Donkey 40k liquid chip based on targeted capture sequencing and application thereof

Abstract

The application discloses a donkey 40K liquid phase chip based on targeted capture sequencing and application thereof. The donkey 40K liquid phase chip is composed of independently packaged probe mixed solution and hybridization capture reagent, wherein the probe mixed solution comprises capture probes designed and synthesized according to 45894 SNP sites screened, and the density of the donkey 40K liquid phase chip can be reduced to low density 1K or can be expanded to high density 200K by flexible adjustment. The problem of lack of donkey high-throughput SNP chip caused by uneven marker density and poor polymorphism is solved, and an efficient genomic typing approach is provided for marker-assisted selection research and application of donkeys.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, specifically to a donkey 40K liquid phase chip based on genotyping by targeted sequencing (GBTS) technology and its application in donkey genotyping. Background Technology

[0002] Single nucleotide polymorphisms (SNPs) are characterized by their large number, wide distribution, ease of rapid and large-scale screening, and convenient genotyping. They represent the third generation of genetic markers, following first-generation restriction fragment length polymorphism (RFLP) markers and second-generation microsatellite markers (i.e., short tandem repeat markers), and hold significant biological importance. Currently, SNP markers have become crucial tools for biological population identification, genetic structure analysis, functional gene mapping, and genome selection. With the development of high-throughput SNP genotyping technology, methods based on whole-genome or simplified genome sequencing have become the mainstream SNP genotyping techniques. For species with reference genome sequences, researchers typically design SNP chips using known sequences. By hybridizing randomly fragmented genomic DNA fragments with oligonucleotide probes on the chip, the genotype of the corresponding SNP locus can be determined. Currently, there are no mature commercial SNP chips for donkeys. Therefore, a genotyping technology is needed to reduce the manufacturing cost of high-throughput SNP chips and to allow for the customization of SNP markers based on specific population conditions, thereby achieving rapid, cost-effective, and convenient genotyping.

[0003] Targeted capture sequencing genotyping (GBTS) is a technique that achieves deep resequencing only at target sites by reducing library abundance, which can significantly reduce the cost of genotyping. However, this technique has not yet been applied to donkeys. Simplified genome sequencing genotyping techniques suffer from uneven marker density and poor polymorphism, hindering the application of high-throughput SNP genotyping technology in basic research and molecular breeding in donkeys. Summary of the Invention

[0004] The purpose of this invention is to provide a donkey 40K liquid phase chip based on targeted capture sequencing and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A donkey 40K liquid phase chip based on targeted capture sequencing is disclosed. The donkey 40K liquid phase chip consists of individually packaged probe mixtures and hybridization capture reagents. The probe mixtures include capture probes designed and synthesized according to each SNP site (genotyping target) being screened. The density of the donkey 40K liquid phase chip can be flexibly adjusted to reduce it to a low density of 1K (the specific locations of 1000 SNP sites on the donkey reference genome in EquAsi 1.0 version are shown in Table II below) or expanded to a high density of 200K.

[0007] Preferably, the genotyping targets of the donkey 40K liquid phase chip also include other SNP sites located in the EquAsi 1.0 version donkey reference genome and matched by capture probes designed and synthesized based on each screened SNP site (the capture probes corresponding to the above 1000 SNP sites can match the following SNP sites located in the EquAsi 1.0 version donkey reference genome: chr15:23191566, chr15:26258385, chr15:24168300, etc.).

[0008] Preferably, each SNP site selected is determined by aligning the whole genome sequencing results of donkeys to the donkey reference genome in EquAsi 1.0 and according to the following principles for selecting SNP sites: even distribution on each chromosome of the donkey reference genome, heterozygosity <15%, and good site polymorphism. The indicator of good site polymorphism is an MAF greater than 0.35 in 18 Chinese donkey breeds, 8 foreign donkey breeds, and wild donkey populations (see Table I below).

[0009] Preferably, the design principles of the capture probe include: probe length of 100-120nt, probe GC content of 30%-80%, and the selected area does not include GAP areas.

[0010] Preferably, two nucleotide sequences that overlap by 60%-70% and cover the selected SNP site are designed based on the selected SNP site. Then, two DNA sequences of 100-120 nt in length with biotinylate modification at the 5' end are synthesized based on the designed nucleotide sequences. These are the capture probes corresponding to the SNP site.

[0011] Preferably, the capture probes corresponding to each screened SNP site are mixed in equal amounts and then diluted to a final volume using a mixture of EDTA and Tris-HCl to obtain the probe mixture.

[0012] A donkey genotyping method based on targeted capture sequencing includes the following steps:

[0013] S1 fabricates donkey liquid phase chips based on targeted capture sequencing, where the chip density can be flexibly adjusted. Based on the donkey 40K liquid phase chip, it can be reduced to a low density of 1K or expanded to a high density of 200K.

[0014] S2 uses the genomic DNA of the donkey to be tested to construct a high-throughput sequencing library of donkey DNA;

[0015] S3 uses the donkey liquid phase chip prepared in S1 to capture DNA fragments containing corresponding SNP sites in the donkey DNA high-throughput sequencing library constructed in S2.

[0016] S4 amplifies and purifies the DNA fragment obtained in S3. After high-throughput sequencing, the sequencing results are compared with the donkey reference genome in EquAsi 1.0 to obtain the genotyping data of the donkey to be tested.

[0017] Preferably, the method for constructing a donkey DNA high-throughput sequencing library in S2 specifically includes the following steps: fragmenting the genomic DNA of the donkey to be tested by ultrasonic breaking or enzyme digestion, and obtaining a donkey DNA high-throughput sequencing library by end-padding, adding A tails, and connecting sequencing adapters; and detecting the concentration of the donkey DNA high-throughput sequencing library using Qubit.

[0018] Preferably, the capture in S3 specifically includes the following steps: using the capture probe contained in the probe mixture to adsorb the target donkey genomic DNA fragment onto magnetic beads covered with streptavidin, and then removing the DNA fragments in the non-target region by elution.

[0019] Preferably, obtaining the genotyping data of the donkey to be tested in step S4 specifically includes the following steps: processing the high-throughput sequencing data using BWA and GATK software to obtain the genotype of the corresponding SNP site of the donkey to be tested.

[0020] The above-mentioned application of the donkey 40K liquid phase chip based on targeted capture sequencing in donkey molecular breeding.

[0021] Preferably, the SNP sites chr15:25408816, chr15:23191566, chr15:26258385 or chr15:24168300 in the genotyping object of the chip are SNP markers that are significantly associated with the coat color trait of donkeys.

[0022] The beneficial effects of this invention are reflected in:

[0023] This invention is based on the development of high-throughput SNP chips for donkeys (such as 40K liquid-phase chips based on targeted capture sequencing genotyping technology). The probe design takes into account the distribution of the captured SNP sites in the whole genome (donkey reference genome) and the need for flexible chip application. It effectively solves the problem of the scarcity of high-throughput SNP chips for donkeys caused by uneven marker density and poor polymorphism, and provides an efficient genotyping approach for genetic diversity analysis, breed identification, marker-assisted breeding research and application in donkeys.

[0024] Furthermore, this invention improves the polymorphism level of genomic loci used for probe design by analyzing genomic sample data from donkey breeds (18 Chinese donkey breeds and 8 foreign donkey breeds) and wild donkey populations from different geographical origins (MAF requirement greater than 0.35, etc.), and provides an important reference for discovering candidate SNP markers.

[0025] Furthermore, this invention designs two single-stranded DNA probes with 60%-70% sequence overlap for each SNP site (GC content controlled between 30%-80%), which can effectively improve the hybridization stability of the genomic fragment containing each SNP, thereby improving the fragment capture efficiency. At the same time, this probe design also fully considers the genomic specificity of the site flanking sequences, ensuring site-specific capture rate, reducing sequencing costs, and facilitating its application in large-scale SNP genotyping using donkey-specific markers.

[0026] Furthermore, the probe designed in this invention (selected region excluding GAP region) has a high tolerance for variations in the flanking sequences of the site. Even when the variation in the flanking sequences is no more than 10%, it can still stably capture the target fragment. In this way, in addition to obtaining the target SNP information, it can also obtain the sequence information of some upstream and downstream regions, providing more information support for donkey molecular breeding at the same cost. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of probe design based on SNP sites.

[0028] Figure 2 This is a distribution map of the number of SNP sites on different chromosomes in a donkey 40K liquid phase chip.

[0029] Figure 3 This is a diagram showing the spacing between adjacent SNP sites on a 40K liquid phase chip.

[0030] Figure 4 PCA diagrams for differentiating nine donkey breeds using a 40K liquid phase chip.

[0031] Figure 5Manhattan plot for genome-wide association analysis of coat color trait in Texas donkeys using the Donkey 40K liquid phase chip. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments described are merely illustrative of the invention and are not intended to limit the scope of protection of the invention.

[0033] (I) Donkey 40K liquid phase chip based on targeted capture sequencing

[0034] This invention aims to develop a novel high-throughput SNP chip for donkeys based on targeted capture sequencing (GBTS) using the donkey 40K liquid phase chip, thereby enabling the large-scale application of GBTS technology in basic research and molecular breeding of donkeys.

[0035] The whole genome sequencing results of donkeys (in the chip development stage, 165 Chinese domestic donkeys, 7 Kyrgyz donkeys, 17 Kenyan donkeys, 6 Nigerian donkeys, 8 Egyptian donkeys, 10 Ethiopian donkeys, 18 Spanish donkeys, 8 Iranian donkeys, 8 Australian donkeys, and 7 wild donkeys were selected, and blood samples and genomic DNA were extracted from some of the Chinese domestic donkeys; see Table I) were compared with the donkey reference genome of EquAsi 1.0 to screen for SNP sites. The principles for screening SNP sites were: (1) uniform distribution on each chromosome, heterozygosity <15%, and good site polymorphism, wherein the index of good site polymorphism was MAF greater than 0.35 in 18 Chinese donkey breeds, 8 foreign donkey breeds, and wild donkey populations; (2) the coverage area of ​​the screened SNP sites also met the set probe design principles (probe length 100-120nt, probe GC content between 30%-80%, and the selected area did not include GAP regions). For each SNP site identified through screening, two nucleotide sequences with 60%-70% overlap covering the identified SNP site were designed (e.g., ...). Figure 1 As shown in the figure, probes are then synthesized according to the designed nucleotide sequence. The two synthesized DNA single strands, each 100-120 nt in length and with a biotinylate group modified at the 5' end, are the probes used to capture the genomic fragment containing the SNP site.

[0036] Probes were designed for donkey high-throughput SNP microarrays based on targeted capture sequencing genotyping technology for a total of 45,894 SNP loci (target loci) obtained through screening. Experiments showed that the probe sequences designed based on the screened SNP loci ensured the accuracy and sensitivity of the resulting donkey 40K liquid-phase microarray. Meanwhile, Figure 2 This indicates that the SNP sites involved in the Donkey 40K liquid phase chip cover every chromosome; Figure 3This indicates that the SNP site spacing involved in the donkey 40K liquid phase chip is reasonable (averaging about 40kb, which is roughly the same as the SNP site spacing on each chromosome).

[0037] Table I. Sources of Genomic Data for Donkey Breeds and Wild Donkey Populations in China and Abroad

[0038]

[0039]

[0040] Note: [1]Kyrgyz; [2]Kenya; [3]Nigeria; [4]Egypt; [5]Ethiopia; [6]Spain; [7]Iran; [8]Australia

[0041] Compared with the mainstream solid-phase chips developed based on the Illumina and Affymetrix platforms, the 40K liquid-phase chip has the following advantages in addition to retaining the advantages of high detection accuracy and low price: (1) The density of the developed SNP chip can be flexibly adjusted. Based on the mainstream 40K, it can be reduced to a low density of 1K (see Table II) or expanded to a high density of 200K; (2) Sites can be added at any time without sample volume limitations; (3) The detection cost is lower than that of current solid-phase chips.

[0042] Table II. Location and mutation information of 1000 SNP sites

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] (II) Donkey genotyping method using donkey 40K liquid phase chip based on targeted capture sequencing

[0053] S1. Preparation of a donkey 40K liquid phase chip based on targeted capture sequencing

[0054] The donkey 40K liquid phase chip consists of individually packaged probe mixtures and hybridization capture reagents.

[0055] Probe mixture: Two single strands of DNA with biotinylate groups at the 5' end, designed and synthesized according to each SNP site being screened, are mixed in equal molar amounts and brought to a final volume using a mixture of EDTA and Tris-HCl (concentration of 3 pmol / mL for each capture probe).

[0056] Hybridization capture reagents: GenoBaits DNA-seqLibrary Prep kit purchased from Shijiazhuang Borui Biotechnology Co., Ltd.; specifically including individually packaged GenoBaits Block I, GenoBaits Block II for ILM / MGI, GenoBaits 2×Hyb Buffer V3.3, GenoBaits Hyb Buffer Enhancer V3.3, GenoBaits 10×Wash Buffer I, GenoBaits 10×Wash Buffer II, GenoBaits 10×Wash Buffer III, and GenoBaits 10×Stringent Wash Buffer.

[0057] S2. Construct a high-throughput sequencing library of donkey DNA using the genomic DNA of the donkey to be tested.

[0058] 2.1 Extraction of genomic DNA from the donkeys to be tested

[0059] The TIANamp Genomic DNA Kit for blood / cell / tissue genomic DNA extraction was used during extraction, and the specific method is as follows:

[0060] (1) Material processing: Use 200 μL of fresh, frozen or anticoagulant-added blood directly. If less than 200 μL, add buffer GA to make up the difference.

[0061] (2) Add 20 μL of Proteinase K solution and mix well.

[0062] (3) Add 200 μL of buffer GB, mix thoroughly by inverting, place at 70°C for 10 min, wait for the solution to become clear, and briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0063] (4) Add 200 μL of anhydrous ethanol and shake well for 15 seconds. At this time, flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0064] (5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (place the adsorption column in the collection tube), centrifuge at 12000 rpm (13400×g) for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0065] (6) Add 500 μL of buffer GD (add anhydrous ethanol before use) to the adsorption column CB3, centrifuge at 12000 rpm (13400×g) for 30 seconds, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

[0066] (7) Add 600 μL of washing solution PW (add anhydrous ethanol before use) to the adsorption column CB3, centrifuge at 12000 rpm (13400×g) for 30 seconds, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

[0067] (8) Repeat step 7 once.

[0068] (9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm (13400×g) for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any residual rinsing liquid in the adsorption material.

[0069] (10) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μL of elution buffer TE to the middle of the adsorption membrane, let it stand at room temperature for 2-5 min, centrifuge at 12000 rpm (13400×g) for 2 min, and collect the solution into the centrifuge tube.

[0070] (11) Detect the length of donkey genomic DNA extracted from blood samples.

[0071] 2.2 Construction of a donkey DNA high-throughput sequencing library

[0072] (1) Prepare a reaction system of 20 μL in a PCR tube: 200 ng of donkey genomic DNA obtained in step 1, 4 μL of GenoBaits End Repair Buffer (Shijiazhuang Borui Biotechnology Co., Ltd.), 3.1 μL of GenoBaits End Repair Enzyme (Shijiazhuang Borui Biotechnology Co., Ltd.), and the remainder is ultrapure water.

[0073] (2) Gently mix the reaction system and briefly centrifuge to collect the reaction liquid to the bottom of the tube.

[0074] (3) Place the PCR tubes in the PCR instrument and react according to the conditions in Table 1. Heat cover at 82℃.

[0075] Table 1. PCR instrument reaction conditions

[0076]

[0077]

[0078] (4) Add the following components directly to the above PCR tube (total amount is 20 μL): 2 μL GenoBaits ULtra DNA Ligase (Shijiazhuang Borui Biotechnology Co., Ltd.), 8 μL GenoBaits ULtra DNA Ligase Buffer (Shijiazhuang Borui Biotechnology Co., Ltd.), 2 μL GenoBaits Adapter for MGI (Shijiazhuang Borui Biotechnology Co., Ltd.), and the remainder is ultrapure water.

[0079] (5) Gently shake the PCR tube to mix the added components thoroughly, and briefly centrifuge to collect the liquid to the bottom of the tube.

[0080] (6) Place the PCR tube in the PCR instrument and react according to the conditions in Table 2, removing the heat cap:

[0081] Table 2. PCR instrument reaction conditions

[0082] temperature time 22℃ 60min 4℃ save

[0083] (7) Add 48 μL of DNA purification magnetic beads that have been equilibrated at room temperature for more than 30 minutes to the above PCR tube, shake to mix, let stand for 5 minutes, and then briefly centrifuge to collect the liquid to the bottom of the tube.

[0084] (8) Place the PCR tube on a magnetic rack for at least 3 minutes until the solution is clear, then remove the supernatant.

[0085] (9) Keep the PCR tube in a magnetic rack, add 100 μL of 80% ethanol; incubate at room temperature for 30 seconds, and remove the supernatant.

[0086] (10) Keep the PCR tube in the magnetic rack, open the lid and let it air dry for 5 minutes until the ethanol evaporates.

[0087] (11) Remove the PCR tube from the magnetic rack and let the magnetic beads inside dry.

[0088] (12) Prepare a reaction system with a total volume of 20 μL: 10 μL GenoBaits PCRMaster Mix (Shijiazhuang Borui Biotechnology Co., Ltd.), 1 μL I5 Barcode (10 μm)-MGI, 5 μL I7 Barcode (2 μm)-MGI, and 4 μL ultrapure water.

[0089] (13) Add the reaction system from step 12 to the PCR tube from step 11, resuspend the dried magnetic beads, and collect the liquid to the bottom of the tube by short centrifugation.

[0090] (14) Place the PCR tubes in the PCR instrument and react according to the conditions in Table 3:

[0091] Table 3. PCR instrument reaction conditions

[0092]

[0093]

[0094] The cycle number in Table 3 is adjusted according to the initial amount of DNA; see Table 4 for details.

[0095] Table 4. Reference number of PCR amplification cycles

[0096] Starting amount Cycle number 1ng-10ng 8 10ng-100ng 6-8 100ng and above 6

[0097] (15) Add 20 μL of DNA purification magnetic beads that have been equilibrated at room temperature for more than 30 minutes to the above PCR tube, shake to mix, let stand for 5 minutes, and then briefly centrifuge to collect the liquid to the bottom of the tube.

[0098] (16) Place the PCR tube on a magnetic rack for at least 3 minutes until the solution is clear, then remove the supernatant.

[0099] (17) Keep the PCR tube in the magnetic rack, add 100 μL of 80% ethanol; incubate at room temperature for 30 seconds, and remove the supernatant.

[0100] (18) Keep the PCR tube in the magnetic rack and let it air dry for 10 minutes.

[0101] (19) Remove the PCR tube from the magnetic rack, add 35 μL of Tris-HCl, vortex to mix, let stand for 5 min, and then briefly centrifuge to collect the liquid to the bottom of the tube.

[0102] (20) Place the PCR tube on a magnetic rack and wait for the solution to clarify (about 3 minutes). Transfer the supernatant to a new tube to complete the construction of the donkey DNA high-throughput sequencing library. Library quality testing: Take 1 μL of the prepared library sample and quantify the library sample using Qubit. The total DNA content of the sequencing library should be above 500 ng. Take 3 μL of the prepared library sample and perform a 1% agarose gel electrophoresis experiment. The DNA fragment range of the electrophoresis result should be 300-500 bp to ensure the effectiveness of subsequent capture experiments. The donkey DNA high-throughput sequencing library can be stored at 4℃ for a short time or at -20℃ for a long time.

[0103] S3. Mix the probe mixture of the donkey 40K liquid phase chip prepared in S1 with the donkey DNA high-throughput sequencing library constructed in S2 to capture DNA fragments containing the target site in the donkey DNA high-throughput sequencing library.

[0104] (1) Equilibrate the streptavidin magnetic beads at room temperature for more than 30 minutes and place them in a 0.2 mL PCR tube.

[0105] (2) Add 500 ng of donkey DNA high-throughput sequencing library, 5 μL of GenoBaits Block I, 2 μL of GenoBaits Block II for ILM / MGI and probe mixture (containing 300 ng of probe) to the above PCR tube to form a mixture containing donkey DNA high-throughput sequencing library and capture probe.

[0106] (3) Centrifuge the PCR tube at 12000 rpm for 1 min.

[0107] (4) Check the dissolution of the GenoBaits hybridization reagent; if crystals are found, heat to 65°C until the crystals are completely dissolved.

[0108] (5) Add GenoBaits 2×Hyb Buffer V3.38μL, GenoBaits Hyb Buffer Enhancer V3.32μL and NucLease-Free Water 6μL to the PCR tube after step 3.

[0109] (6) Mix the PCR tube by pipetting or vortexing, centrifuge at 12000 rpm for 1 min, place at room temperature for 5 min, mix again by pipetting or vortexing, centrifuge slightly, and transfer all the mixture in the PCR tube to a 0.2 mL EP tube.

[0110] (7) Place the EP tube in the PCR instrument and incubate it at 95°C for 10 min (heat cap temperature 105°C).

[0111] (8) Transfer the EP tube to another PCR instrument with a hot cap at 75°C and incubate at 65°C for 1 hour to allow the DNA (the DNA fragment containing the target site in the donkey DNA high-throughput sequencing library) to bind to streptavidin magnetic beads through the corresponding capture probe.

[0112] (9) Add 100 μL of GenoBaits 1×Wash Buffer I preheated at 65℃ to the EP tube, vortex for 5 seconds, centrifuge for 5 seconds; then place it on a magnetic rack, completely separate the magnetic beads from the solution, and discard the supernatant (containing unbound DNA).

[0113] (10) Add 150 μL of preheated (65°C) GenoBaits 1×Stringent Wash Buffer to the EP tube and slowly aspirate and beat it up and down 10 times (avoid generating air bubbles); let it stand for 2 minutes after the sample is mixed; then place it on a magnetic rack to completely separate the magnetic beads from the solution and remove the supernatant.

[0114] (11) Add 150 μL of GenoBaits 1×Wash Buffer I (room temperature) to the EP tube and shake for 2 min; then place it on a magnetic rack to completely separate the magnetic beads from the solution and remove the supernatant.

[0115] (12) Add 150 μL of GenoBaits 1×Wash Buffer II (room temperature) to the EP tube and shake for 1 min; then place it on a magnetic rack to completely separate the magnetic beads from the solution and remove the supernatant.

[0116] (13) Add 150 μL of room temperature GenoBaits 1×Wash Buffer III to the EP tube and shake for 30 s; then place it on a magnetic rack to completely separate the magnetic beads from the solution and remove the supernatant.

[0117] (14) Remove the EP tube from the magnetic rack, add 20 μL of NucLease-Free Water, and pipette 20 times to resuspend the magnetic beads. Use 10 μL of the resuspended solution for subsequent PCR amplification and purification, and keep the remaining 10 μL as a backup.

[0118] For the elution buffers used in the above steps, those requiring preheating to 65°C should be placed in the PCR instrument beforehand; the remaining elution buffers should be stored at room temperature. The elution buffers need to be prepared in advance (see Table 5):

[0119] Table 5. Preparation of eluent

[0120]

[0121]

[0122] S4. The DNA fragments obtained in S3 are amplified and purified. After high-throughput sequencing, the sequencing results are compared with the donkey reference genome to obtain the genotyping data of the donkey to be tested.

[0123] (1) Prepare a total of 30 μL of PCR reagents in a 0.2 mL PCR tube: 15 μL of GenoBaits PCR Master Mix (Shijiazhuang Borui Biotechnology Co., Ltd.), 1.2 μL of GenoBaits Primer Mix for MGI (Shijiazhuang Borui Biotechnology Co., Ltd.), 10 μL of magnetic beads that have been adsorbed with captured DNA, and 3.8 μL of ultrapure water.

[0124] (2) Briefly vortex and centrifuge to ensure that the magnetic beads remain in the solution.

[0125] (3) Place the PCR tubes into the PCR instrument, set the lid temperature to 105℃, and perform PCR amplification according to the program, see Table 6:

[0126] Table 6. PCR instrument reaction conditions

[0127]

[0128] (4) Add 45 μL (1.5 × 100 μL) of DNA purification magnetic beads that have been equilibrated at room temperature for more than 30 minutes to the PCR reaction product, shake to mix, try not to generate bubbles, let stand for 5 minutes, then briefly centrifuge to collect the liquid to the bottom of the tube; place on a magnetic rack for at least 3 minutes until the solution is clear, and remove the supernatant.

[0129] (5) Keep the PCR tube in the magnetic rack, add 100 μL of 80% ethanol, incubate at room temperature for 30 seconds, remove the supernatant, keep the PCR tube in the magnetic rack, and open the lid to air for 10 minutes.

[0130] (6) Remove the PCR tube from the magnetic rack, add 35 μL Tris-HCl, vortex to mix, let stand for 5 min, then briefly centrifuge to collect the liquid to the bottom of the tube; place the PCR tube on the magnetic rack, wait for the solution to become clear (about 3 minutes), transfer the supernatant to a new tube, and store at -20℃. The purified PCR fragment can be stored at -20℃ for one week.

[0131] (7) The captured DNA library was measured using the Qubits FLuorometer and Qubit dsDNA HS Assay Kit. The average fragment length of the captured DNA library on the digital electrophoresis system was measured. The concentration of the captured DNA library was measured using the KAPA Library Quantification Kit.

[0132] (8) The DNA library was captured and sequenced using a sequencer. The sequencing data was processed by BWA and GATK software to obtain the genotype of the corresponding SNP site of the donkey to be tested.

[0133] (III) Evaluation of Genotyping Quality Based on Donkey 40K Liquid Chip Targeted Capture Sequencing

[0134] 1. Detection rate

[0135] SNP detection rate is an important indicator of chip quality, and it is generally measured using loci on autosomes and the X chromosome. Sample testing showed that the average SNP detection rate of the aforementioned donkey 40K liquid phase chip was 98.5%-99.1%, all exceeding the standard detection rate of 95%, indicating that the donkey 40K liquid phase chip has good genotyping detection quality.

[0136] 2. Genetic structure analysis

[0137] For the whole-genome sequencing results of the donkey samples (excluding wild donkeys) in Table I, genotypic annotation was performed using the SNP sites involved in the donkey 40K liquid microarray described above, followed by principal component analysis (PCA). See [link to relevant documentation]. Figure 4 The donkey 40K liquid phase microarray can clearly distinguish Chinese donkey breeds from eight foreign donkey breeds. The results also indicate that the donkey 40K liquid phase microarray can provide SNP markers for genetic structure segmentation.

[0138] (iv) Breeding Application

[0139] Genotyping was performed on 210 Dezhou donkeys using the aforementioned Donkey 40K liquid chromatography chip. Among them, 98 donkeys had a three-pink coat color, and 112 had a black-headed coat color (blood samples were collected in Dong'e County, Shandong Province, in May 2022). Genome-wide association analysis was used to screen for significant molecular markers of the coat color trait. After data quality control, a total of 205 individuals and 44,661 SNP loci were retained. The significance thresholds were defined as 0.05 / N (genome-wide significance level) and 1 / N (chromosomal significance level), where N is the number of SNP loci used. The results are as follows: Figure 5 As shown, a total of 4 SNP loci reached genome-wide significance: chr15:25408816 (P = 1.67 × 10⁻⁶). -34 ), chr15:23191566 (P value = 3.19 × 10⁻⁶) -9 ), chr15:26258385 (P value = 9.94 × 10) -8 ), chr15:24168300 (P value = 1.05 × 10⁻⁶) -6 The most significant SNP locus (chr15:25408816) is the SNP locus corresponding to sequence number 621 in Table II. The genotypic distribution of this locus in the *Trifolium repens* population is: CC 3 individuals, CT 22 individuals, TT 72 individuals; and in the *Aconitum carmichaelii* population, the genotypic distribution is: CC 82 individuals, CT 25 individuals, TT 1 individual. Therefore, selecting individuals with the TT and CC genotypes respectively can rapidly establish the *Trifolium repens* and *Aconitum carmichaelii* populations. According to annotation, the candidate gene affecting coat color is the ASIP (agouti signaling protein) gene.

[0140] In summary, the donkey 40K liquid-phase chip developed in this invention provides SNP variation information containing up to 200K or more for genotyping detection results, and SNP sites can be added at any time. New probes designed based on these new sites only need to be added to the existing probe mixture, and SNP sites that do not need genotyping can also be removed. Furthermore, unlike solid-phase chip technology which requires a certain sample volume (one chip can typically process 12, 24, 48, or even 96 samples), the donkey 40K liquid-phase chip developed in this invention has no sample volume requirement. The entire experiment is conducted in a PCR tube, and the reaction system can be added as needed, so even a single sample can be detected. This flexible sample detection capability overcomes the technical limitations of solid-phase chips and has greater potential for application in donkey genetic structure analysis and molecular breeding. In addition, this invention optimizes the capture hybridization system through screening, reducing the hybridization capture time to only 1 hour (step 8 in S3). Compared to overnight hybridization capture processes that take more than 16 hours, this significantly shortens the genotype acquisition time, allowing the entire library construction and capture process to be completed within one day, saving time for genotype detection.

Claims

1. A target capture sequencing based equine liquid chip, characterized by: The genotyping objects of the chip include 1000 SNP sites located on the donkey reference genome, and the locations of the 1000 SNP sites on the donkey reference genome are as follows: The donkey reference genome is EquAsi1.0 version.

2. The horse liquid chip based on targeted capture sequencing according to claim 1, characterized in that: The genotyping objects of the chip also include other SNP sites located on the donkey reference genome and matched by capture probes designed and synthesized according to the 1000 SNP sites.

3. The horse liquid chip based on targeted capture sequencing according to claim 1, characterized in that: The chip includes capture probes designed and synthesized according to each SNP site screened, and the capture probes are two nucleic acid molecules that are 60%-70% overlapped with each other and both cover the SNP site, wherein the length of each nucleic acid molecule is 100-120 nt.

4. The horse liquid chip based on targeted capture sequencing according to claim 3, characterized in that: The GC content of the capture probes is 30%-80%, and the selected region does not contain a GAP region.

5. The horse liquid chip based on targeted capture sequencing according to claim 3, characterized in that: Each screened SNP site is uniformly distributed on each chromosome of the donkey reference genome, has a heterozygosity of less than 15%, and has a MAF greater than 0.35 in the donkey breed and wild donkey population.

6. A method for equine genotyping based on targeted capture sequencing, characterized in that: The method comprises the following steps: S1. preparing the donkey liquid phase chip based on targeted capture sequencing according to any one of claims 1-5; S2. constructing a donkey DNA high-throughput sequencing library using the genomic DNA of the donkey to be tested; S3. capturing the DNA fragments containing the corresponding SNP sites in the donkey DNA high-throughput sequencing library using the prepared donkey liquid phase chip based on targeted capture sequencing; S4. amplifying and purifying the captured DNA fragments, and after high-throughput sequencing of the product, comparing the sequencing results with the donkey reference genome to obtain the genotyping data of the donkey to be tested.

7. The method of equine genotyping based on targeted capture sequencing as claimed in claim 6, wherein: The method for constructing the donkey DNA high-throughput sequencing library specifically comprises the following steps: fragmenting the genomic DNA of the donkey to be tested by ultrasonic disruption or enzyme digestion.

8. The donkey liquid phase chip based on targeted capture sequencing according to any one of claims 1-5 is applied to donkey molecular breeding.

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