A 10K SNP liquid phase chip for introduced pig breeds based on targeted capture sequencing and its application

Abstract

The present invention discloses a 10K SNP liquid phase chip for imported pig breeds based on targeted capture sequencing and its application, belonging to the field of biomolecular breeding technology. The present invention first collects, selects, and filters target SNP sites to screen out a comprehensive set of SNP sites, designs probes based on the SNP site set, and further prepares a 10K SNP liquid phase chip for imported pig breeds. The SNP sites on the chip have the characteristics of uniformity, polymorphism, compatibility, stability, and functionality, and can cover the pig genome to the greatest extent. At the same time, it can significantly reduce the selection of pig genomes, especially reducing the cost of early selection of pig breeds. In addition, it can ensure the selection efficiency and filling accuracy of chip sites exceeding 50K, meeting the needs of subsequent breed identification, pedigree analysis, kinship identification, genomic selection breeding, and whole-genome association analysis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomolecular breeding, and in particular relates to a 10K SNP liquid phase chip of introduced pig breeds based on targeted capture sequencing and its application. Background Art

[0002] With the rapid development of biomolecular breeding, technologies based on genomic selection have been extensively studied in a variety of organisms, bringing fundamental changes to biobreeding and propelling it towards a more sophisticated direction. Single nucleotide polymorphisms (SNPs) are variations in a single nucleotide in the genome. Known as "third-generation DNA molecular markers," SNPs are base substitutions, transversions, insertions, or deletions caused by variations in a single nucleotide in the genome. They are numerous, densely distributed, easy to detect, and suitable for genotyping.

[0003] With the rapid development of molecular detection technology, SNP chips with high-throughput genotyping capabilities have gradually become widely used. They can be divided into solid-phase chips and liquid-phase chips, both of which perform typing based on different carrier principles. Solid-phase chips utilize complementary hybridization between probes and DNA sequences and perform typing via fluorescent signals. They have advantages such as high accuracy and short cycle times. However, the cost of single-point typing on solid-phase chips is high and difficult to customize. Liquid-phase chips are based on targeted sequencing genotyping (Genotyping By Target Sequencing, GBTS) technology. Because they can enable target probes to bind complementary to target sequences for point-to-point capture, and can rapidly complete thousands of probe hybridization reactions simultaneously in the liquid phase, they have the advantages of platform adaptability, labeling flexibility, detection efficiency, information additivity, support convenience, and broad application spectrum.

[0004] SNP-based targeted sequencing genotyping technology (GBTS) is a simplified sequencing method that can efficiently and accurately detect genotypes at specific loci. Targeted sequencing genotyping technology is suitable for a variety of applications, including genetic selection, breeding, and seed testing. Furthermore, SNP chip technology is also widely used in livestock and poultry research. It can detect tens of thousands of SNP loci at high throughput, accurately assess genetic variation, and conduct population analysis. With its advantages of efficiency, accuracy, and convenience, it is a preferred method for genotyping.

[0005] In the early stages of piglet development, breeding farms use individual piglet genomic data to assess breeding value and calculate a comprehensive selection index. This ranking, based on the results, allows for the selection of superior individuals and the elimination of those who are castrated. This requires large-scale genotyping, typically using low-density microarrays to obtain genomic data. Currently, most mainstream pig SNP microarrays contain over 50K loci, placing a significant cost burden on breeding companies. Using a low-density SNP microarray with a 10K SNP density for early selection can also achieve the same effect of selecting superior individuals, significantly reducing costs and driving the rapid development of pig breeding. Summary of the Invention

[0006] Technical problems to be solved: In response to the above technical problems, the purpose of the present invention is to disclose a 10K SNP liquid phase chip for introduced pig breeds based on targeted capture sequencing and its application, which belongs to the field of biomolecular breeding technology. The present invention first collects, selects and filters the target SNP sites to screen out a comprehensive set of SNP sites, designs probes based on the SNP site set, and further prepares a 10K SNP liquid phase chip for introduced pig breeds. The SNP sites on the chip have the characteristics of uniformity, polymorphism, compatibility, stability and functionality, which can cover the pig genome to the greatest extent, and can significantly reduce the selection of the pig genome, especially reduce the cost of early selection of introduced pig breeds. In addition, it can ensure the selection efficiency and filling accuracy of chip sites of more than 50K, meeting the needs of subsequent breed identification, pedigree analysis, kinship identification, genomic selection breeding, and whole genome association analysis.

[0007] Technical solution: A 10K SNP liquid phase chip of introduced pig breeds based on targeted capture sequencing. The pig 10K SNP liquid phase chip is composed of high-quality SNP 10K probes of introduced pig breeds and reagents and carrier materials for hybridization, washing and signal detection.

[0008] Furthermore, it is characterized in that the method for preparing the high-quality SNP introduced pig breed 10K probe comprises the following steps: S1. collecting a set of target SNP sites;

[0009] S2. Target SNP sites are integrated and filtered based on a merging strategy that prioritizes uniform sites and supplements functional sites;

[0010] S3. Design probes based on the identified SNP locus set;

[0011] S4. Select high-quality mSNPs based on the probe design results, screen out the final probe sequence that meets the requirements, and optimize the probe to obtain a high-quality SNP 10K probe for the introduced pig breed.

[0012] Furthermore, the target SNP site set in step S1 includes a uniform site set that ensures compatibility and a functional site set that ensures specificity.

[0013] Furthermore, the uniform site set is based on the developed liquid phase chip to extract representative uniformly distributed sites according to the LD region; the functional site set includes: ① the top 0.1% sites of the GWAS results based on the genome and phenotype group data of the Shiji biological population, ② the lead SNPs of the GWAS of each trait in PigBioBank, ③ important trait gene sites, and ④ the variety-specific sites of each of the five Shiji varieties.

[0014] Furthermore, the filtering principles in step S2 are: ① maintaining a certain interval between sites, ② ensuring that the sites are polymorphic, and ③ avoiding microsatellite SSR regions, genome assembly gap regions, and telomere regions.

[0015] Furthermore, the method for designing the probe in step S3 is a multiple single nucleotide polymorphism detection technology.

[0016] Furthermore, the probe design principles in step S3 are as follows: ① select all probes with a length of 100-120bp that can cover the region, ② calculate the GC content of the probes that can cover the target region, and ③ calculate the number of homology regions in the probes, where homology is calculated as a length of 40bp or more and identical, a length of 70bp and 95% similarity, and a length of 80bp and 85% similarity. Furthermore, the selection principles in step S4 are as follows: ① select probes with a GC content between 30-70%, ② select probes with a number of homology regions ≤5, and ③ select probe regions that do not contain SSR or N regions.

[0017] The application of the 10K SNP liquid phase chip of introduced pig breeds based on targeted capture sequencing as described in any of the above items in the early selection of introduced pig breeds, breed identification, trait-related gene positioning, genetic diversity analysis, genotyping detection and molecular breeding.

[0018] Beneficial effects:

[0019] 1. The present invention provides a 10K SNP liquid phase chip for introduced pig breeds based on targeted capture sequencing. The chip sites comprehensively consider uniformity, polymorphism, compatibility, stability, and functionality to ensure uniform coverage of the genome and improve the accuracy of genotype filling in the future. At the same time, it reduces the correlation between sites, avoids interference from repetitive sequences and variability, and improves the continuity and reliability of the sites.

[0020] 2. The present invention provides an imputation strategy, which constructs a haplotype reference panel (HRP) based on 50K chip detection data, imputing the 10K liquid phase chip to 50K and then to the WGS level. The imputed genomic data is highly accurate and can meet the needs of many subsequent analyses, such as variety identification, pedigree analysis, kinship identification, and whole-genome association analysis.

[0021] 3. The present invention can significantly reduce costs while ensuring the accuracy of genome selection and genome filling, meet the needs of large-scale genome breeding and selection, improve the efficiency of genetic analysis and molecular breeding of pig breeds, and have a higher cost-effectiveness.

[0022] 4. This invention uses targeted genotyping technology based on sequencing (GBTS), which has higher flexibility than traditional solid-phase chips. Probes can be directly added or removed to adjust the target site during the subsequent development process. At the same time, it can not only genotype the target site, but also genotype sites within a certain range near the target SNP. It has universal platform compatibility and is suitable for all second-generation and third-generation sequencing systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram showing the distribution of SNP marker density in the 10K SNP liquid phase array of introduced pig breeds;

[0024] Figure 2 This is the MAF distribution of SNP sites in the 10K SNP liquid phase array of introduced pig breeds;

[0025] Figure 3 This is a picture of the kit for the 10K SNP liquid phase array for introduced pig breeds;

[0026] Figure 4 To test individual consistency;

[0027] Figure 5 The results of early selection and verification of growth traits of different chips in Example 3 are as follows;

[0028] Figure 6 The correlation between the growth traits GEBV and TBV of different chips in Example 3;

[0029] Figure 7 Correlation between growth trait GEBV and CorrPhe of different chips in Example 3;

[0030] Figure 8 The results of early selection verification of reproductive traits of different chips in Example 3 are as follows;

[0031] Figure 9The correlation between GEBV and TBV of reproductive traits of different chips in Example 3;

[0032] Figure 10 Correlation between reproductive traits GEBV and CorrPhe on different chips in Example 3;

[0033] Figure 11 The results of variety identification verification using Shiji 10K are shown in Figure 1, where ① represents the PCA result; ② represents the NJ tree result; and ③ represents the admixture result. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with examples, which are provided to explain the present invention and are not limited to the following examples:

[0035] Example 1

[0036] Collection and filtering of target SNP loci and probe design and optimization

[0037] Step 1: Collect target SNP loci

[0038] (1) Uniform loci collection: Based on the developed liquid phase microarray and the microarray sequencing data of five introduced pig breeds (Duroc, Duroc S2, Pietrain, Yorkshire Large White, and Yorkshire Landrace) from Shiji Biotechnology, representative uniformly distributed loci were extracted by the degree of linkage disequilibrium between markers. The collection is shown in Table 1.

[0039] Table 1 Collection of samples of five introduced pig breeds by Shiji Biological

[0040] variety Duroc New Duroc strain Large White Pig Landrace pigs Pietrain quantity 1298 530 3982 734 575

[0041] (2) Functional site set:

[0042] ① Based on the GWAS results of the Shiji Bio population genome and phenotype data, the top 0.1% sites were selected; ② The lead SNPs of each trait GWAS were downloaded from the PigBioBank database; ③ The gene loci of important pig traits published to date were collected; ④ The breed-specific loci of each of the five introduced pig breeds of Shiji Bio (Duroc, S2, Pietrain, Large White, and Landrace).

[0043] Step 2. SNP site integration and filtering

[0044] The target SNP site set obtained by merging uniform sites and functional sites was filtered according to the following filtering principles: ① the pruning function of plink1.9 was used to maintain a certain interval between sites, ② the maf parameter of plink1.9 was used to ensure that the sites were polymorphic, and ③ based on the pig 11.1 reference genome, some sites in the microsatellite (SSR), genome assembly gap, telomere and other regions were removed; finally, 9819 SNP sites were obtained, which were evenly distributed on the chromosomes.

[0045] Step 3. Probe design

[0046] Using multiple single nucleotide polymorphism (mSNP) detection technology, a pair of specific amplification primers are designed for each SNP marker. This SNP marker is generated in the obtained amplicon, and one amplicon corresponds to one SNP marker. The probe design principles are as follows: ① Select all probes with a length of 100-120bp that can cover the region, ② Calculate the GC content of the probes that can cover the target region, and ③ Calculate the number of homology regions of the probes, where homology is calculated as a length of more than 40bp and completely identical, a length of 70bp and 95% similarity, and a length of 80bp and 85% similarity.

[0047] Step 4. Probe optimization

[0048] Based on the probe design results, the probe sequences that finally met the requirements were screened out. The probe selection principles are as follows: ① Select probes with a GC content between 30-70%, ② Select probes with a number of homology regions ≤ 5, and ③ Select probe regions that do not contain SSR or N regions; then confirm the coverage of the target sites, and finally successfully design probes for 9668 SNP sites to enter the next round of testing.

[0049] Table 2 Information of 9668 SNP loci

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[0107] Figure 1 The SNP marker density distribution in the Shiji No. 1 10K liquid phase chip is shown. Figure 1 It can be seen that the site density is evenly distributed and can cover the entire genome; Figure 2The MAF (minimum allele frequency) distribution of SNP sites in the Shiji No. 1 10K liquid phase chip is shown. Figure 2 It can be seen that the MAF of most sites is concentrated in a higher frequency range, which can ensure higher filling accuracy and genome selection prediction accuracy.

[0108] Example 2

[0109] A method for preparing a 10K SNP liquid phase chip for introduced pig breeds based on targeted capture sequencing comprises the following steps:

[0110] Step 1. Mixing and drying of reagents

[0111] ① Calculate the library pooling volume based on the library concentration measurement results. For example, if the input volume of a single library is 200 ng, and the library concentration is 20 ng / μL, then 10 μL of the library should be used. Ensure that the input volume of each library is consistent.

[0112] ② Mix the library according to the calculated volume into a new 1.5mL EP tube.

[0113] ③ Add the reagents shown in Table 3 to the above tubes.

[0114] Table 3 Reagents to be mixed and dried

[0115]

[0116] ④ Seal the tube with sealing film, poke 3-5 small holes, and concentrate to dryness using a vacuum concentrator at a temperature of ≤30℃;

[0117] ⑤ After concentration is completed, centrifuge at 12000 rpm for 1 min before proceeding with subsequent operations.

[0118] Step 2. DNA library hybridization capture

[0119] ① Vortex and centrifuge the dissolved hybridization reagents in Table 4 to mix thoroughly. Prepare the reaction system and add it to the test tube in step 1.⑤.

[0120] Table 4 Hybridization capture reaction solution

[0121]

[0122] ② Mix by pipetting or vortexing, centrifuge at 12000 rpm for 1 min, and let stand at room temperature for 5 min.

[0123] ③Pipette or vortex to mix again, and centrifuge at 12000 rpm for 5 seconds.

[0124] ④ Transfer all 32 μL of hybridization capture mix to a 0.2 mL PCR tube.

[0125] ⑤ Set up two PCR instrument programs according to Table 5.

[0126] ⑥Thermal cycle incubation conditions: 95℃ for 10 min (heat cover temperature: 105℃).

[0127] ⑦ When the PCR amplification instrument cools down to 65℃, transfer the cells to another PCR instrument at 65℃ (heated cover temperature 75℃), start timing, and hybridization should last for 2 hours or more.

[0128] Table 5 Procedure for hybridization incubation

[0129] temperature Incubation time Hot cover 95℃ 10min 105℃ 65℃ Hold 75℃

[0130] Step 3. Prepare Wash Buffers

[0131] ① Single capture system, dilute according to Table 6 Make the WashBuffers to 1× working volume.

[0132] Table 6 Dilution method of Wash Buffers (amount per hybridization reaction)

[0133]

[0134] ②100 μL 1× Wash Buffer I and 300 μL 1×StringentWashBufferr needs to be preheated at 65°C in advance.

[0135] ③The rest Store 1× Wash Buffer at room temperature.

[0136] Step 4. Preparation DNA Probe Beads

[0137] ① After equilibration at room temperature Vortex the DNA Probe Beads for 15 seconds to mix thoroughly.

[0138] ② Transfer 50 μL to one reaction ③ Place the PCR tube on a magnetic stand until the solution becomes clear.

[0139] ④ Remove the supernatant with a pipette, retaining the magnetic beads. Remove the PCR tube from the magnetic stand.

[0140] ⑤ Add 150 μL Add 1× Beads Wash Buffer and vortex for 10 seconds. Place the PCR tube on a magnetic rack until the solution is clear. Carefully remove the supernatant and retain the magnetic beads.

[0141] ⑥Repeat step 5 twice, washing 3 times in total.

[0142] Step 5. Hybridize the fragments with DNAProbeBeads binding

[0143] ①After the hybridization process is completed, open the PCR instrument cover, open the PCR tube cover, and transfer 32μL of hybridization capture solution to the prepared DNAProbeBeads.

[0144] ② Vortex and oscillate for 10 seconds to mix thoroughly, and then centrifuge immediately.

[0145] ③ Place the PCR tube in the PCR instrument, 65℃, 45min, hot cover temperature 75℃.

[0146] ④ Every 12 minutes, vortex for 5 seconds and centrifuge briefly.

[0147] Step 6. Elution to remove unbound DNA

[0148] ① Add 100 μL of 65℃ preheated 1×WashBufferⅠ.

[0149] ② Vortex for 5 seconds and centrifuge immediately.

[0150] ③ Place the PCR tube on a magnetic rack until the solution is clear. Carefully remove the supernatant (retain the magnetic beads), making sure not to absorb the magnetic beads.

[0151] ④ Add 150 μL of 65℃ preheated 1× Stringent Wash Buffer, slowly pipette up and down 10 times to mix the magnetic beads thoroughly.

[0152] ⑤ After the last set of samples is mixed, place the PCR tube in a PCR instrument at 65°C for 2 minutes.

[0153] ⑥ Place the PCR tube on a magnetic rack until the magnetic beads are completely separated from the solution. Use a pipette to quickly remove the supernatant, retaining the magnetic beads. ⑦ Repeat steps 4 to 6 in step 6.

[0154] ⑧ Transfer the PCR tube from the magnetic stand and add 150 μL of room temperature 1× Wash Buffer I, vortex for 2 min, and centrifuge briefly.

[0155] ⑨ Place the PCR tube on a magnetic rack until the solution is clear. Carefully remove the supernatant (retain the magnetic beads), making sure not to absorb the magnetic beads.

[0156] ⑩ Transfer the PCR tube from the magnetic stand and add 150 μL of room temperature 1× Wash Buffer II, vortex for 1 min, and centrifuge briefly.

[0157] Place the PCR tube on a magnetic stand until the solution is clear, then carefully remove the supernatant (retaining the magnetic beads), making sure not to aspirate the magnetic beads.

[0158] Add 150 μL of room temperature 1× Wash Buffer III, vortex for 30 seconds, and centrifuge briefly.

[0159] Place the PCR tube on a magnetic stand until the solution is clear, then carefully remove the supernatant (retaining the magnetic beads), making sure not to aspirate the magnetic beads.

[0160] Remove the tube containing the captured DNA beads from the magnetic stand and add 20 μL Nuclease-Free Water.

[0161] Use a pipette to slowly pipette up and down 10 times to ensure that all the magnetic beads are resuspended.

[0162] Step 7. PCR enrichment

[0163] ① Prepare the reaction system in a 0.2 mL low-adsorption PCR tube according to Table 7.

[0164] Table 7 PCR enrichment system (single hybrid system)

[0165]

[0166] ② Vortex to mix and centrifuge briefly to ensure that the magnetic beads are still in the solution.

[0167] ③ Place the PCR tube in the PCR instrument and set the PCR program according to Table 8, with the heated cover temperature at 105°C.

[0168] Table 8 PCR enrichment program

[0169]

[0170] Note: ∞ indicates the reaction mixture is kept at 4°C until removed from the thermal cycler.

[0171] Step 8. PCR product purification

[0172] ① Place the PCR tube on a magnetic rack until the solution becomes clear. It is expected that it will stand at room temperature for at least 3 minutes.

[0173] ② Transfer the supernatant to a new PCR tube.

[0174] ③Add 45 μL DNACleanBeads, vortex to mix.

[0175] ④ After standing at room temperature for 5 minutes, briefly centrifuge.

[0176] ⑤ Place the PCR tube on a magnetic rack until the solution is clear, which is expected to be at room temperature for at least 3 minutes. Carefully remove the supernatant (retaining the magnetic beads), making sure not to aspirate the beads.

[0177] ⑥ While the PCR tube is on the magnetic rack, add 100% ethanol and let it stand at room temperature for 30 seconds. Carefully remove the supernatant (retaining the magnetic beads) and let it stand at room temperature for 5 minutes until the ethanol is completely evaporated.

[0178] ⑦ Remove the PCR tube from the magnetic rack to room temperature, add 35 μL of 10 mM Tris-HCl, and vortex to mix. Let it stand at room temperature for 5 minutes, then briefly centrifuge.

[0179] ⑧Place the PCR tube on the magnetic rack until the solution becomes clear. It is expected to stand at room temperature for at least 3 minutes.

[0180] 9. Transfer the supernatant to a new 0.2 mL low-adsorption tube, making sure not to absorb the magnetic beads. The purified DNA hybridization library can be stored at -20°C until required for sequencing.

[0181] Figure 3 The image of the kit for the Shiji No. 1 10K chip is shown. The Shiji No. 1 10K chip was jointly developed by Shiji Biotechnology and Zhejiang University. The porcine 10K SNP liquid phase chip consists of high-quality SNP-specific introduced pig 10K probes and reagents and carrier materials for hybridization, washing, and signal detection, as shown in Table 9.

[0182] Table 9 Kit Components

[0183]

[0184] Example 3

[0185] Detection rate and genotyping verification test of 10K SNP liquid phase chip for introduced pig breeds

[0186] The Neogen 50K chip and the Shiji I 10K chip were tested simultaneously on 200 individuals to verify the detection rate and genotyping of the chips.

[0187] The following processing was performed on the 9668 candidate SNP sites obtained by screening:

[0188] Step 1. DNA fragmentation and end repair plus A (40-50 min)

[0189] ① Prepare the end-repair reaction system according to the reagents and amounts shown in Table 10;

[0190] ② Vortex the end-repair reaction system to mix thoroughly and centrifuge briefly;

[0191] ③ Place the PCR tube plate containing the reaction system in the PCR instrument and set the PCR amplification program according to Table 11. Set the heated lid temperature to 82°C.

[0192] Table 10 Composition of end repair reaction system

[0193]

[0194] Table 11 End-repair system procedures

[0195] step temperature time 1 37℃ 10 / 20min* 2 72℃ 20min 3 4℃ ∞

[0196] Note: *(10 / 20min) indicates that the incubation time at 37°C can be 10 minutes or 20 minutes. The preferred incubation time depends on the quality and quantity of the DNA, the degree of end repair required, and the specific experimental protocol. High-quality DNA may require only a shorter incubation time, while fragmented or damaged DNA may require a longer incubation time.

[0197] ∞(4°C) means that after the reaction is completed, the reaction system can be stored indefinitely at 4°C, and the DNA will not be significantly degraded at this temperature.

[0198] Step 2. Adapter ligation (70 min): Once the temperature in step 1 reaches 4°C, immediately proceed with the adapter ligation as follows:

[0199] ① Prepare the adapter ligation system according to the reagents and amounts shown in Table 12, and directly add the adapter ligation system to the reaction system in step 1;

[0200] ② Vortex the reaction system containing the adapter ligation system and briefly centrifuge;

[0201] ③ Place the PCR tube containing the reaction system containing the adapter ligation system in the PCR instrument, set the PCR amplification program according to Table 13, and remove the heated cover;

[0202] ④When the PCR amplification program reaches 4°C, immediately proceed to the adapter ligation system purification;

[0203] Table 12 Composition of the joint connection system

[0204]

[0205] Table 13 Joint connection system procedures

[0206] step temperature time 1 22℃ 60 minutes* 2 4℃ ∞

[0207] Note: *(60 min) indicates the recommended incubation time for adapter ligation reaction at room temperature (22°C). This time is sufficient for the ligase to effectively connect the adapter molecules to the DNA fragments. Shorter incubation times may result in incomplete ligation, while longer incubation times may not significantly improve efficiency and may increase the risk of nonspecific ligation.

[0208] ∞(4°C) indicates that after the adapter ligation reaction is completed, the reaction system can be stored indefinitely at 4°C to ensure the stability of the ligated DNA product.

[0209] Step 3. Adapter ligation system purification (30-65 min)

[0210] ① Add 48 μL to the connection system DNACleanBeads, vortex to mix, avoid creating bubbles during vortexing, let stand at room temperature for 5 minutes, and then centrifuge briefly;

[0211] ② Place the PCR tube on a magnetic stand and let it stand at room temperature for at least 3 minutes until the solution is clear. Carefully remove the supernatant (retain the magnetic beads), making sure not to absorb the magnetic beads.

[0212] ③ Keep the PCR tube on the magnetic rack, add 100 μL of 80% ethanol, let it stand at room temperature for 30 seconds, carefully remove all the supernatant (retain the magnetic beads), and let it stand at room temperature for at least 5 minutes until the ethanol is completely evaporated;

[0213] ④ Remove the PCR tube from the magnetic rack, add 32 μL of 10 mM Tris-HCl, shake and mix, let it stand at room temperature for 5 minutes, and then briefly centrifuge;

[0214] ⑤ Place the PCR tube on a magnetic rack and let it stand at room temperature for at least 3 minutes until the solution becomes clear;

[0215] ⑥ Transfer 30 μL of supernatant to a new 0.2 mL low-adsorption PCR tube (keep the supernatant), making sure not to absorb the magnetic beads;

[0216] ⑦ Add 16.5 μL (0.55X) of DNACleanBeads, vortex and shake to make the amplified product Mix DNACleanBeads thoroughly, let stand at room temperature for 5 minutes, and centrifuge briefly;

[0217] ⑧ Place the PCR tube on a magnetic rack and let it stand at room temperature for at least 3 minutes until the solution becomes clear;

[0218] ⑨Carefully pipette the supernatant into a new 0.2 mL low-adsorption PCR tube (keep the supernatant), making sure not to absorb the magnetic beads;

[0219] ⑩ Add 4.5 μL (0.15X) of DNACleanBeads, vortex and shake to make the amplified product Mix DNACleanBeads thoroughly, let stand at room temperature for 5 minutes, and centrifuge briefly;

[0220] Place the PCR tube on a magnetic stand and let it stand at room temperature for at least 2 minutes until the solution is clear. Carefully remove the supernatant (retain the magnetic beads), making sure not to absorb the magnetic beads.

[0221] Keep the PCR tube on the magnetic rack, add 100 μL of 80% ethanol, let it stand at room temperature for 30 seconds, carefully remove the supernatant (retain the magnetic beads), and let it stand at room temperature for 5 minutes until the ethanol is completely evaporated;

[0222] Remove the PCR tube from the magnetic stand and immediately resuspend the magnetic beads using the PCR amplification system;

[0223] Step 4. Library amplification (30 min)

[0224] ① Prepare the PCR amplification system in a PCR tube according to Table 14;

[0225] ② Vortex the PCR amplification system to mix thoroughly and centrifuge briefly;

[0226] ③ Place the PCR tube containing the reaction system into the PCR instrument and set the PCR amplification program according to Table 15. The heated cover should be set to 105°C.

[0227] ④ When the PCR amplification system cycle program reaches 4°C, the PCR amplification product can be stored at -20°C overnight;

[0228] Table 14 Composition of PCR amplification system

[0229]

[0230] Table 15 PCR amplification system cycle program

[0231]

[0232]

[0233] Note: *(2 min) indicates that the initial denaturation step at 98°C is critical for completely separating the double-stranded DNA template into single strands, enabling primer binding. A longer initial denaturation time (2 min instead of the typical 30 s) is used to ensure complete denaturation, especially for difficult-to-denature or long templates.

[0234] ∞(4°C) means that after PCR amplification is complete, the PCR product can be stored indefinitely at 4°C. This temperature helps maintain the stability of the amplified DNA and prevent degradation.

[0235] Step 5. Purification (40 min)

[0236] ① Add 40 μL to the PCR amplification system DNACleanBeads, vortex to mix, avoid creating bubbles during vortexing, let stand at room temperature for 5 minutes, and then centrifuge briefly;

[0237] ② Place the PCR tube on a magnetic stand and let it stand at room temperature for at least 3 minutes until the solution is clear. Carefully remove the supernatant (retain the magnetic beads), making sure not to absorb the magnetic beads.

[0238] ③ Keep the PCR tube on the magnetic rack, add 100 μL of 80% ethanol, let it stand at room temperature for 30 seconds, carefully remove the supernatant (retain the magnetic beads), and let it stand at room temperature for 5 minutes until the ethanol is completely evaporated;

[0239] ④ Remove the PCR tube from the magnetic rack, add 35 μL of 10 mM Tris-HCl, shake and mix, let it stand at room temperature for 5 minutes, and then briefly centrifuge;

[0240] ⑤ Place the PCR tube on a magnetic rack and let it stand at room temperature for at least 3 minutes until the solution becomes clear;

[0241] ⑥ Transfer the supernatant to a new 0.2 mL low-adsorption PCR tube (keep the supernatant), making sure not to absorb the magnetic beads;

[0242] ⑦Sequencing of the purified DNA library.

[0243] In terms of detection rate, the designed 10K chips were tested in the test round. Among the 188 test samples, 0 samples had a detection rate lower than 90%, the detection rate ranged from 99.432% to 99.928%, and the average detection rate was 99.809%. Figure 4 It can be seen that Figure 4 The consistency of the Newgene 50K chip and the Shiji No. 1 liquid phase chip was demonstrated at the individual level and the site level respectively. Compared with the Newgene 50K chip, 188 of the consistency at the individual level were higher than 90%, and 98.66159% of the sites had consistency higher than 90% at the site level.

[0244] Example 4

[0245] Effect verification test of 10K SNP liquid phase chip for introduced pig breeds

[0246] The 10K SNP liquid phase chip of introduced pig breeds prepared in Example 2 was compared with three commercially available chips in terms of the accuracy of genome selection and missing genotype filling to verify the chip application effect. The specific number of chip sites used for verification is shown in Table 16.

[0247] Table 16 Chip name and number of sites used for validation experiments

[0248]

[0249] (1) Early selection verification test

[0250] The early selection validation test was based on the design of 10K loci and three other mainstream chips on the market (geneseek80K chip, geneseek60K chip, and "SMIC No. 1" chip). 5 growth traits of three breeds (Duroc, S2, and Large White pigs) and 5 reproductive traits of Large White pigs were used for early selection through ssGBLUP with 10 five-fold cross-validations. The test was conducted to test how many (%) of the individuals ranked 1% by TBV could be included in the top 50% of the calculated GEBV and make a comparison.

[0251] Figure 5 and Figure 8 The top 50% of individuals in GEBV calculated for growth and reproductive traits can contain how many (%) individuals in TBV ranking 1%, respectively, as shown by Figure 5 and Figure 8 It can be seen that whether it is growth traits or reproductive traits, compared with the other three chips, the Shiji No. 1 10K chip can achieve good early selection effects and successfully screen out excellent individuals.

[0252] (2) Genomic selection verification test

[0253] The genomic selection validation test was based on the design of 10K loci and three other mainstream chips on the market (geneseek80K chip, geneseek60K chip, and "SMIC No. 1" chip). Genomic selection was performed on ssGBLUP using 10 five-fold cross-validations for five growth traits of three breeds (S1, S2, and Large White pigs) and five reproductive traits of Large White pigs. The breeding value (TBV) and corrected phenotypic value (CorrPhe) calculated for each population based on all phenotypes and all pedigrees were used as benchmarks, and the accuracy was evaluated by comparing the genomic selection breeding value (GEBV) of the validation population calculated by cross-validation based on each chip locus with it (i.e., calculating the correlation coefficient).

[0254] The specific quantities of each variety and trait involved in the early selection verification test and the genomic selection verification test are shown in Tables 17 and 18.

[0255] Table 17 Number of verified individuals for 3 varieties

[0256]

[0257] Table 1810 Characters Names

[0258]

[0259]

[0260] Figure 6 and Figure 9 Represent the correlation coefficient between the genomic selection breeding value (GEBV) of the validation population calculated based on each chip site and the breeding value (TBV) calculated based on all phenotypes and all pedigrees, respectively. Figure 6 and Figure 9 It can be seen that in some traits and varieties, such as FCR and BFT of Duroc variety, the prediction effect of Shiji No. 1 10K chip is comparable to that of GeneSeek 80K and GeneSeek 60K chips, and even slightly higher in some cases.

[0261] Figure 7 and Figure 10 Represents the genomic selection breeding value (GEBV) and corrected phenotypic value (CorrPh e ), the correlation coefficient between Figure 7 As shown in Figure 10, the Shiji No. 1 10K chip showed high genetic correlation in multiple species and traits, which shows that the Shiji No. 1 10K chip is competitive in genomic selection.

[0262] (3) Genome filling verification

[0263] ①Fill to 50K accuracy assessment:

[0264] The results of the accuracy evaluation of padding from 10K to 50K in the padding verification are shown in Table 19. As shown in Table 19, the Shiji No. 1 10K chip can be padded to the 50K level with high accuracy, indicating that the Shiji No. 1 10K chip can actually represent the information of the 50K chip and has good compatibility.

[0265] Table 19 Accuracy of filling different chips to 50K

[0266] 10K to 50K Shiji No. 1 10K F-score 0.999l correlation 0.9826

[0267] ②Fill in WGS accuracy assessment:

[0268] The results of the imputation validation from 10K and 20K to WGS are shown in Table 20. As shown in Table 20, two imputation methods were used for the 10K locus imputation: direct imputation from 10K to WGS level and imputation from 10K to 50K before imputation to WGS level. The results show that the 10K of Shiji No. 1 can be imputed to WGS level with high accuracy, indicating that the 10K chip-based imputation scheme with the help of PHARP is sufficient to meet the requirements of subsequent analysis.

[0269] Table 20 Accuracy of different chips filled into WGS

[0270]

[0271] (4) Variety identification and verification

[0272] According to the pedigree records and low-depth sequencing results, pedigree analysis was performed and 50 purebred imported pig breeds were selected as the verification group, as shown in Table 21. After testing with the Shiji No. 1 10K chip, population genetics analysis was performed and the results were shown in Figure 11 In. By Figure 11 It can be seen that Figure 11 ① represents the PCA calculation results. It can be seen that the five groups are clearly separated and stratified at the PCA level. The D (Duroc) and T (S2) with closer ancestry are closer, but can be separated into groups. Figure 11 ② in the middle represents the result of the NJ tree, where the five varieties are located in different branches and there is no confusion between varieties; Figure 11 ③ represents the admixture result, which analyzed the genetic composition ratio of 50 individuals, showing that these individuals have a high bloodline purity, and some individuals have a small amount of other bloodlines.

[0273] Table 21 Status of 5 purebred pig breeds introduced by Shiji Biological

[0274]

[0275] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A 10K SNP liquid phase chip for introduced pig breeds based on targeted capture sequencing, characterized in that: The pig 10K SNP liquid phase chip is composed of high-quality SNP introduced pig 10K probes and reagents and carrier materials for hybridization, washing and signal detection; the SNP site information is as follows: The method for preparing the high-quality SNP introduced pig breed 10K probe comprises the following steps: S1. Collecting a set of target SNP sites; the target SNP site set includes a uniform site set to ensure compatibility and a functional site set to ensure specificity; S2. Target SNP sites are integrated and filtered based on a merging strategy that prioritizes uniform sites and supplements functional sites; S3. Designing probes based on a set of determined SNP sites; the method for designing probes is a multi-single nucleotide polymorphism detection technology; S4. Based on the probe design results, high-quality mSNP probes are selected, and the final probe sequence that meets the requirements is screened and optimized to obtain a high-quality SNP-specific 10K probe for the introduced pig breed. The pig reference genome is pig 11.

1.

2. The 10K SNP liquid phase chip for introduced pig breeds based on targeted capture sequencing according to claim 1, characterized in that: The probe design principles in step S3 are as follows: ① select all probes with a length of 100-120 bp that can cover the region, ② calculate the GC content of the probes that can cover the target region, and ③ calculate the number of homology regions of the probes, where homology is calculated as a length of more than 40 bp and completely identical, a length of 70 bp and 95% similarity, and a length of 80 bp and 85% similarity.

3. The 10K SNP liquid phase chip for introduced pig breeds based on targeted capture sequencing according to claim 1, characterized in that: The selection principles in step S4 are as follows: ① select probes with a GC content between 30-70%, ② select probes with a number of homology regions ≤ 5, and ③ select probe regions that do not contain SSR or N regions.

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