SNP sites of the antibiotic resistance gene tet(Q) and their applications

Abstract

This invention discloses the SNP sites of the antibiotic resistance gene tet(Q) and their applications, belonging to the field of poultry genetic breeding and molecular marker-assisted selection technology. The SNP sites are one of the following: (1) 1:131826194, a C or T polymorphism; (2) 1:131828702, a T or A polymorphism; (3) 1:131829512, an A or G polymorphism; (4) 1:131829538, a G or C polymorphism. The application of the SNP sites and specific primers for detecting the SNP sites in the screening, identification, and marker-assisted breeding of ducks is also discussed. Through large-scale population association analysis, this invention screened and obtained four SNP markers related to the abundance of the duck intestinal antibiotic resistance gene tet(Q) and its host bacterium Phocaeicola. These markers showed a strong correlation with the abundance of the antibiotic resistance gene tet(Q) and its host bacterium Phocaeicola. The four SNP loci of this invention can be used for assisted breeding of domestic ducks, enabling rapid screening of large-scale breeding populations and reducing breeding costs.

Description

Technical Field

[0001] This invention relates to the SNP site of the antibiotic resistance gene tet(Q) and its application, belonging to the field of poultry genetic breeding and molecular marker-assisted selection technology. Background Technology

[0002] The gut of an animal contains a vast and complex microbial ecosystem. The microbial community within this system is known as the "microbial organ" of the host. They not only play a crucial role in the animal's growth and development, nutritional metabolism, and immune barrier, but also serve as an important reservoir of antibiotic resistance genes (ARGs).

[0003] Domestic ducks are one of the world's important sources of animal protein, with their meat and eggs being rich in nutrients, containing a variety of high-quality amino acids and vitamins. The extensive use of antibiotics can easily lead to the emergence and spread of antibiotic resistance genes (ARGs), promoting the diffusion of these genes within the gut microbiota and environment. This can easily disrupt gut ecological stability, increase the risk of opportunistic infections and diseases, and create ecological risks.

[0004] tet(Q) is an antibiotic resistance gene encoding a ribosomal protective protein that mediates high levels of resistance to multiple tetracycline antibiotics through an "antibiotic target protection" mechanism. This gene is often located on mobile genetic elements, allowing it to rapidly transfer between bacteria via conjugative transposons, making it a high-risk ARG.

[0005] Traditional methods to combat the spread of antibiotic resistance genes mainly rely on strict control of antibiotic use and enhanced infection prevention and control. However, given the vast pool of antibiotic resistance genes already widely present in the environment, animal flora, and human symbiotic microbiota, these measures have limitations in blocking horizontal transfer of resistance genes (HGT). They cannot completely curb the continuous flow and spread of resistance genes between different bacteria, or even between different habitats, through mobile genetic elements. To ensure the sustainable development of animal husbandry and the ecological environment, systematically screening and immobilizing genetic markers associated with low antibiotic resistance gene loads using methods such as genomic selection and molecular marker technology, and breeding high-performance, low-antibiotic-resistance-gene-loaded, green and healthy poultry, is a fundamental strategy to control the spread of resistance at the host source and achieve synergistic development of quality improvement, efficiency enhancement, and biosafety. Summary of the Invention

[0006] In view of the above-mentioned prior art, the present invention provides the antibiotic resistance gene tet(Q) and its host bacterium Phocaeicola SNP site and its application.

[0007] This invention is achieved through the following technical solution: The SNP site for the antibiotic resistance gene tet(Q) is one of the following: (1) 1:131826194, located at position 131826194 on chromosome 1 of duck, is associated with gene TBL1X. This SNP is either C (wild type) or T (mutant) polymorphism. (2) 1:131828702, located at position 131828702 on chromosome 1 of duck, is associated with gene TBL1X. This SNP is either T (wild type) or A (mutant type) polymorphism; (3) 1:131829512, located at position 131829512 on chromosome 1 of duck, is associated with gene TBL1X. This SNP is either A (wild type) or G (mutant type) polymorphism. (4) 1:131829538, located at position 131829538 on chromosome 1 of duck, is associated with gene TBL1X. This SNP is either G (wild type) or C (mutant type) polymorphism.

[0008] Application of any one or more combinations of the above-mentioned SNP loci in the screening, identification, and assisted breeding of domestic ducks. Application of specific primers for detecting any one or more of the above-mentioned SNP loci in the screening, identification, and assisted breeding of domestic ducks.

[0009] The nucleotide sequences of the specific primers for detecting SNP site 1:131826194 are shown in SEQ ID NO.1~2.

[0010] The nucleotide sequences of the specific primers for detecting SNP site 1:131828702 are shown in SEQ ID NO.3~4.

[0011] The nucleotide sequences of the specific primers for detecting SNP site 1:131829512 are shown in SEQ ID NO.5~6.

[0012] The nucleotide sequences of the specific primers for detecting SNP site 1:131829538 are shown in SEQ ID NO.5~6.

[0013] Furthermore, the breed of domestic duck is the Suichuan Red-feathered Duck.

[0014] Specific applications include: early screening of green poultry with low antibiotic resistance gene load, optimization of the genetic background of core breeding populations, and cultivation of strains with excellent reproductive performance.

[0015] A specific application method is as follows: Genotyping of selected domestic duck individuals using the aforementioned SNP markers is performed. Individuals with heterozygous genotypes (any one or more of T / C, A / T, G / A, C / G) are selected as breeding stock, while individuals with inferior genotypes are eliminated. This allows for the rapid construction of a breeding population with excellent reproductive performance and accelerates the genetic improvement process. The samples tested are intestinal contents (such as cecal contents) or fecal samples (containing antibiotic resistance genes and host bacteria).

[0016] This invention, through large-scale population association analysis, screened and identified four SNP markers associated with the abundance of the duck intestinal antibiotic resistance gene tet(Q) and its host bacterium Phocaeicola. These markers are located in specific regions of duck chromosome 1 and exhibit a strong association with the abundance of the antibiotic resistance gene tet(Q) and its host bacterium Phocaeicola. Individuals with dominant genotypes showed significantly lower abundances of the antibiotic resistance gene tet(Q) and its host bacterium Phocaeicola compared to other genotypes, enabling precise marking of the target trait. The four SNP loci of this invention can be used for assisted breeding of domestic ducks, enabling rapid screening of large-scale breeding populations and reducing breeding costs.

[0017] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0018] Figure 1 GWAS region map of the antibiotic resistance gene tet(Q).

[0019] Figure 2 GWAS region map of the Phocaeicola genus.

[0020] Figure 3 GWAS region map of P. coprocola.

[0021] Figure 4 GWAS region diagram of strain MAG8727 in the genus Phocaeicola.

[0022] Figure 5 GWAS region diagram of strain MAG19359 in the genus Phocaeicola.

[0023] Figure 6 GWAS region diagram of strain MAG21658 in the genus Phocaeicola.

[0024] Figure 7 : Chained unbalanced blocks among 4 SNPs.

[0025] Figure 8The results of agarose gel electrophoresis of the amplification products are shown. From left to right, the 10 lanes are as follows: lanes 1-3 contain amplification products of duck genomic DNA carrying three genotypes of SNP (1:131826194); lanes 4-6 contain amplification products of duck genomic DNA carrying three genotypes of SNP (1:131828702); lanes 7-9 contain amplification products of duck genomic DNA carrying three genotypes of SNP (1:131829512 and 1:131829538); and lane 10 is the marker.

[0026] Figure 9 Sequencing peak diagram of the C / C genotype of SNP (1:131826194).

[0027] Figure 10 Sequencing peak diagram of the T / C genotype of SNP (1:131826194).

[0028] Figure 11 Sequencing peak diagram of the T / T genotype of SNP (1:131826194).

[0029] Figure 12 Sequencing peak diagram of the T / T genotype of SNP (1:131828702).

[0030] Figure 13 Sequencing peak diagram of the A / T genotype of SNP (1:131828702).

[0031] Figure 14 Sequencing peak diagram of the A / A genotype of SNP (1:131828702).

[0032] Figure 15 Sequencing peak diagrams of A / A and G / G genotypes of SNPs (1:131829512 and 1:131829538).

[0033] Figure 16 Sequencing peak diagrams of G / A and C / G genotypes of SNPs (1:131829512 and 1:131829538).

[0034] Figure 17 Sequencing peak diagrams of G / G and C / C genotypes of SNPs (1:131829512 and 1:131829538).

[0035] Figure 18 Box plot showing the difference in abundance of the antibiotic resistance gene tet(Q) among individuals with different genotypes. The horizontal axis represents the three genotypes corresponding to the SNP (1:131826194), and the vertical axis represents the abundance of tet(Q).

[0036] Figure 19 Box plot showing the difference in abundance of host bacteria g_Phocaeicola among individuals with different genotypes. The horizontal axis represents the three genotypes of the SNP (1:131826194), and the vertical axis represents the abundance of the genus Phocaeicola.

[0037] Figure 20 Box plot showing the difference in abundance of host bacterium s_P. coprocola among individuals with different genotypes, where the horizontal axis represents the three genotypes of SNP (1:131826194) and the vertical axis represents the abundance of P. coprocola.

[0038] Figure 21 Box plot showing the difference in abundance of host bacterium MAG8727 among individuals with different genotypes. The horizontal axis represents the three genotypes of SNP (1:131826194), and the vertical axis represents the abundance of MAG8727.

[0039] Figure 22 Box plot showing the difference in abundance of host bacterium MAG19359 among individuals with different genotypes. The horizontal axis represents the three genotypes of SNP (1:131826194), and the vertical axis represents the abundance of MAG19359.

[0040] Figure 23 Box plot showing the difference in abundance of host bacterium MAG21658 among individuals with different genotypes. The horizontal axis represents the three genotypes of SNP (1:131826194), and the vertical axis represents the abundance of MAG21658.

[0041] Figure 24 Box plot showing the difference in abundance of the antibiotic resistance gene tet(Q) among individuals with different genotypes. The horizontal axis represents the three genotypes corresponding to the SNP (1:131828702), and the vertical axis represents the abundance of tet(Q).

[0042] Figure 25 Box plot showing the difference in abundance of host bacteria g_Phocaeicola among individuals with different genotypes. The horizontal axis represents the three genotypes of SNP (1:131828702), and the vertical axis represents the abundance of the genus Phocaeicola.

[0043] Figure 26 Box plot showing the difference in abundance of host bacterium s_P. coprocola among individuals with different genotypes, where the horizontal axis represents the three genotypes of SNP (1:131828702) and the vertical axis represents the abundance of P. coprocola.

[0044] Figure 27Box plot showing the difference in abundance of host bacterium MAG8727 among individuals with different genotypes. The horizontal axis represents the three genotypes of SNP (1:131828702), and the vertical axis represents the abundance of MAG8727.

[0045] Figure 28 Box plot showing the difference in abundance of host bacterium MAG19359 among individuals with different genotypes. The horizontal axis represents the three genotypes of SNP (1:131828702), and the vertical axis represents the abundance of MAG19359.

[0046] Figure 29 Box plot showing the difference in abundance of host bacterium MAG21658 among individuals with different genotypes. The horizontal axis represents the three genotypes of SNP (1:131828702), and the vertical axis represents the abundance of MAG21658.

[0047] Figure 30 Box plot showing the difference in abundance of the antibiotic resistance gene tet(Q) among individuals with different genotypes. The horizontal axis represents the three genotypes corresponding to the SNP (1:131829512), and the vertical axis represents the abundance of tet(Q).

[0048] Figure 31 Box plot showing the difference in abundance of host bacteria g_Phocaeicola among individuals with different genotypes. The horizontal axis represents the three genotypes of the SNP (1:131829512), and the vertical axis represents the abundance of the genus Phocaeicola.

[0049] Figure 32 Box plot showing the difference in abundance of host bacterium s_P. coprocola among individuals with different genotypes, where the horizontal axis represents the three genotypes of SNP (1:131829512) and the vertical axis represents the abundance of P. coprocola.

[0050] Figure 33 Box plot showing the difference in abundance of host bacterium MAG8727 among individuals with different genotypes. The horizontal axis represents the three genotypes of SNP (1:131829512), and the vertical axis represents the abundance of MAG8727.

[0051] Figure 34 Box plot showing the difference in abundance of host bacterium MAG19359 among individuals with different genotypes. The horizontal axis represents the three genotypes of SNP (1:131829512), and the vertical axis represents the abundance of MAG19359.

[0052] Figure 35Box plot showing the difference in abundance of host bacterium MAG21658 among individuals with different genotypes. The horizontal axis represents the three genotypes of SNP (1:131829512), and the vertical axis represents the abundance of MAG21658.

[0053] Figure 36 Box plot showing the difference in abundance of the antibiotic resistance gene tet(Q) among individuals with different genotypes. The horizontal axis represents the three genotypes corresponding to the SNP (1:131829538), and the vertical axis represents the abundance of tet(Q).

[0054] Figure 37 Box plot showing the difference in abundance of host bacteria g_Phocaeicola among individuals with different genotypes. The horizontal axis represents the three genotypes of the SNP (1:131829538), and the vertical axis represents the abundance of the genus Phocaeicola.

[0055] Figure 38 Box plot showing the difference in abundance of host bacterium s_P. coprocola among individuals with different genotypes, where the horizontal axis represents the three genotypes of SNP (1:131829538) and the vertical axis represents the abundance of P. coprocola.

[0056] Figure 39 Box plot showing the difference in abundance of host bacterium MAG8727 among individuals with different genotypes. The horizontal axis represents the three genotypes of SNP (1:131829538), and the vertical axis represents the abundance of MAG8727.

[0057] Figure 40 Box plot showing the difference in abundance of host bacterium MAG19359 among individuals with different genotypes. The horizontal axis represents the three genotypes of SNP (1:131829538), and the vertical axis represents the abundance of MAG19359.

[0058] Figure 41 Box plot showing the difference in abundance of host bacterium MAG21658 among individuals with different genotypes. The horizontal axis represents the three genotypes of SNP (1:131829538), and the vertical axis represents the abundance of MAG21658. Detailed Implementation

[0059] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0060] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0061] The meanings of some terms involved in this invention are as follows: SNP marker: Single Nucleotide Polymorphism (SNP) refers to DNA sequence polymorphism caused by a single nucleotide variation at the genomic level, and is the most common type of genetic variation.

[0062] MAG: Metagenomic Assembly Genome, refers to a strain-level microbial genome constructed based on community-level metagenomic data from microbiome samples.

[0063] ARG: Antibiotic resistance genes are genes found in the genomes of bacteria or other microorganisms that give them resistance to antibiotics.

[0064] GWAS: Genome-wide association studies, a method for searching for associations between genetic variations (usually single nucleotide polymorphisms, SNPs) and complex traits across the entire genome.

[0065] MAF: Minor allele frequency, refers to the proportion of the second most common allele in a specific population.

[0066] LD: Linkage Disequilibrium, refers to the non-random association between alleles at different loci within a population.

[0067] Primer pair: A pair of artificially synthesized oligonucleotide fragments, complementary to the DNA sequences upstream and downstream of the target SNP site, respectively, used for PCR amplification of specific fragments.

[0068] Experiment 1: Screening and analysis of SNP sites related to the antibiotic resistance gene tet(Q) 1. Extraction of duck genomic DNA and whole-genome resequencing Twenty-seven adult Suichuan Red-feathered Ducks were selected, and cecal tissue samples were collected for genomic DNA extraction and resequencing, followed by variant detection. Host genomic DNA was extracted from the cecal tissue using a DNA extraction kit. The total DNA concentration was determined using a Qubit Fluorometer, and DNA integrity was assessed using 1% agarose gel electrophoresis. Subsequently, whole-genome resequencing (average sequencing depth approximately 14×) was performed on each DNA sample using the BGI DNBSEQ platform. The resulting paired-end reads were aligned to the duck reference genome (IASCAAS_PekinDuck_T2T genome version) using BWA software. Genotype data for each individual was obtained using samtools, GATK, and vcftools. Quality control of the obtained genotype data was performed using plink v1.9, retaining high-quality loci with a minor allele frequency (MAF) >5% and a genotype deletion rate <10%. A total of 12,969,991 molecular marker loci were finally obtained on the duck autosomes.

[0069] 2. Duck metagenomic sequencing and MAG assembly Three hundred adult Suichuan Red-feathered Ducks were selected, and their intestinal contents were collected for metagenomic sequencing and MAG assembly. DNA was extracted from all samples using the MagPure kit, and for samples requiring long-read sequencing, a high-molecular-weight DNA enrichment process was employed. Short-read paired-end sequencing was performed on the BGIDNBSEQ-T7 and Illumina NovaSeq 6000 platforms, respectively; and some samples were selected for long-read sequencing on PromethlON-48 (ONT, UK). After quality control, host removal, and MAG assembly of the raw data, gene and species annotation was performed using GTDB-Tk. A total of 18,442 MAGs and their abundance in each sample were obtained, and the results are expressed as TPM values.

[0070] 3. Annotation and quantification of antibiotic resistance genes and their host species Using the RGI tool, the non-redundant gene catalog obtained from metagenomic sequencing was aligned to the Comprehensive Antibiotic Resistance Database (CARD), retaining alignment results with ≥40% sequence identity. The obtained ARGs were then quantified; the Salmon tool was used to re-attach filtered paired-end reads to the non-redundant gene catalog to obtain the abundance of ARGs in each sample, with results expressed as TPM values. Based on continuous sequences containing ARG open reading frames (ORFs), their host species were traced, and subsequently, species identification was performed using Kraken2 and standard databases.

[0071] 4. Genome-wide association analysis Genome-wide association analysis (GWAS) was performed on the abundance of antibiotic resistance genes and their host bacteria in 297 samples with both genomic and metagenomic data using an ADDO-based dominant-effects model. The model was y = M·β + μ + e, where y is the phenotypic residual vector adjusted for environmental fixed effects and other covariates; M is the matrix encoding the genotype effect of a given SNP; β is the genotype effect vector M based on the encoded SNP; and μ and e are vectors of random genetic background and residual random effects. The results showed that the antibiotic resistance gene tet(Q) was significantly associated with host bacteria g_Phocaeicola, s_P. coprocola, MAG8727, MAG19359, and MAG21658 (all three MAGs belong to the genus Phocaeicola) in the same region of chromosome 1 of the duck genome.

[0072] GWAS results for the antibiotic resistance gene tet(Q) are as follows: Figure 1 As shown, the GWAS results for the genus *Phocaeicola* are as follows: Figure 2 As shown, the GWAS results for P. coprocola are as follows: Figure 3 As shown, the GWAS results for MAG8727 are as follows: Figure 4 As shown, the GWAS results for MAG19359 are as follows: Figure 5 As shown, the GWAS results for MAG21658 are as follows: Figure 6 As shown. Figures 1-6A physical map of duck chromosome 1 was displayed, showing the precise coordinates of the target SNP markers, the distribution of upstream and downstream genes, and the LD value (r²) between the top SNP and other surrounding SNPs, thus clarifying the genomic location of the markers. GWAS results showed that the top SNP sites significantly associated with the abundance of the antibiotic resistance gene tet(Q) and its host bacteria *Phocaeicola*, *P. coprocola*, MAG8727, MAG19359, and MAG21658 were all concentrated on chromosome 1. These six GWAS results shared a common chromosomal significance threshold (1 × 10⁻⁶) on chromosome 1. -6 The region shares four top SNPs, the information of which is shown in Table 1. This information includes physical location, nucleotide variation type, wild-type nucleotides, mutant nucleotides, minor allele frequency, and the gene where the top SNPs are located. Linkage disequilibrium blocks among the four SNPs are shown in Table 1. Figure 7 As shown, there is a strong linkage disequilibrium among these SNPs.

[0073] Table 1 Information on the 4 top SNPs

[0074] 5. Primer design and PCR amplification Download the duck reference genome (IASCAAS_PekinDuck_T2T genome version) from the NCBI database, which contains the sequences of these 4 top SNPs. Use the primer design tool provided by NCBI to design specific PCR primer pairs. The information of the specific primers is shown in Table 2, and the nucleotide sequences of the specific primers are shown in SEQ ID NO.1~6.

[0075] Table 2 Information on specific primers

[0076] Subsequently, using the extracted duck DNA as a template, PCR amplification was performed according to the designed primers. The PCR system consisted of: 1 μL DNA template, 2 μL each of forward and reverse primers, 1 μL DNA polymerase, 1 μL dNTPs, 25 μL 2×PhantaMax Buffer, and 18 μL double-distilled water, prepared to a 50 μL PCR system. The PCR reaction conditions were: 95℃, 3 min; 95℃, 15 s; 56℃, 15 s; 72℃, 60 s; 35 cycles; 72℃, 5 min; 4℃, infinity.

[0077] 6. Agarose gel electrophoresis and Sanger sequencing The amplification products were detected by 1% agarose gel electrophoresis. The results of the agarose gel electrophoresis of the amplification products are as follows: Figure 8As shown in the figure. The results showed that the duck genomic DNA amplification products carrying the three genotypes of SNP (1:131826194) could amplify a specific band of 631 bp, the duck genomic DNA amplification products carrying the three genotypes of SNP (1:131828702) could amplify a specific band of 312 bp, and the duck genomic DNA amplification products carrying the three genotypes of SNP (1:131829512 and 1:131829538) could amplify a specific band of 328 bp, all consistent with expectations. This indicates that specific fragments of the expected size can be amplified from duck genomic DNA, and the primer pairs designed in this invention have good specificity.

[0078] The amplified products were subjected to Sanger sequencing, and the accuracy of the sequences was verified by alignment using Minimap2 software. The genotypes of the four top SNP sites were determined using Chromas software.

[0079] Sequencing results: The nucleotide sequence of the C / C genotype of SNP (1:131826194) is shown in SEQ ID NO.7, the T / C genotype of SNP (1:131826194) is shown in SEQ ID NO.8, and the T / T genotype of SNP (1:131826194) is shown in SEQ ID NO.9. The T / T genotype of SNP (1:131828702) is shown in SEQ ID NO.10, the A / T genotype of SNP (1:131828702) is shown in SEQ ID NO.11, and the A / A genotype of SNP (1:131828702) is shown in SEQ ID NO.12. The A / A and G / G genotypes of SNPs (1:131829512 and 1:131829538) are shown in SEQ ID NO. 13; the G / A and C / G genotypes of SNPs (1:131829512 and 1:131829538) are shown in SEQ ID NO. 14; and the G / G and C / C genotypes of SNPs (1:131829512 and 1:131829538) are shown in SEQ ID NO. 15. It should be noted that individuals with different genotypes exhibit genetic differences, and the signal initiation region of Sanger sequencing may be cluttered, resulting in low reliability. Therefore, the initial sequence needs to be excised. Inconsistent excision sites may also lead to inconsistent lengths of subsequent sequences. In addition, some heterozygous individuals only showed the mutated strand or the wild-type strand in Sanger sequencing. However, the sequencing electrophoresis map showed that the heterozygous site had two peaks, with both the reference genotype and the mutated genotype.

[0080] The sequencing peak diagram of the C / C genotype of SNP (1:131826194) is shown below. Figure 9 As shown, the sequencing peak diagram of the T / C genotype of SNP (1:131826194) is as follows. Figure 10 As shown, the sequencing peak diagram of the SNP (1:131826194) T / T genotype is as follows. Figure 11 As shown in the figure. The sequencing peak diagram of the T / T genotype of SNP (1:131828702) is as follows. Figure 12 As shown, the sequencing peak diagram of the A / T genotype of SNP (1:131828702) is as follows. Figure 13 As shown, the sequencing peak diagram of the A / A genotype of SNP (1:131828702) is as follows. Figure 14 As shown in the figure. The sequencing peak diagrams of the A / A and G / G genotypes of SNPs (1:131829512 and 1:131829538) are as follows. Figure 15 As shown, the sequencing peak diagrams of the G / A and C / G genotypes of SNPs (1:131829512 and 1:131829538) are as follows. Figure 16 As shown, the sequencing peak diagrams of the G / G and C / C genotypes of SNPs (1:131829512 and 1:131829538) are as follows. Figure 17 As shown.

[0081] 7. Association analysis of topSNP site polymorphism with tet(Q) and its host bacterium Phocaeicola The association between the polymorphisms (different genotypes) of four top SNP sites on the TBL1X gene of Suichuan Red-feathered Duck and the abundance of the antibiotic resistance gene tetQ and its host bacteria, Phocaeicola, P. coprocola, MAG8727, MAG19359, and MAG21658, was statistically analyzed using the ggpubr package in R software. Wilcoxon test was used for pairwise comparisons between groups, and p-value < 0.05 was used as the criterion for significance.

[0082] Association analysis results as follows Figures 18-41 As shown, Figures 18-41 The distribution range, median, and significance p-values ​​of the abundance of the antibiotic resistance gene tet(Q) and its host bacterium *Phocaeicola* among individuals with different genotypes are shown. It is evident that the abundance of the antibiotic resistance gene tet(Q), the genus *Phocaeicola*, the species *P. coprocola*, MAG8727, MAG19359, and MAG21658 did not differ significantly between the two homozygous genotypes of the four top SNPs, but significant differences existed between the heterozygous genotype and the two homozygous genotypes. This suggests that the abundance of the antibiotic resistance gene tet(Q) and its host bacterium *Phocaeicola* may be influenced by dominant inheritance effects in ducks.

[0083] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. Use of SNP loci of antibiotic resistance gene tet(Q) in screening, identifying or assisting breeding of domestic ducks, characterized in that, The SNP site is any one or a combination of two or more of the following four SNP sites: (1) 1:131826194, located at position 131826194 on chromosome 1 of duck, is associated with gene TBL1X. This SNP is either C or T polymorphic. (2) 1:131828702, located at position 131828702 on chromosome 1 of duck, is associated with gene TBL1X. This SNP is either T or A polymorphic. (3) 1:131829512: Located at position 131829512 on chromosome 1 of duck, belonging to gene TBL1X. This SNP is either A or G polymorphic. (4) 1:131829538, located at position 131829538 on chromosome 1 of duck, is associated with gene TBL1X. This SNP is either G or C polymorphic.

2. The use of specific primers for SNP sites of antibiotic resistance gene tet(Q) in screening, identifying or assisting breeding of domestic ducks, characterized in that, The SNP site is any one or a combination of two or more of the following four SNP sites: (1) 1:131826194, located at position 131826194 on chromosome 1 of duck, is associated with gene TBL1X. This SNP is either C or T polymorphic. (2) 1:131828702, located at position 131828702 on chromosome 1 of duck, is associated with gene TBL1X. This SNP is either T or A polymorphic. (3) 1:131829512: Located at position 131829512 on chromosome 1 of duck, belonging to gene TBL1X. This SNP is either A or G polymorphic. (4) 1:131829538, located at position 131829538 on chromosome 1 of duck, is associated with gene TBL1X. This SNP is either G or C polymorphic.

3. The application according to claim 2, characterized in that: The nucleotide sequences of the specific primers for detecting SNP site 1:131826194 are shown in SEQ ID NO.1~2.

4. The application according to claim 2, characterized in that: The nucleotide sequences of the specific primers for detecting SNP site 1:131828702 are shown in SEQ ID NO.3~4.

5. The application according to claim 2, characterized in that: The nucleotide sequences of the specific primers for detecting SNP site 1:131829512 are shown in SEQ ID NO.5~6.

6. The application according to claim 2, characterized in that: The nucleotide sequences of the specific primers for detecting SNP site 1:131829538 are shown in SEQ ID NO.5~6.

7. The application according to claim 1 or 2, characterized in that: The breed of domestic duck mentioned is the Suichuan Red-feathered Duck.

8. The application according to claim 1 or 2, characterized in that, Specific applications include: early screening of green poultry with low antibiotic resistance gene load, optimization of the genetic background of core breeding populations, and development of strains with excellent reproductive performance.

9. The application according to claim 1 or 2, characterized in that, The specific application method is as follows: SNP marker genotyping is performed on selected domestic duck individuals, individuals with heterozygous genotypes are selected as breeding individuals, and individuals with inferior genotypes are eliminated, thereby quickly building a breeding population with excellent reproductive performance and accelerating the genetic improvement process. The heterozygous genotypes refer to: the genotype of SNP locus 1:131826194 is T / C; the genotype of SNP locus 1:131828702 is A / T; the genotype of SNP locus 1:131829512 is G / A; and the genotype of SNP locus 1:131829538 is C / G.

10. The application according to claim 9, characterized in that: The object of the test is intestinal contents or feces.

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