5k liquid chip for identifying sheep breed and application thereof

Abstract

The present application relates to the technical field of gene chip, and more particularly to a 5K liquid phase chip for sheep breed identification and application thereof. The 5K liquid phase chip comprises probes of target SNP molecular markers, and the SNP molecular markers are located on 5145 SNP molecular markers on a sheep reference genome Genome assembly ARS-UI_Ramb_v2.0, as shown in Table 1. The liquid phase chip prepared by using the molecular markers can realize genotyping, and has high application value in many fields such as sheep breed identification.

Description

Technical Field

[0001] This invention relates to the field of gene chip technology, and in particular to a 5K liquid phase chip for sheep breed identification and its application. Background Technology

[0002] With the in-depth implementation of the seed industry revitalization action and breed improvement plan, sheep breeding has shifted from traditional binary crossbreeding to ternary and multi-component crossbreeding, from breeding for both wool and meat to breeding for specialized meat breeds, and from single breeding entities to regional joint breeding. In addition, the introduction and transportation of breeds have become more convenient, leading to an increasing demand for breed exchange between regions, especially for high-quality breeding sources. However, since evaluation based solely on body shape and appearance characteristics cannot effectively identify highly backcrossed breeding sheep, cases of inferior breeds being passed off as superior ones and counterfeit breeds occur frequently. This poses a challenge to sheep breeding work. Therefore, it is necessary to strengthen the accurate identification of sheep breeds.

[0003] SNP microarray detection technology is a commonly used technique for detecting genetic diversity, mainly divided into solid-phase microarrays and liquid-phase microarrays. Solid-phase microarrays primarily utilize fixed, allele-specific probes constructed based on known SNP loci. These probes hybridize with DNA from samples stained with fluorescent dyes, and the hybridization signal is read to detect the genotype of genetic diversity loci in a population or individual. Currently, commonly used sheep solid-phase microarrays include the Illumina Ovine SNP50 BeadChip, Illumina Ovine SNP600K BeadChip, Affymetrix Ovine 50K, Affymetrix Ovine 600K HD, and Neogen GGP Ovine 50K, which have been widely used in germplasm resource genetic diversity analysis, genetic and evolutionary analysis, kinship identification, genome-wide association studies, and genome selection.

[0004] Liquid phase microarrays primarily work by capturing probes mixed in a solution for reaction, and then sequencing the results. Due to its efficient detection method, low cost, and simple data reading process, this technology has been widely used in large-scale sheep genetic breeding. In recent years, domestic research institutes and breeding companies have developed a series of liquid phase microarrays. Currently, publicly released models include the "Huayangxin" 45K breeding chip, the "Zhongyang No. 1" 50K breeding chip, the "Baize No. 1" 40K breeding chip, the dairy sheep 20K functional locus liquid phase breeding chip, the Lanping black-bone sheep 50K breeding chip, the "Junkenxin" 55K breeding chip, the "Tanyang No. 1" 50K breeding chip, and the "Yuyangmuxin" 10K breeding chip. Most of these are constructed with the aim of precisely locating key breeding trait-related loci, and their main functions include genome selection, genome-wide association analysis, and pedigree construction.

[0005] However, while existing solid-phase chips offer high SNP density and multiple detection functions, their development costs are significantly higher than those of liquid-phase chips, inevitably increasing the costs of detection and downstream analysis. When used for sheep breed classification, these chips often result in unnecessary financial and data waste. Furthermore, most current liquid-phase chips are breeding-specific, with only a few offering breed identification capabilities, and even then, they can only identify breeds involved in the breeding process. If used for sheep breed classification, these chips often cannot accurately identify breeds outside the chip's capabilities, exhibiting significant limitations. Therefore, there is an urgent need to develop dedicated liquid-phase chips for sheep breed resource identification. Summary of the Invention

[0006] The purpose of this invention is to provide a sheep 5K whole-genome liquid microarray based on targeted capture sequencing technology, which can not only genotype the target locus, but also accurately genotype SNPs within a certain range around the target locus. In particular, it relates to a 5K liquid microarray for sheep breed identification and its applications.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a combination of SNP molecular markers for the entire sheep genome, consisting of 5145 SNP molecular markers located on the sheep reference genome Genome assembly ARS-UI_Ramb_v2.0, as shown in Table 1; Table 1

[0008] The present invention also provides a molecular probe for specifically recognizing the SNP molecular marker combination.

[0009] The present invention also provides a 5K liquid phase chip for sheep breed identification, including the aforementioned molecular probe.

[0010] The present invention also provides a kit for sheep breed identification, comprising the molecular probe or the 5K liquid phase chip.

[0011] This invention also provides the application of the aforementioned SNP molecular marker combination, the aforementioned molecular probe, the aforementioned 5K liquid phase chip, or the aforementioned kit in sheep genotyping detection.

[0012] This invention also provides the application of the aforementioned SNP molecular marker combination, the aforementioned molecular probe, the aforementioned 5K liquid phase chip, or the aforementioned kit in sheep kinship identification or sheep breed identification.

[0013] This invention also provides the application of the aforementioned SNP molecular marker combination, the aforementioned molecular probe, the aforementioned 5K liquid phase chip, or the aforementioned kit in sheep molecular marker-assisted breeding or whole-genome selection breeding.

[0014] This invention also provides the application of the aforementioned SNP molecular marker combination, the aforementioned molecular probe, the aforementioned 5K liquid phase chip, or the aforementioned kit in sheep genetic diversity analysis.

[0015] This invention also provides the application of the aforementioned SNP molecular marker combination, the aforementioned molecular probe, the aforementioned 5K liquid phase chip, or the aforementioned kit in sheep breed improvement.

[0016] Preferably, the sheep is at least one of the following: Altay fat-tailed sheep, Bashibai sheep, Bayinbuluke sheep, Diqing sheep, Dolan sheep, Guide black fur sheep, Guangling fat-tailed sheep, Hotan sheep, Hulunbuir sheep, Hu sheep, Hanzhong sheep, Jinzhong sheep, Kazakh fat-tailed sheep, Lanzhou fat-tailed sheep, Lop sheep, Lanping black-boned sheep, fat-tailed Han sheep, Luzhong mountain sheep, Ninglang black sheep, Cele black sheep, Sunite sheep, Shiping Qing sheep, Sishui fur sheep, small-tailed Han sheep, Tan sheep, Tengchong sheep, Taihang fur sheep, Tibetan sheep, Qinghai Tibetan sheep, Sichuan Tibetan sheep, Tong sheep, Turpan black sheep, Ujumqin fat-tailed sheep, lowland sheep, Weining sheep, western Henan fat-tailed sheep, Zhaotong sheep, Huanghuai meat sheep, Zhongyu meat sheep, Suffolk, Australian Merino sheep, German Merino sheep, Australian white sheep, Dorper sheep, and Texel sheep.

[0017] The present invention has the following beneficial effects: This invention mines sheep breed-specific and selected sites from large-scale resequencing data, discovering and screening 5145 SNP molecular marker sites that can be used for microarray design. Among them, 3468 are SNP sites unique to each of the 45 sheep breeds, and 1677 are SNP sites that are evenly distributed on chromosomes in the 45 sheep breeds, are independent of each other, and have high polymorphism. Microarrays prepared using the above SNP molecular marker sites can achieve genotyping and have high application value in multiple fields of sheep breed identification.

[0018] Furthermore, the sheep liquid-phase microarray (sheep 5K liquid-phase microarray) involved in this invention is based on targeted capture sequencing technology. It can not only genotype the target locus, but also accurately genotype SNPs within a certain range around the target locus, thus obtaining more SNP genotyping information than labeled loci. Compared with traditional solid-phase microarrays, it offers greater flexibility, allowing labeled loci to be added at any time according to application needs. Simultaneously, the liquid-phase microarray relies on a next-generation sequencing platform, resulting in lower genotyping costs and providing a technical means for large-scale genotyping. Attached Figure Description

[0019] Figure 1 This shows the distribution of 5145 SNP loci from a sheep 5K liquid phase chip across the entire genome (the number of SNPs contained within each 1M window of the genome).

[0020] Figure 2 Principal component analysis diagram for sheep breed identification.

[0021] Figure 3 Results of genetic structure analysis for 45 sheep breeds. Detailed Implementation

[0022] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Example 1

[0024] Fabrication of a sheep 5K liquid-phase chip based on targeted capture sequencing

[0025] Resequencing was performed on 202 sheep from 45 sheep breeds. High-quality FASTQ files were obtained through data quality control and filtering. The quality-controlled short sequences were aligned to the sheep reference genome Genome assembly ARS-UI_Ramb_v2.0 using BWA software. All samples from each breed were combined together for comparison and detection of variant sites. After filtering of variant sites, 77,977,365 SNP sites were obtained for subsequent screening.

[0026] The sheep breeds are as follows: 3 Altay fat-tailed sheep (ALT), 4 Bashibai sheep (BAS), 4 Bayinbuluke sheep (BAY), 5 Diqing sheep (DQS), 5 Dolan sheep (DUL), 5 Guide black fur sheep (GBF), 4 Guangling fat-tailed sheep (GLT), 4 Hotan sheep (HET), 4 Hulunbuir sheep (HUL), 3 Hu sheep (HUS), and 3 Hanzhong sheep (HZS). 3 sheep, 5 Jinzhong sheep (JZS), 5 Kazakh fat-tailed sheep (KAZ), 3 Lanzhou fat-tailed sheep (LLT), 5 Lop sheep (LOP), 5 Lanping black sheep (LPB), 4 fat-tailed Han sheep (LTH), 3 Luzhong mountain sheep (LZM), 3 Ninglang black sheep (NLB), 3 Cele black sheep (QIB), 5 Sunite sheep (SNT), 4 Shiping blue sheep (SPG) 5 Sishui Fur Sheep (SSF), 5 Small-tailed Han Sheep (STH), 5 Tan Sheep (TAN), 4 Tengchong Sheep (TCS), 4 Taihang Fur Sheep (THF), 17 Tibetan Sheep (TIB), 5 Qinghai Tibetan Sheep (TIBQ), 4 Sichuan Tibetan Sheep (TIBS), 3 Tong Sheep (TON), 4 Turpan Black Sheep (TUB), 5 Ujimqin Fat-tailed Sheep (UJI), and 10 lowland sheep. 4 WDS sheep, 4 WNS sheep, 5 YFT sheep, 4 ZTS sheep, 5 HHS sheep, 7 ZYS sheep, 6 EFS sheep, 3 AME sheep, 3 GME sheep, 3 AWD sheep, 3 DOP sheep, and 5 TEX sheep.

[0027] The above sites were screened as follows: 1) Site quality control Low-quality variant sites were removed using the following screening criteria: minimum sequencing depth > 2X, site deletion rate < 0.1%, minor allele frequency (MAF) > 0.05, removal of non-secondary SNP sites, and removal of Indel sites. A total of 13,375,274 sites were obtained for further selection.

[0028] 2) Site selection

[0029] ① Variety Identification Locus Screening: 1. Within each subgroup (variety), loci with MAF < 0.05 and deletion rate > 0.1 were filtered out. 2. The ΔRAF for each subgroup was calculated as |RAF_target - RAF_other|, where RAF_target is the reference allele frequency of the target variety SNP locus, and RAF_other is the reference allele frequency calculated by combining the remaining variety SNP loci after removing the target variety. 3. SNP loci were sorted from largest to smallest according to the ΔRAF value, and the top 100 SNP loci were selected as target variety-specific SNP loci. For the quality-controlled SNP loci, the allele frequency difference (ΔRAF) between varieties was calculated. Then, the loci were merged and redundancy was removed to obtain the final loci. A total of 3494 SNP loci were selected as the core loci for variety identification.

[0030] ② Screening of high-frequency background sites: Based on the principle of uniform distribution of the genome and MAF>0.4, a total of 1677 SNP sites that are uniformly distributed on the chromosome, independent of each other, and highly polymorphic were selected.

[0031] 3) Site assessment

[0032] Site evaluation aims to design probes for a given target site or segment and evaluate the coverage of the design results.

[0033] Evaluation principles: Probe length: 110bp, GC content: 30-70% (including boundaries), number of homologous regions: ≤5 (homology calculation: a) completely identical above 40bp; b) similarity 85%, length 80bp; c) similarity 95%, length 70bp).

[0034] All candidate sites were modified by removing duplicate sites and submitted to Shijiazhuang Borui Biotechnology Co., Ltd. for evaluation. Sites that could not be uniquely aligned on the genome and sites containing duplicate sequences in flanking sequences were removed. The inter-site spacing was evaluated again, and sites with an inter-site spacing of less than 100 bp were removed. A total of 26 SNP sites were filtered out, and finally 5145 SNP sites evenly distributed in the sheep genome were obtained. Figure 1The coordinates of these SNP sites on the sheep genome Genomeassembly ARS-UI_Ramb_v2.0 version are shown in Table 1. The locations described in Table 1, for example, 1_297686, are located at position 297686 bp on chromosome 1 of the reference genome.

[0035] Based on the genomic locations of the 5145 SNP loci mentioned above, and using the sheep reference genome, a 110 bp reverse complementary sequence, including the target locus, was selected as the probe sequence for that locus. For example, for the first locus located at 297686 bp on chromosome 1 in the table, the NCBI database shows that this locus contains a total of 110 bp of bases on the sheep Genomeassembly ARS-UI_Ramb_v2.0 genome for probe design. The probe sequences for the remaining loci were determined in the same way, and then synthesized by Shijiazhuang Borui Biotechnology Co., Ltd., resulting in the sheep 5K liquid phase chip.

[0036] Example 2

[0037] Procedure for Genotyping Sheep DNA Samples Using a Sheep 5K Liquid Chromatography Chip

[0038] 1) DNA extraction

[0039] Blood was collected from the jugular vein of sheep, and DNA was extracted using the phenol-chloroform method or a blood genomic extraction kit (Tiangen Biotech Co., Ltd., Beijing).

[0040] 2) Quality Inspection

[0041] The results were detected by 1.2% agarose gel electrophoresis and the gel imaging system (GelDocXRSystem, Bio-Rad, USA) was used to judge the results to ensure the integrity of the genome. The concentration of genomic DNA was measured by a micro-volume ultraviolet spectrophotometer (Q5000, Quawell, USA) or a similar nucleic acid and protein analyzer, and the DNA concentration was adjusted to the working concentration of 10-50 ng / μL.

[0042] 3) Library construction and liquid phase probe capture

[0043] Follow the standard procedure for liquid phase chip testing (http: / / www.molbreeding.com / index.php / Technology / GenoBaits.html).

[0044] 4) Sequencing

[0045] 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.

[0046] 5) Genotype data analysis

[0047] The raw data were quality controlled using FASTP software (CHEN S, ZHOU Y, CHEN Y, et al. FASTP: an ultra-fastall-in-one FASTQ preprocessor. Bioinformatics, 2018, 34(17): i884-i90.). Then, BWA software (LI H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv preprint arXiv:13033997, 2013.) was used to align the quality-controlled clean reads to the sheep reference genome assembly ARS-UI_Ramb_v2.0 (International Sheep Genomics Consortium, Archibald AL, Cockett NE, et al. The sheep genome reference sequence: a work in progress. Animal Genetics, 2010, 41(5):449-453.) generates a BAM file, sorts the BAM file using samtools, and then removes duplicates from all PCR repeats using Picard software. Finally, the standard procedure of GATK software (VAN DER AUWERA GA, CARNEIRO MO, HARTL C, et al. From FastQ data to high-confidence variant calls: the genome analysis toolkit bestpractices pipeline. Current protocols in bioinformatics, 2013, 43(1):11.0.1-.0.33.) is used to detect SNPs and perform genotyping.

[0048] Genotyping analysis was performed on 45 sheep breeds according to this procedure, followed by principal component analysis (PCA) of the genotypes. Specifically, GCTA software was used for PCA analysis to obtain the variance explained by each principal component (PC) and the score matrix of the sample in each PC. Key information extracted from SNPs was categorized into PC1, PC2, PC3, etc., according to effect size from largest to smallest. The results mainly show the pairwise scatter plots of the first three PCs. The three-dimensional plots of the first three PCs are shown below. Figure 2 (The variance explained in parentheses is the variance explained for that PC).

[0049] Genetic structure analysis was performed on genotypes of 45 sheep breeds. Specifically, ADMIXTURE software was used to analyze their genetic structure (STRUCTURE). The analysis was run with different ancestral component numbers K=2-45, and different colors represented the proportions of different ancestral components. When K=45, each breed's lineage could be distinguished by color. The genetic structure diagram is shown below. Figure 3 As shown in the figure, the 5K liquid phase chip can be used to identify sheep breeds.

[0050] Example 3

[0051] Application of 5K liquid phase chip in sheep breed identification

[0052] The samples used for chip design come from 45 sheep breeds in China, which are purebred, widely sourced, and highly representative. They include 38 local breeds, 2 cultivated breeds, and 5 introduced breeds. They have medium- and high-frequency SNP loci unique to sheep breeds, making them more suitable for sheep breed identification research. This is beneficial for sheep breeding work and the research and protection of sheep germplasm resources.

[0053] To verify the practical effectiveness of this chip in breed identification, blood samples were collected from 100 breeding sheep from four breeding farms in Henan and Gansu provinces. Genotyping was performed using a sheep 5K liquid chromatography chip (see Example 2 for specific genotyping methods). Genotyping results were obtained for each test sample. The pedigree composition of the test samples was analyzed using Admixture software. Each row represents the proportion of each breed's pedigree in a sample (summed to 1), and each column represents a single breed's pedigree. Further analysis and calculation yielded the pedigree proportions of the test samples (pedigree ratio 0–1) (Table 2). If the pedigree proportion exceeded 0.9, the test sample was determined to be purebred (e.g., HH95, HH96) and could be directly used for breeding. If the pedigree score was below 0.9, and the proportion of other related breed pedigrees exceeded 0.1, the test sample was determined to be non-purebred (e.g., GH70, GH85) and required further selection and purification. If the test sample was from a crossbreeding population, its pedigree structure and score could be inferred (e.g., DH24, DH26), providing a reference for the next step of breeding program development.

[0054] Table 2 Predicted pedigree scores for test samples

[0055] As can be seen from the above embodiments, the present invention mines sheep breed-specific and selected sites from large-scale resequencing data, discovering and screening 5145 SNP sites that can be used for chip design. Among them, 3468 are SNP sites unique to each of the 45 sheep breeds, and 1677 are SNP sites that are evenly distributed on chromosomes in the 45 sheep breeds, are independent of each other, and have high polymorphism. The designed chip can achieve efficient genotyping and has high application value in multiple fields of sheep breed identification.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A combination of SNP molecular markers for the entire sheep genome, characterized in that, The genome consists of 5145 SNP molecular markers located on the sheep reference genome Genomeassembly ARS-UI_Ramb_v2.0, as shown in Table 1. Table 1 2. A molecular probe, characterized in that, The molecular probe is used to specifically identify the SNP molecular marker combination of claim 1.

3. A 5K liquid chromatography chip for sheep breed identification, characterized in that, Includes the molecular probe described in claim 2.

4. A reagent kit for sheep breed identification, characterized in that, This includes the molecular probe of claim 2 or the 5K liquid phase chip of claim 3.

5. The application of the SNP molecular marker combination of claim 1, the molecular probe of claim 2, the 5K liquid phase chip of claim 3, or the kit of claim 4 in sheep genotyping detection.

6. The application of the SNP molecular marker combination of claim 1, the molecular probe of claim 2, the 5K liquid phase chip of claim 3, or the kit of claim 4 in sheep kinship identification or sheep breed identification.

7. The application of the SNP molecular marker combination of claim 1, the molecular probe of claim 2, the 5K liquid phase chip of claim 3, or the kit of claim 4 in sheep molecular marker-assisted breeding or whole genome selection breeding.

8. The application of the SNP molecular marker combination of claim 1, the molecular probe of claim 2, the 5K liquid phase chip of claim 3, or the kit of claim 4 in the analysis of sheep genetic diversity.

9. The application of the SNP molecular marker combination of claim 1, the molecular probe of claim 2, the 5K liquid phase chip of claim 3, or the kit of claim 4 in sheep breed improvement.

10. The application according to any one of claims 5 to 9, characterized in that, The sheep mentioned are at least one of the following: Altay fat-tailed sheep, Bashibai sheep, Bayinbuluke sheep, Diqing sheep, Dolan sheep, Guide black fur sheep, Guangling fat-tailed sheep, Hotan sheep, Hulunbuir sheep, Hu sheep, Hanzhong sheep, Jinzhong sheep, Kazakh fat-tailed sheep, Lanzhou fat-tailed sheep, Lop sheep, Lanping black-bone sheep, fat-tailed Han sheep, Luzhong mountain sheep, Ninglang black sheep, Cele black sheep, Sunite sheep, Shiping Qing sheep, Sishui fur sheep, small-tailed Han sheep, Tan sheep, Tengchong sheep, Taihang fur sheep, Tibetan sheep, Qinghai Tibetan sheep, Sichuan Tibetan sheep, Tong sheep, Turpan black sheep, Ujumqin fat-tailed sheep, lowland sheep, Weining sheep, Yuxi fat-tailed sheep, Zhaotong sheep, Huanghuai meat sheep, Zhongyu meat sheep, Suffolk, Australian Merino sheep, German Merino sheep, Australian white sheep, Dorper sheep, and Texel sheep.

Images