaaaaaaa caggatgacg ttgactgtat 300
atgataaaaa cttacaagga catttgaaac aagactaaga caaaataaaa tgtagctgac 360
aaaaataaca tgtgctgctt cggtataatt tattgttctg tgtgctgtgc acattttggc 420
ttttattcta atttacattt tataaaatta ttttactcca aaggaaatct gtctttgata 480
agaataattg acctgccatt ttgtgtatca gttacccact gcgtgttacc ttgaattcat 540
gaagaaaacg ttttttctct catgcctcca cggtgaagaa gaat 584
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gtgggaggaa tgctgatgtg 20
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
attcttcttc accgtggagg c 21
Claims (7)
1. A reagent for detecting the genotype of an SNP locus related to the resistance of the nervous necrosis virus of rockfishes is characterized in that the reagent is used for detecting the genotype of the SNP locus, the SNP locus is positioned at the 520 th base at the 5' end of a nucleotide sequence shown in SEQ ID NO.1, and the death probability of the rockfishes with the genotype of the SNP locus being CT infected with the nervous necrosis virus is obviously lower than that of individuals with the genotypes being CC or TT.
2. The reagent as claimed in claim 1, wherein the grouper is grouper akaara.
3. The reagent according to claim 1 or 2, wherein the reagent is a primer having a nucleotide sequence shown in SEQ ID No. 2-3.
4. Use of the reagent of claim 1 or 2 for the preparation of a kit for detecting nervous necrosis virus resistance in grouper.
5. A kit for detecting the nervous necrosis virus resistance of grouper, which comprises the reagent of claim 1.
6. The kit for detecting the resistance to the nervous necrosis virus of the pike according to claim 4, wherein the reagent is a primer having a nucleotide sequence as shown in SEQ ID No. 2-3.
7. Use of the reagent according to claim 1 or the kit for detecting the resistance to the nervous necrosis virus of epinephelus according to claim 5 in the molecular breeding of epinephelus, wherein the epinephelus having the resistance to the nervous necrosis virus of epinephelus is selected.
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| CN115992265A (en) * | 2023-03-22 | 2023-04-21 | 中山大学 | Grouper whole genome liquid phase chip and application thereof |
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| CN109852710A (en) * | 2019-04-11 | 2019-06-07 | 深圳华大海洋科技有限公司 | One kind SNP marker relevant to grouper ammonia tolerance and application thereof |
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| CN109852710A (en) * | 2019-04-11 | 2019-06-07 | 深圳华大海洋科技有限公司 | One kind SNP marker relevant to grouper ammonia tolerance and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| HUI GE 等: "De novo assembly of a chromosome‐level reference genome of red‐spotted grouper(Epinephelus akaara)using nanopore sequencing and Hi‐C", 《MOLECULAR ECOLOGY RESOURCES》, vol. 19, no. 6, 14 August 2019 (2019-08-14), pages 1461 - 1469 * |
| XIANG WANG等: "An SNP-Based Genetic Map and QTL Mappingfor Growth Traits in the Red-Spotted Grouper(Epinephelus akaara)", 《GENES》, vol. 10, no. 793, 12 October 2019 (2019-10-12), pages 1 - 16 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115992265A (en) * | 2023-03-22 | 2023-04-21 | 中山大学 | Grouper whole genome liquid phase chip and application thereof |
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