A mink whole genome 40k snp liquid chip and application thereof

Abstract

The application discloses a mink 40K liquid phase chip, which contains 44,801 SNP sites for detecting 44,801 SNP sites positioned to a mink reference genome ASM_NN_V1; the 44,801 SNP sites include a SNP molecular marker combination for mink breed character related gene positioning, genetic diversity analysis, whole genome association analysis, kinship analysis, breed identification, germplasm resource improvement and protection. The mink 40K liquid phase chip is designed based on the above-mentioned sites by using a targeted capture sequencing technology, and shows high application value in multiple fields such as mink breeding. The mink 40K liquid phase chip provides a scientific basis for mink breed selection and genetic improvement.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of whole genome gene chip, and particularly relates to a mink whole genome 40K SNP liquid chip and application thereof. BACKGROUND

[0002] Farmed mink (Neovison vison) is a semi-aquatic carnivore, and its wild population is traditionally originated from the northern hemisphere. Due to the importance in fur industry and commercial demand of fur production, these animals and their pelage were introduced from western Europe to the Far East, especially China, in the late 20th century. At present, with the development of mink industry in China, the number of farmed mink has reached 33 million. Farmed mink has become one of the most luxurious clothing materials due to its amazing coat color diversity and high-quality fur. Due to the demand of fur market, mink has been domesticated and selectively bred for coat color traits, body size and temperament. Compared with wild type, there are at least 35 coat color mutations and more than 100 combination variation types in mink. Studies have confirmed that coat color traits belong to complex traits and are regulated by multiple genes. Currently, few studies focus on the chromosomal regions or quantitative trait loci (QTL) related to mink coat color.

[0003] The current molecular marker research of mink economic traits (such as body weight, coat color, total litter size) needs to be improved, and a low-cost and high-flexibility breeding chip is urgently needed to support mink local breed protection and precision breeding. However, the existing commercial solid-phase chip of mink (such as Illumina 40K) has the following defects: 1. High cost, not suitable for large-scale typing; 2. Designed based on foreign breeds, lacking of polymorphisms related to Chinese local mink breeds; 3. No sites associated with mink fur color. SUMMARY

[0004] In view of the current technical blank of mink liquid chip in the field, the present application develops a mink 40K liquid chip by targeting 44,801 SNP sites, which is used for breed identification, genetic analysis, etc., and provides technical reference for mink industry development and breeding research.

[0005] In view of the current technical blank of mink liquid chip in the field, the present application develops a mink 40K liquid chip by targeting 44,801 SNP sites, which is used for breed identification, genetic analysis, etc., and provides technical reference for mink industry development and breeding research.

[0006] To achieve the above purpose, the present application adopts the following technical scheme:

[0007] A mink 40K liquid chip, the chip contains 44,801 SNP sites for detecting 44,801 SNP sites are located to the mink reference genome ASM_NN_V1; the 44,801 SNP sites include SNP molecular marker combinations for mink breed trait related gene positioning, genetic diversity analysis, whole genome association analysis, kinship analysis, breed identification, germplasm improvement and protection; the position information of the 44,801 SNP molecular markers on the reference genome ASM_NN_V1 is shown in Table 1.

[0008] The 44,801 SNP sites include 1815 functional sites; the functional sites are conservative functional SNP sites related to mink body length, total litter size, litter survival number and coat color on the mink genome.

[0009] Among them, the SNP site information related to mink body length is 1-677 shown in Table 1.

[0010] The SNP site information related to mink total litter size is 678-743 shown in Table 1.

[0011] The SNP site information related to mink litter survival number is 744-1336 shown in Table 1.

[0012] The SNP site information related to mink coat color is 1337-1815 shown in Table 1.

[0013] The 44,801 SNP sites also include 377 SNP sites related to economic traits of slaughtered minks, and the position information is shown in Table 1 as 1816-2192.

[0014] Table 1. Position of 44,801 SNP molecular markers

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[0112] The mink 40K liquid chip provided by the application also comprises probes designed according to gene sequences covering 44,801 SNP sites; the probes are designed and synthesized by using a targeted capture sequencing technology according to the positions of the 44,801 target SNP sites and the sequence information on both sides; the length of the probes is 110 bp. The probe design mainly evaluates the specificity, complexity, GC content and the like of the upstream and downstream sequences of the target site; the target site is preferentially placed in the middle position of the probe.

[0113] The application also provides a design method of the mink 40K liquid chip, comprising the following steps:

[0114] (1) using PLINK software to perform linear regression analysis on the weight and coat phenotype data of 133 minks of 7 breeds by using whole genome association analysis, screening the SNP (P<1x10 -5 ) significantly associated with the traits; obtaining known SNPs related to mink weight and reproduction genes;

[0115] (2) obtaining uniformly distributed polymorphic sites by the following filtering conditions: minimum allele frequency (MAF)>0.1; genotype missing rate<0.1; Hardy-Weinberg equilibrium test P>0.001; using Haploview software to select tagSNP in the linkage block;

[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 can analyze the genetic structure of mink populations, accurately evaluate the level of genetic diversity, analyze the differences between individuals by SNP sites, and mine genetic sites related to important traits through whole genome association analysis, thereby providing strong technical support for mink genetics research and industry development. It provides a scientific basis for mink breeding and genetic improvement, and effectively improves the economic benefits of mink breeding. BRIEF DESCRIPTION OF DRAWINGS

[0124] Figure 1 : The distribution histogram of the SNP sites of the mink 40K liquid chip on each chromosome.

[0125] Figure 2 : The PCA clustering results of 7 mink breeds based on the mink 40K liquid chip of the application.

[0126] Figure 3 : The phylogenetic tree results of 7 mink breeds based on the mink 40K liquid chip of the application. DETAILED DESCRIPTION

[0127] The application will be further described in detail below in combination with the drawings and examples, and the examples are only used to explain the application, but not to limit the protection scope of the application. The methods described in the application are conventional methods in the art unless otherwise specified; and the reagents are commercially available products unless otherwise specified.

[0128] Example 1, design and preparation of mink 40K low-density SNP liquid chip

[0129] 1. Establishment of mink SNP database

[0130] The application uses whole genome resequencing data of 133 individuals of 7 mink breeds to obtain highly reliable SNP sites for subsequent screening through sequencing analysis.

[0131] Whole genome resequencing data acquisition: Illumina NovaSeq is used to perform whole genome resequencing on 133 minks (Table 2) of 7 breeds (red-eyed white mink, black mink, red mink, coffee mink, silver blue mink), with an average coverage of 9.18x. BWA software is used to align to the mink reference genome

[0132] ASM_NN_V1, GATK4 software detects SNP, and obtains high-quality mutation sites.

[0133] Table 2. List of mink breeds used

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[0135]

[0136] The target SNP sites screened by the application include the following categories: related to the body weight and body length of mink; related to the total number of litters and the number of litters surviving of mink; related to the fur color of mink; and conserved sites on the genome of mink.

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

[0138] (1) Using whole genome association analysis on the body weight and fur color phenotype data of 133 minks of 7 varieties, linear regression analysis is performed using PLINK software to screen SNP (P < 1 x 10 -5 ) significantly associated with traits; obtaining known SNPs related to mink body weight and reproduction genes; obtaining 1,815 trait sites (Table 1, 1-1815);

[0139] (2) Obtain evenly distributed polymorphic sites by the following filtering conditions: minimum allele frequency (MAF) > 0.1; genotype missing rate < 0.1; Hardy-Weinberg equilibrium test P > 0.001; select tagSNP in linkage block using Haploview software;

[0140] (3) Obtain 377 SNP sites related to economic traits of slaughtered mink (Table 1, 1816-2192); integrate the sites obtained in steps (1) and (2), and align the candidate sites with whole genome sequencing data (Neovison_vison-NCBI-ASM_NN_V1) of mink. The screening criteria are MAF ≥ 0.35, missing rate < 0.1, and heterozygosity < 0.5. Sites that do not meet the requirements are excluded; then select sites with a distance of less than 500 bp, and retain sites with good polymorphism and located on genes; determine 34,123 SNP sites (Table 1, 2193-36315) as VCF background sites;

[0141] (4) Based on the whole genome evenly covered SNP sites selected from the resequencing data of 7 varieties of mink, supplement the background SNP sites: divide the genome into intervals with 200 kb as the standard. If there are functional sites in the interval, no sites are supplemented. If the interval lacks functional sites, one background site is supplemented. The screening principle of the background site is MAF > 0.35, missing rate < 0.1, and heterozygosity < 0.5. Finally, 8486 large GAP supplemented sites are supplemented (Table 1, 36316-44801).

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

[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] Mix and centrifuge the reaction system, and then centrifuge it briefly to concentrate the reaction solution at the bottom of the tube.

[0154] End repair reaction conditions: Place the reaction tube in a PCR instrument, set the hot lid at 82°C, and then connect the reaction adapter.

[0155] Mix the reaction system and centrifuge it briefly to concentrate the reaction solution at the bottom of the tube. It is particularly important to mix the system thoroughly, otherwise the library construction may fail.

[0156] Adapter ligation reaction conditions: Place the reaction tube in a PCR instrument and cancel the hot lid.

[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, shake to mix (note to avoid air bubbles), and then centrifuge briefly after standing for 5 min.

[0158] Magnetic bead washing and drying

[0159] Place the PCR tube in the magnetic stand for at least 3 min until the solution is clear, and then remove the supernatant.

[0160] Keep the PCR tube in the magnetic stand, add 100 μL of 80% ethanol, and then remove the supernatant after incubating at room temperature for 30 s.

[0161] Leave the cap open for 5 min until the ethanol is completely evaporated.

[0162] Magnetic bead drying: Take the PCR tube out of the magnetic stand to ensure that the magnetic beads are completely dried.

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

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

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

[0166] Second magnetic bead purification and washing

[0167] Add 20 μL of DNA purification magnetic beads equilibrated at room temperature for more than 30 min, shake well, and let stand for 5 min, then collect the liquid by brief centrifugation.

[0168] Place in the magnetic stand for at least 3 min, and remove the supernatant.

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

[0170] Open the cap and air dry for 10 min.

[0171] Elution and recovery: Take the PCR tube out of the magnetic stand, add 35 μL of Tris-HCl, shake well, let stand for 5 min, and collect the liquid by brief centrifugation. Place in the magnetic stand until the solution is clear (about 3 min), and transfer the supernatant to a new tube to complete the DNA sequencing library construction.

[0172] Library quality detection

[0173] Quantification: Take 1 μL of the library sample and use Qubit for quantification, requiring a total DNA amount of ≥ 500 ng.

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

[0175] Storage: Short-term storage at 4°C, long-term storage at -20°C.

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

[0177] Magnetic bead and probe mixing: Equilibrate the DNA purification magnetic beads at room temperature for more than 30 min, take an appropriate amount of mink 40K probe and magnetic beads, mix well in a 0.2 mL PCR tube, and centrifuge briefly.

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

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

[0180] Hybridization reagent preparation: Dissolve GenoBaits hybridization reagent at room temperature. If 2X Hyb Buffer crystallizes, heat to 65℃ 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, and centrifuge at 12000 rpm for 1 min. Place at room temperature for 5 min, mix again, and transfer to a 0.2 mL EP tube.

[0182] Thermal cycling and incubation

[0183] Incubate at 95℃ for 10 min (heat cap at 105℃).

[0184] When the temperature drops to 65℃, transfer to a PCR instrument with a heat cap at 75℃ for 1 h of incubation.

[0185] Elution buffer preparation

[0186] Preheat the required elution buffer at 65℃, and store the rest at room temperature.

[0187] If 10X Wash Buffer I and 10X S-W Buffer have precipitated, dissolve them at 65℃.

[0188] Magnetic bead pretreatment

[0189] Place the magnetic beads at room temperature for 10 min before use, and mix by vortexing for 15 s.

[0190] Take 50 μL of magnetic beads to an EP tube, and remove the supernatant after magnetic separation.

[0191] Add 150 μL of 1X Bead Wash Buffer, vortex for 10 s, and remove the supernatant after magnetic separation. Repeat the washing 3 times.

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

[0193] Magnetic bead washing steps

[0194] Add 100 μL of 1X Wash Buffer I preheated at 65℃, shake, and remove the supernatant after magnetic separation.

[0195] Add 150 μL pre-warmed 1X Stringent Wash Buffer, pipette up and down 10 times, let stand for 2 min, then magnetically separate and quickly remove supernatant (repeat once).

[0196] Add 150 μL room temperature 1X Wash Buffer I, shake for 2 min, then magnetically separate and remove supernatant.

[0197] Add 150 μL room temperature 1X Wash Buffer II, shake for 1 min, then magnetically separate and remove supernatant.

[0198] Add 150 μL room temperature 1X Wash Buffer III, shake for 30 s, then magnetically separate and remove supernatant.

[0199] Elution of captured DNA

[0200] Remove the tube from the magnetic stand, add 20 μL Nuclease-Free Water, and resuspend the magnetic beads by pipetting up and down 10 times. Take 10 μL for subsequent PCR amplification and purification, and keep the remaining 10 μL as a backup.

[0201] Amplification, purification, and sequencing of captured DNA

[0202] PCR amplification system preparation: In a 0.2 mL PCR tube, prepare 30 μL system: GenoBaits PCR Master Mix 15 μL, GenoBaits Primer Mix for MGI 1.2 μL, 10 μL captured DNA magnetic bead suspension, ultrapure water 3.8 μL.

[0203] Amplification reaction preparation and conditions: After vortexing, place in a PCR instrument, heat cover 105℃ for amplification (the specific program needs to be supplemented), the product can be stored at 4℃ overnight.

[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, then centrifuge and magnetically separate to remove the supernatant.

[0206] Add 100 μL 80% ethanol for washing, incubate at room temperature for 30 s, then remove the supernatant, and open the cover to air dry for 10 min.

[0207] Elution and storage: Add 35 μL Tris-HCl, shake and let stand, then centrifuge, magnetically separate the supernatant to a new tube, and store at -20℃ (can be stored for one week).

[0208] (5) Library quality detection

[0209] Concentration was determined using Qubit FLuorometer and Qubit dsDNA HS Assay Kit, and average length of fragments was detected by digital electrophoresis system.

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

[0211] (6) High-throughput sequencing

[0212] The captured library was subjected to probe sequencing using a sequencer, and the sequencing results were back posted to the mink reference genome for alignment to obtain genotyping.

[0213] Example Three Quality Evaluation of Mink 40K Liquid Chip Genotyping

[0214] (1) Genotyping stability: repeated detection was performed on part of the mink samples, and the results showed that the consistency of the two genotyping of the 15 repeated samples was 99.3%, and the correlation coefficient was 99.7%, indicating that the detection system had good stability. It can be seen from this that the stability of the mink 40K liquid chip genotyping of the present application is very good. It can be seen from the results that the mink 40K liquid chip of the present application can significantly improve the accuracy and stability of large-scale genotyping detection of mink.

[0215] (2) SNP detection rate: the detection data of the sites of autosomes and X chromosomes showed that the average detection rate of SNPs was as high as 99.88% (standard deviation 0.007); the detection rate of individuals was more than 99%, and the average detection rate of individuals was 99.76% (standard deviation 0.0016), fully confirming that the quality level of the detection method is excellent.

[0216] (3) Genetic structure analysis: principal component analysis (PCA) was carried out based on SNP data: PLINK2 software was used to filter biallelic variations (exclude sites with minor allele frequency (MAF) less than 0.05), and linkage disequilibrium (LD) pruning was performed; based on the filtered SNPs, a pair of genetic distance matrix was calculated by PLINK2, and a neighbor-joining (NJ) tree containing 1000 bootstrap repetitions was constructed using PHYLIP software. The genetic structure of different mink populations was explored by comprehensively analyzing the phylogenetic tree construction, PCA and population structure analysis (using ADMIXTURE software).

[0217] The PCA clustering results of 7 mink varieties based on the mink 40K liquid chip of the present application are shown in Figure 2 , and the phylogenetic tree results are shown in Figure 3The results show that the mink whole genome 40K SNP liquid chip can systematically analyze the genetic structure of mink population, accurately evaluate the level of genetic diversity, analyze the differences between individuals by SNP sites, and mine genetic sites related to important traits through whole genome association analysis, thereby providing strong technical support for mink genetics research and industry development.

Claims

1. A mink 40K liquid phase chip, characterized in that, The chip comprises probes designed according to gene sequences covering 44,801 SNP sites; the probes are used for detecting 44,801 SNP sites located to the mink reference genome ASM_NN_V1; the 44,801 SNP sites comprise a combination of SNP molecular markers for mink breed trait-related gene location, genetic diversity analysis, whole genome association analysis, kinship analysis, breed identification, germplasm improvement and protection; the position information of the 44,801 SNP molecular markers on the reference genome ASM_NN_V1 is shown in Table 1 in the specification; the mink reference genome ASM_NN_V1 is the mink whole genome sequencing data Neovison_vison-NCBI-ASM_NN_V1; The probes are designed and synthesized by using targeted capture sequencing technology according to the position and flanking sequence information of the 44,801 target SNP sites; the length of the probes is 110 bp.

2. The mink 40K liquid chip of claim 1, wherein, The 44,801 SNP sites comprise 1815 functional sites; the functional sites are conservative functional SNP sites on the mink genome related to mink body weight, body length, total litter size, litter survival number and fur color.

3. The mink 40K liquid chip of claim 2, wherein, The SNP site information related to mink body weight is shown in Table 1 as the first to 677.

4. The mink 40K liquid chip of claim 2, wherein, The SNP site information related to mink total litter size is shown in Table 1 as 678 to 743.

5. The mink 40K liquid chip of claim 2, wherein, The SNP site information related to mink litter survival number is shown in Table 1 as 744 to 1336.

6. The mink 40K liquid chip of claim 2, wherein, The SNP site information related to mink fur color is shown in Table 1 as 1337 to 1815.

7. The mink 40K liquid chip of claim 1, wherein, The 44,801 SNP sites further comprise 377 SNP sites related to economic traits of slaughtered minks, and the position information is shown in Table 1 as 1816 to 2192.

8. The application of the 40K liquid chip of the mink in claim 1 in mink genotype detection and whole genome association analysis.

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