SNP molecular marker for identifying sheep breed and application thereof

Abstract

The present disclosure relates to the technical field of bioengineering, in particular to SNP molecular markers for identifying sheep breeds and application thereof. The present disclosure relates to a SNP molecular marker combination for identifying sheep breeds, which comprises SNP loci as shown in Table 1. The SNP molecular marker combination of the present disclosure can be applied to sheep breed identification, sheep whole genome breeding and sheep kinship identification, and can effectively identify different breeds of sheep and play an important role in breeding.

Description

Technical Field

[0001] This disclosure relates to the field of bioengineering technology, specifically to SNP molecular markers for identifying sheep breeds and their applications. Background Technology

[0002] Single nucleotide polymorphisms (SNPs) are among the most common forms of genetic variation in the genome, referring to variations in a single base in the genomic DNA sequence. Due to their abundance, wide distribution across the genome, high genetic stability, and ease of automated detection, SNPs have become one of the most ideal molecular markers, widely used in population genetics, disease association analysis, and genome selection breeding.

[0003] Genomic selection (GS), since its introduction in 2001, has revolutionized animal and plant breeding strategies. The core principle of GS is to use genome-wide SNP marker information to build predictive models that directly estimate an individual's genomic estimated breeding value (GEBV), enabling precise selection early in the season or even when phenotypic records are unavailable. Compared to traditional phenotypic selection, GS can significantly shorten generation intervals, improve breeding efficiency, and accelerate genetic improvement.

[0004] While genomic selection has demonstrated significant potential in livestock breeding and has been successfully applied in species such as dairy cattle, its widespread adoption in livestock breeds like sheep remains challenging. Currently available sheep SNP chips, such as high-density solid-phase chips, are expensive. Furthermore, these chips are mostly developed based on European or North American sheep breeds, exhibiting poor adaptability to the genetic background of native Chinese sheep and significant confirmation bias. This leads to reduced genotyping efficiency and accuracy, limiting their large-scale application in sheep molecular breeding in my country.

[0005] Chinese sheep possess abundant genetic resources and unique production performance, but in their breeding process, there is a lack of cost-effective and efficient SNP genotyping tools applicable to local breeds. Therefore, there is an urgent need to develop a low-cost, high-precision SNP chip that can effectively cover the genetic diversity of local Chinese sheep to promote the popularization and development of sheep genome selection and molecular breeding. Summary of the Invention

[0006] The purpose of this invention is to provide SNP molecular marker combinations for identifying sheep breeds, probes for detecting SNP molecular markers, SNP liquid phase chips and their applications, providing technical support for the identification and breeding of sheep breeds.

[0007] According to a first aspect of this disclosure, a combination of SNP molecular markers for identifying sheep breeds is provided, the combination of SNP molecular markers including SNP sites as shown in Table 1.

[0008] In some implementations, the location information of the SNP sites is determined by comparison with a sheep genome reference sequence, which is ARS-UI-Ramb-v3.0 (NCBI accession number: GCF-016772045.2).

[0009] According to a second aspect of this disclosure, there is a use of a reagent for detecting combinations of SNP molecular markers described in the first aspect of this disclosure in the preparation of a chip for identifying sheep breeds.

[0010] In some embodiments, the reagent includes a set of probes for detecting the SNP site.

[0011] In some embodiments, the chip is a liquid-phase chip or a solid-phase chip, and more particularly a liquid-phase chip.

[0012] According to a third aspect of this disclosure, a probe set is provided for detecting the SNP sites described in the first aspect of this disclosure.

[0013] In some embodiments, the probe is modified, and more particularly, it is biotin-modified.

[0014] According to a fourth aspect of this disclosure, an SNP chip for identifying sheep breeds is provided, the SNP chip comprising the probe set described in the third aspect of this disclosure.

[0015] In some embodiments, the SNP chip is a solid-phase chip or a liquid-phase chip, and further, the SNP chip is a liquid-phase chip.

[0016] According to a fifth aspect of this disclosure, a kit for identifying sheep breeds is provided, the kit comprising the probe set described in the third aspect of this disclosure or the SNP chip described in the fourth aspect of this disclosure.

[0017] In some embodiments, the kit further includes streptavidin-modified magnetic beads.

[0018] According to the sixth aspect of this disclosure, applications are provided for the SNP molecular marker assemblies described in the first aspect, the probe sets described in the third aspect, the SNP chips described in the fourth aspect, or the kits described in the fifth aspect, wherein the applications include one or more of the following: 1) Sheep breed identification; 2) Whole-genome breeding of sheep; 3) Sheep kinship testing; 4) Sheep genotyping.

[0019] In some embodiments, the sheep are selected from one or more of the following: Sunite sheep, Tan sheep, Black Tan sheep, Dolan sheep, East Furi sheep, Mongolian sheep, Northern Mongolian sheep, Tibetan sheep, Guide black fur sheep, Hulunbuir short-tailed sheep, Hulunbuir sheep, Small-tailed Han sheep, Ujumqin sheep, and Hu sheep.

[0020] In some embodiments, the sheep are selected from Sunite sheep, Tan sheep, Black Tan sheep, Dolan sheep and / or Hu sheep.

[0021] According to a seventh aspect of this disclosure, a method for identifying sheep breeds is provided, the method comprising the steps of identification using the probe set described in the third aspect of this disclosure, the SNP chip described in the fourth aspect, or the kit described in the fifth aspect.

[0022] In some implementations, the method includes the following steps: S1) Extract genomic DNA from the sheep to be tested; S2) Detect the genomic DNA using the probe set, the SNP chip, or the kit to obtain detection data; and S3) Analyze the detection data to obtain the typing results of the sheep to be tested.

[0023] In some embodiments, the sheep are selected from one or more of the following: Sunite sheep, Tan sheep, Black Tan sheep, Dolan sheep, East Furi sheep, Mongolian sheep, Northern Mongolian sheep, Tibetan sheep, Guide black fur sheep, Hulunbuir short-tailed sheep, Hulunbuir sheep, Small-tailed Han sheep, Ujumqin sheep, and Hu sheep.

[0024] In some embodiments, the sheep are selected from Sunite sheep, Tan sheep, Black Tan sheep, Dolan sheep and / or Hu sheep.

[0025] The beneficial effects of this disclosure include: 1. The SNP liquid phase chip provided in this disclosure has a site design that fully considers the genetic background of Chinese local sheep, and integrates background markers that are evenly distributed throughout the genome, highly differentiated breed identification markers, and functional markers that are significantly associated with key economic traits and adaptive traits, and has high relevance and practicality.

[0026] 2. Compared with traditional solid-phase chips, the liquid-phase chip technology disclosed herein has a significant cost advantage, lowers the economic threshold for large-scale population genotyping, and is more conducive to its promotion and application in my country's livestock production.

[0027] 3. The chip loci disclosed herein are derived from high-depth whole-genome resequencing data of nine representative sheep breeds, ensuring the accuracy and polymorphism of the loci, making them applicable to imported breeds and local improved breeds widely raised in China, and able to meet the dual needs of scientific research and production.

[0028] 4. The SNP liquid phase chip provided in this disclosure can be widely used in many fields such as sheep breed identification, genetic tracing, pedigree correction, genetic diversity assessment, adaptive trait research, early genome selection breeding, and QTL mapping analysis related to target traits. It has high value in both production applications and scientific research. Attached Figure Description

[0029] Figure 1 This diagram shows the distribution of the SNP sites disclosed herein across the entire sheep genome.

[0030] Figure 2 This diagram shows the density distribution of the SNP sites disclosed herein at the whole sheep genome level.

[0031] Figure 3 The results of the LD decay pattern analysis of SNP sites detected by liquid phase chip genotyping in this disclosure for sheep population are shown.

[0032] Figure A shows the LD decay of four major sheep populations, and Figure B shows the LD decay curves of nine specific sheep breeds included in this disclosure.

[0033] Figure 4 The results of the SNP phylogenetic tree for genotyping detection using liquid-phase chip technology disclosed herein are shown. Detailed Implementation

[0034] 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 specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0035] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0036] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0037] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0038] The "SNP (single nucleotide polymorphism)" or "single nucleotide polymorphism" mentioned in this article refers to a class of molecular genetic markers, mainly referring to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. The polymorphisms exhibited by SNPs usually involve variations in only a single base, such as transitions, transversions, insertions, and deletions.

[0039] SNP genotyping chip technology is a high-throughput genotyping platform based on SNP markers, offering advantages such as high throughput, high efficiency, and high accuracy. SNP chips are mainly divided into solid-phase chips and liquid-phase chips. Solid-phase chips, also known as SNP microarrays, utilize the base-pairing reaction between DNA marker sequences immobilized on the chip and target nucleic acid molecules to achieve precise identification of genetic information.

[0040] Liquid-phase microarrays are generally designed based on the principle of DNA complementarity. For each target site, a biotin-labeled probe covering the target SNP is designed. These probes hybridize with the target region of the genome in a liquid state to form a double strand. The adsorption of biotin-coated magnetic beads with streptavidin can be used to elute, amplify, construct libraries, and then perform next-generation sequencing to finally restore the genotypic status of the target site and its surrounding SNPs.

[0041] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0042] The technical solutions of the present invention are not limited to the specific embodiments. All technical modifications made according to the technical solutions of the present invention fall within the protection scope of the present invention.

[0043] Example Example 1: Preparation of a low-density 10K SNP liquid-phase chip for the whole sheep genome 1.1 Acquisition of whole-genome resequencing data and SNP identification This study collected fresh ear tissues from 221 individuals across seven breeds of Chinese sheep (Sunite sheep, Ujumqin sheep, Hulunbuir sheep, Hulunbuir short-tailed sheep, small-tailed Han sheep, and Hu sheep) and the introduced breed Dongfuli sheep. Additionally, 40 whole-genome resequencing data sets (SRR35888771-SRR35888780, SRR35888782-SRR35888791, SRR31508815-SRR31508833, SRR31482347) from Guide black fur sheep and Tibetan sheep were downloaded from the National Center for Biotechnology Information (NCBI) website, representing a total of 261 individuals across nine breeds.

[0044] DNA was extracted from fresh ear tissue samples using magnetic beads and high-depth whole-genome resequencing was performed using the BGI Genomics platform, achieving an average sequencing depth of 12.23×. Read alignment, SNP variant detection, and filtering were performed using the GATK Best Practices workflow, ultimately yielding 55.75 million high-quality SNP loci, laying the data foundation for subsequent chip design.

[0045] 1.2 Chip Site Screening and Design Based on the aforementioned high-quality SNP dataset, this study uses the following strategy to screen and design loci for sheep 10K low-density liquid-phase SNP chips: (1) Uniformly distributed background markers across the entire genome: Priority was given to SNP loci that were uniformly distributed across the 26 autosomes and the X and Y chromosomes, had a high minimum allele frequency (MAF) (>0.05), and exhibited good genotypic quality, to ensure broad coverage of the sheep genome and accuracy of genetic assessment using the microarray. The selection criteria for SNP loci with good genotypic quality were as follows: Raw sequencing data underwent quality control (removal of adapters, reads with N content ≥10%, low-quality reads, etc.) to obtain clean data. The clean data was aligned to the sheep reference genome ARS-UI-Ramb_v3.0 using BWA software. After processing with samtools, SNPs were retrieved in GATK with filtering conditions set (QD<2.0, MQ<40.0, FS>60.0, SOR>2.0, MARankSum<-12.5, ReadPosRankSum<-8.0). A total of 5314 genome-wide loci were obtained.

[0046] (2) High population differentiation (FST) markers: FST values ​​were calculated for different sheep breeds using VCFtools (v0.1.16). FST values ​​were screened based on a deletion rate <= 0.1 and MAF >= 0.05. These loci can effectively distinguish different breeds and are used for breed identification and tracing. 5049 FST loci were obtained. These were merged with the genome-wide loci obtained in step (1) to obtain a total of 10,363 SNP loci.

[0047] Ultimately, this study successfully designed and developed a low-density 10K liquid phase SNP chip containing 10,363 SNP loci in the sheep whole genome. The location information of the SNP loci is shown in Table 1.

[0048] Table 1. Location information of SNP sites

[0049] Note: The location information of the above SNP sites was determined based on the sheep genome reference sequence ARS-UI-Ramb-v3.0.

[0050] 1.3 Chip Features After rigorous screening and design, the sheep 10K liquid-phase SNP chip loci disclosed in this paper exhibit the following characteristics in their distribution across the sheep genome: a. Genome-wide coverage: 10,363 SNP loci are evenly distributed across the 26 autosomes and the X and Y chromosomes of the sheep genome, such as... Figure 1 and Figure 2 As shown, this ensures good coverage of the entire genome.

[0051] b. Locus spacing: The average distance between adjacent SNP marker loci was 254 kb, and the maximum locus spacing was 14.7 Mb. Most locus spacings ranged from 10 kb to 500 kb.

[0052] c. Minimum allele frequency (MAF): The average MAF of the microarray loci is 0.26. A high MAF ensures the polymorphism of the loci in the population, which is beneficial for genotyping and genetic analysis.

[0053] d. Functional enrichment: The chip not only contains uniformly distributed background markers, but also particularly enriches SNP loci associated with key economic traits such as litter size, growth and development, meat quality formation, and environmental adaptability. The distribution and number of SNP chip loci on each chromosome are shown in Table 2.

[0054] Table 2. Distribution and number of SNP loci on each chromosome of the sheep genome in this disclosure.

[0055] As shown in Table 2, the sheep whole genome SNP chip sites disclosed in this paper are evenly distributed, covering 26 autosomes and contigs representing the X and Y chromosomes, and have good whole genome coverage.

[0056] Example 2: Sheep whole genome 10K SNP liquid phase chip detection procedure 1. Sample collection and DNA extraction 1.1 Samples for chip detection can include sheep ear tissue, blood, and hair follicles. The sampling site should be disinfected before collection. Ear tissue should be immediately immersed in 75% ethanol after collection, and anticoagulant blood collection tubes should be used for blood samples. All samples should be stored at -20°C or lower. Genomic DNA was extracted using a standard genomic DNA extraction kit (QIAGEND Neasy Blood & Tissue Kit). The extracted DNA was analyzed for quality and concentration using a Nanodrop spectrophotometer and 1% agarose gel electrophoresis. The required total DNA amount is ≥1.0 μg, concentration is ≥20 ng / μL, OD260 / OD280 is between 1.8 and 2.0, and there should be no significant degradation.

[0057] 1.2 Sheep DNA Library Construction and Targeted Capture BGI Genomics fragmented qualified genomic DNA samples and ligated specific adapters to construct a DNA library. Subsequently, the 10K liquid-phase SNP chip probes designed in Example 1 of this disclosure were used to hybridize and capture the target SNP site regions in the library, enriching the DNA fragments in the target regions.

[0058] 2. High-throughput sequencing and bioinformatics analysis After capture and enrichment, the libraries were amplified and purified by PCR, and then sequenced on a high-throughput sequencing platform. Raw sequencing data underwent quality control, adapter removal, and alignment to the sheep reference genome ARS-UI-Ramb-v3.0. Variation detection and genotyping were then performed using GATK (v4.2.6.1) software. Finally, the detected SNP sites were filtered to remove sites with low detection rates, low MAF, or those not conforming to Hardy-Weinberg equilibrium, resulting in high-quality genotyping data.

[0059] Example 3: Population genetic analysis based on the sheep 10K liquid phase chip of this disclosure. To verify the effectiveness of the microarray disclosed herein in population genetic analysis, the method of Example 2 was used to obtain genotyping data for the sheep population shown in Tables 3 (newly included sheep population) and 4. The average LD coefficient between molecular markers on the genome was calculated using the software PopLDdecay (v3.41) to analyze the LD decay pattern of the sheep population as the distance between markers increased. LD (Linkage Disequilibrium) refers to the phenomenon where alleles at two or more loci on the same chromosome exhibit non-random association. Its decay pattern can reflect historical events in the population, such as effective population size, gene flow, and selection pressure.

[0060] Table 3. Classification and number of major sheep groups

[0061] Table 4. Types and numbers of sheep breeds used in Example 1

[0062] The LD decay mode analysis results of the sheep population in this embodiment are as follows: Figure 3 As shown. Figure 3 A shows the LD decay of four major sheep populations. Observations revealed that the LD decay rate of the East Frye sheep was relatively slow, which may be related to the strong selective pressure or small effective population size experienced during its breeding as a high-yielding dual-purpose breed. In contrast, the LD decay rates of Mongolian sheep, Northern Mongolian sheep, and Tibetan sheep were faster, indicating that these local breeds may have larger effective population sizes and richer genetic diversity. Figure 3 B presents the LD decay curves for the nine specific sheep breeds used in Example 1. The results show that the East Frye sheep still exhibits the slowest LD decay, significantly different from the LD decay patterns of other Chinese local sheep breeds. While the LD decay patterns among Chinese local sheep breeds are generally similar, subtle differences can still be observed at different distances, reflecting the unique population history and breeding strategies of each breed. This provides a reliable molecular tool for sheep breed identification, purity verification, and tracing.

[0063] Example 4: Performance Evaluation of the Disclosed Sheep 10K Liquid Chip To verify the application efficacy of the sheep 10K low-density liquid phase SNP chip developed in this disclosure in population genetic analysis, this embodiment uses the chip to perform genotyping on 117 sheep samples from five breeds (Sunite (22 sheep), Tan (25 sheep), Black-Tan (20 sheep), Duolang (30 sheep), and Hu (20 sheep)) using the method of Example 2. Based on the obtained SNP data, the genetic distance matrix between samples is calculated using Plink software (v1.90b6.21), and a phylogenetic tree is constructed.

[0064] Figure 4The results showed that the phylogenetic tree clearly clustered individuals from different breeds, with similar individuals or individuals of the same breed clustering closely together within their respective branches, and a clear genetic differentiation boundary was observed between breeds. This clustering pattern not only conforms to the known origins and genetic backgrounds of sheep breeds, but also fully verifies that the sheep 10K liquid-phase SNP chip developed in this publication has the ability to accurately distinguish between different sheep breeds, especially demonstrating excellent performance in differentiating Sunite sheep from non-Sunite sheep populations. This indicates that the chip can be effectively used for genetic structure analysis and breed tracing of sheep populations, laying a solid foundation for subsequent sheep molecular breeding using this chip.

Claims

1. A combination of SNP molecular markers for identifying sheep breeds, characterized in that, The SNP molecular marker combinations include SNP sites as shown in Table 1.

2. The SNP molecular marker combination according to claim 1, characterized in that, The location information of the SNP sites was determined by comparison with the sheep genome reference sequence, which is ARS-UI-Ramb-v3.

0.

3. Use of a reagent for detecting the SNP molecular marker combination of claim 1 or 2 in the preparation of a chip for identifying sheep breeds. Preferably, the reagent comprises a probe set for detecting the SNP site; Preferably, the chip is a liquid-phase chip or a solid-phase chip.

4. The use according to claim 3, characterized in that, The chip is a liquid phase chip.

5. A probe assembly, characterized in that, The probe set is used to detect the SNP sites described in claim 1 or 2. Preferably, the probe is modified, more preferably biotin-modified.

6. An SNP chip for identifying sheep breeds, characterized in that, The SNP chip includes the probe group as described in claim 5.

7. The SNP chip according to claim 6, characterized in that, The SNP chip is either a solid-state chip or a liquid-state chip. Preferably, the SNP chip is a liquid phase chip.

8. A reagent kit for identifying sheep breeds, characterized in that, The kit includes the probe set as described in claim 5, or the SNP chip as described in claim 6 or 7. Preferably, the kit further includes magnetic beads modified with streptavidin.

9. The application of the SNP molecular marker combination of claim 1 or 2, the probe set of claim 5, the SNP chip of claim 6 or 7, or the kit of claim 8, wherein the application includes one or more of the following: 1) Sheep breed identification; 2) Whole-genome breeding of sheep; 3) Sheep kinship testing; 4) Sheep genotyping; Preferably, the sheep is selected from one or more of the following: Sunite sheep, Tan sheep, Black Tan sheep, Dolan sheep, East Furi sheep, Mongolian sheep, Northern Mongolian sheep, Tibetan sheep, Guide black fur sheep, Hulunbuir short-tailed sheep, Hulunbuir sheep, Small-tailed Han sheep, Ujumqin sheep, and Hu sheep.

10. A method for identifying sheep breeds, characterized in that, The method includes the steps of identification using the probe set of claim 5, the SNP chip of claim 6 or 7, or the kit of claim 8. Preferably, the method includes the following steps: S1) Extract genomic DNA from the sheep to be tested; S2) Detect the genomic DNA using the probe set, the SNP chip, or the kit to obtain detection data; and S3) Analyze the detection data to obtain the typing results of the sheep to be tested. Preferably, the sheep is selected from one or more of the following: Sunite sheep, Tan sheep, Black Tan sheep, Dolan sheep, East Fulisheng sheep, Mongolian sheep, Northern Mongolian sheep, Guide Black Fur sheep, Hulunbuir Short-tailed sheep, Hulunbuir sheep, Small-tailed Han sheep, Ujumqin sheep, and Hu sheep.

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