SNP marker set for dog identification and dog identification method using same

Abstract

To provide an SNP marker set for dog identification and a dog identification method using the same.SOLUTION: The present disclosure relates to a set of SNP markers for individual identification of dogs and a method for identifying individual dogs using the same. By using 381 single nucleotide polymorphisms (SNPs) on a gene and based on simple results of AA / AB / BB (Homozygote / Heterozygote / Homo mutant), it has been confirmed that individual dogs can be accurately and easily readably identified through the SNP markers and combinations thereof, thereby, the present disclosure enables useful use for animal individual identification.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a SNP marker set for identifying individual dogs and a method for identifying individual dogs using the same, and more particularly to a SNP marker set for identifying individual dogs with improved accuracy and readability and a method for identifying individual dogs using the same. [Background technology]

[0002] Rapid growth of industry and economy has caused social and / or psychological problems. To alleviate such stress and anxiety, an increasing number of households are keeping pets, and the pet industry, including pet insurance, is developing rapidly. However, the growth of the pet industry has brought about social, ethical and industrial problems.

[0003] Among these, the loss and identification of pets is a major problem. When a pet gets lost due to an accident, contrary to the owner's wishes, there is a limit to how much the pet's identity can be confirmed through its appearance and other characteristics. In addition, lost pets cause problems such as aggression, excrement, and littering of general garbage, which has become a social issue. Furthermore, in the United States, approximately 6.5 million pets, including those living outdoors, are left at animal shelters, and approximately 1.5 million are euthanized every year. Furthermore, in the UK, pet insurance fraud increased four-fold in 2011, becoming a social issue.

[0004] The root cause of these problems is the lack of official records of pet ownership. China is currently considering how to create a database of pet identity information at the government level. One method currently being developed is subcutaneous microchip injection, which verifies the identity of pets through a serial number embedded in the chip.

[0005] However, because chip readers are easy to use and purchase, they can be targets for crime, and the information about owners contained in the chips can be easily used for fraud. Also, implanting microchips in other pets can be another route for pet insurance fraud, and there are limitations to holding owners responsible for social problems caused by pets that are caused by chips being removed by their owners if the loss of the pet is arbitrary.

[0006] Yet another method for identifying pets while minimizing the limitations of such microchips is a method based on genetic markers. This method uses the genetic information of an individual, and can identify the pet and confirm the owner only if the individual and the registered record are known. In the method based on genetic markers, various genetic markers are used to prevent genetic polymorphism, heterozygosity, and phylogeny. The markers may include autosomal microsatellites markers, Y chromosome markers, mitochondrial DNA (mtDNA), and single nucleotide polymorphisms (SNPs).

[0007] Among the markers, SNPs are the most abundant in the genome, have a lower mutation rate than polymorphic minisatellite markers and mtDNA, and can be analyzed efficiently once found. In addition to being useful for rapid, large-scale, and economical genotyping, SNPs occur frequently in mammalian genomes and can be used as an efficient tool for ecological, conservation, population, and evolutionary studies.

[0008] As of 2018, the number of pets in Korea is estimated to be about 6.35 million, and the top 20 countries in the world are estimated to have a total of 578.83 million pets. The most commonly used method for identifying individual dogs is using Short Tandem Repeat Markers (STR Markers), which is highly accurate, but has the problem that multiple genotypes can be identified from one marker, and differences in humidity and temperature can occur at the stage of identifying the genotype depending on its length.

[0009] Under such circumstances, the present inventors have conducted intensive research to accurately distinguish and identify specific pet dogs from other dogs. As a result, they have confirmed that individual dogs can be accurately and easily identified through the SNP markers and their combinations based on the simple results of AA / AB / BB (Homozygote / Heterozygote / Homo mutant) using 381 SNPs (Single Nucleotide Polymorphism) on the genes without using STR markers, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention has been made in consideration of the above circumstances, and its object is to provide a SNP marker set for identifying individual dogs, which includes one or more SNPs selected from a group consisting of 381 SNPs in a dog genome dataset.

[0011] Another object of the present invention is to provide a composition for identifying individual dogs, comprising a preparation capable of detecting or amplifying the SNP marker set for identifying individual dogs.

[0012] It is still another object of the present invention to provide a kit or microarray for identifying individual dogs, which comprises the composition for identifying individual dogs.

[0013] Another object of the present invention is to provide a method for identifying individual dogs, comprising the steps of i) isolating nucleic acid from a dog whose individual is to be identified, and ii) confirming the SNP marker set from the nucleic acid. [Means for solving the problem]

[0014] The advantages and features of the present invention, as well as the methods for achieving them, will become apparent from the following detailed description of the embodiments. However, the present invention is not limited to the embodiments disclosed below, and may be embodied in various different forms. However, the embodiments are provided to complete the disclosure of the present invention and to allow those skilled in the art to fully understand the scope of the present invention, and the present invention is defined only by the scope of the claims.

[0015] The terms used in the present specification are for the purpose of describing the embodiments and are not intended to limit the present invention. In the present specification, the singular form includes the plural form unless otherwise specified. The terms "comprises" and / or "comprising" used in the specification do not exclude the presence or addition of one or more other components other than the components mentioned. The terms "and / or" used in the specification include each and every combination of one or more of the components mentioned. Even if "first", "second", etc. are used to describe various components, it is of course not limited to these terms. These terms are used simply to distinguish one component from another component. Therefore, it goes without saying that the first component mentioned below can also be the second component within the technical spirit of the present invention.

[0016] Unless otherwise defined, all terms (including technical and scientific terms) used in this specification are used as meanings commonly understood by ordinary skilled artisans in the technical field to which the present invention belongs. Furthermore, terms defined in commonly used dictionaries are not interpreted ideally or excessively unless clearly and specifically defined.

[0017] The present invention provides a SNP marker set for identifying individual dogs, which includes all 381 SNPs listed in Table 41 in the dog genome dataset.

[0018] The SNP of the present invention is a single nucleotide polymorphism, which means a variation in a DNA sequence that occurs when a single nucleotide (A, T, C, or G) in the genome differs between individuals of the same species or between paired chromosomes of an individual.

[0019] For example, when three DNA fragments from different individuals contain a single base difference (e.g., AAGT[A / A]AG, AAGT[A / G]AG, AAGT[G / G]AG), these are called two alleles (A or G), and generally most SNPs have two alleles.

[0020] Within a population, a SNP can be assigned a minor allele frequency (MAF; the lowest allele frequency at a locus found in a particular population). A single base can be changed (substitution), removed (deleted) or added (inserted) to a polynucleotide sequence, and a SNP can induce a change in the translation frame. In this specification, the "SNP marker" can be described interchangeably with "SNP".

[0021] The marker of the present invention means a biomarker, and in the present invention has the meaning particularly of a label that can identify an individual dog.

[0022] As the number of SNP markers included in the marker set of the present invention increases, the number of cases for individual identification increases and the accuracy can be improved.

[0023] Each SNP marker that constitutes the SNP marker set is an SNP marker described in the table, and the location on the dog genome and its surrounding sequences described in this information can be easily understood by a person skilled in the art through a dog genome database.

[0024] In a specific embodiment of the present invention, it was confirmed that a specific individual can be 100% identified using the SNP marker set of the present invention.

[0025] The nucleotide sequences used in the present invention are also considered to include sequences that show substantial identity to the sequences described, taking into consideration mutations that have biologically equivalent activity. The substantial identity refers to a sequence that shows at least 60% homology, specifically 70% homology, more specifically 80% homology, and even more specifically 90% homology, when the sequences of the present invention are aligned to any other sequence to maximize their correspondence and the aligned sequences are analyzed using algorithms commonly used in the art.

[0026] The alleles of the present invention are base sequences located in pairs at the loci of a pair of homologous chromosomes, and different alleles can often exhibit different traits.

[0027] A base, as used herein, is used synonymously with a nucleotide, which is a deoxyribonucleotide or ribonucleotide that exists in single- or double-stranded form, and can include analogs of naturally occurring nucleotides.

[0028] The breed of the dog is not limited to its type, but may be preferably selected from the group consisting of Beagle, Water dog, Maltese, Pomeranian, Poodle, Yorkshire terrier, Shih Tzu, Dachshund, Chihuahua, American Pit bull terrier, and crossbreeds thereof, and more preferably may be Beagle, Chihuahua, or American Pit bull terrier.

[0029] The present invention also provides a composition for identifying individual dogs, comprising a preparation capable of detecting or amplifying the SNP marker set for identifying individual dogs.

[0030] In the present invention, amplification means that in order to activate the expression of a particular trait in a certain cell, a chromosomal portion containing a gene or group of genes that controls that trait is specifically and repeatedly replicated to increase its number.

[0031] The preparation capable of detecting an SNP marker of the present invention means a preparation capable of specifically binding to and recognizing an SNP marker contained in the SNP marker composition, or capable of detecting and amplifying the SNP.

[0032] The preparation may be a probe capable of specifically binding to a polymorphic site containing the SNP marker, or a primer or primer set capable of characteristically detecting the SNP marker site or specifically detecting and amplifying a polynucleotide containing the SNP marker or a complementary polynucleotide thereto.

[0033] The probe of the present invention refers to a linear oligomer of natural or modified monomers or linkages, which includes deoxyribonucleotides and ribonucleotides and can specifically hybridize to a target nucleotide sequence, and which may be naturally occurring or artificially synthesized. The probe of the present invention may be single-stranded, preferably an oligodeoxyribonucleotide. The probe of the present invention may include natural dNMPs (i.e., dAMP, dGMP, dCMP, and dTMP), nucleotide analogs, or derivatives. The probe of the present invention may also include ribonucleotides.

[0034] The primer of the present invention means a single-stranded oligonucleotide that can act as a starting point for template-directed DNA synthesis under suitable conditions (i.e., four different nucleoside triphosphates and a polymerization reaction enzyme) in a suitable temperature and a suitable buffer solution. The appropriate length of the primer can vary depending on various factors, such as temperature and the use of the primer. In addition, the sequence of the primer does not need to have a sequence that is completely complementary to a part of the sequence of the template, and it is sufficient that the primer has sufficient complementarity within a range in which it can hybridize with the template and act as a primer. Therefore, the primer in the present invention does not need to have a sequence that is completely complementary to the nucleotide sequence of the template gene, and it is sufficient that the primer has sufficient complementarity within a range in which it can hybridize with this gene sequence and act as a primer. Furthermore, the primer according to the present invention is preferably one that can be used in a gene amplification reaction. The amplification reaction means a reaction for amplifying a nucleic acid molecule, and such gene amplification reactions are well known in the art and may include, for example, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), ligase chain reaction (LCR), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), etc.

[0035] In this case, the sequences of the primers can be modified based on PCR conditions and the lengths of the sense and antisense primers known in the art.

[0036] Specific examples may include methylation, capping, nucleotide substitutions or internucleotide modifications, such as modifications to uncharged linkers (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) or charged linkers (e.g., phosphorothioates, phosphorodithioates, etc.).

[0037] The sequence of the primer can be modified to improve the specificity and binding strength to the SNP marker, and multiple primers having different base sequences can be used for the same SNP marker. More specifically, the length of each primer can be adjusted by adding repeated bases to its end, and the type of base can be A, T, G, C, or a combination thereof, but there is no limitation on the type of base as long as it has a repeated sequence.

[0038] The present invention also provides a kit for identifying an individual dog, comprising the composition for identifying an individual dog.

[0039] The kit of the present invention can distinguish and identify individual dogs by detecting and identifying the SNP markers of the present invention.

[0040] The kit may be a PCR kit that includes essential elements required for performing PCR. For example, the PCR kit may include, in addition to the primers specific to each of the SNP markers, a test tube or other suitable container, a reaction buffer (varies in pH and magnesium concentration), deoxynucleotides (dNTPs), dideoxynucleotides (ddNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor, DEPC-water, sterile water, etc. Also, the kit may include a primer pair specific to a gene used as a quantitative control group.

[0041] The present invention also provides a microarray for identifying individual dogs, comprising the composition for identifying individual dogs.

[0042] The microarray of the present invention may contain DNA or RNA polynucleotides. The microarray is a typical microarray, except that the probe polynucleotide contains the polynucleotide of the present invention. Methods for immobilizing the probe polynucleotide on a substrate to produce a microarray are well known in the art.

[0043] The hybridization of nucleic acids on the microarray and the detection of the hybridization result are well known in the art. For example, the detection can be performed by labeling a nucleic acid sample with a labeling substance capable of generating a detectable signal, including a fluorescent substance, hybridizing the sample on the microarray, and detecting the signal generated from the labeling substance to detect the hybridization result. The fluorescent substance can be pyrene, Texas Red, fluorescein, BODYPY, tetramethylrhodamine, Alexa, cyanine, allophycocyanin, Nile Blue, Nile Red, or other fluorescent substances that generate fluorescence. Also, a fluorescent substance with a high quantum yield can be used. Furthermore, the fluorescent substance can be a hydrophilic or hydrophobic dye.

[0044] The present invention also provides a method for identifying individual dogs, comprising the steps of i) isolating nucleic acid from a dog whose individual is to be identified, and ii) confirming the SNP marker set from the nucleic acid.

[0045] The nucleic acid of the present invention can be isolated from a sample obtained from the dog to be identified, and can be isolated from a sample selected from the group consisting of blood, epidermis, urine, feces, hair, saliva, buccal cells, muscle and organs of the dog.

[0046] Genomic DNA can be extracted from the sample using a phenol / chloroform extraction method, SDS extraction method, or CTAB separation method commonly used in the art, or a commercially available DNA extraction kit.

[0047] The isolated nucleic acid can be used as a template to carry out an amplification reaction using a primer set specific thereto to amplify the target sequence. Methods for amplifying the target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, strand displacement amplification, or amplification using Qβ replicase, or any method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method for amplifying a target nucleic acid from a primer set that specifically binds to the target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits can be used. PCR can be carried out using a PCR reaction mixture containing multiple components required for PCR reaction and known in the art.

[0048] The step ii) of the present invention can be carried out by applying various methods known in the art that can be used to identify a specific sequence, i.e., to identify the base at the SNP site. For example, these methods may include, but are not limited to, fluorescence in situ hybridization (FISH), DNA sequencing, PFGE analysis, Southern blot analysis, single-stranded conformational analysis (SSCA), RNase protection analysis, DAT blot analysis, denaturing gradient gel electrophoresis (DGGE), methods using proteins that recognize nucleotide mismatches (e.g., the mutS protein of E. coli), allele-specific probe hybridization, allele-specific amplification, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism, allele-specific PCR, and mass array analysis.

[0049] After step ii), the method may further include a step of confirming one or more pieces of information selected from the group consisting of the dog's blood type, physical characteristics, weight, sex, and age.

[0050] The identification results of individual dogs of the present invention can be communicated to the requester in the usual form and manner known in the art, for example, but not limited to, the test results can be communicated through telephone, letter, email, publication on a web page, etc.

[0051] The test results can provide a numerical representation of the degree of identification, or can provide only the identification result. They can include information about the dog as well as the owner. Effect of the Invention

[0052] The present invention provides a SNP marker set for individual dog identification and a method for identifying individual dogs using the same. More specifically, the present invention confirms that individual dogs can be accurately and easily identified through the SNP markers and their combinations based on the simple results of AA / AB / BB (Homozygote / Heterozygote / Homo mutant) using 381 SNPs (Single Nucleotide Polymorphism) on the gene, and can be usefully used for individual animal identification. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The present invention will now be described in more detail with reference to the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, and includes modifications of the technical ideas equivalent thereto.

[0054] Example 1. Sample preparation and DNA isolation The specimens used in this study consisted of six dog breeds related to one another, and DNA extraction was performed on blood samples from the six dogs using the Exgene Clinic SV kit (Gene All). The six dog breeds were two Chihuahuas, three Beagles, and one American Pit Bull Terrier.

[0055] Example 2. SNP genotyping The DNA isolated in Example 1 was analyzed using i-StarMAX TM Multiplex PCR was performed using the ProFlex PCR System (Applied Biosystems) with the II DNA Polymerase kit (iNtRON).

[0056] For library construction used in NGS, 1 ng of DNA and the ampliseq library kit were used, starting with 95°C for 5 minutes, followed by 30 cycles of 95°C for 30 seconds, 60°C for 75 seconds, and 72°C for 30 seconds, with a final reaction at 72°C for 5 minutes.

[0057] Experimental example 1. Identification of individual dogs using selected SNP markers The products amplified by PCR in Example 2 were subjected to an experiment using an IonS5 sequencing machine to obtain the genotype of each sample, and then the individuals were identified by the genotype.

[0058] 1-1. Preliminary testing of dog population Using 381 SNPs (Single Nucleotide Polymorphisms) on the gene, we confirmed that the accuracy of the results was increased by making the results easier to interpret based on the simple results of AA / AB / BB (Homozygote / Heterozygote / Homo mutant).

[0059] In addition, when using a genotyping assay with TaqMan probe to determine whether any of the three genotypes (AA / AB / BB) were SNPs, the ease of interpretation was increased through NGS (Next Generation Sequencing).

[0060] Therefore, differences between individuals were confirmed as shown in Table 1 below.

[0061] [Table 1]

[0062] 1-2. SNP selection for each individual 381 SNPs in the gene were selected, and the genotypes were obtained from each sample as shown in Tables 2 to 40 below, and it was confirmed that individuals could be identified by the genotypes.

[0063]

Table 2

[0064]

Table 3

[0065]

Table 4

[0066]

Table 5

[0067]

Table 6

[0068]

Table 7

[0069]

Table 8

[0070]

Table 9

[0071]

Table 10

[0072]

Table 11

[0073]

Table 12

[0074]

Table 13

[0075]

Table 14

[0076]

Table 15

[0077]

Table 16

[0078]

Table 17

[0079]

Table 18

[0080]

Table 19

[0081]

Table 20

[0082]

Table 21

[0083]

Table 22

[0084]

Table 23

[0085]

Table 24

[0086]

Table 25

[0087]

Table 26

[0088]

Table 27

[0089]

Table 28

[0090]

Table 29

[0091]

Table 30

[0092]

Table 31

[0093]

Table 32

[0094]

Table 33

[0095]

Table 34

[0096]

Table 35

[0097]

Table 36

[0098]

Table 37

[0099]

Table 38

[0100]

Table 39

[0101]

Table 40

[0102]

Table 41

[0103] As described above, by confirming differences between individuals using 381 SNPs in the gene, individuals can be accurately and easily identified.

[0104] Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments, and can be manufactured in various different forms, and a person skilled in the art to which the present invention belongs can understand that the present invention can be embodied in other specific forms without changing the technical idea or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not restrictive.

Claims

1. A SNP marker set for individual dog identification that includes all 381 SNPs listed in Table 41 in the dog genome data set.

2. The SNP marker set for individual identification of dogs according to claim 1, characterized in that the dog breed is selected from the group consisting of Beagle, Water Dog, Maltese, Pomeranian, Poodle, Yorkshire Terrier, Shih Tzu, Dachshund, Chihuahua, American Pit Bull Terrier, and crossbreeds thereof.

3. A composition for identifying individual dogs, comprising a preparation capable of detecting or amplifying the SNP marker set for identifying individual dogs described in claim 1.

4. The composition for identifying individual dogs according to claim 3, characterized in that the preparation is a primer that specifically binds to the SNP marker or that amplifies a polynucleotide containing the SNP marker.

5. The composition for identifying individual dogs according to claim 3, characterized in that the preparation is a probe that specifically binds to the SNP marker or that amplifies a polynucleotide containing the SNP marker.

6. A kit for identifying individual dogs, comprising the composition for identifying individual dogs according to claim 3.

7. A microarray for identifying individual dogs, comprising the composition for identifying individual dogs according to claim 3.

8. i) isolating nucleic acid from a dog whose individual is to be identified; ii) identifying the SNP marker set according to claim 1 from the nucleic acid; A method for identifying an individual dog, comprising:

9. The method for identifying an individual dog according to claim 8, characterized in that the nucleic acid is isolated from one or more selected from the group consisting of blood, epidermis, urine, feces, hair, saliva, oral cells, muscles and organs of the dog.

10. The method for identifying an individual dog according to claim 8, further comprising, after step ii), a step of confirming one or more pieces of information selected from the group consisting of the dog's blood type, external characteristics, weight, sex, and age.

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