Detection method for streptococcus agalactiae disease resistance of tilapia, KASP primer group and application

Abstract

The invention relates to the technical field of biology, in particular to a detection method for tilapia streptococcus agalactiae resistance, a KASP primer group and application. According to the invention, the key SNP site of the ABCB4 gene, which is obviously associated with the streptococcus agalactiae disease resistance, of the tilapia is identified by a population genetics method, a typing method of the SNP site is developed by a KASP technology, efficient evaluation on disease resistance characters is realized, experimental data shows that the survival time of an AA genotype individual in a challenge test is prolonged compared with that of a GA / GG genotype individual, and the survival time of the AA genotype individual in the challenge test is obviously prolonged compared with that of a GA / GG genotype individual. The proportion of resistant fishes is remarkably increased, and genotype frequency distribution is strongly related to group disease resistance. According to the breeding strategy based on the SNP marker, the disease resistance of bred groups can be integrally improved, the morbidity and death rate are reduced, an efficient genetic improvement tool is provided for breeding of tilapia mossambica for resisting streptococcus agalactiae diseases, the loss of breeding diseases is reduced, and antibiotic dependence is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for detecting resistance of tilapia to Streptococcus agalactiae disease, a KASP primer set and applications thereof. Background Art

[0002] Tilapia (Oreochromis spp.), a staple freshwater fish species in my country, faces significant challenges in its industrial development due to streptococcal disease caused by Streptococcus agalactiae. This pathogen, characterized by explosive spread during hot seasons and a high mortality rate and rapid transmission, has intensified in recent years, and existing prevention and control measures have been unable to effectively halt the spread. While traditional breeding techniques have improved growth performance through the selection of superior strains such as "Jifu," they still face bottlenecks in improving disease resistance traits, manifested in slow genetic progress and inefficient phenotypic selection.

[0003] The rise of molecular marker-assisted breeding (MAS) technology has provided a new avenue for disease-resistant breeding. It enables early selection by screening DNA markers associated with target traits, significantly improving breeding precision. Single nucleotide polymorphism (SNP) markers have become a key tool in MAS due to their high density, high stability, and potential for automated detection. ATP-binding cassette subfamily B member 4 (ABCB4), a member of the ATP-binding cassette (ABC) transporter family, is implicated in tumor resistance in mammals by mediating drug efflux. In aquatic animals, this gene is hypothesized to mediate resistance to various exogenous pollutants through an "efflux pump" mechanism. However, breeding efforts for tilapia resistance to Streptococcus agalactiae have focused primarily on model genes (such as MHC-like genes), with insufficient attention paid to ABC transporter family genes. In particular, the role of ABCB4 in fish disease resistance has not been systematically elucidated, and its potential as a molecular marker has remained largely unexplored. Currently, molecular markers for the agalactiae resistance phenotype remain limited in number and have low interpretability of genetic variation. Screening for single nucleotide polymorphisms (SNPs) in immunity- or resistance-related genes is a key approach for efficiently identifying molecular markers associated with the resistance phenotype. Therefore, this application further explores molecular markers in the ABCB4 gene that are tightly linked to the agalactiae resistance phenotype. Summary of the Invention

[0004] In view of this, the present invention proposes a method for detecting tilapia resistance to Streptococcus agalactiae, a KASP (competitive allele-specific PCR) primer set and its application. Taking ABCB4 gene polymorphism as the entry point, a SNP marker detection method significantly associated with resistance to Streptococcus agalactiae is established through population genetics analysis combined with KASP high-throughput typing technology.

[0005] The technical solution of the present invention is achieved as follows:

[0006] In a first aspect, the present invention provides a KASP primer set related to tilapia resistance to Streptococcus agalactiae, comprising: forward primers SEQ ID NO: 1 and SEQ ID NO: 2, and a reverse primer SEQ ID NO: 3.

[0007] In a second aspect, the present invention provides a kit for detecting resistance to Streptococcus agalactiae in tilapia, the components of which include a primer set as shown in SEQ ID NO: 1-3.

[0008] Furthermore, in some specific embodiments, the components of the detection kit further include at least one of KASP MasterMix, ddH2O and a DNA extraction reagent.

[0009] In a third aspect, the present invention provides use of the KASP primer set or the detection kit in breeding tilapia resistant to Streptococcus agalactiae disease.

[0010] In a fourth aspect, the present invention provides use of the KASP primer set or the detection kit in identifying resistance of tilapia to Streptococcus agalactiae disease.

[0011] In a fifth aspect, the present invention provides a method for detecting resistance to Streptococcus agalactiae in tilapia, comprising the following steps:

[0012] S1, extracting genomic DNA of the tilapia to be tested;

[0013] S2. Perform genotype detection on the SNP site of the ABCB4 gene in the genome; the SNP site is: reference to the Nile tilapia O_niloticus_UMD_NMBU genome version, located at 20310698bp of chromosome 11, the reference base is A, and the mutant base is G; tilapia with a genotype of AA at the SNP site has significantly higher resistance to Streptococcus agalactiae disease than tilapia with a genotype of GA or GG.

[0014] Furthermore, in some specific embodiments, the genomic DNA of step S1 is used as a template, primers are designed according to the position information of the SNP site, a PCR reaction is performed to obtain a PCR amplification product, and the genotype of the SNP site is analyzed.

[0015] Furthermore, in some specific embodiments, the primers are a primer set as shown in SEQ ID NO: 1-3.

[0016] In a sixth aspect, the present invention provides a method for breeding tilapia resistant to Streptococcus agalactiae, comprising the following steps:

[0017] Genotype detection is performed on the SNP site of the ABCB4 gene in the genomic DNA of the tested tilapia; the SNP site is: reference to the genome version of Nile tilapia O_niloticus_UMD_NMBU, located at 20310698bp of chromosome 11, the reference base is A, and the mutant base is G;

[0018] Tilapia with AA genotype is selected as a parent, and the parent is bred to obtain offspring with improved resistance to Streptococcus agalactiae disease.

[0019] Furthermore, in some specific embodiments, the primer set shown in SEQ ID NO: 1-3 is used to perform genotype detection on the SNP site.

[0020] The beneficial effects of the present invention include at least the following:

[0021] The present invention screened out a key SNP site in the ABCB4 gene (the A / G mutation site at 20310698bp on chromosome 11) that is significantly associated with resistance to Streptococcus agalactiae in the tilapia genome, and developed a typing method for the above SNP site using KASP technology, achieving efficient evaluation of disease resistance traits. Experimental data showed that individuals with the AA genotype survived longer in the challenge test than individuals with the GA / GG genotype, the proportion of resistant fish increased significantly, and the genotype frequency distribution was strongly correlated with the disease resistance of the population. The breeding strategy based on this SNP marker can improve the overall disease resistance of the aquaculture population and reduce the incidence and mortality rate, providing an efficient genetic improvement tool for tilapia breeding for resistance to Streptococcus agalactiae, helping to reduce losses from aquaculture diseases and reduce dependence on antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The effect of genotype differences at the LG11_0698 locus of the ABCB4 gene on the structure and function of ABCB4;

[0024] Figure 1 Middle: a is the functional domain prediction of the AA genotype; b is the functional domain prediction of the GG genotype (Note: boxes of different colors represent putative functional domains); c is the secondary structure prediction of the AA genotype; d is the secondary structure prediction of the GG genotype; (Structural annotation: red boxes represent the position of SNP LG11_0698, blue arrows represent chain structures, and red curled boxes represent short helical structures)

[0025] Figure 2 The results are the test results of five tilapia populations (Population 1-5, i.e. P1-P5 populations) against Streptococcus agalactiae disease;

[0026] Figure 2 Middle: a is the survival time of tilapia after infection with Streptococcus agalactiae, with the red dotted boxes representing the peak (left) and trough (right) periods of survival time; b is the statistical results of the mortality rate of five tilapia groups infected with Streptococcus agalactiae; different letters above each bar indicate statistically significant differences in the mortality rates of the corresponding different groups (p < 0.05);

[0027] Figure 3 This is the KASP typing test result of the LG11_0698 locus of the ABCB4 gene. Each dot represents a tested individual, and different colors correspond to different genotypes.

[0028] Figure 4 In the figure, ad represents the survival time of tilapia of different genotypes after infection with Streptococcus agalactiae in the mixed population (Multi-population), P1 population, P4 population, and P5 population, respectively. Fisher's exact test showed that the survival time of AA genotype was statistically significant compared with other genotypes, with a p-value less than 0.05. The mixed population refers to the statistical analysis of all individuals of P1, P2, P3, P4, and P5 without distinguishing between groups. e represents the susceptibility / resistance to Streptococcus agalactiae of tilapia of different genotypes in the mixed population. The chi-square test was used to analyze the resistance ratio of AA genotype compared with other genotypes, with a p-value less than 0.05, indicating statistical significance.

[0029] Figure 5 This is a fan diagram of the allele frequency distribution of SNP LG11_0698 in the P1-P5 population. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the embodiments described are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0031] Table 1 Sequence information

[0032]

[0033] 1. Experimental Methods

[0034] 1. Animal husbandry

[0035] The study used healthy individuals of the GIFT strain of Nile tilapia, 2 to 2.5 cm in length. These individuals were obtained from five commercial populations (designated P1, P2, P3, P4, and P5) representing different geographic origins. Specifically, P1 was from southwestern Guangdong Province, while P2, P3, P4, and P5 were obtained from separate farms in Hainan Province. The parents of each population were independently selected and bred; each population was the offspring of a mixed family. Each population consisted of 500 healthy fish. Each population was housed individually in a 4 m × 3 m × 2 m recirculating cement tank. They were fed a commercial diet twice daily (at 7:00 AM and 7:00 PM) at a rate of 3% of their body weight. Fish were reared under identical conditions across all populations. The water temperature was maintained at 28°C ± 2°C, dissolved oxygen was maintained between 6.0 and 7.5 mg / L, pH was stabilized between 7.5 and 8.5, and total ammonia nitrogen levels were maintained at <0.1 mg / L. All experimental individuals wore Passive Integrated Transponder (PIT) electronic tags.

[0036] 2. Preparation of Streptococcus agalactiae liquid

[0037] The Streptococcus agalactiae WC1535 in the examples can be found in the reference (Zhang Meiyan, Zhu Weijuan, Liu Zhigang, et al. Differential expression analysis of plasma proteomics before and after tilapia infection with Streptococcus agalactiae [J]. Journal of Dalian Ocean University, 2024, 39(04): 559-567. DOI: 10.16535 / j.cnki.dlhyxb.2023-288.). The public can obtain it from the Pearl River Fisheries Research Institute of the Chinese Academy of Fishery Sciences. The applicant promises to release the biological material to the public within 20 years from the date of application.

[0038] The bacterial solution was dissolved at room temperature and streaked on blood agar plates (Hopebio, Qingdao, China) and cultured overnight at 37°C. Then, a single colony was picked and placed in 10 mL of brain heart infusion (BHI, Solarbio Technology, Beijing, China) broth and cultured at 37°C for 24 hours (hours, h). When the bacteria reached the mid-logarithmic phase (OD 600 ≈0.5, about 10 8 The bacteria were washed three times and resuspended in sterile 1× PBS. The concentration of the bacterial suspension was measured using a BioMerieux turbidity meter (BioMerieux, Shanghai, China) and adjusted to 3.8×10 6 CFU / mL. The experimental concentration was determined by the results of the preliminary experiment. The preliminary experiment set up 4 WC1535 concentration gradient challenge groups, which were 10 9 , 10 8 , 10 7 and 10 6 CFU / mL, 10 fish per group. Each fish was intraperitoneally injected with 100uL of bacterial solution, and the number of fish deaths in each group was recorded within 7 days. Then, the online software LD 50 The median lethal dose (LD50) was calculated using the calculator (https: / / www.aatbio.com / tools / ld50-calculator) under default parameters. 50 In this experiment, the bacterial injection concentration was 3.8×10 6 CFU / mL.

[0039] 3. Challenge experiment and phenotypic recording

[0040] (1) Determining the resistance of Nile tilapia to Streptococcus agalactiae in populations P1-P5 through a challenge experiment

[0041] Before the experiment, approximately 160 healthy fish from each group were randomly assigned to two 500L tanks (70-90 fish per tank). Water quality and feeding conditions were the same as in Part 1, but the temperature was elevated to 31±1°C. After two weeks of training, the fish were challenged with Streptococcus agalactiae. 100 μL of the bacterial solution was injected intraperitoneally into the fish. The number of fish that died in each group was recorded, and the cumulative mortality rate was calculated. The calculation formula was: Mortality rate per tank (%) = 100 × (number of fish that died / number of fish vaccinated per tank). The average mortality rate of the two tanks in each group was used as the mortality rate for that group.

[0042] (2) Nile tilapia challenge experiment and phenotypic records for KASP typing

[0043] This experiment involved a control group and an experimental group. In the control group, 50 fish were randomly selected from five populations and intraperitoneally injected with 100 μL of 1× PBS. In the experimental group, 313 healthy fish were challenged with the virus. Survival time was recorded immediately after the last fish was injected, measured in hours post infection (hpi). For example, if a fish died 1 hour after infection, the survival time was recorded as 1 hpi. For the first four days after challenge, survival time was recorded every 0.5 hours. Starting from day 5 post-challenge, survival time was recorded every 12 hours. Individuals killed by S. agalactiae were identified based on typical symptoms such as proptosis, white eye, swirling swimming style, and skin ulceration. From day 10 post-challenge, no individuals died in the experimental group. During this period, the control group remained healthy and showed no signs of illness. Therefore, in this experiment, the survival time of fish that died was recorded until 240 hpi, and a fixed survival time (250 hpi) was assigned to surviving fish. Furthermore, each fish was assigned a susceptible or resistant phenotype. Fish that died within 10 days of infection (hpi ≤ 240 hpi) were classified as susceptible individuals, while fish that survived 10 days after infection were classified as resistant individuals (hpi = 250 hpi).

[0044] The experimental groups included 70 individuals from population P1 (35 susceptible individuals and 35 resistant individuals), 41 individuals from population P2 (17 susceptible individuals and 24 resistant individuals), 54 individuals from population P3 (13 susceptible individuals and 41 resistant individuals), 77 individuals from population P4 (29 susceptible individuals and 48 resistant individuals), and 71 individuals from population P5 (23 susceptible individuals and 48 resistant individuals).

[0045] 4. DNA Extraction

[0046] Genomic DNA was extracted from the caudal fin according to the instructions of the HiPure Tissue DNA Micro Kit (Magen, Guangzhou, China). DNA integrity and concentration were determined by 1.5% agarose gel electrophoresis and Nanodrop 2000 spectrophotometry.

[0047] 5. SNP scanning, functional domain prediction, protein secondary structure prediction

[0048] Aspera (v2.0) was used to download 494 tilapia resequencing data from the CNGB database (Submit ID: sub071023). After quality control using fastQC (v3.0) with default parameters, the raw data were mapped back to the Nile tilapia reference genome, O_niloticus_UMD_NMBU (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_001858045.2 / ).

[0049] Then, Samtools (v 1.21) was used to index the replied files. GATKHaplotypeCaller (v4.0) was used to call SNPs and generate gVCF files for each individual. High-quality SNPs were extracted from each individual gVCF file according to the filtering conditions of QD < 2.0, MQ < 40.0, FS > 60.0, QUAL < 30.0, MQrankSum < -12.5, ReadPosRankSum < -8.0, -clusterSize 2, -clusterWindowSize 5. Finally, GATKGenomicsDBImport was used to merge all gVCF files, and GATK GenotypeGVCF was used to genotype the merged gVCF data to generate a single VCF file containing the detected variants of all samples. Finally, Bcftools software (v3.6c) was used to extract the ABCB4 gene mutation SNP sites.

[0050] 6. KASP classification

[0051] Two allele-specific forward primers and one universal reverse primer were designed for each SNP using Primer 5.0. The final bases of the two forward primers were the reference base and the mutant base, respectively. As shown in Table 2, the underlined sequence in LG11-0698-1FX corresponds to the reference base fluorescent tag sequence, and the underlined sequence in primer LG11-0698-FY corresponds to the mutant base fluorescent tag sequence. Primers were synthesized by Beijing Sangon Biotechnology Co., Ltd. All three SNP primers were diluted to 10 μmol and mixed in a volume ratio of 12:12:30.

[0052] Table 2 KASP typing primer sequences

[0053]

[0054] The fin ray DNA samples of 313 tilapia in the above method 3(2) were genotyped for SNP LG11-0698 using KASP typing. The typing experiment was completed on the high-throughput genotyping platform of LGC in Teddington, UK. The PCR reaction system is shown in Table 3. The amplification reaction was carried out in a high-throughput water bath system Hydrocycler. The PCR program was set as follows: first, pre-denaturation at 94℃ for 15 minutes; then 10 cycles of touchdown program (denaturation at 94℃ for 20 seconds, annealing and extension between 61℃ and 55℃, with a decrease of 0.6℃ per cycle for 1 minute); then 26 standard cycles (denaturation at 94℃ for 20 seconds, extension at 55℃ for 60 seconds). After amplification, the fluorescence signal was detected using a BMG PHERAstar multifunctional microplate reader produced by Olsberg, Germany, and the typing was performed. The typing results were statistically analyzed using SNPviewer2 software (v1.123) provided by LGC.

[0055] Table 3 PCR amplification reaction system

[0056] Each component 234 reactions 2× PCR Mastermix 422μL Primermix 11.7μL <![CDATA[Double distilled water (ddH2O)]]> 422μL DNA dry powder (DNApower) 8-10ng Reaction system / sample 3μL

[0057] 7. Data Statistics

[0058] After KASP typing, Minitab software (v21.0) was used for significance testing. The disease resistance of different groups was tested with the Student's t two-tailed test. When statistically analyzing the correlation between phenotype and genotype, P1, P2, P3, P4, P5 and mixed groups (multiple population, multi-population) were analyzed separately. Mixed population refers to the statistical analysis of all individuals of P1, P2, P3, P4 and P5 without distinguishing the groups. Among them, Fisher's exact test was used to perform statistical significance on survival time between different genotypes. The chi-square test was used to perform statistical significance on the number of sensitive / resistant individuals between different genotypes. The chi-square test was used to perform statistical significance on the differences in genotype frequency between groups. p < 0.05 was considered statistically significant.

[0059] 2. Experimental Results

[0060] 1. Screening of sense mutations in the ABCB4 gene of Nile tilapia

[0061] This application found 20 SNP mutation sites in the ABCB4 gene (NCBI database Gene ID: 100534453, https: / / www.ncbi.nlm.nih.gov / ) from the published resequencing screening of 494 Nile tilapia (Table 3). Among them, the SNP LG11_20310698 (LG11_0698, A / G) at 20310698bp on chromosome 11 is located in exon 18 of the ABCB4 gene. This gene encodes 1,273 amino acids and contains 8 potential functional domains. LG11_0698 is located in the fifth domain ( Figure 1 When LG11_0698 mutates from A to G, the encoded amino acid changes from threonine (T) to alanine (A) ( Figure 1 c, d). When LG11_20310698 encodes threonine, amino acids 750-777 of ABCB4 contain three short helical structures and a single-stranded structure ( Figure 1 When the SNP encodes alanine, amino acids 750-777 of ABCB4 contain only two helical structures of different lengths.

[0062] Table 3 Mutation sites of ABCB4 gene in Nile tilapia

[0063] chromosome Location SNP_ID Reference sites mutation site Notes NC_031976.2 20302161 NC_031976.2__20302161 G A nonsense mutation NC_031976.2 20303748 NC_031976.2__20303748 T C nonsense mutation NC_031976.2 20304328 NC_031976.2__20304328 A C nonsense mutation NC_031976.2 20304584 NC_031976.2__20304584 C T nonsense mutation NC_031976.2 20307764 NC_031976.2__20307764 A G nonsense mutation NC_031976.2 20309671 NC_031976.2__20309671 C A nonsense mutation NC_031976.2 20310698 NC_031976.2__20310698 A G Sense mutation NC_031976.2 20314307 NC_031976.2__20314307 C T nonsense mutation NC_031976.2 20316224 NC_031976.2__20316224 T G nonsense mutation NC_031976.2 20319550 NC_031976.2__20319550 C T nonsense mutation NC_031976.2 20321284 NC_031976.2__20321284 G A nonsense mutation NC_031976.2 20326797 NC_031976.2__20326797 C G nonsense mutation NC_031976.2 20329009 NC_031976.2__20329009 C T nonsense mutation NC_031976.2 20331810 NC_031976.2__20331810 C T nonsense mutation NC_031976.2 20335810 NC_031976.2__20335810 C G nonsense mutation NC_031976.2 20344277 NC_031976.2__20344277 T G nonsense mutation NC_031976.2 20344980 NC_031976.2__20344980 G A nonsense mutation NC_031976.2 20349585 NC_031976.2__20349585 C T nonsense mutation NC_031976.2 20354640 NC_031976.2__20354640 G A nonsense mutation NC_031976.2 20357182 NC_031976.2__20357182 A G nonsense mutation

[0064] 2. Results of Streptococcus agalactiae resistance test in Nile tilapia populations P1-P5

[0065] After infection with Streptococcus agalactiae, the survival time of 335 susceptible fish and 557 surviving fish from five Nile tilapia populations was recorded. Survival time peaked at 52.5 to 67.5 hours after infection and reached a trough at 187.5 to 202.5 hours after infection ( Figure 2 The mortality rate of the P3 group was the highest among all the groups, followed by the P2 and P5 groups, while the mortality rate of the P1 and P4 groups was the lowest ( Figure 2 (b)

[0066] 3. KASP typing verification of SNP LG11_20310698 (LG11_0698)

[0067] The KASP typing test results (313 fish) of this application verified the correlation between LG11_0698 and the resistance of tilapia to Streptococcus agalactiae. Figure 3 As shown, 265 (86.5%) fish were successfully typed by KASP typing. Two alleles (A / G) and three genotypes (AA / GA / GG) were observed for LG11_0698.

[0068] like Figure 4 As shown in Figures ad, in the analysis of P1, P4, P5 and mixed populations, the AA genotype significantly prolonged the survival time compared with other genotypes (Fisher test p < 0.05). At the same time, the chi-square test found that the proportion of resistant fish of the AA genotype (the proportion of fish resistant to Streptococcus agalactiae disease in the total number of fish) was significantly higher than that of the GG genotype, and the genotype frequency was significantly correlated with the sensitive / resistant binary trait ( Figure 4 These results indicate that LG11_0698 is associated with resistance to Streptococcus agalactiae in tilapia, with the AA genotype being a resistance-associated genotype.

[0069] 4. Association of SNP LG11_0698 genotype frequency distribution and resistance to Streptococcus agalactiae in Nile tilapia P1-P5 populations

[0070] The statistical results of the frequency distribution of three genotypes (AA / GA / GG) in the Nile tilapia P1-P5 population are as follows: Figure 5 As shown. Among the five populations, AG was the main genotype in each population. The chi-square test showed that the frequency of the AA genotype was significantly higher in the P1 (27%), P2 (32%), P4 (41%), and P5 (34%) populations with better disease resistance than in the P3 population with the worst disease resistance (9%); the frequency of the GG genotype was significantly higher in the P3 population (32%) than in the other populations (P1, P2, P4, and P5: 8%-15%) (χ 2 =0.003). The above results further confirmed that AA was associated with the resistance of Nile tilapia to Streptococcus agalactiae, while GG genotype was associated with the susceptibility to Streptococcus agalactiae.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A KASP primer set associated with tilapia resistance to Streptococcus agalactiae, characterized in that: include: The forward primers are SEQ ID NO: 1 and SEQ ID NO: 2, and the reverse primer is SEQ ID NO:

3.

2. A detection kit for resistance to Streptococcus agalactiae in tilapia, characterized in that: The components include the KASP primer set according to claim 1.

3. The detection kit according to claim 2, characterized in that The components also include at least one of KASP MasterMix, ddH2O, and a DNA extraction reagent.

4. Use of the KASP primer set according to claim 1 or the detection kit according to any one of claims 2 and 3, characterized in that: Used for breeding tilapia resistant to Streptococcus agalactiae disease.

5. Use of the KASP primer set according to claim 1 or the detection kit according to any one of claims 2 and 3, characterized in that: Used to identify resistance of tilapia to Streptococcus agalactiae disease.

6. A method for detecting resistance to Streptococcus agalactiae in tilapia, characterized in that: The steps include: S1, extracting genomic DNA of the tilapia to be tested; S2. Perform genotype detection on the SNP site of the ABCB4 gene in the genome; the SNP site is: reference to the Nile tilapia O_niloticus_UMD_NMBU genome version, located at 20310698bp of chromosome 11, the reference base is A, and the mutant base is G; tilapia with a genotype of AA at the SNP site has significantly higher resistance to Streptococcus agalactiae disease than tilapia with a genotype of GA or GG.

7. The detection method according to claim 6, characterized in that Using the genomic DNA from step S1 as a template, primers are designed according to the position information of the SNP site, a PCR reaction is performed to obtain a PCR amplification product, and the genotype of the SNP site is analyzed.

8. The detection method according to claim 7, characterized in that The primers are the primer set shown in SEQ ID NO: 1-3.

9. A method for breeding tilapia resistant to Streptococcus agalactiae disease, characterized in that: The steps include: Genotype detection is performed on the SNP site of the ABCB4 gene in the genomic DNA of the tested tilapia; the SNP site is: reference to the genome version of Nile tilapia O_niloticus_UMD_NMBU, located at 20310698bp of chromosome 11, the reference base is A, and the mutant base is G; Tilapia with AA genotype is selected as a parent, and the parent is bred to obtain offspring with improved resistance to Streptococcus agalactiae disease.

10. The breeding method according to claim 9, characterized in that: The genotype of the SNP site was detected using the primer set shown in SEQ ID NO: 1-3.

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