A detection method for resistance of rohu to streptococcus agalactiae, a kasp primer set and application thereof

Abstract

The present application relates to the field of biotechnology, and more particularly to a detection method for resistance of tilapia to streptococcosis, a KASP primer set and application. The present application identifies a key SNP site of the ABCB4 gene significantly associated with resistance of tilapia to streptococcosis by the method of population genetics, and develops a genotyping method for the SNP site by KASP technology, thereby realizing efficient evaluation of the disease-resistant trait. Experimental data show that the survival time of AA genotype individuals in the challenge test is longer than that of GA / GG genotype individuals, the proportion of resistant fish is significantly improved, and the genotype frequency distribution is strongly correlated with the disease resistance of the population. The breeding strategy based on the SNP marker can improve the overall disease resistance of the breeding population and reduce the mortality rate, thereby providing an efficient genetic improvement tool for breeding of tilapia resistant to streptococcosis, and helping to reduce the loss of breeding diseases and reduce the dependence on antibiotics.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and in particular to a detection method for resistance of tilapia to streptococcosis, a KASP primer set and application. BACKGROUND

[0002] Oreochromis spp. is a major freshwater economic fish in China, and its industry scale is severely restricted by streptococcosis caused by Streptococcus agalactiae. The pathogenic bacteria spread explosively in the high-temperature season, with high mortality and fast transmission speed, and the epidemic trend has intensified in recent years, while the existing prevention and control measures cannot effectively block it. Although traditional breeding techniques have improved the growth performance through breeding of "Jihe" and other excellent strains, they still face bottlenecks in the improvement of disease resistance traits, such as slow genetic progress and low efficiency of phenotype selection.

[0003] The rise of molecular marker assisted breeding (MAS) technology provides a new path for disease resistance breeding, which realizes early selection by screening DNA markers associated with target traits, and significantly improves the accuracy of breeding. Single nucleotide polymorphism (SNP) markers have become a key tool in the field of MAS due to their high density, high stability and potential for automation detection. ATP binding cassette subfamily B member 4 (ABCB4) belongs to the ATP binding cassette (ABC) transporter family, and in mammals it is involved in tumor drug resistance regulation by mediating drug efflux. In aquatic animals, this gene is speculated to mediate resistance to multiple types of exogenous pollutants through the "efflux pump" mechanism. However, in the practice of breeding tilapia resistant to Streptococcus agalactiae, much attention has been focused on model genes (such as MHC class genes), and there has been insufficient attention to ABC transporter family genes, especially the role of ABCB4 gene in fish disease resistance mechanism has not been systematically analyzed, and its development value as a molecular marker has not been explored. Currently, there are still few molecular markers for Streptococcus agalactiae resistance phenotype, and the genetic variation explanation is low. SNP screening through immune or resistance-related genes is one of the main ways to efficiently obtain molecular markers related to disease resistance phenotype. Therefore, the present application further develops molecular markers in the ABCB4 gene that are tightly linked to the Streptococcus agalactiae resistance phenotype. SUMMARY

[0004] Therefore, the application provides a detection method for streptococcus agalactiae resistance of rohu, a KASP (Kompetitive Allele Specific PCR) primer group and application thereof, which takes polymorphism of an ABCB4 gene as a breakthrough point, and establishes a SNP marker detection method significantly associated with streptococcus agalactiae resistance by population genetics analysis combined with KASP high-throughput genotyping technology.

[0005] The technical scheme of the application is implemented as follows:

[0006] In a first aspect, the application provides a KASP primer group related to streptococcus agalactiae resistance of rohu, which comprises: forward primers of SEQ ID NO: 1 and SEQ ID NO: 2, and a reverse primer of SEQ ID NO: 3.

[0007] In a second aspect, the application provides a detection kit for streptococcus agalactiae resistance of rohu, which comprises a primer group as shown in SEQ ID NO: 1-3.

[0008] Further, in some specific embodiments, the components of the detection kit further comprise at least one of KASP MasterMix, ddH2O and DNA extraction reagents.

[0009] In a third aspect, the application provides application of the KASP primer group or the detection kit in breeding rohu with streptococcus agalactiae resistance.

[0010] In a fourth aspect, the application provides application of the KASP primer group or the detection kit in identifying streptococcus agalactiae resistance of rohu.

[0011] In a fifth aspect, the application provides a detection method for streptococcus agalactiae resistance of rohu, which comprises the following steps:

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

[0013] S2, detecting genotypes of an ABCB4 gene SNP site in the genome; the SNP site is located at 20310698 bp of the 11th chromosome in the reference Nile rohu O_niloticus_UMD_NMBU genome version, the reference base is A, and the mutant base is G; the streptococcus agalactiae resistance of rohu with the SNP site genotype AA is significantly higher than that of rohu with the genotypes GA or GG.

[0014] Further, in some specific embodiments, the primer is a primer group as shown in SEQ ID NO: 1-3.

[0015] Further, in some specific embodiments, the primer is a primer group as shown in SEQ ID NO: 1-3.

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

[0017] The genotype of the SNP site of the ABCB4 gene in the genome DNA of the tilapia to be tested is detected; the SNP site is located at 20310698bp of chromosome 11, and the reference base is A and the mutant base is G according to the O_niloticus_UMD_NMBU genome version of Nile tilapia;

[0018] The tilapia with genotype AA is selected as the parent, and the parent is bred to obtain offspring with improved resistance to streptococcosis.

[0019] Further, in some specific embodiments, the primer group as shown in SEQ ID NO: 1-3 is used for genotype detection of the SNP site.

[0020] The present application has at least the following beneficial effects:

[0021] The present application screens the key SNP site (A / G mutation site at 20310698bp of chromosome 11) of the ABCB4 gene significantly associated with streptococcosis resistance in the genome of tilapia, and develops a genotyping method for the SNP site through KASP technology, thereby realizing efficient evaluation of the disease resistance trait. Experimental data show that the survival time of AA genotype individuals in the challenge test is longer than that of GA / GG genotype individuals, the proportion of resistant fish is significantly improved, and the genotype frequency distribution is strongly correlated with the disease resistance of the population. The breeding strategy based on the SNP marker can improve the overall disease resistance of the breeding population and reduce the mortality rate, thereby providing an efficient genetic improvement tool for tilapia breeding against streptococcosis, and helping to reduce disease loss and reduce antibiotic dependence. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0023] Figure 1 is the effect of ABCB4 gene LG11_0698 genotype difference on the structure and function of ABCB4;

[0024] Figure 1 a is the functional domain prediction of AA genotype; b is the functional domain prediction of GG genotype (Note: different colored boxes represent the predicted functional domains); c is the secondary structure prediction of AA genotype; d is the secondary structure prediction of GG genotype; (structure note: the red box represents the position of SNP LG11_0698, the blue arrow represents the chain structure, and the red curly box represents the short helix structure)

[0025] Figure 2 is the detection result of the resistance of five populations of tilapia (Population 1-5, i.e. P1-P5 populations) to streptococcosis;

[0026] Figure 2 a is the survival time of tilapia after being infected with streptococcus, and the red dotted box represents the peak period (left) and the trough period (right) of the survival time; b is the statistical result of the mortality rate of the five populations of tilapia after being infected with streptococcus; the different letters above each column chart represent that there is a statistically significant difference (p<0.05) between the mortality rates of the corresponding different populations;

[0027] Figure 3 is the KASP genotyping detection result of the LG11_0698 locus of the ABCB4 gene, and each dot represents a detection individual, and different colors correspond to different genotypes;

[0028] Figure 4 a-d are the survival time of tilapia with different genotypes after being infected with streptococcus in the mixed population (Multi-population), P1 population, P4 population and P5 population, respectively; Fisher's exact test analysis shows that the survival time of AA genotype has statistical significance compared with other genotypes, and the p value is less than 0.05; the mixed population refers to the statistical analysis of mixing all individuals of P1, P2, P3, P4 and P5 without distinguishing the population; e is the streptococcosis susceptibility / resistance trait of tilapia with different genotypes in the mixed population; the chi-square test analysis shows that the resistance proportion of AA genotype has statistical significance compared with other genotypes, and the p value is less than 0.05;

[0029] Figure 5 is the distribution sector diagram of the allele frequency of SNP LG11_0698 in P1-P5 populations. DETAILED DESCRIPTION

[0030] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the protection scope of the present application. If specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are adopted. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased on the market.

[0031] Table 1 Sequence information table

[0032]

[0033] I. Experimental methods

[0034] 1. Raising of experimental animals

[0035] Healthy GIFT Nile tilapia individuals with a body length of 2-2.5 cm were used for the test. These individuals were from five commercial populations representing different geographical origins (labeled as P1, P2, P3, P4 and P5). Specifically, P1 was from the southwestern part of Guangdong Province, while P2, P3, P4 and P5 were from different independent breeding 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 contained 500 healthy fish. Each population was individually raised in a 4m x 3m x 2m circulating water cement tank. Commercial feed was fed twice a day (7:00 am and 7:00 pm) at a rate of 3% of their body weight. The fish in each population were raised under the same conditions. The water temperature was maintained at 28℃±2℃, the dissolved oxygen was maintained at 6.0-7.5 mg / L, the pH value was stabilized at 7.5-8.5, and the total ammonia nitrogen level was maintained at <0.1 mg / L. All experimental individuals were equipped with Passive Integrated Transponder (PIT) electronic tags.

[0036] 2. Preparation of Streptococcus agalactiae bacterial solution

[0037] In the examples, Streptococcus agalactiae WC1535 is described in the reference (Zhang MY, Zhu WJ, Liu ZG, et al. Differential expression analysis of plasma proteomics of GIFT Nile tilapia before and after 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 Chinese Academy of Fishery Sciences, and the applicant promises to issue biological materials to the public for twenty years from the filing date.

[0038] Bacteria were dissolved at room temperature and streaked on blood agar plates (Hopebio, Qingdao, China) and incubated at 37°C overnight. Then, single colonies were picked and put into 10 mL brain heart infusion (BHI, Solarbio Technology, Beijing, China) broth and incubated at 37°C for 24 hours (h). When the bacteria proliferated to mid-log phase (OD 600 ≈0.5, about 10 8 CFU / mL), the bacteria were washed 3 times and resuspended in sterile 1x PBS. The concentration of the bacterial suspension was measured using a BioMerieux turbidimeter (BioMerieux, Shanghai, China) and adjusted to 3.8x10 6 CFU / mL. This experimental concentration was determined from the results of a pre-experiment. The pre-experiment set 4 WC1535 concentration gradient challenge groups at 10 9 , 10 8 , 10 7 , and 10 6 CFU / mL, with 10 fish per group. Each fish was injected intraperitoneally with 100 uL of the bacterial solution, and the number of dead fish in each group was recorded for 7 days. Then, the Median lethal dose (LD 50 ) was calculated using the online software LD 50 calculator (https: / / www.aatbio.com / tools / ld50-calculator) under the default parameters. In this experiment, the concentration of the bacterial solution injected was 3.8x10 6 CFU / mL.

[0039] 3. Challenge experiment and phenotype recording

[0040] (1) Determining the resistance of P1-P5 populations of Nile tilapia to S. agalactiae by challenge experiment

[0041] Before the experiment, about 160 healthy fish from each population were randomly assigned to two 500 L tanks (70-90 fish per tank), with the same water quality and feeding conditions as in 1, but the temperature was increased to 31 ± 1°C. After two weeks of training and feeding, the S. agalactiae challenge experiment was performed. 100 uL of the bacterial solution was injected intraperitoneally into the fish, and the number of dead fish in each population was recorded, and the cumulative mortality rate was calculated. The formula is: mortality rate (%) = 100 x (number of dead fish / number of fish inoculated per tank), and the average mortality rate of two tanks of fish from each population was taken as the mortality rate of that population.

[0042] (2) Challenge experiment and phenotype recording of Nile tilapia for KASP typing

[0043] The experiment was divided into control group and experimental group. The control group randomly selected 50 fish from 5 groups and injected 100 uL of 1xPBS intraperitoneally. The experimental group selected 313 healthy fish for challenge experiment. After the last fish completed injection, the survival time was recorded immediately, and the unit of survival time was (hour post infection, hpi). For example, a fish died 1 hour after infection, and the survival time was recorded as 1 hpi. After challenge, the survival time was recorded every 0.5 hours for the first 4 days. From the 5th day after challenge, the survival time was recorded every 12 hours. The death of individuals caused by S. agalactiae was identified according to typical symptoms such as exophthalmos, white eyeballs, rotating swimming posture, and skin ulcers. From the 10th day after challenge, there were no deaths in the experimental group. And in this period, the control group of fish was healthy, with no signs of illness. Therefore, the survival time of the dead fish was recorded to 240 hpi, and the survival time of the surviving fish was given (250 hpi). At the same time, each fish was given a susceptible and resistant phenotype. Among them, the fish that died within 10 days of infection (hpi≤240 hpi) were recorded as susceptible individuals. While the fish that survived after 10 days of infection were recorded as resistant individuals (hpi=250 hpi).

[0044] The experimental group included 70 individuals of population P1 (35 susceptible individuals and 35 resistant individuals), 41 individuals of population P2 (17 susceptible individuals and 24 resistant individuals), 54 individuals of population P3 (13 susceptible individuals and 41 resistant individuals), 77 individuals of population P4 (29 susceptible individuals and 48 resistant individuals), and 71 individuals of 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 detected using 1.5% agarose gel electrophoresis and Nanodrop 2000 spectrophotometry.

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

[0048] The CNGB database 494 re-sequencing data of Oreochromis niloticus (Submit ID: sub071023) was downloaded using Aspera (v2.0). After quality control by fastQC (v3.0) under the default parameters, the raw data was mapped to the Nile Tilapia reference genome O_niloticus_UMD_NMBU (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_001858045.2).

[0049] The files of the post-threads were then indexed using Samtools (v 1.21). GATK HaplotypeCaller (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 criteria of QD < 2.0, MQ < 40.0, FS > 60.0, QUAL < 30.0, MQrankSum < -12.5, ReadPosRankSum < -8.0, -clusterSize 2, -clusterWindowSize 5. Finally, all gVCF files were merged using GATK GenomicsDBImport, and the combined gVCF data was genotyped using GATK GenotypeGVCF to generate a single VCF file containing the detected variants of all samples. Finally, the ABCB4 gene mutation SNP sites were extracted using Bcftools software (v3.6c).

[0050] 6. KASP genotyping

[0051] Two allele-specific forward primers and one reverse universal primer were designed for each SNP using Primer5.0. The last base of the two forward primers was the reference base and the mutant base, respectively. As shown in Table 2, the underlined sequence of primer LG11-0698-1FX corresponds to the reference base fluorescent tag sequence, and the underlined sequence of primer LG11-0698-FY corresponds to the mutant base fluorescent tag sequence. The primers were synthesized by Beijing Sunhyvesyn Biological Engineering Co., Ltd. The three SNP primers were diluted to 10 pmol and mixed according to the volume ratio of 12:12:30.

[0052] Table 2. KASP genotyping primer sequences

[0053]

[0054] The KASP genotyping was used to genotype the SNP LG11-0698 in the fin DNA samples of 313 tilapia individuals from the above method 3(2). The genotyping experiment was performed on the high-throughput genotyping platform of LGC company in Teddington, UK. The PCR reaction system is shown in Table 3, and the amplification reaction was performed in the 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 (94℃ denaturation for 20 seconds, annealing and extension between 61℃ and 55℃, decreasing by 0.6℃ per cycle, for 1 minute); followed by 26 standard cycles (94℃ denaturation for 20 seconds, 55℃ extension for 60 seconds). After amplification, the fluorescence signal was detected using the BMG PHERAstar multifunctional microplate reader produced by Olsberg company in Germany, and the genotyping was interpreted. The genotyping results were statistically analyzed by the SNPviewer2 software (v1.123) provided by LGC company.

[0055] Table 3 PCR amplification reaction system

[0056] Components 234 reactions 2x PCR master mix 422 μL Primer mix 11.7 μL double distilled water (ddH2O) 422 μL DNA power 8-10 ng Reaction system / sample 3 μL

[0057] 7、Data statistics

[0058] After KASP genotyping, the Minitab software (v21.0) was used for significance test. The Student's t two-tailed test was used for the test of disease resistance of different populations. In the statistical analysis of the correlation between phenotype and genotype, P1, P2, P3, P4, P5 and mixed population (Multi-population) were analyzed respectively. The mixed population refers to the statistical analysis of all individuals of P1, P2, P3, P4 and P5 without distinguishing the population. Among them, Fisher's exact test was used for the significance statistics of survival time between different genotypes. Chi-square test was used for the significance statistics of the number of sensitive / resistant individuals between different genotypes. Chi-square test was used for the significance statistics of the difference of genotype frequency between populations. p<0.05 was considered statistically significant.

[0059] II、Experimental results

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

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

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

[0063] Chromosome Position SNP ID Reference site Mutation site Annotation 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 Nonsense 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. The detection results of Streptococcus agalactiae disease resistance of P1-P5 groups of Nile tilapia

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

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

[0067] The KASP genotyping detection results (313 fish) of the present application verified the correlation between LG11_0698 and the resistance of tilapia to Streptococcus agalactiae disease, and the results are shown in Table 3. Through KASP genotyping, 265 (86.5%) fish were successfully genotyped. Two alleles (A / G) and three genotypes (AA / GA / GG) of LG11_0698 were observed. Figure 3

[0068] ​​​​​​As Figure 4 In the P1, P4, P5 and mixed population analysis, AA genotype significantly prolonged the survival time compared with other genotypes (Fisher test p<0.05) as shown in Table 2a-d. Meanwhile, Chi-square test found that the proportion of resistant fish (the proportion of fish without streptococcosis in the total number of fish) of AA genotype was significantly higher than that of GG genotype, and the genotype frequency was significantly correlated with the sensitive / resistant binary trait (χ2=0.003) as shown in Table 2e). These results indicate that LG11_0698 is associated with the resistance of tilapia to streptococcosis. Among them, AA genotype is the resistance-related genotype. Figure 4

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

[0070] The statistical results of the frequency distribution of three genotypes (AA / GA / GG) in P1-P5 populations of Nile tilapia in the present application are shown in Table 3. Figure 5 In the five populations, AG is the main genotype of each population. Chi-square test showed that the frequency of AA genotype in P1 (27%), P2 (32%), P4 (41%) and P5 (34%) populations with better resistance was significantly higher than that in P3 population (9%) with the worst resistance; the frequency of GG genotype in P3 population (32%) was significantly higher than that in other populations (P1, P2, P4 and P5: 8%-15%) (χ2=0.003). The above results further verify that AA is related to the resistance of Nile tilapia to streptococcosis, and GG genotype is associated with susceptibility to streptococcosis. 2

[0071] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.​​

Claims

1. The application of a KASP primer set or a detection kit including the KASP primer set in the breeding of tilapia resistant to streptococcal disease, characterized in that, The KASP primer set includes: a forward primer with sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, and a reverse primer with a sequence as shown in SEQ ID NO:

3.

2. The application of the KASP primer set or a detection kit including the KASP primer set in identifying the resistance of tilapia to Streptococcus agalactiae disease, characterized in that, The KASP primer set includes: a forward primer with sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, and a reverse primer with a sequence as shown in SEQ ID NO:

3.

3. A method for detecting resistance to Streptococcus agalactiae in tilapia, characterized in that, Includes the following steps: S1. Extract genomic DNA from the tilapia to be tested; S2. Genotyping of the ABCB4 gene SNP site in the genomic DNA; the SNP site is located at 20310698 bp on chromosome 11, referring to the Nile tilapia O_niloticus_UMD_NMBU genome version, with reference base A and mutant base G; tilapia with the SNP site genotype AA have significantly higher resistance to streptococcal disease than tilapia with the genotypes GA or GG.

4. The detection method according to claim 3, characterized in that, Using the genomic DNA from step S1 as a template, primers were designed based on the location information of the SNP sites, and a PCR reaction was performed to obtain PCR amplification products. The genotypes of the SNP sites were then analyzed.

5. The detection method according to claim 4, characterized in that, The primers are primer sets with sequences as shown in SEQ ID NO: 1-3.

6. A method for breeding tilapia resistant to agalactiae streptococcal disease, characterized in that, Includes the following steps: Genotyping was performed on the ABCB4 gene SNP site in the tilapia genomic DNA to be tested; the SNP site was located at 20310698 bp on chromosome 11, with reference base A and mutant base G, based on the Nile tilapia O_niloticus_UMD_NMBU genome version. Tilapia with genotype AA were selected as parents, and the parents were bred to obtain offspring with enhanced resistance to Streptococcus agalactiae disease.

7. The breeding method according to claim 6, characterized in that, Genotyping of the SNP sites was performed using primer sets with sequences as shown in SEQ ID NO: 1-3.

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