Molecular marker combination for analyzing wool fineness and application
By combining 1601 SNP loci and using high-throughput genomics technology, the problems of insufficient marker accuracy and sample diversity in the genetic improvement of wool fineness were solved, and efficient breeding and variety management of wool fineness were achieved.
Patent Information
- Application Number
- CN202511677130.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-14
- Publication Date
- 2026-02-06
AI Technical Summary
Existing technologies for genetic improvement of wool fineness suffer from limited marker accuracy, insufficient sample size and diversity, and limited application of genetic markers, resulting in low efficiency in genetic improvement.
Using a combination of 1601 SNP loci, based on a high-quality T2T-sheep1.0 genome reference, and combined with high-throughput genomics technology, we developed molecular probe combos, gene chips, and kits for wool fineness analysis and breeding.
It improves the accuracy and breeding efficiency of wool fineness trait improvement, enabling rapid and accurate evaluation of wool fineness, variety screening, variety identification, variety tracing and breeding, reducing costs and expanding the market benefits of genetic improvement.
Smart Images
Figure CN121472418A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to the field of biodetection technology, and more specifically to wool fineness SNP site combinations and their applications. Background Technology
[0002] Wool fineness, as a key trait determining wool quality, has always been an important research area of great interest in sheep farming. Wool fineness directly affects the market value of wool and the quality of textiles; therefore, developing sheep breeds with finer wool has become an important goal of genetic improvement.
[0003] In recent years, with the development of genomics technologies, especially the maturity of genome-wide association studies (GWAS), numerous studies have reported multiple genes associated with wool traits. For example, research has found that the FOXQ1 gene is closely related to hair morphology and growth rate, becoming a key target in wool fineness research. In addition, other genes such as KRTAP3-3, KRTAP5-5, and FAS1 also play important roles in traits such as wool fineness and fiber growth. Domestically, significant progress has also been made in research on wool fineness. Chinese scholars have proposed several genetic markers for wool fineness through genomic analysis of different sheep breeds. However, these studies mostly focus on a few candidate genes, and the genetic effects of these markers have not yet been widely validated in the practice of wool fineness improvement.
[0004] Although existing research has shown that genes such as FOXQ1 play an important role in wool fineness, current research on the genetic improvement of wool fineness still has certain limitations: 1) Limited marker precision: Most existing molecular markers focus on a few genes or a single phenotypic trait, lacking consideration of the comprehensive effects of multiple sites, which limits the accuracy and efficiency of genetic improvement. For example, although the KRTAP3-3 gene marker proposed by Sun et al. (2024) can predict some changes in wool fineness, the scope and stability of its genetic effect have not been fully verified.
[0005] 2) Insufficient sample size and diversity: Most current studies rely on small samples or specific breeds of sheep, neglecting the genetic variation between sheep breeds and the differences between populations. For example, Ma et al. (2023) mainly focused on a single breed of sheep and failed to fully explore the diversity of different sheep breeds.
[0006] 3) Limited application of genetic markers: Although existing research has provided a variety of SNP loci, their application has not been effectively promoted in actual breeding work. The screening and validation process of markers is complex, and some research methods have not been applied in a wide range of populations, which has affected their promotion in breeding. Summary of the Invention
[0007] Existing methods for improving wool fineness primarily rely on phenotypic selection, which is heavily influenced by environmental factors and has relatively low efficiency. This invention, through genomic methods, particularly the application of SNP loci, provides a molecular marker, molecular probe combination, gene chip, reagent kit, and application for analyzing wool fineness. Utilizing the locus information provided by this invention, the accuracy and breeding efficiency of wool fineness trait improvement are enhanced, thus offering the wool industry a more efficient genetic improvement tool. It enables rapid and accurate evaluation of wool fineness, variety screening, variety identification, variety tracing, and breeding, which is beneficial for germplasm resource protection and improvement, and is time-efficient, low-cost, and has broad market benefits.
[0008] To achieve the technical objective of this invention, the following technical solution is provided: In a first aspect, the present invention provides an application of a combination of 1601 SNP sites in the analysis of wool fineness, the physical locations of which are shown in Table 1: Table 1. Location information of 1601 site combinations
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[0018] ; Its physical location was determined based on the T2T-sheep1.0 genome sequence alignment of the reference genome.
[0019] The second aspect provides a method for analyzing wool fineness by comparing the genotypes of 1601 SNP sites in the genomic DNA to be tested with the genotypes of the same 1601 SNP sites in the control genomic DNA. The 1601 SNP sites mentioned above are the 1601 SNP sites listed in Table 1.
[0020] The third aspect provides a molecular probe combination for analyzing wool fineness, wherein the molecular probe combination detects the SNP site combination shown in Table 1 in the sample to be tested, and the physical location information of the site combination in Table 1 is determined based on the T2T-sheep1.0 genome sequence alignment of the reference genome.
[0021] Fourthly, a gene chip for analyzing wool fineness, wherein the gene chip is loaded with the molecular probe combination described in the third aspect.
[0022] Fifthly, a kit for analyzing wool fineness, having the molecular probe combination described in the third aspect or the gene chip described in the fourth aspect.
[0023] The sixth aspect is the method for analyzing wool fineness, which involves using the molecular probe combination described in the third aspect, the gene chip described in the fourth aspect, or the kit described in the fifth aspect to detect the sample to be tested.
[0024] The seventh aspect, the molecular probe combination described in the third aspect, the gene chip described in the fourth aspect, or the kit described in the fifth aspect have any of the following uses: (1) Application in wool quality evaluation; (2) Application in variety selection; (3) Application in variety identification; (4) Application in variety traceability; (5) Applications in breeding; (6) Application in germplasm resource conservation; (7) Application in germplasm resource improvement; (8) Application in genealogy reconstruction.
[0025] Beneficial effects 1. The SNP site detection and screening of this invention is based on the high-quality T2T-sheep1.0 genome as a reference. Compared with the current chromosome-level reference genome, the SNP site detection in this study has higher single-base quality, alignment performance, and variant detection capability, resulting in greater applicability and ensuring the universality and stability of the analysis results. Because the integrity and accuracy of the T2T genome are significantly improved compared to the currently used reference genome, not only in the increase of telomere and centromere sequences, but also in the unlocking of some complex regions not resolved in the currently widely used reference genome, and the core SNPs provided by this invention are located in these newly resolved regions, which cannot be obtained through previous reference genomes, the SNP sites provided by this invention have high innovation and are irreplaceable.
[0026] 2. Large-scale SNP screening: Through high-throughput genomics technology, we can comprehensively identify multiple SNP sites associated with wool fineness, covering a wider range of genes and regulatory regions. Compared to studies using single genes or a few markers, our marker set has higher predictive power.
[0027] 3. Molecular Marker-Assisted Breeding: By molecularly marker-assisted breeding of these SNP sites, we have developed a set of molecular marker-assisted breeding tools that can be widely applied to wool fineness improvement. These markers can accurately predict wool fineness traits, providing a theoretical basis and practical support for the rapid selection of high-quality sheep breeds. Attached Figure Description
[0028] Figure 1 The Manhattan plot is a selection elimination analysis of the fine-wool group and the hairy non-hair sheep. Figure 2 This is a Manhattan plot of selective elimination analysis performed on the fine-wool and medium-wool hair groups. Figure 3 Manhattan plot of coarse-wool and hairy non-wool sheep selected for elimination analysis; Figure 4 This is a Manhattan plot of the selection elimination analysis performed on the medium-wool semi-fine wool group and the hairy non-wool sheep group. Figure 5 This is a graph showing the significance test results of the group threshold analysis. Detailed Implementation
[0029] The present invention will be further illustrated below with reference to a detailed description of specific embodiments. However, these embodiments are merely illustrative and should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and can be referred to the third edition of the original book "Bioinformatics and Functional Genomics" or related books. The bioinformatics software and products used are also commercially available. Various processes and methods not described in detail are conventional methods known in the art. The source of materials used, trade names, and components that need to be listed are indicated upon their first appearance. Unless otherwise specified, the same reagents used thereafter are considered the same as those initially indicated.
[0030] Furthermore, it should be noted that the site combinations and applications provided by this invention are the result of the inventors' arduous creative work and optimization efforts.
[0031] The features and advantages described in the site combination section above also apply to molecular probe combinations, gene chips, kits, and their applications based on site combinations, and will not be repeated here.
[0032] It should be noted that the wool fineness referred to in this invention refers to the fineness of the wool, and is classified according to whether or not the wool fineness is present.
[0033] The SNP referred to in this invention is Single Nucleotide Polymorphism, which mainly refers to DNA sequence polymorphism caused by a single nucleotide variation at the genomic level. The single nucleotide variation includes variations caused by a single base conversion, transversion, insertion, or deletion.
[0034] It should be noted that the molecular markers referred to in this invention are all heritable and detectable DNA sequences or proteins, including but not limited to molecular markers based on molecular hybridization, such as RFLP and Minisatellite DNA; molecular markers based on PCR technology, such as RAPD, STS, SSR, and SCAR; DNA markers based on restriction enzyme digestion and PCR technology; molecular markers based on DNA microarray technology, such as SNP; and analytical marker technologies developed based on EST databases. The molecular markers provided by this invention can be used for genome mapping, gene localization studies, map-based gene cloning, species phylogenetics, and systematic classification.
[0035] It should be noted that the probe referred to in this invention is a nucleic acid sequence (DNA or RNA) that is complementary to the target gene, has a known sequence and carries a detection marker, such as the Taqman-MGB probe.
[0036] It should be noted that the kit referred to in this invention is any kit commonly used in the art that contains reagents for detection or experimentation, freeing operators from the cumbersome process of reagent preparation and optimization. In one embodiment of this invention, it includes primers for amplifying the site information provided by this invention, molecular markers or probes or gene chips for detecting the site information provided by this invention, as well as enzymes and buffers for amplification, or fluorescent labels for detection.
[0037] Example 1: Obtaining the SNP site combination for wool fineness 1. Acquisition of genetic data Collect biological samples (e.g., blood or tissue samples) from coarse-wool and fine-wool sheep populations, specifically from the following breeds: Chinese Merino (fine-wool type), Chinese Merino (extra-fine-wool type), Azerbaijani Mountain Merino, Kazakh Fine-wool Sheep, Romanov Sheep, Welsh Mountain Sheep, Bonga Sheep, Afar Sheep, Mbororo Sheep, Yankasasheep, West African Dwarf Sheep, Uda Sheep, Djiallonke Sheep, Sahelian Sheep, Cameroon Sheep, Kazakh Arkhar-Merino Sheep, Jaffna Local Sheep, Vembur Sheep, Mecheri Sheep, Madras Red Sheep, Nellore Sheep, Pattanam Sheep, Kilakarsalsheep, Dagestan Mountain Merino Sheep, Transbaikal Finewool Sheep, Ouessant, Rhoen Sheep, Ryelands Sheep, Swedish Finewool Sheep, Feral Sheep, Gotland, Morada Nova Sheep, Brazilian Somali Sheep, and Santa Inês Sheep.
[0038] Based on the market value of wool fineness, this application divides the samples into four groups from fine to coarse: fine-wool, medium-wool, coarse-wool, and hairy.
[0039] It should be noted that some of the above-mentioned variety names do not yet have official Chinese translations and are currently still using their English names.
[0040] Samples containing genetic information from individual sheep are collected using conventional methods in the field. These samples include, but are not limited to, blood, cells, tissues, skin, hair, and excrement.
[0041] Genetic information (e.g., DNA) was extracted from the samples and subjected to high-depth sequencing. Raw data from 810 population samples were aligned to the T2T-sheep1.0 genome using BWA, and approximately 133 million SNP loci were detected using a standardized GATK variant detection process. Compared to variant detection results based on the reference genome Oar_rambouillet_v1.0 (obtained from NCBI), approximately 2.66 million new SNP loci were obtained in newly resolved regions of T2T-sheep1.0. Variants related to the coarse and fine wool sheep samples were extracted from the total variant set, and deleted and low-frequency loci were filtered using a MIS (deletion rate) threshold of 0.1 and a MAF (minor allele frequency) threshold of 0.05 to obtain a high-quality SNP dataset. Furthermore, based on this dataset, selection signal analysis was performed between coarse-wool and fine-wool sheep populations using XP-CLR and π methods to obtain SNP loci and candidate genes related to wool-producing traits. Specifically, selection elimination analysis was conducted between the fine-wool group and the hairy non-wool sheep group (Manhattan diagram shown). Figure 1 As shown), a selective elimination analysis was performed on the fine-wool ultrafine hair group and the medium-wool semifine hair group (Manhattan plot as shown). Figure 2 As shown in the figure), a selection elimination analysis was performed on coarse-wool sheep and hairy non-wool sheep (Manhattan plot as shown). Figure 3 (As shown in the figure), a selection elimination analysis was performed on the medium-wool semi-fine wool group and the hairy non-wool sheep (Manhattan plot as shown). Figure 4 (As shown).
[0042] It should be noted that the genetic information referred to in this invention refers to the information passed from parent to offspring in order for an organism to replicate itself, or the information passed from cell to cell during each cell division.
[0043] It should be noted that the extraction of genetic information (e.g., DNA) from samples for high-depth sequencing can be performed by biotechnology companies, such as BGI Genomics and Illumina. The high-depth sequencing method adopts conventional methods in the field or the methods of biotechnology companies. In one embodiment of the present invention, an average sequencing depth of ~25.7× is used, and a resequencing analysis process is applied for high-depth sequencing.
[0044] 3. Phenotypic Analysis Using statistical methods and combining wool fineness phenotypic data, selection signal analysis was performed on whole-genome data to screen out SNP loci associated with wool fineness. Specifically, these loci were functionally annotated to analyze their possible genetic roles, which are: influencing the morphology and function of the inner root sheath by regulating the expression of keratin and related proteins, thereby leading to a curly and soft wool phenotype in animals.
[0045] 4. SNP loci acquisition Validation experiments confirmed the genetic effects of the selected SNP loci. Molecular markers suitable for these SNP loci were designed, and preliminary marker detection was performed to verify their stability and reliability. Specifically, the allele frequencies of these loci were examined in sheep populations with different wool fineness, revealing significant differences.
[0046] Using bedtools, we searched for SNP sites corresponding to the functional regions of the candidate genes identified in step 3 in the total SNP set, resulting in a wool fineness site combination containing only 1601 SNP sites.
[0047] Example 2: Using wool fineness SNP site combinations to prepare primer and probe combinations. Those skilled in the art can design primers based on the sequence information of each site in the wool fineness SNP site combination provided by the present invention, and conduct secondary structure evaluation and Tm value evaluation on the designed primers to finally obtain primers with good specificity, high sensitivity, and the ability to achieve the detection purpose under the same reaction conditions.
[0048] The secondary structure assessment and Tm value assessment can be performed using any method commonly used in the field. For example, the secondary structure can be assessed using DNA folding form (see http: / / unafold.rna.albany.edu / ?q=mfold / DNA-Folding-Form), and the Tm value can be assessed using the software RaW-Probe.
[0049] The above methods are all conventional methods. Based on the site information in the SNP site combination of wool fineness provided in this application, they can be obtained without any creative effort. Therefore, the primers obtained by the SNP site combination provided in this invention also fall within the protection scope of this invention.
[0050] Similarly, probes prepared using the SNP site combinations provided by this invention, such as the Tanqman probe, also fall within the scope of protection of this invention.
[0051] Example 3: Combination of SNP sites for analyzing wool fineness was used to prepare a gene chip. The SNP gene chip of the present invention uses conventional methods to fix the primers or probes obtained in Example 2 onto a polymer substrate, such as a nylon membrane, nitrocellulose membrane, plastic, silicone wafer, micro magnetic beads, etc., or fix the probes onto a glass plate, or directly synthesize the primers or probes obtained in Example 2 on a hard surface such as glass. The method of using the SNP gene chip of this application is the same as the conventional method.
[0052] It should be noted that those skilled in the art can prepare SNP gene chips for detecting wool fineness using any of the methods described herein, or they can entrust a biotechnology company to prepare them. However, SNP gene chips prepared based on the SNP locus combinations for wool fineness analysis provided in this application are all within the scope of protection of this invention.
[0053] Example 4: Wool Fineness Analysis Kit The SNP detection kit for analyzing the fineness of sheep wool provided in this application includes primers, probes, or gene chips obtained based on the SNP site combinations obtained in Example 1. Depending on the type of use, it also includes corresponding detection reagents. For example, when the SNP site combinations obtained based on Example 1 are TaqMan probes, it also includes buffers, ligases, AceQ Universal U+ Probe Master Mix V2, TaqMan Probe, etc., commonly used in real-time PCR reactions.
[0054] Those skilled in the art can configure different SNP kits for wool fineness detection according to different usage methods, but all wool fineness SNP detection kits configured based on the wool fineness SNP site combinations provided in this application are within the protection scope of this invention.
[0055] Example 5: Detection of wool fineness Based on the SNP site combination for analyzing wool fineness provided in Embodiment 1 of this application, the known wool fineness is detected. Based on the detection results and in conjunction with the known wool fineness, the accuracy of the detection is determined, specifically as follows: Peripheral blood was collected using conventional methods, and whole-genome DNA was extracted from it to obtain whole-genome DNA samples; Gene chips were designed using conventional methods based on the site information in the SNP site combination provided by the present invention. Whole genome DNA samples were tested to obtain the genotyping results of each site (i.e., whether each site is homozygous, heterozygous, mutant homozygous, or has a base deletion). The frequency value of the genotyping results of each site was calculated and compared with the population threshold. The comparison results showed that the gene detection results were consistent with the corresponding wool fineness phenotype.
[0056] It should be noted that the group threshold mentioned in this application is obtained by analyzing the size groups of wool fineness, namely the hairy non-wool sheep and the wool fine wool group, using the same method as above.
[0057] This application conducted a significance test (independent samples Man-Whitney U test) on the analysis results of the large wool fineness group and the small wool fineness group. The results are as follows: Figure 5 As shown in the figure, the results indicate that P < 0.01, which is highly significant. This demonstrates that the results obtained using the method of this invention are accurate and effective.
[0058] Industrial applications Based on the SNP locus combination for analyzing wool fineness provided in this application, which consists of only 1601 SNP loci, those skilled in the art can prepare SNP probe combinations, gene chips, and kits for analyzing wool fineness at the genomic level, assessing genetic information, screening and identifying varieties, controlling the breeding process, and can also be applied to variety tracing, pedigree reconstruction, germplasm resource protection, and germplasm resource improvement.
[0059] The above description is merely a preferred embodiment to aid in understanding the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any alterations or modifications made by those skilled in the art based on this description without departing from the spirit of the present invention should also fall within the scope of the present invention.
Claims
1. 1 Application of the 1601 SNP locus combinations in the analysis of wool fineness, the physical locations of the 1601 SNP locus combinations are shown in Table 1; Its physical location was determined based on the genome sequence alignment of the sheep reference genome T2T-sheep1.
0.
2. A method for analyzing wool fineness involves comparing the genotypes of 1601 SNP sites in the genomic DNA to be tested with the genotypes of the same 1601 SNP sites in the control genomic DNA; in, The 1601 SNP sites are the 1601 SNP sites described in claim 1.
3. A molecular probe combination for analyzing wool fineness, wherein the molecular probe combination detects the SNP site combination shown in Table 1 in the sample to be tested, and the physical location information of the site combination in Table 1 is determined based on the ARS1 genome sequence alignment of the reference genome.
4. A gene chip for analyzing wool fineness, wherein the gene chip is loaded with the molecular probe combination as described in claim 3.
5. A kit for analyzing wool fineness, comprising the molecular probe combination of claim 3 or the gene chip of claim 4.
6. A method for analyzing wool fineness, comprising using the molecular probe combination of claim 3, the gene chip of claim 4, or the kit of claim 5 to detect the sample to be tested.
7. The molecular probe combination of claim 3, the gene chip of claim 4, or the kit of claim 5 has the following uses: (1) Application in wool fineness evaluation; (2) Application in screening wool fineness varieties; (3) Application in the identification of wool fineness varieties; (4) Application in the traceability of wool fineness varieties; (5) Application in breeding wool fineness varieties; (6) Application in the conservation of wool fineness germplasm resources; (7) Application in the improvement of wool fineness germplasm resources; (8) Application in the reconstruction of wool fineness pedigree.