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CN118166113A - SNP marker v20CT of WSSV resistance of litopenaeus vannamei and application thereof - Google Patents

SNP marker v20CT of WSSV resistance of litopenaeus vannamei and application thereof Download PDF

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CN118166113A
CN118166113A CN202410257193.2A CN202410257193A CN118166113A CN 118166113 A CN118166113 A CN 118166113A CN 202410257193 A CN202410257193 A CN 202410257193A CN 118166113 A CN118166113 A CN 118166113A
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litopenaeus vannamei
v20ct
primer
population
wssv
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李旭鹏
孟宪红
隋娟
栾生
孔杰
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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Abstract

The invention belongs to the technical field of aquatic animal genetic and molecular marker assisted selective breeding, and particularly relates to a SNP marker v20CT of WSSV resistance of litopenaeus vannamei and application thereof. The invention provides a nucleotide sequence containing the SNP marker v20CT, which is shown as SEQ ID NO.1 or SEQ ID NO. 2; the base of the SNP marker locus v20CT is T or C. The SNP marker locus v20CT has high resistance recognition on WSSV of the Litopenaeus vannamei, and can rapidly and efficiently screen out a population with excellent WSSV resistance, so that the designed primer group is not limited by the growth stage of the Litopenaeus vannamei in the screening process, a scientific basis is provided for breeding the Litopenaeus vannamei with WSSV resistance, and the situation that the Litopenaeus vannamei cannot become a parent due to death of individuals in the disease resistance character acquisition process is avoided.

Description

SNP marker v20CT of WSSV resistance of litopenaeus vannamei and application thereof
Technical Field
The invention belongs to the technical field of aquatic animal genetic and molecular marker assisted selective breeding, and particularly relates to a SNP marker v20CT of WSSV resistance of litopenaeus vannamei and application thereof.
Background
Litopenaeus vannamei (Penaeusvannamei), also known as Penaeus vannamei Boone, pacific white shrimp, is the type of prawn with the highest culture yield in China. However, along with the expansion of the cultivation scale, the threat of diseases to the litopenaeus vannamei is also increasing, so that the high-yield and disease-resistant litopenaeus vannamei improved variety has important significance for the healthy development of the industry.
The White Spot Syndrome Virus (WSSV) is one of main pathogens seriously threatening the healthy development of the prawn industry, the transmission speed is high, a virus carrier can directly transmit the virus to offspring vertically, and meanwhile, the virus has strong pathogenicity and lethality, and causes great harm to the prawn breeding industry in China and even the world. However, currently, there is little research on SNP markers with Guan Fanna penaeus vannamei WSSV resistance, and there is a lack of SNP markers that are directly used for screening and assisted breeding.
Disclosure of Invention
The invention aims to provide a SNP marker v20CT of WSSV resistance of litopenaeus vannamei and application thereof, which can efficiently screen WSSV resistant populations of litopenaeus vannamei and is beneficial to culturing and obtaining the litopenaeus vannamei with WSSV resistance.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a SNP marker v20CT of WSSV resistance of litopenaeus vannamei, wherein the nucleotide sequence of the SNP marker v20CT is shown as SEQ ID NO.1 or SEQ ID NO. 2;
The base of the SNP marker locus v20CT is T or C.
Amplifying the primer set of the SNP molecular marker v20CT in the technical scheme, wherein the primer set comprises an amplification primer P1, an amplification primer P2 and an extension primer P3;
the nucleotide sequence of the amplification primer P1 is shown as SEQ ID NO. 3;
the nucleotide sequence of the amplification primer P2 is shown as SEQ ID NO. 4;
the nucleotide sequence of the extension primer P3 is shown as SEQ ID NO. 5.
The SNP marker v20CT or the primer set in the technical scheme is applied to screening and/or cultivating the litopenaeus vannamei colony with WSSV resistance.
Preferably, the screening is performed at the frequency of CT genotypes in the Litopenaeus vannamei population.
The invention also provides a method for screening the WSSV resistant litopenaeus vannamei population by using the primer group in the technical scheme, which comprises the following steps:
Taking the DNA of the Litopenaeus vannamei population to be detected as a template, and carrying out PCR amplification on the template DNA by using the amplification primers P1 and P2 to obtain a PCR amplification product; performing extension treatment on the PCR amplification product by using the extension primer P3 to obtain an extended PCR amplification product; analyzing the extended PCR amplification product by using a time-of-flight mass spectrometry to obtain genotyping data;
When the CT genotype frequency in genotyping data of SNP marker v20CT in the Litopenaeus vannamei population to be detected is more than or equal to 20.41%, the Litopenaeus vannamei population to be detected has WSSV resistance;
When the CT genotype frequency in genotyping data of SNP marker v20CT in the Litopenaeus vannamei population to be detected is less than 20.41%, the Litopenaeus vannamei population to be detected does not have WSSV resistance.
Preferably, the number of the litopenaeus vannamei in the litopenaeus vannamei population to be detected is more than or equal to 30.
Preferably, the PCR amplification system comprises, in 5. Mu.L: 10 to 30 ng/. Mu.L of template DNA, 10. Mu.M of amplification primer P, 10.25. Mu.L of amplification primer P, 10. Mu.M of amplification primer P, 25mM of dNTPs 0.1. Mu.L, 10 XPCRBufer 0.5. Mu.L, 25mM of MgCl 2, 0.4. Mu.L, 5U/. Mu.L of HotStarTaq 0.1. Mu.L, ddH 2 O.
Preferably, the reaction conditions for the PCR amplification include: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 56℃for 25s, elongation at 72℃for 30s,40 cycles; extending at 72 ℃ for 3min; preserving at 4 ℃.
Preferably, the extension treatment comprises an iilex extension reaction reagent; the iCalex extension reaction reagent was 2. Mu.L in weight, and included: 10 xiLexBuffer 0.2. Mu.L, 10 xiLexTerminationMix 0.2. Mu.L, 2U/. Mu.L iLexenzyme 0.041. Mu.L, 10. Mu.M extension primer P30.804. Mu.L and ddH 2 O0.755. Mu.L.
Preferably, the reaction conditions of the extension treatment include: pre-denaturation at 94 ℃ for 30s; denaturation at 94℃for 5s, annealing at 52℃for 5s, extension at 80℃for 5s,40 cycles; extending at 72 ℃ for 3min; preserving at 4 ℃.
The beneficial effects are that:
The invention provides a SNP marker v20CT of WSSV resistance of litopenaeus vannamei, wherein the nucleotide sequence of the SNP marker v20CT is shown as SEQ ID NO.1 or SEQ ID NO. 2; the base of the SNP marker locus v20CT is T or C. The SNP marker locus v20CT has high resistance recognition on WSSV of litopenaeus vannamei, and can rapidly and efficiently screen out a colony with excellent WSSV resistance; in addition, the invention designs a corresponding primer group according to the SNP marker locus v20CT, is not limited by the growth stage of the litopenaeus vannamei in the screening process, and provides scientific basis for breeding WSSV resistance litopenaeus vannamei, thereby avoiding the situation that individuals cannot become parents due to death in the disease resistance character acquisition process. Therefore, the SNP marker v20CT provided by the invention improves the utilization efficiency of the high-quality litopenaeus vannamei parents, not only is beneficial to accelerating the breeding process of the litopenaeus vannamei, but also is beneficial to promoting the development of the breeding industry of the species.
Based on the technical advantages, the invention also provides a method for screening WSSV resistant litopenaeus vannamei populations by using the primer set in the technical scheme, which comprises the following steps: taking the DNA of the Litopenaeus vannamei population to be detected as a template, and carrying out PCR amplification on the template DNA by using the amplification primers P1 and P2 to obtain a PCR amplification product; performing extension treatment on the PCR amplification product by using the extension primer P3 to obtain an extended PCR amplification product; and analyzing the extended PCR amplification product by using a time-of-flight mass spectrometry method to obtain genotyping data. Experiments prove that when the CT genotype frequency in genotyping data of SNP marker v20CT in the litopenaeus vannamei population to be detected is more than or equal to 20.41%, the litopenaeus vannamei population to be detected has WSSV resistance; when the CT genotype frequency in genotyping data of SNP marker v20CT in the Litopenaeus vannamei population to be detected is less than 20.41%, the Litopenaeus vannamei population to be detected does not have WSSV resistance, so that the Litopenaeus vannamei with WSSV resistance can be accurately and rapidly screened.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a graph showing 3 genotype discrimination examples of the result of time-of-flight mass spectrometry of SNP molecular marker v20CT in a detection population sample in example 2. Wherein A: CC genotype. B: CT genotype. C: TT genotype.
Detailed Description
The invention provides a SNP marker v20CT of WSSV resistance of litopenaeus vannamei, wherein the nucleotide sequence of the SNP marker v20CT is shown as SEQ ID NO.1 or SEQ ID NO. 2; the specific nucleotide sequence of SEQ ID NO.1 is ：5'-CTTCCTCTTTCTCTTTCCCTTCCTCTCCCACTCCCTCTCCTCTATCTTGCTTTGGGATTAACAAAAGGTTTAACATCCCATAAAACTTTCATCCTGGCGCGTTTTGGCGGCGATAAACTTGGCGATCTCAAGAACTCGAAATCTTGATCTCTCGGCGAGTGTAACGTCTTTGATTCATCCACTTGTTCCCTCCTTTATTCCCCTTTTGATCTTCTCCCTCTTTCTCTTCTTCTCTTCCGAGTCTGTTCTATCGGCACTTTGGCGGATTCCTCGTTCGTTAAAGAAACCAGTAAAAGGGTAAGTTTTATTTTTGAGTAAATGAGGAGGAATTATTATTATTATTTTCTTCTCTCTCTCCTTCTAACACTTCTGTAATCACTCGGGGAGATATTTTGATTCCT-3';, the specific nucleotide sequence of SEQ ID NO.2 is ：5'-CTTCCTCTTTCTCTTTCCCTTCCTCTCCCACTCCCTCTCCTCTATCTTGCTTTGGGATTAACAAAAGGTTTAACATCCCATAAAACTTTCATCCTGGCGCGTTTTGGCGGCGATAAACTTGGCGATCTCAAGAACTCGAAATCTTGATCTCTCGGCGAGTGTAACGTCTTTGATTCATCCACTTGTTCCCTCCTTTATTCTCCTTTTGATCTTCTCCCTCTTTCTCTTCTTCTCTTCCGAGTCTGTTCTATCGGCACTTTGGCGGATTCCTCGTTCGTTAAAGAAACCAGTAAAAGGGTAAGTTTTATTTTTGAGTAAATGAGGAGGAATTATTATTATTATTTTCTTCTCTCTCTCCTTCTAACACTTCTGTAATCACTCGGGGAGATATTTTGATTCCT-3';, and the base of the SNP marker locus v20CT is T or C (namely, the nucleotide sequence is shown as the underlined position in SEQ ID NO.1 or SEQ ID NO. 2). The SNP marker locus v20CT has high resistance recognition on WSSV of litopenaeus vannamei, and can rapidly and efficiently screen out a colony with excellent WSSV resistance.
The invention provides a primer group for amplifying SNP molecular marker v20CT in the technical scheme, which comprises an amplification primer P1, an amplification primer P2 and an extension primer P3; the nucleotide sequence of the amplification primer P1 is shown as SEQ ID NO.3, and is specifically 5'-ACGTTGGATGCGATAGAACAGACTCGGAAG-3'; the nucleotide sequence of the amplification primer P2 is shown as SEQ ID NO.4, and is specifically 5'-ACGTTGGATGTTCATCCACTTGTTCCCTCC-3'; the nucleotide sequence of the extension primer P3 is shown as SEQ ID NO.5, and is specifically 5'-TAATAGGGAGAAGATCAAAAGG-3'. The amplification primer and the extension primer can specifically amplify the SNP marker v20CT of the WSSV resistance of the litopenaeus vannamei, thereby realizing the screening of the WSSV resistant population of the litopenaeus vannamei.
The invention also provides application of the SNP marker v20CT or the primer set in screening and/or culturing the Litopenaeus vannamei population with WSSV resistance, preferably including application in screening and culturing the Litopenaeus vannamei population with WSSV resistance. The screening is preferably carried out at the frequency of CT genotypes in the litopenaeus vannamei group; the frequency of the CT genotype is obtained according to the number of the populations in the actual screening process, and the higher the proportion of individuals with the CT genotype in the populations, the better the WSSV resistance of the populations is indicated.
The invention provides a method for screening a WSSV resistant litopenaeus vannamei population by using the primer group in the technical scheme, which comprises the following steps: taking the DNA of the Litopenaeus vannamei population to be detected as a template, and carrying out PCR amplification on the template DNA by using the amplification primers P1 and P2 to obtain a PCR amplification product; performing extension treatment on the PCR amplification product by using the extension primer P3 to obtain an extended PCR amplification product; analyzing the extended PCR amplification product by using a time-of-flight mass spectrometry to obtain genotyping data; when the CT genotype frequency in genotyping data of SNP marker v20CT in the Litopenaeus vannamei population to be detected is more than or equal to 20.41%, the Litopenaeus vannamei population to be detected has WSSV resistance; when the CT genotype frequency in genotyping data of SNP marker v20CT in the Litopenaeus vannamei population to be detected is less than 20.41%, the Litopenaeus vannamei population to be detected does not have WSSV resistance.
The invention preferably extracts genome DNA of the Litopenaeus vannamei population to be detected to obtain template DNA. In the invention, the number of the Litopenaeus vannamei groups to be detected is preferably more than or equal to 30; the source of the template DNA preferably comprises muscle tissue of Litopenaeus vannamei; the extraction mode of the template DNA has no special requirement, and the technology well known in the field is adopted; in a specific embodiment of the invention, genomic DNA is extracted with a high throughput DNA extraction kit to obtain template DNA.
After the template DNA is obtained, the invention utilizes the amplification primers P1 and P2 to carry out PCR amplification on the template DNA to obtain a PCR amplification product. In the present invention, the PCR amplification system preferably comprises, in 5. Mu.L: 10 to 30 ng/. Mu.L of template DNA, 10. Mu.M of amplification primer P1.25. Mu.L, 10. Mu.M of amplification primer P2.25. Mu.L, 25mM dNTPs 0.1. Mu.L, 10 XPCR Buffer 0.5. Mu.L, 25mM MgCl 2 0.4.4. Mu.L, 5U/. Mu.L of HotStarTaq 0.1. Mu.L, ddH 2 O.
The reaction conditions for PCR amplification according to the present invention preferably include: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 56℃for 25s, elongation at 72℃for 30s,40 cycles; extending at 72 ℃ for 3min; preserving at 4 ℃.
After the PCR amplification product is obtained, the PCR amplification product is preferably subjected to alkaline phosphatase treatment, so that the PCR amplification product after the alkaline phosphatase treatment is obtained. The alkaline phosphatase-treated reaction system of the present invention is preferably: 5. Mu.L of PCR amplification product, 0.17. Mu.L of 10 XSP Buffer, 1.7U/. Mu.L of SAP Enzyme 0.3. Mu. L, ddH 2 O1.53. Mu.L; the programmed reaction of the alkaline phosphatase treatment is preferably: 40min at 37 ℃;85 ℃ for 5min; preserving at 4 ℃. The removal of dNTPs in the reaction is facilitated by adding SAP (SHRIMP ALIKAIINE phosphtase, shrimp alkaline phosphatase) to the PCR product.
After the PCR amplified product treated by alkaline phosphatase is obtained, the extension primer P3 is utilized to carry out extension reaction on the PCR amplified product treated by alkaline phosphatase, so that the extended PCR amplified product is obtained. In the present invention, the extended reaction system preferably includes: 2. Mu.L of iPLex reagent and 7. Mu.L of alkaline phosphatase-treated PCR amplification product; preferably, the iPlex reagent comprises, per 2 μl of the iPlex reagent: 10×iPerex Buffer 0.2. Mu.L, 10× iPlex Termination mix 0.2.2. Mu.L, 2U/. Mu.L iPerex enzyme 0.041. Mu.L, 10. Mu.M extension primer P3.804. Mu.L and ddH 2 O0.755. Mu.L.
The reaction conditions for the extension of the present invention preferably include: pre-denaturation at 94 ℃ for 30s; denaturation at 94℃for 5s, annealing at 52℃for 5s, extension at 80℃for 5s,40 cycles; extending at 72 ℃ for 3min; preserving at 4 ℃. The apparatus for the extension reaction of the present invention is not particularly limited, and may be any apparatus known in the art.
After the extended PCR amplification product is obtained, the present invention preferably purifies the extended PCR amplification product to obtain a purified PCR amplification product. In the present invention, the purification method and reagent are not particularly limited, and the techniques well known in the art may be employed. In a specific embodiment of the present invention, the PCR amplified product after extension is purified with a resin.
After the purified PCR amplification product is obtained, the method utilizes a time-of-flight mass spectrometry to analyze the purified PCR amplification product to obtain genotyping data. When the CT genotype frequency in genotyping data of SNP marker v20CT in the Litopenaeus vannamei population to be detected is more than or equal to 20.41%, the Litopenaeus vannamei population to be detected has WSSV resistance; when the CT genotype frequency in genotyping data of SNP marker v20CT in the Litopenaeus vannamei population to be detected is less than 20.41%, the Litopenaeus vannamei population to be detected does not have WSSV resistance. The present invention has no particular requirements for the time-of-flight mass spectrometry, and can employ techniques well known in the art. In a specific embodiment of the invention, the instrument for mass spectrometry detection isMALDI-TOFSystem; the software used for data analysis was TYPER.
Experiments prove that after the technical scheme provided by the invention is adopted, the screening process is not limited by the growth stage of the litopenaeus vannamei, and the situation that the litopenaeus vannamei cannot become a parent due to death of individuals in the disease resistance property acquisition process is avoided. Therefore, the technical scheme provided by the invention not only improves the utilization efficiency of the high-quality litopenaeus vannamei parents, but also is beneficial to promoting the development of the litopenaeus vannamei breeding industry and accelerating the breeding and development of the species.
For further explanation of the present invention, the following describes in detail a SNP marker v20CT of WSSV resistance of Litopenaeus vannamei and its application, provided by the present invention, with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
WSSV resistance tests were carried out in the yellow sea aquatic products institute of China aquatic products science, WSSV resistance test laboratory, using Litopenaeus vannamei from the Cooperation base Potentilla Utili industry technology Co., ltd, feeding a total of 293 Litopenaeus vannamei from 15 different families with the same dose of WSSV poison. The infected litopenaeus vannamei was then kept in a round pool of 6m 3, and the dead individuals were noted per hour, with death defined as: the prawn loses balance, falls to one side, and loses response to external stimulus. And the dead individuals store muscle tissues for subsequent experiments, the test is continued until 296 hours are finished, and the remaining 7 surviving litopenaeus vannamei are dead after the test is finished.
The v20CT genotyping and anti-WSSV association analysis steps are as follows:
(1) DNA extraction: DNA extraction is carried out on muscle tissues of each litopenaeus vannamei by using TIANGEN marine animal tissue DNA extraction kit, so as to obtain high-quality genome DNA.
(2) Genotyping: detecting the purity of the genome DNA sample in the step (1) by a micro-spectrophotometer, detecting the integrity of the genome DNA sample by agarose gel electrophoresis, and precisely quantifying the genome DNA sample by fluorescence quantification (Qubit) to obtain a DNA sample with qualified quality.
And carrying out whole genome resequencing analysis on the DNA sample with qualified quality. The resequencing was performed using a platform IlluminaHiSeq and a sequencing depth of 10×, and the sequencing strategy was PE-150. After sequencing was completed, the filtered reads were aligned to the Litopenaeus vannamei genome (ASM 378908v 1) using BWA 0.7 software. The average CLEAN READS numbers 205962265, the average Cleanbases numbers 30662176690, all sample coverage greater than 72.43% and sequencing depth greater than 8.26. Then, the sample is subjected to population SNP detection by GATK4, SNP loci are filtered by PLINK 1.9, samples with deletion rate more than 20% are removed, SNP loci which deviate from Hardy-Weinberg (HWE) balance remarkably, deletion rate more than 15% and Minimum Allele Frequency (MAF) less than 0.05 are removed, and finally, the analysis is carried out by 18137179 high-quality SNP loci and 293 high-quality samples. And after the quality control is finished, filling the genotype by using the bell software to obtain SNP variation sites of the complete genotype.
(3) Correlation analysis: as a result of genome-wide correlation analysis (genome-wideassociationstudy, GWAS) based on the survival time of Litopenaeus vannamei infected with WSSV, the SNP effect value of SNP molecular marker v20CT was 34.517, and the P value was less than 1e-5 (i.e., 0.00001). Namely, the SNP molecular markers provided by the invention have obvious correlation with WSSV resistance of the Litopenaeus vannamei.
Then, 7 penaeus monodon which survived after 296 hours and 23 penaeus monodon which died last (survival time of 23 penaeus monodon is 226.3+ -33.3 hours), namely 30 penaeus monodon in total, were taken as a high-resistance group, and 30 penaeus monodon which died earliest in the test (survival time of 30 penaeus monodon is 45.4+ -3.1 hours) were taken as a low-resistance group. The typing results of SNP molecular markers v20CT in the high resistance group and the low resistance group are shown in Table 1.
TABLE 1 typing results of SNP molecular markers v20CT in high resistance and Low resistance groups
The results in table 1 were subjected to chi-square test and showed P <0.01. It can be seen that the frequencies of the three genotypes of the SNP molecular marker v20CT are very significantly different between the high-resistance group and the low-resistance group. Then, the chi-square test is carried out on the distribution number of CT genotypes in the table 1, and the CT genotype 10:2 ratio and 1:1 ratio significantly different, P <0.05, i.e. the number of CT genotype individuals in the high resistance group was significantly higher (P < 0.05) than in the low resistance group.
Example 2
The litopenaeus vannamei used is from the national institute of aquatic products, yellow sea, and the institute of aquatic products, the national institute of aquatic products, the yellow sea, and the institute of aquatic products, the WSSV resistance test laboratory, developed the following WSSV resistance test.
The prawns to be tested were from 32 families (and different from the 15 families used for the GWAS analysis in example 1). Each family to be tested has 32 test prawns, and 1024 prawns are tested in total. The 32 test prawns of each family to be tested are equally divided into 4 parallel experimental groups, and 256 prawns of each parallel experimental group are cultivated in a circulating water cultivation system of 0.6m 3 water body. Each shrimp is fed with WSSV poison bait with the same dosage, and observed every 6 hours, and information such as survival time, family number, water temperature and the like of dead individuals is recorded. 2 times of feeding baits every 1 day, 1 time of water changing, and 100% of water changing amount each time.
Collecting dead individuals, and taking muscle tissues of the dead individuals to preserve at-80 ℃ for subsequent use. Death is defined as: the prawn loses balance, falls to one side, and loses response to external stimulus. After the test 12d is finished, the muscle tissues of the surviving prawns are collected and stored at the temperature of-80 ℃ so as to be convenient for subsequent use.
The 49 penaeus vannamei which still survived after 12d was tested as the high resistance group and the earliest dead 48 penaeus vannamei (survival time of 48 penaeus vannamei is 145.1.+ -. 36.5 h) was tested as the low resistance group. The SNP marker v20CT genotyping information of the prawns is obtained by a time-of-flight mass spectrometry, and the specific experimental flow is as follows:
(1) DNA extraction: high quality DNA of high resistance group 49 tail shrimp and low resistance group 48 tail shrimp muscle tissue was obtained as PCR template DNA using a high throughput DNA extraction kit.
(2) PCR amplification to obtain PCR amplified product:
PCR reactions were performed in 384 well plates using the amplification primer P1 of SEQ ID NO.3 and the amplification primer P2 of SEQ ID NO.4 in a total volume of 5. Mu.L each, comprising: 1. Mu.L of template DNA (10 ng/. Mu.L), 0.25. Mu.L of amplification primer P1 (10. Mu.M), and adding ddH 2 O to the amplification primer P2(10μM)0.25μL、dNTPs(25mM)0.1μL、PCR Buffer(10×)0.5μL、MgCl2(25mM)0.4μL、HotStar Taq(5U/μL)0.1μL, to a total volume of the reaction system of 5. Mu.L;
Reaction conditions: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 56℃for 25s, elongation at 72℃for 30s,40 cycles; extending at 72 ℃ for 3min; preserving at 4 ℃.
(3) Alkaline phosphatase treatment of PCR products
After the completion of the PCR reaction, the PCR product was treated with Agena Bioscience SAP (SHRIMP ALIKAIINE phosphtase, shrimp alkaline phosphatase) to remove dNTPs in the reaction. SAP Buffer (10X) 0.17. Mu.L, SAP Enzyme (1.7U/. Mu.L) 0.3. Mu. L, ddH 2 O1.53. Mu.L was added to each PCR reaction of step (2). And pressing at 37deg.C for 40min;85 ℃ for 5min; and (3) carrying out a reaction by a reaction program stored at 4 ℃ to obtain a PCR product after alkaline phosphatase treatment.
(4) Extension reaction
The iCalex reagent was prepared and was purchased from Agena Bioscience, inc. at 2. Mu.L each containing the following components: iPLex Buffer (10×) 0.2. Mu.L, iPlex Termination mix (10×) 0.2. Mu.L, iPLex enzyme (2U/. Mu.L) 0.041. Mu.L, extension primer P3 (10. Mu.M) shown in SEQ ID NO.5 0.804. Mu. L, ddH 2 O0.755. Mu.L. Adding an iPLex reaction reagent to the PCR product obtained after the alkaline phosphatase treatment in the step (3). The treatment was performed by the following procedure: pre-denaturation at 94 ℃ for 30s; denaturation at 94℃for 5s, annealing at 52℃for 5s, extension at 80℃for 5s,40 cycles; extending at 72 ℃ for 3min; preserving at 4 ℃.
(5) Purification of the product
6Mg of cation exchange resin is taken and evenly covered on a 384-hole resin scraper, redundant resin is scraped off, and the resin is placed for 20min. Centrifuging the 384-well plate subjected to the extension reaction in the step (4) at 1000rpm for 1min, adding 25 mu L of deionized water into each well, inverting the 384-well plate on a resin plate, reversely fastening the resin plate on the 384-well plate, knocking to enable the resin to fall into the 384-well plate, and sealing the membrane. Turning over the 384-well plate for 20min by taking the long axis of the 384-well plate as the axis, and centrifuging for 5min at 3500rpm to obtain a purified PCR product for later use.
(6) Mass spectrometry detection
Samples were spotted with Nanodispenser SpectroCHIP chips, and the purified PCR product detection samples were transferred from 384-well reaction plates to MASSARRAY SPECTROCHIP chips with surface-coated substrates. And carrying out mass spectrum detection by MASSARRAY ANALYZER COMPAC, transferring the sample to a SpectroCHIP chip, and then placing the sample into a mass spectrometer for detection, wherein each detection point only needs 3-5 s, and full-automatic analysis is carried out. And then analyzing the experimental result by TYPER software to obtain typing data, wherein the results are shown in table 2 and figure 1 (figure 1 is 3 genotype discrimination examples of the flight time mass spectrometry typing result of SNP molecular marker v20CT in the detection population sample, wherein A represents CC genotype experimental result, B represents CT genotype experimental result, and C represents TT genotype experimental result).
TABLE 2 SNP molecular marker v20CT typing results in high-resistance and Low-resistance groups
Chi-square test was performed on the results of table 2, which showed P <0.01. That is, the frequencies of three genotypes of the SNP molecular markers v20CT have extremely significant differences between the high-resistance group and the low-resistance group. Chi square test was continued for each genotype distribution number in table 2, CC genotypes 20:17 ratio and 49: the 48 ratios were not significantly different, P >0.05; CT genotype 10:1 ratio and 49: significant differences in 48 ratios, P <0.05; TT genotype 19:30 ratio and 49: the 48 ratios were not significantly different, P >0.05. It can be seen that the number of CT genotype individuals in the high resistance group was significantly higher (P < 0.05) than in the low resistance group.
Therefore, the proportion of CT genotype individuals of the SNP molecular marker v20CT provided by the invention in the high-resistance group is 20.41%. When the CT genotype frequency of SNP molecular marker v20CT in the litopenaeus vannamei population is more than or equal to 20.41%, the population is selected as a parent for culturing WSSV resistant litopenaeus vannamei. The ratio of 20.41% is the data obtained by combining 2 resistance correlation analyses, and can be used as a corresponding reference value, while in actual implementation, the higher the ratio of CT genotype individuals in the population, the better the WSSV resistance.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (10)

1. The SNP marker v20CT of the WSSV resistance of the litopenaeus vannamei is characterized in that the nucleotide sequence of the SNP marker v20CT is shown as SEQ ID NO.1 or SEQ ID NO. 2;
The base of the SNP marker locus v20CT is T or C.
2. The primer set for amplifying the SNP molecular marker v20CT of claim 1, wherein the primer set comprises an amplification primer P1, an amplification primer P2 and an extension primer P3;
the nucleotide sequence of the amplification primer P1 is shown as SEQ ID NO. 3;
the nucleotide sequence of the amplification primer P2 is shown as SEQ ID NO. 4;
the nucleotide sequence of the extension primer P3 is shown as SEQ ID NO. 5.
3. Use of the SNP marker v20CT of claim 1 or the primer set of claim 2 for screening and/or breeding a population of litopenaeus vannamei with WSSV resistance.
4. The use according to claim 3, wherein said screening is performed at the frequency of CT genotypes in said population of litopenaeus vannamei.
5. A method for screening a WSSV resistant population of litopenaeus vannamei using the primer set of claim 2, comprising the steps of:
Taking the DNA of the Litopenaeus vannamei population to be detected as a template, and carrying out PCR amplification on the template DNA by using the amplification primers P1 and P2 to obtain a PCR amplification product; performing extension treatment on the PCR amplification product by using the extension primer P3 to obtain an extended PCR amplification product; analyzing the extended PCR amplification product by using a time-of-flight mass spectrometry to obtain genotyping data;
When the CT genotype frequency in genotyping data of SNP marker v20CT in the Litopenaeus vannamei population to be detected is more than or equal to 20.41%, the Litopenaeus vannamei population to be detected has WSSV resistance;
When the CT genotype frequency in genotyping data of SNP marker v20CT in the Litopenaeus vannamei population to be detected is less than 20.41%, the Litopenaeus vannamei population to be detected does not have WSSV resistance.
6. The method according to claim 5, wherein the number of litopenaeus vannamei in the litopenaeus vannamei population to be tested is not less than 30.
7. The method of claim 5, wherein the PCR amplification system is 5 μl, comprising: 10 to 30 ng/. Mu.L of template DNA, 10. Mu.M of amplification primer P, 10.25. Mu.L of amplification primer P, 10. Mu.M of amplification primer P, 25mM of dNTPs, 0.1. Mu.L of 10 XPCR Buffer, 0.5. Mu.L of 25mM of MgCl 2, 0.4. Mu.L of HotStarTaq, 5U/. Mu.L of ddH 2 O.
8. The method of claim 5, wherein the reaction conditions for PCR amplification comprise: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 56℃for 25s, elongation at 72℃for 30s,40 cycles; extending at 72 ℃ for 3min; preserving at 4 ℃.
9. The method of claim 5, wherein the extension treatment comprises an iPlex extension reagent; the iCalex extension reaction reagent was 2. Mu.L in weight, and included: 10×iPerex Buffer 0.2. Mu.L, 10× iPlex Termination mix 0.2.2. Mu.L, 2U/. Mu.L iPerex enzyme 0.041. Mu.L, 10. Mu.M extension primer P30.804. Mu.L and ddH 2 O0.755. Mu.L.
10. The method of claim 5, wherein the reaction conditions of the extension treatment comprise: pre-denaturation at 94 ℃ for 30s; denaturation at 94℃for 5s, annealing at 52℃for 5s, extension at 80℃for 5s,40 cycles; extending at 72 ℃ for 3min; preserving at 4 ℃.
CN202410257193.2A 2024-03-07 2024-03-07 SNP marker v20CT of WSSV resistance of litopenaeus vannamei and application thereof Pending CN118166113A (en)

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