CN114540510A - SNP marker on LvFREP2 gene related to disease resistance of litopenaeus vannamei, detection primer and application of SNP marker - Google Patents
SNP marker on LvFREP2 gene related to disease resistance of litopenaeus vannamei, detection primer and application of SNP marker Download PDFInfo
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Abstract
The invention discloses an SNP marker on an LvFREP2 gene related to the disease resistance of litopenaeus vannamei, a detection primer and application thereof. The mutation site disclosed by the invention is positioned in the coding region of the LvFREP2 gene of litopenaeus vannamei, the base of the mutation site is C, and when the genotype of the mutation site is CC individual, the survival rate under the condition of vibrio infection is higher. The invention also discloses a primer combination for detecting the mutation site and a breeding application method thereof. In the breeding work of the disease resistance of the litopenaeus vannamei, the method disclosed by the invention can be used for screening a backup parent group or a breeding family, and eliminating individuals susceptible to vibrio infection as soon as possible, thereby laying a foundation for rapidly breeding the good disease resistance variety of the litopenaeus vannamei.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to an SNP marker on an LvFREP2 gene related to the disease resistance of litopenaeus vannamei, a detection primer and application thereof.
Background
Litopenaeus vannamei (Litopenaeus vannamei) is the most important cultured prawn, accounting for more than 80% of the total yield of all cultured prawns (FAO, 2020). By 2021 years, the annual seed production of litopenaeus vannamei in China is 1.56 trillion, the annual culture yield exceeds 180 million tons, the litopenaeus vannamei culture industry becomes the supporting industry for the development of fishery in China, and the expanding propagation of improved seeds has great significance for the litopenaeus vannamei culture industry.
However, the artificial culture of litopenaeus vannamei in China has been seriously troubled by various viruses represented by White Spot Syndrome Virus (WSSV), various bacteria represented by Vibrio parahaemolyticus (Vibrio parahaemolyticus) and Vibrio harveyi (V.harveyi) and parasites represented by shrimp Enterocytozoon hepatopenaei (Enterococcyon hepatopenaei) for a long time. In recent years, outbreaks of various diseases bring huge economic damage to the breeding industry. Therefore, the breeding for disease resistance is an urgent need for sustainable development of the litopenaeus vannamei breeding industry in China.
Fibrinogen-related proteins (FREPs), also known as fibrinogen-like domain-containing immunoagglutinins (FBs), are widespread in vertebrates and invertebrates and participate in the endogenous immune response of the body as pattern recognition proteins that play important roles in cell adhesion, phagocytosis and encapsulation. Therefore, the invention develops the SNP marker related to the disease resistance of the litopenaeus vannamei aiming at the LvFREP2 gene of the litopenaeus vannamei, and provides the excellent molecular marker for the disease-resistant auxiliary breeding of the litopenaeus vannamei, thereby improving the breeding efficiency of the disease-resistant variety of the litopenaeus vannamei.
Disclosure of Invention
The invention aims to provide an SNP marker on an LvFREP2 gene related to the disease resistance of litopenaeus vannamei, a detection primer and application thereof. The molecular marker can be applied to molecular marker-assisted selective breeding of the disease resistance of the litopenaeus vannamei, and provides useful information for SNP (single nucleotide polymorphism) variation sites related to the disease resistance, so that the breeding efficiency of the disease-resistant excellent variety of the litopenaeus vannamei is improved.
In order to realize the aim, the genomic DNA of the litopenaeus vannamei dead and living after virus attack is used as a template, the templates and the genotype of each SNP locus contained in the LvFREP2 gene are detected by designing primers and PCR amplification, finally, the SNP markers related to disease resistance are determined by correlation analysis, the SNP locus amplification primers are provided, a technical system for backup parent population or alternative family molecular breeding is established, and a foundation is laid for accelerating the breeding of excellent disease-resistant varieties of the litopenaeus vannamei.
The first purpose of the invention is to provide a SNP marker related to the disease resistance of litopenaeus vannamei, wherein the SNP marker is positioned in a sequence shown in SEQ ID NO.1, the 11 th base position from the 5' end is C or T, and is represented by Y (C/T). The genotype of the variation site is obviously related to the vibrio resistance of the litopenaeus vannamei. The base sequence of SEQ ID NO.1 is: 5'-GCTGGTGGTA (C/T) AACGTGTGTC-3'.
The second purpose of the invention is to provide a detection primer of an SNP marker related to the disease resistance of litopenaeus vannamei, which comprises the following primers:
KG-F: 5'-TGTAAAACGACGGCCAGTGAGGGAATATCATTTGTATGATGC-3' (shown in SEQ ID NO. 2);
KG-R: 5'-CAGGAAACAGCTATGACCAGAGAGACGCGGAGAGAGG-3' (shown as SEQ ID number 3).
The third purpose of the invention is to provide the application of the product for identifying the SNP marker in the preparation of the reagent for detecting the disease resistance of the litopenaeus vannamei.
The fourth purpose of the invention is to provide the application of the SNP marker or the detection primer in the preparation of the detection kit for the disease resistance of the litopenaeus vannamei.
The fifth purpose of the invention is to provide a litopenaeus vannamei disease resistance detection kit, which comprises the detection primer.
The sixth purpose of the invention is to provide the application of the SNP marker, the detection primer or the detection kit in the detection of the disease resistance of the litopenaeus vannamei. When the genotype of the SNP marker is CC, the disease resistance of the litopenaeus vannamei is high.
The seventh purpose of the invention is to provide the application of the SNP marker, the detection primer or the detection kit in the molecular marker-assisted selective breeding of the litopenaeus vannamei. In particular to application in breeding of disease-resistant variety of litopenaeus vannamei.
The eighth purpose of the invention is to provide a breeding method of a litopenaeus vannamei disease-resistant variety, which comprises the following steps:
a) extracting the genomic DNA of the litopenaeus vannamei to be detected;
b) carrying out PCR amplification on the genomic DNA of the litopenaeus vannamei to be detected by using the detection primer KG-F/KG-R of claim 2;
c) sequencing the amplified product, determining the genotype of the SNP marker according to claim 1, and selecting a CC genotype individual as a backup parent to breed the litopenaeus vannamei disease-resistant variety.
The SNP marker disclosed by the invention is positioned in a coding region of a Litopenaeus vannamei LvFREP2 gene, when the mutation site is C and the genotype of the mutation site is CC, the survival rate of the Litopenaeus vannamei under a vibrio infection environment is obviously higher.
The invention also discloses a primer combination for detecting the mutation site and a breeding application method thereof. In the disease-resistant breeding of litopenaeus vannamei individuals on vibrio parahaemolyticus, the method disclosed by the invention can be used for screening a backup parent population or a breeding family, and a foundation is laid for rapidly breeding a good disease-resistant variety of litopenaeus vannamei.
Drawings
FIG. 1 is a diagram of LvFREP2-1354633 locus genotype peaks.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.
The experimental procedures in the following examples were carried out in a conventional manner or according to the kit instructions unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Clone sequencing and primer Synthesis Guangzhou Tianyihui Gene technology, Inc.
Example 1
1. Collection of susceptible and disease-resistant litopenaeus vannamei samples
1.1 challenge experiment
Temporarily culturing Litopenaeus vannamei whole sibling family with weight of about 2g for one day, and injecting into Litopenaeus vannamei individual with injection concentration of 10710 μ L of cfu/mL Vibrio parahaemolyticus (Vibrio parahaemolyticus) was used to exclude death due to injection factors, and dead individuals were recorded 12 hours after challenge. The results are shown in Table 1:
TABLE 1 shrimp mortality at different challenge times
1.2 Collection of susceptible and disease-resistant Litopenaeus vannamei samples
The subjects who do not die after 48 hours of virus attack are taken as disease-resistant samples, and the subjects who die after 12-48 hours of virus attack are taken as susceptible samples.
2. Development of disease-resistant SNP markers
2.1 extraction of susceptible and disease-resistant genomic DNA of Litopenaeus vannamei
Selecting 56 susceptible Litopenaeus vannamei individuals and 50 disease-resistant individuals, respectively taking muscle tissues, extracting the genome DNA by using a marine animal tissue genome DNA extraction kit (Tiangen Biochemical technology Co., Ltd., Beijing), and performing an operation method according to the kit and the kit use instructions.
2.2SNP PCR primer design
Based on the mRNA sequence (GenBank No: MF418127.1) of the LvFREP2 gene of the litopenaeus vannamei obtained from NCBI, a section of genome sequence of the LvFREP2 is designed and amplified by a primer, and the requirement of the design of the primer is as follows: the length of the primer is 18-22bp, the GC content is 40-60%, the Tm value is 50-62 ℃, the difference between the Tm values of the upstream primer and the downstream primer is not more than 5, and primer dimer, hairpin structure, mismatching and the like are avoided as much as possible. The primer sequences are as follows:
KG-F(1354397～1354420)：
5'-TGTAAAACGACGGCCAGTGAGGGAATATCATTTGTATGATGC-3' (shown in SEQ ID NO. 2);
KG-R(1354849～1354867)：
5'-CAGGAAACAGCTATGACCAGAGAGACGCGGAGAGAGG-3' (shown in SEQ ID NO. 3).
The numbers in parentheses represent the position of the primers in the LvFREP2 genome.
2.3PCR amplification and sequencing
Randomly selecting a template from the genomic DNA of the litopenaeus vannamei extracted in the step 2.1, and carrying out PCR amplification according to the primer designed in the step 2.2, wherein the reaction system is 25 mu L and comprises: containing Mg2+10 XPCR buffer 2.5. mu.L, 10mM dNTPs 1. mu. L, Taq enzyme 2U, 10. mu.M forward primer 1. mu.L, 10. mu.M reverse primer 1. mu. L, DNA template 10ng, the remainder being nothingThe solution is distilled to 25 mu L. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds, annealing (temperature from 48 ℃ to 65 ℃) for 30 seconds, and extension at 72 ℃ for 30 seconds for 35 cycles; the extension was continued for 10 minutes at 72 ℃. Sequencing the PCR result.
2.4SNP locus genotyping
The genotype peak map of LvFREP2-1354633 locus is shown in figure 1.
The results of the genotype analysis of the 56 susceptible litopenaeus vannamei and 50 disease resistant litopenaeus vannamei SNP loci are shown in Table 2.
TABLE 2 different phenotype idiotypes
Note: in the table, susceptible individuals represent individuals who died 12 to 48 hours after Vibrio parahaemolyticus infection, and disease-resistant individuals represent individuals who survived 48 hours after Vibrio parahaemolyticus infection.
Statistical vibrio parahaemolyticus disease resistance association analysis was performed on the results in table 2, and the results are shown in table 3:
TABLE 3 genotype frequency and Vibrio parahaemolyticus disease resistance Association analysis of SNP loci
As can be seen from Table 3, the number of CC genotypes appeared in the disease-resistant individuals was 26, which accounted for 52% of the total number of the disease-resistant individuals, the number of C genes was 67, and the frequency of appearance was 67%, indicating that the individuals with CC genotypes were more tolerant in the case of Vibrio infection. The frequency of TT genotype in susceptible individuals is high, 21 individuals account for 38 percent, the number of T genes is 64 and accounts for 57 percent, which indicates that individuals with TT genotype have weak disease resistance under vibrio infection. The results show that the genotype of the SSR site located in the coding region of the LvFREP2 gene is obviously related to the vibrio resistance of the litopenaeus vannamei, the SNP marker is located at the 11 th base from the 5' end of the sequence shown in SEQ ID NO.1, and the base is C or T. When the mutation site is C and the genotype of the mutation site is CC, the survival rate of the litopenaeus vannamei under a vibrio infection environment is obviously higher, so that the genotype disclosed by the invention can be used as a good molecular marker for the assisted breeding of the litopenaeus vannamei in a vibrio resistant breeding process, and the breeding efficiency of the disease resistant variety of the litopenaeus vannamei is improved.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.
Sequence listing
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Claims (10)
1. An SNP marker related to the disease resistance of litopenaeus vannamei, which is characterized in that the SNP marker is positioned at the 11 th base from the 5' end of a sequence shown in SEQ ID NO.1, and the base is C or T.
2. A detection primer of an SNP marker related to the disease resistance of Litopenaeus vannamei is characterized in that the primer is as follows:
KG-F：5'-TGTAAAACGACGGCCAGTGAGGGAATATCATTTGTATGATGC-3'；
KG-R：5'-CAGGAAACAGCTATGACCAGAGAGACGCGGAGAGAGG-3'。
3. the application of the product for identifying the SNP marker of claim 1 in preparing a reagent for detecting the disease resistance of Litopenaeus vannamei.
4. The use of the SNP marker according to claim 1 or the detection primer according to claim 2 for preparing a detection kit for the disease resistance of litopenaeus vannamei.
5. A detection kit for the disease resistance of Litopenaeus vannamei, which is characterized by comprising the detection primer of claim 2.
6. The SNP marker according to claim 1, the detection primer according to claim 2 or the detection kit according to claim 5, for use in the detection of the disease resistance of Litopenaeus vannamei.
7. The use of claim 6, wherein the disease resistance of Litopenaeus vannamei is high when the genotype of the SNP marker is CC.
8. The use of the SNP marker according to claim 1, the detection primer according to claim 2 or the detection kit according to claim 5 in molecular marker assisted selection breeding of litopenaeus vannamei.
9. The application of claim 8, which is applied to breeding of disease-resistant varieties of litopenaeus vannamei.
10. A breeding method of a disease-resistant variety of Litopenaeus vannamei is characterized by comprising the following steps:
a) extracting the genomic DNA of the litopenaeus vannamei to be detected;
b) carrying out PCR amplification on the genomic DNA of the litopenaeus vannamei to be detected by using the detection primer KG-F/KG-R of claim 2;
c) sequencing the amplified product, determining the genotype of the SNP marker as set forth in claim 1, and selecting CC genotype individual as the backup parent for breeding disease-resistant variety of Litopenaeus vannamei.
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| WO2024027254A1 (en) * | 2023-03-13 | 2024-02-08 | 中国科学院南海海洋研究所 | Molecular marker combination for enterocytozoon hepatopenaei resistance trait of litopenaeus vannamei, kasp detection primers, and use |
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| WO2024027254A1 (en) * | 2023-03-13 | 2024-02-08 | 中国科学院南海海洋研究所 | Molecular marker combination for enterocytozoon hepatopenaei resistance trait of litopenaeus vannamei, kasp detection primers, and use |
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