A SNP site combination for identifying resistance to streptococcosis of rohu and application thereof

Abstract

The application provides a SNP site combination for identifying resistance to streptococcosis of rohu and application thereof. Resequencing is performed on tail fin tissue DNA of rohu after streptococcus agalactiae attack, resistance and sensitive individuals of the rohu population are screened, GWAS analysis is performed, SNPs sites related to the resistance of rohu to streptococcosis are identified, functional gene annotation and enrichment analysis are performed, and two SNP sites LG13_20881657 and LG13_20548718 are screened out. The GG genotype of LG13_20881657 and the TT genotype of LG13_20548718 are dominant genotypes related to the resistance of rohu to streptococcosis. The two SNP sites are used to successfully screen rohu with resistance to streptococcosis, provide effective SNPs for carrying out molecular marker assisted breeding of rohu against streptococcosis, ensure excellent traits of parents, and improve breeding efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection technology, and in particular to a SNP site combination for identifying Streptococcus rofii resistance and application thereof. BACKGROUND

[0002] Tilapia is one of the main freshwater fish varieties in China. In 2023, the tilapia yield accounted for 6.76% of the total freshwater fish yield in China, ranking second only to grass, silver carp, bighead carp, carp, crucian carp and other major aquatic products. With the expansion of the scale and density of aquaculture, Streptococcus agalactiae caused Streptococcus disease has a particularly significant impact on tilapia in China.

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

[0004] In recent years, molecular marker-assisted breeding based on families (or natural populations) has been gradually applied to the improvement of high-quality traits and the cultivation of new varieties of various aquatic economic animals due to its more efficient selection scheme and reduced breeding process. Especially for disease resistance breeding, molecular marker-assisted breeding can reduce the consumption of breeding material quality due to individual pathogen challenge. However, the genetic basis of tilapia resistance to Streptococcus disease is complex. Population type, population size, and analysis model can all affect the accuracy of identifying molecular markers related to tilapia resistance to Streptococcus disease, resulting in problems such as multiple molecular markers related to disease resistance, low degree of explanation of identified molecular markers for tilapia disease resistance, and few effective molecular markers in genetic mapping analysis based on Best Linear Unbiased Prediction (BLUP) and Genome-wide association analysis (GWAS). Therefore, it is still necessary to expand the population size, develop diversified detection and verification methods, and accelerate the excavation of candidate molecular markers for this trait.

[0005] Therefore, it is urgent to expand the genetic variation sites and regulatory genes of tilapia resistance to Streptococcus disease, which will help the development of molecular markers for tilapia resistance to Streptococcus disease and marker-assisted selection, and is of great significance for the selection breeding of tilapia resistant to Streptococcus disease. SUMMARY

[0006] In order to solve the problem that there are few effective molecular markers of tilapia against streptococcosis, and the selection effect of tilapia against streptococcosis is poor, the application provides a SNP site combination for identifying the streptococcosis resistance of tilapia and application thereof.

[0007] According to a first aspect of the application, a SNP site combination for identifying the streptococcosis resistance of tilapia is provided, the SNP site combination comprises two SNP sites, and the positions and base information of the two SNP sites on the chromosome are as follows:

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

[0009] The application takes Nile tilapia after streptococcus attack as the research object, extracts DNA from the tail fin tissue, resequences, and performs genome-wide association analysis (GWAS) on the resistance (survival) and sensitivity (death) phenotypes, identifies the molecular markers (SNPs sites) related to the streptococcosis resistance of tilapia, and performs functional gene annotation on the identified molecular markers by bioinformatics methods, and performs enrichment analysis on the genes annotated by the SNPs sites by using GO and KEGG, and determines the relationship with the immune and inflammatory pathways. Two candidate SNPs sites LG13_20881657 and LG13_20548718 related to the streptococcosis resistance of tilapia are screened out by the above-mentioned mode, both of the two SNPs sites are located on the 13th chromosome, the two candidate SNPs can be applied to the identification and screening of the streptococcosis resistance of tilapia, and the tilapia with the streptococcosis resistance can be successfully screened out, which provides effective SNPs for the molecular marker assisted breeding of tilapia against streptococcosis, ensures the disease resistance dominant traits of the parents, and improves the 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] It is proved by the related experiment verification that the SNP site (abbreviated as LG13_20881657) corresponding to the 20881657th base on the 13th chromosome (NC_031978.2) of tilapia and the SNP site (abbreviated as LG13_20548718) corresponding to the 20548718th base are the dominant genotypes related to the streptococcosis resistance of tilapia, and the dominant genotypes of the two SNPs sites provide theoretical support for the screening of the streptococcosis resistance of tilapia and the breeding of tilapia against streptococcosis.

[0012] Preferably, the SNP site combination further comprises 7 SNP sites, and the positions and base information of the 7 SNP sites on the chromosome are as follows:

[0013] SNP locus name Chromosome Position 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 the two candidate SNPs sites related to the resistance to Streptococcus disease of the tilapia, the present application further screens seven other candidate SNPs sites LG13_20910218, LG13_20884023, LG19_245783, LG22_566327, LG13_20863685, LG17_25373146 and LG17_25373231 which are significantly related to the resistance to Streptococcus disease, and the seven candidate SNPs sites are mainly located on the 13th, 17th, 19th and 22nd chromosomes (LG), and can also be applied to the identification and screening of the tilapia resistant to Streptococcus disease, and in combination with the two verified candidate SNPs sites, the tilapia resistant to Streptococcus disease can be more comprehensively identified and screened.

[0015] Preferably, the SNP site combination further comprises 787 SNP sites, and the positions and base information of the 787 SNP sites on the chromosome are shown in Table 3.

[0016] In addition to the nine candidate SNPs sites with relatively large effects (p<2.50E-06) related to the resistance to Streptococcus disease of the tilapia (hereinafter referred to as large-effect SNPs), the present application further screens 787 candidate SNPs sites with relatively small effects (2.50E-06

[0017] According to the second aspect of the present application, the application of the SNP site combination for identifying the resistance to Streptococcus disease of the tilapia in the preparation of a reagent or a liquid chip for identifying the resistance to Streptococcus disease of the tilapia is provided.

[0018] According to the third aspect of the present application, a genomic liquid chip for identifying the resistance to Streptococcus disease of the tilapia is provided, and the genomic liquid chip comprises a probe combination for identifying the SNP site combination for identifying the resistance to Streptococcus disease of the tilapia.

[0019] ​According to a fourth aspect of the present application, a KASP primer set for identifying the resistance of Streptococcus agalactiae disease of 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 as SEQ ID NO: 1, and the nucleotide sequence of the upstream primer B is shown as 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 as SEQ ID NO: 3, and the nucleotide sequence of the upstream primer D is shown as SEQ ID NO: 4.

[0020] Based on the two candidate SNPs LG13_20881657 and LG13_20548718 associated with the resistance of Streptococcus agalactiae disease of Tilapia screened by the present application, KASP primer development is carried out on the two candidate SNPs by using the method of competitive allele-specific PCR (KASP), and the first KASP primer pair for detecting LG13_20881657 and the second KASP primer pair for detecting LG13_20548718 are obtained, the first KASP primer pair and the second KASP primer pair both contain two upstream primers, and the primer set containing the first KASP primer pair and the second KASP primer is used for SNP genotyping of Tilapia, which can successfully screen out the dominant genotype of Tilapia against Streptococcus agalactiae disease, and is of great significance for molecular marker assisted breeding of Tilapia against Streptococcus agalactiae disease.

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

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

[0023] According to a fifth aspect of the present application, the KASP primer set for identifying the resistance of Streptococcus agalactiae disease of Tilapia described above is applied in the identification of the resistance of Streptococcus agalactiae disease of Tilapia or the typing detection of Tilapia.

[0024] According to a sixth aspect of the present application, a kit for identifying the resistance of Streptococcus agalactiae disease of Tilapia is provided, the kit comprising reagents for detecting the SNP site combination for identifying the resistance of Streptococcus agalactiae disease of Tilapia described above or the KASP primer set for identifying the resistance of Streptococcus agalactiae disease of Tilapia described above.

[0025] According to a seventh aspect of the present application, a method for identifying the resistance of Streptococcus agalactiae disease of Tilapia is provided, comprising the following steps:

[0026] S1. Obtaining genomic DNA of the tilapia sample;

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

[0028] S3. Analyzing the raw data to obtain the results of the resistance of different tilapia to streptococcosis. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Figure 1 is a SNP density plot of resequencing data of tilapia after challenge for Example 1.

[0030] Figure 2 Figure 2 is a result plot of PCA analysis of tilapia population structure for Example 1.

[0031] Figure 3 Figure 3 is a result plot of identification of candidate molecular markers of tilapia resistance to streptococcosis by GWAS analysis for Example 2.

[0032] Figure 4 Figure 4 is a result plot of annotation of functional genes of small-effect SNP sites by functional enrichment analysis for Example 3.

[0033] Figure 5 Figure 5 is a haplotype analysis result plot of SNPs (p<2.50E-06) related to the resistance of tilapia to streptococcosis for Example 4.

[0034] Figure 6 Figure 6 is a result plot of SNP genotyping of 234 tilapia samples by KASP typing primers for Example 4. DETAILED DESCRIPTION

[0035] The technical features of the technical solutions provided by the present application will be further described clearly and completely in combination with the specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0036] Example 1

[0037] The Nile tilapia of GIFT strain (Oreochromis niloticus, GIFT) was used as the experimental object in this embodiment. The Nile tilapia was from Guangdong Tilapia Breeding Farm, with an average weight of 150±10 g, and had been passively integrated transponder (PIT) electronic marked. The Nile tilapia was temporarily raised in an experimental tank (length x width x height = 2.5 m x 1.0 m x 0.8 m) for standby, and the water temperature was kept at 31℃±0.5℃ during the period. The Nile tilapia was fed twice a day, and the feeding amount was about 3% of the body weight. The Nile tilapia was conventionally raised and managed. The experimental site was the experimental base of the Pearl River Fisheries Research Institute of the Chinese Academy of Fishery Sciences.

[0038] The Streptococcus agalactiae WC1535 from the Nile tilapia was thawed and plated on a Brain-Heart Infusion Broth (BHI) solid medium and cultured at 37℃ overnight. Two single colonies were picked and cultured in a BHI liquid medium at 37℃ overnight (grown to the logarithmic phase). The bacteria were centrifuged, washed with sterile PBS three times at a speed of 7000 rpm / min for 5 minutes each time, and then the concentration of the washed bacteria was determined by an electronic turbidimeter DensiCHEK Plus (BioMerieux, Shanghai, China). The bacteria were diluted to 1.03×10 7 CFU / mL (obtained from a pre-experiment) to obtain the Streptococcus agalactiae bacteria solution.

[0039] Before the challenge, the tail fin tissues of the Nile tilapia were collected and completely immersed in anhydrous ethanol and stored at -20℃ for a long time for DNA extraction. The Nile tilapia population after the collection of the tail fin was placed in an experimental water tank for two weeks of feeding, and the water temperature was kept at 31℃±1℃. Then, the Streptococcus agalactiae bacteria solution was injected into the abdominal cavity of the Nile tilapia (challenged) by intraperitoneal injection at a dose of 100 μL per fish. After the injection, the other breeding conditions were unchanged, and the phenotype record observation was continuously performed. The individuals that died within 7 days (d) after the infection were defined as streptococcus-sensitive individuals, and the individuals that still survived after 14 d after the infection were defined as resistant individuals. The sensitive and resistant phenotypes were counted. After the challenge, the number of streptococcus-sensitive individuals within the first 7 days after the challenge was continuously recorded. The number of resistant individuals was counted on the 14th day after the challenge. The resistance / sensitivity phenotype record method was as follows: “0” represented a sensitive individual, and “1” represented a resistant individual. The resistance / sensitivity phenotype data were used for GWAS.

[0040] Further, the collected tail fin tissues of the Nile tilapia were sent to Beijing Comfor Biotech Co., Ltd. for DNA extraction. After the quality inspection, re-sequencing was performed. The sequencing platform was DNBseq manufactured by Huada, and the read length was 150 bp. The average sequencing depth of each sample was about 5×.

[0041] The raw sequencing data was quality controlled with fastqc under default parameters, and the quality controlled raw data was aligned to the Nile tilapia reference genome O_niloticus_UMD_NMBU (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_001858045.2 / ) with bwa (v0.7.17) under default parameters, then, the aligned BAM files were sorted and indexed with samtools, after that, SNP calling was performed with GATK's HaplotypeCaller parameter to generate gVCF files for each sample, and integrated into a single VCF file with GenomicsDBImport and GenotypeGVCFs parameters.

[0042] The VCF file was SNP filtered with plink 1.9, and the quality control parameters were: --geno 0.1, --maf 0.05, --hwe 0.000001; the quality controlled VCF file was used by vcftools to calculate the number of SNPs within each 100 kb SNP according to the position of the SNP on the chromosome with a step of 10 kb and draw the SNP density distribution curve; GWAS analysis was not allowed to have missing sites, and for individual sites with individuals not detected SNPs, Beagle (v5.2) was used to fill in under default parameters; the filled VCF file was converted into a plink format file, and GCTA was used for population structure analysis and genetic force analysis; first, the GRM matrix was generated using the --make-grm parameter. The generated matrix was used to calculate the Principal Component Analysis (PCA) PCA and genetic force under the --grm snp.gcta and --grm qc parameters, respectively.

[0043] The model used in this GWAS analysis is 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 the polygenic effect, e ~ N(0, Iσ 2 ) is the residual effect. K in the polygenic effect is the kinship matrix inferred from markers.

[0046] The analysis software is GEMMA (v0.98.5). The software parameters are as follows: -miss 1, -maf 0, -r2 1, -hwe 0, -cgemma_cov.txt. Among them, gemma_cov.txt is the calculation result of the first three PCs in the above PCA result as a covariate added to the GWAS analysis. The number of SNPs is obtained by using PLINK 1.9 --indep-pairwise 100 10 0.2 to remove redundancy. In addition, since the threshold value calculated by the Bonferroni method (0.05 / SNP number) is too strict, the significance threshold is calculated by 1 / SNP number.

[0047] Table 1 shows the results of estimating the genetic parameters of the SNP-based resistance phenotype of tilapia.

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

[0049] The genetic parameters of the susceptibility / resistance phenotype of tilapia based on whole genome SNPs after challenge are shown in Table 1. As can be seen from Table 1, after tilapia were infected with S. agalactiae, 20 sensitive and 20 resistant individuals were taken, and the heritability of the susceptibility / resistance phenotype was 0.727, which had a high genetic heritability.

[0050] At the same time, the DNA of the above 40 tilapia after challenge was subjected to whole genome resequencing. The SNP density results after quality control and SNP calling of the raw data are shown in Table 2. Figure 1 As can be seen from Table 2, 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 the SNP was most distributed on chromosome 3 with the largest density, followed by the distribution density on chromosomes 7 and 23. Figure 1 The results of analyzing the population structure of tilapia by PCA are shown in Table 3.

[0051] As can be seen from Table 3, the PCA analysis using the first three principal components shows that the variances of the first principal component PC1, the second principal component PC2 and the third principal component PC3 are 18.87%, 10.52% and 9.83% respectively, indicating that most of the samples in the tilapia population are uniformly distributed, but there are 9 outlying samples, indicating that there is population stratification. Therefore, in the whole genome association analysis, the first three principal components are used as covariates and applied to the linear mixed model to explain the population stratification. Figure 2 Example 2 Screening of SNP site combination for identifying tilapia streptococcosis resistance

[0052]

[0053] ​The present example is based on the GWAS analysis of the anti-streptococcus agalactiae trait in Example 1, and the identification results of the candidate molecular markers of the anti-streptococcus agalactiae trait in the rohu are shown in Figure 3 Table 2, and the small-effect SNP sites are shown in Table 3, wherein, Figure 3 a Manhattan plot represents the distribution of SNPs significantly associated with the resistance of rohu to streptococcus agalactiae on the chromosome, Figure 3 b Quantile-quantile plot represents the probability distribution of expected p-value and observed p-value of SNPs, Figure 3 c Pie chart represents the proportion of small-effect candidate SNPs on each chromosome.

[0054] Table 2 Nine large-effect SNP sites in the GWAS analysis of the binary phenotype of the anti-streptococcus agalactiae trait in rohu

[0055]

[0056] From Figure 3 , Table 2 and Table 3, based on the mixed linear model of the sensitive / resistance phenotype, 9 large-effect SNPs (p<2.50E-06) and 787 small-effect candidate SNPs (2.50E-06

[0057] Table 3 787 small-effect SNP sites in the GWAS analysis of the sensitive / resistance phenotype of the anti-streptococcus agalactiae trait in rohu

[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-mentioned SNPs sites is summarized in Table 4.

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

[0071] Chromosome Chromosome name Chromosomes of the Nile 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] This example aims to perform functional gene annotation on the 9 large-effect SNPs and 787 small-effect SNPs sites associated with the resistance of tilapia to S. agalactiae disease screened in Example 2. Specifically, using annnovar (v 4.7) software, taking O_niloticus_UMD_NMBU as a reference genome, and based on the SNP region (-regionanno), the coding genes within the 100 kb range of the linkage disequilibrium (LD) decay distance upstream and downstream of the SNP sites in the candidate genes obtained by association analysis are annotated by the function of annotate_variation.pl.

[0074] The large-effect SNPs annotation genes are artificially annotated with gene functions. The candidate gene set annotated by the small-effect SNPs is simultaneously subjected to GO and KEGG enrichment analysis. After all the coding genes in the tilapia O_niloticus_UMD_NMBU are annotated by EGGNOG (http: / / eggnog-mapper.embl.de / ), a background annotation set is formed, and then the GO and KEGG enrichment of the candidate genes in the background annotation set is analyzed on the OmicShare platform.

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

[0076]

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

[0078] Moreover, the significant SNPs on chromosome LG13 are located in the intron region of WDR27, THBS2 and COL19A1. WDR27 belongs to the WD-repeat (WDR) family members, although the family is less reported in bacterial diseases, but multiple members thereof have been used as candidate target genes for various tumor suppressor drugs and are involved in the regulation of tumor immune microenvironment. For example, WDR4 has been reported to up-regulate the chromatin accessibility of TNFα, increase the number of myeloid-derived suppressor cells in tumors, reduce CD8 + T cell infiltration, thereby promoting the progression of hepatocellular carcinoma, on the contrary, TNFα recruits NF-κB to activate the transcription of WDR6, establishing a WDR6-TNFα feedback loop. THBS2 belongs to the thrombospondin family and is related to immune cell infiltration, immune regulation and other functions. Methylation of the promoter region thereof is considered as a prognostic marker for diseases such as endometrial adenocarcinoma and ovarian cancer. In fish, it is found that the gene plays an important role in the immune response of the fish against lymphocystis disease virus. COL19A1 gene is an extracellular matrix protein, and high expression of COL19A1 gene is related to the infiltration of immune effector cells (memory B cells, immature B cells and activated B cells). The significant SNP on LG22 is located in the intron region of LOC112841710. The gene encodes class I histocompatibility antigen, F10 alpha chain, and is involved in the presentation of foreign antigens to the immune system. The significant correlation SNPs on LG17 are annotated to the new gene LOC109195059, and the biological function thereof needs to be further studied.

[0079] The functional gene annotation results of the above-mentioned 9 large-effect SNPs show that the 9 candidate SNPs identified in Example 2 are related to the resistance of rohu streptococcosis.

[0080] 787 small-effect SNPs sites Figure 4As shown in FIG. 1, Figure 4 a, b respectively represent GO and KEGG enrichment analysis of small-effect SNP site annotated genes, respectively. From Figure 4 It can be seen that the small-effect SNP sites are annotated to 356 genes, and the GO enrichment result shows that the most significantly enriched biological function of the small-effect SNP site annotated genes is the urokinase plasminogen activator signaling pathway, and is significantly related to the biological function of synapse assembly, which regulates the activation of NF-κB in macrophages and the expression of cytokines Figure 4 a), and the top 5 enrichment pathways include Cyanoamino acid metabolism involved in the immune system and bacterial infection, Glycosaminoglycan degradation involved in the inflammatory response and the formation of tumor microenvironment, Ras signal pathway related to the down-regulation of anti-inflammatory response, Arachidonic acid metabolism related to pro-inflammatory response and inducing oxidative stress, stimulating immune response, Taurine and hypotaurine metabolism that can alleviate the apoptosis induced by Helicobacter pylori Figure 4 b).

[0081] The functional gene annotation result of the above-mentioned 787 small-effect SNP sites shows that the small-effect SNP sites identified in Example 2 are related to the resistance to Streptococcus agalactiae disease of the rohu.

[0082] Example 4

[0083] In order to further verify the relationship between the 9 large-effect SNP sites and 787 small-effect SNPs identified in Example 2 and the resistance to Streptococcus agalactiae disease, in this embodiment, the large-effect SNP sites are taken as an example, first, the haplotype analysis of the large-effect SNP sites is carried out, specifically, the upstream and downstream 100 kb of the large-effect SNP sites are extracted from the VCF file by using plink 1.9--recode HV, and a linkage format file is generated, then, the linkage file generated is subjected to LD decay and haplotype analysis by using Haploview software, the sites linked to the large-effect SNPs are taken as a collection, and the representative SNPs in the haplotype corresponding to each large-effect SNP are further generated by using the Tagger function.

[0084] The haplotype analysis result of the large-effect SNPs is as shown inFigure 5 As shown in the figure, Figure 5 a, b, c respectively show the haplotype analysis results of LG13_20881657, LG13_20863685 and LG17_25373146, LG17_25373231 sites, and the target SNP is in the Block (polymorphic sites in the haplotype block are completely linked to the imbalance region) region.

[0085] In combination with Table 5 and Figure 5 It can be seen that there is a linkage disequilibrium region in LG13_20881657, LG13_20863685 and LG17_25373146, and they are the core SNPs of the corresponding block region. On this basis, the present 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 genotyping primer sequences

[0087]

[0088]

[0089] Note: The first 15 bases (italic part) of primer_X and primer_Y are 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 mutation of the SNP.

[0090] Before KASP marker genotyping of tilapia using KASP primers as shown in Table 6, 300 Nile tilapia were subjected to streptococcus agalactiae challenge experiment, and the tail fin DNA of 133 resistant and 101 sensitive individuals (total 234) obtained was subjected to KASP molecular marker genotyping.

[0091] To develop the genotyping method of representative SNPs, this embodiment is centered on the target SNPs (LG13_20881657 and LG13_20548718), extracts the 300 bp genome sequences upstream and downstream of the two SNPs, then designs primers to PCR amplify the 200 bp genome sequences upstream and downstream thereof, respectively selects 10 tails of the surviving tilapia after the attack and 10 tails of the dead tilapia after the attack and performs Sanger sequencing, and through DNAMAN sequence alignment, the mutation information of the sequences upstream and downstream of the target SNPs is determined. Based on the mutation information, KASP primers are developed for the candidate molecular markers, and three KASP genotyping primers for the two target SNPs of LG13_20881657 and LG13_20548718 are designed as shown in Table 5, which are synthesized by Beijing Shengong Bioengineering Co., Ltd., and the three SNP primers are diluted to 10 μmol, and mixed according to the volume ratio of 12:12:30.

[0092] Further, 234 Nile tilapia are selected in the expanded population for the attack experiment. As described above, the tail fin tissues of the sensitive and resistant fish are collected, and the genomic DNA is immediately extracted using the blood / cell / tissue genomic DNA extraction kit (DP304, Tiangen Biochemical Technology, Beijing) according to the instructions. The extracted DNA is subjected to 1% agarose electrophoresis and Nanodrop 2100 quality control (the quality control requirements are as follows: the electrophoretic band is single and the Nanodrop 2100 detects A260 / 280 between 1.8 and 2.0), and the DNA stock solution after quality control is diluted with sterile double distilled water to 10-20 ng / μL for standby.

[0093] Table 7 PCR amplification reaction system

[0094]

[0095]

[0096] Based on the KASP technology and with the help of the LGC high-throughput genotyping system, the KASP genotyping primers shown in Table 6 are used for SNP genotyping of 234 tilapia samples, and the PCR reaction system involved is shown in Table 7. The PCR reaction is carried out in the high-throughput water bath system Hydrocycler, and the specific procedure is as follows: 94°C pre-denaturation for 15 minutes; 94°C, 20 seconds (denaturation); 55-61°C, 1 minute (annealing & extension); 10 cycles of amplification with touch down program, each cycle decreasing by 0.6°C; 94°C, 20 seconds (denaturation); 55°C, 60 seconds for 26 cycles of continuous amplification. After amplification, the BMG PHERAstar instrument is used to detect the fluorescence signal and check the genotyping, and the SNPviewer2 software is used for statistics.

[0097] The KASP typing primer was used for SNP genotyping of 234 tilapia samples, and the results are shown in Table 1. Figure 6 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] As shown in Table 1, Figure 6 232 (99.1%) of the fish were successfully genotyped at the L1657 locus, of which the number of susceptible fish with TT genotype was more than that of resistant fish (susceptible fish: 31; resistant fish: 23), and the number of susceptible fish with GG genotype was less than that of resistant fish (susceptible fish: 17; resistant fish: 35) (a, b); Figure 6 223 (95.3%) of the fish were successfully genotyped at the L8718 locus, of which the number of susceptible fish with TT genotype was less than that of resistant fish (susceptible fish: 9; resistant fish: 27), and the number of susceptible fish with AA genotype was more than that of resistant fish (susceptible fish: 46; resistant fish: 33) (c, d). Figure 6

[0099] As shown in Table 1,

[0100] The above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the above examples, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently, and these modifications or replacements are within the protection scope of the present application.​​

Claims

1. Use of a combination of SNP loci for identifying resistance to Streptococcus agalactiae disease in making a reagent or a liquid chip for identifying resistance to Streptococcus agalactiae disease, characterized in that, The SNP site combination comprises 2 SNP sites, the positions and base information of the 2 SNP sites on the chromosome are as follows: ； The SNP site combination further comprises 7 SNP sites, the positions and base information of the 7 SNP sites on the chromosome are as follows:

2. The use of the SNP site combination for identifying the resistance to Streptococcus agalactiae disease of the Oreochromis niloticus according to claim 1 in the preparation of a reagent or a liquid chip for identifying the resistance to Streptococcus agalactiae disease, characterized in that, The SNP site combination further comprises 787 SNP sites, the positions and base information of the 787 SNP sites on the chromosome are as follows: 。 3. The use of the SNP site combination for identifying the resistance to Streptococcus agalactiae disease of the Oreochromis niloticus according to claim 2 in the preparation of a reagent or a liquid chip for identifying the resistance to Streptococcus agalactiae disease of the Oreochromis niloticus, characterized in that, The KASP primer set comprises a first KASP primer pair and a second KASP primer pair; 。 4. A KASP primer set for identifying resistance to Streptococcus rofii disease in fish, characterized in that: The first KASP primer pair comprises an upstream primer A, an upstream primer B and a universal reverse primer E, the nucleotide sequence of the upstream primer A is shown as SEQ ID NO: 1, the nucleotide sequence of the upstream primer B is shown as SEQ ID NO: 2, and the nucleotide sequence of the universal reverse primer E is shown as SEQ ID NO: 5; The second KASP primer pair comprises an upstream primer C, an upstream primer D and a universal reverse primer F, the nucleotide sequence of the upstream primer C is shown as SEQ ID NO: 3, the nucleotide sequence of the upstream primer D is shown as SEQ ID NO: 4, and the second KASP primer pair further comprises a universal reverse primer F, the nucleotide sequence of the universal reverse primer F is shown as SEQ ID NO:

6.

5. The KASP primer set for identifying the resistance of Streptococcus agalactiae disease of Tilapia for use in identifying the resistance of Streptococcus agalactiae disease of Tilapia according to claim 4. The kit comprises the KASP primer set for identifying the resistance of Streptococcus agalactiae disease of Tilapia according to claim 4.

6. A kit for identifying resistance to Streptococcus rofita disease, characterized by: ​

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