Molecular marker combinations and their applications in analyzing sheep breeds

Abstract

The present invention discloses a molecular marker combination and application for analyzing sheep breeds, relating to the field of biotechnology. The present invention provides 10,082 molecular markers capable of analyzing sheep, wherein the physical location information of the molecular markers is determined based on genome sequence alignment of the sheep reference genome Oar_rambouillet_v1.0. A molecular probe combination, a gene chip, and a kit prepared using the 10,082 SNP molecular markers can be used to perform genetic assessment on individual sheep, perform individual selection on sheep breeds that were difficult to measure in the early stages, shorten the generation interval, accelerate the breeding process, and thus save a large amount of breeding costs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, specifically to the field of biological detection technology, and more specifically to analyzing SNP site combinations of sheep breeds and applications thereof. Background Art

[0002] Sheep not only provide high-quality livestock products for humans but are also commonly used in biomedical research. Approximately 8,000 to 10,000 years ago, sheep were domesticated in the Mesopotamian and Fertile Crescent regions of the Middle East (Ryder 1984; Zeder 2008, 2015; Stiner et al. 2014), and subsequently spread to diverse climates around the world through migration. According to a recent report from the Food and Agriculture Organization of the United Nations, there are currently over 1,400 breeds worldwide, each adapted to diverse climates, including alpine, desert, and humid environments (Scherf 2000; Lv et al. 2014). Identification of sheep breed resources is crucial for the conservation of local sheep breeds, sustainable breeding strategies, traceability of lamb, and registration of local breeds. Local sheep breeds, as important genetic resources, possess significant genetic diversity across their genomes. However, most local breeds are at risk of extinction due to multiple challenges, including emerging diseases, climate change, and competition from commercial breeds. Accurate sheep breed identification is one of the most important measures to address these risks.

[0003] Through centuries, even millennia, of natural selection and artificial breeding, sheep have developed breed-specific genetic structures, which facilitate the use of genetic information for breed identification (Yang et al. 2016; Zhao et al. 2017; Li et al. 2020). Molecular genetic markers such as short tandem repeats (STRs) and single-nucleotide polymorphisms (SNPs) can be used to identify local breeds by identifying genetic heterogeneity among breeds. Previous studies have reported that highly variable STRs can be used for livestock and poultry breed identification (Ozerov et al. 2013). With the continuous advancement of high-throughput sequencing and genotyping technologies, a large number of SNPs have been confirmed to be useful for livestock and poultry breed identification and may replace STRs in animal genetic variation studies (Wilkinson et al. 2011). Breed-specific SNPs, known as breed informative markers, can be used for breed identification, assessing genetic composition, and characterizing selection signals. Commercial sheep SNP arrays primarily include the Illumina Ovine SNP50 Beadchip (50K) and the Illumina Sheep HD Genotyping Beadchip (680K). The Illumina Ovine SNP50 Beadchip, developed jointly by Illumina and multiple research teams, covers over 54K SNPs across the entire sheep genome and can be used for research in genetic breeding, genome-wide association studies, quantitative trait loci mapping, gene optimization, and comparative genomics. The Illumina Sheep HD Genotyping Beadchip (680K), also produced by Illumina, is a high-density array amplified from the Illumina Ovine SNP50 Beadchip by the International Sheep Genome Consortium (ISGC). However, to distinguish important sheep breeds, it is necessary to select a more specific SNP subset. Compared to commercial SNP arrays, selecting breed-specific SNPs can eliminate noisy and redundant SNPs and reduce breed identification costs. Summary of the Invention

[0004] To meet the needs of sheep breed research, the present invention provides a SNPs and molecular probe combination, gene chip, kit and application for analyzing sheep breeds. Utilizing the site information provided by the present invention, sheep breed screening, identification, traceability and breeding can be achieved quickly and accurately, which is beneficial to the protection and improvement of germplasm resources, with short time consumption, low cost and broad market benefits.

[0005] In order to achieve the technical purpose of the present invention, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides an application of a 10,082 SNP locus combination in analyzing sheep breeds. The physical locations of the 10,082 SNP locus combinations are shown in Table 1:

[0007] Table 1 Position information of 10,082 site combinations

[0008]

[0009] Its physical location was determined based on the genome sequence alignment with the sheep reference genome Oar_rambouillet_v1.0.

[0010] In a second aspect, the present invention provides a method for analyzing sheep breeds, comprising comparing the genotypes of 10,082 SNP sites in the genomic DNA of a test sheep with the genotypes of the 10,082 SNP sites in the genomic DNA of a control sheep;

[0011] Among them, the 10082 SNP sites are the 10082 SNP sites mentioned above.

[0012] In a third aspect, the present invention provides a molecular probe combination for analyzing sheep breeds, wherein the molecular probe combination detects the SNP site combination shown in Table 1 in the sample to be tested, and the physical position information of the site combination in Table 1 is determined based on the genome sequence comparison of the sheep reference genome Oar_rambouillet_v1.0.

[0013] In a fourth aspect, the present invention provides a gene chip for analyzing sheep breeds, wherein the gene chip is loaded with the above-mentioned molecular probe combination.

[0014] In a fifth aspect, the present invention provides a kit for analyzing sheep breeds, which comprises the above-mentioned molecular probe combination or gene chip.

[0015] In a sixth aspect, the present invention provides a method for analyzing sheep breeds, using the above-mentioned molecular probe combination or gene chip or kit to detect the sample to be tested.

[0016] In a seventh aspect, the present invention provides uses of the above-mentioned molecular probe combination, gene chip, or kit in the following aspects:

[0017] (1) Application in sheep breed screening;

[0018] (2) Application in sheep breed identification;

[0019] (3) Application in sheep breed traceability;

[0020] (4) Application in sheep breeding;

[0021] (5) Application in germplasm resource conservation;

[0022] (6) Application in germplasm resource improvement. Beneficial effects

[0023] 1. A SNP set derived from sufficient breed and sample data provides more comprehensive and representative information. The SNP set selected for the chip design in this invention is based on resequencing data from 676 domestic sheep individuals representing 163 breeds worldwide, with an average sequencing depth of ~25.7x. This data, encompassing both local and improved breeds, covers the world's major sheep-producing regions and countries. The sample data is highly reliable and representative, meeting the requirements for global sheep breed resource identification.

[0024] 2. Compared with other sheep SNP chips, the present invention uses PCA and NJ tree methods to select effective SNPs sets, clarify the genetic relationship between sheep breeds, and enable the chip to be directly applied to sheep breed identification.

[0025] 3. The present invention includes a rich variety of Chinese local sheep breeds, which greatly improves the applicability of the chip to Chinese native sheep breeds and is beneficial to the protection and utilization of China's sheep germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 To analyze the location of 10,082 SNPs on various chromosomes in sheep breeds;

[0027] Figure 2 Figure 1 shows the PCA analysis results for 10,082 SNPs in sheep breeds.

[0028] Figure 3 A phylogenetic tree was constructed for the analysis of 10,082 SNPs in sheep breeds. DETAILED DESCRIPTION

[0029] The present invention is further illustrated below with reference to the detailed description of specific embodiments, but these embodiments are merely illustrative and should not be construed as limiting the present invention. If not otherwise specified, the technical means adopted in the embodiments are conventional means well known to those skilled in the art, and can be carried out with reference to the third edition of the original work "Bioinformatics and Functional Genomics" or related books, and the bioinformatics software and products adopted are also commercially available. Various processes and methods not described in detail are conventional methods well known in the art, and the materials used, trade names, and those that are necessary to list their components are all indicated when first occurring, and thereafter, same reagents used, if not otherwise specified, are identical with the content indicated for the first time.

[0030] In addition, it should be noted that the site combinations and applications provided by the present invention were completed by the inventors through arduous creative labor and optimization work.

[0031] The features and advantages described in the site combination section above are also applicable to the molecular probe combination, gene chip, kit and their applications formed based on the site combination, and will not be repeated here.

[0032] The SNP referred to in the present invention refers to single nucleotide polymorphism (SNP), which mainly refers to DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level. The variation of the single nucleotide includes variation caused by conversion, transversion, insertion or deletion of a single base.

[0033] It should be noted that the molecular markers referred to in the present 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 chip technology, such as SNP; analytical marker technology based on EST database development, etc. The molecular markers provided by the present invention can be used for genome mapping, gene localization research, map-based gene cloning, species relationship and systematic classification, etc.

[0034] It should be noted that the probe referred to in the present invention is a nucleic acid sequence (DNA or RNA) with a detection label and a known sequence that is complementary to the target gene, such as a Taqman-MGB probe.

[0035] It should be noted that the kit referred to in the present invention is any box commonly used in the art that contains reagents for detection or experimentation, which can relieve the operator of the tedious process of reagent preparation and optimization. In one embodiment of the present invention, it contains primers for amplifying the site information provided by the present invention, molecular markers or probes or gene chips for detecting the site information provided by the present invention, enzymes and buffers for amplification, and optionally fluorescent markers for detection.

[0036] Example 1 Analysis of SNPs locus combinations in sheep breeds

[0037] To meet the current needs of sheep germplasm resources and breed identification, this paper, based on high-depth whole-genome resequencing data from 676 domestic sheep individuals from 163 breeds worldwide, used the Oar_rambouillet_v1.0 genome as a reference and combined with existing research on sheep breed resources, designed a 10K sheep SNP array for global sheep breed identification. The specific method is as follows:

[0038] 1. Selection of individual sheep

[0039] To achieve more comprehensive coverage of domestic and international sheep breeds, the present invention resequenced 676 individuals from 163 sheep breeds worldwide. The specific breeds are:

[0040] Table 2 Information on 163 sheep breeds

[0041]

[0042]

[0043] It should be noted that some of the above-mentioned sheep breed names do not yet have official Chinese translations and are still using English names.

[0044] 2. Obtaining the total SNP set of the sheep genome

[0045] Samples containing genetic information from the individual sheep in step 1 are collected using conventional methods in the art. Such samples include, but are not limited to, blood, cells, tissues, skin, hair, excrement, etc. The genetic information (e.g., DNA) extracted from the samples is sequenced at a depth of 25.7×. The samples are aligned to the sheep reference genome released in 2016 (obtained from NCBI) using SAMtools and GATK. The combined results of the two alignments form a SNP set containing a total of 28.36 million single nucleotide variant sites at the whole genome level. Specifically:

[0046] The specific steps are to use the mem algorithm in the Bwa (v0.7.17) software to align the high-quality gene sequences of each population to the Oar_rambouillet_v1.0 (https: / / www.ncbi.nlm.nih.gov / genome / ?term=Oar_rambouillet_v1.0) reference genome. Specifically, call the configuration tool in the operating system, which contains the code -t 32 -k 32 -M –R "@RG\tID:'${i}'\tSM:'${i}'\tPL:illumina\tLB:'${i}'\tPU:'${i}'", where -t 32 specifies 32 threads, -k 32 sets the minimum seed length to 32bp, -M marks the secondary alignment, -R adds the Read Group header information to the output BAM file, and ${i} is a variable in the Shell script, usually the sample name, \t refers to the tab key space, ID refers to the unique identifier of the read group, SM refers to the sample name, PL:illumina refers to the sequencing platform as Illumina, LB refers to the library name, and PU refers to the sequencing unit; the obtained bam file was sorted using SAMtools (v1.6); PCR duplicates were removed from the bam file using the MarkDuplicate module in GATK (v4.1.2.0), and the obtained bam file was used for downstream variant detection; the GATK HaplotypeCaller module was used to detect variants in the sample to obtain GVCF files, and the GVCF files of each sample were merged using the GATK CombineGVCFs module. Finally, the gatkGenotypeGVCFs module was used to convert the GVCF files of each population into vcf files to obtain SNP sites; the GATK software SelectVariants was used to retain only SNP variant sites; the obtained VCF files were hard filtered using gatk VariantFiltration with the specific parameters "QUAL < 30.0 | QD < 2.0 | MQ < 40.0 | FS > 60.0 | SOR > 3.0 | MQRankSum < -12.5 | ReadPosRankSum < -8.0; vcftools (v0.1.16) was used to perform advanced filtering on the vcf file obtained by hard filtering with the following parameters: remove polymorphic SNP sites, retain dimorphic SNP sites, --min-alleles 2 --max-alleles 2.

[0047] To avoid the impact of data quality on the analysis results, the present invention also uses PLINK v1.07 software to perform strict quality control on individuals and SNPs in the data set. In the data set, individuals or SNPs that do not meet the following criteria are deleted: missing chromosome number and physical location information; minimum allele frequency (MAF) < 0.05; individual detection rate < 1.0; SNP genotyping missing rate > 0.05; Hardy-Weinberg equilibrium shift probability P < 0.000001, resulting in 10082 SNPs as shown in Table 1. The specific locations are as follows Figure 1 As shown in the figure, the marker positions of SNPs on chromosomes 1 to 26, and the white vertical bars are SNPs.

[0048] It should be noted that the genetic information referred to in the present invention refers to the information that is passed from parent to offspring in order for an organism to replicate itself, or that is passed from cell to cell each time a cell divides.

[0049] It should be noted that the extraction of genetic information (such as DNA) from samples for high-depth sequencing can be completed by biological companies, such as BGI, Illumina, etc. The high-depth sequencing method adopts conventional methods in the field or methods of biological 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.

[0050] 3. Genetic distance analysis

[0051] The present invention analyzed the genetic relationships among sheep populations using principal component analysis (PCA) and neighbor-joining trees (NJ trees). PCA analysis was performed using the SmartPCA package in EIGENSOFT v6.0 software (Patterson et al., 2006), and the results were visualized using R v3.3.0 software (http: / / www.r-project.org / ). Figure 2 The allele frequency of each locus was calculated using PEAS v.1.0 software, and then the population NJ tree was developed using the subroutine in PHYLIP v.3.695 software. Finally, the results were graphically displayed using FigTree v.1.4.2 software, as shown in the figure. Figure 3 shown.

[0052] according to Figure 2The molecular marker sets based on screening are shown in the screened molecular marker sets. It can be seen that the present invention performed principal component analysis (PCA) on 676 individuals (representing 163 varieties), and the results showed that PC1 and PC2 can clearly distinguish all varieties. Figure 3 The phylogenetic tree constructed using the same molecular marker set further verified this result, and each variety formed an independent branch.

[0053] Example 2: Using SNPs from sheep breeds for the preparation of primer and probe combinations

[0054] Those skilled in the art design primers based on the sequence information of each site in the SNPs analyzed in the sheep breeds provided by the present invention, and perform secondary structure evaluation and Tm value evaluation on the designed primers, and finally obtain primers with good specificity and high sensitivity that can achieve the detection purpose under the same reaction conditions.

[0055] The secondary structure and Tm value evaluation can be performed using any method commonly used in the art, such as using a DNA folding form to evaluate the secondary structure, see (http: / / unafold.rna.albany.edu / ?q=mfold / DNA-Folding-Form) for details, and then using the software RaW-Probe to evaluate the Tm value.

[0056] The above methods are all conventional methods. According to the site information in the SNPs provided in this application, they can be obtained without any creative work. Therefore, the primers obtained according to the SNPs provided by the present invention also fall within the scope of protection of the present invention.

[0057] Likewise, the use of the SNPs provided by the present invention to prepare probes, such as tanqman probes, also falls within the scope of protection of the present invention.

[0058] Example 3 Analysis of Gene Chips of Sheep Breeds

[0059] The SNP gene chip of the present invention is prepared by fixing the primers or probes obtained in Example 2 on a polymer substrate, such as a nylon membrane, nitrocellulose membrane, plastic, silica gel wafer, micro magnetic beads, etc., using conventional methods, or fixing the probes on a glass plate, or directly synthesizing the primers or probes obtained in Example 2 on a hard surface such as glass. The method of using the SNP gene chip of the present application is the same as the conventional method.

[0060] It should be noted that those skilled in the art can prepare SNP gene chips for detecting sheep breeds in any manner, and can also entrust a biological company to prepare them. However, SNP gene chips prepared based on the SNP site combination for sheep breed analysis provided in this application all fall within the scope of protection of the present invention.

[0061] Example 4 Kit for analyzing sheep breeds

[0062] The present application provides a SNP detection kit for analyzing sheep breeds, including primers, probes, or gene chips obtained based on the SNPs obtained in Example 1. Depending on the type of use, corresponding detection reagents may also be included. For example, when the SNPs obtained in Example 1 are prepared as TaqMan probes, the kit may also include buffers, ligases, AceQUniversal U+ Probe Master Mix V2, TaqMan Probes, and the like conventionally used in fluorescent quantitative PCR reactions.

[0063] Those skilled in the art can configure different SNP kits for detecting sheep breeds according to different usage modes, but the sheep breed SNP detection kits configured for analyzing sheep breed SNPs provided in this application all fall within the scope of protection of the present invention.

[0064] Example 5 Detection of Sheep Breeds

[0065] Phylogenetic trees were constructed based on the sheep genome-wide SNP marker combination data, and it was found that 163 domestic and foreign sheep breeds could be separated according to breed. The relevant numbers representing the breeds are shown in Table 2.

[0066] PCA analysis based on genome-wide SNP marker data revealed that PC1 and PC2 clearly separated 163 domestic and international sheep breeds. The phylogenetic tree and PCA analysis results were consistent and mutually supportive, demonstrating the reliability of the breed identification results.

[0067] Industrial Applications

[0068] Based on the SNP site combination for analyzing sheep breeds composed of 10,082 SNPs provided in this application, technicians in this field can prepare SNP probe combinations, gene chips, and kits for analyzing sheep breeds, which can be used to screen and identify sheep breeds at the genomic level, control the breeding process, and can also be applied to sheep breed tracing, sheep pedigree reconstruction, germplasm resource protection, and germplasm resource improvement.

[0069] The above description is only a preferred example to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can be subject to various changes and modifications. Without violating the spirit of the present invention, various changes or modifications made by those skilled in the art to the present invention on this basis should also fall within the scope of the present invention.

Claims

1. Application of 10082 SNP loci combination in analyzing sheep breeds. The physical locations of the 10082 SNP loci combination are shown in Table 1: Table 1 Position information of 10,082 site combinations ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； Its physical location was determined based on genome sequence alignment with the sheep reference genome, Oar_rambouillet_v1.0; in, The sheep breeds are shown in Table 2: Table 2 Information on 163 sheep breeds ； 。 2. A method for analyzing sheep breeds, wherein the genotypes of 10,082 SNP sites in the genomic DNA of the tested sheep are compared with the genotypes of the 10,082 SNP sites in the genomic DNA of the control sheep; in, The 10082 SNP sites are the 10082 SNP sites described in claim 1, and the sheep breed is the sheep breed shown in Table 2 in claim 1.

3. A molecular probe combination for analyzing sheep breeds, wherein the molecular probe combination detects the SNP site combination shown in Table 1 in the test sample, wherein the physical location information of the site combination in Table 1 is determined based on the genome sequence alignment of the sheep reference genome Oar_rambouillet_v1.0; in, The sheep breed is the sheep breed shown in Table 2 of claim 1.

4. A gene chip for analyzing sheep breeds, wherein the gene chip is loaded with the molecular probe combination according to claim 3; in, The sheep breed is the sheep breed shown in Table 2 of claim 1.

5. A kit for analyzing sheep breeds, comprising the molecular probe combination of claim 3 or the gene chip of claim 4; in, The sheep breed is the sheep breed shown in Table 2 of claim 1.

6. A method for analyzing sheep breeds, comprising: detecting a sample using the molecular probe combination of claim 3, the gene chip of claim 4, or the kit of claim 5; wherein: The sheep breed is the sheep breed shown in Table 2 of claim 1.

7. The molecular probe assembly according to claim 3, the gene chip according to claim 4, or the kit according to claim 5 has the following uses: (1) Application in sheep breed screening; (2) Application in sheep breed identification; (3) Application in sheep breed traceability; (4) Application in sheep breeding; (5) Application in the protection of sheep breed germplasm resources; (6) Application in improving sheep germplasm resources; in, The sheep breed is the sheep breed shown in Table 2 of claim 1.

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