Tilapia mossambica streptococcus agalactiae resistance related KASP molecular marker and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a tilapia mossambica streptococcus agalactiae resistance related KASP molecular marker and application thereof. According to the invention, two SNP (Single Nucleotide Polymorphism) loci, namely SNP-1 and SNP-2, which are remarkably associated with resistance are screened based on an intron region of an EGFRX2 gene of tilapia mossambica, a typing method of the loci is developed through a KASP technology, and relevance between the genotype of the SNP-1 and a resistance phenotype is verified. Furthermore, streptococcus agalactiae resistance dominant diplotype individuals are screened in combination with double SNP loci, and the survival rate of a challenge experiment is remarkably improved. The detection kit provided by the invention can realize high-throughput and high-precision typing, is suitable for tilapia mossambica resistance genotype identification and molecular marker-assisted breeding, provides an efficient genetic improvement tool for tilapia mossambica disease-resistant breeding, and is beneficial to reduction of breeding disease loss and reduction of antibiotic dependence.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to KASP molecular markers related to the resistance of tilapia to Streptococcus agalactiae and their applications. Background Art

[0002] Tilapia (Oreochromis spp.) is the largest exported aquaculture fish species in China, with an industrial scale exceeding one million tons and an annual output value exceeding ten billion yuan. However, streptococcosis caused by Streptococcus agalactiae has become the core bottleneck restricting the sustainable development of the industry. This disease is characterized by a fast transmission speed, a high lethality rate (30% - 90%), and is prone to mixed infections with other pathogens. Moreover, traditional drug treatments face three risks: drug resistance, environmental pollution, and food safety. Therefore, creating disease-resistant new varieties through genetic improvement is an important way to meet the needs of the healthy development of the industry and the national seed industry revitalization strategy.

[0003] In recent years, genetic parameter evaluations have shown that the resistance of tilapia to streptococcosis has significant additive genetic variation, and the estimated heritability ranges from 0.11 ± 0.02 to 0.52 ± 0.12 (medium to high heritability), indicating the theoretical feasibility of selective breeding. However, traditional breeding techniques require individuals to be challenged by pathogens, resulting in a reduction in the number of breeding individuals and a decline in quality. There are also problems such as low breeding efficiency, a long cycle, and being easily affected by the environment. Although single nucleotide polymorphism (SNP) molecular markers and combinations obtained based on genome-wide association analysis (GWAS) and best linear unbiased prediction (BLUP) can effectively break through the limitations of traditional breeding. However, in the case of tilapia, there are still problems such as a small number of molecular markers for the phenotype of resistance to Streptococcus agalactiae controlled by minor polygenes and a low degree of genetic variation explanation, which are difficult to support the efficient application of marker-assisted selection (MAS) or genome selection (GS) in the breeding of tilapia resistant to Streptococcus agalactiae. Therefore, there is still a need to develop diverse detection and verification methods to accelerate the discovery of candidate molecular markers for this trait.

[0004] SNP identification based on functional genes is one of the main means to discover effective SNPs. As a transmembrane tyrosine kinase receptor, Epidermal growth factor receptor (EGFR) plays an important role in the regulation of immune homeostasis in vertebrates by activating intracellular signal transduction pathways such as Mitogen-activated protein kinase (MAPK). Functional studies have shown that inhibiting EGFR expression in zebrafish significantly inhibits the expression of Chemokines (CXCL) 1 / 8, leading to a decrease in the efficiency of pathogen clearance. Knockout of the mouse EGFR gene exacerbates tissue damage by upregulating the expression of pro-inflammatory chemokines C-C Motif Chemokine Ligand (CCL2) and CXCL10. These results suggest that EGFR may have different immune regulation mechanisms in different species, but it is still an essential inflammatory response regulatory gene in the body after pathogen infection. However, in the research on the disease resistance breeding of tilapia, the molecular regulation mechanism of the EGFR gene has not been clarified. In particular, the research on the association mechanism between EGFR gene mutation and Streptococcus agalactiae resistance, the development and application of functional SNP markers are still in the blank stage, which seriously restricts the precise application of MAS or GS technology in the breeding of tilapia resistant to Streptococcus agalactiae. Summary of the Invention

[0005] In view of this, the present invention takes tilapia as the research object. First, based on the analysis of expression characteristics, it is confirmed that the EGFRX2 gene plays an immune regulation function in tilapia infected with Streptococcus agalactiae. Furthermore, a genotyping method for two SNP loci (SNP-1, SNP-2) of the EGFRX2 gene is developed based on Kompetitive Allele Specific PCR (KASP). The present invention develops a genotyping method for the above loci by KASP technology and verifies the association between the SNP-1 genotype (AA is the dominant type, GG is the recessive type) and the phenotype of Streptococcus agalactiae resistance. Further, the resistant dominant diplotype (AA / AA) individuals are screened by combining double SNP loci, and the survival rate of the challenge test is significantly improved. The detection kit provided by the present invention can achieve high-throughput and high-precision genotyping, is suitable for the identification of the genotype of tilapia resistant to Streptococcus agalactiae and molecular marker-assisted selection, provides an efficient genetic improvement tool for the breeding of tilapia resistant to Streptococcus agalactiae, helps to reduce the loss of aquaculture diseases, and reduces the dependence on antibiotics.

[0006] The technical solution of the present invention is realized as follows:

[0007] In the first aspect, the present invention provides a method for detecting the resistance of tilapia to Streptococcus agalactiae, which includes the following steps:

[0008] S1. Extract the genomic DNA of the tilapia to be tested;

[0009] S2. Detect the genotype of the SNP-1 site of the EGFRX2 gene in the genome; the SNP-1 site is: referring to the O_niloticus_UMD_NMBU genome version of Nile tilapia, located at 19925361 bp on chromosome 18, the reference base is A, and the mutant base is G;

[0010] S3. When the genotype of the SNP-1 site is detected as GG, determine that this genotype is a resistance-disadvantaged genotype, and the tilapia to be tested is a susceptible individual to Streptococcus agalactiae; when the genotype of the site is detected as AA, determine that this genotype is a resistance-dominant genotype, and the tilapia to be tested is an individual resistant to Streptococcus agalactiae disease.

[0011] In some specific embodiments, the genotype detection uses a reagent / kits containing a primer set; the primer set includes SEQ ID NO: 9-11. Preferably, the reagent / kits components further include 2×PCR premix.

[0012] In a second aspect, the present invention provides the detection method for any of the following applications: (1) identifying the resistance of tilapia to Streptococcus agalactiae disease; (2) breeding tilapia resistant to Streptococcus agalactiae disease.

[0013] In a third aspect, the present invention provides a detection product for the resistance of tilapia to Streptococcus agalactiae disease, and the components of the detection product include the primer set shown in SEQ ID NO: 9-11. Preferably, the detection product components further include 2×PCR premix.

[0014] In a fourth aspect, the present invention provides the application of the detection product for any of the following applications: (1) identifying the resistance of tilapia to Streptococcus agalactiae disease; (2) breeding tilapia resistant to Streptococcus agalactiae disease.

[0015] In a fifth aspect, the present invention provides a method for identifying the resistance-dominant diplotype of tilapia to Streptococcus agalactiae disease, including the following steps:

[0016] S1. Extract the genomic DNA of the tilapia to be tested;

[0017] S2. Respectively detect the genotypes of the SNP-1 site and the SNP-2 site of the EGFRX2 gene in the genome;

[0018] The SNP-1 site is: referring to the O_niloticus_UMD_NMBU genome version of Nile tilapia, located at 19925361 bp on chromosome 18, the reference base is A, and the mutant base is G;

[0019] The SNP-2 locus is as follows: Referring to the O_niloticus_UMD_NMBU genome version of Nile tilapia, it is located at 19918668 bp on chromosome 18. The reference base is C and the mutant base is A;

[0020] S3. When it is detected that the genotype of the SNP-1 locus is AA and the genotype of the SNP-2 locus is AA, the diplotype is the resistant dominant genotype, and the tilapia to be tested is an individual resistant to Streptococcus agalactiae disease.

[0021] In some specific embodiments, in the above step S2, genotype detection is performed using a reagent / kits containing a KASP genotyping primer set; the KASP genotyping primer set includes SEQ ID NOs: 9-14.

[0022] Sixthly, the present invention provides an application of the identification method of the resistant dominant diplotype of tilapia to Streptococcus agalactiae disease, which is used for breeding tilapia resistant to Streptococcus agalactiae disease.

[0023] The SEQ ID NOs: 9-14 are shown in the following table respectively:

[0024]

[0025] Note: The first 21 bases (italic part) of primer-1FX (SEQ ID NOs: 9 and 12) and primer-1FY (SEQ ID NOs: 10 and 13) are adapter sequences. Primer-1FX is used to detect the base site in the reference genome corresponding to the SNP, and primer-1FY is used to detect the base site after SNP mutation.

[0026]

Term Explanation

[0027] In some specific embodiments of the present invention, the meanings of relevant terms include the following:

[0028] Resistance to Streptococcus agalactiae disease: Refers to the natural or acquired immune ability of the host after infection with Streptococcus agalactiae, that is, the ability to limit bacterial colonization, invasion or pathogenicity, manifested as the individual not dying, having no abnormal swimming posture, and normal feeding.

[0029] Resistant dominant genotype to Streptococcus agalactiae disease: A genotype related to a significant increase in the survival rate after Streptococcus agalactiae infection, enhanced immune response or improved virulence clearance ability;

[0030] Susceptible inferior genotype to Streptococcus agalactiae disease: A genotype related to enhanced susceptibility to Streptococcus agalactiae, immunosuppression or aggravated pathological damage, manifested as high mortality or immune escape after infection.

[0031] KASP genotyping primer set: A primer combination designed based on competitive allele-specific PCR technology for SNP genotyping.

[0032] The primer combination includes two competitive forward primers (each linked with FAM / HEX fluorescent tags) and one common reverse primer. The 3'-end of the forward primer contains SNP-specific bases to ensure allele-specific amplification. After PCR amplification, the fluorescent signal is released by the allele-matched primer, and the genotyping result is determined by the fluorescence intensity.

[0033] Superior haplotype: Refers to the genotype formed by the combination of two pairs of alleles (diplotype) under specific phenotypes or environmental stresses, showing significant adaptability or functional advantages (such as disease resistance). The superior haplotype can be used as a target for MAS or GS to accelerate the genetic improvement of excellent traits. Brief Description of the Drawings

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0035] Figure 1 It shows the expression distribution of the EGFRX2 gene in different tissues of healthy tilapia in the embodiments of the present invention;

[0036] Figure 2 It shows the expression changes of the EGFRX2 gene in various tissues of tilapia after infection with Streptococcus agalactiae in the embodiments of the present invention;

[0037] Figure 3 It shows the expression changes of the tilapia EGFRX2 gene in various tissues at different times after Poly I:C stimulation in the embodiments of the present invention;

[0038] Figure 4 It shows the specificity of the EGFRX2 polyclonal antibody by Western blot analysis in the embodiments of the present invention; among them, Figure 4 In A: Lane 1 is the EGFR polyclonal antibody (4-fold dilution); Lane M is the protein molecular weight standard (kDa); Figure 4 In B: Lane 1 is 50 ng of EGFR incubated with 2 μg / mL of pre-immune rabbit antibody (using the pre-immune rabbit antibody as a control); Lane 2 is 50 ng of EGFR incubated with 2 μg / mL of the EGFR polyclonal antibody; M: Protein molecular weight standard (kDa);

[0039] Figure 5In the embodiments of the present invention, the positive area ratio and density difference of EGFR protein in different tissue layers; wherein, Figure 5 A shows the difference in the positive area ratio of EGFRX2 protein distribution in the intestine of the healthy group; Figure 5 B is the density difference diagram of EGFRX2 protein distribution in the intestine of the healthy group; Figure 5 C is the difference diagram of the positive area ratio of EGFRX2 protein distribution in the intestine before and after challenge with Streptococcus agalactiae; Figure 5 D is the density difference diagram of the positive area distribution of EGFRX2 protein in the intestine before and after challenge with Streptococcus agalactiae;

[0040] Figure 6 In the embodiments of the present invention, the positive expression of EGFRX2 protein in goblet cells, columnar cells and lymphocytes in the midgut of tilapia; wherein, Figure 6 A and 6B are fluorescence immunohistochemical sections of the midgut of healthy tilapia; 6C and 6D are fluorescence immunohistochemical sections of the midgut of tilapia 24 hours after challenge with Streptococcus agalactiae; Labels: s indicates the serosa; m indicates the muscular layer; sm indicates the submucosa; mu indicates the mucous layer; white arrows indicate goblet cells;

[0041] Figure 7 In the embodiments of the present invention, the detection results of SNPs related to Streptococcus agalactiae resistance of the EGFRX2 gene in Nile tilapia; wherein: 7A is the sequencing peak diagram of the LG18_8668 product; 7B is the sequencing peak diagram of the LG18_5361 product;

[0042] Figure 8 In the embodiments of the present invention, the KASP genotyping results, wherein 8A and 8B respectively correspond to the KASP genotyping results of LG18_8668 and LG18_5361 (each point represents a tested individual, and different colors correspond to different genotypes). Detailed implementation manners

[0043] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0044] Table 1 Sequence information table

[0045]

[0046]

[0047] In some specific embodiments, the present invention confirmed the correlation between the EGFRX2 gene and Streptococcus agalactiae infection in tilapia by detecting the expression and localization of the EGFRX2 gene before and after Streptococcus agalactiae challenge.

[0048] In some specific embodiments, the present invention amplified and scanned the SNP sites of a part of the EGFRX2 gene by PCR.

[0049] In some specific embodiments, the present invention genotyped and verified the above-mentioned SNP sites of the EGFRX2 gene by using KASP (Kompetitive Allele Specific PCR) in an enlarged population.

[0050] I. Experimental methods

[0051] 1. Rearing of experimental fish and collection of tissue samples

[0052] Five hundred healthy and non-deformed Nile tilapia (Oreochromis niloticus, GIFT strain) with a body weight of 80 ± 20 g and a body length of 12 ± 2 cm were selected. They were acclimated for two weeks before the experiment, and the water temperature was maintained at (30 ± 1) °C. They were fed commercial feed once in the morning and once in the evening every day and managed according to conventional conditions. All experimental tilapia were from the cooperative enterprise, Guangdong Tilapia Breeding Farm.

[0053] After the acclimation period, eight healthy tilapia were randomly selected. The fish were immersed in 0.3 mL / L ethyl 3-aminobenzoate (MS-222) (E10521, Sigma, USA) for 1 minute (min) to be anesthetized. Then, their hearts, gills, brains, livers, spleens, mid-kidneys, mid-intestines, stomachs, skins, muscles and blood were quickly taken, frozen in liquid nitrogen, and stored at -80 °C for later use. The above tissue materials were used for the analysis of the tissue expression characteristics of EGFRX2.

[0054] 2. Culture of Streptococcus agalactiae, artificial challenge of tilapia and sample collection

[0055] In the example, Streptococcus agalactiae WC1535 can be found in the reference (Molecular characterization, expression and functional analysis of NOD1, NOD2 and NLRC3 in Nile tilapia (Oreochromis niloticus)[J]. Fish&shellfish immunology, 2018, 73: 207-219.). The public can obtain it from the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences. The applicant undertakes to distribute the biological material to the public within twenty years from the filing date. The revived WC1535 was inoculated into a blood agar plate and placed in an incubator at 37 °C. After culturing for 16 hours (Hours, h), the bacteria were washed down with PBS. Then, the concentration of the washed bacterial solution was measured using an electronic turbidimeter DensiCHEK Plus (BioMerieux, Shanghai, China), and the concentration of the bacterial suspension was adjusted to 5×10 6 CFU / mL for standby (the final concentration of the bacterial solution was obtained from preliminary experiments). 100 healthy tilapia were selected from Method 1 and randomly divided into a control group and an experimental group, with 50 biological replicates in each group. Each tilapia in the control group was intraperitoneally injected with 100 μL of PBS solution, and each tilapia in the experimental group was intraperitoneally injected with 100 μL of Streptococcus agalactiae suspension. At 0 h, 8 h, 24 h, 48 h, 72 h, and 96 h after injection, 6 surviving individuals were randomly selected from the experimental group and the control group, and gill, kidney, spleen, liver, and intestinal tissues were collected under sterile conditions according to Method 1. After quick-freezing in liquid nitrogen, they were transferred to -80 °C for storage and standby.

[0056] 3. Intraperitoneal injection of Poly I:C (31852-29-6, Sigma, USA) and sample collection

[0057] 100 healthy tilapia were selected and randomly divided into two groups, namely the Poly I:C experimental group and the control group, with 50 fish in each group. Each tilapia in the control group was injected with 100 μL of PBS solution, and each fish in the Poly I:C experimental group was injected with 100 μL of 1 mg / mL Poly I:C prepared with PBS. At 0 h, 8 h, 24 h, 48 h, and 72 h after injection, 6 fish were selected from the experimental group and the control group, and the intestine, gill, kidney, and spleen of the fish were collected according to Method 2, quickly frozen in liquid nitrogen, and then transferred to -80 °C for storage and standby.

[0058] 4. Total RNA extraction and complementary DNA (cDNA) synthesis

[0059] According to the instructions of MagZol Reagent one-step RNA extraction reagent (Trizol) (R4801, Maigen Biotech, Shanghai), total RNA was extracted from each of the above tissue samples. The purity and concentration of RNA were detected using a UV spectrophotometer, and its integrity was detected using 1% agarose gel electrophoresis. After passing the detection, it was reverse-transcribed into cDNA using the All-in-One First-Strand Synthesis Master Mix (with dsDNase) kit (XKL0511, Xinkailai Biotech, Guangzhou), and the cDNA was stored at -20 °C for later use.

[0060] 5. Real-time quantitative PCR (RT-qPCR) and data processing

[0061] According to the cDNA sequence of the EGFRX2 gene (GenBank accession number XM_025900552.1), specific primers EGFRX2-F and EGFRX2-R were designed using NCBI primer-BLAST (Table 1). Using the reverse-transcribed cDNA as a template, RT-qPCR was performed, with the elongation factor-1 complex alpha gene (EF-1α) as the internal reference gene (Table 1). RT-qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Q711, Novoprotein, Nanjing) according to the instructions. The reaction system was as follows: 10 μL of SYBR mixed reagent, 8.2 μL of ddH2O, 0.4 μL each of the upstream and downstream primers (10 nM), and 1 μL of cDNA template. The RT-qPCR reaction program was: pre-denaturation at 95 °C for 30 seconds (s); denaturation at 95 °C for 10 s, annealing at 56 °C for 10 s, final extension at 72 °C for 30 s, for a total of 40 cycles; extension at 72 °C for 5 min. The relative expression levels of the EGFRX2 gene in each tissue were calculated using the 2 －ΔΔCt method.

[0062] 6. Construction of EGFRX2 expression plasmid, induction expression of recombinant protein, and preparation of polyclonal antibody

[0063] The tilapia EGFRX2 gene was ligated into the pET-B2M vector (purchased from Wuhan Jinkairui Bioengineering Co., Ltd.) to construct the recombinant plasmid pET-B2M-EGFRX2. The EGFRX2 amplification primers were as follows:

[0064] Upstream primer:

[0065] Downstream primer:

[0066] Among them, the regular text is the target sequence, and the italic text is the homologous arms on the vector. Homologous recombination was carried out according to the instructions of the ClonExpress Ultra OneStep Cloning Kit V3 (C117, Novoprotein, Nanjing). The recombinant pET-B2M-EGFRX2 was transferred into Escherichia coli TOP10 competent cells, screened on 1% streptomycin LB solid medium, positive colonies were picked, and colony PCR identification was performed. The constructed recombinant fusion expression plasmid pET-B2M-EGFRX2 was heat-shock transformed into Rosetta competent cells, 800 μL of pre-warmed LB liquid medium was added, and the cells were cultured with shaking at 158 rpm for 50 min, centrifuged at 6000 rpm for 4 min to remove the supernatant, and the remaining bacterial solution was spread on an LB plate containing 1% kanamycin and cultured at 37 °C for 12 h.

[0067] Positive single colonies were picked and inoculated into 3 mL of LB culture medium containing 1% kanamycin, and cultured at 37 °C until the logarithmic growth phase (OD 600 = 0.5), and the bacterial concentration was measured using an electronic turbidimeter DensiCHEK Plus (BioMerieux). A part of the bacterial solution was taken as the control group, and the remaining bacterial solution was added to an inducer with a final concentration of 1 mM isopropyl-β-D-thiogalactoside and cultured at 37 °C for 3 h. The cells were centrifuged at 12000 g for 2 min to collect the cells, and SDS-PAGE gel was used to detect protein expression.

[0068] 100 μL of the successfully induced bacterial solution was taken and inoculated into 200 mL of LB liquid medium, and cultured at 37 °C until the OD 600 reached 0.6, IPTG inducer was added to a final concentration of 0.5 mM, and centrifuged at 8000 rpm for 3 min. The precipitate was collected and resuspended with pre-cooled Ni-NTA buffer. The resuspended matter was ice-bathed for 30 min, and then the bacteria were lysed by sonication at 200 W (working for 3 s, pausing for 4 s, for a total of 25 min). Centrifuged at 16000 rpm for 50 min at 4 °C, the supernatant and precipitate were collected respectively, and SDS-PAGE electrophoresis was performed for detection.

[0069] The recombinant protein was determined to exist in the form of inclusion bodies according to the size of the gel electrophoresis bands, and the precipitate was subjected to inclusion body protein purification. It was resuspended in 50 mL of Ni-NTA buffer, and dithiothreitol was added to a final concentration of 1 mM. Then, the bacteria were lysed on an ultrasonic disruptor according to the following parameters: 200 W, working for 3 s, pausing for 3 s, and the time was 10 min. The lysed bacteria were centrifuged at 4°C and 10,000 rpm for 10 min to remove the supernatant (repeated three times until the supernatant was clear). The inclusion bodies were resuspended in 3 mL of 6 M guanidine hydrochloride, and dithiothreitol was added to a final concentration of 5 mM. It was shaken at 37°C and 220 rpm for 3 h until all the inclusion bodies were dissolved. It was centrifuged at 4°C and 10,000 rpm for 10 min, and the supernatant was taken for concentration determination, and then sent to Wuhan Kingcare to prepare rabbit anti-EGFRX2 polyclonal antibody.

[0070] 7. Immunofluorescence detection

[0071] According to the experimental steps of Method 2, 24 hours after artificial challenge of tilapia with Streptococcus agalactiae, 6 fish were randomly selected from each of the challenged group and the control group (injected with PBS), and the midgut tissues of the fish were collected. The collected midgut was fixed in 4% paraformaldehyde. After 24 hours, the tissue was trimmed flat and placed in an embedding frame, and then subjected to conventional dehydration, wax infiltration, and embedding. After the wax block solidified, the solidified wax block was taken out of the embedding frame and trimmed. A tissue section with a thickness of 4 μm was cut using a paraffin slicer (RM2016, Leica Instruments, Shanghai). The section was spread at 40°C, dried at 60°C, and stored at room temperature for later use.

[0072] The above sections were dewaxed in turn and washed with distilled water. The sections were placed in a 10mM citric acid antigen retrieval solution for antigen retrieval, and then placed in a microwave oven (high heat) for 5 minutes, and allowed to cool naturally. The sections were placed in PBS and washed by shaking on a decolorizing shaker, and repeated 3 times, 5 minutes each time. After the sections were slightly dried, circles were drawn around the tissue with a tissue pen, and 3% bovine serum albumin solution was added for blocking for 30 minutes. The diluted primary antibody was added to the sections at a concentration of 3.34μg / mL, and the sections were placed flat in a wet box and incubated overnight at 4°C. After that, the sections were placed in PBS, shaken on a decolorizing shaker, and washed 3 times, 5 minutes each time. Next, the secondary antibody Alexa Fluor 488-labeled goat anti-rabbit IgG (A0423, Biyuntian, Shanghai) was added and incubated at room temperature in the dark for 50 minutes. After incubation, the sections were placed in PBS and washed 3 times, 5 minutes each time, on a decolorizing shaker. DAPI nuclear stain was added and incubated at room temperature in the dark for 10 min. Afterwards, anti-fluorescence quenching sealing agent was added for sealing. The location of the fluorescent positive signal in the tissue was photographed and analyzed using a fluorescence microscope (Nikon Eclipse C1, Nikon, Japan) and software Aipathwell (V2) and slideviewer (V2.6). Among them, the parameters related to the positive area ratio and the positive surface distribution density were analyzed as follows: positive area ratio = positive area / tissue area, reflecting the amount of positive area; positive surface distribution density = cumulative optical density value / tissue pixel area, reflecting the average depth of positivity in the tissue area to be measured.

[0073] 8. Screening of SNP loci associated with resistance to Streptococcus agalactiae in the EGFRX2 gene of tilapia

[0074] Randomly select the caudal fin tissues of 10 Nile tilapia, extract the genomic DNA of the caudal fin according to the operation process of HiPure Universal DNA Kit (D3018, Meiji Bio, Guangzhou), and store it at -20°C for SNP detection of the EGFRX2 gene. Mix the genomic DNA of 10 individuals together and use it as a template for PCR reaction. The primers used in the PCR reaction were designed by the NCBI primerblast online software, and the sequence information is shown in Table 1 (5-6), and it was synthesized by Sangon Biotech. The PCR reaction system is shown in Table 2. The PCR reaction program was pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at an appropriate temperature (54.6°C for LG18-5361; 61.2°C for LG18-8668) for 10 s, extension at 72°C for 10 s, for a total of 30 cycles; final extension at 72°C for 5 min. After the amplification was completed, the PCR products were subjected to 1.5% agarose gel electrophoresis. After obtaining a single and bright band, the remaining PCR products were sent to Sangon Biotech for Sanger sequencing. According to the sequencing result peak map, the SNP sites existing in the EGFRX2 gene were judged.

[0075] Table 2 PCR reaction system

[0076] Each component Reaction system 2×PCR Premix (2×Mastermix, P111, Novoprotein, Nanjing) 10 μL Forward primer 0.5 μL Reverse primer 0.5 μL Sterile double-distilled water 8 μL DNA template 1 μL

[0077] 9. Genotyping verification of SNP sites related to Streptococcus agalactiae resistance in the tilapia EGFRX2 gene

[0078] According to the experimental fish specifications, breeding conditions in Method 1 above and the artificial challenge steps in Method 2 above, a Streptococcus agalactiae challenge experiment was carried out on 113 tilapia individuals. Referring to the criteria for judging sensitive and resistant samples in previous studies, individuals that died within 7 days (Day, d) after infection were regarded as sensitive individuals, and individuals that still survived 14 d after infection were regarded as resistant individuals. A total of 77 caudal fin samples of resistant individuals and 36 caudal fin samples of sensitive individuals were collected, placed in absolute ethanol, and stored at -20°C for later use. Extract the genomic DNA of the caudal fin according to the operation process of HiPure Universal DNA Kit (D3018, Meiji Bio, Guangzhou), and store it at -80°C for KASP genotyping verification.

[0079] Two allele-specific forward primers and one reverse universal primer were designed for each SNP using Primer5.0. The last bases of the two forward primers were the reference base and the mutant base, respectively. As shown in Table 1 (SEQ ID NO: 9-14), the italicized LG18-5361-1FX and LG18-8668-1FX (the italic part represents the adapter sequence) corresponded to the reference base fluorescent tag sequence (the reference base fluorescent tag sequence is used to detect the base site in the reference genome corresponding to the SNP), and the italicized LG18-5361-1FY and LG18-8668-1FY (the italic part represents the adapter sequence) corresponded to the mutant base fluorescent tag sequence (the mutant base fluorescent tag sequence is used to detect the base site after SNP mutation). The primers were synthesized by Beijing Sunbiotech Co., Ltd. The three SNP primers were all diluted to 10 μmol and mixed according to a volume ratio of 12:12:30.

[0080] The SNP genotyping of tilapia fin DNA samples was performed using the KASP technology. The SNP genotyping based on KASP was carried out on the LGC high-throughput genotyping system (LGC, Teddington, UK). The PCR reaction system required for the experiment is shown in Table 3. The PCR reaction was carried out in the high-throughput water bath system Hydrocycler. The specific procedure was pre-denaturation at 94 °C for 15 min; denaturation at 94 °C for 20 s, annealing at 61 °C - 55 °C for 1 min (amplified for 10 cycles with a touchdown PCR program, decreasing 0.6 °C per cycle), extension at 72 °C for 30 s; denaturation at 94 °C for 20 s, annealing at 55 °C for 60 s, extension at 72 °C for 30 s, for 26 cycles; final extension at 72 °C for 5 min. After the amplification was completed, the fluorescence signal was detected using a BMG PHERAstar multimode microplate reader (Orlsberg, Germany) and the genotyping results were viewed. The genotyping results were statistically analyzed using SNPviewer2 (v1.123) software (LGC).

[0081] 10. Data Statistics

[0082] Statistical analysis was performed using Minitab software (v21.0). After KASP genotyping, significant statistical analyses were performed on the dominant genotypes of sensitive and resistant phenotypes, respectively. The test model was the chi-square test. The remaining statistical tests were one-way analysis of variance. P < 0.05 was considered statistically significant.

[0083] Table 3 KASP genotyping PCR batch reaction system

[0084] Each component 234 reactions 2×PCR Premix (2×Mastermix, P111, Novoprotein, Nanjing) 422 μL Primer Premix 11.7 μL Sterile double-distilled water 422 μL DNA dry powder 8 - 10 ng Reaction system / per sample 3 μL

[0085] II. Experimental Results

[0086] 1. Tissue expression distribution of EGFRX2 in healthy tilapia

[0087] The expression distribution pattern of EGFRX2 in different tissues of healthy tilapia is shown in Figure 1 , and this gene is expressed in tissues such as brain, heart, mesonephros, intestine, stomach, skin, gill, spleen and muscle. Among them, the expression level in skin is the highest, followed by muscle, intestine, stomach and spleen, while the expression levels in liver and blood are the lowest.

[0088] 2. Expression changes of EGFRX2 gene in tilapia after artificial challenge with Streptococcus agalactiae

[0089] The expression changes of EGFRX2 gene in tilapia artificially infected with Streptococcus agalactiae at 8h, 24h, 48h, 72h and 96h are shown in Figure 2 . Compared with the expression level of the control group, the expression levels of EGFRX2 gene in spleen tissue were significantly decreased at 48h and 72h after challenge (P<0.05). In gill and kidney tissues, there were no significant differences in the expression levels of EGFRX2 gene before and after challenge. While in liver and intestine tissues, the expression levels of EGFRX2 gene were significantly increased at 24h after infection with Streptococcus agalactiae (P<0.01).

[0090] 3. Tissue expression changes of EGFRX2 gene in tilapia after stimulation with Poly I:C

[0091] After stimulation with Poly I:C, the expression level changes of EGFRX2 gene in various tissues of tilapia are shown in Figure 3 . In kidney tissue, there were no significant differences in EGFRX2 expression at each time point. In intestinal tissue, Poly I:C could significantly promote the up-regulation of EGFRX2 gene expression at 8h after stimulation, but then the expression level showed a downward trend, and there was no significant difference compared with the expression level at 0h. In gill tissue, the expression level of EGFRX2 showed a trend of first increasing (8h), then decreasing (24 - 48h), and then increasing again (72h). Among them, the increasing levels at 8h and 72h were significantly different compared with the control group (0h) (P<0.05). In spleen tissue, the change trend of EGFRX2 gene expression level was similar to that of gill tissue.

[0092] 4. Western blot verification of EGFRX2 polyclonal antibody

[0093] The results of SDS-PAGE electrophoresis showed that the recombinant expression protein of EGFRX2 was at the position of 62kDa, which was consistent with the expected size of the expressed protein. The results of Western blot of the recombinant antigen showed that the concentration of the primary antibody (EGFRX2 polyclonal antibody) was 2μg / mL, and there were bands at 62kD, verifying the specificity of the EGFRX2 polyclonal antibody, which was suitable for subsequent EGFRX2-related detections.

[0094] 5. Immunofluorescence localization analysis of EGFRX2 protein in intestinal tissues

[0095] (1) Localization analysis of EGFRX2 protein in tilapia intestinal tissues

[0096] Immunofluorescence detection found that in the healthy tilapia intestine, the EGFRX2 gene was positively expressed in the serosa layer, muscular layer, submucosa layer and mucosa layer ( Figure 5 ), and there were significant differences in the positive area ratio and distribution density of EGFRX2 protein between the serosa layer and the muscular layer (P<0.05). Among them, the positive area ratio (58.11%) and distribution density (0.0658) of the muscular layer were larger, the positive area ratio of the serosa layer was 32.94%, and the distribution density was 0.0393. In addition, there were no significant differences in the distribution density and positive area ratio of EGFRX2 protein between the submucosa layer and the mucosa layer of the healthy tilapia intestine.

[0097] After artificial infection with Streptococcus agalactiae, compared with the control group, the distribution density and positive area ratio of EGFRX2 protein in the serosa layer of tilapia intestine showed a significant upward trend (P<0.05). However, there were no significant changes in the distribution density and area ratio of EGFRX2 protein in the muscular layer, submucosa layer and mucosa layer ( Figure 5 ). The results showed that the serosa layer EGFRX2 protein played an important role in tilapia's response to Streptococcus agalactiae infection.

[0098] (2) Expression changes of EGFRX2 protein at the cellular level in tilapia intestine

[0099] Immunofluorescence detection found that EGFRX2 protein was expressed in each cell of tilapia intestinal tissues in the control group and 24 h after challenge, as Figure 6 shown. Among them, columnar epithelial cells, goblet cells, lymphocytes, etc. in the mucosa layer, nerve cells, lymphocytes, etc. in the submucosa layer, and muscle cells, nerve cells, etc. in the muscular layer were expressed before and after challenge. Among them, the goblet cells in the mucosa layer of the fish intestinal tissues increased significantly after challenge, and the positive expression signal of EGFRX2 protein in the goblet cells was significantly enhanced compared with the control group.

[0100] 6. Screening and application of SNP markers related to anti-Streptococcus agalactiae disease of EGFRX2 gene

[0101] (1) Identification of SNPs in partial introns of EGFRX2 gene

[0102] This application screened SNP markers related to Streptococcus agalactiae resistance in the EGFRX2 gene of tilapia, specifically as follows: This application used the mixed fin DNA of Nile tilapia as a template to amplify the 21st and 27th intron regions of the EGFRX2 gene. The tilapia EGFRX2 sequence was derived from the Nile tilapia O_niloticus_UMD_NMBU reference genome, and the GenBank accession number is XM_025900552.1. SNP sites were confirmed through sequencing chromatograms. The results showed that there was 1 SNP in each of the 21st and 27th introns, located at 19918668 bp (LG18_8668) on chromosome 18 and 19925361 bp (LG18_5361) on chromosome 18, respectively. LG18_8668 was a C / A mutation ( Figure 7 A), while LG18_5361 was an A / G mutation ( Figure 7 B).

[0103] (2) Screening of SNP markers related to Streptococcus agalactiae resistance in the EGFRX2 gene and KASP genotyping verification

[0104] This application verified the correlation of the above-screened SNP markers related to Streptococcus agalactiae resistance in the EGFRX2 gene of tilapia, specifically as follows:

[0105] To further verify the relationship between the above 2 SNPs and Streptococcus agalactiae resistance in tilapia, based on the above 2 SNPs, this application designed KASP genotyping primers. Both LG18-5361 and LG18-8668 were successfully genotyped ( Figure 8 ).

[0106] In the expanded population, the fin DNA of 79 resistant individuals and 36 susceptible individuals was genotyped. The results showed that the individuals to be detected were clearly divided into three genotypes. Among them, 113 (98.26%) fish at the LG18-5361 locus were successfully divided into AA, AG, and GG genotypes (Table 4). Chi-square test found that the genotype frequencies and allele frequencies of LG18-5361 were extremely significantly correlated with the trait of Streptococcus agalactiae resistance. Among them, the AA genotype was significantly correlated with Streptococcus agalactiae resistance, while the GG genotype was significantly correlated with Streptococcus agalactiae susceptibility (Table 4). In addition, 113 (98.26%) fish at the LG18-8668 locus were successfully divided into AA, AC, and CC genotypes (Table 4). Chi-square test found that the genotype frequencies and allele frequencies of LG18-8668 were not correlated with the trait of Streptococcus agalactiae resistance.

[0107] Table 4 Statistical analysis of the correlation of SNPs in the tilapia EGFRX2 gene between the Streptococcus agalactiae-resistant population and the susceptible population

[0108]

[0109] (3) Association analysis of the diploid combinations of LG18-8668 and LG18-5361 with Streptococcus agalactiae resistance in tilapia

[0110] This application analyzed the association between the diploids formed by different SNPs and Streptococcus agalactiae resistance in tilapia, as follows:

[0111] According to the data in Table 4, the three pairs of alleles of LG18-8668 and LG18-5361 were paired pairwise. A total of 111 individuals (total number of individuals) were combined into 9 diploids, namely D1-D9. At the same time, the number of disease-resistant and susceptible individuals with paired genotypes was accumulated to form the number of resistant / susceptible individuals corresponding to each diploid (Table 5). First, this study calculated the survival rate of fish corresponding to each diploid. The results showed that there were significant differences in survival rates among diploids (0.1 - 0.21) (Table 5). Among them, the survival rate of the diploid LG18-8668:AA / LG18-5361:AA (AA / AA) composed of D1 was the highest (Table 5). Therefore, this study compared the differences in resistant / susceptible phenotypes between D1 and the other 8 diploids through chi-square test. The results showed that the number of resistant individuals in D1 was significantly more than that in D3, D4, and D6 (Table 5). This result indicates that D1 is the dominant diploid for Streptococcus agalactiae resistance in tilapia, while D3, D4, and D6 are the inferior genotypes. During breeding, fish with the D1 diploid should be selected as much as possible, and fish with the D3, D4, and D6 diploids should be eliminated.

[0112] Table 5 Association analysis of the diploids formed by the combination of LG18-8668 and LG18-5361 with disease-resistant traits

[0113]

[0114] Note: NA indicates that due to too small expected count, the chi-square approximation may be invalid.

[0115] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting the resistance of tilapia to Streptococcus agalactiae, characterized in that, It includes the following steps: S1. Extract the genomic DNA of the tilapia to be tested; S2. Detect the genotype of the SNP-1 site of the EGFRX2 gene in the genome; the SNP-1 site is: referring to the O_niloticus_UMD_NMBU genome version of Nile tilapia, located at 19925361 bp on chromosome 18, the reference base is A, and the mutant base is G; S3. When the genotype of the SNP-1 site is detected as GG, determine that this genotype is a resistance-disadvantaged genotype, and the tilapia to be tested is a susceptible individual to Streptococcus agalactiae; when the genotype of the site is detected as AA, determine that this genotype is a resistance-advantaged genotype, and the tilapia to be tested is a resistant individual to Streptococcus agalactiae disease.

2. The detection method according to claim 1, wherein In step S2: The genotype detection uses a reagent / kits containing a primer set; the primer set includes SEQ ID NO: 9-11.

3. The detection method according to claim 2, characterized in that, The reagent / kits components also include 2×PCR premix.

4. Use of the detection method according to any one of claims 1 to 3, characterized in that For any of the following applications: (1) Identify the resistance of tilapia to Streptococcus agalactiae disease; (2) Breed tilapia resistant to Streptococcus agalactiae disease.

5. A detection product for the resistance of tilapia to Streptococcus agalactiae, characterized in that, The detection product components contain the primer set shown in SEQID NO: 9-11.

6. The detection product according to claim 5, characterized in that, The detection product components also include 2×PCR premix.

7. Use of the detection product according to claim 5 or 6, characterized in that For any of the following applications: (1) Identify the resistance of tilapia to Streptococcus agalactiae disease; (2) Breed tilapia resistant to Streptococcus agalactiae disease.

8. A method for identifying a diplotype with resistance advantage to Streptococcus agalactiae disease in tilapia, characterized in that, It includes the following steps: S1. Extract the genomic DNA of the tilapia to be tested; S2. Respectively detect the genotypes of the SNP-1 site and SNP-2 site of the EGFRX2 gene in the genome; The SNP-1 site is: referring to the O_niloticus_UMD_NMBU genome version of Nile tilapia, located at 19925361 bp on chromosome 18, the reference base is A, and the mutant base is G; The SNP-2 site is: referring to the O_niloticus_UMD_NMBU genome version of Nile tilapia, located at 19918668 bp on chromosome 18, the reference base is C, and the mutant base is A; S3. When the genotype of the SNP-1 site is detected as AA and the genotype of the SNP-2 site is AA, the tilapia is a resistance-advantaged diplotype to Streptococcus agalactiae disease.

9. The identification method according to claim 8, wherein, In step S2, the genotype detection uses a reagent / kits containing a KASP genotyping primer set; the KASP genotyping primer set includes SEQ ID NO: 9-14.

10. Use of the identification method according to claim 8 or 9, characterized in that, For breeding tilapia resistant to Streptococcus agalactiae disease.

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