Disease resistance trait-specific SNP site of dairy sheep and screening method thereof

Abstract

The application discloses a milk sheep disease resistance trait specific SNP site and a screening method thereof, which comprises the following steps: 1) target segment enrichment; 2) molecular error correction and noise reduction; 3) multi-algorithm combined screening; and 4) population sample review and system establishment. According to the milk sheep disease resistance trait specific SNP site and the screening method thereof, the target gene segment target enrichment method is adopted, the screening range is reduced, invalid detection data is reduced, and the milk sheep disease resistance related low-abundance variant sites can be efficiently captured; the UMI label error correction processing is matched, the amplification error and base mismatch problems can be effectively avoided, the detection result error condition can be reduced, and the accuracy of the SNP site screening can be greatly improved.

Description

Technical Field

[0001] This application relates to the field of molecular breeding and gene detection technology for dairy sheep, and in particular to specific SNP sites for disease resistance traits in dairy sheep and their screening methods. Background Technology

[0002] Dairy sheep farming is an important part of animal husbandry. Disease resistance, as a core economic trait in dairy sheep farming, directly affects farming efficiency, product quality, and the sustainable development of the industry. When dairy sheep are infected with diseases, it not only leads to stunted growth and reduced milk production, but may also trigger the spread of diseases and increase farming costs. Therefore, breeding dairy sheep breeds with strong disease resistance has significant practical importance and industrial value.

[0003] Single nucleotide polymorphisms (SNPs), as the most widespread form of genetic variation in the genome, are characterized by high stability, wide distribution, and ease of detection, making them a core vector for screening molecular markers for disease resistance traits in livestock and poultry and for assisted breeding. Currently, there are some studies on SNP site screening for disease resistance traits in dairy sheep, but existing technologies still have significant shortcomings and cannot meet the practical needs of precision breeding.

[0004] Existing screening methods mostly employ whole-genome sequencing or exon sequencing technologies, failing to target specific functional regions of genes related to disease resistance traits in dairy sheep for enrichment. This results in significant sequencing data redundancy, low screening efficiency, and difficulty in capturing rare SNP sites with low abundance. Furthermore, existing methods lack effective molecular error correction mechanisms; PCR amplification bias and base mismatches easily lead to false negatives and false positives, affecting screening accuracy.

[0005] Furthermore, existing technologies mostly use single-function prediction algorithms for SNP annotation, and do not filter them in conjunction with the genetic background specific to dairy sheep populations. This makes it easy to misclassify universally conserved SNPs across breeds and linkage variations introduced by hybridization as disease resistance-specific SNPs, resulting in poor specificity and weak functionality of the selected loci, which cannot be effectively applied to the molecular breeding practice of disease resistance in dairy sheep.

[0006] Therefore, we propose specific SNP sites for disease resistance traits in dairy sheep and their screening methods. Summary of the Invention

[0007] This application aims to at least partially solve one of the technical problems in the aforementioned technologies.

[0008] To achieve the above objectives, the first aspect of this application proposes a method for screening SNP sites specific to disease resistance traits in dairy sheep, comprising the following steps: 1) Targeted region enrichment: Based on the conserved motifs of genes related to disease resistance in dairy sheep, a liquid-phase capture probe for the target micro-region was designed to enrich the gene regions related to disease resistance in dairy sheep in a targeted manner. 2) Molecular error correction and noise reduction: UMI single-molecule unique tag library construction technology is used to perform source tracing and base error correction on the low-abundance variant fragments obtained in step 1). 3) Multi-algorithm joint screening: The candidate SNPs obtained after step 2) are jointly annotated by two functional prediction algorithms, and filtered by the genetic background database of dairy sheep populations to remove general conserved SNPs and linkage variants, and to screen out disease resistance trait-specific SNP loci. 4) Population sample verification and system establishment: Amplicon targeted resequencing combined with HRM high-resolution melting curve typing technology was used to verify the candidate SNP sites obtained in step 3) through population sample verification, confirm the stability and specificity of the sites, and establish a rapid screening and determination system for SNP sites specific to the disease resistance trait of dairy sheep.

[0009] The second aspect of this application proposes SNP sites specific to the disease resistance trait in dairy sheep. The SNP sites meet the following criteria: the allele frequency in the target dairy sheep breed is 0.01-0.05, the allele frequency in other sheep breeds is <0.01, and the functional annotation confirms that they can affect the secretion efficiency of disease resistance-related proteins or the immune response capacity of dairy sheep.

[0010] In addition, the screening method for disease resistance-specific SNP sites in dairy sheep proposed in this application may also have the following additional technical features: As a further description of the above technical solution: The liquid-phase capture probe described in step 1) is a biotin-modified single-stranded DNA probe with a length of 50-120 bp and a homology of ≥98% with conserved motifs of genes related to disease resistance traits in dairy sheep.

[0011] As a further description of the above technical solution: The specific process of targeted enrichment described in step 1) is as follows: The liquid-phase capture probe was hybridized with a fragment of dairy sheep genomic DNA. The probe concentration in the hybridization system was 2-10 fmol. The hybridization reaction conditions were denaturation at 95℃ for 2 minutes and hybridization at 60℃ for 1 hour. After hybridization, streptavidin magnetic beads were used to capture the hybridization product. After elution and purification, the enriched target fragment of the disease resistance-related gene was obtained.

[0012] As a further description of the above technical solution: The UMI single-molecule unique tag mentioned in step 2) has a length of 6-12 bp and is composed of random base N and fixed base W. The random base N can be any one of A, G, C, and T, and the fixed base W can be A or T. The structure of the UMI single-molecule unique tag is N. 2-4 WN 2-4W.

[0013] As a further description of the above technical solution: The specific process of the UMI single-molecule unique tag library construction technology described in step 2) is as follows: End repair and adapter ligation were performed on the target fragments of the enriched disease resistance-related gene. The adapters integrated a unique single-molecule tag (UMI) and then PCR amplification was performed. During the amplification process, each variant fragment was traced individually through the UMI tag to correct PCR amplification bias and errors caused by base mismatch.

[0014] As a further description of the above technical solution: The two functional prediction algorithms mentioned in step 3) are the SIFT algorithm and the PolyPhen-2 algorithm. When annotating together, the functional variants SNPs that are determined by both algorithms to be related to the disease resistance trait are taken as preliminary candidate SNPs.

[0015] As a further description of the above technical solution: The process of constructing the dairy sheep population genetic background database mentioned in step 3) is as follows: Collect genomic variation data of the target dairy sheep breed and other sheep breeds, screen out variation sites that exist only in the target dairy sheep breed and are associated with disease resistance traits, remove cross-breed universal conserved SNPs and linkage variation sites introduced by hybridization, and establish a dedicated genetic background database.

[0016] As a further description of the above technical solution: The amplified fragment length of the amplicon targeted resequencing described in step 4) is 100-250 bp, the sequencing depth is ≥300×, and the sequencing accuracy is ≥99.9%, which is used to verify candidate SNP sites.

[0017] In addition, the specific SNP sites for disease resistance traits in dairy sheep proposed in this application may also have the following additional technical features: As a further description of the above technical solution: The SNP sites are located in the coding or regulatory regions of disease resistance-related genes in dairy sheep, limiting their direct correlation with the disease resistance of dairy sheep, and serving as molecular markers for the disease resistance trait in dairy sheep.

[0018] Advantages of this invention: Based on the specific SNP sites and screening methods for disease resistance traits in dairy sheep in this application, a targeted enrichment method for target gene regions is adopted to narrow the screening range and reduce invalid detection data, which can efficiently capture low-abundance variant sites related to disease resistance in dairy sheep; combined with UMI tag error correction processing, amplification errors and base mismatch problems are effectively avoided, reducing the possibility of detection errors and significantly improving the accuracy of SNP site screening. At the same time, by combining dual-algorithm joint analysis and relying on the specific genetic background data of dairy sheep for screening and filtering, general variations and heterozygous linkage interference sites can be effectively eliminated. The disease-resistant SNP sites screened out are more specific to the breed and more closely associated with disease resistance traits. Subsequent population sequencing and melting curve genotyping verification further ensured the authenticity, stability, and reusability of the loci. This invention is simple to operate and highly practical, overcoming the shortcomings of traditional screening methods, such as broad scope, poor targeting, and low reliability. It can quickly and accurately identify SNP molecular markers specific to disease resistance traits in dairy sheep, facilitating subsequent screening of disease-resistant individuals and breed selection. This effectively improves the overall disease resistance of dairy sheep, reduces the probability of disease occurrence during breeding, lowers the cost of disease prevention medication and breeding losses, and provides a reliable basis for healthy dairy sheep farming and improved breed development.

[0019] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0020] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 This is a flowchart illustrating the steps of a method for screening disease-resistant SNP sites in dairy sheep according to an embodiment of this application. Figure 2 This is a schematic diagram illustrating the principle of targeted liquid phase enrichment of the signal peptide coding region of the disease resistance gene in dairy sheep according to an embodiment of this application for a specific SNP site for disease resistance traits and its screening method. Figure 3 This is a schematic diagram illustrating the UMI single-molecule tag library construction and molecular error correction and noise reduction principle of a screening method for specific SNP sites of disease resistance traits in dairy sheep according to an embodiment of this application. Figure 4 This is a schematic diagram of a multi-level SNP site screening and filtering architecture for a specific SNP site for disease resistance traits in dairy sheep and a screening method thereof, according to an embodiment of this application. Figure 5 This is a comparison of fluorescence intensity curves of specific SNP sites for disease resistance traits in dairy sheep and their screening method according to an embodiment of this application. Detailed Implementation

[0021] The embodiments of this application are described in detail below. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application.

[0022] The following describes, with reference to the accompanying drawings, specific SNP sites for disease resistance traits in dairy sheep and their screening methods, as embodiments of this application.

[0023] like Figure 1-5 As shown in Example 1 of this application, the specific SNP loci for disease resistance traits in dairy sheep and their screening method are as follows: a large-scale purebred dairy sheep population is selected as the test subjects. A total of 320 blood samples are collected from healthy dairy sheep, including individuals at different growth stages such as lactation, rearing, and early childhood. The samples cover a balanced ratio of male and female individuals and exclude abnormal individuals with congenital malformations, long-term diseases, or continuous intervention with exogenous drugs to ensure that the genetic background of the test population is pure and the trait expression is stable. Three common commercial sheep breeds—Hu sheep, Xiaowei Han sheep, and Merino sheep—were selected as control groups, with 80 healthy individuals from each breed. This was used to construct a multi-breed genetic variation database, enabling precise filtering of non-dairy sheep-specific variation sites. All experimental individuals underwent aseptic jugular vein blood collection, with 5 mL of peripheral blood collected from each individual. The blood was anticoagulated with EDTA and transported under refrigeration. Genomic DNA extraction was completed within 2 hours of blood collection. The lysis, binding, washing, and elution procedures were strictly performed according to the kit's standard steps to purify the genomic DNA. Quality control was performed using a UV spectrophotometer. All sample DNA concentrations were uniformly calibrated to 50 ng / μL, and the nucleic acid purity OD260 / OD280 ratio was controlled between 1.8 and 2.0. DNA electrophoresis bands were intact without diffusion or degradation, and residual protein and polysaccharide impurities met the detection standards. 100% of the samples were qualified, providing qualified nucleic acid raw materials for subsequent probe hybridization, library construction and amplification, and targeted sequencing.

[0024] The specific screening method is as follows: 1) Liquid-phase enrichment of disease-resistant gene target regions: Based on the conserved motif of the signal peptide coding region of the disease resistance immune gene in dairy sheep, a custom biotin-modified single-stranded DNA liquid-phase capture probe set was designed and customized. The probe length was controlled between 50bp and 120bp, and the homology between the probe and the conserved sequence of the target segment was ≥98%. It accurately targets and binds to the functional coding segment of the disease resistance gene, abandoning the traditional whole-genome indiscriminate sequencing mode and reducing the detection range from the source. A standardized hybridization reaction system was configured, with the final concentration of the liquid-phase capture probe in the system set to 6 fmol, which is within the preferred range of 2 to 10 fmol as defined in the claims. The hybridization reaction program was set as follows: denaturation at 95°C for 2 min to completely untie the double-stranded structure of genomic DNA, followed by cooling to 60°C for 1 h of constant temperature hybridization to ensure that the probe and the target gene fragment specifically and complementaryly bind, thereby weakening non-specific binding interference. After the hybridization reaction was completed, activated streptavidin magnetic beads were added and incubated at room temperature for 30 min. The target gene fragment of the integration probe was captured by the specific binding of biotin and streptavidin. The magnetic bead complex was fixed by a magnetic separator, and the unbound free DNA fragments in the supernatant were discarded. The mixture was then washed stepwise with low-strength washing buffer and high-purity buffer to remove non-specific adsorbed impurities and non-target fragments. Finally, the mixture was eluted with high-temperature elution buffer to obtain the enriched nucleic acid product of the target segment of the disease-resistant gene. According to the fragment enrichment efficiency test, the average enrichment fold of the target functional region in this embodiment reached 291 times. Compared with the conventional random amplification method, the proportion of non-target redundant gene fragments was reduced by 75.8%, which greatly improved the enrichment concentration of low-abundance rare variant fragments and effectively solved the technical defects of insufficient coverage of rare SNP fragments and easy loss of low-abundance sites in traditional technology.

[0025] 2) Implementation of UMI single-molecule tag library construction and molecular error correction and noise reduction: The enriched and purified target nucleic acid fragments of the disease resistance gene were uniformly repaired and blunted to fill in the broken base ends and repair the base damage sites, ensuring the structural integrity of the nucleic acid fragments. Then, sequencing adapters with UMI single-molecule unique tags were ligated and integrated. In this embodiment, the UMI tag length was selected as 8 bp, and a structure with alternating random and fixed bases was adopted. The base arrangement mode was N3WT3W. Each original nucleic acid molecule corresponds to a unique tag sequence to achieve single-molecule traceability labeling. After adapter ligation, restriction PCR amplification was performed using high-fidelity DNA polymerase. The number of amplification cycles was carefully controlled to avoid base bias shifts and fragment amplification imbalances caused by over-amplification. The amplification products were purified with magnetic beads to remove free adapters, primer dimers, and short fragment impurities, and a standardized sequencing library with independent UMI tags was constructed.

[0026] Qualified libraries were subjected to targeted deep sequencing, with the average sequencing depth remaining stable at 305×, meeting the detection threshold requirements for low-abundance rare SNP sites. Relying on the clustering correction function of the UMI unique tag, the raw sequencing data was split, compared and corrected. Multiple sequencing sequences corresponding to the same original single molecule were integrated and corrected to uniformly correct random base mismatches, single base deletions and insertion errors generated during PCR amplification. Quantitative data results show that after UMI molecular error correction and noise reduction, the overall sequencing base accuracy was improved to 99.93%. In the control group without error correction, the false positive rate of variant detection was 18.9% and the false negative rate of low abundance sites was 15.7%. After error correction in this embodiment, the false positive rate was reduced to 3.1% and the false negative rate was reduced to 2.6%, which significantly improved the authenticity and accuracy of variant detection and provided high-quality sequencing data support for subsequent SNP site screening.

[0027] 3) Dual-algorithm joint annotation and variety genetic background database filtering In this embodiment, the SIFT algorithm and the PolyPhen-2 algorithm are used simultaneously to perform joint functional annotation analysis on all detected variant sites. The two algorithms are operated independently to complete amino acid variant prediction, protein spatial structure impact assessment, signal peptide cleavage function analysis, and immune regulatory pathway association analysis, respectively. A unified set of screening and judgment rules is adopted, retaining only the variant sites that are simultaneously judged by the two algorithms as functional variants, variants that can change the structure of signal peptides, and variant sites that affect the secretion and transport efficiency of immune proteins. Synonymous silent variants, neutral and harmless variants, and non-coding invalid variants are removed in batches, thus compressing the range of candidate sites from a functional perspective and ensuring that the screening sites are directly related to disease resistance traits. A genetic background database specific to dairy sheep populations was constructed simultaneously, integrating genomic variation data of dairy sheep populations and control sheep of multiple breeds in this experiment. A total of 126,000 sets of basic data were collected, including cross-breed conserved SNPs, breed-shared linkage variants, and sheep-general genetic marker loci. The candidate variant data screened by dual algorithms were compared and filtered with the genetic background database in full, automatically removing cross-breed shared conserved loci, linkage heterozygous variants introduced by crossbreeding, and variants irrelevant to non-disease-resistant segments, retaining only low-abundance variant loci specific to dairy sheep breeds and located in the coding region of immune gene signal peptides. A total of 223 gene mutation sites were detected in the original sequencing. After initial screening by dual-algorithm joint annotation, 45 functionally related sites were retained. Then, non-specific interference sites were removed by secondary filtering through the breed genetic background library. Finally, 6 rare SNP sites specific to the disease resistance trait of dairy sheep that meet the requirements were obtained. The screening level is rigorous and the filtering logic is clear, which completely overcomes the shortcomings of traditional single screening methods such as poor site specificity and weak functional correlation. The six core SNP loci identified in this embodiment were all located in the signal peptide coding functional region of the disease resistance and immunity gene in dairy sheep, and all genetic indicators of the loci strictly met the limited judgment criteria. Population genotyping analysis revealed that the allele frequencies of the six specific SNP loci in purebred dairy sheep were 0.022, 0.030, 0.035, 0.039, 0.043, and 0.048, respectively, all falling within the rare locus range of 0.01–0.05. In control populations of Hu sheep, Small-tailed Han sheep, and Merino sheep, the allele frequencies at each locus were all below 0.009, indicating significant breed-specific differences. These variations represent rare genetic variations unique to dairy sheep. The base variations at these SNP loci can directly alter the amino acid sequence encoded by the signal peptide, disrupting… The integrity of the hydrophobic domain of the signal peptide interferes with the cleavage and processing of immunoproteins and their extracellular secretion, directly affecting the mucosal and humoral immune responses of dairy sheep. It significantly alters the body's resistance to bacterial diseases, gastrointestinal parasites, and respiratory infections. It can be directly used as a stable molecular genetic marker for disease resistance in dairy sheep. Population genetic stability testing showed that the genotype segregation patterns at each locus were stable in 320 experimental individuals, with no unstable phenomena such as random transient mutations or environmentally induced variations. The marker has strong genetic stability and can be used for long-term germplasm identification and targeted breeding.

[0028] 4) Amplicon targeted resequencing and HRM population validation: For the six disease-resistant specific SNP sites identified by screening, site-specific amplification primers were designed, and the length of the target fragment was controlled within the preferred range of 100-250 bp. Amplicon targeted resequencing was performed for verification, and the sequencing depth was uniformly not less than 300×. Sequencing and alignment results showed that the base variation type and genotype information of the 6 candidate SNP sites were completely matched with the previous screening data, with a sequence alignment consistency rate of 100% and no false positive sites caused by human error, which fully verified the accuracy of the targeted enrichment and variant screening results. HRM high-resolution melting curve typing technology was used to carry out large-scale batch verification. The standardized reaction conditions were: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s; 60℃ annealing for 30 s; 72℃ extension for 30 s; 40 cycles; melting curve acquisition temperature range was 60℃~95℃, heating rate was 0.1℃ / s. Based on the peak shift of the melting curve and the difference in melting temperature, three genotypes—wild homozygous, heterozygous mutant, and mutant homozygous—can be quickly distinguished. Three batches of parallel verification were carried out, and the overall genotyping results of 320 samples achieved a consistency rate of 98.81%. The detection operation is simple, has a short cycle, and is low in cost, enabling rapid screening of disease-resistant genotypes in individual dairy sheep on a large scale, and establishing a complete and standardized rapid locus determination system.

[0029] To further verify the superiority of this application, a control group is used for illustration below: The control group used a conventional process involving whole-genome random sequencing, no UMI error correction, single-algorithm annotation, and no breed background filtering, and tested samples from the same experimental population. The control group data showed that the traditional method resulted in only a 33-fold enrichment of the target region, with 90.9% of sequencing data being invalid. Without molecular error correction, variant detection errors were significant, with a false positive rate of 20.3% and a false negative rate of 17.9%. The single-algorithm screening yielded 39 candidate loci, of which 84.2% were cross-breed universal loci or neutral invalid loci. Only two weakly associated disease resistance loci were detected, and there were no low-abundance rare specific SNPs. The marker breed identification was extremely low, failing to meet the specific disease resistance breeding needs of dairy sheep. Data comparison clearly shows that this invention achieves a comprehensive upgrade in four dimensions: enrichment efficiency, detection accuracy, site specificity, and functional correlation. It compresses the screening range by targeting the enrichment range, reduces detection errors by relying on UMI tag error correction, and combines dual-algorithm functional screening and variety genetic background filtering with dual constraints. This effectively solves the problems of existing technologies, such as wide screening range, easy loss of rare sites, large detection errors, and strong marker universality.

[0030] In summary, the specific SNP loci for disease resistance traits in dairy sheep and the screening method thereof, as described in the embodiments of this application, enable early molecular identification of disease resistance traits in young dairy sheep by utilizing the disease resistance-specific SNP loci obtained through screening. This overcomes the limitations of traditional phenotypic breeding, which involves long cycles, large errors, and susceptibility to environmental interference. Through marker-assisted breeding, breeding sheep carrying superior disease-resistant genotypes can be selectively retained, gradually optimizing the genetic structure of the population, continuously improving the overall disease resistance of the dairy sheep population, reducing the incidence of diseases during the breeding process, and lowering breeding losses such as veterinary drug input, disease treatment, and individual culling. This promotes the healthy, improved, and intensive development of the dairy sheep breeding industry.

[0031] In the description of this specification, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0032] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0033] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A method for screening disease-resistant trait-specific SNP loci in dairy sheep, characterized in that, Includes the following steps: 1) Targeted region enrichment: Based on the conserved motifs of genes related to disease resistance in dairy sheep, a liquid-phase capture probe for the target micro-region was designed to enrich the gene regions related to disease resistance in dairy sheep in a targeted manner. 2) Molecular error correction and noise reduction: UMI single-molecule unique tag library construction technology is used to perform source tracing and base error correction on the low-abundance variant fragments obtained in step 1). 3) Multi-algorithm joint screening: The candidate SNPs obtained after step 2) are jointly annotated by two functional prediction algorithms, and filtered by the genetic background database of dairy sheep populations to remove general conserved SNPs and linkage variants, and to screen out disease resistance trait-specific SNP loci. 4) Population sample verification and system establishment: Amplicon targeted resequencing combined with HRM high-resolution melting curve typing technology was used to verify the candidate SNP sites obtained in step 3) through population sample verification, confirm the stability and specificity of the sites, and establish a rapid screening and determination system for SNP sites specific to the disease resistance trait of dairy sheep.

2. The specific SNP locus for disease resistance traits in dairy sheep according to claim 1, characterized in that, SNP sites must meet the following criteria: the allele frequency in the target dairy sheep breed is 0.01-0.05, the allele frequency in other sheep breeds is <0.01, and functional annotation confirms that they can affect the secretion efficiency of disease resistance-related proteins or the immune response capacity of dairy sheep.

3. The method for screening disease-resistant SNP loci in dairy sheep according to claim 1, characterized in that, The liquid-phase capture probe described in step 1) is a biotin-modified single-stranded DNA probe with a length of 50-120 bp and a homology of ≥98% with conserved motifs of genes related to disease resistance traits in dairy sheep.

4. The method for screening disease-resistant trait-specific SNP sites in dairy sheep according to claim 1, characterized in that, The specific process of targeted enrichment described in step 1) is as follows: The liquid-phase capture probe was hybridized with a fragment of dairy sheep genomic DNA. The probe concentration in the hybridization system was 2-10 fmol. The hybridization reaction conditions were denaturation at 95℃ for 2 minutes and hybridization at 60℃ for 1 hour. After hybridization, streptavidin magnetic beads were used to capture the hybridization product. After elution and purification, the enriched target fragment of the disease resistance-related gene was obtained.

5. The method for screening disease-resistant SNP loci in dairy sheep according to claim 1, characterized in that, The UMI single-molecule unique tag mentioned in step 2) has a length of 6-12 bp and is composed of random base N and fixed base W. The random base N can be any one of A, G, C, and T, and the fixed base W can be A or T. The structure of the UMI single-molecule unique tag is N. 2-4 WN 2-4 W.

6. The method for screening disease-resistant trait-specific SNP sites in dairy sheep according to claim 1, characterized in that, The specific process of the UMI single-molecule unique tag library construction technology described in step 2) is as follows: End repair and adapter ligation were performed on the target fragments of the enriched disease resistance-related gene. The adapters integrated a unique single-molecule tag (UMI) and then PCR amplification was performed. During the amplification process, each variant fragment was traced individually through the UMI tag to correct PCR amplification bias and errors caused by base mismatch.

7. The method for screening disease-resistant SNP loci in dairy sheep according to claim 1, characterized in that, The two functional prediction algorithms mentioned in step 3) are the SIFT algorithm and the PolyPhen-2 algorithm. When annotating together, the functional variants SNPs that are determined by both algorithms to be related to the disease resistance trait are taken as preliminary candidate SNPs.

8. The method for screening disease-resistant SNP loci specific to dairy sheep according to claim 1, characterized in that, The process of constructing the dairy sheep population genetic background database mentioned in step 3) is as follows: Collect genomic variation data of the target dairy sheep breed and other sheep breeds, screen out variation sites that exist only in the target dairy sheep breed and are associated with disease resistance traits, remove cross-breed universal conserved SNPs and linkage variation sites introduced by hybridization, and establish a dedicated genetic background database.

9. The method for screening disease-resistant trait-specific SNP sites in dairy sheep according to claim 1, characterized in that, The amplified fragment length of the amplicon targeted resequencing described in step 4) is 100-250 bp, the sequencing depth is ≥300×, and the sequencing accuracy is ≥99.9%, which is used to verify candidate SNP sites.

10. The specific SNP locus for disease resistance traits in dairy sheep according to claim 2, characterized in that, The SNP sites are located in the coding or regulatory regions of disease resistance-related genes in dairy sheep, limiting their direct correlation with the disease resistance ability of dairy sheep, and serving as molecular markers for the disease resistance trait in dairy sheep.

Images