A method for fabricating 10K SNP liquid-phase chips for commercial pig breeds and its application

Abstract

This invention discloses a method for preparing a 10K SNP liquid-phase microarray for commercial pig breeds and its application, belonging to the field of molecular breeding and genomics technology. Based on representative samples from the core breeding population of Chinese commercial pigs, this invention employs liquid-phase probe capture technology to develop a high-performance pig genome SNP genotyping microarray containing 10,055 rigorously screened high-quality SNP loci. The probes are 110 bp in length, with a GC content of 30%–70% and ≤5 homologous regions. Each locus is captured using two probes with 60%–70% overlap for dual targeting, significantly improving hybridization efficiency and anti-interference ability, and enabling stable detection of low-quality DNA samples. Simultaneously, this invention sets dual quality control standards to ensure genotyping accuracy. Verification shows that the detection rate of this microarray ranges from 98.17% to 99.602%, with an average detection rate of 99.161%. This invention has significant value for improving the breeding efficiency of commercial pigs in my country.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a method for preparing a 10K SNP liquid phase chip for commercial pig breeds and its application. Background Technology

[0002] The swine industry is a pillar industry of my country's agriculture, and molecular breeding has become a core technology for improving breeding efficiency. With the development of molecular biology, genomic selection breeding has become a core technology for pig genetic improvement, and its effective implementation depends on large-scale, low-cost, and highly accurate genotyping of breeding populations. Single nucleotide polymorphism (SNP) markers, due to their wide distribution and abundance throughout the genome and suitability for high-throughput detection, have become the most important molecular markers in genomic selection.

[0003] With the rapid development of molecular biology techniques, high-throughput genotyping technology based on single nucleotide polymorphisms (SNPs) has become a core tool in modern livestock and poultry breeding. Currently, mainstream SNP genotyping technologies mainly include solid-phase microarrays and liquid-phase microarrays. Solid-phase microarrays, with their advantages of high accuracy and short processing time, have been widely used in the past, such as the 50K solid-phase microarray commonly used in pig genome selection. However, this technology has inherent drawbacks, including high cost per genotype, poor flexibility in site customization, throughput limited by fixed sample sizes (such as multiples of 12 or 24), and detection speed that is difficult to match the fast-paced requirements of modern breeding. These inherent limitations severely restrict its widespread application in large-scale populations (especially in the early selection of replacement breeding pigs).

[0004] Unlike solid-phase microarrays, liquid-phase microarrays based on genotyping by target sequencing (GBTS) technology demonstrate enormous application potential due to their advantages such as platform versatility, label flexibility, detection efficiency, and low cost. Liquid-phase microarrays achieve efficient and accurate sequencing of specific genomic regions by performing thousands of probe-target sequence hybridization captures in a single operation within a liquid system. Although GBTS-based liquid-phase microarrays (including 50K density) effectively reduce costs, the expenses still constitute a heavy burden for breeding companies with large annual testing volumes.

[0005] Utilizing low-density microarrays (such as 10K) combined with genotype filling technology is a strategic approach to further significantly reduce the cost of molecular breeding. However, the development and application of existing low-density microarrays still face a key bottleneck: their genotyping detection rate often falls short of ideal levels. Detection rate is a core indicator for evaluating microarray performance; a low detection rate leads to a significant loss of genetic information, introducing data bias and severely impacting the accuracy of subsequent genotype filling and the reliability of genome selection. Therefore, developing a low-density liquid chromatography microarray that can maintain a stable and extremely high detection rate even with large sample sizes has become a crucial technical challenge that urgently needs to be addressed to promote the large-scale application of molecular breeding in pigs.

[0006] Existing 10K liquid chromatography-mass spectrometry (LC-MS) chips for pigs generally suffer from problems such as being designed for foreign breeding pigs, poor adaptability to Chinese commercial pig populations, and large fluctuations in detection rates. This is particularly true for mainstream commercial pig breeds such as Duroc, Landrace, and Large White, where the typing accuracy is insufficient to meet breeding requirements. Furthermore, existing probe designs often employ a single-probe strategy, making them susceptible to GC shifts and homologous sequence interference, resulting in insufficient site capture efficiency. Therefore, developing a highly stable 10K SNP LC-MS chip for core breeding populations of Chinese commercial pigs is of practical significance. Summary of the Invention

[0007] To address the aforementioned limitations of existing technologies, the present invention aims to provide a method for preparing a 10K SNP liquid-phase microarray for commercial pig breeds and its application. This microarray fully utilizes the flexibility of GBTS technology, employing precise probe design and optimized experimental procedures to reduce detection costs to a fraction of those of 50K microarrays while ensuring an average detection rate of no less than 99%. This provides a reliable solution for achieving low-cost, high-efficiency, and high-accuracy pig genome breeding. All loci in this microarray are based on screening of commercial pig populations from the Hongxing Pig Farm in Heze, Shandong, China. Locus polymorphism, MAF distribution, and flanking sequences are highly adapted to the genetic background of mainstream Chinese commercial pigs, demonstrating stable and excellent performance in large-scale sample testing, thus resolving the issue of foreign microarrays being incompatible with local conditions. Based on this invention, problems such as high genotyping costs, large required sample sizes, inflexible customization, lack of functional loci, and slow detection speed in mainstream pig breeds like Landrace and Duroc can be solved.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a 10K SNP liquid phase chip for commercial pig breeds, comprising a highly stable 10K SNP probe, reagents for hybridization, washing and signal detection, and carrier materials; The 10K SNP liquid-phase chip covers 10,055 SNP sites; the reference genome for the 10K SNP liquid-phase chip is the porcine reference genome Sus_scrofa_v11.1.

[0009] The loci in this chip were obtained from a core population of 5,928 commercial pigs in Shandong Province, including 1,477 Duroc, 1,306 Landrace, and 3,145 Large White. High-quality meta-GWAS functional loci were integrated, and the locus polymorphism, MAF distribution, and flanking sequences are highly adapted to the genetic background of mainstream commercial pigs in China. Performance was further validated using 184 independent test samples, with SNP detection rates ranging from 98.17% to 99.602%, and an average detection rate of 99.161%. The chip demonstrated stable and excellent performance in large-scale sample testing, resolving the issue of foreign chips not being compatible with local conditions.

[0010] The 10,055 core SNP sites were derived from the initial 10,079 original sites. After feasibility and site evaluation, they were successfully synthesized and used for chip fabrication with a conversion rate of approximately 99.76% and almost no site loss.

[0011] Furthermore, the average detection rate is no less than 99%.

[0012] Furthermore, the SNP sites in the 10K SNP liquid-phase chip are evenly distributed on chromosomes 1–18, with no obvious gap regions, and more than 98% of the sites have a MAF ≥ 0.05.

[0013] A second aspect of the present invention provides a method for fabricating the 10K SNP liquid-phase chip, comprising the following steps: (1) Target SNP locus information collection and quality control screening: Collect sample population sequencing data and perform quality control. The quality control steps include: removing adapter sequences; removing paired reads with more than 10 N bases; removing paired reads with low-quality bases accounting for more than 40% of the read length; the low-quality bases Q≤20; (2) Sequence alignment and SNP detection: The clean reads after quality control were aligned with the pig reference genome Sus_scrofa_v11.1 to extract genotype information of core SNP sites; a double quality control threshold was set to ensure the accuracy of genotyping results: SNP site coverage depth ≥5X, and ≥4 reads of both alleles of heterozygous sites were supported. (3) SNP site screening: The initial site list is evaluated by bioinformatics, and sites that do not meet the probe design criteria are removed; the probe design criteria include: number of flanking sequence homologous regions ≤ 5, GC content 30%-70%, and not located in simple repeat sequence regions. (4) Design and synthesis of liquid phase probes: Based on the SNP sites obtained in step (3), probes are designed with a length of 110 bp, a GC content of 30%-70%, and a number of homologous regions ≤5; two probes are designed for each site, and the nucleotide sequences of the probes have 60%-70% overlap covering the SNP site. The dual-target capture structure significantly improves hybridization efficiency and anti-interference ability, enabling stable and efficient site detection even for degraded or low-quality DNA samples.

[0014] (5) Determine the final probe and chip preparation: Test the probe synthesized in step (4) to obtain the final chip SNP site set. Mix all probes with hybridization, washing and signal detection reagents and carrier materials to obtain probe mixture, i.e. 10K SNP liquid phase chip.

[0015] To prepare the chip, all probes were equimolarly mixed to form a probe pool covering all target sites. This probe pool was then quantitatively and precisely diluted with a specific pH EDTA-Tris-HCl buffer system (TE buffer) to prepare a ready-to-use premix. This premix is ​​stable and can be directly used in downstream hybridization capture experiments.

[0016] The preparation method also includes library construction and hybridization capture procedures, specifically including: (1) Sample DNA extraction, fragmentation, end repair, A-tailing, adapter ligation, and construction of sequencing libraries; (2) The probe mixture prepared in step (1) is hybridized and captured with the constructed library, and the hybridization is carried out at 65°C for 2 to 16 hours; (3) Use streptavidin-coated magnetic beads to capture probe-library hybridization complexes and perform a series of washes to remove non-specific bindings; (4) Using specific primers with sample barcodes, PCR amplification was performed on the target DNA fragments captured by the magnetic beads to specifically enrich the target library that successfully hybridized with the probe. A unique sequence tag was added to each sample. After amplification, the PCR products were purified using a magnetic bead purification method to effectively remove primer dimers, enzyme residues, and other impurities from the reaction system, ultimately obtaining a high-purity final capture library suitable for high-throughput sequencing on mainstream platforms such as Illumina or MGI.

[0017] Furthermore, the concentration of the probe mixture is 2-4 pmol / mL.

[0018] A third aspect of the invention provides the application of the liquid phase chip in any of the following (a)-(f): (a) Genotyping of commercial pigs; (b) Population genetic structure and kinship analysis; (c) Genome-wide association analysis; (d) Genome-wide selection breeding; (e) Germplasm resource identification and genetic improvement; (f) Early selection of commercial pigs.

[0019] Genotype data obtained using the 10K SNP liquid-phase chip of this invention can be used as a base genotype. Through genotype imputation technology, this data can be effectively filled into a high-density chip (e.g., 50K) genotype map covering tens of thousands of loci. Genomic selection based on this imputation data shows no significant difference in accuracy compared to results obtained directly from the high-density chip, thus ensuring the reliability of molecular breeding selection while reducing detection costs by several times.

[0020] The beneficial effects of this invention are: (1) Extremely high detection rate and stability: The 10K liquid phase chip provided by this invention achieved an average detection rate of 99.161% in 184 commercial pig samples, with all samples having a detection rate higher than 98.17%; the SNP genotypic consistency rate of two replicate experiments for 12 samples (last 4 digits of the number) was 0.9959, and the coefficient of variation was 0.12% (far below the 1% instability standard), and all samples met the stability requirements (see Appendix). Figure 8 This superior performance ensures the integrity and reliability of genotyping data, providing a high-quality data foundation for subsequent analysis.

[0021] (2) Excellent coverage and polymorphism: The chip contains 10,055 SNP loci that evenly cover all 18 autosomes of pigs, with moderate average distances between loci and reasonable distribution in gene functional regions (such as introns, exons, UTR regions, etc.). MAF analysis shows good locus polymorphism (MAF≥0.05), making it suitable for population genetic analysis.

[0022] (3) Significant cost-effectiveness: Compared with the mainstream 50K chip, the detection cost of the 10K chip of this invention is greatly reduced. At the same time, through its high detection rate and high data quality, combined with genotype filling technology, it can greatly promote the large-scale implementation of pig molecular breeding without reducing the accuracy of genome selection.

[0023] (4) Process standardization and reliability: The complete experimental process provided by this invention, from DNA extraction and library construction to hybridization capture, has been rigorously optimized and quality controlled to ensure the stability of chip performance and the repeatability of experimental results.

[0024] (5) Dual probe overlapping capture design, with stronger compatibility: The present invention adopts a dual probe overlapping target capture structure, which significantly improves site hybridization efficiency and anti-interference ability. It can still achieve stable detection of degraded and low-quality DNA samples, and is suitable for actual sampling and detection scenarios in breeding farms.

[0025] (6) Adopting a dual quality control standard with a coverage depth of ≥5X and ≥4 reads of bicelestem at heterozygous sites, the accuracy of genotypes is guaranteed from the data source, and the typing accuracy is better than that of existing conventional chips. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the experimental workflow for a 10K SNP liquid-phase chip for commercial pigs. In the diagram, a represents the DNA library preparation workflow; b represents the DNA library hybridization and capture workflow.

[0027] Figure 2 A statistical chart showing the distribution of 10,055 SNP markers across the 18 autosomes of pigs.

[0028] Figure 3 A continuous distribution map of SNP markers on pig chromosomes.

[0029] Figure 4 This is a gap analysis diagram of the chip sites.

[0030] Figure 5 A statistical graph showing the distribution of SNP markers in different functional regions of a gene.

[0031] Figure 6 A statistical graph showing the distribution of minor allele frequencies (MAF) at SNP loci. Figure 7 This is a statistical graph of genetic diversity among samples calculated based on the target site.

[0032] Figure 8 This is a graph showing the genotypic consistency rate of SNPs in the chip sample. Detailed Implementation

[0033] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0034] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0035] Unless otherwise specified, all experimental materials used in the embodiments of this invention are conventional experimental materials in the art and can be purchased through commercial channels. All domestic pig nucleotide sequences in this application are located and annotated with reference to the NCBI domestic pig reference genome Sscrofa11.1 (i.e., Sus_scrofa_v11.1, NCBI accession number GCF_000003025.6).

[0036] Example 1: Collection and filtering of target SNP sites and design and optimization of probes 1.1 Steps for collecting and screening target SNP loci information The target SNP locus information was derived from the whole-genome resequencing data of 5928 commercial pigs in a core breeding population in Shandong Province, including 1477 Duroc pigs, 1306 Landrace pigs, and 3145 Large White pigs. High-quality meta-GWAS functional loci were also included to enhance their breeding application value. An additional 184 independent test samples (61 Duroc, 61 Landrace, and 62 Large White) were used for chip performance verification. Based on the above population data, core SNP loci suitable for liquid-phase chips were mined and screened. Specific capture probes were then designed for these loci, and the final probe sequences were determined through multiple rounds of optimization. The steps included preprocessing the raw sequencing sequences to remove invalid and low-quality data, obtaining clean reads. Specific quality control steps are as follows: (1) Remove the adapter sequence; (2) When the number of N bases in a sequencing read exceeds 10, remove the paired reads; (3) When the number of low-quality (Q≤20) bases in a sequencing read exceeds 40% of the length of the read, remove the paired reads.

[0037] 1.2 Sequence Preprocessing and SNP Detection The clean reads after quality control were aligned with the pig reference genome Sus_scrofa_v11.1 using BWA software to generate SAM files, which were then converted to BAM format using SAMtools. Subsequent processing was performed using the Sentieon workflow: the BAM files were sorted using `sentieon util sort`; PCR repetitive sequences were marked and removed using the Sentieon driver command, and Base Quality Score Recalibration (BQSR) was performed to generate quality-corrected BAM files for variant detection. Variance detection was performed using the Haplotyper model of the Sentieon driver, generating gVCF files, which were then combined with the GVCFtyper model to generate individual VCF files. Using 184 commercial pigs from Heze Hongxing Pig Farm as the research subjects, after whole-genome sequencing, data quality control, alignment, and variant detection, 10079 candidate SNPs were screened, and ultimately 10055 high-quality loci were obtained. To ensure the highest accuracy of genotyping results, two key quality control standards are set: First, the sequencing coverage depth of the SNP locus must be ≥5X; if the coverage depth is insufficient, the genotype of that locus is considered missing and marked as "NA" in the genotyping results; Second, for heterozygous loci, both of its alleles must be supported by at least 4 sequencing reads; if the supporting reads of either allele do not reach this threshold, the genotype of that locus is also considered missing and marked as "NA" in the genotyping results.

[0038] 1.3 SNP site screening Based on the initial list of sites, a rigorous screening process was conducted to determine the feasibility of the probe design, including the following principles: (1) The GC content of the flanking sequence of the site should be between 30% and 70%.

[0039] (2) The number of homologous regions of the flanking sequence of the site on the reference genome is no more than 5.

[0040] (3) The site is not located in a simple repeat sequence region.

[0041] (4) The loci should be evenly distributed across the genome to avoid excessive concentration, which would affect the effectiveness of subsequent association analysis.

[0042] 1.4 Design and Synthesis of Liquid Phase Probes Based on the SNP sites obtained through the above steps, probes should be designed to meet the following requirements: (1) The probe length is 110 bp.

[0043] (2) The GC content is controlled between 30% and 70% to ensure stability and hybridization efficiency.

[0044] (3) The number of homologous regions should be ≤5. The selected regions should avoid simple repetitive sequences and unknown base regions as much as possible to reduce non-specific binding.

[0045] (4) For each selected SNP site, two specific capture probes are designed. These two probes are spatially staggered, with 60% to 70% overlap between their sequences, to ensure continuous and gapless dual targeting coverage of the SNP site and its flanking sequences, thereby maximizing the success rate and reliability of hybridization capture.

[0046] All nucleotide sequences designed according to the above principles were synthesized and purified into two 110 bp DNA probes modified with biotin at the 5' end. Subsequently, all synthesized probes were precisely mixed in equimolar amounts and diluted to a final concentration of 3 pmol / mL using EDTA-Tris-HCl buffer (TE buffer). The resulting solution served as the probe mixture for the porcine 10K SNP liquid-phase chip and was stored at -20°C for subsequent hybridization capture experiments. Probes were successfully designed for 10055 SNP sites, and the next round of testing commenced.

[0047] Example 2: Preparation of a 10K SNP liquid phase chip for commercial pigs with high detection rate The specific preparation method is as follows: Genomic DNA was extracted from porcine tissue according to standard procedures and quality controlled by agarose gel electrophoresis and Qubit quantification. The quality-controlled DNA was used as the input template, and the end-repair, A-addition, adapter ligation, and library amplification steps were strictly followed according to the manufacturer's instructions for the commercially available high-throughput sequencing library preparation kit (GenoBaits DNA-seq Library Prep Kit) to complete the construction of a high-throughput sequencing library suitable for subsequent hybridization capture. The specific steps of library construction are as follows: Step 1, DNA fragmentation and end repair with A: Total volume 20 μL, 37℃ for 20 min, 72℃ for 20 min.

[0048] The second step is adapter connection: connecting the adapter to both ends of the DNA fragment undergoing end repair.

[0049] The third step is to purify the adapter ligation system: 48 μL DNA Clean Beads, for about 30 min.

[0050] Step 4, Library amplification: The purified adapter ligation product is enriched by PCR amplification, and barcodes for sample differentiation are added at the same time. The process takes about 40 minutes.

[0051] Step 5, purification: 40 μL DNA Clean Beads, approximately 40 min.

[0052] Step 6, Library Quality Control: The constructed library is precisely quantified using a Qubit 2.0 Fluorometer, and the fragment size distribution is analyzed by agarose gel electrophoresis. A qualified library must meet the following criteria: sufficient total fragment size, and its major fragments concentrated within the expected range of 300-500 bp. Only libraries that pass this quality control process can proceed to subsequent hybridization capture experiments. The library preparation experimental procedure is described below. Figure 1 a.

[0053] After library preparation, hybridization capture was performed. The concentrated and dried DNA library and probe mixture (GenoBaits) were then prepared. ® Panel) and blocking reagent (GenoBaits) ® Block I and GenoBaits ® Block II) mixing was performed to bind the probe mixture to the target library. Wash Buffers were then diluted to a 1× working concentration, followed by GenoBaits. ® Probe beads were washed three times with 1×BeadsWash Buffer, followed by capture using prepared beads, a process that took approximately 30 minutes. Unbound DNA was then eluted to remove it, and the target fragment was enriched and purified by PCR amplification. Finally, the concentration of the purified library was determined using qPCR and qubit / qPCR techniques, and sequencing was performed on an MGISEQ-2000 Illumina sequencing platform. The DNA library hybridization capture workflow is described below. Figure 1 b.

[0054] Example 3: Performance Verification To verify the actual performance of the liquid-phase chip described in this invention, a systematic test and analysis were conducted on a 10K SNP liquid-phase chip (containing 10055 core SNP sites) based on meta-GWAS functional sites screened and integrated from 5928 commercial pigs (1477 Duroc, 1306 Landrace, and 3145 Large White) in Shandong Province. The test sample consisted of 184 commercial pig individuals from independent sources, and the standard hybridization capture and sequencing procedures were strictly followed. The testing process is as follows: (1) Test Samples and Core Performance Indicators: 184 commercial pig samples were used for testing. Genotyping results showed that the SNP detection rate of all samples ranged from 98.17% to 99.602%, with an average detection rate as high as 99.161%. This result far exceeds the industry standard, fully demonstrating that the liquid phase chip of this invention has extremely high and stable detection sensitivity and reliability, providing a high-quality data foundation for subsequent genetic analysis.

[0055] (2) Analysis of genome coverage uniformity and continuity SNP marker chromosome distribution statistics are shown in [see...] Figure 2 The 10,055 SNP loci contained in the chip of this invention are evenly distributed throughout the entire genome. The number of loci on each chromosome is proportional to its physical length, showing no significant bias, thus providing balanced marker support for whole-genome analysis. The chromosome distribution of SNP markers is shown in [see figure]. Figure 3 By using fine-tuned window scanning, the continuous distribution of SNP sites on the genome was demonstrated, further verifying that the microarray markers had no large deletion regions and good coverage continuity.

[0056] To ensure coverage continuity, we performed a dedicated gap analysis. The gap analysis of loci on the genome is shown in [link to genome analysis]. Figure 4 No values ​​exceeding the theoretical value (genome size / number of loci) were found across the entire genome. 10) Significant gaps. Data from the adjacent site distance statistics table (Table 1 below) also confirms that the average distance between sites is reasonable. These results collectively demonstrate that the chip design ensures the continuity and unbiasedness of genomic marker coverage.

[0057] Table 1. Statistics on the distance between adjacent sites (3) Evaluation of site functional representativeness and polymorphism: Gene structure annotation analysis of SNP sites, the results of which are as follows: Figure 5 As shown, the loci are widely distributed across various functional regions, including intergenic regions, intronic regions, exon regions, ncRNAs, and upstream and downstream regulatory regions (UTRs), indicating that the chip can effectively capture genetic variations with potential biological functions and possesses good representativeness of functional regions. The assessment of locus polymorphism is crucial for evaluating the application value of the chip. The MAF distribution statistics are shown below. Figure 6 As shown, the minor allele frequency (MAF) at the vast majority of loci is greater than 0.05, exhibiting rich genetic polymorphism. This characteristic ensures that the microarray provides high information content in population genetic analysis, linkage disequilibrium mapping, and genome selection, forming the basis for obtaining reliable analytical results.

[0058] (4) Population genetic analysis practice verification: Using the genotype data generated by the chip, we conducted an in-depth genetic analysis of the test population. Figure 7 The statistical results of inter-sample genetic diversity based on the target locus revealed extensive genetic differences among samples, demonstrating that the microarray has excellent resolution to distinguish different individuals. Furthermore, we performed principal component analysis (PCA) based on the microarray data, and the population PCA distribution based on the target locus is shown below. Figure 8 As shown, the first three principal components effectively distinguish the substructures within the population, and the sample points are clearly distributed. This result strongly demonstrates the powerful ability of the chip of this invention to analyze the genetic structure of complex populations and assess kinship, fully meeting the needs of precise population management in molecular breeding.

[0059] The performance of the chip of this invention is compared with that of existing commercial 10K chips, and the results are shown in Table 2.

[0060] Table 2 Performance Comparison of the Invention Chip with Existing Commercial 10K Chips In summary, this invention, through precise chip design, standardized preparation process, and optimized experimental methods, has successfully developed a commercial pig 10K SNP liquid phase chip with high detection rate, high stability, excellent genome coverage, and rich polymorphism, providing a low-cost, high-efficiency, and high-precision reliable technical solution for molecular breeding of pigs.

[0061] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A 10K SNP liquid phase chip for commercial pig breeds, characterized in that, Includes highly stable 10K SNP probes, reagents and carrier materials for hybridization, washing and signal detection; The 10K SNP liquid-phase chip covers 10,055 SNP sites; the reference genome for the 10K SNP liquid-phase chip is the pig reference genome Sus_scrofa_v11.

1.

2. The 10K SNP liquid phase chip according to claim 1, characterized in that, The average detection rate is no less than 99%.

3. The 10K SNP liquid phase chip according to claim 1, characterized in that, The SNP loci in the 10K SNP liquid-phase chip are evenly distributed on chromosomes 1–18, with no obvious gap regions, and more than 98% of the loci have a MAF ≥ 0.

05.

4. The method for fabricating a 10K SNP liquid-phase chip according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Target SNP locus information collection and quality control screening: Collect sample population sequencing data and perform quality control. The quality control steps include: removing adapter sequences; removing paired reads with more than 10 N bases; removing paired reads with low-quality bases accounting for more than 40% of the read length; the low-quality bases Q≤20; (2) Sequence alignment and SNP detection: The clean reads after quality control were aligned with the pig reference genome Sus_scrofa_v11.1 to extract genotype information of core SNP sites; a double quality control threshold was set to ensure the accuracy of genotyping results: SNP site coverage depth ≥5X, and ≥4 reads of both alleles of heterozygous sites were supported. (3) SNP site screening: The initial site list is evaluated by bioinformatics, and sites that do not meet the probe design criteria are removed; the probe design criteria include: number of flanking sequence homologous regions ≤ 5, GC content 30%-70%, and not located in simple repeat sequence regions. (4) Design and synthesis of liquid phase probes: Based on the SNP sites obtained in step (3), probes are designed with a length of 110 bp, a GC content of 30%-70%, and a number of homologous regions ≤5; two probes are designed for each site, and the nucleotide sequences of the probes have 60%-70% overlap covering the SNP site. (5) Determine the final probe and chip preparation: Test the probe synthesized in step (4) to obtain the final chip SNP site set. Mix all probes with hybridization, washing and signal detection reagents and carrier materials to obtain probe mixture, i.e. 10K SNP liquid phase chip.

5. The method for fabricating a 10K SNP liquid-phase chip according to claim 4, characterized in that, The concentration of the probe mixture is 2-4 pmol / mL.

6. The use of the liquid phase chip according to any one of claims 1-3 in any one of the following (a)-(f): (a) Genotyping of commercial pigs; (b) Population genetic structure and kinship analysis; (c) Genome-wide association analysis; (d) Genome-wide selection breeding; (e) Germplasm resource identification and genetic improvement; (f) Early selection of commercial pigs.

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