Multiple single-nucleotide polymorphism-based 10k liquid phase chip for pig, and use thereof

Abstract

A multiple single-nucleotide polymorphism (mSNP)-based 10K liquid phase chip for a pig, and a use thereof. Under the condition that the basic principle of liquid phase chip design is maintained, a new marker is added to an existing chip, and an mSNP technology is used to optimize a probe, so as to generate more SNP markers having high typing quality and medium linkage disequilibrium with a target site SNP in a probe area, thereby increasing the number of effective markers of the chip. According to the mSNP-based liquid phase chip, the number of detectable SNPs can be increased to be 1.5-2 times of the number of target site SNP markers, thereby solving the problem of existing chips only containing the target site SNP and being unable to provide an mSNP marker, without increasing the cost. By means of a genotype imputation technology, the genotype data of the chip is filled into a mainstream 50K chip for genome selection, thereby obtaining molecular seed selection accuracy identical or similar to that of the 50K chip, and reducing the molecular breeding cost of the pig.

Description

A pig 10K liquid phase chip based on multiple single nucleotide polymorphisms and its application Technical Field

[0001] The present invention relates to the field of biological breeding, and in particular to a pig 10K liquid phase chip based on multiple dispersed nucleotide polymorphism (mSNP) and applications thereof. Background Art

[0002] With the rapid advancement of molecular biology, biobreeding technologies centered around genomic selection have been extensively researched and applied in dairy cows, pigs, chickens, crops, forest trees, and aquatic species. This has brought about a major revolution in livestock and poultry breeding and has become a powerful driving force for its advancement. Currently, genomic selection is a breeding technology being vigorously developed within my country's seed industry revitalization plan. A prerequisite for genomic selection is rapid, high-throughput genotyping of individuals. Single nucleotide polymorphisms (SNPs) are considered the best molecular markers currently available due to their large number, wide distribution across the genome, and ease of large-scale, rapid screening and genotyping. Genomic selection utilizes genome-wide SNP markers to calculate an individual's genomic breeding value and select superior individuals. With the advancement of molecular detection technology, SNP chips capable of high-throughput genotyping have emerged.

[0003] Theoretical research and breeding practice have demonstrated that the 50K microarray, containing approximately 50,000 SNP markers, is highly effective in molecular breeding and is currently the mainstream approach for genomic selection. However, animal breeding, particularly pig populations, is large, necessitating early selection of piglets to identify individuals for performance testing and ultimately select the best individuals for inclusion in the breeding core herd. However, only parental information is available for early selection of piglets, making it difficult to distinguish genetically superior individuals from the same litter due to the shared parental information. Research suggests that genomic selection can improve the accuracy of early selection of piglets, just as each child is unique. Genomic information can be used to select the best individuals from the same litter. This necessitates large-scale genotyping of piglets. For example, for a breeding farm with 1,000 sows, achieving optimal genomic selection requires testing at least 22,000 piglets annually. Solid-phase 50K microarrays and liquid-phase 50K microarrays would cost 3.96 million and 2.97 million yuan, respectively. While liquid-phase arrays have significantly reduced the cost of microarray testing, it remains a significant burden, significantly limiting the implementation of molecular breeding in pigs in my country. To save costs, some companies only test a small number of individuals, which introduces significant randomness and significantly reduces breeding effectiveness. Using a low-density chip, such as a 10K chip (containing approximately 10,000 markers), for early testing can significantly reduce the cost of early selection genotyping due to its low cost. Furthermore, using genotype filling technology, the marker genotypes of a 10K chip can be filled into a 50K chip with high accuracy, ensuring the same accuracy in early piglet selection as testing with a 50K chip.

[0004] However, there is currently no commercial 10K microarray for pig breeding. The inventors developed a low-density 10K microarray for pigs based on solid-phase microarray technology and have obtained a patent (Patent No.: ZL201711190317.6, containing 8,846 markers). However, the lack of flexibility, strict sample size requirements (must be multiples of 12 or 24), and high customization costs of solid-phase microarrays have hindered their widespread adoption. Unlike solid-phase microarrays, liquid-phase microarrays based on genotyping by target sequencing (GBTS) technology offer advantages such as easy marker addition and removal, no sample size requirements, and low customization costs. Figure 1 shows that GBTS technology primarily performs multiple sequencing of the target locus and its upstream and downstream sites to ensure the quality of the target locus genotyping. Furthermore, while liquid-phase microarrays genotype the target locus, they also genotype polymorphic sites upstream and downstream of the target locus, known as multiple single nucleotide polymorphism clusters (mSNPs or multiple dispersed nucleotide polymorphisms (MNPs)). For mSNPs with moderate linkage disequilibrium, markers upstream and downstream of the target site can provide additional information. Therefore, without increasing the number of target SNPs or testing costs, multi-SNP technology can clearly add more effective mSNP markers, improve the information content of liquid-phase microarrays, and fully utilize the characteristics of GBTS technology, which is not possible with solid-phase microarrays.

[0005] Summary of the Invention

[0006] Therefore, based on the developed pig 9K solid-phase SNP chip (patent number: ZL201711190317.6), the present invention combines the characteristics of the pig genome, utilizes the multiple single nucleotide polymorphism technology, optimizes the target site probe design, and develops the pig 10K liquid-phase chip based on multiple single nucleotide polymorphism (abbreviated as 10K liquid-phase chip) for pig genome breeding, reducing the cost of pig molecular breeding.

[0007] The present invention has developed a pig 10K liquid phase chip and provided a chip target site SNP marker screening and probe preparation method. The chip designed by the present invention can save the cost of pig genome selection and maximize the use of mSNP marker information upstream and downstream of the target site, thereby improving the efficiency of pig genetic analysis and molecular breeding.

[0008] Specifically, the purpose of the present invention is to provide the following aspects:

[0009] First, a method for SNP marker screening and probe preparation is provided. Based on the whole-genome sequencing data of three pig breeds (Duroc, Large White, and Landrace) (reference genome version 11.1), 10,200 target site SNPs were mined and screened. Then, based on probe design principles, probes were designed and optimized for the target site SNPs, ultimately designing 15,572 probes.

[0010] The method comprises the following steps:

[0011] Step 1: Target site SNP marker screening

[0012] (1) Preliminary screening of target site SNP markers. Referring to the method described in ZL201711190317.6, based on the whole genome sequencing data of three pig breeds, Duroc, Large White, and Landrace, and aligned to the pig 11.1 reference genome, markers were selected based on marker spacing and gene function annotation to screen target capture sites, namely target site SNPs. The principles of target site SNP screening are: ① uniform distribution on each chromosome and dense distribution at both ends of each chromosome; ② marker spacing greater than 280Kb; ③ important candidate sites for growth, reproduction, feed conversion, meat quality, and body size traits; ④ QTLdb database comparison, SNP markers should fall as much as possible in the QTL region of economic traits. As described in ZL201711190317.6, 8846 markers were obtained as target sites.

[0013] (2) Target site SNP supplementation and optimization. Based on the initial screening, markers with high polymorphism and moderate regional linkage disequilibrium were supplemented and optimized. The principles are as follows: ① Polymorphism mainly considers MAF>0.35 in Duroc, Landrace, and Large White pigs; ② Average linkage disequilibrium with upstream and downstream SNP markers r 2 Below 0.85.

[0014] In the area where the marker spacing of 9K solid-phase chip is greater than 280Kb (the average spacing of 9K solid-phase chip is 279Kb), markers that meet the above-mentioned SNP principles are selected to supplement and optimize the target site SNPs. Compared with the pig 9K solid-phase SNP chip, a total of 2243 target site SNPs that meet the requirements are added.

[0015] Step 2: Design probes based on target site SNP

[0016] Probes were optimized and designed using multiplex single nucleotide polymorphism (SNP) detection technology. One to four 110bp probes were designed, each covering the target site. The total probe coverage, centered around the target site, was 165bp. Probe design principles were: 1) select probes with a target content between 30% and 80%; 2) select probes with ≤5 homology regions; and 3) select probes that do not contain SSR or N regions.

[0017] Step 3: Select probes containing high-quality mSNPs and optimize probes by multiple sequencing and hybridization of probes

[0018] Probes were sequenced and hybridized to test the genotyping quality of mSNPs within the target locus. Standard criteria for screening mSNPs were set as NA < 0.1, minimum allele frequency (MAF) >= 0.05, and heterozygosity (Het) < 0.5. Unsatisfactory mSNPs were removed. If a probe did not contain an mSNP that met genotyping quality control requirements, the probe and the target locus SNP were deleted. mSNPs that met quality control requirements were ultimately selected as 10K mSNP liquid phase array sites.

[0019] The probe designed in step 3 of the method helps to ensure that the number and quality of mSNP markers (including the target site) detected are optimized under the same target site SNP.

[0020] According to the aforementioned probe and target site SNP quality requirements (missing frequency NA < 0.1, minimum allele frequency MAF >= 0.05, heterozygosity Het < 0.5), 1089 target site SNPs on the 9K solid-phase array did not meet the requirements and were deleted. The newly added 2443 target site SNPs and mSNPs met the requirements. The present invention ultimately includes 10,200 target site SNPs and 15,572 probes.

[0021] From the above, it can be seen that the target site SNPs screened by the present invention are optimized and updated based on 9K. The target site SNPs with low probe quality and small number of mSNP markers in the upstream and downstream intervals of the probe in the pig 9K solid-phase SNP chip are eliminated, and new target sites are added in the interval where the marker spacing in the pig 9K solid-phase SNP chip is greater than 280KB.

[0022] In the second aspect, based on the 15,572 probes designed in the first aspect, the present invention mixes the synthesized pig 10K probes in equal molar mass, and uses a mixture of EDTA and Tris-HCl to adjust the volume to 3 pmol / mL of the pig 10K probe mixture for subsequent probe sequencing and hybridization, which is called an mSNP liquid phase chip.

[0023] Step 1: Probe preparation: The porcine 10K probes synthesized above were mixed in equal molar masses and fixed to a porcine 10K probe mixture of 3 pmol / mL using a mixture of EDTA and Tris-HCl.

[0024] Step 2: Preparation of probe hybridization solution. Based on the library type, the present invention uses Pooled, barcoded library, GenoBaits Block I, GenoBaits Block II for ILM / MGI, and the probe of the present invention to prepare pig 10K probe hybridization solution for subsequent DNA library hybridization capture.

[0025] Thirdly, the liquid phase chip of the second aspect is used to detect the individual genotypes of pigs.

[0026] The method comprises the following steps:

[0027] Step 1: Obtain pig samples and extract genomic DNA;

[0028] Step 2: Construction of pig cDNA library;

[0029] Step 3: hybridizing and sequencing the library using the probe of the present invention;

[0030] Step 4: According to the sequencing data operation process, mSNP genotyping is performed to complete the genotyping of all liquid phase chip marker sites of the individual.

[0031] Compared with the existing technology, the development of the pig 10K liquid phase chip provided by the present invention has the following beneficial effects:

[0032] This invention provides a porcine SNP 10K liquid-phase microarray probe for genotyping based on targeted capture sequencing. The probe design considers the genome-wide distribution of captured SNPs, site polymorphism, and mSNP marker quality. The target site MAF requirement is greater than 0.35 in Duroc, Landrace, and Large White pig populations, effectively avoiding the issues of uneven marker density and poor polymorphism that can result from simplified genome sequencing-based genotyping techniques.

[0033] This method addresses linkage disequilibrium between mSNP markers (1-4) within a probe at the same target locus. While maintaining the fundamental principles of liquid-phase microarray design, it generates more mSNPs with high typing quality and moderate linkage disequilibrium with target SNPs within the probe region. This increases the overall polymorphism of SNP markers within the probe region, expanding the number of detectable SNPs to 1.5-2 times the number of loci. This solves the problem of limited high-quality and effective mSNP markers in conventional liquid-phase microarrays without increasing costs.

[0034] The present invention helps reduce the cost of pig genomic selection while maintaining the accuracy of genomic selection. Compared to the current mainstream 50K chip, the detection fee of the present invention is 1 / 3 of that of the 50K solid-phase chip and 1 / 2 of that of the 50K liquid-phase chip. The present invention takes into account compatibility with 50K solid-phase chips and 50K liquid-phase chips. Through genotype filling technology, the genotype data of the 10K liquid-phase chip can be filled into the 50K chip with high accuracy. The filled genotype data is used for genomic selection, which can achieve the same genomic selection accuracy as the 50K chip, greatly saving the cost of molecular breeding and can be used for genomic breeding on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of solid-phase chip and liquid-phase chip technology.

[0036] Figure 2 Distribution of the number of pig 10K mSNP liquid microarray target sites (dark gray) and mSNPs (light gray) on each chromosome.

[0037] Fig. 3 Chromosome density distribution of the 10K mSNP liquid microarray target site (A) and all mSNPs including the target site (B).

[0038] Figure 4. Number of SNP and mSNP markers at target sites of 10K liquid phase array after genotype quality control.

[0039] Figure 5 Distribution of target sites (A) and all mSNPs (including target sites) (B) on each chromosome after quality control.

[0040] Figure 6. Filling accuracy of different chromosomes (A) and MAF intervals (B) when 10K liquid-phase array genotypes were filled to 50K. DETAILED DESCRIPTION

[0041] The following embodiments of the present invention are described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0042] Example 1: SNP marker screening method and probe preparation required by the present invention

[0043] Step 1: Target site SNP marker screening.

[0044] (1) Preliminary screening of target site SNP markers. Referring to the method described in ZL201711190317.6, based on the whole genome sequencing data of three pig breeds, Duroc, Large White, and Landrace, and aligned to the pig 11.1 reference genome, markers were selected based on marker spacing and gene function annotation to screen for targeted capture sites, namely target site SNPs. The principles of target site SNP screening are: ① uniform distribution on each chromosome and dense distribution at both ends of each chromosome; ② marker spacing greater than 280Kb; ③ important candidate sites for growth, reproduction, feed conversion, meat quality, and body size traits; ④ QTLdb database comparison, SNP markers should fall as much as possible in the QTL region of economic traits. As described in ZL201711190317.6, 8846 markers were obtained as target sites.

[0045] (2) Target site SNP supplementation and optimization

[0046] On the basis of the initial screening, markers with high polymorphism and moderate regional linkage disequilibrium were supplemented and optimized according to the following principles: ① Polymorphism was mainly considered in Duroc, Landrace, and Large White pigs with MAF>0.35; ② Average linkage disequilibrium with upstream and downstream SNP markers (r 2 ) is lower than 0.85.

[0047] In the area where the marker spacing of 9K solid-phase chip is greater than 280Kb (the average spacing of 9K solid-phase chip is 279Kb), markers that meet the above SNP principles are selected to supplement and optimize the target site SNPs, and a total of 2243 target site SNPs that meet the requirements are added.

[0048] Step 2: Design probes based on the target site SNP.

[0049] Probes were optimized and designed using multiplex single nucleotide polymorphism (SNP) detection technology. One to four 110bp probes were designed, each covering the target site. The total probe coverage, centered around the target site, was 165bp. Probe design principles were: 1) select probes with a target content between 30% and 80%; 2) select probes with ≤5 homology regions; and 3) select probes that do not contain SSR or N regions.

[0050] Step 3: Through multiple sequencing and hybridization of probes, select probes containing high-quality mSNPs and optimize the probes.

[0051] Probes were hybridized and sequenced to assess the genotyping quality of the target SNP and mSNPs within the coverage of upstream and downstream probes. mSNPs were screened using criteria such as missingness (NA) < 0.1, minimum allele frequency (MAF) >= 0.05, and heterozygosity (Het) < 0.5. Unsatisfactory mSNPs were removed. If a probe did not identify an mSNP that met genotyping quality control requirements, the probe and the corresponding target SNP were deleted. SNPs that met these requirements were ultimately selected as target sites for the 10K mSNP liquid phase array.

[0052] The pig 9K solid phase chip adopts solid phase chip technology, which only considers the genotype detection quality of the target site SNP, and does not consider the quality of the mSNP upstream and downstream of the target site SNP. 1089 target sites did not meet the probe quality and mSNP screening standards and were deleted. The newly added 2443 target site SNPs all met the probe and mSNP quality requirements. The pig 10K mSNP liquid phase chip developed by the present invention has 10,200 target sites, and 15,572 probes are designed. Table 1 shows the number of target site SNPs deleted and newly added on the 9K solid phase chip on each chromosome. In addition, the present invention adopts multi-single nucleotide polymorphism technology. There are multiple mSNPs within the 200bp region of each target site SNP. The 15,572 probes produce a total of approximately 20,580 high-quality mSNPs. The distribution of target sites and mSNP sites (including target sites) on each chromosome is shown in Figure 2.

[0053] Table 1 Number of SNPs at deleted and newly added target sites on 9K solid-phase microarray on each chromosome

[0054] Example 2: Preparation of 10K liquid phase chip

[0055] This example is used to demonstrate the preparation process of the 10K liquid phase chip of the present invention. The details are as follows:

[0056] Step 1: Probe preparation

[0057] According to Example 1, the porcine 10K probe was synthesized and mixed in equal molar mass. The volume was fixed with a mixture of EDTA and Tris-HCl (TE buffer) to prepare a 3 pmol / mL porcine 10K probe mixture for subsequent sequencing and hybridization.

[0058] 1. Preparation of TE buffer

[0059] 1×TEBuffer

[0060] Composition concentration: 10mMTris-HCl 1mM EDTA, pH = 8.0

[0061] Preparation volume: 500ml

[0062] Preparation method: Measure the following solution into a 500ml beaker

[0063] 1M Tris-HCl Buffer PH=8.0,5ml

[0064] 0.5M EDTA PH=8.0, 1ml

[0065] Add approximately 400 ml of dd H2O to the beaker and mix thoroughly. After diluting the solution to 500 ml, sterilize it by high-temperature and high-pressure sterilization; store it at room temperature. 2. Use the prepared TE buffer to dilute the porcine 10K probe to a 3 pmol / mL porcine 10K probe mixture for subsequent sequencing and hybridization.

[0066] Step 2: Preparation of probe hybridization solution

[0067] 1. Based on the library type, mix the following reagents in a 1.5 mL PCR tube. Composition and volume:

[0068] 2. Use a vacuum concentrator to concentrate to dryness at a temperature of ≤60°C;

[0069] 3. After concentration is completed, centrifuge at 12000 rpm for 1 min. The prepared probe can be stored at room temperature (15-25°C) overnight for subsequent DNA library hybridization capture.

[0070] Example 3: Use and detection method of 10K liquid phase chip

[0071] This example is used to demonstrate the operational process of genotype detection using the 10K liquid phase chip of the present invention. The details are as follows:

[0072] Step 1: Obtain pig samples and extract genomic DNA.

[0073] We selected 133 Large White and Landrace pigs from a certain pig farm and extracted genomic DNA from their ear tissues. The specific method is as follows:

[0074] 1. Cut the pig ear tissue dehydrated with an appropriate amount of ethanol into small pieces and place them in a 96-deep-well plate (if the amount is small, a 2.0 mL centrifuge tube can be used), add a 5 mm steel ball, freeze it in liquid nitrogen, and grind it in a grinder for 1-2 minutes.

[0075] 2. Add 500 μL of Buffer PL2 and 5 μL of Proteinase K (the diluted Proteinase K currently used in the laboratory) to the deep-well plate. Secure the lid tightly and oscillate using a shaker to mix thoroughly.

[0076] 3. Place in a 65°C water bath for 30 minutes. Take out in the middle of the water bath and mix thoroughly by inversion.

[0077] 4. Add 500uL of phenol chloroform isoamyl alcohol to the deep-well plate, mix thoroughly by pipetting up and down with an oscillator or pipette, and let it stand for 5 minutes.

[0078] 5. Centrifuge at 4000 rpm for 10 minutes and transfer 400 μL of the supernatant to a new 96-well deep-well plate. (Be careful not to aspirate the precipitate in the middle.)

[0079] 6. Add 800uL PW solution and mix thoroughly.

[0080] 7. Transfer the supernatant from step 6 into 96-well centrifuge columns twice and vacuum filter.

[0081] 8. Add 600 μL of WBI to a 96-well spin column, let stand at room temperature for 2 minutes, and then vacuum filter. (Make sure the volume of anhydrous ethanol specified on the bottle has been added to the WBI.)

[0082] 9. Add 600 μL of WB II to a 96-well spin column and vacuum filter. (Make sure the volume of anhydrous ethanol specified on the bottle has been added to the WB II.)

[0083] 10. Add 600uL WB II to the 96-well spin column and vacuum filter.

[0084] 11. Place the 96-well spin column in an empty collection plate and centrifuge at 4000 rpm for 5 minutes. Place the 96-well spin column in a new 96-well PCR plate and allow to dry at room temperature.

[0085] 12. Add 60-100 μL of 65°C preheated TE to a 96-well centrifuge column, let it stand at room temperature for 2 minutes, and centrifuge at 4000 rpm for 5 minutes (preheating TE to 65°C will help improve DNA elution efficiency and detect the length of the target fragment by gel electrophoresis).

[0086] Step 2: Construction of pig cDNA library.

[0087] 1. Probe mixture

[0088] a) Prepare the following reaction in a PCR tube using the reagent of the present invention: 1 ng-200 ng DNA, 4 μL GenoBaits end repair buffer, 3.1 μL GenoBaits end repair enzyme, and add nuclease-free water to 20 μL.

[0089] b) Gently mix the reaction system and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0090] c) Place the reaction tube in a PCR instrument and perform the following reaction: 37°C for 20 minutes, 72°C for 20 minutes, and then hold at 4°C. Once completed, set the heated lid to 82°C:

[0091] 2. Connector connection

[0092] a) Add the following components directly to the reaction system in step 1: 2 μL of GenoBaits ULtra DNA Ligase, 8 μL of GenoBaits ULtra DNA Ligase Buffer, 2 μL of GenoBaits Adapter for MGI, and 8 μL of nuclease-free water to a total volume of 20 μL.

[0093] b) Gently mix the reaction system and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0094] Note: The system must be thoroughly mixed, otherwise library construction may fail.

[0095] c) Place the reaction tube in a PCR instrument and perform the following reaction program: 22°C for 60 minutes, hold at 4°C. Remove the heated lid after completion:

[0096] 3. DNA Purification

[0097] a) Remove the GenoPrep DNA Clear Beads in advance, equilibrate at room temperature for at least 30 minutes, and vortex to mix before use.

[0098] b) Add 48 μL of GenoPrep DNA Clear Beads to the ligation system in step 2, vortex to mix thoroughly, avoiding bubbles. Let stand for 5 minutes, then briefly centrifuge to collect the liquid at the bottom of the tube.

[0099] c) Place on a magnetic stand for at least 3 minutes until the solution is clear, and remove the supernatant.

[0100] d) While the PCR tube is still in the magnetic rack, add 100 μL of 80% ethanol. Incubate at room temperature for 30 seconds and remove the supernatant.

[0101] e) Place the PCR tube in the magnetic rack with the lid open and allow to air dry for 5 minutes until the ethanol evaporates.

[0102] f) Remove the PCR tube from the magnetic stand and resuspend the magnetic beads using the PCR system from step 4.

[0103] 4. Library Amplification

[0104] When the starting amount is 1ng-10ng, the recommended number of amplification cycles is 8; when the starting amount is 10ng-100ng, the recommended number of amplification cycles is 6-8; when the starting amount is 100ng and above, the recommended number of amplification cycles is 6.

[0105] a) Prepare the following reaction in a new tube: 10 μL of GenoBaits PCR Master Mix, 1 μL of I5 Barcode (10 μm)-MGI, 5 μL of I7 Barcode (2 μm)-MGI, and add 4 μL of nuclease-free water to a total volume of 20 μL.

[0106] b) Add the above system to the magnetic beads dried in step 3, resuspend the beads, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0107] c) Place the reaction tube in a PCR instrument and perform the following reactions:

[0108] 5. Purification

[0109] a) Add 20 μL of GenoPrep DNA Clear Beads to the tube in step 4. Vortex to mix thoroughly, avoiding bubbles. Let stand for 5 minutes, then centrifuge briefly to collect the liquid at the bottom of the tube.

[0110] b) Place on a magnetic stand for at least 3 minutes until the solution is clear, and remove the supernatant.

[0111] c) While keeping the PCR tube in the magnetic rack, add 100 μL of 80% ethanol. Incubate at room temperature for 30 seconds and remove the supernatant.

[0112] d) Place the PCR tube in the magnetic rack with the lid open and allow to air dry for 10 minutes.

[0113] e) Remove the PCR tube from the magnetic rack, add 35 μL of Tris-HCl, vortex to mix, let stand for 5 minutes, and briefly centrifuge to collect the liquid at the bottom of the tube.

[0114] f) Place on a magnetic rack and wait for the solution to clarify (approximately 3 minutes). Transfer the supernatant to a fresh tube and store at -20°C.

[0115] g) The library needs to undergo further quality testing (such as concentration and distribution determination) before it can be used for the machine or the next experiment.

[0116] Step 3: hybridize the pig genome fragment with the probe of the present invention, perform PCR on the sample, purify it, and sequence it.

[0117] 1. Melt the mixed probe at room temperature (15-25°C), mix well and centrifuge briefly. 2. GenoBaitsBLock II, GenoBaitsBLock

[0118] a) Depending on the library type, the following reagents were mixed in a 1.5 mL PCR tube: 0.6-1 μg of mixed library, 5 μg (5 μL) of GenoBaitsBLock I, 2 μL of GenoBaitsBLock II for ILM / MGI, and 300 ng of the probe of the present invention.

[0119] b) concentrating to dryness using a vacuum concentrator at a temperature of ≤60°C;

[0120] c) After concentration is complete, centrifuge at 12,000 rpm for 1 minute before proceeding with subsequent operations.

[0121] 3. DNA library hybridization capture

[0122] a) Dissolve all GenoBaits hybridization reagents at room temperature;

[0123] b) adding reagents to the test tube;

[0124] c) Mix by pipetting or vortexing, centrifuge at 12,000 rpm for 1 min, let stand at room temperature for 5 min, mix again by pipetting or vortexing, centrifuge briefly, and transfer the entire mixture to a 0.2 mL EP tube;

[0125] d) Thermal cycle incubation conditions: 95°C for 10 min (heated lid temperature: 105°C);

[0126] e) When the PCR amplification instrument cools down to 65°C, transfer the cells to another PCR instrument with a heated lid at 75°C. Note: If the experiment requires, hybridization can be performed overnight at 65°C (14-16 hours). *65°C hybridization temperature helps improve capture efficiency.

[0127] 4. Prepare Wash Buffer

[0128] For a single capture system, dilute GenoBaits buffer to a 1× solution.

[0129] *1× system can be stored at room temperature for 4 weeks

[0130] *10× Wash Buffer I and 10× SW Buffer will form a precipitate at room temperature. The precipitate can be dissolved by storing at 65°C.

[0131] 5. Prepare GenoBaits DNA Probe Beads

[0132] a) GenoBaits Probe Beads were placed at room temperature for 10 minutes before use.

[0133] b) Vortex and mix for 15 seconds;

[0134] c) Prepare 50 μL of GenoBaits Probe Beads for one reaction and place them in a 0.2 mL EP tube;

[0135] d) Place the test tube on a magnetic rack to allow the magnetic beads to completely separate from the solution.

[0136] e) Remove the supernatant and retain the magnetic beads

[0137] f) Elution: Add 150 μL of GenoBaits 1X Bead Wash Buffer to each reaction, vortex for 10 seconds, transfer the tube to a magnetic rack, completely separate the beads from the solution, and discard the supernatant.

[0138] g) Repeat step 6 above twice, for a total of 3 washes.

[0139] 6. Hybridization fragments bind to GenoBaits DNA Probe Beads

[0140] a) Transfer 16 μL (all) of the hybridization solution to the prepared beads

[0141] b) Vortex and mix thoroughly for 10 seconds, and centrifuge for 5 seconds.

[0142] c) Place the EP tube in a PCR instrument, 65°C, 45 min, with the heated lid at 75°C (DNA binds to the magnetic beads)

[0143] Every 12 minutes, shake for 5 seconds

[0144] 7. Elute to remove unbound DNA (using 1× Wash Buffer in step 4)

[0145] a) Prepare 65°C elution buffer (done on a PCR instrument)

[0146] b) Prepare the room temperature eluent

[0147] c) Resuspend the magnetic beads and use the suspension in step 8, leaving 10 μL for backup.

[0148] 8. PCR enrichment

[0149] a) Prepare PCR reagents in a 0.2 mL PCR tube according to the library type.

[0150] b) Vortex briefly and centrifuge to ensure the beads remain in solution

[0151] c) Place the PCR tube in a PCR instrument with a heated cover at 105°C for PCR amplification.

[0152] 9. PCR product purification

[0153] a) Add 45 μL (1.5X volume) to each PCR reaction

[0154] GenoPrep DNA Clean Beads, vortex to mix, try not to produce bubbles, let it stand for 5 minutes, and then centrifuge briefly to collect the liquid at the bottom of the tube.

[0155] b) Place on a magnetic stand for at least 3 minutes until the solution is clear, and remove the supernatant.

[0156] c) While keeping the PCR tube in the magnetic rack, add 100 μL of 80% ethanol. Incubate at room temperature for 30 seconds and remove the supernatant.

[0157] d) Place the PCR tube in the magnetic rack with the lid open and allow to air dry for 10 minutes.

[0158] e) Remove the PCR tube from the magnetic rack, add 35 μL of Tris-HCl, vortex to mix, let stand for 5 minutes, and briefly centrifuge to collect the liquid at the bottom of the tube.

[0159] f) Place on a magnetic rack and wait for the solution to clarify (approximately 3 minutes). Transfer the supernatant to a fresh tube and store at -20°C.

[0160] g) The library needs to undergo further quality testing (such as concentration and distribution determination) before it can be used for the machine or the next experiment.

[0161] 10. Library detection

[0162] a) Measure the library using the Qubits FLuorometer and Qubit dsDNA HS Assay Kit

[0163] b) Measure the average length of captured DNA library fragments on the digital electrophoresis system

[0164] c) Measure library concentration using the KAPA Library Quantification Kit

[0165] 11. Sequencing

[0166] Follow the sequencer's instructions. The average sequencing depth for the target SNP was 105.88X.

[0167] Step 4: mSNP genotyping

[0168] Obtain the genotypes of all mSNP sites according to the sequencing data processing process. The specific steps are as follows:

[0169] 1. Use Trimmomatic software to remove adapters and low-quality reads.

[0170] 2. Use BWA software to align the reads of each individual to the pig reference genome Sscrofa11.1 (GCA_000003025.6);

[0171] 3. Use SAMtools to generate BAM and sorted BAM files;

[0172] 4. Use the GATK pipeline to generate a VCF file containing all mSNPs (including target site SNPs).

[0173] Example 3 demonstrates the operational procedures for using the liquid phase chip of the present invention, and Example 4 evaluates the quality of liquid phase chip genotyping detection on pig farm samples.

[0174] Example 4: Quality Assessment of Pig 10Km SNP Liquid Chip Genotyping

[0175] Blood samples were collected from 133 Landrace and Large White pigs in a pig farm. Genotyping was performed using the present invention as in Example 3 to evaluate the quality of the mSNP markers of the present invention.

[0176] 1. Target site and mSNP marker number statistics

[0177] Based on the statistical results of 10K chip genotyping of 133 pig samples, the actual measurement, as shown in the present invention, shows that the 10KmSNP liquid phase chip has a total of 10,200 target SNP sites. After genotyping, a total of 20,585 SNPs were found, including 10,200 target site SNPs and 10,385 mSNP markers for their probes. This shows that the mSNP liquid phase chip designed by the present invention can significantly increase the number of SNPs, and compared with solid-phase chips that only consider target sites, it can significantly increase the number of effective SNP markers without increasing sequencing costs.

[0178] 2. Distribution density statistics of target sites and mSNP markers on each chromosome

[0179] Figures A and B in Figure 3 show the chromosomal distribution of the target site on the 10K liquid-phase array and the distribution density of mSNPs (including the target site SNP) on each chromosome. The two distributions are similar. The mSNP only increases the density of SNPs based on the target site SNPs and does not change the overall SNP distribution. This demonstrates that in practical applications, mSNP detection conforms to the theoretical characteristics of multiple single nucleotide polymorphism detection technology, thereby improving the efficiency of capturing effective mSNP markers.

[0180] 3. Missing rate and MAF statistics of target sites and mSNP markers

[0181] The genotype data of 133 samples showed that there was almost no difference in the detection rate and minimum allele frequency (MAF) between the 10,200 target sites in the mSNP liquid phase array and the additional 10,385 mSNPs (Table 2), indicating that the increase in the number of SNPs in the mSNP liquid phase array does not reduce the quality of the array data.

[0182] Table 2 Genotype quality of target site SNP and mSNP

[0183] Note: The standard deviation is in brackets

[0184] 4. After genotype quality control, the number of target sites and mSNP markers was counted;

[0185] Genotype quality control is a routine operation after high-throughput genotyping, mainly used to eliminate markers and individuals with low genotype detection quality for subsequent genetic analysis or molecular breeding. The quality control steps are usually as follows:

[0186] a) Remove sites with unknown positions

[0187] b) Remove SNPs with a call rate lower than 90% or a missing rate higher than 10%;

[0188] c) removing SNPs with a minimum allele frequency (MAF) lower than 0.05;

[0189] d) Remove the samples that seriously deviate from Hardy-Weinberg equilibrium (P<10 -6 ) SNP.

[0190] After quality control, some SNPs were removed from the target site SNPs and all mSNP markers (including the target site) of the 10KmSNP liquid phase array. After quality control, a total of 16,001 mSNP markers were found, including 8,423 target sites (Figure 4). For target sites that did not meet quality control requirements, the mSNPs within their probes also did not meet quality control requirements. After quality control, the number of mSNP markers was still approximately twice that of target sites, essentially the same as before quality control. The density distribution of target sites and all mSNP markers on each chromosome did not change significantly (Figure 5, A and B), consistent with the distribution of the 10K target sites before quality control (Figure 3, A and B). This indicates that after quality control, mSNP can significantly increase the number of SNPs detected without compromising data quality, which helps preserve more genetic variation.

[0191] This invention is primarily intended to reduce the cost of pig molecular breeding. Fewer markers means lower costs. The testing fee for this invention is one-third that of a 50K solid-phase chip and one-half that of a 50K liquid-phase chip (ZL202110359470.7). While genomic selection is still ideal with 50,000 markers, 10K liquid-phase chips are primarily populated with genotype data from a 50K chip through genotype populating technology. Example 5 illustrates the accuracy of genotype populating from a 10K chip to a 50K chip and the effectiveness of genomic selection.

[0192] Example 5: 10K mSNP liquid phase array genotype filling into 50K array and genome selection effect

[0193] The present invention performed genotyping detection on the above 133 individuals using the liquid chip of the present invention, and simultaneously performed genotyping detection using the invented 50K liquid chip (ZL202110359470.7) already obtained by the applicant, so that each individual had pig liquid 10K and liquid 50K genotyping data.

[0194] Theoretical research and breeding practice show that the 50K chip takes into account both genomic selection effect and breeding cost and is the most widely used. In order to ensure the genomic selection effect of the 10K chip, it is necessary to utilize genotype filling technology to fill the 10K chip data into the 50K chip genotype data. Therefore, it is necessary to construct a 50K chip population of a certain scale as a filling reference population, and then perform 10K chip filling based on this population. This embodiment uses 4126 individuals accumulated from previous studies as a reference population, including three varieties of Changbai, Dabai and Duroc, and is detected using a 50K liquid phase chip. It should be noted that the genotype filling reference population does not need to be too large, and usually 4000 can ensure high filling accuracy. 133 10K liquid phase chip individuals were filled into the 50K liquid phase chip according to the operating instructions using the classic genotype filling software Beagle4.1. Since these 133 individuals also underwent 50K liquid phase chip testing (original 50K chip genotype), the correlation coefficient between the 50K chip genotype filled at each locus of the 133 samples and the original 50K chip genotype was calculated as a measure of filling accuracy. As shown in Figure 6, the average accuracy of the genotype filling at all loci on all chromosomes was 0.964 (Figure 6). When the marker MAF was lower than 0.05, the filling accuracy was low, and these markers were generally removed during genotype quality control. Therefore, after genotype quality control, the accuracy of the 10K liquid phase chip filling to the 50K liquid phase chip will be further improved to 0.98. Since the proportion of markers with filling errors is not high, it has no effect on the genomic selection accuracy of the 50K liquid phase chip.

[0195] On the basis of filling 133 10K chips into 50K chips, this embodiment further selected a 10K liquid phase chip of a larger group size, filled it into a 50K liquid phase chip, and evaluated the effect of genomic selection. The embodiment selected 2991 large white individuals from the same pig farm, of which 1199 individuals contained growth trait (age at 100 kg body weight, back fat thickness of 100 kg body weight) phenotypic records, 804 had reproductive trait (total number of offspring) phenotypic records, and a total of 42281 pedigree records. All individuals were detected using the above-mentioned 50K liquid phase chip and the 10K liquid phase chip of the present invention at the same time. The 10K liquid phase chip was filled with the 50K chip genotype data using the above-mentioned 4126 50K liquid phase chip reference group. Validation cohorts of 198 and 108 youngest individuals from populations with recorded growth and reproductive phenotypes were selected, respectively. The remaining population served as a reference cohort. Genomic breeding values ​​for three traits were estimated using the classic one-step GBLUP (ssGBLUP) method. Pearson correlation coefficients between the trait-corrected phenotypic values ​​of validation cohort individuals and their estimated genomic breeding values ​​were used to assess genomic selection accuracy. The results, as shown in Table 3, showed no difference in genomic selection accuracy for the three traits using the filled core 50K array and the original 50K array. This suggests that genomic selection using only a 10K liquid phase array and then filling the array to a 50K liquid phase array achieves the same results as using only the 50K liquid phase array.

[0196] Table 3. Genome selection accuracy of 10K liquid phase chip filled to 50K liquid phase chip

[0197] During the implementation of genomic selection, a large number of individual genotype tests are required. If a 50K liquid phase chip is used, the cost of genotype testing is very high. However, through genotype filling, only a certain scale of individuals need to be tested with a 50K liquid phase chip for genotype filling of the reference group, and the 10K chip of the present invention is used more frequently. The present invention achieves the purpose of significantly reducing the cost of genomic selection molecular breeding by using a 10K liquid phase chip without reducing the accuracy of genomic selection through genotype filling technology. This solves a bottleneck in my country's pig molecular breeding, thereby enabling large-scale implementation of molecular breeding and becoming a booster for the revitalization of my country's seed industry.

Claims

1. A method for SNP marker screening and probe preparation for a 10K liquid phase chip for multiple single nucleotide polymorphisms, which uses whole genome sequencing data of pig breeds to mine and screen target site SNPs, then designs and optimizes probes for the target site SNPs, and finally obtains a determined probe, the method comprising the following steps: Step 1: Determine the target site SNP: The target site SNP was screened and obtained as the confirmed target site SNP by the following method: (1) Preliminary screening of target site SNP markers: based on the whole genome sequencing data of three pig breeds, Duroc, Large White and Landrace, aligned to the pig 11.1 reference genome, markers were selected according to marker spacing and gene function annotation to screen the targeted capture site, i.e., target site SNP. The principles of target site SNP screening are: ① uniform distribution on each chromosome and dense distribution at both ends of each chromosome; ② marker spacing is greater than 280Kb; ③ important candidate sites for growth, reproduction, feed conversion, meat quality, and body size traits; ④ QTLdb database comparison, SNP markers are placed in the QTL region of economic traits as much as possible; the markers obtained in this way are used as target site SNPs; (2) Target site SNP supplementation and optimization: Based on the target site SNP obtained in the initial screening step, markers with high polymorphism and medium regional linkage disequilibrium are supplemented and optimized. The principles are as follows: ① Polymorphism mainly considers MAF>0.35 in Duroc, Landrace, and Large White pigs; ② The average linkage disequilibrium degree with upstream and downstream SNP markers r 2 Less than 0.85; Step 2: Design probes based on the determined target site SNP: Using the multiple single nucleotide polymorphism detection technology, with each target site SNP as the core, design 1-4 110bp length probes, each probe covers the target site, and the probes with the target site as the core cover a total length interval of 165bp; The principles of probe design are: 1) select probes with a content between 30% and 80%; 2) select probes with a number of homology regions ≤ 5; 3) select probe regions that do not contain SSR or N regions; Step 3. Select probes containing high-quality mSNPs and optimize the probes: hybridize and sequence the probes to detect the quality of mSNP genotyping including the target site. Set the missing rate NA < 0.1, the minimum allele frequency MAF > = 0.05, and the heterozygosity Het < 0.5 as the standard for screening mSNPs and remove mSNPs that do not meet the requirements. If the probe does not have an mSNP that meets the standard requirements, delete the probe and the target site. The mSNP that meets the quality inspection requirements will eventually be used as the target site for the 10K mSNP liquid phase chip.

2. The method according to claim 1, characterized in that The whole genome sequencing data of the pig breed is the 11.1 reference genome; the principles of the target site SNP screening are: ① uniform distribution on each chromosome and dense distribution at both ends of each chromosome; ② polymorphism considering MAF>0.35 in Duroc, Landrace pigs, and Large White pigs; ③ the average linkage disequilibrium degree with upstream and downstream SNP markers r 2 Lower than 0.85; ④ Compared with the QTLdb database, the SNP markers should fall into the QTL region of economic traits as much as possible; ⑤ Overlap with some sites of the known pig 50K chip.

3. The method according to claim 2, characterized in that The known pig 50K chip is the 50KSNP liquid phase chip, the Neogen GGP50K or the SMIC-1.

4. A pig 10KmSNP liquid phase chip based on multiple single nucleotide polymorphisms, characterized in that: The method is prepared based on the probe obtained by the method according to any one of claims 1 to 3.

5. The pig 10KmSNP liquid phase chip according to claim 4, characterized in that: After the probes are synthesized, they are mixed in equal molar mass, the volume of the buffer is fixed to 1-5 pmol / mL, and then the probe hybridization solution is prepared.

6. The pig 10KmSNP liquid phase chip according to claim 5, characterized in that: The buffer solution is a mixture of EDTA and Tris-HCl.

7. The pig 10KmSNP liquid phase chip according to claim 5, characterized in that: The method further comprises using Pooled, barcoded library, GenoBaits Block I, GenoBaits Block II for ILM / MGI to prepare a probe hybridization solution, the composition of which is as follows:

8. The pig 10KmSNP liquid phase chip according to claim 7, characterized in that: The probe hybridization solution was concentrated to dryness using a vacuum concentrator at a temperature of ≤60°C.

9. A method for detecting individual genotypes of pigs using the pig 10KmSNP liquid phase chip according to any one of claims 4 to 8, the method comprising the following steps: Obtaining samples from the tested pigs and extracting genomic DNA; Construction of pig cDNA library; hybridization and sequencing of the constructed library with the pig 10KmSNP liquid phase chip; According to the sequencing data operation process, mSNP genotyping is performed to complete the genotype determination of all liquid phase chip marker sites of the individual.

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