SNP site combination for identifying streptococcicosis resistance of tilapia mossambica and application of SNP site combination

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) site combination for identifying streptococcicosis resistance of tilapia and application of the SNP site combination. DNAs of tilapia tail fin tissues attacked by streptococcus agalactiae are re-sequenced, resistant and sensitive individuals of a tilapia population are screened, GWAS analysis is carried out, SNPs sites related to tilapia streptococcus agalactiae disease resistance are identified, functional gene annotation and enrichment analysis are carried out, two SNP sites LG1320881657 and LG1320548718 are screened, and the SNP sites related to tilapia streptococcus agalactiae disease resistance are obtained. The GG genotype of the LG1320881657 and the TT genotype of the LG1320548718 are the dominant genotypes related to the streptococcus agalactiae disease resistance of the tilapia mossambica. The tilapia mossambica resistant to streptococcosis is successfully screened by utilizing the two SNPs sites, effective SNPs are provided for developing tilapia mossambica anti-streptococcosis molecular marker-assisted breeding, excellent characters of parents are ensured, and the breeding efficiency is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a SNP site combination for identifying tilapia streptococcal disease resistance and an application thereof. Background Art

[0002] Tilapia is one of the main freshwater fish species in my country. In 2023, tilapia production accounted for 6.76% of my country's total freshwater fish production, second only to major aquatic products such as grass carp, silver carp, bighead carp, carp and crucian carp. With the expansion of aquaculture scale and density, the impact of streptococcal disease caused by Streptococcus agalactiae on tilapia in my country is particularly prominent.

[0003] In recent years, with the development of genetic research on tilapia streptococcal disease resistance, it was found that tilapia streptococcal disease resistance has significant additive genetic variation, and its heritability is between 0.11 ± 0.02 and 0.52 ± 0.12. In family selection, selective breeding based on sensitivity or resistance can reduce the risk of death of Nile tilapia due to streptococcal disease by 54-65%. These results show that it is feasible to improve tilapia streptococcal disease resistance through selective breeding to reduce the harm of streptococcal disease.

[0004] In recent years, molecular marker-assisted breeding based on family (or natural population) has been gradually applied to the improvement of high-quality traits and breeding of new varieties of various aquatic economic animals due to its more efficient selection scheme and shortened breeding process. Especially for the selection of disease resistance, molecular marker-assisted breeding will reduce the consumption of breeding material quality due to individual pathogen challenges. However, the genetic basis of tilapia's resistance to streptococcal disease is complex. Population type, population size, analysis model, etc. will affect the accuracy of identification of molecular markers related to tilapia's resistance to streptococcal disease, resulting in genetic positioning analysis based on Best Linear Unbiased Prediction (BLUP) and Genome-wide association analysis (GWAS). There are problems such as diverse molecular markers related to disease resistance, low explanatory power of the identified molecular markers for tilapia disease resistance, and few effective molecular markers. Therefore, it is still necessary to accelerate the mining of candidate molecular markers for this trait by expanding the population size and developing diversified detection and verification methods.

[0005] Therefore, there is an urgent need to expand the genetic variation sites and regulatory genes of tilapia streptococcal disease resistance traits, which will help in the development of molecular markers for tilapia streptococcal disease resistance and marker-assisted selection, and is of great significance for the selective breeding of tilapia resistant to streptococcal disease. Summary of the invention

[0006] In order to solve the problem that there are few effective molecular markers for tilapia resistance to streptococcus disease, resulting in poor selection effect of tilapia resistance to streptococcus disease, the present invention provides a SNP site combination for identifying tilapia resistance to streptococcus disease and application thereof.

[0007] According to a first aspect of the present invention, a SNP site combination for identifying resistance to streptococcal disease in tilapia is provided, the SNP site combination comprising two SNP sites, the positions and base information of the two SNP sites on the chromosome are as follows:

[0008] SNP site name chromosome Location on chromosome Reference / mutant base LG13_20881657 NC_031978.2 20881657 T / G LG13_20548718 NC_031978.2 20548718 T / A .

[0009] The present invention uses Nile tilapia challenged with Streptococcus agalactiae as the research object, extracts DNA from its caudal fin tissue and performs resequencing, and performs genome-wide association analysis (GWAS) on the resistant (survival) and sensitive (death) phenotypes to identify molecular markers (SNPs sites) associated with tilapia resistance to Streptococcus agalactiae disease, and performs functional gene annotation on the identified molecular markers through bioinformatics methods, and simultaneously performs enrichment analysis on the genes annotated to the SNPs sites using GO and KEGG to clarify their relationship with immune and inflammatory pathways. Two candidate SNPs sites LG13_20881657 and LG13_20548718 related to tilapia resistance to streptococcal disease were screened out by the above method. Both SNPs sites are located on chromosome 13. These two candidate SNPs can be used in the identification and screening of tilapia resistance to streptococcal disease, and tilapia resistant to streptococcal disease can be successfully screened out, providing effective SNPs for molecular marker-assisted breeding of tilapia resistance to streptococcal disease, ensuring the disease-resistant advantage of the parents, and improving breeding efficiency.

[0010] Preferably, the dominant genotype of LG13_20881657 against Streptococcus agalactiae is GG, and the dominant genotype of LG13_20548718 against Streptococcus agalactiae is TT.

[0011] Relevant experiments have proved that the GG genotype corresponding to the SNP site at the 20881657th base (LG13_20881657 for short) on chromosome 13 of tilapia (NC_031978.2) and the TT genotype corresponding to the SNP site at the 20548718th base (LG13_20548718 for short) are the dominant genotypes associated with the resistance to Streptococcus agalactiae in tilapia. The dominant genotypes of these two SNPs sites provide theoretical support for the screening of tilapia resistance to streptococcal disease and the breeding of tilapia against streptococcal disease.

[0012] Preferably, the above SNP locus combination further includes 7 SNP loci, and the positions and base information of these 7 SNP loci on the chromosome are as follows:

[0013] SNP site name chromosome Location on chromosome Reference / mutant base LG13_20910218 NC_031978.2 20910218 G / A LG13_20884023 NC_031978.2 20884023 C / T LG19_245783 NC_031983.2 245783 G / A LG22_566327 NC_031985.2 566327 G / T LG13_20863685 NC_031978.2 20863685 C / G LG17_25373146 NC_031981.2 25373146 C / A LG17_25373231 NC_031981.2 25373231 T / C 。

[0014] In addition to screening out the above 2 candidate SNPs loci related to the resistance of tilapia to streptococcosis, the present invention also screens out another 7 candidate SNPs loci significantly related to the resistance of tilapia to streptococcosis, namely LG13_20910218, LG13_20884023, LG19_245783, LG22_566327, LG13_20863685, LG17_25373146 and LG17_25373231. These 7 candidate SNPs loci are mainly located on chromosomes 13, 17, 19 and 22 (LG), and can also be applied to the identification and screening of tilapia resistant to streptococcosis. After combining with the above 2 verified candidate SNPs loci, they can more comprehensively identify and screen tilapia resistant to streptococcosis.

[0015] Preferably, the SNP locus combination further includes 787 SNP loci, and the positions and base information of these 787 SNP loci on the chromosome are shown in Table 3.

[0016] In addition to screening out the above 9 relatively large-effect candidate SNPs loci related to the resistance of tilapia to streptococcosis (p<2.50E-06) (hereinafter referred to as large-effect SNPs), the present invention also screens out 787 relatively small-effect candidate SNPs loci related to the resistance of tilapia to streptococcosis (2.50E-06<p<1.00E-04, hereinafter referred to as small-effect SNPs). These small-effect candidate SNPs loci can also be applied to the identification and screening of the resistance of tilapia to streptococcosis. After combining with the above 9 large-effect candidate SNPs loci, they can more comprehensively identify and screen tilapia resistant to streptococcosis.

[0017] According to the second aspect of the present invention, there is provided an application of the above SNP locus combination for identifying the resistance of tilapia to streptococcosis in the preparation of a reagent or a liquid-phase chip for identifying the resistance of tilapia to streptococcosis.

[0018] According to the third aspect of the present invention, there is provided a genomic liquid-phase chip for identifying the resistance of tilapia to streptococcosis, which genomic liquid-phase chip includes a probe combination for identifying the above SNP locus combination for identifying the resistance of tilapia to streptococcosis.

[0019] According to a fourth aspect of the present invention, a KASP primer set for identifying resistance to streptococcal disease in tilapia is provided, the KASP primer set comprising a first KASP primer pair and a second KASP primer pair; the first KASP primer pair comprises an upstream primer A and an upstream primer B, the nucleotide sequence of the upstream primer A is shown in SEQ ID NO: 1, and the nucleotide sequence of the upstream primer B is shown in SEQ ID NO: 2; the second KASP primer pair comprises an upstream primer C and an upstream primer D, the nucleotide sequence of the upstream primer C is shown in SEQ ID NO: 3, and the nucleotide sequence of the upstream primer D is shown in SEQ ID NO: 4.

[0020] Based on two candidate SNPs sites LG13_20881657 and LG13_20548718 related to the streptococcal disease resistance of tilapia screened out by the present invention, KASP primers are developed for the two candidate SNPs sites by using a competitive allele-specific PCR (KASP) method, and a first KASP primer pair for detecting LG13_20881657 and a second KASP primer pair for detecting LG13_20548718 are obtained. Both the first KASP primer pair and the second KASP primer pair contain two upstream primers. SNP genotyping of tilapia is performed by using a primer set containing the first KASP primer pair and the second KASP primer, and a superior genotype of the streptococcal disease resistance of tilapia can be successfully screened out, which is of great significance to molecular marker-assisted breeding of tilapia against streptococcal disease.

[0021] Preferably, the first KASP primer pair further includes a universal reverse primer E, and the nucleotide sequence of the universal reverse primer E is shown in SEQ ID NO:5.

[0022] Preferably, the second KASP primer pair further includes a universal reverse primer F, and the nucleotide sequence of the universal reverse primer F is shown in SEQ ID NO:6.

[0023] According to a fifth aspect of the present invention, there is provided a use of the above-mentioned KASP primer set for identifying resistance to streptococcal disease in tilapia in identification of resistance to streptococcal disease in tilapia or in typing detection of tilapia.

[0024] According to a sixth aspect of the present invention, a kit for identifying resistance to streptococcal disease in tilapia is provided, the kit comprising a reagent for detecting the above-mentioned SNP site combination for identifying resistance to streptococcal disease in tilapia or the above-mentioned KASP primer set for identifying resistance to streptococcal disease in tilapia.

[0025] According to a seventh aspect of the present invention, there is provided a method for identifying resistance to streptococcal disease in tilapia, comprising the following steps:

[0026] S1. Obtain genomic DNA of tilapia samples;

[0027] S2. Detecting genomic DNA using the above-mentioned genomic liquid phase chip for identifying tilapia streptococcal disease resistance or the above-mentioned KASP primer set for identifying tilapia streptococcal disease resistance to obtain raw data;

[0028] S3. Analyze the original data and obtain the results of the resistance of different tilapia to Streptococcus agalactiae disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the SNP density map of the resequencing of the tilapia after infection in Example 1 and the quality control of the resequencing data.

[0030] Figure 2 This is a graph showing the results of analyzing the population structure of tilapia using PCA in Example 1.

[0031] Figure 3 This is a diagram showing the results of identifying candidate molecular markers for tilapia resistance to Streptococcus agalactiae disease using GWAS analysis in Example 2.

[0032] Figure 4 This is a graph showing the results of annotating the functional genes of small-effect SNPs sites using functional enrichment analysis in Example 3.

[0033] Figure 5 The haplotype analysis results of SNPs (p<2.50E-06) associated with resistance to Streptococcus agalactiae in tilapia are provided for Example 4.

[0034] Figure 6 This is the result of SNP genotyping of 234 tilapia samples using KASP typing primers in Example 4. DETAILED DESCRIPTION

[0035] The following is a further clear and complete description of the technical features in the technical solution provided by the present invention in conjunction with the specific implementation methods. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0036] Example 1

[0037] This embodiment uses healthy Nile tilapia GIFT strain (Oreochromis niloticus, GIFT) as the experimental object, which comes from the Guangdong Province Tilapia breeding farm, with an average body weight of 150±10g, and has been electronically marked with a passive integrated transponder (PIT), temporarily kept in an experimental water tank (length×width×height=2.5m×1.0m×0.8m) for standby, during which the water temperature is maintained at 31°C±0.5°C, and fed twice a day, with a feeding amount of about 3% of body weight, and conventional feeding management. The experimental site is the experimental base of the Pearl River Fisheries Research Institute of the Chinese Academy of Fishery Sciences.

[0038] Tilapia-derived Streptococcus agalactiae WC1535 was thawed and plated on brain-heart infusion broth (BHI) solid medium at 37°C overnight. Two monoclonal colonies were picked and cultured overnight in BHI liquid medium at 37°C (grow to logarithmic phase). Centrifugation was performed and washed three times with sterile PBS, each time at 7000 rpm / min for 5 minutes. The concentration of the washed bacterial solution was then measured using an electronic turbidimeter DensiCHEK Plus (BioMerieux, Shanghai, China), and the bacterial solution concentration was diluted to 1.03×10 with sterile PBS buffer. 7 CFU / mL (obtained from the preliminary experiment), and the Streptococcus agalactiae liquid was obtained.

[0039] Before the challenge, the tail fin tissue of tilapia was collected and completely immersed in anhydrous ethanol, and stored at -20℃ for a long time for DNA extraction. After the tail fin was collected, the tilapia group was placed in an experimental water tank for two weeks, and the water temperature was kept at 31℃±1℃. Then, during the challenge, the Streptococcus agalactiae liquid was injected into the abdominal cavity of tilapia at a dosage of 100μL per fish by intraperitoneal injection (challenge). After the injection, other breeding conditions remained unchanged, and phenotypic records were continuously observed. Individuals that died within 7 days (day, d) after infection were defined as streptococcal sensitive individuals, and individuals that survived 14d after infection were defined as resistant individuals, and sensitive and resistant phenotypes were counted. After the challenge, the number of streptococcal sensitive individuals within the first 7 days after the challenge was continuously recorded. And on the 14th day after the challenge, the number of resistant individuals was counted. The binary phenotype recording method of anti-streptococcal disease: "0" represents sensitive individuals and "1" represents resistant individuals. The resistance / sensitive phenotype data are used for GWAS.

[0040] Furthermore, the collected tilapia tail fin tissue was sent to Beijing Compson Agricultural Technology Co., Ltd. for DNA extraction. After passing the quality inspection, it was resequenced. The sequencing platform was DNBseq manufactured by BGI, the sequencing read length (reads) was 150bp, and the average sequencing depth of each sample was about 5×.

[0041] The raw sequencing data were quality controlled using fastqc under default parameters, and the quality-controlled raw data were aligned to the Nile tilapia reference genome O_niloticus_UMD_NMBU (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_001858045.2 / ) using bwa (v0.7.17) under default parameters. Then, samtools was used to sort and index the aligned BAM files. After that, SNP identification used the HaplotypeCaller parameters of GATK to generate gVCF files for each sample, and then integrated into a single VCF file using the GenomicsDBImport and GenotypeGVCFs parameters.

[0042] The VCF file was filtered by SNP using plink 1.9, and the quality control parameters were: --geno 0.1, --maf 0.05, --hwe0.000001; the quality-controlled VCF file was used by vcftools to calculate the number of SNPs in every 100kb SNP according to the position of the SNP on the chromosome, with a step length of 10kb, and to draw the SNP density distribution curve; GWAS analysis does not allow missing sites, and for individual sites with undetected SNPs, Beagle (v5.2) was used to fill in the default parameters; the filled VCF file was converted into a plink format file, and GCTA was used for population structure analysis and heritability analysis; first, the GRM matrix was generated using the --make-grm parameter. The generated matrix was used to calculate the principal component analysis (PCA) and heritability under the --grm snp.gcta and --grm qc parameters, respectively.

[0043] The model used in this GWAS analysis was a mixed linear model.

[0044] y=Xβ+Z K γ K +ξ+e

[0045] y is the phenotype vector, Xβ is the population structure fixed effect, Z k γ k is the marker effect to be tested, ξ~N(0,KΦ 2 ) is a polygenic effect, e~N(0,Iσ 2 ) is the residual effect. K in the polygenic effect is the kinship matrix inferred by the markers.

[0046] The analysis software was GEMMA (v0.98.5). The software parameters were -miss 1, -maf 0, -r2 1, -hwe 0, -cgemma_cov.txt. Among them, gemma_cov.txt is the calculation results of the first three PCs in the above PCA results, which were added as covariates in the GWAS analysis. The number of SNPs was obtained by removing redundancy using --indep-pairwise 100 10 0.2 of PLINK 1.9. In addition, because the calculation threshold of the Bonferroni method (0.05 / number of SNPs) was too strict, the significance threshold was calculated by 1 / number of SNPs.

[0047] Table 1 Genetic parameter estimation results of tilapia disease resistance phenotype based on SNPs

[0048] Genetic parameters Coefficient of variation Standard error Additive variance components 0.180889 0.181752 Residual variance components 0.06798 0.168824 Phenotypic variance components 0.248868 0.057065 Heritability 0.726845 0.68537

[0049] The results of genetic parameter estimation of sensitive / resistant phenotype of tilapia based on whole genome SNPs after infection are shown in Table 1. As shown in Table 1, after tilapia was infected with Streptococcus agalactiae, 20 sensitive and resistant individuals were selected, and the heritability of sensitive / resistant phenotype was 0.727, which had medium and high heritability.

[0050] At the same time, the whole genome resequencing of the tail fin DNA of the above 40 tilapia infected with the virus was performed. The SNP density results after quality control and SNP calling of the original data are as follows: Figure 1 As shown. Figure 1 It can be seen that after resequencing and quality control, a total of 6,592,274 (58.51%) SNP sites were retained in the tilapia population, the average length of the SNP distribution distance was 0.142 kb, and SNPs were most distributed on chromosome 3 with the highest density, followed by chromosome 7 and chromosome 23 with a higher distribution density.

[0051] The results of PCA analysis on the population structure of tilapia are as follows: Figure 2 As shown, the PCA analysis using the first three principal components showed that the variances of the first principal component PC1, the second principal component PC2 and the third principal component PC3 were 18.87%, 10.52% and 9.83%, respectively, indicating that most samples of the tilapia population were uniformly distributed, but there were 9 outlier samples, indicating the existence of population stratification. Therefore, in the whole genome association analysis, the first three principal components were used as covariates and applied to the linear mixed model to explain the population stratification.

[0052] Example 2 Screening of SNP loci combinations for identifying resistance to streptococcal disease in tilapia

[0053] Based on Example 1, this example conducts a GWAS analysis on the trait of resistance to Streptococcus agalactiae. The identification results of candidate molecular markers for the trait of resistance to Streptococcus agalactiae in tilapia are as follows Figure 3 shown. In the GWAS analysis of the sensitive / resistant phenotype, the major-effect SNP loci are shown in Table 2, and the minor-effect SNP loci are shown in Table 3. Among them, Figure 3 the Manhattan plot in a shows the distribution of SNPs significantly associated with Streptococcus agalactiae resistance in tilapia on chromosomes, Figure 3 the quantile-quantile plot in b shows the probability distribution of the expected p-values and observed p-values of SNPs, Figure 3 and the pie chart in c shows the distribution ratio of minor-effect candidate SNPs on each chromosome.

[0054] Table 2 Nine major-effect SNP loci in the GWAS analysis of the binary phenotype of resistance to Streptococcus agalactiae in tilapia

[0055]

[0056] From Figure 3 , Table 2 and Table 3, it can be seen that based on the mixed linear model of the sensitive / resistant phenotype, nine major-effect SNPs (p < 2.50E-06) and 787 minor-effect candidate SNPs (2.50E-06 < p < 1.00E-04) associated with Streptococcus agalactiae resistance in tilapia were discovered. These are the SNP locus combinations used to identify Streptococcus agalactiae resistance in tilapia. Moreover, among the major-effect SNPs, 5 SNPs are distributed on chromosome 13, 2 SNPs are distributed on chromosome 17, and 1 SNP is distributed on each of chromosomes 19 and 22. The minor-effect SNPs are concentrated on chromosomes 17, 1, and 13. The contributions of the major-effect SNPs and minor-effect SNPs to the variance of the resistant / sensitive phenotype are 0.51 ± 0.042 and 0.40 ± 0.003, respectively.

[0057] Table 3 Seven hundred and eighty-seven minor-effect SNP loci in the GWAS analysis of the sensitive / resistant phenotype of resistance to Streptococcus agalactiae in tilapia

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069] In order to more clearly understand the chromosome information of the 9 large-effect SNPs sites shown in Table 2 and the 787 small-effect SNPs sites shown in Table 3, the chromosome information of the above SNPs sites is summarized in Table 4.

[0070] Table 4 Chromosome information of the 9 large-effect SNPs and the 787 small-effect SNPs shown in Table 3

[0071] chromosome Chromosome name Chromosomes of Tilapia Chromosome length (bp) NC_031965.2 LG1 chromosome 1 40,673,430 NC_031966.2 LG2 chromosome 2 36,523,203 NC_031969.2 LG4 chromosome 4 35,549,522 NC_031970.2 LG5 chromosome 5 39,714,817 NC_031971.2 LG6 chromosome 6 42,433,576 NC_031972.2 LG7 chromosome 7 64,772,279 NC_031973.2 LG8 chromosome 8 30,527,416 NC_031974.2 LG9 chromosome 9 35,850,837 NC_031975.2 LG10 chromosome 10 34,704,454 NC_031976.2 LG11 chromosome 11 39,275,952 NC_031977.2 LG12 chromosome 12 38,600,464 NC_031978.2 LG13 chromosome 13 34,734,273 NC_031979.2 LG14 chromosome 14 40,509,636 NC_031980.2 LG15 chromosome 15 39,688,505 NC_031987.2 LG16 chromosome 16 36,041,493 NC_031981.2 LG17 chromosome 17 38,839,487 NC_031982.2 LG18 chromosome 18 38,636,442 NC_031983.2 LG19 chromosome 19 30,963,196 NC_031984.2 LG20 chromosome 20 37,140,374 NC_031985.2 LG22 chromosome 22 39,199,643 NC_031986.2 LG23 chromosome 23 45,655,644 NC_031967.2 LG3 chromosome 3 87,567,345

[0072] Example 3

[0073] The purpose of this example is to perform functional gene annotation on the 9 large-effect SNPs and 787 small-effect SNPs sites associated with tilapia resistance to Streptococcus agalactiae disease screened in Example 2. Specifically, using annnovar (v 4.7) software, with O_niloticus_UMD_NMBU as the reference genome, the annotate_variation.pl function is based on the SNP region (-regionanno) to annotate the position of the coding gene within the 100kb range of the linkage disequilibrium (LD) attenuation distance upstream and downstream of the SNP site in the candidate gene obtained by association analysis.

[0074] Genes annotated with large-effect SNPs were artificially annotated with gene functions. GO and KEGG enrichment analysis were performed on the candidate gene sets annotated with small-effect SNPs at the same time. Among them, all coding genes in tilapia O_niloticus_UMD_NMBU were annotated with EGGNOG (http: / / eggnog-mapper.embl.de / ) to form a background annotation set. Then, GO and KEGG enrichment of candidate genes under this background annotation set were analyzed on the OmicShare platform.

[0075] Table 5 Gene annotation results of large effect SNPs

[0076]

[0077] The gene annotation results of the 9 large-effect SNPs are shown in Table 5. As shown in Table 5, a total of 5 candidate genes were annotated to the large-effect SNPs, but none of them were located in the exon region of the gene. Among them, chromosome 13 LG13_20881657, LG13_20910218 and LG13_20884023 were annotated to the WDR27 gene, LG13_20863685 and LG13_20548718 were annotated to the THBS2 gene and COL19A1 gene, respectively, chromosome 17 LG17_25373146 and LG17_25373231 were annotated to the new gene LOC109195059, and chromosome 22 LG22_566327 was annotated to the gene LOC112841710 (class I histocompatibility antigen, F10 alpha chain).

[0078] Moreover, the significant SNPs on chromosome LG13 are located in the intronic regions of WDR27, THBS2, and COL19A1. WDR27 belongs to the WD-repeat sequence (WDR) family. Although there are few reports on this family in bacterial diseases, many of its members have been used as candidate target genes for a variety of tumor suppressor drugs and are involved in the regulation of the tumor immune microenvironment. For example, WDR4 has been reported to upregulate the chromatin accessibility of TNFα, increase the number of myeloid-derived suppressor cells in tumors, and reduce CD 8 + T cell infiltration promotes the progression of hepatocellular carcinoma. Conversely, TNFα recruits NF-κB to activate the transcription of WDR 6, establishing a WDR6-TNFα feedback loop. THBS2 belongs to the thrombopoietin family and is related to immune cell infiltration and immune regulation. Methylation of its promoter region is considered to be a prognostic marker for diseases such as endometrial adenocarcinoma and ovarian cancer. In fish, this gene was found to play an important role in the immune response of flounder against lymphocystic disease virus. The COL19A1 gene is an extracellular matrix protein. Highly expressed COL19A1 gene is associated with the infiltration of immune effector cells (memory B cells, immature B cells and activated B cells). The significant SNP of LG22 is located in the intron region of LOC112841710. This gene encodes class I histocompatibility antigen, F10α chain, and is involved in presenting foreign antigens to the immune system. The significant associated SNPs on LG17 are annotated to the new gene LOC109195059, and its biological function needs further study.

[0079] The functional gene annotation results of the above 9 large-effect SNPs sites showed that the 9 candidate SNPs identified in Example 2 were related to the resistance of tilapia to streptococcal disease.

[0080] The functional gene enrichment results of 787 small effect SNPs are as follows Figure 4As shown, Figure 4 a, b represent GO and KEGG enrichment analysis of annotated genes at small effect SNPs sites, respectively. Figure 4 It can be seen that a total of 356 genes were annotated to the small-effect SNPs sites. The GO enrichment results showed that the most significantly enriched biological function of the genes annotated to the small-effect SNPs sites was the urokinase plasminogen activator signaling pathway, which was significantly correlated with the biological functions related to synapse assembly. This pathway regulates NF-κB activation and cytokine expression in macrophages ( Figure 4 a), while the top 5 enriched pathways included cyanamine metabolism involved in the immune system and bacterial infection, glycosaminoglycan degradation involved in inflammatory response and the formation of tumor microenvironment, Ras signaling pathway associated with anti-inflammatory response downregulation, arachidonic acid metabolism associated with pro-inflammatory response and inducing oxidative stress and stimulating immune response, and taurine and hypotaurine metabolism that can alleviate Helicobacter pylori-induced apoptosis ( Figure 4 b).

[0081] The functional gene annotation results of the above 787 small-effect SNPs sites showed that the small-effect SNPs sites identified in Example 2 were related to the resistance of tilapia to streptococcal disease.

[0082] Example 4

[0083] In order to further verify the relationship between the 9 large-effect SNPs sites and 787 small-effect SNPs identified in Example 2 and the resistance to streptococcal disease in tilapia, this example takes the large-effect SNPs site as an example, and first performs haplotype analysis on the large-effect SNPs site. Specifically, plink 1.9--recode HV is used to extract 100 kb upstream and downstream of the large-effect SNPs site from the VCF file, and a linkage format file is generated. Then, Haploview software is used to perform LD attenuation and haplotype analysis on the generated linkage file, and the sites linked to the large-effect SNPs are taken as a collection, and the Tagger function is continued to generate representative SNPs in the haplotype corresponding to each large-effect SNP.

[0084] The haplotype analysis results of large effect SNPs are as follows Figure 5 As shown, Figure 5 a, b, and c show the haplotype analysis results of LG13_20881657, LG13_20863685 and LG17_25373146, LG17_25373231 loci, respectively. The inverted triangle in the figure is the block where the target SNP is located (the polymorphic site in this haplotype block is a region of complete linkage disequilibrium).

[0085] Combining Table 5 and Figure 5 It can be seen that LG13_20881657, LG13_20863685 and LG17_25373146 have linkage disequilibrium regions and serve as core SNPs in their corresponding block regions. On this basis, this embodiment develops KASP primers for candidate molecular markers LG13_20881657 (L1657) and LG13_20548718 (L8718) based on the KASP method.

[0086] Table 6 KASP molecular marker typing primer sequences

[0087]

[0088]

[0089] Note: The first 15 bases of primer_X and primer_Y (italic part) are the linker sequences. Primer_X is used to detect the base site in the reference genome corresponding to the SNP, and primer_Y is used to detect the base site after the SNP mutation.

[0090] Before using the KASP primers shown in Table 6 to perform KASP marker typing on tilapia, 300 Nile tilapia were challenged with Streptococcus agalactiae, and the tail fin DNA of 133 resistant individuals and 101 sensitive individuals (a total of 234 individuals) were subjected to KASP molecular marker typing.

[0091] In order to develop a genotyping method for representative SNPs, this example takes the target SNP (LG13_20881657 and LG13_20548718) as the center, extracts the 300bp genomic sequence upstream and downstream of these two SNPs, then designs primers for PCR amplification of the 200bp genomic sequence upstream and downstream thereof, selects 10 tilapia that survived after the poisoning and 10 tilapia that died after the poisoning respectively, and performs Sanger sequencing, and compares the sequences by DNAMAN to clarify the mutation information of the upstream and downstream sequences of the target SNPs. Based on the mutation information, KASP primers were developed for candidate molecular markers, and three KASP typing primers for the two target SNPs LG13_20881657 and LG13_20548718 as shown in Table 5 were designed, and Beijing Shenggong Biotechnology Co., Ltd. was commissioned to synthesize them, and the three SNP primers were diluted to 10 μmol and mixed according to a volume ratio of 12:12:30.

[0092] Then, 234 Nile tilapia were selected from the expanded group for the challenge experiment. As mentioned above, the tail fin tissues of sensitive and resistant fish were collected, and genomic DNA was immediately extracted using the blood / cell / tissue genomic DNA extraction kit (DP304, Tiangen Biochemical Technology, Beijing) according to the instructions. The extracted DNA was quality controlled by 1% agarose electrophoresis and Nanodrop2100 (the quality inspection qualification requirements are as follows: the electrophoresis band is single and the A260 / 280 detected by Nanodrop2100 is between 1.8 and 2.0), and the quality-controlled DNA stock solution was diluted to 10-20 ng / μL with sterile double distilled water for use.

[0093] Table 7 PCR amplification reaction system

[0094]

[0095]

[0096] Based on KASP technology and with the help of LGC high-throughput genotyping system, 234 tilapia samples were subjected to SNP genotyping using KASP typing primers as shown in Table 6. The PCR reaction system involved is shown in Table 7. The PCR reaction was carried out in a high-throughput water bath system Hydrocycler. The specific procedure was as follows: 94°C pre-denaturation, 15 minutes; 94°C, 20 seconds (denaturation); 55-61°C, 1 minute (renaturation & extension); 10 cycles of amplification with touch down program, with 0.6°C lowered per cycle; 94°C, 20 seconds (denaturation); 55°C, 60 seconds, and continued amplification for 26 cycles. After amplification, the fluorescence signal was detected and the typing was checked using the BMG PHERAstar instrument, and the typing results were statistically analyzed using the SNPviewer2 software.

[0097] The results of SNP genotyping of 234 tilapia samples using KASP typing primers are shown in the figure. Figure 6 As shown, Figure 6 a, b represent the KASP typing results and statistical analysis of LG13_20881657 (L1657), respectively. Figure 6 c, d represent the KASP typing results and statistical analysis of LG13_20548718 (L8718), respectively.

[0098] Depend on Figure 6 It can be seen that the L1657 locus of 232 (99.1%) fish was successfully typed, among which the number of sensitive fish with TT genotype was more than the number of resistant fish (sensitive fish: 31; resistant fish: 23), and the number of sensitive fish with GG genotype was less than the number of resistant fish (sensitive fish: 17; resistant fish: 35) ( Figure 6 a, b); 223 (95.3%) fish were successfully typed at the L8718 locus, among which the number of sensitive fish with TT genotype was less than that of resistant fish (sensitive fish: 9; resistant fish: 27), and the number of sensitive fish with AA genotype was more than that of resistant fish (sensitive fish: 46; resistant fish: 33) ( Figure 6 c, d).

[0099] From the above KASP typing results, it can be seen that the GG genotype of L1657 and the TT genotype of L8718 are dominant resistance genotypes, and the detection success rate of the typing primers designed in this example by the KASP method is above 95%.

[0100] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. A SNP locus combination for identifying resistance to streptococcal disease in tilapia, characterized in that: The SNP site combination includes 2 SNP sites, and the positions and base information of the 2 SNP sites on the chromosome are as follows: 。 2. The SNP locus combination for identifying resistance to streptococcal disease in tilapia according to claim 1, characterized in that: The dominant genotype of LG13_20881657 against Streptococcus agalactiae is GG and / or the dominant genotype of LG13_20548718 against Streptococcus agalactiae is TT.

3. The SNP site combination for identifying resistance to streptococcal disease in tilapia according to claim 1, characterized in that: The SNP site combination also includes 7 SNP sites, and the positions and base information of the 7 SNP sites on the chromosome are as follows: 。 4. The SNP site combination for identifying resistance to streptococcal disease in tilapia according to claim 3, characterized in that: The SNP site combination also includes 787 SNP sites, and the positions and base information of the 787 SNP sites on the chromosome are as follows:

5. Use of the SNP locus combination for identifying the resistance to streptococcal disease in tilapia according to any one of claims 1 to 4 in the preparation of a reagent or liquid phase chip for identifying the resistance to streptococcal disease in tilapia.

6. A genomic liquid phase chip for identifying resistance to streptococcal disease in tilapia, characterized in that: The genomic liquid phase chip comprises a probe combination, and the probe combination is used to identify the SNP site combination for identifying the resistance to streptococcal disease in tilapia as claimed in any one of claims 1 to 4.

7. A KASP primer set for identifying resistance to streptococcal disease in tilapia, characterized in that: The KASP primer set includes a first KASP primer pair and a second KASP primer pair; The first KASP primer pair includes an upstream primer A and an upstream primer B, wherein the nucleotide sequence of the upstream primer A is shown in SEQ ID NO: 1, and the nucleotide sequence of the upstream primer B is shown in SEQ ID NO: 2; The second KASP primer pair includes an upstream primer C and an upstream primer D. The nucleotide sequence of the upstream primer C is shown in SEQ ID NO:3, and the nucleotide sequence of the upstream primer D is shown in SEQ ID NO:

4.

8. The KASP primer set for identifying the resistance of tilapia to streptococcal disease as claimed in claim 7, characterized in that: The first KASP primer pair further includes a universal reverse primer E, the nucleotide sequence of which is shown in SEQ ID NO: 5; The second KASP primer pair further includes a universal reverse primer F, and the nucleotide sequence of the universal reverse primer F is shown in SEQ ID NO:

6.

9. Use of the KASP primer set for identifying resistance to streptococcal disease in tilapia as claimed in claim 7 or 8 in identification of resistance to streptococcal disease in tilapia or typing detection of tilapia.

10. A kit for identifying resistance to streptococcal disease in tilapia, characterized in that: The kit comprises a reagent for detecting the SNP site combination for identifying the resistance to streptococcal disease in tilapia as claimed in any one of claims 1 to 4 or a KASP primer set for identifying the resistance to streptococcal disease in tilapia as claimed in any one of claims 7 to 8.

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