Mink whole genome 40K SNP liquid phase chip and application thereof

Abstract

The invention discloses a mink 40K liquid phase chip. The chip contains SNP (Single Nucleotide Polymorphism) sites which are used for detecting 44, 801 and are positioned to a mink reference genome ASMNNV1; the 44 and 801 SNP loci comprise SNP molecular marker combinations for mink variety character related gene positioning, genetic diversity analysis, whole genome association analysis, genetic relationship analysis, variety identification and germplasm resource improvement and protection. The mink 40K liquid phase chip disclosed by the invention is designed on the basis of the sites by adopting a targeted capture sequencing technology, and shows relatively high application value in multiple fields such as mink breeding and the like. And a scientific basis is provided for mink variety breeding and genetic improvement.

Description

Technical Field

[0001] This invention relates to the field of whole genome gene chips, specifically to a mink whole genome 40K SNP liquid phase chip and its application. Background Technology

[0002] Farmed mink (Neovison vison) are semi-aquatic carnivores whose wild populations traditionally originated in the Northern Hemisphere. Due to their importance in the fur industry and the commercial demand for fur production, these animals and their pelts were introduced to the Far East, particularly China, from Western Europe in the late 20th century. Currently, with the development of China's mink industry, the number of farmed mink has reached 33 million. Farmed mink, with its remarkable coat color diversity and high-quality fur, has become one of the most luxurious clothing materials. Driven by the fur market demand, mink have been domesticated and selectively bred for coat color traits, body shape, and temperament. Compared to the wild type, mink exhibit at least 35 coat color mutations and over 100 combinatorial variations. Research has confirmed that coat color is a complex trait, regulated by multiple genes. Currently, very few studies focus on the chromosomal regions or quantitative trait loci (QTLs) associated with mink coat color.

[0003] Current research on molecular markers for economic traits in mink (such as weight, coat color, and total litter size) urgently needs improvement. A low-cost, highly flexible breeding microarray is urgently needed to support the protection and precision breeding of local mink breeds. However, existing commercially available solid-phase microarrays for mink (such as Illumina 40K) have the following drawbacks: 1. High cost, unsuitable for large-scale genotyping; 2. Based on foreign breeds, lacking sufficient polymorphism related to local Chinese mink breeds; 3. Lack of loci associated with mink coat color. Summary of the Invention

[0004] Addressing the current gap in mink liquid phase chip technology, this invention achieves targeted capture of 44,801 mink cells.

[0005] We developed a 40K liquid phase chip for mink using SNP loci for breed identification, genetic analysis, and other purposes, providing technical reference for the development of the mink industry and breeding research.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A mink 40K liquid phase chip contains 44,801 SNP loci located in the mink reference genome ASM_NN_V1. These 44,801 SNP loci include combinations of SNP molecular markers used for mink breed trait-related gene mapping, genetic diversity analysis, genome-wide association analysis, phylogenetic analysis, breed identification, and germplasm resource improvement and protection. The location information of these 44,801 SNP molecular markers on the reference genome ASM_NN_V1 is shown in Table 1.

[0008] The 44,801 SNP sites include 1,815 functional sites; the functional sites are conserved functional SNP sites in the mink genome that are related to mink body weight, body length, total number of litters, number of litters surviving, and coat color.

[0009] Among them, the SNP loci information related to mink weight and body length are shown in Table 1, numbered 1-677.

[0010] The SNP loci information related to the total number of mink offspring are shown in Table 1, numbered 678-743.

[0011] The SNP loci information related to the number of mink offspring survivals are shown in Table 1, from 744 to 1336.

[0012] The SNP loci related to mink coat color are numbered 1337-1815 as shown in Table 1.

[0013] The 44,801 SNP loci also include 377 SNP loci associated with the economic traits of slaughter mink, and their location information is shown in Table 1 as numbers 1816-2192.

[0014] Table 1.44, 801 SNP molecular marker positions

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[0112] The mink 40K liquid phase chip of this invention further includes probes designed based on gene sequences covering 44,801 SNP sites. The probes are designed and synthesized using targeted capture sequencing technology based on the location of the 44,801 target SNP sites and flanking sequence information. The probe length is 110 bp. The probe design primarily evaluates the specificity, complexity, and GC content of the upstream and downstream sequences of the target sites; priority is given to placing the target sites in the middle of the probe.

[0113] This invention also provides a design method for the mink 40K liquid phase chip, comprising the following steps:

[0114] (1) Using genome-wide association analysis, linear regression analysis was performed on the weight and coat color phenotypic data of 133 mink from 7 breeds using PLINK software to screen for SNPs that were significantly associated with the traits (P<1×10⁻⁶). -5 ); Obtain known SNPs related to mink weight and reproductive genes;

[0115] (2) Obtain uniformly distributed polymorphic sites by the following filtering conditions: minimum allele frequency (MAF) > 0.1; genotype deletion rate < 0.1; Hardy-Weinberg equilibrium test P > 0.001; use Haploview software to select tag SNPs in linkage blocks;

[0116] (3) After integrating 377 SNP loci related to the economic traits of slaughter mink with the loci obtained in steps (1) and (2), the candidate loci were compared with the mink whole genome sequencing data (Neovison_vison-NCBI-ASM_NN_V1). Loci that did not meet the requirements were removed based on the screening criteria of MAF≥0.35, deletion rate<0.1, and heterozygosity rate<0.5. Then, loci with an interval of less than 500 bp were selected and loci with good polymorphism and located on genes were retained. 34,123 SNP loci were determined as VCF background loci. The location information of the 34,123 SNP loci is shown in Table 1, from 2193 to 36315.

[0117] (4) Based on the resequencing data of 7 mink breeds, SNP sites with uniform coverage of the whole genome were selected, and background SNP sites were added: The genome was divided into several intervals with 200kb as the standard. If a functional site exists in an interval, no site is added. If a functional site is missing in an interval, one background site is added. The selection criteria for background sites are MAF>0.35, deletion rate<0.1, and heterozygosity rate<0.5. Finally, 8486 large gap supplement sites were added. The location information of the 8486 SNP sites is shown in Table 1, from 36316 to 44801.

[0118] The present invention also provides the application of the mink 40K liquid phase chip in mink genotyping, genome-wide association analysis, breed identification, kinship analysis, and breeding of new mink strains for economic traits.

[0119] The beneficial effects of this invention are reflected in:

[0120] (1) This invention successfully obtained key functional loci and breed-specific loci of mink through in-depth mining of large-scale sequencing data, and screened out 44,801 SNP loci that can be used for chip design. The chip designed based on the above loci can realize genotyping and has shown high application value in multiple fields such as mink breeding.

[0121] (2) The mink 40K liquid phase chip of the present invention uses targeted capture sequencing technology, which can not only accurately genotype the target site, but also accurately genotype SNPs within a certain range around the target site, thereby obtaining more SNP genotyping information than the labeled site.

[0122] (3) Compared with traditional solid-phase chips, this liquid-phase chip has significant advantages: it is more flexible and can add marker sites at any time according to actual application needs; and relying on the second-generation sequencing platform, it effectively reduces the cost of genotyping and provides reliable technical support for large-scale genotyping.

[0123] (4) The chip of this invention can systematically analyze the genetic structure of mink populations and accurately assess the level of genetic diversity; it can analyze the differences in SNP loci among individuals; and through genome-wide association analysis, it can discover genetic loci related to important traits, providing strong technical support for mink genetics research and industrial development. It provides a scientific basis for mink breed selection and genetic improvement, and effectively improves the economic benefits of mink farming. Attached Figure Description

[0124] Figure 1 Histogram of the distribution of SNP sites on each chromosome of the 40K liquid phase chip of mink in this invention.

[0125] Figure 2 PCA clustering results of seven mink breeds based on the 40K liquid phase chip of this invention.

[0126] Figure 3 Phylogenetic tree results of seven mink breeds based on the 40K liquid phase chip of this invention. Detailed Implementation

[0127] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. These embodiments are only for explaining the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the methods described in this invention are conventional methods in the art; the reagents described are commercially available products unless otherwise specified.

[0128] Example 1: Design and fabrication of a mink 40K low-density SNP liquid-phase chip

[0129] 1. Establishment of the Mink SNP Database

[0130] This invention utilizes whole-genome resequencing data from 133 individuals across 7 mink breeds to obtain highly reliable SNP loci for subsequent screening through sequencing analysis.

[0131] Whole-genome resequencing data acquisition: Whole-genome resequencing was performed on 133 minks of 7 breeds (red-eyed white mink, black mink, red mink, brown mink, and silver-blue mink) using Illumina NovaSeq (Table 2). The average coverage was 9.18×. The genomes were aligned to the mink reference genome using BWA software.

[0132] ASM_NN_V1 was used to detect SNPs using GATK4 software, and high-quality mutation sites were obtained.

[0133] Table 2. List of mink breeds used

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[0135]

[0136] The target SNP sites screened in this invention include the following categories: those related to mink body weight and length; those related to total litter size and litter survival rate; and conserved sites on the mink genome related to mink coat color.

[0137] The specific screening process for the sites is as follows:

[0138] (1) Using genome-wide association analysis, linear regression analysis was performed on the weight and coat color phenotypic data of 133 mink from 7 breeds using PLINK software to screen for SNPs that were significantly associated with the traits (P<1×10⁻⁶). -5 ); Obtain known SNPs related to mink body weight and reproductive genes; Obtain 1,815 phenotypic loci (Table 1, pp. 1-1815);

[0139] (2) Obtain uniformly distributed polymorphic sites by the following filtering conditions: minimum allele frequency (MAF) > 0.1; genotype deletion rate < 0.1; Hardy-Weinberg equilibrium test P > 0.001; use Haploview software to select tag SNPs in linkage blocks;

[0140] (3) 377 SNP loci related to the economic traits of slaughter mink were obtained (Table 1, pp. 1816-2192). After integrating the loci obtained in steps (1) and (2), the candidate loci were compared with the mink whole genome sequencing data (Neovison_vison-NCBI-ASM_NN_V1). Loci that did not meet the requirements were removed based on the screening criteria of MAF≥0.35, deletion rate<0.1, and heterozygosity rate<0.5. Then, loci with an interval of less than 500 bp were selected and loci with good polymorphism and located on genes were retained. 34,123 SNP loci (Table 1, pp. 2193-36315) were identified as VCF background loci.

[0141] (4) Background SNP sites were supplemented based on the SNP sites with uniform coverage of the whole genome selected from the resequencing data of 7 mink breeds: The genome was divided into several intervals with 200kb as the standard. If a functional site exists in an interval, no site is supplemented. If a functional site is missing in an interval, one background site is supplemented. The selection criteria for background sites are MAF>0.35, deletion rate<0.1, and heterozygosity rate<0.5. Finally, 8486 large gap supplement sites were added (Table 1, pp. 36316-44801).

[0142] 2. Preparation of 40K liquid phase chip by mink

[0143] Mink 40K probes were designed for 44,801 SNP sites, resulting in a mink 40K liquid-phase chip. Based on the location and flanking sequence information of the 44,801 SNP sites, primers were designed and probes were synthesized using targeted capture sequencing technology to obtain the mink 40K SNP liquid-phase chip. The probe design primarily evaluated the specificity, complexity, and GC content of the upstream and downstream sequences of the target site; priority was given to placing the target site in the middle of the probe. The probes were 110 bp long DNA nucleotide sequences with a biotinylated group modified at the 5' end.

[0144] The distribution of all 44,801 SNP loci in the mink genome chromosome of this invention's liquid-phase chip is shown in the figure. Figure 1 As shown. Figure 1 This indicates that the SNP markers screened by the mink 40K liquid phase chip of the present invention uniformly cover each chromosome, strictly following the chip design principles; it also shows that the SNP spacing layout of the chip of the present invention is reasonable, with an average spacing of about 40kb, and the SNP spacing on each chromosome is basically consistent.

[0145] Example 2: Application of Mink 40K Liquid Chromatography Chip in Detecting Genetic Diversity in Local Mink Species

[0146] (1) Extraction of genomic DNA from the mink to be tested: In this embodiment, the hybridization capture reagent was provided by a biotechnology company. The specific extraction method was as follows: using the phenol-chloroform method or a commercially available blood genomic DNA extraction kit.

[0147] DNA extraction.

[0148] (2) DNA sample quality testing: Agarose gel electrophoresis with a mass fraction of 1%–1.5% was used for detection. The electrophoresis results were judged using the GelDocXRSystem gel imaging system to ensure the integrity of the genome. The genome was measured using a Q5000 micro-ultraviolet spectrophotometer or a similar nucleic acid and protein analyzer.

[0149] The concentration of DNA was adjusted to a working concentration of 10–50 ng / μL.

[0150] (3) Construction of high-throughput sequencing library for mink DNA

[0151] Preparation of the end-repair reaction system: Prepare a 20 μL reaction system in a PCR tube, containing 200 ng of mink DNA obtained in step 1, 4 μL of GenoBaits EndRepair Buffer, and 3.1 μL of GenoBaits End Repair Enzyme. Make up the remaining volume with ultrapure water. The reagents used in this example are commercially available.

[0152] GenoBaits DNA-seq Library Prep Kit.

[0153] System mixing and centrifugation: After gently mixing the reaction system, perform a brief centrifugation operation to concentrate the reaction solution to the bottom of the tube.

[0154] End-repair reaction conditions: Place the reaction tube in the PCR instrument and set the lid to 82°C for reaction adapter ligation system preparation: Add the following components directly to the above reaction tube to make the total volume reach 20 μL: GenoBaits Ultra DNA Ligase 2 μL, GenoBaits Ultra DNA Ligase Buffer 8 μL, GenoBaits Adapter for MGI 2 μL, and make up the remaining volume with ultrapure water.

[0155] Mixing and Precautions for Connecting the System: After gently mixing the reaction system, briefly centrifuge to collect the reaction solution to the bottom of the tube. Special attention must be paid to ensuring thorough mixing; otherwise, library construction may fail.

[0156] Adapter connection reaction conditions: Place the reaction tube in the PCR instrument and remove the heat cap to allow the reaction to proceed.

[0157] Magnetic bead purification (first time): Add 48 μL of DNA purification magnetic beads that have been equilibrated at room temperature for more than 30 min to the system, vortex to mix (be careful to avoid generating bubbles), let stand for 5 min and then briefly centrifuge.

[0158] Magnetic bead washing and drying

[0159] Place on a magnetic rack for at least 3 minutes until the solution becomes clear, then remove the supernatant.

[0160] Keep the PCR tube in a magnetic rack, add 100 μL of 80% ethanol, incubate at room temperature for 30 seconds, and then remove the supernatant.

[0161] Open the lid and let it air dry for 5 minutes until the ethanol has completely evaporated.

[0162] Drying the magnetic beads: Remove the PCR tube from the magnetic rack and ensure that the magnetic beads are completely dry.

[0163] PCR amplification system preparation: Prepare a 20 μL reaction system in a new PCR tube, including 10 μL GenoBaits PCR MasterMix, 1 μL I5 Barcode (10 μM)-MGI, 5 μL I7 Barcode (2 μM)-MGI, and 4 μL ultrapure water.

[0164] Magnetic bead resuspension and mixing: Add the above system to a PCR tube containing dried magnetic beads, resuspend the magnetic beads, and then briefly centrifuge to collect the reaction solution.

[0165] PCR amplification conditions: Place the reaction tube in a PCR instrument for amplification (adjust the number of amplification cycles according to the initial amount of DNA; the initial amount and recommended number of cycles need to be supplemented).

[0166] Second magnetic bead purification and washing

[0167] Add 20 μL of DNA purification magnetic beads that have been equilibrated at room temperature for at least 30 min, vortex to mix, let stand for 5 min, and then briefly centrifuge to collect the liquid.

[0168] Place on a magnetic rack for at least 3 minutes, then remove the supernatant.

[0169] Keep the PCR tube in a magnetic rack, add 100 μL of 80% ethanol, incubate at room temperature for 30 seconds, and then remove the supernatant.

[0170] Open the lid and let it air dry for 10 minutes.

[0171] Elution and recovery: Remove the PCR tube from the magnetic rack, add 35 μL of Tris-HCl, vortex to mix, let stand for 5 min, and briefly centrifuge to collect the liquid. Place on the magnetic rack until the solution becomes clear (about 3 min), transfer the supernatant to a new tube, and complete the construction of the DNA sequencing library.

[0172] Document Quality Inspection

[0173] Quantification: Take 1 μL of library sample and quantify using Qubit. The total DNA amount should be ≥500 ng.

[0174] Fragment size detection: Take 3μL of library sample and perform 1% agarose gel electrophoresis. The DNA fragment range should be 300-500bp.

[0175] Storage: Store at 4℃ for short periods, and at -20℃ for long periods.

[0176] (4) Liquid-phase chip and sequencing library mixing and target fragment capture

[0177] Mixing magnetic beads and probes: Equilibrate DNA-purified magnetic beads at room temperature for at least 30 minutes. Take an appropriate amount of mink 40K probe and mix it with the magnetic beads in a 0.2 mL PCR tube. Centrifuge briefly.

[0178] Preparation of the mixture: Add the following reagents to the PCR tube: 500 ng mink DNA high-throughput sequencing library, 5 μg (5 μL) GenoBaits Block I, 2 μL GenoBaits Block II for ILM / MGI, and 300 ng probe to form a liquid chip mixture.

[0179] Vacuum concentration and centrifugation: Concentrate to dryness using a vacuum concentrator at ≤60℃. After concentration, centrifuge at 12000rpm for 1min. It can be stored overnight at room temperature (15-25℃).

[0180] Hybridization reagent preparation: Dissolve GenoBaits hybridization reagent at room temperature. If crystals appear in 2X Hyb Buffer, heat to 65°C and shake until completely dissolved.

[0181] Hybridization system preparation and mixing: Add the hybridization reagent system to the PCR tube, mix by pipetting or vortexing, centrifuge at 12000 rpm for 1 min, let stand at room temperature for 5 min, mix again, and transfer to a 0.2 mL EP tube.

[0182] Heat circulation and hot bath

[0183] Incubate at 95℃ for 10 minutes (heated lid 105℃).

[0184] When the temperature drops to 65°C, transfer the sample to a PCR instrument with a 75°C hot cover and incubate for 1 hour.

[0185] Preparation of elution solution

[0186] Preheat the required eluent at 65°C, and store the rest at room temperature.

[0187] If precipitates form in 10×Wash Buffer I and 10×SW Buffer, they can be dissolved at 65°C.

[0188] Magnetic bead pretreatment

[0189] Before use, place the magnetic beads at room temperature for 10 minutes and then vortex for 15 seconds to mix them thoroughly.

[0190] Take 50 μL of magnetic beads into the EP tube, separate them with a magnetic rack, and remove the supernatant.

[0191] Add 150 μL of 1X Bead Wash Buffer, vortex for 10 seconds, separate with a magnetic rack, discard the supernatant, and repeat the washing process 3 times.

[0192] Hybridization solution transfer and binding reaction: Transfer 16 μL of hybridization solution from a 65℃ incubator for 1 h to a pretreated EP tube, vortex to mix, centrifuge, and incubate in a PCR instrument at 65℃ for 45 min (75℃ with hot lid), shaking for 5 s every 12 min.

[0193] Magnetic bead washing steps

[0194] Add 100 μL of 1X Wash Buffer I preheated at 65°C, shake, and then separate using a magnetic rack to remove the supernatant.

[0195] Add 150 μL of preheated 1X Stringent Wash Buffer, slowly aspirate and aspirate 10 times, let stand for 2 minutes, then separate with a magnetic rack and quickly remove the supernatant (repeat once).

[0196] Add 150 μL of 1X Wash Buffer I at room temperature, shake for 2 min, and then separate the supernatant using a magnetic rack.

[0197] Add 150 μL of 1X Wash Buffer II at room temperature, shake for 1 min, and then separate the supernatant using a magnetic rack.

[0198] Add 150 μL of 1X Wash Buffer III at room temperature, shake for 30 seconds, and then separate the supernatant using a magnetic rack.

[0199] DNA capture elution

[0200] Remove the test tube from the magnetic rack, add 20 μL of Nuclease-Free Water, and resuspend the magnetic beads 10 times. Use 10 μL for subsequent PCR amplification and purification, and keep the remaining 10 μL as a backup.

[0201] Capture DNA amplification, purification and sequencing

[0202] PCR amplification system preparation: Prepare a 30 μL system in a 0.2 mL PCR tube: 15 μL GenoBaits PCR Master Mix, 1.2 μL GenoBaits Primer Mix for MGI, 10 μL DNA capture magnetic bead resuspension, and 3.8 μL ultrapure water.

[0203] Preparation and conditions for amplification reaction: After vortexing and centrifugation, place the product in a PCR instrument and amplify at 105°C with the lid on (specific procedures need to be supplemented). The product can be stored overnight at 4°C.

[0204] Magnetic bead purification and washing

[0205] Add 45 μL (1.5 times the volume) of magnetic beads, shake to mix, let stand for 5 min, centrifuge, and then separate the supernatant using a magnetic rack.

[0206] Add 100 μL of 80% ethanol to wash, incubate at room temperature for 30 seconds, remove the supernatant, and let it air dry for 10 minutes.

[0207] Elution and storage: Add 35 μL Tris-HCl, shake and let stand, then centrifuge. Separate the supernatant into a new tube using a magnetic rack and store at -20°C (can be stored for one week).

[0208] (5) Document Quality Inspection

[0209] Concentrations were determined using a Qubit FLuorometer and a Qubit dsDNA HS Assay Kit, and the average fragment length was detected using a digital electrophoresis system.

[0210] Library concentration was measured using the KAPA Library Quantification Kit.

[0211] (6) High-throughput sequencing

[0212] The captured library was sequenced using a sequencer, and the sequencing results were compared with the mink reference genome to obtain genotyping.

[0213] Example 3: Quality Evaluation of Genotyping of Mink Using 40K Liquid Chip Array

[0214] (1) Genotyping stability: Repeated testing was performed on some mink samples. The results showed that the consistency of genotyping between two tests of 15 replicate samples reached 99.3%, and the correlation coefficient was 99.7%, indicating that the detection system has good stability. Therefore, the 40K liquid phase chip genotyping of mink described in this invention has excellent stability. These results demonstrate that the 40K liquid phase chip of mink described in this invention can significantly improve the accuracy and stability of large-scale genotyping detection in mink.

[0215] (2) SNP detection rate: The detection data of autosomal and X chromosome loci showed that the average SNP detection rate was as high as 99.88% (standard deviation 0.007); the individual detection rate was over 99%, and the individual average detection rate was 99.76% (standard deviation 0.0016), which fully confirms the excellent quality of the detection method.

[0216] (3) Genetic structure analysis: Principal component analysis (PCA) was performed based on SNP data. PLINK2 software was used to filter biallelic variations (excluding loci with minor allele frequencies (MAF) below 0.05) and linkage disequilibrium (LD) pruning was performed. Based on the filtered SNPs, the pairwise genetic distance matrix was calculated using PLINK2, and a neighbor-joining (NJ) tree with 1000 bootstrap repeats was constructed using PHYLIP software. The genetic structure of different mink populations was explored by combining phylogenetic tree construction, PCA, and population structure analysis (using ADMIXTURE software).

[0217] The PCA clustering results of seven mink breeds based on the mink 40K liquid phase chip of this invention are as follows: Figure 2 As shown, the phylogenetic tree results are as follows: Figure 3As shown in the figure. The results indicate that the 40K SNP liquid-phase chip for the entire mink genome of this invention can systematically resolve the genetic structure of mink populations and accurately assess the level of genetic diversity; it can analyze the differences in SNP sites among individuals; and through genome-wide association analysis, it can discover genetic loci associated with important traits, providing strong technical support for mink genetics research and industrial development.

Claims

1. A mink 40K liquid phase chip, characterized in that, The chip contains 44,801 SNP loci located in the mink reference genome ASM_NN_V1. These 44,801 SNP loci include combinations of SNP molecular markers used for mink breed trait-related gene mapping, genetic diversity analysis, genome-wide association analysis, phylogenetic analysis, breed identification, and germplasm resource improvement and protection. The location information of these 44,801 SNP molecular markers on the reference genome ASM_NN_V1 is shown in Table 1 of the specification.

2. The mink 40K liquid phase chip as described in claim 1, characterized in that, The 44,801 SNP sites include 1,815 functional sites; the functional sites are conserved functional SNP sites in the mink genome that are related to mink body weight, body length, total number of litters, number of litters surviving, and coat color.

3. The mink 40K liquid phase chip as described in claim 2, characterized in that, The SNP loci related to mink weight and body length are listed as numbers 1-677 in Table 1 of the specification.

4. The mink 40K liquid phase chip as described in claim 2, characterized in that, The SNP loci information related to the total number of mink offspring are shown in Table 1, numbered 678-743.

5. The mink 40K liquid phase chip as described in claim 2, characterized in that, The SNP loci associated with the number of mink offspring survivals are shown in Table 1, numbered 744–1336.

6. The mink 40K liquid phase chip as described in claim 2, characterized in that, The SNP loci related to mink coat color are numbered 1337-1815 as shown in Table 1.

7. The mink 40K liquid phase chip as described in claim 1, characterized in that, The 44,801 SNP loci also include 377 SNP loci associated with the economic traits of slaughter mink, the location information of which is shown in Table 1 of the specification as numbers 1816-2192.

8. The mink 40K liquid phase chip as described in claim 1, characterized in that, It also includes probes designed based on gene sequences covering 44,801 SNP sites; the probes are designed and synthesized using targeted capture sequencing technology based on the location of the 44,801 target SNP sites and the flanking sequence information; the probes are 110 bp in length.

9. The design method of the mink 40K liquid phase chip as described in claim 1 or 8, characterized in that, Includes the following steps: (1) Using genome-wide association analysis, linear regression analysis was performed on the weight and coat color phenotypic data of 133 mink from 7 breeds using PLINK software to screen for SNPs that were significantly associated with the traits (P<1×10⁻⁶). -5 ); Obtain known SNPs related to mink weight and reproductive genes; (2) Obtain uniformly distributed polymorphic sites by the following filtering conditions: minimum allele frequency (MAF) > 0.1; genotype deletion rate < 0.1; Hardy-Weinberg equilibrium test P > 0.001; use Haploview software to select tag SNPs in linkage blocks; (3) After obtaining 377 SNP loci associated with the economic traits of slaughter mink and integrating them with the loci obtained in steps (1) and (2), the candidate loci were combined with the mink whole genome sequencing data. The (Neovison_vison-NCBI-ASM_NN_V1) algorithm was compared, and sites that did not meet the requirements were removed based on the screening criteria of MAF≥0.35, deletion rate<0.1, and heterozygosity rate<0.

5. Subsequently, sites with an interval of less than 500 bp were selected, and sites with good polymorphism and located on genes were retained. 34,123 SNP sites were identified as VCF background sites. The location information of the 34,123 SNP sites is shown in Table 1 of the specification, from 2193 to 36315. (4) Based on the resequencing data of 7 mink breeds, SNP sites with uniform coverage of the whole genome were selected, and background SNP sites were added: the genome was divided into several intervals with 200kb as the standard. If a functional site exists in the interval, no site is added. If a functional site is missing in the interval, one background site is added. The selection principle for background sites is MAF>0.35, deletion rate<0.1, and heterozygosity rate<0.

5. Finally, 8486 large gap supplement sites were added. The location information of the 8486 SNP sites is shown in Table 1 of the specification as 36316-44801.

10. The application of the mink 40K liquid phase chip of claim 1 in mink genotyping, genome-wide association analysis, breed identification, kinship analysis, and breeding of new mink strains for economic traits.

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