KR101766273B1

KR101766273B1 - Composition for Breed Discrimination of Dog comprising mitochondria SNP Markers and Diagnosing Method using the Same

Info

Publication number: KR101766273B1
Application number: KR1020150147527A
Authority: KR
Inventors: 최봉환; 임다정
Original assignee: 대한민국
Priority date: 2015-10-22
Filing date: 2015-10-22
Publication date: 2017-08-10
Also published as: KR20170047469A

Abstract

The present invention relates to a composition for identifying dog breeds containing SNP markers of mitochondrial DNA and a method for identifying dog breeds using the same, and the present invention provides a method for identifying dog breeds quickly, accurately and economically, including identification of domestic dogs and foreign breed dogs There is an advantage that it can be done.

Description

Technical Field [0001] The present invention relates to a composition for identifying a dog breed including a SNP marker of mitochondrial DNA, and a method for identifying a dog breed using the same.

The present invention relates to a composition for identifying dog breeds containing SNP markers of mitochondrial DNA, and a method for identifying the breeds using the same. More specifically, the present invention relates to a method for identifying dog breeds quickly and accurately and economically And a SNP marker of a mitochondrial DNA which can be used as a marker for identifying a dog breed.

Dogs are world-class animals, with about 200 varieties. In order to protect dog breeds and domestic dogs, which are highly utilized as special dogs, selective breeding and utilization are possible through genome studies.

In 2008, the market size of dogs in the United States was about $ 43.2 billion, with about 77 million dogs and 39% of the total feed market. Shepard is a varietal made in 1899, characterized by learning ability, endurance, and body shape. Dogs, dogs, special dogs, dogs, Shepard is one of the top dogs in dog sales, led by 500,000 enthusiasts and 50,000 dogs produced annually. The market size is about 12 trillion won, and China is about 680 million yuan of pet food market in 2008 when 11 million dogs are raised in Japan.

In addition, the pet dog industry is growing in Korea, but the number of dogs is increasing accordingly. As the number of dogs increases, problems such as disease infections such as rabies and natural destruction are emerging. Therefore, to reduce the incidence of dogs, it is being implemented by introducing a system to register dogs. The companion dog registration system includes an embedded wireless identification device, i.e., inserting the microchip into the animal's body, attaching an external wireless identification device, or attaching a registration identification tag. However, in the case of the dog companion dog registration system, there is a concern about side effects caused by inserting the foreign object into the dog dog 's body. In addition, when it is an external type, it can be easily separated, so its effectiveness is questionable.

Therefore, there is a need for more stable and practical breeding and breeding identification markers that can be used for foreign dog identification, breed identification and individual identification.

Korean Patent Registration No. 10-1156047 (Jun. 26, 2012) discloses a supersatellite marker composition and a method of identifying an individual using the supersatellite marker composition according to the prior art of the present invention.

Korea Patent Registration No. 10-1156047 (June 26, 2012)

It is an object of the present invention to provide a composition for identifying breeds of dogs, which comprises a single base polymorphism (SNP) marker of mitochondrial DNA (mtDNA) of SEQ ID NO: 1.

Another object of the present invention is to provide a method for identifying breeds of dogs using the composition.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a composition for identification of individual breeds comprising a single nucleotide polymorphism (SNP) marker of mitochondrial DNA (mtDNA) having the nucleotide sequence shown in SEQ ID NO: 1.

According to one embodiment of the present invention, the SNP marker comprises a nucleotide C at position 15595, a nucleotide T at position 15611, a nucleotide T at position 15612, a nucleotide C at position 15595, a nucleotide T at position 15612, The nucleotide A at position 15618 is G, the nucleotide T at position 15620 is C, the nucleotide A at position 15627 is G, the nucleotide C at position 15632 is T, the nucleotide T at position 15639 is A or G, the nucleotide A at position 15643 is G, The nucleotide T at position 15650 is C, the nucleotide G at position 15652 is A, the nucleotide T at position 15665 is C, the nucleotide C at position 15750 is C, the nucleotide T at position 15800 is C, the nucleotide A at position 15811 is G, Nucleotide T at position 15815 is C, nucleotide C at position 15912 is at T, nucleotide C at position 15955 is at T, nucleotide A at position 16003 is G, position A nucleotide A of position 16084 is C, a nucleotide A of position 16083 is G, a nucleotide A of position 16248 is G, a nucleotide A of position 16268 is G, a nucleotide G of position 16276 is A, a nucleotide G of position 16306 is A, At least one SNP marker selected from the group consisting of a nucleotide G is A, a nucleotide G at position 16338 is A, a nucleotide G at position 16346 is A, and a nucleotide T at position 16671 is C.

According to another aspect of the present invention, there is provided a kit for identifying an individual breed comprising the composition.

According to still another aspect of the present invention, there is provided a microarray for identifying breeds of dogs comprising the composition.

According to another aspect of the present invention, there is provided a method for producing a recombinant vector comprising the steps of: Amplifying the gene using the separated genomic DNA as a template and using primers set forth in SEQ ID NOS: 2 and 3; And purifying the amplified gene product to analyze the base sequence or hybridizing the marker with the marker of claim 2.

According to one embodiment of the present invention, identification of individual breeds, identification of individuals, and identification of parents can be performed quickly, accurately, and economically using SNP markers in mitochondrial DNA.

In particular, according to the present invention, there is provided a highly economical assay method for tracking maternal heredity using SNP markers of mitochondrial DNA.

Also, according to an embodiment of the present invention, it is possible to distinguish between domestic dogs and foreign dogs using specific expression of SNP markers of mitochondrial DNA.

According to one embodiment of the present invention, breed identification, individual identification, and parent identification can be performed in a faster, more accurate and economical manner.

In addition, the composition of the present invention can be very usefully used for the preparation of a scientific basis for a dog breeding regimen and for supplementation of a tracking history system.

1 is a view showing SNP markers of mitochondrial DNA according to an embodiment of the present invention.

The present invention is capable of various modifications and various embodiments, and specific embodiments are described in detail in the description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. Hereinafter, the present invention will be described in more detail.

Certain terms are hereby defined for convenience in order to facilitate a better understanding of the present invention. Unless otherwise defined herein, scientific and technical terms used in the present invention shall have the meanings commonly understood by one of ordinary skill in the art. Also, unless the context clearly indicates otherwise, the singular form of the term also includes plural forms thereof, and plural forms of the term should be understood as including its singular form.

The present invention can provide a composition for identifying an individual breed which is highly economical to track the maternal heredity using SNP markers of mitochondrial DNA.

According to one embodiment of the present invention, the SNP marker comprises a nucleotide C at position 15595, a nucleotide T at position 15611, a nucleotide T at position 15612, a nucleotide C at position 15595 of the mitochondrial DNA (mtDNA) The nucleotide A at position 15618 is G, the nucleotide T at position 15620 is C, the nucleotide A at position 15627 is G, the nucleotide C at position 15632 is T, the nucleotide T at position 15639 is A or G, the nucleotide A at position 15643 is G, The nucleotide T at position 15650 is C, the nucleotide G at position 15652 is A, the nucleotide T at position 15665 is C, the nucleotide C at position 15750 is C, the nucleotide T at position 15800 is C, the nucleotide A at position 15811 is G, Nucleotide T at position 15815 is C, nucleotide C at position 15912 is at T, nucleotide C at position 15955 is at T, nucleotide A at position 16003 is G, position A nucleotide A of position 16084 is C, a nucleotide A of position 16083 is G, a nucleotide A of position 16248 is G, a nucleotide A of position 16268 is G, a nucleotide G of position 16276 is A, a nucleotide G of position 16306 is A, At least one SNP marker selected from the group consisting of a nucleotide G is A, a nucleotide G at position 16338 is A, a nucleotide G at position 16346 is A, and a nucleotide T at position 16671 is C.

The present invention can utilize all 65 SNPs to identify alleles by specific SNPs that can appear in the breeds and to use them for breed identification, individual identification, and paternity discrimination in a faster, more accurate and economical manner.

According to another aspect of the present invention, there is provided a kit for identifying breeds of dogs comprising the composition.

According to one embodiment of the present invention, the kit comprises an agent capable of detecting or amplifying the SNP marker.

In the present invention, the kit may be, but is not limited to, an RT-PCR kit or a microarray chip kit including a preparation capable of detecting or amplifying SNP markers.

The RT-PCR kit can comprise a respective pair of primers specific for the marker gene and can be used in combination with other test tubes or other appropriate containers, reagents necessary for PCR amplification, such as buffers, DNA polymerases (e.g., Thermus aquaticus Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermisflavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs.

The microarray chip kit may be a microarray kit for identifying individual breeds containing the DNA markers.

In the present invention, a microarray means a group of polynucleotides immobilized on a substrate at a high density, and the polynucleotide group means a microarray immobilized in a constant region. Such microarrays are well known in the art. The microarrays are described, for example, in U.S. Patent Nos. 5,445,934 and 5,744,305, the contents of which are incorporated herein by reference.

The kit may be made from a number of separate packaging or compartments containing the reagent components described above.

According to another embodiment of the present invention, the kit comprises a set of primers represented by SEQ ID NOS: 2 and 3.

According to another aspect of the present invention, Amplifying the gene using the separated genomic DNA as a template and using primers set forth in SEQ ID NOS: 2 and 3; And purifying the amplified gene product to analyze the base sequence or hybridizing the marker with the marker of claim 2.

The present invention separates SNP markers that are unique to Korean domestic dogs and foreign breeds as well as SNP markers that are unique to each breed, and quickly, accurately, and economically discriminates dog breeds, individual identities, and siblings using the markers .

The target sequence can be amplified by performing amplification reaction using the separated genomic DNA as a template and the primer set of SEQ ID NOS: 2 and 3. Methods for amplifying a 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 with Q [beta] replicase, or any other suitable method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used. The PCR can be carried out using a PCR reaction mixture containing various components known in the art necessary for the PCR reaction.

In the present invention, hybridization refers to a process in which two complementary strands of a nucleic acid are combined to form a double stranded molecule (hybrid). In the method of the present invention, the hybridization is carried out under high stringency hybridization conditions.

To detect the degree of hybridization, the target sequence may be labeled with a detectable labeling substance. In this embodiment, the labeling substance may be a fluorescent, phosphorescent or radioactive substance, but is not limited thereto. Preferably, the labeling substance is Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. In addition, if the radioactive isotope such as P32 or S35 is added to the PCR reaction solution during the PCR, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive.

According to one embodiment of the present invention, in the amplified gene product, when the nucleotide A at the position 16248 of SEQ ID NO: 1 contains at least one of the G SNP marker and the nucleotide A nucleotide A at position 16268, do.

According to another embodiment of the present invention, in the amplified gene product, the nucleotide G at position 16276 of SEQ ID NO: 1 is A, the nucleotide G at position 16306 is A, the nucleotide G at position 16326 is A, and the nucleotide G at position 16338 is A, a nucleotide G at position 16346 is A, and a nucleotide T at position 16671 is C, it is judged that it is an annihilator if it contains at least one SNP marker selected from the group consisting of SNP markers.

According to another embodiment of the present invention, in the amplified gene product, the nucleotide A at position 15627 of SEQ ID NO: 1 is G, the nucleotide T at position 15639 is A, the nucleotide at nucleotide 15814 is T, and the nucleotide at position 16025 is T Of SNP markers are included.

According to another embodiment of the present invention, in the amplified gene product, if it contains at least one of the SNP marker at position 15618 of nucleotide A in G at position G and the SNP marker at position 15811 in nucleotide A at position 15811 .

According to another embodiment of the present invention, in the amplified gene product, the nucleotide A at position 15618 of SEQ ID NO: 1 is G, the nucleotide T at position 15665 is C, the nucleotide A at position 15811 is G, and the nucleotide T at position 16025 is C, the nucleotide A at position 16248 is G, the nucleotide A at position 16268 is G, the nucleotide G at position 16276 is A, the nucleotide G at position 16306 is A, the nucleotide G at position 16326 is A, the nucleotide G at position 16338 is A, At least one SNP marker selected from the group consisting of a nucleotide G at position 16346 of A and a nucleotide T at position 16671 of C is C is considered to be a domesticated species.

According to a specific embodiment of the present invention, it is judged that the SNP marker contains only SNP markers having nucleotide T at position 16025 of SEQ ID NO: 1.

According to another embodiment of the present invention, in the amplified gene product, the nucleotide T at position 15620 of SEQ ID NO: 1 is C, the nucleotide A at position 15627 is G, the nucleotide T at position 15639 is A, and the nucleotide C at position 15814 is T and the nucleotide C at position 15955 contains only SNP markers, it is judged to be a Labrador retriever.

According to another embodiment of the present invention, in the amplified gene product, a group consisting of SNP markers in which the nucleotide T at position 15611 of SEQ ID NO: 1 is C, the nucleotide A at position 15643 is G, and the nucleotide at position 15650 is C It is judged that it is a German shepter.

According to another embodiment of the present invention, in the amplified gene product, at least one SNP marker among the SNP markers having nucleotide C at position 15750 and nucleotide A at position 16083 is included in the amplified gene product .

According to another embodiment of the present invention, in the amplified gene product, the nucleotide C at position 15595 of SEQ ID NO: 1 is T, the nucleotide T at position 15611 is C, the nucleotide T at position 15612 is C, and the nucleotide T at position 15620 is C, nucleotide A at position 15643 is G, nucleotide T at position 15650 is C, nucleotide C at position 15750 is T, nucleotide T at position 15815 is C, nucleotide A at position 16003 is G, and nucleotide A at position 16083 is G Of the SNP markers are included in the SNP markers.

According to a specific embodiment of the present invention, it is judged that a Labrador Retriever is included if only the SNP marker having the nucleotide T at position 15620 of SEQ ID NO: 1 among the SNP markers is included.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example

Disclosure Materials and Genomic DNA ( Genomic DNA) extraction

Materials used in this study include 32 JinDo Dogs, 45 Sapsal Dogs, 20 German Shepherd, 20 Labrador Retrievers collected from the Jindo Doge Office, Sparrow Shelter, Detection Dog Center, (Labrador Retriever) 8, PoongSan Dog 19, DongKangEi 43, and Belgian Malinois 181 strains of blood DNA were used. Table 1 shows the population by breed.

Polymerase chain reaction ( PCR ) primer Production and gene amplification

For mtDNA sequencing, genomic information (D-loop 15,458-16,055 site of U96639) registered in National Center for Biotechnology Information (NCBI) GenBank was used, and Primer3 program (http://bioinfo.ut.ee/primer3- 0.4.0 /). The prepared primer was designated as forward primer F (5'-TCC CTG ACA CCC CTA CAT TC-3 '; SEQ ID NO: 2) and reverse primer R (5'-GCG ACT CAT CTT GGC ATT TT-3' The nucleotide sequence was confirmed by overlapping the nucleotide sequences. mtDNA amplification PCR was performed by adding 1 μg of gDNA, 1 μl of 10 × buffer, 0.8 μl of 2.5 mM dNTP, 0.2 μl of primer (10 pmol / μl) and 0.1 μl of Hot start Taq DNA polymerase (2 U / μl) The solution was adjusted to 20 μl. The mixture was first denatured at 94 ° C for 1 min, bound for 1 min at 60 ° C, elongated at 72 ° C for 2 min, initiated at 95 ° C for 11 min using a Verti 96-well Thermal Cycler (Applied Biosystem, CA, USA) After 25 cycles, final elongation was carried out at 60 ° C for 60 minutes.

Amplification product Validation using purification and sequencing

MultiScreen HTS ^™ PCR (MILLIPORE, Seoul, Korea) was used for amplification products. The PCR product was purified using isopropanol and ethanol, and 10 μl of Hi-Di formamide was added to the 96-well plate pellet. . After denaturation at 95 ° C for 5 minutes, sequencing was performed using a sequencing analyzer ABI 3730 XL (Applied Biosystems, Foster City, Calif., USA) equipped with a 36 cm capillary.

Table 2 shows the location and number of mtDNA mutations in Korean native dogs and overseas varieties.

As shown in Table 2, A total of 43 dogs were found at the five locations, 15639, 15814, 16025, 16248, and 16268, respectively. The specific SNPs found only in Tokyo dogs were found to be 16248 and 16268, totaling 2 SNPs.

A total of 45 spermatozoa were found in 11 positions, at positions 15627, 15639, 15652, 15665, 15814, 16276, 16306, 16326, 16338, 16346 and 16671. A total of 6 SNPs were found at the positions 16276, 16306, 16326, 16338, 16346,

A total of 19 Poongsan dogs were found in four locations, 15627, 15639, 15814 and 16025. There was no specific SNP that appeared only in P. aeruginosa, but SNP was also found in P. aeruginosa at the site 16025 of the SNP found only in the red canopy.

The total number of Jindo dogs was 32, and mutations were found at 11 sites in total at positions 15618, 15627, 15632, 15639, 15652, 15800, 15811, 15814, 15912, 15955 and 16025. The specific SNPs found only in Jindo dogs were located at positions 15618 and 15811, totaling two SNPs.

There are a total of 12 places where mutations only occur in Korean native breeds, namely, 15618, 15665, 15811, 16025, 16248, 16268, 16276, 16306, 16326, 16338, 16346, and 16671.

A total of eight Labrador retrievers were found in five locations, at positions 15620, 15627, 15639, 15814 and 15955.

The mutation was found at a total of sixteen positions in a total of 20 German shepherds at positions 15595, 15611, 15612, 15620, 15627, 15632, 15639, 15643, 15650, 15652, 15800, 15814, 15815, 15912, 15955 and 16003. A total of 3 SNPs were found at the positions of 15611, 15643 and 15650,

A total of 14 maloneoons were found at 13 positions in total at positions 15595, 15612, 15632, 15639, 15652, 15750, 15800, 15814, 15815, 15912, 15955, 16003, 16083. Specific SNPs found only in Mala Noise were found at positions 15750 and 16083, totaling 2 SNPs.

There are 10 places where mutations occur only in overseas breed dogs, and they are 15595, 15611, 15612, 15620, 15643, 15650, 15750, 15815, 16003, and 16083.

As described above, by using a total of 65 SNPs according to the present invention, it is possible to analyze alleles by specific SNPs that can appear in the breeds, and to use them for rapid identification, individual identification and paternity discrimination by faster, more accurate and economical methods Do. In addition, it can be very useful for the establishment of the scientific basis of the dog companion registration system and the supplement of the traceability system.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

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acaatctcat atgaagtaac 3180 gctagcaatt attcttctat cagtcctcct aataaacggg tcatttacac tatccacgct 3240 aattattacc caagaacata tatgattaat ctttccggcc tgacccctag ccatgatatg 3300 attcatctct accctagcag aaactaatcg agcccccttc gacttaactg aaggagaatc 3360 cgaactagtc tctggattta acgtagagta tgcagcaggt cctttcgccc tattctttct 3420 agcagagtac gcaaatatta ttataataaa catcctcaca acaattctgt tcttcggcgc 3480 attccacaac ccattcatac cagaactcta ctctattaac ttcactataa aaaccctctt 3540 attaaccatc tgcttcctat gaattcgagc atcataccct cgattccgct acgatcagtt 3600 aatacactta ttatgaaaaa attttctacc cttaacttta gccctatgca tatgacatgt 3660 tgccttaccc attatcaccg caagtatccc accccaaaca taagaaatat gtctgataaa 3720 agagttactt tgatagagta aataatagag gtttaaatcc tcttatttct agaataatag 3780 gcttcgaacc taatcttaag aattcaaaga tcttcgtgct accaaactta cactatattc 3840 tacagtaagg tcagctaaat taagctatcg ggcccatacc ccgaaaatgt tggtttatac 3900 ccttcccgta ctaataaaac cccctattct cattatcatc atagcaacta tcatgacagg 3960 aaccataatc gtcatactaa gctcgcactg attactgatc tgaattggat tcgaaataaa 4020 catgctagcc atcatcccta ttctcataaa aaagtacaat ccacgagcca tagaggcctc 4080 tacaaaatat tttcttacac aagctacagc ctcaatatta ctaataatag gagtcactat 4140 caacctcctt tactccggcc aatgggtaat ctcaaaaatc tcaaacccca tcgcatccat 4200 catgataacc actgccctaa caataaaact aggcctatct ccattccact tctgagttcc 4260 cgaagtaaca cagggaatta cgctcatatc aggaataatc ctactaacat gacaaaaaat 4320 cgcacctata tccatcctat atcaaatctc tccatcaatt aacactaacc ttcttatact 4380 aatagccctt acatccgttc tagtaggagg ctgaggcgga ctaaatcaaa ctcaactacg 4440 aaaaatcata gcatactcct ccattgccca cataggctga atagccgcta tcattactta 4500 taaccctaca ataatagttc taaacttaac tttatatatt ctaataacac tatctacctt 4560 catactattt atattaaact catccaccac gaccctatct ttatcccaca tatgaaacaa 4620 atttccccta atcacttcca taatcttaat cttaatacta tccctaggag gactaccccc 4680 attatctggc ttcatcccca aatgaataat tattcaagaa ttaacgaaaa ataacataat 4740 tattattcca acctaatgg ctatcaccgc tctacttaac ttatatttct acctgcgact 4800 cacatatagc accgcactta ccatatttcc atccacaaac aacataaaaa taaaatgaca 4860 gttcgaatac acaaaaaagg caaccctatt acccccctta attattacct caactatact 4920 actcccacta acacctatat tatcagtctt ggactaggag tttaggttag accagaccaa 4980 gagccttcaa agctctaagc aagtgctaca cacttaaccc ctgatcaaat cacctctaag 5040 ggctgcaaga atctatctta catcaattga atgcaaatca aacactttaa ttaagctaag 5100 ccctccctag attggtgagc ttctacctca cgaaatttta gttaacagct aaatacccta 5160 gtaactggct tcaatctacc ttctcccgcc gcgtagaaaa aaaaggcggg agaagccccg 5220 gcggcgtcta ggctgcttct ttgaatttgc aattcaatat gaaaattcac cacggagctt 5280 ggcaaaaaga ggacttaaac ccctatcttt agatttacag tctaatgctt ttatcagcca 5340 ttttacctat gttcattaac cgatgactgt tctccactaa tcacaaggat attggtactt 5400 tatacttact atttggagca tgagccggta tagtaggcac tgctttgagc ctcctcatcc 5460 gagccgaact aggtcagccc ggtactttac taggtgacga tcaaatttat aatgtcatcg 5520 taaccgccca tgctttcgta ataatcttct tcatagtcat gcccatcata attgggggct 5580 ttggaaactg actagtgccg ttaataattg gtgctccgga catggcattc ccccgaataa 5640 ataacatgag cttctgactc cttcctccat cctttcttct actattagca tcttctatgg 5700 tagaagcagg tgcaggaacg ggatgaaccg tatacccccc actggctggc aatctggccc 5760 atgcaggagc atccgttgac cttacaattt tctccttaca cttagccgga gtctcttcta 5820 ttttaggggc aattaatttc atcactacta ttatcaacat aaaaccccct gcaatatccc 5880 agtatcaaac tcccctgttt gtatgatcag tactaattac agcagttcta ctcttactat 5940 ccctgcctgt actggctgct ggaattacaa tacttttaac agaccggaat cttaatacaa 6000 cattttttga tcccgctgga ggaggagacc ctatcctata tcaacaccta ttctgattct 6060 tcggacatcc tgaagtttac attcttatcc tgcccggatt cggaataatt tctcacattg 6120 tcacttacta ctcagggaaa aaagagcctt tcggttatat aggaatagta tgagcaataa 6180 tatctattgg gtttttaggc tttatcgtat gagctcacca tatgtttacc gtaggaatag 6240 atgtagacac acgagcgtac tttacgtccg ccactataat tatcgctatt ccaacgggag 6300 taaaagtatt tagttgactg gcaacacttc atggaggcaa tattaaatga tctccagcta 6360 tgctatgagc tttagggttt attttcttat ttacagtagg cgggttaaca ggtattgtcc 6420 tagctaattc gtccttagac atcgttcttc atgatacata ttatgttgtg gctcattttc 6480 actatgtgct ttcaatagga gcagtttttg ccattatggg aggatttgcc cactgattcc 6540 ctttattctc aggttatact cttaacgata cttgagcaaa gattcacttt acaattatgt 6600 ttgtgggagt aaatataact ttcttccctc aacatttcct aggtttatct ggaatacctc 6660 gtcgatactc tgactaccca gatgcatata ctacctgaaa taccgtctcc tctataggat 6720 cgtttatctc gcttacagcg gtgatgctta taatttttat gatctgggaa gcctttgcat 6780 ccaaacgaga agttgctata gtagaactta ctacaactaa cattgagtga ctacatggat 6840 gtccccctcc ataccacacg ttcgaagaac ctacatatgt gatccaaaaa taagaaagga 6900 aggaatcgaa ccccctaaaa ttggtttcaa gccaatgtca taaccattat gtctttctca 6960 atcaggaggt attagtaaaa cattacatga ctttgtcaaa gttaaattat aggtgaaacc 7020 cctatatatc tctatggcgt acccatttca actcggatta caggacgcaa cctcccctat 7080 tatagaggag ctacttcatt ttcatgacca tacactaata attgtattct taatcagttc 7140 tttagttctc tatatcattt cactaatatt gactacaaaa ttaacccata caagcacaat 7200 agacgcacaa gaagtggaaa cagtatgaac cattctaccc gccattatcc taatcctaat 7260 cgctctacct tccctccgaa tcctttatat aatggacgaa attaataacc cctctttaac 7320 cgtgaaaaca ataggccacc aatgatactg aagctatgaa tatactgact atgaagactt 7380 aaactttgac tcctacataa tcccaacaca agaattaaag ccaggagaac tccgactatt 7440 agaagtagac aaccgagttg tcctcccaat agaaataacc atccgaatac ttatctcttc 7500 agaagacgtt ttgcattcat gagccgttcc atcactaggt ctaaaaactg acgctattcc 7560 aggacgacta aaccaaacca cccttatagc catacgacca ggactgtact atggccagtg 7620 ctctgaaatc tgcggatcta accacagctt tatacccatt gttcttgaaa tagtccccct 7680 atcttacttt gagacctgat ctgccttaat agtataacct agactagatc tactcattaa 7740 gaagctataa agcattaacc ttttaagtta aagactggga gttttaacct ctccttaatg 7800 aaatgccaca gctagataca tccacctgat ttattataat cttttcaata tttctcaccc 7860 tcttcatcct atttcaacta aaaatttcaa atcactacta cccagaaaac ccgataacca 7920 aatctgctaa aattgctggt caacataatc cttgagaaaa caaatgaacg aaaatctatt 7980 cgcttctttc gctgccccct caataatagg tctccctatt gtggtactga tcgtcatatt 8040 cccttccatt ttattcccaa cacccagtcg cctaatcaat aatcggttaa tctccattca 8100 gcaatgacta attcaactaa catcaaaaca aatactagca attcataacc aaaagggacg 8160 aacctgagct ctcatactta tatcactaat tctatttatt ggctcaacta atctacttgg 8220 actattacct cactcattta cgcccacaac acaactctct ataaacctcg gaatagcaat 8280 tcccctatga gcagggacag taattaccgg tttccgctat aaaaccaaag catccttagc 8340 acctttcta ccccaaggca cccctctccc cctaattcca atactagtag tcatcgaaac 8400 tattagtcta tttattcaac ccatggctct agccgttcga ttaaccgcca atattactgc 8460 aggacacctc ctaatccatt tgattggagg ggctacctta gctcttatca atattagcgc 8520 gaccacagct tttatcactt ttattattct aatcctactt acgatcctag aatttgctgt 8580 tgccttaatt caagcctatg tttttacctt actagtgagt ctatacttac atgacaacac 8640 ctaatgaccc accaaactca cgcttaccac atagtcaacc caagcccatg accgctgaca 8700 ggggcccttt ctgccctcct tataacatcg ggtcttatca tatgatttca ctataactca 8760 atagccctac ttacattagg attcacaacc aacctgttaa ccatatgcca gtgatgacga 8820 gatgtgatcc gagaaggcac attccaagga catcataccc ctattgtaca aaaaggacta 8880 cgatacggaa tagttctttt tatcgtatca gaagtatttt tctttgcagg cttcttctga 8940 gccttttacc actccagcct agcccctact cctgaacttg ggggttgctg acctcctacc 9000 ggcattattc ctcttaaccc attagaagtg cctctactca acacctcagt cctcctagcc 9060 tccggagtat ctattacttg agcccatcat agtttaatag aaggtaatcg caaacatata 9120 cttcaagcct tattcattac aatctcctta ggcgtatatt ttacgctatt acaggcctcc 9180 gaatactatg agacatcttt tacaatctcc gatggggtat acggatctac cttttttata 9240 gccactggat ttcacggatt acacgtaatt attggctcta cattcctcat cgtgtgcttc 9300 ctccgacagc tatactacca cttcacatca aaccaccact tcggatttga agccgctgca 9360 tgatattgac actttgttga tgtagtctgg ctattcttgt atgtatctat ttattgatga 9420 ggatcctatt tctttagtat aactagtaca attgacttcc aatcagttag ctccagatca 9480 acctggaaag aagtaataaa cgttatatta actttgataa ctaatgtaac cctagcatcc 9540 ttacttgtac taatcgcatt ctgacttccc cagctaaata tctatacaga caagacaagc 9600 ccctacgaat gtggttttga ccccatggga tctgctcgcc tacctttctc tataaaattt 9660 ttcctagttg ccatcacatt tctgcttttc gacctagaaa ttgcactcct actcccactt 9720 ccctgagcgt cacaaaccaa caagctaaca acaatactta tcatagcact cctactaatc 9780 tccctcctag ctgcgagcct agcgtatgaa tggaccgaaa aggggctaga atgaaccgaa 9840 tatgataatt agtttaagcc aaaaacaaat gatttcgact cattagatta tgatttatct 9900 cataattatc atgtccatag tatatattaa tatctttctg gcattcattc tttctctaat 9960 aggtatactt gtttatcgat cacacctaat atcatcgcta ctatgcttag agggcataat 10020 attatcacta tttgtaataa tatctgtaac tattctcaac aatcacctca cattagccag 10080 catgatacca atcgtactac tagtatttgc tgcctgcgaa gcagcattag gactatctct 10140 actagttata gtatctaaca cctacgggac tgattacgta caaaacctaa accttttaca 10200 atgctaaaaa ttatcatccc tactatcata ctaatccccc ttacatgaat atcaaagcct 10260 aacataatct gaattaacac gacaacatat ggtttgctaa tcagcttaat cagcttattc 10320 tacctaaacc aaccaaatga taatacattg aactcctcct taatattttt ctctgattcc 10380 ctatcggcac cactattagc actcacaaca tgacttctgc cccttatact tatagcaagt 10440 caacaccatt tatcaaaaga acccttaact cgaaaaaaac tatatatttc aatactaatt 10500 cttctccaat tgttcctaat tataactttc acagcctctg aactcatctt cttttacatc 10560 ctatttgaag caacactgat tccgaccctg atcattatta cccgatgagg aaatcaaact 10620 gaacgactaa acgcaggact ctacttctta ttttatactt taataggatc cctcccactc 10680 ctagtagctc tcctttatat ccacaatttc atgggctccc taaattttct cataattcaa 10740 tactgaatcc agcctctgcc aaactcctga tctaatattt tcctatgact ggcatgcata 10800 atagcattca tagtaaagat acctctatac ggcctccact tgtgactacc aaaagcacac 10860 gtagaggccc ctattgccgg ctccatagta cttgccgctg tactcctaaa actagggggc 10920 tatggcatga tacgaattac aaccctacta aatcccctga ccaatttcat agcatacccc 10980 ttcataatat tgtctctatg aggcataatc ataacaagct ctatctgtct ccgtcaaaca 11040 gatctaaaat ccctaattgc atactcctca gttagtcata tggcactggt tatcgtagcg 11100 gttcttattc aaacaccatg aagttatata ggtgcaacag ctctaataat tgcccatggt 11160 ttaacatcct caatactatt ctgcttagcc aactccaatt acgaacgaat ccatagccgt 11220 actataattc tcgcacgagg acttcaaact ctccttcccc taatagcagc ctgatgacta 11280 ttagcaagcc tcacaaatct ggctctccct ccaacaatta atcttatcgg agaactattt 11340 gtagtgatat cctcattctc atgatccaac attactatta tcctaatagg aattaacatt 11400 atcatcaccg ccctatactc actttatata ttaatcacca cacaacgtgg taaatattcc 11460 caccatatca aaaatatcaa accatcattc acacgagaaa atgccctaat gaccttgcac 11520 ctactgcccc tactcctcct atcccttaac cctaaaatta ttctcggtcc catctactgt 11580 aagcatagtt taacaaaaac attagattgt gagtctaaca ataaaagctc aaaccttttt 11640 gcttaccgaa aaagtactgc aagaactgct aattcatgct cccatgcata agaacatggc 11700 tttttcaact tttataggat agaagtaatc cgttggtctt aggaaccaaa aaattggtgc 11760 aactccagat aaaagtaata aatatatttt cttcatgcat aatcacagcc ctagttattc 11820 ttactttgcc catcattata tcctctacta aactttacaa gaataagcta tacccatact 11880 atgtaaaaac cgctacttct tacgcgttca taattagcat aattcccaca ataatattca 11940 tctactcagg acaggaaaca atcatttcaa actggcattg aataacaatc caaactataa 12000 aactatccat gagtttcaaa ttagactact tctcaataat ctttgtacct gtagcccttt 12060 ttgtcacgtg gtctatcata gaattctcta tatgatatat acactccgat ccttacatta 12120 accggttttt caagtacctt ctcttattcc tcatcactat aatagtccta gttaccgcaa 12180 acaacatatt ccaactattc attggttgag aaggagttgg cattatatca ttcctactta 12240 ttggatgatg gtacggccga accgatgcaa atacagccgc cctacaagcc gtcctctaca 12300 accgtattgg agatgtaggc ttcattataa ccatagcatg atttctacta aacttaaaca 12360 catgagacct tcaacaaatc ttcattacga caaacgataa ttttaatctg ccactacttg 12420 gcctactact agcagctacc ggtaaatctg ctcaattcgg cctacatccc tgactcccct 12480 cagccataga aggccccact cctgtatcag ccctacttca ctcaagcaca atagttgtag 12540 caggagtatt tcttcttatc cgctttcatc cactaataga gcataaccaa actattcaaa 12600 ccctcacttt atgcttaggg gccattacta cactatttac cgcaatctgc gctcttacac 12660 agaatgatat caaaaaaatt gtagcgttct ctacctcaag ccaactaggc ctaataatag 12720 taacaattgg cattaaccag ccctacttag ccttcttaca catctgcact cacgcatttt 12780 ttaaagctat actattcata tgctcagggt cagttatcca cagcctaaac gatgaacaag 12840 acattcgaaa aatgggtggc ctatttaaag tccttccctt caccacaacc tcccttatta 12900 tcggaagcct cgcattaaca ggcatgcctt tccttacagg attctactcc aaagacctga 12960 tcatcgagtc cgctaacacg tcgaatacca acgcctgagc cctcttaatt acactcgttg 13020 ccacatccct aaccgctgcc tacagcaccc gaattatatt ctttgctcta ctaggccagc 13080 cccgcttctc ccctataatc cttatcaacg agaataatcc tctcctaatt aactctatta 13140 aacgactcct tatcggaagt gtatttgcag ggtatattat ctcccacagc atcacaccca 13200 ctaccatccc acagataact atgcctcatt atctaaaaaat gacagccctt gcagtaacca 13260 ccttgggttt catcctggca ctagaactaa accttacctc acaaggactc aaatttaact 13320 atccttctaa ttactttaaa ttctccagcc tccttggcta ctatccaacc attatgcacc 13380 gcctcacacc taaaacaagt ttaaccatta gccagaaatc agcatctata cttctggact 13440 ccatctgact agaaaacatc ctacccaaat caatctcata tttccaaata aaatcttcta 13500 cccttatttc aaatcaaaaa ggtctcatca aactctattt cctatcgttc atactaacta 13560 taatcctcag tctactaatc cttaattacc acgggtaact tccatgataa ccaacacacc 13620 acatagata aatacccata gcctcttcac taaaaaaccc agagtccccc gtatcataga tcactcaatc 13740 ccctattcca ttaaacttta atactacctc cacctcgtca tccttcaaaa tatagcaagc 13800 agtcaacaat tcagacaaca aaccagtaat aaaagccgct agaacagcct tatttgaaac 13860 tcacacctca gggtattgct cagtagccat agcagttgta taaccaaata ctactaatat 13920 accccccaaa taaattaaaa atactatcaa ccctaaaaaa gaacccccaa aattcagaac 13980 aatcgcacaa ccaatcccac cgctaataat tagcacaagc ccaccataaa taggagatgg 14040 tttagtggca aaacccacaa aactcatcac aaaaacgata cttaaaataa atacaatgta 14100 tgttatcatt attcctacat ggaatttaac catgactaat gacatgaaaa atcatcgttg 14160 tatttcaact ataagaacat taatgaccaa cattcgaaaa acccacccac tagccaaaat 14220 tgttaataac tcattcattg acctcccagc gccgtctaac atctctgctt gatggaactt 14280 cggatcctta ctaggagtat gcttgattct acagattcta acaggtttat tcttagctat 14340 gcactataca tcggacacag ccacagcttt ttcatcagtc acccacatct gccgagacgt 14400 taactacggc tgaattatcc gctatatgca cgcaaatggc gcttccatat tctttatctg 14460 cctattccta catgtaggac gaggcctata ttacggatcc tatgtattca tagaaacatg 14520 aaacattgga attgtactat tattcgcaac catagccaca gcattcatgg gctatgtact 14580 accatgagga caaatatcat tttgaggagc aactgtaatc actaatcttc tctctgccat 14640 cccttatatc ggaactgact tagtagaatg gatctgaggc ggcttctcag tggacaaagc 14700 aaccctaaca cgattctttg cattccattt catcctccct ttcatcatcg cagctctagc 14760 aatagtacac ctcctatttc tacacgaaac cggatccaac aacccttcag gaatcacatc 14820 agactcagac aaaattccat ttcaccctta ctacacaatc aaggatatcc taggagcctt 14880 actcctactc ctaatcctaa tatcactagt tttattttca cctgacctat taggagaccc 14940 agataactac acccctgcaa accccctaaa cacccctcca catattaaac ctgagtgata 15000 ttttctattc gcctatgcta tcctacgatc cattcctaat aaattaggag gtgtactcgc 15060 cctagtattc tccatcctaa tcttggcatt cattccactc ctccacacat ctaagcaacg 15120 cagcataata ttccggcccc ttagccaatg cctattctga cttttagtcg ccgatcttct 15180 cactttaaca tgaattggag gacaaccagt tgagcaccct ttcatcatta tcggacaagt 15240 cgcttcaatc ttatatttca ccatcttatt gatcctaata ccaacagtta gcgttatcga 15300 aaacaacctt ctaaaatgaa gagtctttgt agtataatca ttaccttggt cttgtaaacc 15360 aaaaatggag agtaaccgcc ctccctaaga ctcaaggaag aagctcttgc tccaccatca 15420 gcacccaaag ctgagattct tcttaaacta ttccctgaca cccctacatt catatattga 15480 atcaccccta ctgtgctatg tcagtatctc caggtaaacc cttctcccct cccctatgta 15540 cgtcgtgcat taatggtttg ccccatgcat ataagcatgt actaatatta tatccttaca 15600 taggacatat taactcaatc tcataattca ctgatctttc aacagtaatc gaatgcatat 15660 cacttagtcc aataagggct taatcaccat gcctcgagaa accatcaacc cttgctcgta 15720 atgtccctct tctcgctccg ggcccatact aacgtggggg ttactatcat gaaactatac 15780 ctggcatctg gttcttactt cagggccata accttattta ctccaatcct actaattctc 15840 gcaaatggga catctcgatg gactaatgac taatcagccc atgatcacac ataactgtgg 15900 tgtcatgcat ctggtatctt ttaattttta gggggggaat ctgctatcac tcacctacga 15960 ccgcaacggc actaactcta acttatcttc tgctctcagg gaatatgccc gtcgcggccc 16020 taatgcagtc aaataacttg tagctggact tattcattat catttatcaa ctcacgcata 16080 aaatcaaggt gctattcagt caatggtttc aggacatata gttttagggt acacgtacgt 16140 acacgtacgt acacgtacgt acacgtacgt acacgtacgt acacgtgcgt acacgtgcgt 16200 acacgtacgt acacgtgcgt acacgtgcgt acacgtgcgt acacgtacgt acacgtgcgt 16260 acacgtacgt acacgtgcgt acacgtacgt acacgtacgt acacgtgcgt acacgtacgt 16320 acacgtgcgt acacgtgcgt acacgtgcgt acacgtacgt acacgtgcgt acacgtgcgt 16380 acacgtacgt acacgtacgt acacgtgcgt acacgtacgt acacgtacgc acgcgcgtaa 16440 gacattaagt taacttatac aaacccccct taccccccgt aaactcatgt catctattat 16500 acacttattt atgtcccgcc aaaccccaaa aacaggacta agtgcataca atactcacaa 16560 gctttattta aattatatac aaatgtattg ctactctagt taacttaaca caacagtctt 16620 acacgcattt ggtctcgtag tctatctata gatagcattc cctttttttt ccctctcata 16680 tttactatgt attttattta ttacgcacac tacaatttca gtataa 16726

Claims

A single nucleotide polymorphism (SNP) marker of mitochondrial DNA (mtDNA) present in the nucleotide sequence shown in SEQ ID NO: 1,
The SNP marker has nucleotide A at position 15618, nucleotide T at position 15665, nucleotide A at position 15811, nucleotide A at position 15825, position C at position 15625 of mitochondrial DNA (mtDNA) in SEQ ID NO: The nucleotide A of position 16328 is G, the nucleotide A of position 16268 is G, the nucleotide G of position 16276 is A, the nucleotide G of position 16306 is A, the nucleotide G of position 16326 is A, the nucleotide G of position 16338 is A, Wherein the nucleotide G is A and the nucleotide T at position 16671 is C, and a composition for identifying an overseas breed dog.

The method according to claim 1,
The nucleotide T at position 15611 is C, the nucleotide T at position 15612 is C, the nucleotide T at position 15620 is C, and the nucleotide C at position 15612 is C A SNP marker 15643, a nucleotide A at position 15650, a nucleotide T at position 15650, a nucleotide C at position 15750, a nucleotide T at position 15815, a nucleotide A at position 16003, and a nucleotide A at position 16083 And a composition for identifying a foreign breed dog.

The method according to claim 1,
The SNP marker has nucleotide A at position 15627, nucleotide C at position 15632, nucleotide T at position A or G at position 15639, nucleotide G at position 15652 in position A of the mitochondrial DNA (mtDNA) of SEQ ID NO: , At least one SNP marker selected from the group consisting of a nucleotide T at position 15800 C, a nucleotide C at position 15814, a nucleotide C at position 15912, and a nucleotide C at position 15955, wherein T is C, is T Compositions for identification of domestic dogs and foreign breed dogs.

A kit for identifying a domestic dog and an overseas breed dog comprising the composition according to any one of claims 1 to 3.

5. The kit of claim 4, wherein the kit comprises an agent capable of detecting or amplifying the SNP marker.

5. The kit for identification of foreign dogs according to claim 4, wherein said kit comprises a primer set represented by SEQ ID NOS: 2 and 3.

Isolating genomic DNA from the blood of the dog;
Amplifying the gene using the separated genomic DNA as a template and using primers set forth in SEQ ID NOS: 2 and 3; And
Wherein the amplified gene product is purified to analyze the base sequence or to hybridize with the marker according to any one of claims 1 to 3.

8. The method of claim 7,
When the amplified gene product contains at least one of an SNP marker having a nucleotide A at position 16248 of SEQ ID NO: 1 and a SNP marker having a nucleotide A of G at position 16268, .

8. The method of claim 7,
In the amplified gene product, the nucleotide G at position 16276 of SEQ ID NO: 1 is A, the nucleotide G at position 16306 is A, the nucleotide G at position 16326 is A, the nucleotide G at position 16338 is A, and the nucleotide G at position 16346 is A And a SNP marker having a nucleotide T at position 16671, wherein the SNP marker comprises at least one SNP marker selected from the group consisting of SNP markers.

8. The method of claim 7,
In the amplified gene product, if the nucleotide A at position 15627 of SEQ ID NO: 1 is G, the nucleotide T at position 15639 is A, the nucleotide at nucleotide 15814 is T, and the nucleotide at position 16025 is C, How to identify domestic dogs and foreign breed dogs to judge.

8. The method of claim 7,
When the amplified gene product contains at least one of a SNP marker having nucleotide A at position 15618 of SEQ ID NO: 1 and a SNP marker having nucleotide A at position 15811 at position 15811, .

8. The method of claim 7,
In the amplified gene product, the nucleotide A at position 15618 in position 1 of SEQ ID NO: 1 is G, the nucleotide T at position 15665 is C, the nucleotide A at position 15811 is G, the nucleotide T at position 16025 is C, and the nucleotide A at position 16248 is G The nucleotide A at position 16268 A is G, the nucleotide G at position 16276 is A, the nucleotide G at position 16306 is A, the nucleotide G at position 16326 is A, the nucleotide G at position 16338 is A, the nucleotide G at position 16346 is A, And at least one SNP marker selected from the group consisting of SNP markers having nucleotide T at position 16671 is identified as a domesticated dog species.

13. The method of claim 12,
Wherein said SNP markers include only SNP markers having a nucleotide T of C at position 16025 of SEQ ID NO: 1.

8. The method of claim 7,
In the amplified gene product, the nucleotide T at position 15620, the nucleotide A at position 15627 is G, the nucleotide T at position 15639 is T, the nucleotide C at position 15814 is T, and the nucleotide C at position 15955 is C T SNP markers to determine if they are Labrador Retrievers.

8. The method of claim 7,
In the amplified gene product, at least one SNP marker selected from the group consisting of a nucleotide T at position 15611 of SEQ ID NO: 1 is C, a nucleotide A at position 15643 is G, and a nucleotide at position 15650 is C is C Identify domestic dogs and overseas breed dogs that are deemed to be German sheep if included.

8. The method of claim 7,
In the above amplified gene product, if a SNP marker having a nucleotide C at position 15750 of SEQ ID NO: 1 and at least one SNP marker of nucleotide A of G at position 16083 is included in the amplified gene product, Identification of breed dogs.

8. The method of claim 7,
In the amplified gene product, the nucleotide C at position 15595, the nucleotide T at position 15611, the nucleotide T at position 15612, the nucleotide T at position 15620, the nucleotide T at position 15620, the nucleotide A at position 15643, A nucleotide T at position 15650 is C, a nucleotide C at position 15750 is T, a nucleotide T at position 15815 is C, a nucleotide A at position 16003 is G, and a nucleotide A at position 16083 is G Identification of domestic dogs and overseas breed dogs that contain at least one SNP marker and are deemed to be of foreign breeds.

18. The method of claim 17,
Among the above SNP markers, if the SNP marker having the nucleotide T at position 15620 of SEQ ID NO: 1 contains only the SNP marker, it is determined that it is a Labrador retriever.