Application of sheep molecular marker combination, chip, kit, method and application

Abstract

The invention relates to the technical field of molecular detection, and particularly discloses application, a chip, a kit, a method and application of a sheep molecular marker combination, and the application comprises population genetics analysis and economic character prediction of local sheep; the local sheep comprise small-tailed han sheep, western Henan fat-tailed sheep, big-tailed han sheep, opisthopause fur sheep, Tong sheep, Zhongyu mutton sheep and Huanghuai mutton sheep; the sheep molecular marker combination comprises 12363 SNP (Single Nucleotide Polymorphism) sites and 18 Indel (Indel) sites; the 12363 SNP loci and the 18 Indel loci are determined on the basis of comparison of a whole genome sequence of a sheep reference genome, and the version number of the whole genome sequence of the sheep reference genome is AR-UIRamv3.0; wherein the 12363 SNP loci are as shown in a table 1; and 18 Indel loci are as shown in a table 2. The method can be used for population genetics analysis and economic character prediction of local sheep.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, specifically to the uses, chips, reagent kits, methods, and applications of sheep molecular marker combinations. Background Technology

[0002] With the continuous development of global animal husbandry, sheep, as an important economic animal, have received increasing attention for the research and utilization of their genetic resources. my country ranks first in the world in terms of sheep inventory, slaughter volume, and mutton production. Henan Province, as a major agricultural and animal husbandry province, possesses abundant sheep and goat breed resources and is one of the five major mutton sheep producing areas in China. Henan Province currently has four main sheep breeds: Small-tailed Han sheep, Western Henan fat-tailed sheep, Large-tailed Han sheep, and Taihang fur sheep. The Huanghuai mutton sheep and Zhongyu mutton sheep are breeds developed using the Small-tailed Han sheep as the maternal line. Among them, the Huanghuai mutton sheep, with its high reproductive rate, fast growth rate, tolerance to roughage, and excellent meat quality, became the first new mutton sheep breed in Henan Province to be approved by the state. Its main production performance has reached the international advanced level and has significant value for promotion and application.

[0003] Local sheep genetic resources are valuable breeding materials, and their protection and development are crucial for the discovery of superior germplasm genes and livestock genetic improvement programs. With the continuous development of the industry, in-depth exploration of the germplasm characteristics of local sheep, scientific evaluation, and utilization must shift towards precise molecular-level assessment. In the era of genomic breeding, the application of genomic microarrays can effectively identify sheep flocks carrying specific genes, thereby accelerating the selection of superior breeds.

[0004] The lack of local sheep genome research and microarray development in Henan Province has resulted in current breeding techniques still relying primarily on traditional methods, mainly depending on human experience and physical characteristics for evaluation, leading to slow breeding progress. Therefore, developing a gene chip suitable for Henan's local sheep populations will not only improve the genetic improvement of sheep and promote the sustainable development of animal husbandry, but also provide a scientific basis for the protection and development of local sheep breeds, offering a solid guarantee for the revitalization of Henan's sheep breeding industry. Summary of the Invention

[0005] The purpose of this invention is to provide the uses, chips, kits, methods and applications of sheep molecular marker combinations, which can be used for population genetic analysis and economic trait prediction of local sheep.

[0006] This invention is achieved through the following technical solution: The applications of sheep molecular marker combinations include population genetic analysis and economic trait prediction for local sheep breeds. These local sheep breeds include those from Henan and Shaanxi provinces, specifically including the Small-tailed Han sheep, Western Henan Fat-tailed sheep, Large-tailed Han sheep, Taihang Fur sheep, Tong sheep, Huanghuai meat sheep, and Central Henan meat sheep. The sheep molecular marker combinations include 12,363 SNP loci and 18 Indel loci. These 12,363 SNP loci and 18 Indel loci were determined based on whole-genome sequence alignment of a sheep reference genome, version number ARS-UI_Ramb_v3.0. The 12,363 SNP loci are shown in Table 1, and the 18 Indel loci are shown in Table 2. Table 1 ; Note: In Table 1, the numbers before the underscore represent chromosomes, and the numbers after the underscore represent the specific location on the corresponding chromosome.

[0007] Table 2

[0008] In the physical location section, the number before the underscore represents the chromosome, and the number after the underscore represents the physical location of the locus on the corresponding chromosome.

[0009] Among them, population genetic analysis includes sheep genetic diversity analysis, population structure analysis, kinship identification and breed identification; economic traits include birth weight, weaning weight, 6-month-old body weight, 6-month-old body height, 6-month-old body length, 6-month-old cannon circumference and 6-month-old chest width.

[0010] The 12,363 SNP loci and 18 Indel loci of this invention were obtained by whole-genome resequencing, breed-specific locus screening, GWAS analysis and screening based on five local sheep breeds (Small-tailed Han sheep, Yuxi fat-tailed sheep, large-tailed Han sheep, Taihang fur sheep, and Tong sheep) and the cultivated breed Yu meat sheep. The obtained 12,363 SNP loci and 18 Indel loci can be used for genotyping of Henan local sheep and can be used for population genetic analysis and economic trait prediction of Henan local sheep.

[0011] The sheep low-density 10K liquid phase chip genotyping includes the aforementioned 12,363 SNP loci and 18 Indel loci.

[0012] In a preferred embodiment, the sheep low-density 10K liquid phase chip also includes probes designed based on gene sequences covering 12,363 SNP sites and 18 Indel sites.

[0013] In a preferred embodiment, a sheep low-density 10K liquid phase chip is used, with a GC content ratio of 30-70% for the probe sequence, a probe length of 110 bp, a maximum upper limit of less than 5 for the number of specific similar fragments on the reference genome, and a maximum distance of less than 10 bp from the designed region.

[0014] The kit includes a sheep low-density 10K liquid phase chip or includes probes and / or primers for detecting 12,363 SNP sites as shown in Table 1 and 18 Indel sites as shown in Table 2.

[0015] The design method for a low-density 10K liquid phase chip for sheep includes the following steps: S1. Obtaining high-quality SNP sites: S11. Sample Collection: Obtain DNA samples from local sheep breeds. Local sheep breeds include Small-tailed Han sheep, Western Henan Fat-tailed sheep, Large-tailed Han sheep, Taihang Fur sheep, Tong sheep, and Central Henan meat sheep. Among them, Small-tailed Han sheep, Western Henan Fat-tailed sheep, Large-tailed Han sheep, and Taihang Fur sheep are local sheep breeds in Henan, Tong sheep is a local sheep breed in Shaanxi, and Central Henan meat sheep is a cultivated breed. S12. Obtaining the original SNP sites: Perform whole-genome resequencing on the DNA sample to obtain the original SNP sites; S13. Data quality control and alignment: Align the original SNP loci to the sheep reference genome and perform quality control filtering to obtain high-quality SNP loci. S2. Screening of variety-specific sites: Calculate the absolute difference ΔRAF of allele frequencies among varieties, sort them from high to low according to the ΔRAF value, and finally screen out 805 specific SNP sites with the most significant differentiation among varieties from the high-quality SNP sites obtained in step S13. S3, Functional site screening: S21. Obtain phenotypic data related to sheep economic traits in local sheep samples from step S11 and perform quality control. S22. Perform GWAS analysis on the high-quality SNP loci obtained in step S13 and the phenotypic data after quality control in step 21; based on the significance of association, candidate SNP loci for each economic trait are screened, and then redundancy is removed to obtain 3534 functional SNP loci that are significantly associated with important economic traits. S4. Sheep 1K site screening: High-quality site screening was obtained in step S13 using the GenoBaits® sheep 1K liquid phase chip, and a total of 1510 SNP sites and 18 Indel sites were screened. S5. Background site screening: 7334 background SNP sites were obtained by screening SNP sites obtained through sheep reference genome alignment. S6. After merging 805 specific SNP sites, 3534 functional SNP sites, 1510 SNP sites and 7334 background SNP sites, duplicates were removed and optimized, resulting in 12363 SNP sites and 18 Indel sites.

[0016] In other words, in the chip design of this invention, in addition to the local sheep breeds of Henan Province, some genomic information of local sheep (Tongyang) of Shaanxi Province was also introduced to improve the versatility and coverage of the chip.

[0017] Specifically, in step S13, the conditions for quality control filtration include: The minimum sequencing depth is less than 2X, the site deletion rate is greater than 10%, and the minor allele frequency is less than or equal to 0.05.

[0018] Specifically, in step S21, the sheep's economic traits include birth weight, weaning weight, 6-month-old body weight, 6-month-old body height, 6-month-old body length, 6-month-old cannon bone circumference, and 6-month-old chest width.

[0019] Specifically, in step S5, the filtering criteria include: Low sequencing depth greater than or equal to 5X, deletion rate less than 0.1 and MAF greater than 0.05.

[0020] Application of low-density 10K liquid phase chip in genotyping of local sheep.

[0021] Application of low-density 10K liquid phase microarrays in the analysis of genetic diversity, population structure, kinship identification, or breed identification of local sheep.

[0022] Application of low-density 10K liquid phase chip in predicting the economic morphology of local sheep, including birth weight, weaning weight, 6-month-old weight, 6-month-old height, 6-month-old length, 6-month-old cannon circumference, and 6-month-old chest width.

[0023] Application of the kit in genotyping of local sheep.

[0024] Application of the kit in the analysis of genetic diversity, population structure, kinship identification or breed identification of local sheep.

[0025] The application of the reagent kit in predicting the economic morphology of local sheep, which includes birth weight, weaning weight, body weight at 6 months of age, body height at 6 months of age, body length at 6 months of age, cannon bone circumference at 6 months of age, and chest width at 6 months of age.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. This invention collects DNA samples from five local sheep breeds—Small-tailed Han sheep, Western Henan fat-tailed sheep, Large-tailed Han sheep, Taihang fur sheep, and Tong sheep—as well as the cultivated breed, Zhongyu meat sheep. Through whole-genome resequencing, breed-specific site screening, and GWAS analysis, a molecular marker combination including 12,363 SNP loci and 18 Indel loci is obtained. Based on these 12,363 SNP loci and 18 Indel loci, a low-density 10K liquid phase chip and kit for sheep are developed. The molecular marker combination, the sheep low-density 10K liquid phase chip, and the kit can be used for genotyping, population genetic analysis, and prediction of economic traits in local sheep breeds. Applicable local sheep breeds include Small-tailed Han sheep, Western Henan fat-tailed sheep, Large-tailed Han sheep, Taihang fur sheep, Tong sheep, Zhongyu meat sheep, and Huanghuai meat sheep.

[0027] 2. The sheep low-density 10K liquid phase chip of the present invention is of great significance for improving the utilization efficiency of germplasm resources, accelerating the breeding process of superior traits, and enhancing the economic value of livestock breeds. The sheep low-density 10K liquid phase chip of the present invention adopts the latest GBTS targeted sequencing genotype detection technology for marker genotype detection, which has the advantages of low cost, high accuracy, and high detection sensitivity. Attached Figure Description

[0028] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings: Figure 1 Manhattan plots of SNP sites in GWAS analysis of each trait in Example 1; Figure 2 This is a graph showing the results of principal component analysis of the seven sheep breeds in Example 4; Figure 3 This is a diagram showing the genetic structure analysis results of the seven sheep breeds in Example 4; Figure 4 Phylogenetic trees of the seven sheep breeds in Example 4; Figure 5 This is a correlation diagram between the genotype score of weaning weight and the measured weaning weight of individual sheep in Example 5. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The illustrative embodiments and descriptions of this invention are for illustrative purposes only and are not intended to limit the invention. The embodiments described below are some, but not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0030] In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that these specific details are not necessary to practice the invention. In other embodiments, well-known structures, materials, or methods are not specifically described to avoid obscuring the invention. Unless otherwise specified, the materials, instruments, and reagents used in the following embodiments are commercially available. Unless otherwise specified, the techniques used in the embodiments are conventional methods well known to those skilled in the art.

[0031] Example 1: Screening of sheep molecular marker combinations: This embodiment uses whole-genome resequencing of 296 local sheep samples to obtain raw loci (raw sequencing data), and then filters based on the raw data; 12,363 SNP loci are shown in Table 1 and 18 Indel loci are shown in Table 2. The 296 local sheep samples consist of the following: 5 Small-tailed Han sheep, 5 Yuxi Fat-tailed sheep, 4 Large-tailed Han sheep, 4 Taihang Fur sheep, 3 Tong sheep, and 275 Central Henan meat sheep. Among them, the 275 Central Henan meat sheep samples were used for background locus selection, and the remaining samples were used to select breed identification loci.

[0032] The specific screening process includes the following steps: S1. Obtaining high-quality SNP sites: S11. Sample Collection: Obtain DNA samples from local sheep; details are shown above. S12. Obtaining the original SNP sites: Perform whole-genome resequencing on the DNA sample to obtain the original SNP sites; S13. Data Quality Control and Alignment: The original SNP loci were aligned to the sheep reference genome (ARS-UI_Ramb_v3.0), yielding 45,013,111 loci. Quality control filtering was then performed to obtain 1,202,664 high-quality SNP loci. The quality control filtering involved rigorously controlling the original variant detection file (VCF) and removing loci that did not meet the following criteria: 1) Minimum sequencing depth (Min DP) < 2X; 2) Missing rate (MissingRate) ≥ 10%; 3) Minor allele frequency (MAF) ≤ 0.05; 4) Non-diallelic SNPs and all Indel sites.

[0033] S2. Screening of variety-specific SNPs: Calculate the absolute difference ΔRAF of allele frequencies among varieties, sort them from high to low according to the ΔRAF value, and finally screen out 805 specific SNPs with the most significant differentiation among varieties from the high-quality SNPs obtained in step S13.

[0034] S3, Functional site screening: S31. Obtain phenotypic data related to sheep economic traits in local sheep samples from step S11 and perform quality control; the economic traits involved include birth weight, weaning weight, 6-month-old body weight, 6-month-old body height, 6-month-old body length, 6-month-old cannon circumference, and 6-month-old chest width.

[0035] S32. Perform GWAS analysis on the high-quality SNP loci obtained in step S13 and the phenotypic data after quality control in step 21; select the candidate SNP loci with the strongest association significance (i.e., the smallest P-value). For example, select the top 850 loci from traits such as body height, cannula circumference, and chest width at 6 months of age, and select the loci with the smallest P-value from traits such as birth weight and weaning weight, initially obtaining a total of 3630 candidate SNPs; Then, redundancy was removed, candidate loci from all trait sources were merged, and redundancy was removed again, ultimately retaining 3535 SNP loci significantly associated with important economic traits, forming a functional locus set; the Manhattan plots of SNP loci in the GWAS analysis of each trait are shown below. Figure 1 As shown in Table 3, the phenotypes and p-values ​​corresponding to the 3535 functional SNP sites are as follows: Table 3 .

[0036] S4. Sheep 1K Locus Screening: The high-quality loci obtained in step S13 were screened using the GenoBaits® Sheep 1K liquid phase chip (product number PHR0171_Oa1K). The screening was based on the company's internal standards. The main functions of the chip are kinship analysis, breed identification, and functional locus identification. A total of 1510 SNP loci and 18 Indel loci were screened.

[0037] S5. Background site screening: 7334 background SNP loci were identified through screening of SNP loci obtained from sheep reference genome alignment; specifically: 1) Data and quality control: High-quality variant screening was performed on SNP sites obtained from sheep reference genome alignment and screening, with the following criteria: Min DP ≥ 5X, Missing Rate < 0.1, MAF > 0.05, and non-diaryl SNPs and indels were removed; 2) Uniform distribution screening: Based on their physical location on the 26 autosomes and X chromosome of sheep, a total of 7334 SNP loci were screened out according to the principle of uniform distribution.

[0038] S6. After merging 805 specific SNP sites, 3535 functional SNP sites, 1510 SNP sites, and 7334 background SNP sites, duplicate sites were removed. Finally, the physical distance between sites, linkage disequilibrium, probe design feasibility, and evolutionary conservation score were comprehensively considered for optimization. The final result is 12363 SNP sites as shown in Table 1 and 18 Indel sites as shown in Table 2.

[0039] The composition and function of the core SNP site combination in this embodiment are summarized in Table 4: Table 4 .

[0041] Example 2: A sheep low-density 10K liquid phase chip, which includes a probe combination for identifying genotypes at 12,363 SNP loci and 18 Indel loci.

[0042] The purpose of this embodiment is to apply the 12,363 SNP sites in Table 1 and the 18 Indel sites in Table 2 to probe design. Using GenoBaits Probe Designer software, probe design was performed based on the evaluation results of the upstream and downstream sequences of the target site. The evaluation mainly focused on the complexity of the upstream and downstream sequences, GC content, etc., prioritizing the placement of the target site in the middle of the probe. The designed probe was 110 bp in length (centered on the target SNP / Indel site, extending upstream and downstream). Generally, probe performance was comprehensively evaluated based on GC content (30%-70%), Tm value (60-80℃), and the number of alignments to the reference genome (≤5). The probe was 110 bp in length and contained a biotin-modified DNA nucleotide sequence at its 5' end.

[0043] Example 3: The method for genotyping 12363 SNP loci in Table 1 and 18 Indel loci in Table 2 using the sheep low-density 10K liquid phase chip developed in Example 2 includes the following steps: 1. DNA extraction Sheep blood samples were selected, and DNA was extracted from the samples using the GenoPrep® Blood DNA Rapid Extraction Kit (Magnetic Bead Method) (DN004004).

[0044] 2. Quality Inspection Qubit enables precise quantification of DNA concentration: The dsDNA HS Assay Kit for Qubit is used to precisely quantify samples. Agarose gel electrophoresis to detect genome integrity: Take the DNA sample to be tested, spot it on a 1.5% agarose gel, and electrophoresis at 150V for 25 min.

[0045] Qubit ≥ 50 ng / µL, 1% agarose gel integrity > 20 kb.

[0046] 3. Library Construction A quantitative amount of DNA was collected and fragmented using GenoBaits® End Repair Enzyme. The fragmented DNA was then end-repaired and ligated with A-tails. The A-tailed DNA fragments were ligated to sequencing adapters using GenoBaits® UltraDNA Ligase, and the ligation products were purified using carboxyl-modified magnetic beads (GenoPrep® DNA Clean Beads). The ligation products were then added to barcoded sequencing primers and a high-fidelity PCR reaction system for PCR amplification. Different barcodes were used to distinguish different samples. The amplified products, purified by the carboxyl-modified magnetic beads, were then ready for probe hybridization experiments.

[0047] 4. Liquid phase probe capture Take 500 ng of the constructed DNA library, add a mixture of GenoBaits® Panel, GenoBaits® Block I, and GenoBaits® Block II, and concentrate to dryness. Add GenoBaits® 2X Hyb Buffer and GenoBaits® Hyb Buffer Enhancer, and incubate at 65°C for 2 hours to complete the hybridization reaction. Add GenoBaits® DNA Probe Beads to elute and remove unbound DNA, retaining only the target DNA fragment that hybridizes with the GenoBaits® Panel. Enrich and purify the target fragment by PCR amplification to complete the construction of the hybridization capture library.

[0048] 5. Sequencing After library construction, preliminary quantification was performed using Qubit 2.0, followed by accurate quantification of the effective library concentration using qPCR to ensure library quality. Once the library passed the initial testing, it proceeded to the sequencing stage. A DNBSEQ-T7 sequencer was used, with the PE150 sequencing strategy selected.

[0049] 6. Genotype data analysis The raw data were quality controlled using FastQC. Then, the sequencing data were aligned to the sheep reference genome ARS-UI_Ramb_v3.0 using the default parameters of BWA software. SNPs were detected and genotyping was performed using the standard procedure of GATK software.

[0050] Site quality assessment: Using 35 newly collected local sheep samples (specifically: 8 large-tailed Han sheep, 8 small-tailed Han sheep, 7 Taihang fur sheep, 6 Yuxi fat-tailed sheep, and 6 Tong sheep), the sheep low-density 10K liquid phase chip developed in Example 2 was used for testing with the detection method in Example 3. The average detection rate was 99.7%, which has the advantage of high detection rate.

[0051] The above test results demonstrate that the sheep low-density 10K liquid phase chip, based on the above detection method, can perform genotyping and DNA detection on local sheep samples.

[0052] Example 4: Application of low-density 10K liquid phase microarray in genetic analysis of local sheep populations Blood samples were collected from 59 sheep (specifically: 11 from Huanghuai mutton sheep (HHS), 15 from small-tailed Han sheep (STS), 10 from large-tailed Han sheep (LTS), 5 from Taihang fur sheep (TFS), 3 from Tong sheep (TS), 5 from Yuxi fat-tailed sheep (YXS), and 10 from Zhongyu mutton sheep (ZYS). Among these, Huanghuai and Zhongyu mutton sheep are bred breeds, and both breeds used small-tailed Han sheep as the maternal line; the gene loci of small-tailed Han sheep are also present in both Huanghuai and Zhongyu mutton sheep). Genotyping was performed on these 59 samples using the sheep low-density 10K liquid phase chip developed in Example 2 and the detection method of Example 3. Based on the genotyping results, a local sheep population genetic analysis was conducted. 1) Genetic diversity analysis: The genetic diversity assessment of seven sheep breeds (Huanghuai meat sheep, large-tailed Han sheep, small-tailed Han sheep, Taihang fur sheep, Yuxi fat-tailed sheep, Tong sheep, and Zhongyu meat sheep) using PLINK v2.0 (Table 5) showed significant differences among the breeds in terms of observed heterozygosity (Ho), expected heterozygosity (He), nucleotide polymorphism (Pi), effective allele count (Na), and percentage of polymorphic sites (PPL). Among them, Tong sheep had the highest observed heterozygosity (0.3823), but its expected heterozygosity (0.0899) and percentage of polymorphic sites (56.89%) were the lowest. Furthermore, data on nucleotide polymorphism and effective allele count were missing, suggesting that this population may have inbreeding depression or population stratification, resulting in an inflated observed heterozygosity and a lack of true genetic variation. In comparison, the Small-tailed Han sheep exhibited the highest expected heterozygosity (0.2446) and polymorphic site percentage (93.36%), while the Large-tailed Han sheep ranked high in effective allele count (22.07) and nucleotide polymorphism (0.3182). Both have the broadest genetic base and are ideal germplasm resources and breeding materials. Although the Yuxi Fat-tailed Sheep possessed the highest nucleotide polymorphism (0.3336) and effective allele count (22.34), its expected heterozygosity (0.1449) and polymorphic site percentage (63.52%) were relatively low, with Ho significantly higher than He, suggesting a potentially complex genetic structure within the population or the influence of selection. The Zhongyu and Huanghuai meat sheep, as bred breeds, had relatively low effective allele counts (7.30 and 7.39) and polymorphic site percentages (84.87% and 88.94%), consistent with the expectation of narrowing the genetic base due to artificial selection. Small-tailed Han sheep and large-tailed Han sheep have the richest genetic diversity and are the advantageous groups for subsequent molecular breeding; while Tong sheep and Yuxi fat-tailed sheep need further evaluation of population structure and data integrity in order to clarify their genetic characteristics and formulate scientific conservation strategies.

[0053] Table 5 .

[0054] 2) Principal Component Analysis (PCA) Principal component analysis was performed on autosomal SNPs of seven sheep breeds using GCTA (v1.94). The results are as follows: Figure 2 As shown, PC1 and PC2 contributed a total of 11.21%. PC1 separated the Zhongyu mutton sheep from the other six breeds, and PC2 separated the Huanghuai mutton sheep from the other six breeds. There are some genetic differences among the five sheep breeds: large-tailed Han sheep, small-tailed Han sheep, Taihang fur sheep, Yuxi fat-tailed sheep, and Tong sheep, but the differences are relatively small.

[0055] 3) Group structure analysis ADMIXTURE (v1.3.0) was used to infer the population structure of seven varieties, and cluster analysis was performed with K=2 to K=7. The results are as follows: Figure 3 As shown, with the increase of K value, the genetic components within each variety gradually become more refined, exhibiting different degrees of mixing and differentiation characteristics.

[0056] 4) Phylogenetic tree construction Based on autosomal SNP data, a phylogenetic tree was constructed using the Neighbor-Joining method and the Tajima-Nei model in MEGA X (v10.2.6). The results are as follows: Figure 4 As shown, individuals of the same variety are basically clustered in the same branch, while there is obvious differentiation between different varieties, supporting that each variety has an independent genetic background.

[0057] 5) The genetic differentiation coefficient (FST) was used to assess the degree of genetic differentiation among breeds, and the results are shown in Table 6. Based on the FST assessment results of the seven sheep breeds, the genetic distances among the breeds showed significant differences. Among them, the FST value between Zhongyu meat sheep and Yuxi fat-tailed sheep was the highest (0.16699), indicating that the genetic differentiation between these two breeds was the most obvious and the gene flow was the least; followed by Zhongyu meat sheep and Tongyang (0.14423), Huanghuai meat sheep and Yuxi fat-tailed sheep (0.11305), and Huanghuai meat sheep and Zhongyu meat sheep (0.11598), all of which reached a moderate to high level of differentiation. In stark contrast, the lowest FST value was observed between the large-tailed Han sheep and the small-tailed Han sheep (0.012981), indicating a very similar genetic background, possibly stemming from a common ancestral population or frequent interbreeding. The FST values ​​between the Taihang fur sheep and the Tong sheep (0.020851), the Taihang fur sheep and the small-tailed Han sheep (0.01771), and the large-tailed Han sheep and the Taihang fur sheep (0.021136) were also generally low, all below 0.03, suggesting extensive genetic infiltration or a relatively short history of breed differentiation among these northern sheep breeds. Notably, the FST values ​​between the Huanghuai meat sheep and the five local breeds (large-tailed Han sheep, small-tailed Han sheep, Taihang fur sheep, Tong sheep, and Yuxi fat-tailed sheep) ranged from 0.06 to 0.11, all indicating a moderate level of differentiation. This reflects that the Huanghuai meat sheep, as a bred breed, has retained some of the genetic background of local breeds while also developing unique genetic characteristics during the breeding process. In summary, the FST analysis results reveal the genetic differentiation pattern of sheep breeds: the large-tailed Han sheep, small-tailed Han sheep, Taihang fur sheep, and the same breed constitute a closely related group; the Yuxi fat-tailed sheep occupies a relatively independent intermediate position; while the Central Henan meat sheep and the Huanghuai meat sheep, as modern bred breeds, show the greatest genetic distance from other local breeds. This provides a key theoretical basis for subsequent germplasm resource protection and hybridization utilization.

[0058] Table 6

[0059] In summary, the sheep low-density 10K liquid phase chip of the present invention can be used for genetic analysis of local sheep populations and can be applied in the identification of kinship in local sheep.

[0060] Example 5: Application of low-density 10K liquid phase chip in predicting local sheep economic traits 1) Experimental materials and phenotypic determination This embodiment selected 56 F1 generation lambs from the same sheep, with similar feeding and management conditions and birth dates (±5 days), as the experimental group. Weaning weight was used as an example for illustration; the prediction methods for other traits were the same as for weaning weight.

[0061] To assess weaning weight, all lambs underwent accurate weaning weight measurement before morning feeding at a uniform weaning age (30 days old), and the results were recorded as phenotypic data for the target trait. Descriptive statistics of weaning weight for this group are shown in Table 7. Table 7 .

[0062] 2) Genotyping test: Ear tissue or blood samples were collected from all 56 sheep, and genomic DNA was extracted using the conventional phenol-chloroform method or a commercially available kit. After DNA extraction, quality control was performed to ensure an A260 / A280 ratio between 1.8 and 2.0 and a concentration ≥50 ng / μL. Subsequently, the sheep low-density 10K liquid chromatography chip developed in Example 2 was used for testing using the detection method of Example 3. The specific procedure is as follows: 1. Library construction: Construct sequencing libraries using fragmented DNA; 2. Targeted capture: Hybridization capture is performed using the liquid-phase chip probe pool designed in this invention to enrich the genomic region where the target SNP site is located; 3. High-throughput sequencing: Perform paired-end sequencing on the Illumina platform, with an average sequencing depth of ≥10× for each sample; 4. Bioinformatics analysis: The Clean Reads obtained from sequencing were compared with the sheep reference genome (ARS-UI_Ramb_v3.0), and variant detection was performed using the GATK standard procedure. Finally, high-quality genotyping data (genotyping detection rate >99%) of each sample at all target SNP sites were obtained.

[0063] 3) Genotype scoring calculation and individual screening Based on previous genome-wide association studies (GWAS) and literature reports, it has been confirmed that there are "favorable alleles" significantly associated with weaning weight in sheep among the 153 SNP loci. The Weaning Weight Genotype Score (WWGS) was calculated for each sheep using the following method: Genotype "homozygous for favorable allele" scores 2 points; genotype "heterozygous" scores 1 point; genotype "homozygous for unfavorable allele" scores 0 points. An individual's WWGS score is the sum of scores from all 153 loci. All 56 individuals are ranked in descending order based on their WWGS scores. See Table 8 for details. Table 8

[0064] As shown in Table 8: The top 10 individuals by genotype score had a significantly higher average weaning weight (17 kg) than the population average (14.3 kg). The correlation between the two can be established through... Figure 5 Visually intuitive.

[0065] 4) Validation of model prediction accuracy To objectively evaluate the predictive power of this SNP combination, a cross-validation strategy is adopted: 1. Randomly divide the 56 samples into a training set (n=40) and a validation set (n=16); 2. Using the training set data, establish a linear regression model between "weaning weight genotype score (WWGS)" and "measured weaning weight"; 3. Apply this model to the validation set of individuals and use their WWGS to predict their weaning weight; 4. Calculate the correlation coefficient (r) between the predicted and measured values ​​as an indicator of prediction accuracy. In this embodiment, the prediction accuracy r = 0.869, indicating that the model has high predictive ability.

[0066] In summary, the sheep molecular marker combination and sheep low-density 10K liquid phase chip of this invention can genotype local sheep and effectively identify individuals with excellent weaning weight trait by calculating the weaning weight genotype score (WWGS). This method has high predictive accuracy (up to 86.9%), enabling early, non-destructive, and efficient prediction of this important growth trait, providing a direct and reliable technical tool for molecular-assisted selection and genomic selection breeding of sheep.

[0067] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. 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. The application of sheep molecular marker combinations, characterized in that, The applications include population genetic analysis and economic trait prediction of local sheep breeds; these local sheep breeds include Small-tailed Han sheep, Western Henan Fat-tailed sheep, Large-tailed Han sheep, Taihang Fur sheep, Tong sheep, Central Henan meat sheep, and Huanghuai meat sheep; the sheep molecular marker combination includes 12363 SNP loci and 18 Indel loci; the 12363 SNP loci and 18 Indel loci were determined based on whole-genome sequence alignment of a sheep reference genome, the version number of which is ARS-UI_Ramb_v3.0; the 12363 SNP loci are shown in Table 1; the 18 Indel loci are shown in Table 2. Table 1 Table 2 。 2. A sheep-based low-density 10K liquid phase chip, characterized in that, The genotyping of the sheep low-density 10K liquid phase chip includes the 12,363 SNP sites and 18 Indel sites as described in claim 1.

3. The sheep low-density 10K liquid phase chip according to claim 2, characterized in that, It also includes probes designed based on gene sequences covering 12,363 SNP sites and 18 Indel sites.

4. The sheep low-density 10K liquid phase chip according to claim 3, characterized in that, The probe sequence has a GC content of 30-70%, a probe length of 110 bp, a maximum upper limit of less than 5 specific similar fragments on the reference genome, and a maximum distance of less than 10 bp from the designed region.

5. A reagent kit, characterized in that, Includes the sheep low-density 10K liquid phase chip as described in claim 3 or 4, or includes probes and / or primers for detecting 12,363 SNP sites as shown in Table 1 and 18 Indel sites as shown in Table 2.

6. The design method for a sheep low-density 10K liquid phase chip as described in claim 3 or 4, characterized in that, Includes the following steps: S1. Obtaining high-quality SNP sites: S11. Sample Collection: Obtain DNA samples from local sheep; local sheep include small-tailed Han sheep, western Henan fat-tailed sheep, large-tailed Han sheep, Taihang fur sheep, Tong sheep, and central Henan meat sheep. S12. Obtaining the original SNP sites: Perform whole-genome resequencing on the DNA sample to obtain the original SNP sites; S13. Data quality control and comparison: The original SNP sites are compared to the sheep reference genome and quality control filtering is performed to obtain high-quality SNP sites. S2. Screening of variety-specific sites: Calculate the absolute difference ΔRAF of allele frequencies among varieties, sort them from high to low according to the ΔRAF value, and finally screen out 805 specific SNP sites with the most significant differentiation among varieties from the high-quality SNP sites obtained in step S13. S3, Functional site screening: S31. Obtain phenotypic data related to sheep economic traits in local sheep samples from step S11 and perform quality control. S32. Perform GWAS analysis on the high-quality SNP sites obtained in step S13 and the phenotypic data after quality control in step 21; screen candidate SNP sites for each economic trait based on the significance of association, and then remove redundancy to obtain 3534 functional SNP sites that are significantly associated with important economic traits. S4. Sheep 1K site screening: High-quality site screening was obtained in step S13 using the GenoBaits® sheep 1K liquid phase chip, and a total of 1510 SNP sites and 18 Indel sites were screened. S5. Background site screening: 7334 background SNP sites were obtained by screening SNP sites obtained through sheep reference genome alignment. S6. After merging 805 specific SNP sites, 3534 functional SNP sites, 1510 SNP sites and 7334 background SNP sites, duplicates were removed and optimized, resulting in 12363 SNP sites and 18 Indel sites.

7. The design method according to claim 6, characterized in that, In step S13, the conditions for quality control filtration include: The minimum sequencing depth is less than 2X, the site deletion rate is greater than 10%, and the minor allele frequency is less than or equal to 0.

05.

8. The design method according to claim 6, characterized in that, In step S21, the sheep's economic traits include birth weight, weaning weight, 6-month-old body weight, 6-month-old body height, 6-month-old body length, 6-month-old cannon bone circumference, and 6-month-old chest width.

9. The design method according to claim 6, characterized in that, In step S5, the filtering criteria include: Low sequencing depth greater than or equal to 5X, deletion rate less than 0.1 and MAF greater than 0.

05.

10. The application of the sheep low-density 10K liquid phase chip as described in claim 3 or 4 in the genotyping of local sheep.

11. The application of the sheep low-density 10K liquid phase chip as described in claim 3 or 4 in the analysis of genetic diversity, population structure, kinship identification or breed identification of local sheep.

12. The application of the sheep low-density 10K liquid phase chip as described in claim 3 or 4 in the prediction of local sheep economic patterns, characterized in that, The local sheep economic traits include birth weight, weaning weight, 6-month-old body weight, 6-month-old body height, 6-month-old body length, 6-month-old cannon circumference, and 6-month-old chest width.

13. The application of the kit as described in claim 5 in the genotyping of local sheep.

14. The application of the kit as described in claim 5 in the analysis of genetic diversity, population structure, kinship identification, or breed identification of local sheep.

15. The application of the reagent kit as described in claim 5 in predicting the economic morphology of local sheep, characterized in that, The local sheep economic traits include birth weight, weaning weight, 6-month-old body weight, 6-month-old body height, 6-month-old body length, 6-month-old cannon circumference, and 6-month-old chest width.

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