Sheep anti-stress snp molecular marker combination, gene chip and application thereof

Abstract

The application discloses a sheep anti-adversity SNP molecular marker combination, a gene chip and application thereof, and relates to the technical field of livestock genetics and breeding. The application comprises 14000 SNP molecular markers. The sheep anti-adversity SNP molecular marker combination provided by the application realizes balance of site specificity and whole genome coverage, can focus on anti-adversity more, and can improve the detection accuracy by more than 90%. The 14K anti-adversity gene chip developed by the application has an average interval of 186 kilobases, supports genome selection and kinship analysis, and supports large-scale sheep breeding application. Compared with the detection method of the 50K gene chip and whole genome sequencing, the 14K gene chip provided by the application has low manufacturing cost, and can improve the productivity of sheep flocks by 5-10%.

Description

Technical Field

[0001] This invention relates to the field of animal husbandry genetics and breeding technology, and more specifically, to combinations of SNP molecular markers for sheep stress resistance, gene chips, and their applications. Background Technology

[0002] Sheep, as important economic animals, play a crucial role in global livestock farming. However, with the intensification of climate change, the prevalence of parasitic infections, and increased disease pressure, sheep's stress resistance (including cold stress tolerance, parasite resistance, disease resistance, and environmental adaptation) has become a core challenge in breeding. Current sheep gene chips are mostly general-purpose (e.g., sheep single nucleotide polymorphism (SNP) chips), lacking specific designs for stress resistance, leading to low efficiency in genome-wide association studies (GWAS) and genome selection. Existing studies (such as the sheep GWAS, the Egyptian sheep cold stress GWAS, the Australian sheep parasite resistance GWAS, the Corriedale sheep gastrointestinal nematode resistance single-sample GWAS, and the sheep quantitative trait locus database) have identified various stress resistance-related SNP sites, but these have not yet been integrated into dedicated chips.

[0003] These stress-resistance-related single nucleotide polymorphisms (SNPs) involve traits such as somatic cell count, lactation persistence, body condition score, cold stress tolerance, and gastrointestinal nematode resistance. Candidate genes include PLCB1, STEAP3, KSR2, GALNTL6, PALLD, TLR5, TLR9, LEPR, MYO5A, PRKG1, NTAQ1, ZHX1, CD80, GSK3B, RBP7, FSTL1, GRID2, FAIM, CCSER1, GRIP1, HMGA2, POT1, TMEM229A, and SCN3A. These genes are primarily involved in immune responses, metabolic adaptation, cell signal transduction, and developmental processes. For example, PLCB1 regulates intracellular signal transduction and stress adaptation; STEAP3 catalyzes the reduction of metal ions, promoting cellular iron homeostasis and stress response; KSR2 regulates the RAS / MAPK pathway, affecting energy homeostasis; GALNTL6 participates in the initiation of O-glycosylation, promoting vitamin A stability; PALLD maintains the cytoskeleton and stress response; TLR5 and TLR9 recognize pathogens and activate immunity; LEPR regulates energy balance; MYO5A is responsible for intracellular transport; PRKG1 mediates cGMP signaling; NTAQ1 regulates protein degradation; ZHX1 acts as a transcriptional repressor; CD80 activates T cells; GSK3B regulates glycogen metabolism; RBP7 promotes vitamin A metabolism; FSTL1 regulates TGF-β signaling; GRID2 regulates synaptic plasticity; FAIM inhibits apoptosis; CCSER1 regulates cell proliferation; GRIP1 regulates neuronal development; HMGA2 alters chromatin structure; POT1 protects telomeres; TMEM229A participates in development; and SCN3A regulates neural excitability. These features are closely related to sheep's stress resistance traits and support the design and application of chips.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a combination of SNP molecular markers for sheep stress resistance, a gene chip, and their applications, thereby improving the efficiency of sheep stress resistance breeding.

[0006] This invention is implemented as follows: In a first aspect, the present invention provides a combination of SNP molecular markers for sheep stress resistance, comprising 14,000 SNP molecular markers, the site information of which includes the following:

[0007] In the locus number, 1 to 26 and X on the left indicate the chromosome where the locus is located, the middle value indicates the position of the locus on the chromosome, and the base type on the right indicates the SNP base of that locus in the reference genome. The version number of the whole genome sequence of the reference genome is: Oar_rambouillet_v1.0.

[0008] In the above SNP molecular marker combination, N / A ≠ simple "deletion", but means "there is no available SNP at this position due to genomic conservation and other reasons, so it is used as a placeholder marker for space filling" and will not be retained in the chip product in the end.

[0009] Relevant research data were collected from public databases and literature, such as the National Center for Biotechnology Information (NCBI) Genome Database, the Sheep Quantitative Trait Loci Database (Sheep QTLdb), and the SNP dataset from the International Sheep Genome Consortium (ISGC). The screening criteria were significant variants with a p-value less than 1e-5 in genome-wide association studies (GWAS), or high-density flanking loci within quantitative trait loci regions. Specifically, PLINK software was used for data filtering. Somatic cell count and lactation persistence-related loci were extracted from sheep GWAS, cold tolerance-related loci were extracted from previous GWAS reports on cold stress tolerance, parasite resistance loci were extracted from GWAS on parasite resistance in Australian sheep, gastrointestinal nematode resistance loci were extracted from single-sample GWAS on Gastrointestinal nematode resistance in Corriedale sheep, and functional lifespan-related loci were extracted from the SMARTER GWAS. After integration, at least 47 specific loci associated with stress resistance were obtained. Based on the Oar_rambouillet_v1.0 reference genome, the proportions of each chromosome were calculated, and a specific proportion of SNP sites were allocated to each chromosome, generating 13,953 single nucleotide polymorphism (SNP) sites that could be uniformly covered across the genome. Integrating 47 specific sites and 13,953 uniformly distributed sites yielded 14,000 SNP sites. Through quality verification and optimization of the site combinations, 14K SNP sites were finally obtained.

[0010] Compared with existing technologies, the SNP molecular marker combination for sheep stress resistance developed in this invention has the following beneficial effects: (1) High specificity: Focuses on stress resistance traits, improving detection accuracy to over 90%.

[0011] (2) Comprehensive coverage: with an average spacing of 186 dry bases, it supports genome selection and kinship analysis.

[0012] (3) Low cost: 14K density is suitable for large-scale breeding, and the manufacturing cost is lower than that of whole genome sequencing.

[0013] (4) Wide range of applications: It can increase sheep productivity by 5-10% and promote sustainable animal husbandry.

[0014] Secondly, this invention provides a combination of SNP molecular markers for sheep stress resistance. It includes 47 SNP molecular markers, and the site information of these 47 SNP molecular markers includes the following:

[0015] In the locus number, 1 to 26 and X on the left indicate the chromosome where the locus is located, the middle value indicates the position of the locus on the chromosome, and the base type on the right indicates the SNP base of that locus in the reference genome. The version number of the whole genome sequence of the reference genome is: Oar_rambouillet_v1.0.

[0016] 47 SNP molecular markers were identified as molecular markers specifically associated with sheep stress resistance.

[0017] Thirdly, the present invention provides a probe for detecting 14,000 SNP molecular marker combinations for sheep stress resistance.

[0018] Design specific probes for each single nucleotide polymorphism (SNP) site, using oligonucleotide probes (50-60 bases in length). Design 2-4 probes for each SNP site.

[0019] Fourthly, the present invention provides a probe for detecting a combination of 47 SNP molecular markers for detecting stress resistance in sheep.

[0020] Fifthly, the present invention provides a kit comprising: the probe described above.

[0021] Sixthly, the present invention provides a sheep stress resistance 14K gene chip, comprising: the gene chip including the probes described above. The probes are fixed on the chip surface to form a high-density array (density of approximately 14K sites / square centimeter). The manufacturing process includes quality control: using fluorescent labeling to test probe specificity to ensure hybridization efficiency greater than 95%.

[0022] In a seventh aspect, the present invention provides a method for analyzing the stress resistance of sheep, which includes the following steps: taking a nucleic acid sample to be tested and performing chip hybridization with the above-mentioned sheep 14K gene chip, reading the fluorescence signal, and performing data analysis.

[0023] In one implementation, before performing microarray hybridization on the nucleic acid sample to be tested, nucleic acid amplification is included, with the following procedure: PCR technology is used to amplify the target region, repeated 35 times, with denaturation at 95°C, annealing at 55°C, and extension at 72°C. Primers are specific primers designed and synthesized based on the target region sequence.

[0024] In a preferred embodiment of the present invention, hybridization involves taking the nucleic acid sample to be tested and hybridizing it with a sheep stress resistance 14K gene chip at 42°C for 16 hours.

[0025] Eighthly, the present invention provides the use of the probe, kit, or sheep stress resistance 14K gene chip in at least one of the following: (1) Genome-wide association analysis of sheep; (2) Sheep genotyping; (3) Assessment of genetic diversity in sheep; (4) Analysis of sheep kinship; (5) Sheep stress resistance breeding; (6) Sheep genome selection: The whole genome of the candidate sheep population is scanned by chip, and the obtained genetic marker information is used in combination with the phenotypic data and statistical models of the population to predict the "genome estimated breeding value" of individuals, so as to achieve early, rapid and accurate screening of breeding sheep with excellent stress resistance traits.

[0026] In a preferred embodiment of the present invention, sheep stress resistance is selected from: lactation duration, cold stress resistance, parasite resistance, mastitis resistance, heat stress resistance, feed utilization, disease resistance, reproductive performance, nutritional stress adaptability, fur adaptability, or behavioral adaptability.

[0027] The present invention has the following beneficial effects: This invention provides a high-efficiency, low-cost 14K stress resistance gene chip. By integrating data from multiple sources (the National Center for Biotechnology Information (NCBI) genome database, the Sheep Quantitative Trait Locus Database (Sheep QTLdb), and the SNP dataset from the International Sheep Genome Consortium (ISGC), it achieves a balance between locus specificity and whole-genome coverage, enabling a more focused approach to stress resistance traits and improving detection accuracy by more than 90%. The developed 14K stress resistance gene chip has an average spacing of 186 kilobases, supporting genomic selection and kinship analysis, and facilitating large-scale sheep breeding applications. Compared to 50K gene chips and whole-genome sequencing detection methods, the 14K gene chip provided by this invention has lower manufacturing costs, can improve sheep productivity by 5-10%, and promotes sustainable animal husbandry. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a schematic diagram of the distribution of 14K gene chip loci for sheep stress resistance (the left side is labeled with the stress resistance trait category (e.g., cold stress resistance, parasite resistance, mastitis resistance), and the right side shows examples of single nucleotide polymorphism (SNP) loci distribution under the corresponding trait. Different colored / shaped squares represent specific loci (black) and uniformly covered loci (colored), and the example loci are numbered). Figure 2 This is a density distribution map of single nucleotide polymorphism sites on each chromosome. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0031] Definitions: Linkage disequilibrium refers to the non-random association between alleles of two or more genetic markers (such as SNPs, microsatellites, etc.) in a population.

[0032] "Detection capture rate" usually refers to the proportion of the target region that is actually sequenced out of the total target region during the target region enrichment process.

[0033] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0034] Example 1 This embodiment provides the design process for sheep stress resistance 14K gene chip loci, including data collection, locus extraction, generation, merging, validation, and file output. The entire process utilizes bioinformatics tools and programming scripts to ensure the scientific validity and practicality of the loci.

[0035] The specific steps are as follows: (1) Collection and extraction of stress-resistance-specific single nucleotide polymorphisms (SNPs): First, relevant research data were collected from public databases and literature, such as the National Center for Biotechnology Information (NCBI) genome database, the Sheep Quantitative Trait Loci Database (Sheep QTLdb), and the SNP dataset from the International Sheep Genome Consortium (ISGC). The screening criteria were significant variants with a p-value less than 1e-5 in genome-wide association studies, or high-density flanking loci within the quantitative trait locus region.

[0036] In the specific operation, PLINK software was used for data filtering. Somatic cell count and lactation persistence-related loci were extracted from the sheep genome-wide association study, such as rs403061409 (chromosome 9, position 30159232, alleles A / G, associated genes NTAQ1 and ZHX1, functioning to regulate protein degradation and transcriptional repression, involved in mastitis resistance); cold tolerance-related loci were extracted from the team's genome-wide association study on cold stress, such as OAR7_60704536.1 (chromosome 7, position 54927841, alleles G / A, associated gene MYO5A, functioning to regulate intracellular transport and stress response, involved in low-temperature adaptation); and parasite resistance loci were extracted from the Australian sheep parasite resistance genome-wide association study. For example, rs421630816 (chromosome 2, location 110800000, allele N / A, associated gene PALLD, function is to maintain the cytoskeleton and immune response); gastrointestinal nematode resistance loci were extracted from a single-sample genome-wide association study of resistance to *Gastrointestinal nematode* in Corridor sheep, such as rs429546187 (chromosome 12, location 24624977, allele N / A, associated gene TLR5, function is to recognize pathogens and activate immunity); functional lifespan-related loci were extracted from the SMARTER genome-wide association study, such as OAR2_152193248.1 (chromosome 2, location 155993633, allele N / A, associated gene SCN3A, function is to regulate neural excitability and development). After integration, at least 47 loci were obtained, ensuring coverage of multiple candidate genes such as TLR5 (immune activation), TLR9 (pathogen recognition), and MYO5A (cell transport). This step also includes multi-population validation, using data from 1,000 sheep samples to examine locus polymorphisms, ensuring that the minimum allele frequency is greater than 0.01, and assessing the role of gene function in stress resistance, such as the metabolic adaptation of PLCB1 in signal transduction.

[0037] (2) Calculate and generate genome-uniformly covered single nucleotide polymorphism sites: based on Oar _The rambouillet_v1.0 reference genome (approximately 2.6 Gb in total length) was used to calculate the proportions of each chromosome. For example, chromosome 1, with a length of 272,617,460 base pairs, was allocated approximately 1,400 loci; chromosome x, with a length of 129,731,715 base pairs, was allocated approximately 660 loci. A Python script (with a fixed random seed of 42 to ensure reproducibility) was used to generate loci: the script defined a chromosome length dictionary, calculated intervals (approximately 180-200 K bases), and randomly shifted the generated positions within each interval. Alleles were randomly selected from common pairs such as A / G and C / T. For example, sim_OAR1-11823456 (chromosome 1, position 11823456, allele A / T, no specific association, but covers the genome to support overall analysis) was generated. Variants with a minimum allele frequency greater than 0.05 from public databases were preferentially selected to avoid linkage disequilibrium. The script also handled N / A position padding, deleted missing position rows, ensured no covered blanks, and generated a total of 13,953 loci. At the same time, gene function integration should be considered, for example, prioritizing coverage of regions involved in metabolic adaptation (such as KSR2).

[0038] (3) Merging all locus information: 47 specific loci and 13953 uniform loci were integrated. Duplicates were removed and sorted using Excel or the Python pandas library. A comma-separated value file (sheep_14k_snps.csv) was output, with the format including single nucleotide polymorphism (SNP) site identifier, chromosome, location (base pairs), allele, associated trait / gene, and origin, totaling 14001 rows (including header). For example, the first row header: SNP site identifier, chromosome, location (base pairs), allele, associated trait / gene, and origin; subsequent rows include rs403061409, 9.30159232, A / G, somatic cell count / NTAQ1, ZHX1 (regulating protein degradation), Frizarta elastic genome-wide association study. Gene functions were briefly annotated during merging, such as the cellular transport role of MYO5A.

[0039] (4) Quality Validation and Optimization: A virtual sheep population sample of 1000 was generated using simulation software (e.g., GCTA) for genome-wide association studies. The capture rate was checked to be greater than 99%, the call rate greater than 95%, and the minimum allele frequency greater than 0.01. Linkage disequilibrium (target r) was checked using LD Score Regression. 2 <0.8). If bias is found, iteratively adjust the sites, such as removing high-LD sites or adding new sites. After optimization, cross-validate on real sheep samples to ensure the error rate is below 5%. Validation also includes functional annotation checks, such as confirming whether the role of TLR5 in immune activation is reflected in the simulation.

[0040] (5) Generate chip design files: Based on the integrated file, use Illumina DesignStudio software or a custom script to output the array layout, including site coordinates, probe sequences (2-4 probes per site), and a format compatible with liquid capture sequencing platforms. The file supports subsequent manufacturing, ensuring a capture density of 5-10 sites per quantitative trait site region.

[0041] Schematic diagram of the distribution of 14K gene chip loci in sheep stress resistance (refer to...) Figure 1 As shown in the diagram, the density distribution of single nucleotide polymorphism sites on each chromosome is referenced. Figure 2 As shown. Figure 1 SNPs in the genome are: Single nucleotide polymorphism sites, which are single base variations in the genome; SNP0: Uniformly covered background sites, which are non-functional specific sites generated according to a uniform distribution strategy in the genome; SNPPa: Adversity-related SNPs, which are sites that are significantly associated with adversity traits and selected by GWAS; SNPc: Conserved functional region sites, which are functional candidate sites located in the coding or regulatory regions of genes; TNA: Target Not Amplified, which are location markers of sites that have not been assigned because the region is highly conserved and there are no known SNPs.

[0042] Example This embodiment provides the manufacturing process of a sheep stress resistance 14K gene chip and its practical application in sheep breeding, including sample preparation, chip operation, data analysis, breeding selection, and effect evaluation. The specific operations are as follows: (1) Chip Fabrication: Based on the design documents, the chip was fabricated using the Illumina BeadChip platform or liquid capture sequencing technology. First, oligonucleotide probes (50-60 bases in length) were synthesized, and allele-specific probes were designed for each single nucleotide polymorphism site, such as the A and G allele probes for rs403061409 (probe specificity was optimized considering the protein degradation function of NTAQ1). The probes were immobilized on the chip surface to form a high-density array (approximately 14K sites / cm²). The fabrication process included quality control: probe specificity was tested using fluorescent labeling to ensure hybridization efficiency greater than 95%. Simultaneously, gene function annotations were integrated, such as adding transport function annotations for the MYO5A site to guide subsequent analysis. The final chip was compatible with standard scanners, such as the Illumina iScan system.

[0043] (2) Sample preparation and microarray manipulation: Application in a sheep population (500 samples, covering breeds such as Small-tailed Han sheep). First, deoxyribonucleic acid (DNA) was extracted from sheep blood or ear tissue samples and purified using the QIAamp DNA Mini Kit, with the concentration adjusted to 50 ng / μL and a purity A260 / 280 > 1.8. Microarray manipulation included: sample amplification (using PCR technology to amplify the target region, 35 cycles, with denaturation at 95℃, annealing at 55℃, and extension at 72℃); hybridization (samples and microarrays were hybridized at 42℃ for 16 hours to ensure uniform distribution); washing to remove non-specific bindings (using washing buffer 3 times, 5 minutes each time); scanning using the iScan system to read fluorescence signals at a resolution of 0.5 μm. The entire process was completed in a cleanroom laboratory, taking approximately 48 hours, with environmental temperature monitored to simulate cold stress conditions.

[0044] (3) Data Analysis: GenomeStudio software was used to process the scan data and generate genotype files (in .ped and .map formats), with a call rate > 95%. Genome-wide association studies were performed, and P-values ​​were calculated using PLINK software to identify loci associated with cold stress, such as confirming a significant association between OAR7_60704536.1 and low-temperature adaptation (P < 1e-6, considering the cellular transport function of MYO5A). Heritability was further estimated using GCTA software (h2 > 0.3), and estimated breeding values ​​were calculated. The analysis also included genetic diversity assessment, using software to calculate heterozygosity (He > 0.5) and integrating gene functions, such as assessing the impact of TLR5's role in immune activation on parasite resistance. Data visualization used R ggplot to draw Manhattan plots, highlighting significant loci.

[0045] (4) Breeding selection and verification: Based on the analysis, select individuals with high stress resistance, such as rams carrying favorable alleles as breeding sheep (based on LEpR energy balance function screening).

[0046] Breeding trials: Sheep flock performance was monitored under cold climate conditions (temperature -10℃), comparing the chip-selected group (total n=200) with the control group (ordinary sheep without chip selection, total n=200). Cold stress tolerance was assessed through a low-temperature exposure test: sheep were placed in a -10℃ environment for 72 hours, rectal temperature, serum cortisol levels, and stress behavior scores were monitored, and survival rates were calculated. Parasite infection rates were assessed using a combination of fecal egg count (FEC, McMaster method) and serum antiparasitic IgG titer (ELISA method). All the above indicators were direct phenotypic measurements, not locus-based calculations. Specific locus information is listed in the 14,000 SNP loci in the instruction manual, and the breeding value prediction used the BLUP model.

[0047] Serum collection method: 5 mL of blood was collected from the jugular vein and centrifuged at 3000 rpm for 15 min to separate the serum. Cortisol assay: ELISA (CUSABIO kit). Sensitivity: 0.1 ng / mL, intra-assay CV < 5%. IgG assay: Immunoturbidimetric assay, performed using a fully automated biochemical analyzer, calibrated with standards. Monitoring time points: Blood samples were collected once each at baseline, 24 h, 48 h, and 72 h after exposure to low temperature. Statistical results are expressed as mean ± SD, and t-tests were used for comparisons between groups.

[0048] Productivity assessment indicators: daily gain (ADG, kg / d), dressing percentage (%), and net meat percentage (%). Measurement methods: During the 90-day trial period, the animals were weighed every 30 days on an empty stomach; carcass weight and net meat weight were measured after slaughter.

[0049] The results showed that sheep bred using the chip of this invention had a 15% higher cold stress tolerance (95% vs 80% survival rate) and a 10% lower parasite infection rate.

[0050] Validation included monitoring physiological indicators such as serum stress hormone levels (cortisol < 50 ng / mL) and immune parameters (IgG > 10 g / L). Economic benefit assessment results showed that the ADG (adverse gastric rate) of the chip-fed breeding group was 8% higher than that of the control group, the slaughter rate was 3 percentage points higher, and the net meat yield was 2 percentage points higher. Based on the current market price of 15 yuan / kg for live sheep, the annual income per flock (200 sheep) increased by approximately 20%.

[0051] Experimental Example 1 This experimental example demonstrates the efficiency and accuracy of correlation analysis and selective signal localization detection based on 14K gene chips for simulated or actual sheep samples.

[0052] This experiment used GCTA software to simulate 1000 individual samples and performed association analysis based on 14K microarray genotype data. The data are statistical results based on GCTA (v1.94.1) -- Simu-Qt functional simulation. Specifically: 1. Simulation parameter settings: 100 QTL loci are preset (effect sizes are sampled from an exponential distribution, λ = 1), heritability h 2 =0.3, generating 1000 individual genotypes based on the 14K chip LD structure.

[0053] 2. Detection rate calculation: P-link association analysis was considered significant if P < 5e-8. The detection rate was calculated among 100 pre-defined QTLs, with an average of 87% across 5 replicates.

[0054] 3. Positioning accuracy calculation: Using the QTL true location ±1Mb as the candidate interval, the proportion of significant SNPs falling into this interval was statistically analyzed, and the average of 5 repetitions was 91%.

[0055] Results: The detection rate of known QTL sites was >85%, and the localization accuracy (candidate interval <1Mb) was >90%, which is better than that of random 5K chips (detection rate 65%) and close to that of 50K chips (detection rate 90%). Selected signal detection used CMS comprehensive statistics, and the 14K chip could effectively capture the selected region.

[0056] Experimental Example 2 This experiment, based on a 14K gene chip, used the BLUP model to predict the comprehensive breeding value of stress resistance in 200 experimental sheep.

[0057] Accuracy verification was performed using 5-fold cross-validation: 200 experimental sheep were randomly divided into 5 groups, with 4 groups serving as the training set and 1 group as the validation set in turn, and the BLUP model was used to predict the comprehensive breeding value of stress resistance.

[0058] 1. The following stress resistance index testing methods were used to test the stress resistance of each experimental sheep.

[0059] The resilience index system and observation period are as follows: (1) The breeding experiment was based on the process of cold-region sheep developing resistance, comprehensively considering cold stress, production performance stability, and physiological metabolism. For each experimental sheep, the following indicators were recorded during the cold-region stress period: Relevant physiological indicators were measured, including rectal temperature, respiratory rate, and heart rate. Production performance stability: daily weight gain and feed intake variation; Blood sample indicators: Serum biochemical indicators (glucose, free fatty acids, triglycerides, red blood cell count, etc.) detection.

[0060] (2) Observation period: Adaptation period: ensuring that the individual enters the experimental environment and remains stable for a period of time; Stress period: Record key indicators under cold conditions for subsequent calculation of comprehensive stress resistance index (i.e., measured value).

[0061] 2. Conclusion: The prediction accuracy (Pearson correlation coefficient between predicted and measured values) r = 0.92 (>90%). The CV error of 5 replicates was <3%. Data source: Records of the 2024-2025 Cold Region Sheep Stress Resistance Breeding Experiment.

[0062] The results show that the 14K gene chip provided by this invention can improve the efficiency and accuracy of detection applications such as association analysis and selective signal localization.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A combination of SNP molecular markers for sheep stress resistance, characterized in that, It includes 14,000 SNP molecular markers, and the site information of these 14,000 SNP molecular markers includes the following: In the locus number, 1 to 26 and X on the left indicate the chromosome where the locus is located, the middle value indicates the position of the locus on the chromosome, and the base type on the right indicates the SNP base of that locus in the reference genome. The version number of the whole genome sequence of the reference genome is: Oar_rambouillet_v1.

0.

2. A combination of SNP molecular markers for sheep stress resistance, characterized in that, It includes: The 47 SNP molecular markers, and their site information includes the following: In the locus number, 1 to 26 and X on the left indicate the chromosome where the locus is located, the middle value indicates the position of the locus on the chromosome, and the base type on the right indicates the SNP base of that locus in the reference genome. The version number of the whole genome sequence of the reference genome is: Oar_rambouillet_v1.

0.

3. A probe for detecting the SNP molecular marker combination of sheep stress resistance as described in claim 1.

4. A probe for detecting the SNP molecular marker combination for sheep stress resistance as described in claim 2.

5. A reagent kit, characterized in that, It includes: The probe as described in claim 3 or 4.

6. A sheep stress resistance 14K gene chip, characterized in that, It includes: The gene chip includes the probe described in claim 3 or 4.

7. A method for analyzing the stress resistance of sheep, characterized in that, It includes the following steps: taking the nucleic acid sample to be tested and performing chip hybridization with the sheep 14K gene chip described in claim 6, reading the fluorescence signal, and performing data analysis.

8. The method for analyzing sheep stress resistance according to claim 7, characterized in that, The hybridization process involves taking the nucleic acid sample to be tested and hybridizing it with the sheep stress resistance 14K gene chip at 42°C for 16 hours.

9. Use of the probe as described in claim 3 or 4, the kit as described in claim 5, or the sheep stress resistance 14K gene chip as described in claim 6 in at least one of the following: (1) Genome-wide association analysis of sheep; (2) Sheep genotyping; (3) Assessment of genetic diversity in sheep; (4) Analysis of sheep kinship; (5) Sheep stress resistance breeding; (6) Sheep genome selection.

10. The use according to claim 9, characterized in that, The sheep's stress resistance is selected from: lactation duration, cold stress resistance, parasite resistance, mastitis resistance, heat stress resistance, feed utilization, disease resistance, reproductive performance, nutritional stress adaptability, fur adaptability, or behavioral adaptability.

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