SNP (Single Nucleotide Polymorphism) molecular marker related to drug resistance of Eimeria to diclazuril and application of SNP molecular marker

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker related to drug resistance of Eimeria coccidiosis to diclazuril and application of the SNP molecular marker. The SNP site aiming at the eimeria tenella is selected from at least one of the 574th site, the 1303rd site and the 1394th site of the DNA fragment with the nucleotide sequence of SEQ ID NO: 1; the SNP site aiming at the eimeria heap is selected from at least one of the 556 site, the 1210 site and the 1301 site of the DNA fragment with the nucleotide sequence of SEQ ID NO: 2. The invention provides an SNP molecular marker related to the drug resistance of Eimeria to diclazuril and a primer for detecting the genotype of an SNP site, and is suitable for identification or auxiliary identification of the drug resistance of Eimeria to diclazuril, and the identification result has high accuracy; a reliable basis is provided for screening and detecting diclazuril drug-resistant strains, and scientific prevention and control of coccidiosis and improvement of production benefits of farms are facilitated.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a SNP molecular marker associated with diclazuril resistance in Eimeria coccidia and its application. Background Technology

[0002] Coccidiosis is caused by Eimeria coccidia ( Eimeria spp. Eimeria is an important intestinal parasitic disease in poultry. It poses a serious economic threat to the poultry industry and is one of the most economically damaging chicken diseases. Currently, seven species of Eimeria have been identified and recognized: *Eimeria tenella*, *Eimeria toxicaria*, *Eimeria spurulenta*, *Eimeria prematurei*, *Eimeria gianti*, *Eimeria brucellae*, and *Eimeria lenienti*. Different species of Eimeria parasitize different parts of the intestine, primarily damaging intestinal epithelial cells, leading to impaired digestion and absorption, reduced feed conversion ratio, and decreased production performance.

[0003] Current prevention and control of coccidiosis primarily relies on drug prophylaxis, supplemented by vaccination. Anticoccidial drugs are mainly divided into two categories: polyether ionocarrier drugs and chemically synthesized drugs. With the widespread use of these drugs, drug-resistant strains have been reported. To date, almost all anticoccidial drugs have resulted in drug-resistant strains, which exhibit some degree of cross-resistance to other drugs of the same class or with the same mechanism of action. The development of drug resistance reduces the effectiveness of anticoccidial drugs and seriously challenges the control of coccidiosis.

[0004] Diclazuril, a polyether-based triazine phenylacetonitrile chemically synthesized drug, is currently the least used anticoccidial drug, possessing high efficacy, low toxicity, and a broad spectrum of anticoccidial activity. Diclazuril has good control effects on coccidiosis in various livestock and poultry, including chickens, turkeys, ducks, geese, peacocks, quails, and rabbits. Diclazuril began widespread field application in 1990, but reports of diclazuril resistance emerged in 1994. Currently, the detection method for diclazuril resistance still relies on cage experiments. While this method yields relatively reliable results, it is complex, time-consuming, and requires a large number of fresh coccidia oocysts and experimental animals. Therefore, there is an urgent need to develop a rapid method for detecting diclazuril-resistant coccidia to better guide coccidiosis medication and reduce the economic losses caused by coccidiosis infection in the livestock industry. Summary of the Invention

[0005] This invention provides SNP molecular markers related to diclazuril resistance in Eimeria coccidia and their applications.

[0006] In a first aspect, the present invention provides a composition for identifying or assisting in the identification of SNP site genotypes associated with diclazuril resistance in Eimeria coccidia, wherein when the Eimeria coccidia includes Eimeria tenella, the SNP site is selected from at least one of positions 574, 1303, and 1394 of the DNA fragment whose nucleotide sequence is SEQ ID NO:1; and when the Eimeria coccidia includes Eimeria spurious, the SNP site is selected from at least one of positions 556, 1210, and 1301 of the DNA fragment whose nucleotide sequence is SEQ ID NO:2.

[0007] Taking *Eimeria tenella*, a species sensitive to diclazuril, as an example, the nucleotide sequence of the detected fragment is SEQ ID NO:1. The SNP sites are selected from at least one of positions 574, 1303, and 1394 of the DNA fragment shown in SEQ ID NO:1, specifically the sites marked with underline as follows: 5'-ATGTCGTCTGCGGATCCGTCGCTGCGTGCGTCGCCCGCGTGGCTGGCACACAGCTCGGAGCTGAGCAGCAGCAGCAACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCTGCAGCAGCAGCAGCAGCAGCAGCAGCTGGCGG TGGGGGGGCGTGTGCGCGCTGCTGGGGGGCGTGCTGCTGCACGTGGCTCTGGGGACAGTTTATTGTTTGGGAGTGTCTTCAATTTATTTTATTTCTTTTTTAAAAAGCAGCAGCAGCAGCAGCATCACTGCAGCAGCAGCAGCA GCAATAACAGCAGCAAGCTTCAGCGCCATGGCCCTCGGCATGCCCCTCGGCGGGGCCCTCGAAAGAAAGCTCGGACCCAGGCCGGTGAGTCTGCTGGGGGGTTTGCTGTTCGTGGCGGGTGTCTATACATCTCAGCTAACCATC AAATTAGGGTTTAGGGTTTTCCTTCTTTCCTACGGAGTCCTTTACGGGTTTGGCCTCGGCGTGGCCTACACAAGTCCCTTAGTTGCTGCGCTGCGCTGGTTCCCCCAACAAGGGCTGGTGAGCGGGCTGATCACTGCAGGG TTTGGGCTGGGGAGTGTTGTGTTCTCGCCGCTGCAGCAGCAGCTGCTAAACCCTCGGGGGGCCCCCCCCACGTGGGCAGCAGCAGCAGCAGCAGCAGCAGCAGAGGGGCCCCCCGAAATGTACTACAAAGACCCCGAAATCCTGCAAAGAGTTCCTGCTGCTTTCTCCACTCTTGCTGCTGTTTATTTGGTTTTGGTGCTGCTGGCGGTTTGTCTCATTTCAAACCCCCCCGAGCTGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGAAGGCGCTCGCGCAGGAGGAGGAGGGGCAGCTGCTGCAGCGCCAGCTGCAGCCGCGGCAGCAGCAGCAGCAGCAGCAGCAGCAGCCACCGCTGGCTGCTGTGCTGCAGTCTCCCCAGTTCCTGCTGCTGTGGGGCAGCTTTTTGCTGCAGGGCATCAGCATTATCTTTGTGGCCAGCTTCTGGAAGCCCCTTGCTGCTGCTGCTGCTGCTGCAGGGCTGCAGCAGCTAAGCGAAACCCAACTTGCTGCTGTTGGTGCTGCAGCAAGTGCATGCAACGCCCTGGGCAGAATTCTCTGGGGAAAATATGCAGACGCTCATTCCTTTCAACAGGCTCTAGTGCTGCTCTCGAGCCTTTGGTTTGCTGCTCTGCTGCTGCTGTCTGTTGCAGCAGCAATTAACGGGGCCCTTTATGCTGCAGCAGTTTGTCTAAACTTCTTTTGTCTTGGAGGAAAC T TTGCACTTTTCCCTGCAGGCACTGCTGCTCTCTTTGGCAGACAAGCCTTCGGGCGCGTGTACGGGTTTGTGTTTTTGGCCCAGCTGGGCT C TTCTTTGGTGGGGGCTTTTGTGTTGCCGCTGCTGACGAGACAGCTGGGAGTTTCGGGCACCTGCCGAGCGGTGTCTCTTTGCTGCCTCTCCGTTGTCGCTTTGCTGCTGTCCCTAAAAGGCTCTCGCGCTTACCAGCTGTTCAAGCCTTAG-3’。

[0008] Taking *Eimeria tenella* resistant to diclazuril as an example, the nucleotide sequence of the detected fragment is SEQ ID NO:2. The SNP sites are selected from at least one of positions 556, 1210, and 1301 of the DNA fragment shown in SEQ ID NO:2, specifically the sites marked with underline as follows: 5'-ATGCCTTCATCTGACCCCGCGCTATCGCTGGGCCGCTGTCCCCCTTCGTGGGTGCATGACGGAGAAGGGTTTAAATATGCTAGCAGCAGCAGCAGCAGCAGCAGGAGCAGCAGGAGCACTCTAATAAAGGGTTGTT TGTGCGTTTTAGGGGGTGTATTGCTGCACGTAGCCTTGGGAACTGTCTATACACTCGGGGCTTCTGCTGTTTATTTTATTTCTTATCTAAGAGACAGCAGCAACTCCTCGCTGCGCTTAGCTGCTGCTGCTAAGATAACA GCAGCAGGGTTTAGCAGCATGGCTATAGGGATGCCTCTCGGTGGGGCCTTAGAGAGAAAATTAGGACCCAGGGTGGTGTCTCTGACAGGGGGTCTTATATTTATTTTGGGTGTCTACACTTCTTCAGTAACAATAAAAA TTAATTTTCTTGCTTTTCTTCTTCTTACGGACTCGTCTGCGGCTTTGGGCTCGGCGTTGCATACACCAGCCCCCTTGTTGCTGCTCTTCGATGGTTTCCTGATCAACAAGGGCTTATTAGTGGTTTAATAACAGCAGGA CTTGGCTTAGGCGCTGTTGTGTTTTCGCCGCTGCAGCAGCAGCTGCTAAACCCTTCGGGTGTATCTGCAGCATATCAACCTTATGCTGACTCTCCTCAAGAATTATATTATACAGATAAAACTATTTTAGCTGCTGTACCCTCTGTCTTTCGTCGTCTTGCTGCTGCTTATCTCCTTCTTGTTGTTGCTGCTGTTTGCCTCCTGTCCACCCCTGCCCCAGCGGAGACCAAGCAGCAGCAGCAGCAGCAGCAGCAGCAGGAGCAGCAGATCATGCTGCAGCAGCAGCAGCAGCTAAGGCTGAACGTCTTGCTGCTGTCTCCTCAGTTTCTGCTGCTGTGGAGTACATTCTTGCTTGAGGGGATCAGCATTATATTTGTTGCAAGCTGCTGGAAGCCTCTTGCTGCTGCTGCTGCACCGCAGCAGCAACTCACGGAGCCGCAGCTAGCTGCTGTTGGTGCTGCAGCAAGTGTATTCAATGCTCTAGGAAGAATTGCGTGGGGGAAGGTTGCTGACAGCAGCACTTTTCAGCATGCACTTTTGCTGCTGTCTAGCAGCTGGTGCGTAGGTTTGTTGCTGCTATCGAACGCAGCAAATATTCACTGGTGCGCGTACACCGCAGCAGTTTGCTTAAACTTTTTTTGTTTAGGCGGCAAC T TTGCTCTCTTTCCGGCGGGCACCTCATTCCTCTTCGGGAGACAGTCCTTTGGTCGGGTGTATGGTTTTGTATTTACTGCACAGCTGGGAT C TTCTCTTGTTGGGGCCTTCGCCATCCCCGCTTTAACTCAACACCTGGGTCTTGCTGCTGCTTGCCGCATTGTCTCCGTTTGTTGTTTTGCTGTTGCTGCTCTCCTCTTCTCTCTTAGAGGCAGTTCTGTGTGGAAGCAATTTAAGGCCTAG-3’。

[0009] The composition as described above includes a first primer pair for identifying or assisting in the identification of Eimeria tenella SNP site combinations and / or a second primer pair for identifying or assisting in the identification of Eimeria spp. SNP site combinations. The first primer pair comprises a single-stranded DNA molecule with the nucleotide sequence SEQ ID NO:4 and a single-stranded DNA molecule with the nucleotide sequence SEQ ID NO:5; The second primer pair comprises a single-stranded DNA molecule with the nucleotide sequence SEQ ID NO:6 and a single-stranded DNA molecule with the nucleotide sequence SEQ ID NO:7.

[0010] In a second aspect, the present invention provides a product comprising any of the compositions described above.

[0011] The product described above can be a kit.

[0012] As described above, the kit may also include other reagents or PCR tubes required for PCR.

[0013] Thirdly, the present invention provides a composition for identifying or assisting in the identification of SNP locus genotypes of Eimeria coccidia resistance to diclazuril, or the application of the above-described products, wherein the application is selected from at least one of A1)-A2): A1) Application in identifying or assisting in the identification of diclazuril resistance in Eimeria coccidia; A2) Application in the preparation of products for the identification or auxiliary identification of diclazuril resistance in Eimeria coccidia.

[0014] Fourthly, the present invention provides a method for identifying or assisting in the identification of resistance of Eimeria coccidia to diclazuril, comprising: RNA samples were obtained from the Eimeria coccidia to be tested, and cDNA samples were obtained by reverse transcription. Detect the genotype of the above SNP sites in the cDNA sample; The genotype of the SNP locus was used to determine whether *Eimeria* coccidia were resistant to diclazuril.

[0015] As described above, when the Eimeria coccidia includes Eimeria tenella, when the genotype at position 574 of the DNA fragment of SEQ ID NO:1 is T, it indicates that the Eimeria tenella is sensitive to diclazuril; when the genotype at position 574 of the DNA fragment of SEQ ID NO:1 is C, it indicates that the Eimeria tenella is resistant to diclazuril. When the genotype at position 1303 of the DNA fragment of SEQ ID NO:1 is T, it indicates that the *Eimeria tenella* is sensitive to diclazuril; when the genotype at position 1303 of the DNA fragment of SEQ ID NO:1 is C, it indicates that the *Eimeria tenella* is resistant to diclazuril. When the genotype at position 1394 of the DNA fragment of SEQ ID NO:1 is C, it indicates that the *Eimeria tenella* is sensitive to diclazuril; when the genotype at position 1394 of the DNA fragment of SEQ ID NO:1 is A, it indicates that the *Eimeria tenella* is resistant to diclazuril.

[0016] As described above, when the Eimeria coccidia includes Eimeria spp., if the genotype at position 556 of the DNA fragment of SEQ ID NO:2 is T, it indicates that Eimeria spp. is sensitive to diclazuril; if the genotype at position 556 of the DNA fragment of SEQ ID NO:2 is C, it indicates that Eimeria spp. is resistant to diclazuril. When the genotype at position 1210 of the DNA fragment of SEQ ID NO:2 is T, it indicates that the *Eimeria tenella* is sensitive to diclazuril; when the genotype at position 1210 of the DNA fragment of SEQ ID NO:2 is C, it indicates that the *Eimeria tenella* is resistant to diclazuril. When the genotype at position 1301 of the DNA fragment of SEQ ID NO:2 is C, it indicates that the *Eimeria tenella* species is sensitive to diclazuril; when the genotype at position 1301 of the DNA fragment of SEQ ID NO:2 is A, it indicates that the *Eimeria tenella* species is resistant to diclazuril.

[0017] Fifthly, the present invention provides a method for screening Eimeria coccidia that are sensitive to diclazuril, comprising: RNA samples of Eimeria coccidia to be screened were obtained and cDNA samples were obtained by reverse transcription. Detect the genotype of the above SNP sites in the cDNA sample; Genotype T in the DNA fragment with nucleotide sequence SEQ ID NO:1 at position 574 and / or genotype T in the DNA fragment with nucleotide sequence SEQ ID NO:1 at position 1303 and / or genotype C in the DNA fragment with nucleotide sequence SEQ ID NO:1 at position 1394 was selected from Eimeria species to be screened. The selected Eimeria species are sensitive to diclazuril. Genotype T of the DNA fragment with nucleotide sequence SEQ ID NO:2 at position 556 and / or genotype T of the DNA fragment with nucleotide sequence SEQ ID NO:2 at position 1210 and / or genotype C of the DNA fragment with nucleotide sequence SEQ ID NO:2 at position 1301 was selected from Eimeria species to be screened. The selected Eimeria species were sensitive to diclazuril.

[0018] In a sixth aspect, the present invention provides a method for screening Eimeria coccidia resistant to diclazuril, comprising: RNA samples of Eimeria coccidia to be screened were obtained and cDNA samples were obtained by reverse transcription. Detect the genotype of the above SNP sites in the cDNA sample; From the Eimeria coccidia to be screened, Eimeria tenella with genotype C at position 574 of the DNA fragment with nucleotide sequence SEQ ID NO:1 and / or genotype C at position 1303 of the DNA fragment with nucleotide sequence SEQ ID NO:1 and / or genotype A at position 1394 of the DNA fragment with nucleotide sequence SEQ ID NO:1 were screened. The screened Eimeria tenella were resistant to diclazuril. Genotype C of the DNA fragment with nucleotide sequence SEQ ID NO:2 at position 556 and / or genotype C of the DNA fragment with nucleotide sequence SEQ ID NO:2 at position 1210 and / or genotype A of the DNA fragment with nucleotide sequence SEQ ID NO:2 at position 1301 was selected from Eimeria species to be screened. The selected Eimeria species were resistant to diclazuril.

[0019] The method described above, for detecting the genotype of the SNP site in a cDNA sample, includes: using the cDNA sample as a template, performing PCR amplification using any of the compositions described above, sequencing the amplification products, and obtaining the genotype of the SNP site based on the sequencing results.

[0020] As described above, the RNA sample is derived from the oocysts of the *Eimeria* coccidia.

[0021] As described above, the *Eimeria* coccidia may include a single species of *Eimeria*, or multiple species of *Eimeria*, such as at least one of *Eimeria tenella*, *Eimeria toxicaria*, *Eimeria scabra*, *Eimeria precocious*, *Eimeria giant*, *Eimeria bryonica*, and *Eimeria lenientis*. Further, the *Eimeria* coccidia may include at least *Eimeria tenella* and / or *Eimeria scabra*.

[0022] This invention provides a SNP molecular marker associated with diclazuril resistance in Eimeria coccidia and primers for detecting the genotype of the SNP loci. It is suitable for the identification or auxiliary identification of diclazuril resistance in Eimeria coccidia, and the identification results have high accuracy and are completely consistent with the identification results of traditional cage experiments. It provides a reliable basis for screening and detecting diclazuril-resistant strains, which helps to scientifically control coccidiosis and improve the production efficiency of farms. Attached Figure Description

[0023] Figure 1 To achieve ΔSNP through genome resequencing The results of index analysis of candidate regions for drug resistance genes in different infiltrating strains; where A is the location result of drug resistance gene infiltrating strain BC1F1; B is the location result of drug resistance gene infiltrating strain BC2F2; C is the location result of drug resistance gene infiltrating strain BC3F2; D is the location result of drug resistance gene infiltrating strain BC4F2; the red arrows indicate the candidate regions for drug resistance genes located on chromosome 9.

[0024] Figure 2 The results are shown in the gel electrophoresis of the PCR amplification products in Example 1; where M is the DNA marker, DL5000; lane 1 is the amplification product using the Ea primer pair for detecting Eimeria tenella as primers and Eimeria tenella cDNA as template; lane 2 is the amplification product using the Et primer pair for detecting Eimeria tenella as primers and Eimeria tenella cDNA as template.

[0025] Figure 3 The protein sequences of homologous drug resistance genes of Eimeria tenella, Eimeria scabra, and Toxoplasma gondii in Example 4 of this invention were compared. Et, Ea1, and Tg are the drug resistance SNP mutation sites corresponding to the three strains, respectively. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0028] Example 1: Construction of a diclazuril resistance gene-introduced strain of Eimeria coccidia This embodiment uses *Eimeria tenella* as the research object and constructs drug-resistant gene-introduced strains using genetic hybridization and backcrossing strategies. The specific methods are as follows: 1. Determination of parent strain drug resistance: The experiment was divided into two groups, each consisting of three coccidia-free AA broilers. The first group was inoculated with a laboratory-preserved drug-resistant strain of Eimeria tenella (Rd strain), and the second group was inoculated with the Eimeria tenella Howton strain (H strain). Each chicken was infected with 50,000 oocysts, and diclazuril (1 ppm) was added to the drinking water. Medication was administered starting two days before inoculation. Fecal samples were collected on day 5, and fresh feces from each group were collected daily from day 6 to day 12 using the "five-point sampling method" and tested using the saturated saline flotation method. If oocysts were detected, the strain was considered drug-resistant; if no oocysts were detected, the strain was considered susceptible.

[0029] 2. Crossbreeding of drug-resistant and susceptible parents: Three 3-week-old AA broilers were inoculated with 500 fresh Rd strain oocysts and 5000 fresh H strain oocysts, respectively. Fecal oocysts were collected on days 6-9 and marked as F1 generation. Three 4-week-old AA broilers were then infected with 10,000 oocysts per F1 generation broiler, and 1 ppm diclazuril was added to their drinking water. Fecal oocysts were collected on days 6-9. Fecal oocysts collected at 9 days were designated as F2 generation. F2 generation oocysts exhibited diclazuril resistance. The experimental design included 3 replicates.

[0030] 3. Backcrossing and self-pollination of hybrid offspring: Simultaneously inoculate 3 four-week-old AA broiler chickens with 1000 F2 generation oocysts and 10000 fresh H strain oocysts. Collect the 6th... Fecal oocysts collected on day 9 were designated as generation BC1F1. Three 4-week-old AA broilers were infected with 10,000 oocysts per broiler, and diclazuril was added to their drinking water. Oocysts were collected on day 6. Fecal oocysts were collected after 9 days and marked as generation BC1F2. The backcrossing and selfcrossing process was repeated three times to obtain drug-resistant gene-infiltrated strains (F1, F2, BC1F1, BC1F2, BC2F1, BC2F2, BC3F1, BC3F2, BC4F1, BC4F2).

[0031] Example 2: Identification of SNP molecular markers associated with drug resistance 1. DNA Template Preparation: Following standardized laboratory procedures, oocysts from the diclazuril resistance gene-infiltrating line were collected. Glass beads (1 mm in diameter) were added to the oocyst suspension, and the mixture was thoroughly shaken and centrifuged, retaining the precipitate. 500 mL of CTAB lysis buffer and 40 μL of proteinase K were added to the precipitate, and the precipitate was dispersed. The centrifuge tube was placed in a 55°C metal bath for digestion for 2-3 h. After the suspension returned to room temperature, 20 μL of RNase was added, and the centrifuge tube was placed in a 37°C metal bath for further digestion for 30 min. After returning to room temperature, an equal volume of DNA extraction buffer was added, and the centrifuge tube was vigorously shaken. The supernatant was collected after centrifugation. The liquid was transferred to a new centrifuge tube, and an equal volume of pre-chilled isopropanol was added. The mixture was repeatedly inverted and mixed thoroughly. Incubate at 20°C for at least 30 minutes. Centrifuge at 4°C, retain the precipitate, wash twice with 75% ethanol, and after complete evaporation, dissolve the DNA precipitate in preheated (50°C) enzyme-free water. Place the DNA sample in... Store at 80℃.

[0032] 2. Genome Resequencing: Samples were sequenced and library constructed by Shanghai Paisenno Biotechnology Co., Ltd. Sequencing depth for each sample was 100x. After extracting the genome from all infiltrating strains, the DNA samples were tested. Once the DNA samples passed the tests, they were randomly fragmented using a Covaris ultrasonic disruptor. The entire library preparation process included end repair, A-tailing, sequencing adapter addition, purification, and PCR amplification. After library construction, preliminary quantification was performed using Qubit 2.0 to dilute the library. Then, the insert fragments were detected using an Agilent 2100. Once the insert fragment size met expectations, Q... PCR was used to accurately quantify the effective concentration of the libraries to ensure library quality. After the libraries passed the detection, different libraries were pooled into flow cells according to the effective concentration and the required amount of data for sequencing. After cBOT clustering, sequencing was performed using the Illumina HiSeq PE150 high-throughput sequencing platform.

[0033] 3. Bioinformatics Analysis: After filtering the data, quality control is performed. High-quality clean reads are aligned to the reference genome to extract reliable SNP marker information. Parameters are set, and SNP calls are used to generate VCF files. ΔSNPs are then analyzed using the QTLseqr software package. Index analysis was used to locate candidate regions of drug resistance genes on chromosomes. Figure 1The mutations were eventually located at 2.7Mb-2.9Mb on chromosome 9. The mutated genes within this region were then annotated using SnpEff software. When analyzing mutation sites within candidate regions, mutations with low mutation rates, mutations located in intergenic and intronic regions, and synonymous mutations were excluded. Only non-synonymous mutations located in exon regions with high mutation rates were ultimately selected (Table 1).

[0034] Table 1. Candidate genes screened by whole-genome resequencing

[0035] 4. Candidate Gene PCR Detection: Genes within the candidate regions were amplified using high-fidelity DNA polymerase. A 1.0% agarose gel was prepared, and electrophoresis was performed at 120 V for 15 min. After electrophoresis, the results were observed using gel imaging. The target band was excised from the gel, and its purity was recovered and purified using a DNA gel extraction kit. The target band was then ligated into pEASy... BluntSimple cloning vector (purchased from Beijing TransGen Biotech Co., Ltd.) was then transformed into competent Trans1 cells. T1 (purchased from Beijing TransGen Biotech Co., Ltd.). After being plated on ampicillin-resistant plates and cultured overnight, single clones were picked for PCR identification, and positive clones were selected and sequenced by Beijing Ruiboxingke Biotechnology Co., Ltd. Single clones with correct sequencing results were selected by sequence alignment.

[0036] 5. Gene Editing to Verify Candidate Gene Function: To verify whether the aforementioned candidate genes exist that are related to diclazuril resistance in *Eimeria tenella*, overexpression strains carrying the mutant genes were constructed or homologous substitutions were achieved using CRISPR / Cas9. These strains were then screened under drug stress. Based on the phenotypic results of the transgenic strains under diclazuril drug stress, the diclazuril resistance gene in *Eimeria tenella* was determined.

[0037] Based on the above experimental results, the mutation in the coccidia ETH2_0944800 gene is associated with diclazuril resistance. A SNP mutation exists at nucleotide 1394 of the coccidia ETH2_0944800 gene; at this site, C corresponds to diclazuril sensitivity, while A corresponds to diclazuril resistance (Table 2). The cDNA cDNA of the ETH2_0944800 gene has the nucleotide sequence shown in SEQ ID NO:1, and its SNP sites are indicated by underlined positions.

[0038] Table 2. Sequence alignment results of susceptible and resistant strains

[0039] Example 3: Identification of SNP molecular markers related to drug resistance in *Eimeria tenella* 1. Screening for drug-resistant genes: The drug-resistant gene ETH2_0944800 discovered in Example 2 was subjected to homology comparison, and the homology gene of this gene in *Eimeria tenella* was found to be EaM00000004936. Following the method in Example 2, this gene was amplified in the diclazuril-resistant strain of *Eimeria tenella* (EaR, laboratory-preserved), and compared with the gene in the susceptible strain (EaWT). An SNP mutation was found at nucleotide position 556 of the EaM00000004936 gene. At this position, T corresponds to the diclazuril-susceptible strain, while C corresponds to the diclazuril-resistant strain. The cDNA nucleotide sequence of the EaM00000004936 gene is the sequence shown in SEQ ID NO:2. The SNP mutation site of the ETH2_0944800 gene in Eimeria tenella from Example 2 was mapped to nucleotide 1301 of EaM00000004936 in Eimeria sclerotium.

[0040] 2. Gene Editing to Verify Candidate Gene Function: To verify whether the above candidate genes are related to diclazuril resistance in *Eimeria tenella*, overexpressing strains carrying the mutant genes were constructed and then screened under drug stress. Based on the phenotypic results of the transgenic strains under diclazuril drug stress, the diclazuril resistance gene in *Eimeria tenella* was identified.

[0041] Based on the above experimental results, the EaM00000004936 gene mutation in *Eimeria tenella* is associated with diclazuril resistance. An SNP mutation exists at nucleotide 556 of the *EaM00000004936* gene; at this site, T corresponds to diclazuril sensitivity, while C corresponds to diclazuril resistance (Table 3).

[0042] The SNP mutation at nucleotide 1301 of the EaM00000004936 gene of Eimeria tenella can lead to diclazuril resistance. At this site, C corresponds to diclazuril sensitivity, while A corresponds to diclazuril resistance (Table 3).

[0043] 3. The nucleotide mutation site at position 556 of the *Eimeria tenella* gene (EaM00000004936) was mapped to nucleotide 574 of the *Eimeria tenella* gene (ETH2_0944800). After mutating the T-site of the susceptible strain to the C-site of the resistant strain, an overexpression strain of the *Eimeria tenella* mutant gene was constructed and screened under drug stress. Based on the phenotypic results of the transgenic strains under diclazuril drug stress, it was determined whether this mutation site was also a diclazuril resistance site in *Eimeria tenella*.

[0044] Based on the above results, there is an SNP mutation at nucleotide 574 of the Eimeria tenella ETH2_0944800 gene. At this site, T corresponds to diclazuril sensitivity, while C corresponds to diclazuril resistance (Table 3).

[0045] Table 3. Sequence alignment results of susceptible and resistant strains.

[0046] Example 4: Identification of SNP molecular markers related to drug resistance in Toxoplasma gondii 1. Screening for drug-resistant genes: After homology comparison of the drug-resistant gene ETH2_0944800 found in Example 2, the homolog of this gene in Toxoplasma gondii was identified as TGGT1_233540 (SEQ ID NO:3). This gene was amplified in diclazuril-resistant Toxoplasma gondii strains and compared with the gene in susceptible strains. It was found that the TGGT1_233540 gene has an SNP mutation at nucleotide position 1384. At this position, T corresponds to diclazuril-susceptible strains, while C corresponds to diclazuril-resistant strains.

[0047] 2. Gene editing operation to verify the function of candidate genes: To verify whether the mutation sites of the above candidate genes are SNP mutation sites related to diclazuril resistance in Eimeria tenella, the TGGT1_233540 gene was compared with the protein sequences of Eimeria tenella and Eimeria spp., and it was found that the mutation sites were all located on conserved domains of the three genes. Figure 3 The Toxoplasma gondii mutation sites were mapped to Eimeria tenella, specifically nucleotide 1303 of the ETH2_0944800 gene and nucleotide 1210 of the EaM00000004936 gene. T corresponds to diclazuril-sensitive strains, while C corresponds to diclazuril-resistant strains. Overexpression strains carrying the mutant gene were constructed and then screened under drug stress. Based on the phenotypic results of the transgenic strains under diclazuril drug stress, it was determined whether the SNP mutation site of the Toxoplasma gondii TGGT1_233540 gene could induce diclazuril resistance in Eimeria tenella.

[0048] Based on the above experimental results, the SNP mutation at nucleotide 1303 of the *Eimeria tenella* ETH2_0944800 gene can lead to diclazuril resistance. At this site, T corresponds to diclazuril sensitivity, while C corresponds to diclazuril resistance (Table 4). Similarly, the SNP mutation at nucleotide 1210 of the *Eimeria spp.* EaM00000004936 gene can also lead to diclazuril resistance. At this site, T corresponds to diclazuril sensitivity, while C corresponds to diclazuril resistance.

[0049] Table 4. Sequence alignment results of susceptible and resistant strains

[0050] Example 5: Primer design for detecting SNP molecular markers associated with diclazuril resistance in Eimeria coccidia. Taking *Eimeria tenella* as an example, during primer design, this invention discovered that the *Eimeria tenella* ETH2_0944800 gene contains multiple intron sequences. To efficiently and specifically amplify DNA fragments containing these SNP sites, the template used should be a reverse-transcribed cDNA sample. Therefore, primers were subsequently designed targeting the cDNA sequence. In this embodiment, two pairs of primers targeting this gene from two different *Eimeria* species were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd. The specific primer sequences are shown in Table 5.

[0051] Table 5. Primers for PCR amplification of two types of coccidia genes

[0052] The method for identifying or assisting in the identification of diclazuril resistance in Eimeria coccidia using the above primer composition includes the following steps: 1. RNA Extraction: Take an appropriate amount of *Eimeria* coccidia oocysts, add 1 mm diameter glass beads (purchased from Sigma) to the oocyst suspension, shake thoroughly, centrifuge and retain the precipitate; add 1 mL of TRIzol lysis buffer to the precipitate after glass bead shaking for thorough lysis, add 200 μL of chloroform to the lysis buffer, shake vigorously and let stand. Centrifuge at 4℃, collect the supernatant, add 500 μL of pre-chilled isopropanol, invert and mix well, then... Incubate at 20°C for 30 minutes. Centrifuge at 10,000 rpm for 15 minutes at 4°C. After centrifugation, wash the precipitate twice with 75% ethanol. Discard the supernatant, allow it to evaporate at room temperature until odorless, then add RNase-free water and mix well. Place the extracted RNA sample in... Store at 80℃.

[0053] 2. cDNA sample acquisition: Take 10 pg A 1 μg RNA sample was reverse transcribed into cDNA using the Novizan reverse transcription kit. The resulting cDNA sample was then placed in... Store at 20℃. The specific reverse transcription system is shown in Table 6.

[0054] Table 6. cDNA Reverse Transcription System

[0055] 3. PCR Amplification: Using cDNA as a template, PCR amplification was performed using the primers in Table 3. The specific PCR reaction system and program are as follows: PCR reaction system: 2 μL each of upstream and downstream primers (10 μmol / L), 2 μL of cDNA template, 25 μL of 2×Rapid TaqMaster Mix (purchased from Novizan Biotechnology Co., Ltd.), and deionized water to make up to 50 μL. PCR reaction program: 95℃, 30s; 95℃, 30s; 64℃, 60s; 72℃, 60s, 35 cycles; 72℃, 10min.

[0056] Taking *Eimeria tenella* and *Eimeria filamentosa* as examples, the PCR amplification products obtained using the above method were detected by gel electrophoresis. The detection results are as follows: Figure 2 As shown in Table 5, the target fragments of the lengths obtained by PCR amplification are as shown in Table 5.

[0057] 4. Sequencing and drug resistance identification: Sequencing the amplified products (e.g., nanopore sequencing) and obtaining the genotype of the SNP site based on the sequencing results.

[0058] When the genotype at position 574 of the DNA fragment in SEQ ID NO:1 is T, it indicates that the *Eimeria tenella* species is susceptible to diclazuril; when the genotype at position 574 of the DNA fragment in SEQ ID NO:1 is C, it indicates that the *Eimeria tenella* species is resistant to diclazuril. When the genotype at position 1303 of the DNA fragment in SEQ ID NO:1 is T, it indicates that the *Eimeria tenella* species is susceptible to diclazuril; when the genotype at position 1303 of the DNA fragment in SEQ ID NO:1 is C, it indicates that the *Eimeria tenella* species is resistant to diclazuril. When the genotype at position 1394 of the DNA fragment in SEQ ID NO:1 is C, it indicates that the *Eimeria tenella* species is susceptible to diclazuril. When the genotype at position 1394 of the DNA fragment NO:1 is A, it indicates that the *Eimeria tenella* species is resistant to diclazuril.

[0059] When the genotype at position 556 of the DNA fragment in SEQ ID NO:2 is T, it indicates that the *Eimeria* species is susceptible to diclazuril; when the genotype at position 556 of the DNA fragment in SEQ ID NO:2 is C, it indicates that the *Eimeria* species is resistant to diclazuril. When the genotype at position 1210 of the DNA fragment in SEQ ID NO:2 is T, it indicates that the *Eimeria* species is susceptible to diclazuril; when the genotype at position 1210 of the DNA fragment in SEQ ID NO:2 is C, it indicates that the *Eimeria* species is resistant to diclazuril. When the genotype at position 1301 of the DNA fragment in SEQ ID NO:2 is C, it indicates that the *Eimeria* species is susceptible to diclazuril. When the genotype at position 1301 of the DNA fragment NO:2 is A, it indicates that the described Eimeria coccidia is resistant to diclazuril.

[0060] Example 6: Detection of diclazuril resistance in mixed chicken coccidia samples collected in the field based on SNP molecular markers. Fecal samples were collected from five chicken farms in different counties and districts of four cities in Zhejiang Province (the samples were named mixed populations 1-5 because they contained multiple Eimeria species). Diclazuril resistance was detected using the diclazuril resistance SNP molecular markers described in Examples 2-4. The specific methods are as follows: 1. Template preparation: Coccidia samples isolated from 5 chicken farms were orally inoculated into coccidia-free chickens. Feces were collected on the 5th day after inoculation, and 6 samples were collected. Nine-day-old oocysts were purified 48 hours after sporulation. The potassium dichromate containing the oocysts was poured into a clean centrifuge tube and centrifuged at 3600 rpm for 5 min. The precipitate was resuspended in sodium hypochlorite solution, placed on ice for 2 min, and centrifuged at 3600 rpm for 5 min. The supernatant containing the oocysts was then transferred to another clean centrifuge tube, 10 volumes of PBS were added, and the mixture was centrifuged again. This process was repeated three times. Repeat 5 times until there is no residual sodium hypochlorite odor; the precipitate is the purified oocyst. For subsequent RNA sample preparation, refer to Example 5.

[0061] 2. PCR amplification system and reaction conditions The PCR amplification primers are shown in Table 5, and the reaction system and procedure are the same as in Example 5.

[0062] 3. PCR product sequence alignment The amplified bands were recovered from the gel and sent to Beijing Qingke Biotechnology Co., Ltd. for Ultra (nanopore) sequencing.

[0063] 4. Accuracy assessment of diclazuril resistance SNP site detection The PCR products from the samples obtained in step 3 were sequence aligned, and the results are shown in Tables 7 and 8. The target band was amplified in all five field samples. Ultra (nanopore) sequencing of the PCR products showed that mutations in the diclazuril resistance gene were detected in all five chicken farms.

[0064] Table 7. Sequence alignment results of Eimeria tenella in susceptible strains, drug-resistant strains, and mixed populations.

[0065] Table 8. Sequence alignment results of *Eimeria tenella* in susceptible strains, drug-resistant strains, and mixed populations.

[0066] Meanwhile, all the above-mentioned field samples were used for drug resistance assessment in cage experiments. The method included adding 1 mg / kg of diclazuril to the drinking water before inoculation. On the second day after drug administration, chickens were orally inoculated with the field samples. Feces were collected on the fifth day after inoculation, and oocysts were collected on the tenth day. Microscopic examination was performed to determine whether oocysts were expelled, thereby determining whether the samples exhibited a diclazuril resistance phenotype. The drug resistance phenotype verification index was referenced from: Guangdong Provincial Association of Animal Husbandry and Veterinary Medicine: T / GDAAV 0214-2025 Method for Detection of Coccidiosis Resistance in Chickens (S). Guangzhou: Guangdong Provincial Association of Animal Husbandry and Veterinary Medicine, 2025.

[0067] The results showed that all five field-mixed strains were resistant to diclazuril, indicating that the SNP molecular marker detection results were consistent with the cage-rearing experiment results. Therefore, the SNP molecular marker-based PCR detection method of this invention can accurately reflect whether a strain possesses a diclazuril resistance phenotype. It can be used not only for detecting diclazuril resistance in purebred Eimeria coccidia but also effectively applied to the detection of clinical samples (mixed strains).

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for identifying or assisting in the identification of SNP loci genotypes associated with diclazuril resistance in Eimeria coccidia, characterized in that, When the Eimeria species includes Eimeria tenella, the SNP site is selected from at least one of positions 574, 1303, and 1394 of the DNA fragment whose nucleotide sequence is SEQ ID NO:1; when the Eimeria species includes Eimeria sclerotium, the SNP site is selected from at least one of positions 556, 1210, and 1301 of the DNA fragment whose nucleotide sequence is SEQ ID NO:

2.

2. The composition according to claim 1, characterized in that, The composition includes a first primer pair for identifying or assisting in the identification of the SNP locus genotype of Eimeria tenella and / or a second primer pair for identifying or assisting in the identification of the SNP locus genotype of Eimeria sclerotium. The first primer pair includes a single-stranded DNA molecule with the nucleotide sequence SEQ ID NO:4 and a single-stranded DNA molecule with the nucleotide sequence SEQ ID NO:5; The second primer pair comprises a single-stranded DNA molecule with nucleotide sequence SEQ ID NO:6 and a single-stranded DNA molecule with nucleotide sequence SEQ ID NO:

7.

3. The product, characterized in that, Includes the composition according to any one of claims 1-2.

4. The composition for identifying or assisting in the identification of SNP loci genotypes of Eimeria coccidia resistance to diclazuril as described in any one of claims 1-2, or the application of the product described in claim 3, characterized in that, The application is selected from at least one of A1)-A2): A1) Application in identifying or assisting in the identification of diclazuril resistance in Eimeria coccidia; A2) Application in the preparation of products for the identification or auxiliary identification of diclazuril resistance in Eimeria coccidia.

5. A method for identifying or assisting in the identification of resistance of Eimeria coccidia to diclazuril, characterized in that, include: RNA samples were obtained from the Eimeria coccidia to be tested, and cDNA samples were obtained by reverse transcription. Detecting the genotype of the SNP site described in the composition of any one of claims 1-2 in a cDNA sample; The genotype of the SNP locus was used to determine whether Eimeria coccidia were resistant to diclazuril.

6. The method according to claim 5, characterized in that, When the Eimeria coccidia include Eimeria tenella, if the genotype at position 574 of the DNA fragment of SEQ ID NO:1 is T, it indicates that the Eimeria tenella is sensitive to diclazuril; if the genotype at position 574 of the DNA fragment of SEQ ID NO:1 is C, it indicates that the Eimeria tenella is resistant to diclazuril. When the genotype at position 1303 of the DNA fragment of SEQ ID NO:1 is T, it indicates that the *Eimeria tenella* is sensitive to diclazuril; when the genotype at position 1303 of the DNA fragment of SEQ ID NO:1 is C, it indicates that the *Eimeria tenella* is resistant to diclazuril. When the genotype at position 1394 of the DNA fragment of SEQ ID NO:1 is C, it indicates that the *Eimeria tenella* is sensitive to diclazuril; when the genotype at position 1394 of the DNA fragment of SEQ ID NO:1 is A, it indicates that the *Eimeria tenella* is resistant to diclazuril.

7. The method according to claim 5, characterized in that, When the Eimeria coccidia include Eimeria spp., if the genotype at position 556 of the DNA fragment of SEQ ID NO:2 is T, it indicates that Eimeria spp. is sensitive to diclazuril; if the genotype at position 556 of the DNA fragment of SEQ ID NO:2 is C, it indicates that Eimeria spp. is resistant to diclazuril. When the genotype at position 1210 of the DNA fragment of SEQ ID NO:2 is T, it indicates that the *Eimeria tenella* is sensitive to diclazuril; when the genotype at position 1210 of the DNA fragment of SEQ ID NO:2 is C, it indicates that the *Eimeria tenella* is resistant to diclazuril. When the genotype at position 1301 of the DNA fragment of SEQ ID NO:2 is C, it indicates that the *Eimeria tenella* species is sensitive to diclazuril; when the genotype at position 1301 of the DNA fragment of SEQ ID NO:2 is A, it indicates that the *Eimeria tenella* species is resistant to diclazuril.

8. A method for screening Eimeria coccidia susceptible to diclazuril, characterized in that, include: RNA samples of Eimeria coccidia to be screened were obtained and cDNA samples were obtained by reverse transcription. Detecting the genotype of the SNP site described in the composition of any one of claims 1-2 in a cDNA sample; Genotype T in the DNA fragment with nucleotide sequence SEQ ID NO:1 at position 574 and / or genotype T in the DNA fragment with nucleotide sequence SEQ ID NO:1 at position 1303 and / or genotype C in the DNA fragment with nucleotide sequence SEQ ID NO:1 at position 1394 was selected from Eimeria species to be screened. The selected Eimeria species are sensitive to diclazuril. Genotype T of the DNA fragment with nucleotide sequence SEQ ID NO:2 at position 556 and / or genotype T of the DNA fragment with nucleotide sequence SEQ ID NO:2 at position 1210 and / or genotype C of the DNA fragment with nucleotide sequence SEQ ID NO:2 at position 1301 was selected from Eimeria species to be screened. The selected Eimeria species were sensitive to diclazuril.

9. A method for screening Eimeria coccidia resistant to diclazuril, characterized in that, include: RNA samples of Eimeria coccidia to be screened were obtained and cDNA samples were obtained by reverse transcription. Detecting the genotype of the SNP site described in any one of claims 1-2 in a cDNA sample; From the Eimeria coccidia to be screened, Eimeria tenella with genotype C at position 574 of the DNA fragment with nucleotide sequence SEQ ID NO:1 and / or genotype C at position 1303 of the DNA fragment with nucleotide sequence SEQ ID NO:1 and / or genotype A at position 1394 of the DNA fragment with nucleotide sequence SEQ ID NO:1 were screened. The screened Eimeria tenella were resistant to diclazuril. Genotype C of the DNA fragment with nucleotide sequence SEQ ID NO:2 at position 556 and / or genotype C of the DNA fragment with nucleotide sequence SEQ ID NO:2 at position 1210 and / or genotype A of the DNA fragment with nucleotide sequence SEQ ID NO:2 at position 1301 was selected from Eimeria species to be screened. The selected Eimeria species were resistant to diclazuril.

10. The method according to any one of claims 5-9, characterized in that, The RNA sample was derived from the oocysts of the *Eimeria* coccidia.

Images