KASP molecular marker related to resistance of rohu to streptococcus agalactiae and application thereof

Abstract

The present application relates to the field of biotechnology, and particularly relates to a KASP molecular marker related to the resistance of tilapia to streptococcus agalactiae disease and an application thereof.Two SNP sites (SNP-1 and SNP-2) significantly associated with the resistance are screened from the intron region of EGFRX2 gene of tilapia, a typing method of the above sites is developed through KASP technology, and the correlation between the SNP-1 genotype and the resistance phenotype is verified.Further, an advantage haplotype individual with the resistance to streptococcus agalactiae disease is screened by combining the two SNP sites, and the survival rate of the individual in the challenge experiment is significantly improved.The detection kit provided by the present application can realize high-throughput and high-precision typing, is suitable for the identification of the resistance genotype of tilapia and the marker-assisted selection, provides an efficient genetic improvement tool for the disease-resistant breeding of tilapia, and is helpful 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 particularly relates to a KASP molecular marker related to resistance to Streptococcus agalactiae disease in Oreochromis niloticus and application thereof. BACKGROUND

[0002] Oreochromis niloticus, as the first large export aquaculture fish in China, has an industry scale of over one million tons, and an annual output value of over ten billion yuan. However, Streptococcus agalactiae disease caused by Streptococcus agalactiae has become a core bottleneck restricting the sustainable development of the industry. The disease has the characteristics of fast transmission, high mortality (30% to 90%), easy mixed infection with other pathogens, and the like, and traditional drug treatment faces the triple risks of drug resistance, environmental pollution and food safety. Therefore, creating a new disease-resistant variety through genetic improvement is an important way to meet the needs of the industry for benign development and the national seed industry revitalization strategy.

[0003] In recent years, genetic parameter evaluation shows that the resistance of Oreochromis niloticus to Streptococcus agalactiae disease has significant additive genetic variation, and the heritability is estimated to be in the range of 0.11±0.02 to 0.52±0.12 (medium to high heritability), indicating that selection breeding is theoretically feasible. However, traditional breeding techniques require individuals to be challenged by pathogens, thereby reducing the number of breeding individuals and reducing the quality; and there are problems such as low efficiency, long cycle, and being easily affected by environmental interference. 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 terms of Oreochromis niloticus, the current molecular markers of Streptococcus agalactiae disease resistance phenotype controlled by multiple genes with small effects still have problems such as small number, low genetic variation explanation, and the like, which makes it difficult to support the efficient application of marker-assisted selection (MAS) or genome selection (GS) in the breeding of Oreochromis niloticus against Streptococcus agalactiae. Therefore, it is still necessary to develop diversified detection and verification methods to accelerate the mining of candidate molecular markers for this trait.

[0004] SNP identification based on functional genes is one of the main means to explore 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 can significantly inhibit the expression of neutrophil chemokines (Chemokines, CXCL) 1 / 8, leading to a decrease in pathogen clearance efficiency. Mice EGFR gene knockout can exacerbate tissue damage by up-regulating pro-inflammatory chemokines C-C motif chemokine ligand 2 (CCL2) and CXCL10 expression. These results suggest that EGFR may have different immune regulation mechanisms in different species, but it is still an indispensable gene for regulating inflammatory responses after pathogen infection. However, the molecular regulation mechanism of the EGFR gene in tilapia disease resistance breeding has not been elucidated, especially the correlation mechanism between EGFR gene mutation and streptococcosis 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 tilapia breeding for streptococcosis resistance. SUMMARY

[0005] In view of this, the present application takes tilapia as the research object, first based on expression characteristic analysis, it is proved that EGFRX2 gene plays an immune regulation function in tilapia streptococcosis. Further, the genotyping method of two SNP sites (SNP-1, SNP-2) of EGFRX2 gene is developed based on Kompetitive Allele Specific PCR (KASP). The present application develops the genotyping method of the above-mentioned sites by KASP technology, and verifies the correlation between SNP-1 genotype (AA is the dominant type, and GG is the inferior type) and streptococcosis resistance phenotype. Further, the resistant dominant haplotype (AA / AA) individual is screened out by combining the two SNP sites, and the survival rate of the individual in the challenge test is significantly improved. The detection kit provided by the present application can realize high-throughput and high-precision genotyping, and is suitable for tilapia streptococcosis resistance genotyping and molecular marker assisted selection, which provides an efficient genetic improvement tool for tilapia breeding for streptococcosis resistance, helps to reduce the loss of breeding diseases, and reduces the dependence on antibiotics.

[0006] The technical scheme of the present application is as follows:

[0007] In a first aspect, the present application provides a detection method for streptococcosis resistance of tilapia, comprising the following steps:

[0008] S1, extracting the genomic DNA of the test tilapia;

[0009] S2, detecting the genotype of SNP-1 site of EGFRX2 gene in the genome; the SNP-1 site is located at 19925361 bp of chromosome 18 in the reference O. niloticus UMD_NMBU genome version, with reference base A and mutant base G;

[0010] S3, when the genotype of the SNP-1 site is detected as GG, it is determined that the genotype is a disadvantageous genotype for resistance, and the test tilapia is a susceptible individual to S. agalactiae; when the genotype of the site is detected as AA, it is determined that the genotype is a dominant genotype for resistance, and the test tilapia is a resistant individual to S. agalactiae.

[0011] In some specific embodiments, the genotype detection uses a reagent / kit containing a primer set; the primer set comprises SEQ ID NO: 9-11. Preferably, the components of the reagent / kit further comprise 2x PCR premix.

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

[0013] In a third aspect, the present application provides a detection product for the resistance of tilapia to S. agalactiae, wherein the components of the detection product comprise the primer set shown in SEQ ID NO: 9-11. Preferably, the components of the detection product further comprise 2x PCR premix.

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

[0015] In a fifth aspect, the present application provides a method for identifying a dominant haplotype for the resistance of tilapia to S. agalactiae, comprising the following steps:

[0016] S1, extracting the genomic DNA of the test tilapia;

[0017] S2, detecting the genotype of SNP-1 site and SNP-2 site of EGFRX2 gene in the genome, respectively;

[0018] The SNP-1 site is located at 19925361 bp of chromosome 18 in the reference O. niloticus UMD_NMBU genome version, with reference base A and mutant base G.

[0019] The SNP-2 site is: located at 19918668 bp of chromosome 18 of the reference Nile tilapia O_niloticus_UMD_NMBU genome version, the reference base is C, and the mutant base is A;

[0020] S3, when the genotypes of the SNP-1 site and the SNP-2 site are AA, the haplotype is a resistant dominant genotype, and the tilapia to be tested is a Streptococcus agalactiae resistant individual.

[0021] In some specific embodiments, in the step S2, the genotype detection uses a reagent / reagent kit containing a KASP typing primer set; the KASP typing primer set comprises SEQ ID NO: 9-14.

[0022] In a sixth aspect, the present application provides an application of the method for identifying the resistant dominant haplotype of the tilapia to Streptococcus agalactiae, which is used for breeding the tilapia resistant to Streptococcus agalactiae.

[0023] The SEQ ID NO: 9-14 are shown in the following table respectively:

[0024]

[0025] Note: The first 21 bases (italic part) of primer-1FX (SEQ ID NO: 9 and 12) and primer-1FY (SEQ ID NO: 10 and 13) are linker sequences, primer-1FX is used for detecting the base site in the reference genome corresponding to the SNP, and primer-1FY is used for detecting the base site after mutation of the SNP.

[0026]

Terminology Explanation

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

[0028] Streptococcus agalactiae resistance: refers to the natural or acquired immunity ability of a host after infection with Streptococcus agalactiae, i.e. the ability to limit bacterial colonization, invasion or pathogenicity, which is manifested as survival of the individual, no abnormal swimming posture, and normal feeding.

[0029] Streptococcus agalactiae resistance dominant genotype: a genotype associated with significantly increased survival rate after Streptococcus agalactiae infection, enhanced immune response, or improved virulence clearance ability;

[0030] Streptococcus agalactiae resistance disadvantageous genotype: a genotype associated with increased susceptibility to Streptococcus agalactiae, immune suppression, or aggravated pathological damage, which is manifested as high mortality or immune escape after infection.

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

[0032] Two competitive forward primers (FAM / HEX fluorescent tags attached respectively) and one universal reverse primer are included in the primer combination. The 3' end of the forward primer contains SNP-specific bases to ensure allele-specific amplification. After PCR amplification, the fluorescent signal is released through the allele-matched primer, and the genotyping result is determined by the fluorescence intensity.

[0033] Dominant haplotype: refers to the genotype formed by two pairs of alleles (heterozygote) under specific phenotypes or environmental pressures, showing significant adaptive or functional advantages (such as disease resistance). Dominant haplotype can be used as a target for MAS or GS to accelerate genetic improvement of excellent traits. BRIEF DESCRIPTION OF DRAWINGS

[0034] 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 below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained according to these drawings without creative labor for those skilled in the art.

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

[0036] Figure 2 EGFRX2 gene expression changes in each tissue of tilapia after infection with Streptococcus agalactiae in the embodiments of the present application;

[0037] Figure 3 EGFRX2 gene expression changes in each tissue of tilapia at different times after Poly I:C stimulation in the embodiments of the present application;

[0038] Figure 4 Western blot analysis of the specificity of EGFRX2 polyclonal antibody in the embodiments of the present application; wherein, Figure 4 A: Lane 1 is EGFR polyclonal antibody (4-fold dilution); M is protein molecular weight marker (kDa); Figure 4 B: Lane 1 is 50 ng EGFR incubated with 2 μg / mL preimmune antibody (rabbit preimmune antibody is used as a control); Lane 2 is 50 ng EGFR incubated with 2 μg / mL EGFR polyclonal antibody; M: protein molecular weight marker (kDa);

[0039] Figure 5For the embodiment of the present application, the positive area ratio and density difference of EGFR protein in different tissue layers; wherein, Figure 5 A is the difference of the positive area ratio of EGFRX2 protein distribution in the intestinal tract of the healthy group; Figure 5 B is the difference of the density of EGFRX2 protein distribution in the intestinal tract of the healthy group; Figure 5 C is the difference of the positive area ratio of EGFRX2 protein distribution in the intestinal tract before and after Streptococcus agalactiae challenge; Figure 5 D is the difference of the density of EGFRX2 protein distribution in the intestinal tract before and after Streptococcus agalactiae challenge;

[0040] Figure 6 For the embodiment of the present application, the positive expression of EGFRX2 protein in the intestinal goblet cells, columnar cells and lymphocytes of tilapia; wherein, Figure 6 A and 6B are fluorescence immunohistochemical sections of the intestinal tract of healthy tilapia; 6C and 6D are fluorescence immunohistochemical sections of the intestinal tract of tilapia 24 hours after Streptococcus agalactiae challenge; the labels: s indicates the serosa layer; m indicates the muscular layer; sm indicates the submucosa layer; mu indicates the mucous layer; the white arrow indicates the goblet cells;

[0041] Figure 7 For the embodiment of the present application, the detection results of SNPs related to Streptococcus agalactiae disease resistance of Nile tilapia EGFRX2 gene; wherein: 7A is the product sequencing peak chart of LG18_8668; 7B is the product sequencing peak chart of LG18_5361;

[0042] Figure 8 For the embodiment of the present application, the KASP typing results, wherein 8A and 8B correspond to the KASP typing results of LG18_8668 and LG18_5361 respectively (each dot represents a detection individual, and different colors correspond to different genotypes). DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the present application will be described clearly and completely 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 skilled in the art without creative labor fall within the scope of protection of the present application. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0044] Table 1 Sequence information table

[0045]

[0046]

[0047] In some specific embodiments, the present application proves that the EGFRX2 gene is related to the Streptococcus agalactiae infection of the Nile Tilapia by detecting the expression and localization of the EGFRX2 gene before and after the Streptococcus agalactiae challenge.

[0048] In some specific embodiments, the present application amplifies and scans the SNP site of the EGFRX2 gene by PCR.

[0049] In some specific embodiments, the present application verifies the genotyping of the SNP site of the EGFRX2 gene by KASP (Competitive Allele-Specific PCR) in an expanded population.

[0050] I. Experimental methods

[0051] 1. Experimental fish feeding and tissue sample collection

[0052] 500 healthy and non-deformed Nile Tilapia GIFT strain (Oreochromis niloticus, GIFT strain) with a body weight of 80±20 g and a body length of 12±2 cm were selected. The fish were temporarily raised for two weeks before the experiment, with water temperature maintained at (30±1)℃, and fed with commercial feed twice a day. The fish were raised and managed under normal conditions. The experimental tilapia were obtained from a cooperative enterprise, Guangdong Tilapia Breeding Farm.

[0053] After the temporary raising, 8 healthy tilapia were randomly selected, and the fish were soaked in 0.3 mL / L of 3-aminobenzoic acid ethyl ester (MS-222) (E10521, Sigma, USA) for 1 minute (min) to anesthetize the fish. Then, the heart, gills, brain, liver, spleen, mesonephros, midgut, stomach, skin, muscle, and blood were quickly taken, and the samples were quickly frozen in liquid nitrogen and stored at -80℃ for later use. The above tissue materials were used for EGFRX2 tissue expression characteristic analysis.

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

[0055] Streptococcus agalactiae WC1535 in the example is seen 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, and the applicant promises to issue the biological material to the public for twenty years from the date of application. The recovered WC1535 is inoculated into a blood plate and placed in a 37°C incubator for 16 hours (h) of culture, and then the bacteria are washed down with PBS. Next, the concentration of the washed bacterial solution is determined using an electronic turbidimeter DensiCHEK Plus (BioMerieux, Shanghai, China), and the bacterial suspension concentration is adjusted to 5 x 10 6 CFU / mL (the final concentration of the bacterial solution is obtained from the pre-experiment). 100 healthy tilapia are selected from the healthy tilapia of method 1 and randomly divided into a control group and an experimental group, each with 50 biological replicates. The control group of tilapia is injected with 100 μL of a PBS solution per tail, and the experimental group is injected with 100 μL of a S. agalactiae suspension per tail. At 0 h, 8 h, 24 h, 48 h, 72 h, and 96 h after injection, 6 surviving individuals are randomly selected from the experimental and control groups, respectively, and the gills, kidneys, spleens, livers, and intestinal tissues are collected in a sterile environment according to method 1. After quick freezing in liquid nitrogen, they are transferred to -80°C for storage.

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

[0057] 100 healthy tilapia are selected and randomly divided into two groups, namely a Poly I:C experimental group and a control group, each with 50 tilapia. Each tilapia in the control group is injected with 100 μL of a PBS solution, and each tilapia in the Poly I:C experimental group is 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 tilapia are selected from the experimental and control groups, respectively, and the intestinal, gill, kidney, and spleen tissues are collected according to method 2, quickly placed in liquid nitrogen for quick freezing, and then transferred to -80°C for storage.

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

[0059] According to the MagZol Reagent one-step RNA extraction reagent (Trizol) (R4801, MagZol, Shanghai) instructions, total RNA of each tissue sample was extracted. The purity and concentration of RNA were detected by ultraviolet spectrophotometer, and its integrity was detected by 1% agarose gel electrophoresis. After detection, the cDNA was reversely transcribed using All-in-One First-Strand Synthesis Master Mix (with dsDNase) kit (XKL0511, XinKailai Biological, Guangzhou), and the cDNA was stored at -20℃ for standby.

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

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

[0062] 6、EGFRX2 expression plasmid construction, recombinant protein induction expression and polyclonal antibody preparation

[0063] The tilapia EGFRX2 gene was linked to the pET-B2M vector (purchased from Wuhan Jin Kai Rui Biological Engineering Co., Ltd.) to construct the recombinant plasmid pET-B2M-EGFRX2. The EGFRX2 amplification primers were as follows:

[0064] Upstream primer:

[0065] Downstream primer:

[0066] Wherein, the normal body is the target sequence, and the italic is the homologous arm on the vector. According to the instructions of ClonExpress Ultra OneStep Cloning Kit V3 (C117, Novagen, Nanjing), homologous recombination was carried out. The recombined pET-B2M-EGFRX2 was transferred into E. coli TOP10 competent cells, screened by 1% streptomycin LB solid medium, and positive colonies were picked and identified by colony PCR. The constructed recombinant fusion expression plasmid pET-B2M-EGFRX2 was heat-shocked and transformed into Rosetta competent cells, and 800 μL of preheated LB liquid medium was added, and the culture was shaken at 158 rpm for 50 min, and the supernatant was removed by centrifugation at 6000 rpm for 4 min, and the remaining bacterial liquid was inoculated on LB plates 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 to the logarithmic growth phase (OD 600 = 0.5), and the bacterial liquid concentration was determined by electronic turbidimeter DensiCHEK Plus (BioMerieux). Part of the bacterial liquid was used as a control group, and the remaining bacterial liquid was added with an inducer of isopropyl-β-D-thio-glycoside at a final concentration of 1 mM, and cultured at 37°C for 3 h, and the bacterial cells were collected by centrifugation at 12000 g for 2 min, and the protein expression was detected by SDS-PAGE gel.

[0068] 100 μL of successfully induced bacterial liquid was inoculated into 200 mL of LB liquid medium, and cultured at 37°C to OD 600 0.6, and IPTG inducer was added to a final concentration of 0.5 mM, and centrifuged at 8000 rpm for 3 min. The precipitate was resuspended with pre-cooled Ni-NTA buffer. The resuspension was ice-bathed for 30 min, and then the bacterial cells were broken by ultrasonic treatment (working for 3 s, pause for 4 s, time for 25 min) at 200 W. The supernatant and precipitate were collected by centrifugation at 4°C and 16000 rpm for 50 min, and SDS-PAGE electrophoresis detection was performed.

[0069] According to the gel electrophoresis band size determination of recombinant protein in the form of inclusion bodies, the precipitate was subjected to inclusion body protein purification. Resuspended with 50 mL Ni-NTA buffer, added dithiothreitol to a final concentration of 1 mM. Then, break the bacteria on the ultrasonic disrupter according to the following parameters: 200 W, work for 3 s, pause for 3 s, time for 10 min. The broken bacteria were centrifuged at 4°C, 10000 rpm for 10 min, and the supernatant was removed (repeated three times until the supernatant was transparent). Resuspend the inclusion bodies with 3 mL 6M guanidine hydrochloride, and add dithiothreitol to a final concentration of 5 mM. Shake at 37°C, 220 rpm for 3 h until the inclusion bodies are completely dissolved. Centrifuge at 4°C, 10000 rpm for 10 min, take the supernatant, and determine the concentration, then send to Wuhan Jin Kai Rui to prepare rabbit anti-EGFRX2 polyclonal antibody.

[0070] 7. Immunofluorescence detection

[0071] According to the experimental steps of method 2, 24 hours after artificial infection of rohu with Streptococcus agalactiae, 6 fish were randomly selected from each of the infected group and the control group (injected with PBS) for collection of midgut tissue from the fish body. The collected midgut was fixed in 4% paraformaldehyde, and after 24 hours, the tissue was smoothed and placed in an embedding frame, followed by routine dehydration, wax immersion, and embedding treatment. After the wax block solidified, the solidified wax block was removed from the embedding frame and trimmed. A paraffin microtome (RM2016, Leica Instruments, Shanghai) was used to cut tissue sections with a thickness of 4 μm. The sections were placed at 40°C and dried at 60°C, and then stored at room temperature for later use.

[0072] After the above-mentioned slices were sequentially dewaxed, they were washed with distilled water. The slices were placed in a 10 mM citric acid antigen repair solution for antigen repair, and were cooked in a microwave oven (high heat) for 5 min, and then were naturally cooled. The slices were placed in PBS and were shaken and washed on a decolorizing shaker for 3 times, 5 min each time. After the slices were slightly dried, they were circled with a histological pen at the periphery of the tissue, and 3% bovine serum albumin solution was added dropwise for blocking for 30 min. The diluted first antibody was added dropwise to the slices, at a concentration of 3.34 μg / mL, and the slices were placed in a wet box and were incubated at 4°C overnight. Then, the slices were placed in PBS and were shaken and washed on a decolorizing shaker for 3 times, 5 min each time. Next, goat anti-rabbit IgG labeled with Alexa Fluor 488 (A0423, Biyun Tian, Shanghai) was added, and the slices were incubated in the dark at room temperature for 50 min. After the incubation was completed, the slices were placed in PBS and were washed on a decolorizing shaker for 3 times, 5 min each time. DAPI nuclear staining solution was added, and the slices were incubated in the dark at room temperature for 10 min. Then, anti-fluorescence quenching mounting medium was added for mounting. The localization of the fluorescent positive signals in the tissue was photographed and analyzed using a fluorescence microscope (Nikon Eclipse C1, Nikon, Japan) and software Aipathwell (V2) and slideviewer (V2.6). The positive area ratio and positive surface distribution density related parameter analysis are 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 the positive in the tissue region to be measured.

[0073] 8. Screening of SNPs of Tilapia EGFRX2 gene against S. agalactiae related disease

[0074] Ten tail fin tissues of Nile tilapia were collected, and genomic DNA was extracted according to the operation procedure of HiPure Universal DNA Kit (D3018, Meibio, Guangzhou). The genomic DNA was stored at -20℃ and used for SNP detection of EGFRX2 gene. Genomic DNA of 10 individuals was mixed together as a template for PCR reaction. The primers used in PCR reaction were designed by NCBI primer blast online software, and the sequence information is shown in Table 1 (5-6). The PCR reaction system is shown in Table 2. The PCR reaction program was 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, appropriate temperature (LG18-5361 was 54.6℃; LG18-8668 was 61.2℃) annealing for 10 s, 72℃ extension for 10 s, for a total of 30 cycles; final extension at 72℃ for 5 min. After the amplification, 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 Shengong Bio for Sanger sequencing. According to the sequencing result peak graph, the SNP site of EGFRX2 gene was determined.

[0075] Table 2 PCR reaction system

[0076] Components Reaction system 2x PCR Mastermix (2x Mastermix, P111, Nanjing, Qiagen) 10 μL Upstream primer 0.5 μL Downstream primer 0.5 μL Sterile double distilled water 8 μL DNA template 1 μL

[0077] 9. Verification of SNP site typing of tilapia EGFRX2 gene against S. agalactiae disease

[0078] According to the fish size, breeding conditions of Method 1 and the artificial infection steps of Method 2, 113 tilapia individuals were subjected to S. agalactiae infection experiment. According to the standard for judging sensitive and resistant samples in the previous study, individuals that died within 7 days (d) after infection were considered as sensitive individuals, and individuals that survived after 14 d after infection were considered as resistant individuals. Tail fin samples of 77 resistant individuals and 36 sensitive individuals were collected and stored in anhydrous ethanol at -20℃ for standby. Genomic DNA was extracted according to the operation procedure of HiPure Universal DNA Kit (D3018, Meibio, Guangzhou), and stored at -80℃ for KASP typing verification.

[0079] Two allele-specific forward primers and one reverse universal primer were designed for each SNP using Primer 5.0. The last base of the two forward primers was the reference base and the mutant base, respectively. As shown in Table 1 (SEQ ID NOs: 9-14), the italicized LG18-5361-1FX, LG18-8668-1FX (the italicized part represents the linker sequence) corresponds to the reference base fluorescent tag sequence (which is used to detect the base site in the reference genome corresponding to the SNP), and the italicized LG18-5361-1FY, LG18-8668-1FY (the italicized part represents the linker sequence) corresponds to the mutant base fluorescent tag sequence (which is used to detect the base site after mutation of the SNP). The primers were synthesized by Beijing Sunhybrid Biological Engineering Co., Ltd. The three SNP primers were each diluted to 10 μmol and mixed in a volume ratio of 12:12:30.

[0080] The KASP technology was used to genotype the DNA samples of the tilapia fin strips, and the KASP-based SNP genotyping was performed in 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 performed in a high-throughput water bath system Hydrocycler, and the specific program was 94°C pre-denaturation, 15 min; 94°C denaturation, 20 s, 61°C-55°C annealing, 1 min (10 cycles of amplification by drop PCR program, each cycle decreasing by 0.6°C), 72°C extension for 30 s; 94°C denaturation, 20 s, 55°C annealing, 60 s, 72°C extension for 30 s, 26 cycles; 72°C final extension for 5 min. After amplification, the BMG PHERAstar multifunctional enzyme labeler (Olsberg, Germany) was used to detect the fluorescence signal and check the genotyping. The genotyping results were statistically analyzed using the SNPviewer2 (v1.123) software (LGC).

[0081] 10. Data statistics

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

[0083] Table 3 KASP genotyping PCR batch reaction system

[0084] Components 234 reactions 2x PCR Mastermix (2x Mastermix, P111, Nanjing, Qiagen) 422 μL Primer mix 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. Expression distribution of EGFRX2 in healthy tilapia

[0087] The expression distribution of EGFRX2 in different tissues of healthy Oreochromis niloticus was shown in Figure 1. The gene was expressed in brain, heart, kidney, intestine, stomach, skin, gill, spleen and muscle. The expression level in skin was the highest, followed by muscle, intestine, stomach, spleen, etc. The expression level in liver and blood was the lowest. Figure 1

[0088] 2. Changes in expression of EGFRX2 gene after artificial infection of Streptococcus agalactiae in Oreochromis niloticus

[0089] Changes in expression of EGFRX2 gene in Oreochromis niloticus after artificial infection of Streptococcus agalactiae for 8h, 24h, 48h, 72h and 96h were shown in Figure 2. Compared with the expression level in the control group, the expression level of EGFRX2 gene in the spleen tissue was significantly reduced at 48h and 72h after infection (P<0.05). In the gill and kidney tissues, there was no significant difference in the expression level of EGFRX2 gene before and after infection. In the liver and intestinal tissues, the expression level of EGFRX2 gene was significantly increased at 24h after infection (P<0.01). Figure 2 3. Changes in tissue expression of EGFRX2 gene in Oreochromis niloticus after stimulation by Poly I:C

[0090] Changes in the expression level of EGFRX2 gene in each tissue of Oreochromis niloticus after stimulation by Poly I:C were shown in Figure 3. In the kidney tissue, there was no significant difference in the expression of EGFRX2 at each time point. In the intestinal tissue, Poly I:C could significantly promote the up-regulation of EGFRX2 gene expression at 8h after stimulation, but the expression level showed a downward trend, and there was no significant difference compared with the expression level at 0h. In the gill tissue, the expression level of EGFRX2 showed an upward trend at 8h, a downward trend at 24-48h, and an upward trend at 72h. The expression levels at 8h and 72h were significantly different from those in the control group (0h) (P<0.05). In the spleen tissue, the change trend of the expression level of EGFRX2 gene was similar to that in the gill tissue.

[0091] Figure 3 4. Western blot verification of EGFRX2 polyclonal antibody

[0092] The results of SDS-PAGE electrophoresis showed that the recombinant expression protein of EGFRX2 was located at 62kDa, which was consistent with the expected expression protein size. The results of Western blot of the recombinant antigen showed that the primary antibody (EGFRX2 polyclonal antibody) at a concentration of 2μg / mL had a band at 62kD, which verified the specificity of the EGFRX2 polyclonal antibody and was suitable for subsequent detection of EGFRX2 related detection.

[0093]

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

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

[0096] Immunofluorescence detection found that in the healthy intestinal tract of tilapia, EGFRX2 gene was positively expressed in the serosa layer, muscle layer, submucosa layer and mucosa layer Figure 5 ), and the positive area ratio and distribution density of EGFRX2 protein in the serosa layer and muscle layer were significantly different (P < 0.05), among which the positive area ratio (58.11%) and distribution density (0.0658) of the muscle layer were larger, the positive area ratio of the serosa layer was 32.94%, and the distribution density was 0.0393. In addition, there was no significant difference in the distribution density and positive area ratio of EGFRX2 protein in the submucosa layer and mucosa layer of the healthy intestinal tract of tilapia.

[0097] After artificial infection with S. agalactiae, the distribution density and positive area ratio of EGFRX2 protein in the serosa layer of the intestinal tract of tilapia showed a significant upward trend (P < 0.05) compared with the control group. However, there was no significant change in the distribution density and area ratio of EGFRX2 protein in the muscle layer, submucosa layer and mucosa layer Figure 5 . The results showed that the serosa layer EGFRX2 protein played an important role in the process of tilapia responding to S. agalactiae infection.

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

[0099] Immunofluorescence detection found that EGFRX2 protein was expressed in each cell of the intestinal tract tissue of tilapia in the control group and 24 hours after challenge, as shown in Figure 6 . Among them, the columnar epithelial cells, goblet cells, lymphocytes in the mucosa layer, nerve cells, lymphocytes in the submucosa layer, muscle cells, nerve cells in the muscle layer, etc. were expressed before and after challenge, among which the goblet cells in the mucosa layer of the intestinal tract tissue of fish after challenge increased significantly, 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 EGFRX2 gene anti-S. agalactiae disease related SNP markers

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

[0102] The application screens SNPs markers in EGFRX2 gene related to the resistance of tilapia to S. agalactiae, as follows: The application amplifies the 21st and 27th intron regions of EGFRX2 gene by taking the tail fin DNA of Nile tilapia as a template. The sequence of EGFRX2 of tilapia is from Nile tilapia O_niloticus_UMD_NMBU reference genome with GenBank accession number XM_025900552.1. The SNP sites are confirmed by sequencing peak map, and it is found that there is one SNP in each of the 21st and 27th introns, which are located at 18th chromosome 19918668bp (LG18_8668) and 18th chromosome 19925361bp (LG18_5361) respectively. LG18_8668 is C / A mutation ( Figure 7 A), and LG18_5361 is A / G mutation ( Figure 7 B).

[0103] (2) Screening of SNPs markers in EGFRX2 gene related to the resistance of tilapia to S. agalactiae and KASP typing verification

[0104] The application verifies the correlation of the above screened SNPs markers in EGFRX2 gene related to the resistance of tilapia to S. agalactiae, as follows:

[0105] To further verify the relationship between the above two SNPs and the resistance of tilapia to S. agalactiae, KASP typing primers are designed based on the above two SNPs. LG18-5361 and LG18-8668 are successfully typed ( Figure 8 ).

[0106] The tail fin DNA of 79 resistant individuals and 36 sensitive individuals in the expanded population is typed. The results show that the individuals to be tested are clearly divided into three genotypes. Among them, 113 (98.26%) tail fish are successfully divided into AA, AG and GG genotypes at LG18-5361 site (Table 4). Chi-square test finds that the genotype frequency and allele frequency of LG18-5361 are significantly related to the resistance to S. agalactiae. Among them, AA genotype is significantly related to the resistance to S. agalactiae, and GG genotype is significantly related to the susceptibility to S. agalactiae (Table 4). In addition, 113 (98.26%) tail fish are successfully divided into AA, AC and CC genotypes at LG18-8668 site (Table 4). Chi-square test finds that the genotype frequency and allele frequency of LG18-8668 are not related to the resistance to S. agalactiae.

[0107] Table 4 Statistical analysis of the correlation of SNPs in EGFRX2 gene of tilapia in the population resistant to S. agalactiae and the susceptible population

[0108]

[0109] (3) Association analysis of LG18-8668 and LG18-5361 haplotype combination with resistance to Streptococcus agalactiae in Nile tilapia

[0110] The present application analyzes the association of different SNP combinations into haplotypes with resistance to Streptococcus agalactiae in Nile tilapia, as follows:

[0111] According to the data in Table 4, three pairs of alleles of LG18-8668 and LG18-5361 were paired with each other, respectively, and 111 individuals (total number of individuals) were combined into D1-D9 haplotypes. At the same time, the number of resistant and susceptible individuals of each haplotype was accumulated to form the number of resistant / susceptible individuals corresponding to each haplotype (Table 5). First, the survival rate of fish corresponding to each haplotype was calculated. The results showed that there was a large difference in survival rate among haplotypes (0.1-0.21) (Table 5). Among them, the survival rate of LG18-8668: AA / LG18-5361: AA (AA / AA) haplotype composed of D1 was the highest (Table 5). Therefore, the present study compared the difference in resistant / susceptible phenotype between D1 and the other eight haplotypes by chi-square test. The results showed that the number of resistant individuals of D1 was significantly more than that of D3, D4 and D6 (Table 5). This result indicated that D1 was the dominant haplotype for resistance to Streptococcus agalactiae in Nile tilapia, while D3, D4 and D6 were inferior genotypes. In breeding, fish with D1 haplotype should be selected as much as possible, and fish with D3, D4 and D6 haplotype should be removed.

[0112] Table 5 Association analysis of haplotypes composed of LG18-8668 and LG18-5361 with resistance to Streptococcus agalactiae

[0113]

[0114] Note: NA indicates that the chi-square approximation may not be valid due to the small expected count.

[0115] The above only describes the preferred embodiments of the present application and should not be used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A detection kit for the resistance of rohu to Streptococcus agalactiae disease, characterized by, The kit components comprise nucleotide sequences such as primers shown in SEQ ID NO: 9-11; the detection method of the kit comprises the following steps: S1, extracting the genome DNA of the to-be-tested tilapia; S2, detecting the genotype of the SNP-1 site in the genome; the SNP-1 site is located at 19925361 bp of the 18th chromosome of the reference Nile tilapia O_niloticus_UMD_NMBU genome version, the reference base is A, and the mutant base is G; S3, when the genotype of the SNP-1 site is detected as GG, it is determined that the genotype is a disadvantageous resistance genotype, and the to-be-tested tilapia is a Streptococcus agalactiae susceptible individual; when the genotype of the site is detected as AA, it is determined that the genotype is a dominant resistance genotype, and the to-be-tested tilapia is a Streptococcus agalactiae resistant individual.

2. The test kit of claim 1, characterized in that, The kit components further comprise 2x PCR premix.

3. Use of a test kit according to claim 1 or 2, characterised in that, The kit is used in any of the following applications: (1) identifying the resistance of tilapia to Streptococcus agalactiae; (2) breeding tilapia resistant to Streptococcus agalactiae.

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