Method for evaluating streptococcus agalactiae resistance of genetically improved farmed tilapia

Abstract

The invention discloses a method for evaluating streptococcus agalactiae resistance of genetically improved farmed tilapia. According to the invention, firstly, DNA methylation difference analysis is carried out aiming at the streptococcus agalactiae resistance traits of the genetically improved farmed tilapia, a DNA methylation molecular marker lcp2a308-485 associated with the traits is developed on the basis, and a pair of methylation marker detection primers associated with the streptococcus agalactiae resistance traits of the genetically improved farmed tilapia is provided. The methylation degree of the DNA methylation molecular marker lcp2a308-485 of a plasma cfDNA sample of Oreochromis niloticus has good correlation with the resistance of Streptococcus lactis, and the DNA methylation molecular marker can be used for noninvasive or minimally invasive detection of the resistance of Streptococcus lactis and can be used for breeding of sex Streptococcus lactis resistant Oreochromis niloticus. The breeding efficiency can be greatly improved, the practical problems of long breeding period, high input cost and the like in traditional disease resistance breeding can be solved, and the method has important significance on industrial development of genetically improved farmed tilapia mossambica.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to a method for evaluating the resistance of GIFT tilapia to Streptococcus agalactiae. Background Art

[0002] Tilapia (Oreochromis spp) is a Cichlid fish native to Africa. It is the second largest freshwater farmed fish in the world, second only to carp. Gifu Tilapia belongs to the Percifoxrnes, Percoidei, Cichlidae, and Oreochromis. It is generally believed to be a hybrid of Nile Tilapia (O.niloticus) and Mozambique Tilapia (O.mossambicus) with disease resistance, and is a superior variety bred through multiple generations.

[0003] Tilapia agalactiae has always been a major disease that restricts the development of the tilapia industry and causes huge economic losses to farmers. Currently, the main ways to deal with this disease include vaccination, drug prevention and control, standardized breeding, and breeding of new disease-resistant varieties, among which disease-resistant breeding is considered to have the most potential.

[0004] DNA methylation is an important part of epigenetics, mainly including three processes: de novo methylation, maintenance methylation, and demethylation. The dynamic changes of DNA methylation are closely related to the physiological conditions of the organism, and it is also actively promoted in the breeding of fine varieties. Previous studies have shown that DNA methylation plays a regulatory role in the complex traits of tilapia. For example, the increase in DNA methylation levels during the sexual differentiation of tilapia, and the sexually dimorphic methylation regions make an important contribution to the sex specificity of tilapia (PMID: 27486872; PMID: 34576333). Under saline-alkali or heavy metal stress, abnormal expression of DNA methyltransferase and a decrease in the overall DNA methylation level in tilapia (PMID: 34739975; PMID: 32439378).

[0005] Studies have shown that the methylation levels of the promoter regions of hoxa4a, lcp2a and ccr9a in the spleen of GIF tilapia are related to infection with Streptococcus agalactiae. However, since the sampling of spleen tissue is very harmful to GIF tilapia, the individuals after sampling are basically unsuitable for subsequent breeding of GIF tilapia resistant to Streptococcus agalactiae.

[0006] cfDNA in plasma is often used in medicine for low-invasive, non-invasive or minimally invasive liquid biopsies as an alternative to complex and invasive tissue biopsies. However, due to the tissue specificity of DNA methylation, there is no methylation detection target that can be directly applied to cfDNA in plasma. Summary of the invention

[0007] The purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art and provide a method for breeding GIFT tilapia with high resistance to Streptococcus agalactiae.

[0008] The first object of the present invention is to provide an application of a DNA methylation molecular marker in plasma cfDNA of GIFT tilapia in non-invasive or minimally invasive selection of GIFT tilapia resistant to Streptococcus agalactiae or breeding of GIFT tilapia resistant to Streptococcus agalactiae.

[0009] The second object of the present invention is to provide a non-invasive or minimally invasive method for evaluating the resistance of GIFT tilapia to Streptococcus agalactiae.

[0010] The third object of the present invention is to provide an application of a product for non-invasive or minimally invasive detection of the methylation degree of DNA methylation molecular markers of GIFT tilapia.

[0011] The fourth object of the present invention is to provide a primer for non-invasive or minimally invasive detection of the methylation level of DNA methylation molecular markers associated with resistance to Streptococcus agalactiae in GIFT tilapia.

[0012] The fifth object of the present invention is to provide a kit for non-invasive or minimally invasive detection of the methylation level of DNA methylation molecular markers associated with resistance to Streptococcus agalactiae in GIFT tilapia.

[0013] The sixth object of the present invention is to provide a method for breeding GIFT tilapia with high resistance to Streptococcus agalactiae.

[0014] The above-mentioned purpose of the present invention is achieved by the following scheme:

[0015] The present invention found that in the plasma cfDNA samples of GIFT tilapia, the methylation degree of the methylation marker lcp2a_308-485 was well correlated with the resistance to Streptococcus lactis, and could be used for non-invasive or minimally invasive detection of Streptococcus lactis resistance, and could be used for breeding of GIFT tilapia resistant to Streptococcus lactis.

[0016] Therefore, the present invention claims to protect the use of a DNA methylation molecular marker in the plasma cfDNA of GIFT tilapia in the non-invasive or minimally invasive selection of GIFT tilapia resistant to Streptococcus agalactiae or the breeding of GIFT tilapia resistant to Streptococcus agalactiae. The DNA methylation molecular marker is located at LG10: 32,939,308-32,939,485, LG10 is NC_031975.2, and the methylation level of the DNA methylation molecular marker in GIFT tilapia resistant to Streptococcus agalactiae is lower than that in GIFT tilapia susceptible to Streptococcus agalactiae.

[0017] The genome version is from: NCBI Oreochromis niloticus Annotation Release 104 (i.e., NCBI RefSeq assembly: GCF_001858045.2, Submitted GenBank assembly: GCA_001858045.3, https: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCF / 001 / 858 / 045 / GCF_001858045.2_O_niloticus_UMD_NMBU / GCF_001858045.2_O_niloticus_UMD_NMBU_genomic.fna.gz).

[0018] Also claimed is a non-invasive or minimally invasive method for evaluating GIF tilapia's resistance to Streptococcus agalactiae, which detects the methylation level of the DNA methylation molecular marker located at LG10:32,939,308-32,939,485 in the plasma cfDNA of individual GIF tilapia, the methylation level of the DNA methylation molecular marker in GIF tilapia resistant to Streptococcus agalactiae is lower than that in GIF tilapia susceptible to Streptococcus agalactiae.

[0019] Preferably, the method comprises the following steps: drawing blood from the GIFT tilapia to be tested and separating the plasma, extracting the plasma cfDNA, performing bisulfite conversion on the plasma cfDNA, using the plasma cfDNA after sulfite conversion as a template, performing PCR amplification using specific amplification primers to obtain an amplification product; and detecting the amplification product, wherein the methylation level of the DNA methylation molecular marker in the GIFT tilapia resistant to Streptococcus agalactiae is lower than that in the GIFT tilapia resistant to Streptococcus agalactiae.

[0020] The invention also claims a product for non-invasively or minimally invasively detecting the methylation degree of DNA methylation molecular markers of GIFT tilapia, wherein the application is to breed GIFT tilapia resistant to Streptococcus agalactiae, select GIFT tilapia resistant to Streptococcus agalactiae, or evaluate the resistance of GIFT tilapia to Streptococcus agalactiae.

[0021] The DNA methylation molecular marker is located at LG10: 32,939,308-32,939,485, and LG10 is NC_031975.2.

[0022] The genome was obtained from NCBI Oreochromis niloticus Annotation Release 104 (i.e., NCBI RefSeq assembly: GCF_001858045.2, Submitted GenBank assembly: GCA_001858045.3, https: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCF / 001 / 858 / 045 / GCF_001858045.2_O_niloticus_UMD_NMBU / GCF_001858045.2_O_niloticus_UMD_NMBU_genomic.fna.gz). The methylation level of the DNA methylation molecular markers in the GIF tilapia resistant to S. agalactiae was lower than that in the GIF tilapia susceptible to S. agalactiae.

[0023] Preferably, the product is a primer or a kit.

[0024] The invention also claims a primer for non-invasively or minimally invasively detecting the methylation level of a DNA methylation molecular marker associated with resistance to Streptococcus agalactiae in GIFT tilapia, and the nucleotide sequence thereof is shown in SEQ ID NOs: 1 and 2.

[0025] And a kit for non-invasive or minimally invasively detecting the methylation level of DNA methylation molecular markers associated with resistance to Streptococcus agalactiae in GIFT tilapia, containing the primers.

[0026] Preferably, it also contains one or more of a plasma cfDNA extraction reagent, a bisulfite conversion kit, a PCR reagent, an agarose gel electrophoresis reagent, a DNA nucleic acid dye reagent, a DNA gel recovery kit and a TA cloning reagent.

[0027] Further preferably, the reaction system of the PCR reagents in the kit is 20-30 μL of 2×PCR DS Mix, 1.8-2.2 μL of each primer, 3.6-4.4 μL of template DNA, and 15.3-18.7 μL of water to make a total volume of 50 μL.

[0028] More preferably, the reaction system of the PCR reagents in the kit is 2×PCR DS Mix 25 μL, 2 μL of each primer, 3.6-4.4 μL of template DNA, and 15.3-18.7 μL of water to make a total volume of 50 μL.

[0029] Further preferably, the reaction conditions of the PCR reagents in the kit are pre-denaturation at 94-95°C for 2 min, denaturation at 94-95°C for 15 s, annealing at 58°C for 30 s, extension at 70-72°C for 30 s, 40 cycles, and extension at 70-72°C for 5 min.

[0030] More preferably, the reaction conditions of the PCR reagents in the kit are pre-denaturation at 95°C for 2 min, denaturation at 95°C for 15 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 40 cycles, and extension at 72°C for 5 min.

[0031] The invention also claims a method for breeding GIFT tilapia with high resistance to Streptococcus agalactiae, wherein cfDNA of GIFT tilapia is collected, and GIFT tilapia with high resistance to Streptococcus agalactiae is used as a parent for breeding according to the method.

[0032] Compared with the prior art, the present invention has the following beneficial effects: The present invention develops a DNA methylation molecular marker lcp2a_308-485 related to the resistance of GIFT tilapia to Streptococcus agalactiae, and provides a pair of detection primers for the methylation marker related to the resistance of GIFT tilapia to Streptococcus agalactiae. In the plasma cfDNA sample of GIFT tilapia, the methylation degree of the DNA methylation molecular marker lcp2a_308-485 has a good correlation with the resistance to Streptococcus lactis, can be used for non-invasive or minimally invasive detection of resistance to Streptococcus lactis, and can be used for breeding of GIFT tilapia resistant to Streptococcus lactis. It can greatly improve the breeding efficiency, which will help solve the practical problems of long breeding cycle and high input cost in traditional disease resistance breeding, and is of great significance to the development of the GIFT tilapia industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The WGBS method used in Example 1 uses a window size of 300bp, a step size of 200bp, a significance threshold of 1e-27, and a difference of 40% to identify the chromosomal distribution of DMRs in spleen tissue (A) and the regional distribution of their associated genes (DMGs) (B).

[0034] Figure 2 The WGBS method was used for the present invention, with a window size of 300 bp, a step size of 200 bp, a significance threshold of 1e-27, and a difference of 40%. The chromosomal distribution of DMRs identified in brain tissue (A) and the regional distribution of their associated genes (DMGs) (B) were shown.

[0035] Figure 3The present invention uses a dual luciferase experiment to identify the core promoter region in the first 2000bp of lcp2aTSS. Figure A is a diagram of the lcp2a gene structure and DMR, a predicted core promoter position, and a schematic diagram of the position of the truncated fragment selected in the promoter truncation experiment; Figure B is the BSP detection results of M.SssI-treated cells and 5-AzaC-treated cells using the cfDNA methylation marker lcp2a_308-485, black dots represent methylated sites, and white dots represent unmethylated sites; Figure C is the fluorescence value of each group of cells measured by the dual luciferase experiment, and the higher the value, the higher the fragment promoter activity.

[0036] Figure 4 These are the results of BSP detection of spleen and brain tissues for lcp2a_308-485; black dots represent methylated sites, and white dots represent unmethylated sites.

[0037] Figure 5 These are the results of BSP detection of plasma cfDNA for lcp2a_308-485; black dots represent methylated sites, and white dots represent unmethylated sites.

[0038] Figure 6 These are the BSP test results for plasma cfDNA for ccr9a_821-053; black dots represent methylated sites, and white dots represent unmethylated sites.

[0039] Figure 7 These are the results of BSP detection of plasma cfDNA for hoxa4a_215-410; black dots represent methylated sites, and white dots represent unmethylated sites. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0041] Example 1 WGBS analysis of the ability of individuals from mixed families to resist Streptococcus agalactiae

[0042] 1. Experimental Methods

[0043] 1. Evaluation of disease resistance of GIFT tilapia and sample collection and preservation

[0044] (1) In March 2023, 700 GIFT tilapia (sample group 1) with an average weight of 50 g were selected from the Guangdong Tilapia Breeding Farm. The THN0901 strain of Streptococcus agalactiae (SA strain, Su Youlu, Wang Baotun, Xie Yundan, et al. cel-EIIB protein regulates the expression pattern of virulence-related genes of virulent and weak strains of Streptococcus agalactiae in tilapia [J]. Chinese Fisheries Science, 2020, 27(11): 1360-1370. DOI: 10.3724 / SP.J.1118.2020.20089.) was provided by Professor Li Anxing of Sun Yat-sen University. After two weeks of acclimation, each fish was intraperitoneally injected with SA (10 7 CFU / mL, 0.1mL). After injection, the fish were kept in a cement tank (1m×3m×3m) with a water temperature range of 30-32°C for 21 days. In order to eliminate the error caused by the injection process, the number of fish deaths was recorded every day starting from the third day.

[0045] The reference standard for grading the disease resistance of GIFT tilapia populations is as follows: the first 40 dying individuals recorded are classified as individuals with weak disease resistance to form a susceptible group, and the disease resistance trait score is set as degree 1. In contrast, those individuals that survived for more than 21 days without any phenotypic symptoms were identified as high-quality GIFT tilapia individuals with strong disease resistance, and the disease resistance trait score was set as degree 2, from which 40 individuals were randomly selected to form a resistant and susceptible group.

[0046] The spleen and brain tissues of each individual in the resistant and susceptible groups were collected and stored in anhydrous ethanol for DNA extraction in preparation for WGBS sequencing, with 40 tails in each group.

[0047] 2. Construction of Tilapia DNA Library

[0048] The genomic DNA of the samples was extracted using a DNA extraction kit, the quality of all individual DNA was tested using agarose gel electrophoresis, and the DNA concentration was tested using Qubit 3.0. After equal mixing, the extreme trait DNA was submitted to Wuhan Aijibaike Company for WGBS library construction. The final DNA fragments were subjected to high-throughput sequencing.

[0049] 3. High-throughput sequencing of mixed DNA libraries

[0050] Illumina HiSeqTM2500 was used for high-throughput sequencing. The original sequencing sequence obtained by sequencing, i.e., raw data, was filtered to remove low-quality reads, adapters, and excessive N reads to obtain clean data.

[0051] 4. Identification of DMR (DMP) located in the DMG promoter region

[0052] The clean data were compared with the NCBI Oreochromis niloticus Annotation Release 104 (NCBI RefSeq assembly: GCF_001858045.2, Submitted GenBank) published on NCBI using bismark. assembly: GCA_001858045.3, (https: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCF / 001 / 858 / 045 / GCF_001858045.2_O_niloticus_UMD_NMBU / GCF_001858045.2_O_niloticus_UMD_NMBU_genomic.fna.gz) were used for alignment, and then analyzed using bioinformatics software such as samtools, BatMeth2, and methylKit to obtain DMRs. The obtained DMRs were then genetically associated to obtain differentially methylated genes (DMGs), and DMRs (DMPs) located in the promoter region of DMGs were screened out based on whether the DMRs were located in the promoter region of DMGs.

[0053] 2. Experimental Results

[0054] Through high-throughput sequencing, about 188Gb of raw data were obtained. After quality control, alignment and difference analysis, a large number of DMRs were obtained.

[0055] There were 3932 up-regulated DMRs, 1505 down-regulated DMRs, 1376 up-regulated DMGs and 573 down-regulated DMGs in the spleen, of which 268 genes (DMPs) with DMRs located in the promoter region of DMGs were Figure 1 .

[0056] There were 1553 up-regulated DMRs, 359 down-regulated DMRs, 619 up-regulated DMGs, and 153 down-regulated DMGs in the brain, of which 81 genes (DMPs) had DMRs located in the promoter region of DMGs. Figure 2 .

[0057] There were 479 differentially methylated genes (DMGs) shared among tissues, including 51 shared DMPs.

[0058] Example 2 Increased methylation level in the lcp2a core promoter region leads to a significant decrease in promoter activity

[0059] 1. Experimental Methods

[0060] 1. Selection of candidate functional genes

[0061] According to the results of Example 1, spleen and common DMPs were selected, and genes related to immune response (lcp2a, hoxa4a and ccr9a) were screened according to the gene annotation and enrichment analysis results of the DMPs. The promoters of these genes were predicted using ConTra V3 (PMID: 28472390), TSSG (PMID: 20827586) and promoter2.0 (PMID: 10366655), and IBS (PMID: 35580044) was used to describe the spatial relationship between the predicted promoters and DMRs and their annotated genes.

[0062] The promoter region of the lcp2a gene (NCBI gene number is 100693828, mRNA is XM_005461790.4) has a DMR (LG10: 32,939,201-32,939,500, LG10 is NC_031975.2) that overlaps with the predicted promoter position.

[0063] The promoter region of the hoxa4a gene (NCBI gene number is 100694782, mRNA is XM_025902218.1) has a DMR (LG22: 25342201-25342500, LG22 is NC_031985.2) that overlaps with the predicted promoter position.

[0064] There is a DMR (LG18: 34567801-34568100, LG18 is NC_031982.2) in the promoter region of the ccr9a gene (NCBI gene number is 100691147, mRNA is XM_019348351.2) that overlaps with the predicted promoter position.

[0065] A promoter truncation experiment was performed on lcp2a to confirm the promoter core region; the predicted promoter positions of hoxa4a and ccr9a did not overlap with DMR to a high degree, so truncation experiments were not performed.

[0066] 2. Construction of truncated plasmid for determining the core region of the lcp2a promoter

[0067] Primers for cloning the first 2 kb, first 450 bp, first 300 bp and first 150 bp fragments of lcp2a TSS (sequences are shown in Table 1), which were synthesized by Aiji Biotechnology (Guangzhou) Trading Co., Ltd. PCR amplification was performed using the primer pairs in Table 1. The reaction system for PCR amplification was: 2×PCR Mix 25 μL, 2 μL of upstream and downstream primers, 4 μL of DNA template, and 17 μL of water to a total volume of 50 μL. The DNA template was any genomic DNA of tilapia.

[0068] The reaction conditions of PCR amplification were as follows: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 15 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 40 cycles, and extension at 72°C for 5 min.

[0069] The PCR amplification products were subjected to 1.5% agarose gel electrophoresis using TAE electrophoresis buffer, and the target bands were recovered using the Omega Bio-tek gel recovery kit ( The amplified products were recovered by gel extraction with a Gel Extraction Kit to obtain the first 2 kb, first 450 bp, first 300 bp and first 150 bp fragments of lcp2aTSS, which were then ligated into the luc4.10 plasmid using a homologous recombination kit to obtain plasmids of various truncated lengths.

[0070] Table 1:

[0071]

[0072] 3. Transfect cells with plasmids of various truncated lengths

[0073] The plasmids of each truncated length of each gene were divided into three parts to construct a fully methylated group, a completely unmethylated group and a control group:

[0074] Completely methylated group (M.SssI group): each truncated length plasmid was methylated using M.SssI methyltransferase to obtain a methylated truncated length plasmid of 450 bp, and each methylated truncated length plasmid and prl-tk plasmid were co-transfected into cells;

[0075] Completely unmethylated group (5-Aza group): cells were treated with methyltransferase inhibitor 5-Aza (2.5 mM / mL) (to inhibit cellular CpG methyltransferase) to obtain treated cells, and the treated cells were co-transfected with a 450 bp truncated plasmid and a prl-tk plasmid;

[0076] Control group: cells were co-transfected with plasmids of each truncated length and prl-tk plasmid.

[0077] Each group was paralleled by 4 groups. 24 hours after transfection, one group was selected to extract the cell DNA of M.SssI group and 5-Aza group using HiPure Tissue DNA Mini Kit (Magen, China) and perform BSP test. The other three groups used fluorescence detector to measure the promoter activity by measuring the ratio of firefly fluorescence to sea cucumber fluorescence.

[0078] 4. BSP inspection

[0079] (1) Bisulfite conversion

[0080] The DNA was methylated using the one-step DNA denaturation and bisulfite conversion kit (EZDNA Methylation-Gold TM The cell DNA of the M.SssI group and the 5-Aza group were subjected to bisulfite conversion.

[0081] (2) Primer design and PCR amplification of methylation marker primers

[0082] BSP primers were designed for lcp2a, see Table 2.

[0083] Table 2:

[0084]

[0085] The reaction system for PCR amplification was: 25 μL of 2×PCR Mix, 2 μL of each of the upstream and downstream primers of the primer pair in Table 2, 4 μL of DNA template, and 17 μL of water to make a total volume of 50 μL.

[0086] The reaction conditions of PCR amplification were as follows: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 15 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 40 cycles, and extension at 72°C for 5 min.

[0087] The PCR amplification products were subjected to 1.5% agarose gel electrophoresis using TAE electrophoresis buffer, and the target bands were recovered using the Omega Bio-tek gel recovery kit ( Gel Extraction Kit) was used to recover the amplified product.

[0088] Then, the concentration of the DNA solution recovered from the gel was detected using a Nanodrop 2000 ultra-micro volume UV spectrophotometer (Thermo Scientific, USA).

[0089] (3) Construction and transformation of cloning vector

[0090] The target fragments recovered from the gel were ligated by TA cloning using the TSV-007S TA cloning kit (pClone007 Simple Vector Kit) of Qingke Biotechnology to obtain a cloning vector, which was then transformed into Escherichia coli.

[0091] Afterwards, the selected single clones were subjected to bacterial liquid PCR using M13 primers upstream and downstream of the vector insertion site (Table 3) to detect recombinants.

[0092] Table 3:

[0093] Primer name Sequence (5-3) M13_F GTTGTAAAACGACGGCCAG M13_R CAGGAAACAGCTATGAC

[0094] The reaction system for PCR amplification was: 5 μL of 2×PCR Mix, 0.4 μL of each of the upstream and downstream primers of the M13 primer pair, 0.8 μL of template DNA, and 3.4 μL of water to make a total volume of 10 μL.

[0095] The reaction conditions of PCR amplification were as follows: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 30 cycles, and extension at 72°C for 5 min.

[0096] The PCR amplification products were subjected to electrophoresis, and 5 bacterial cultures with qualified bands were selected and submitted to Aiki Biotechnology (Guangzhou) Trading Co., Ltd. for Sanger sequencing. The sequencing results were analyzed for DNA methylation levels using BiQ Analyzer software.

[0097] 5. Use a fluorescence detector to measure the ratio of firefly fluorescence to sea cucumber fluorescence to measure promoter activity

[0098] Aspirate the culture medium, rinse once with PBS buffer, aspirate the buffer with a pipette, immediately add 1× lysis buffer at 100 μL / well, gently shake to cover the bottom with buffer, and oscillate at 37℃500rpm for 15min. Transfer the lysis solution to a 1.5mL centrifuge tube, centrifuge at 13000g for 1min at room temperature, take 20 μL of supernatant, use GloMaxTM96-well plate luminometer to detect firefly fluorescence and sea cucumber fluorescence, and calculate the ratio of the two to measure promoter activity.

[0099] 2. Experimental Results

[0100] The results are as follows Figure 3 B in the figure shows that except for the first 150bp of lcp2aTSS, which has no promoter activity, the other truncated sequences all have significant promoter activity, among which the first 450bp fragment has the strongest promoter activity, indicating that the lcp2a core promoter region is located between 450bp and 150bp before TSS (LG10:32,939,156~32,939,456), which is highly consistent with the DMR obtained by WGBS analysis (LG10:32,939,201~32,939,500).

[0101] The results are as follows Figure 3 Figure C shows that for the first 450bp fragment of lcp2aTSS, the fully methylated group showed a significant decrease in methylation level compared with the control group, and the fully unmethylated group showed a significant increase in methylation level compared with the control group, indicating that the increase in methylation level in this region will lead to a significant decrease in promoter activity. Figure 3 .

[0102] Example 3 Bisulfite sequencing (BSP) detection of the correlation between the degree of CpG island methylation and the disease resistance of GIFT tilapia

[0103] 1. Experimental Methods

[0104] 1. Prediction of CpG islands

[0105] Based on the results in Example 2, the online methylation primer design tool MethPrimer2 (http: / / www.urogene.org / cgibin / methprimer2 / MethPrimer.cgi) was used to predict the CpG island of the sequence. The prediction results showed that:

[0106] There is a CpG island in the DMR fragment associated with the lcp2a gene (LG10:32,939,201-32,939,500), located at LG10:32,939,308-32,939,485 (i.e., 121-298 before the lcp2a TSS, lcp2a_308-485);

[0107] There is a CpG island in the fragment of the DMR (LG22: 25,342,201-25,342,500) associated with the hoxa4a gene, located at LG22: 32,939,215-32,939,410 (i.e., 179-374 after the lcp2a TSS, hoxa4a_215-410);

[0108] There is a CpG island in the DMR (LG18: 34,567,801-34,568,101) fragment associated with the ccr9a gene, located at LG18: 34567821-34568053 (i.e., 52-284 after the lcp2a TSS, ccr9a_821-053).

[0109] The methylation degree of the above CpG islands was detected by bisulfite sequencing (BSP).

[0110] 2. Bisulfite conversion

[0111] The DNA was methylated using the one-step DNA denaturation and bisulfite conversion kit (EZDNA Methylation-Gold TM The spleen and brain genomic DNA of the resistant and susceptible groups of Example 1 were subjected to bisulfite conversion using a PCR amplification kit (100 μg / ml).

[0112] 3. Primer design and PCR amplification of methylation marker primers

[0113] The methyl primers for CpG islands (lcp2a_308-485, hoxa4a_215-410, and ccr9a_821-053) are the same as those in Table 4 .

[0114] Table 4:

[0115]

[0116] The reaction system for PCR amplification was: 25 μL of 2×PCR Mix, 2 μL of each of the upstream and downstream primers of the primer pair in Table 1, 4 μL of template DNA, and 17 μL of water to make a total volume of 50 μL.

[0117] The reaction conditions of PCR amplification were as follows: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 15 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 40 cycles, and extension at 72°C for 5 min.

[0118] The PCR amplification products were subjected to 1.5% agarose gel electrophoresis using TAE electrophoresis buffer, and the target bands were recovered using the Omega Bio-tek gel recovery kit ( Gel Extraction Kit) was used to recover the amplified product.

[0119] Then, the concentration of the DNA solution recovered from the gel was detected using a Nanodrop 2000 ultra-micro volume UV spectrophotometer (Thermo Scientific, USA).

[0120] 3. Construction and transformation of cloning vector

[0121] Then, the TSV-007S TA cloning kit (pClone007 Simple Vector Kit) of Qingke Biotechnology was used to perform TA cloning on the target fragments recovered from the gel to obtain a cloning vector, and then the cloning vector was transformed into Escherichia coli. After that, the selected single clones were subjected to bacterial liquid PCR using M13 primers upstream and downstream of the vector insertion site (same as Table 3) to detect the recombinants.

[0122] The reaction system for PCR amplification was: 5 μL of 2×PCR Mix, 0.4 μL of each of the upstream and downstream primers of the M13 primer pair, 0.8 μL of template DNA, and 3.4 μL of water to make a total volume of 10 μL.

[0123] The reaction conditions of PCR amplification were as follows: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 30 cycles, and extension at 72°C for 5 min.

[0124] The PCR amplification products were subjected to electrophoresis, and 5 bacterial cultures with qualified bands were selected and submitted to Aiki Biotechnology (Guangzhou) Trading Co., Ltd. for Sanger sequencing. The sequencing results were analyzed for DNA methylation levels using BiQ Analyzer software.

[0125] 2. Experimental Results

[0126] The target product of lcp2a_308-485 is between 100bp and 200bp. The BSP result is as follows Figure 4 The results showed that in the spleen, the methylation level of the susceptible group was 18.33%, and the methylation level of the resistant group was 75%, with a difference of 56.67%. After Fisher's exact test, the significant difference P-value was 1.57e-13. In the brain, the methylation level of the susceptible group was 10%, and the methylation level of the resistant group was 93.33%, with a difference of 83.3%. After Fisher's exact test, the significant difference P-value was 7.28e-17. This shows that the DNA methylation of lcp2a in the spleen and brain tissue of the resistant group of GIFT tilapia is higher than that of the susceptible group.

[0127] In the spleen, the methylation levels of hoxa4a_215-410 and ccr9a_821-053 were significantly different between the susceptible group and the resistant group (P-value < 0.001); however, in the brain, the methylation levels of hoxa4a_215-410 and ccr9a_821-053 were 6.67% (p-value = 0.142) and 33.33% (p-value = 0.022) different between the susceptible group and the resistant group, respectively, and the differences were not significant.

[0128] Example 4 Correlation between DNA methylation molecular markers in plasma cfDNA and Streptococcus agalactiae in GIFT tilapia

[0129] 1. Experimental Methods

[0130] 1. Evaluation of disease resistance of GIFT tilapia, sample collection and cfDNA extraction

[0131] In September 2023, another 100 GIFT tilapia (sample group 2) with an average weight of 150 g were selected from the Guangdong Tilapia Breeding Farm. The Streptococcus agalactiae THN0901 strain (SA) was provided by Professor Li Anxing of Sun Yat-sen University. After two weeks of acclimation, each fish was intraperitoneally injected with SA (10 7 CFU / mL, 0.1mL). After injection, the fish were kept in a cement tank (1m×3m×3m) with a water temperature range of 30-32°C for 21 days. In order to eliminate the error caused by the injection process, the number of fish deaths was recorded every day starting from the third day.

[0132] The reference standard for grading the disease resistance of GIFT tilapia populations is as follows: the first five dying individuals recorded are classified as individuals with weak disease resistance to form a susceptible group, and the disease resistance trait score is set as degree 1. In contrast, those individuals that survived for more than 21 days without any phenotypic symptoms were identified as high-quality GIFT tilapia individuals with strong disease resistance, and the disease resistance trait score was set as degree 2, from which five individuals were randomly selected to form a resistant and susceptible group.

[0133] All individuals had 0.5 mL of blood drawn from the blood, and the plasma was immediately separated and the plasma cfDNA was extracted using a plasma cfDNA extraction kit. The quality of cfDNA of all individuals was tested by agarose gel electrophoresis technology, and the cfDNA concentration was tested by Qubit 3.0. The samples were then equally mixed to obtain cfDNA of the susceptible group and the resistant group, and stored at -80°C.

[0134] 3. BSP detection of methylation levels of lcp2a_308-485, hoxa4a_215-410, and ccr9a_821-053

[0135] The DNA methylation levels of lcp2a_308-485, hoxa4a_215-410, and ccr9a_821-053 in the mixed cfDNA of the susceptible group and the resistant group were determined according to the method and primers in Example 4.

[0136] 2. Experimental Results

[0137] BSP results of LCP2A, CCR9A and HOXA4A in plasma cfDNA Figures 5 to 7 The results showed that for lcp2a, the methylation level of the susceptible group was 3.33%, and that of the resistant group was 70%, with a difference of 56.67%. After Fisher's exact test, the significant difference P-value was 2.49e-15. The DNA methylation level of the resistant group was significantly higher than that of the susceptible group, basically reaching 50%.

[0138] For lCCR9, the methylation rate of the resistant group was 89.1%, and that of the susceptible group was 92.7%. The Fisher's exact test showed that the significant difference P-value was 0.742; there was no significant difference in the methylation level between the resistant group and the susceptible group.

[0139] The methylation rate of HOX4 in the resistant group was 95%, and that in the susceptible group was 95%. The difference was statistically significant with a P-value of 1 after Fisher's exact test.

[0140] Therefore, screening for GIFT tilapia with a DNA methylation level of lcp2a higher than 50% can effectively obtain a GIFT tilapia population with stable disease resistance.

[0141] Finally, it should be noted that 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. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An application of a DNA methylation molecular marker in plasma cfDNA of GIFT tilapia in non-invasive or minimally invasive selection of GIFT tilapia resistant to Streptococcus agalactiae or breeding of GIFT tilapia resistant to Streptococcus agalactiae, characterized in that: The DNA methylation molecular marker is located at LG10:32,939,308-32,939,485, LG10 is NC_031975.2, and the methylation level of the DNA methylation molecular marker in GIF tilapia resistant to Streptococcus agalactiae is lower than that in GIF tilapia susceptible to Streptococcus agalactiae.

2. A non-invasive or minimally invasive method for evaluating the resistance of GIFT tilapia to Streptococcus agalactiae, characterized in that: The methylation level of the DNA methylation molecular marker located at LG10:32,939,308-32,939,485 in the plasma cfDNA of individual GIFT tilapia was detected. The methylation level of the DNA methylation molecular marker in GIFT tilapia resistant to Streptococcus agalactiae was lower than that in GIFT tilapia susceptible to Streptococcus agalactiae.

3. The method according to claim 2, characterized in that The following steps are involved: Blood is drawn from the GIFT tilapia to be tested and the plasma is separated, the plasma cfDNA is extracted, the plasma cfDNA is subjected to bisulfite conversion, and PCR amplification is performed using the plasma cfDNA after sulfite conversion as a template using specific amplification primers to obtain an amplification product; the amplification product is detected, and the methylation level of the DNA methylation molecular marker in the GIFT tilapia resistant to Streptococcus agalactiae is lower than that in the GIFT tilapia susceptible to Streptococcus agalactiae.

4. The method according to claim 3, characterized in that The nucleotide sequences of the specific amplification primers are shown in SEQ ID NOs: 1 and 2.

5. An application of a product for non-invasive or minimally invasive detection of the methylation degree of DNA methylation molecular markers of GIFT tilapia, characterized in that: The application is: breeding GIFT tilapia resistant to Streptococcus agalactiae, non-invasively or minimally invasively selecting GIFT tilapia resistant to Streptococcus agalactiae, or non-invasively or minimally invasively evaluating the resistance of GIFT tilapia to Streptococcus agalactiae. The DNA methylation molecular marker is located at LG10: 32,939,308-32,939,485, and LG10 is NC_031975.

2. The methylation level of the DNA methylation molecular marker in GIF tilapia resistant to Streptococcus agalactiae was lower than that in GIF tilapia susceptible to Streptococcus agalactiae.

6. The use according to claim 5, characterized in that: The product is a primer or a kit.

7. A primer for non-invasive or minimally invasive detection of the methylation level of a DNA methylation molecular marker associated with resistance to Streptococcus agalactiae in GIFT tilapia, characterized in that: The nucleotide sequences thereof are shown in SEQ ID NOs: 1 and 2.

8. A kit for non-invasive or minimally invasive detection of the methylation level of DNA methylation molecular markers associated with resistance to Streptococcus agalactiae in GIFT tilapia, characterized in that: Containing the primer according to claim 7.

9. The kit according to claim 1, characterized in that It also contains one or more of a plasma cfDNA extraction reagent, a bisulfite conversion kit, a PCR reagent, an agarose gel electrophoresis reagent, a DNA nucleic acid dye reagent, a DNA gel recovery kit, and a TA cloning reagent.

10. A method for breeding GIFT tilapia with high resistance to Streptococcus agalactiae, characterized in that: Collect GIFT tilapia cfDNA, and use GIFT tilapia with high resistance to Streptococcus agalactiae as a parent for breeding according to the method of claim 2.

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