KR20110030808A - Microsatellite marker and methods for identification of dogs - Google Patents

Abstract

PURPOSE: A microsatellite marker and a method for identifying dog individual using the same are provided to perform multiplex PCR and to perform parental testing. CONSTITUTION: A microsatellite marker which is able to distinguish dog individual is a combination of one or more sets among: a first set having FH3005, FH2537, FH3921, FH3381, FH3116, FH3372, REN62M06, REN277O05, REN51C16, FH3399, and FH3027; a second set having FH2097, REN204K13, FH1014, FH3058, FH2834, FH2712, REN112C08, FH2998, FH2584, and REN197E16; and a third set having FH2054, REN181K04, FH2079, REN01O23, FH2582, and FH2790. A method for identifying dog individual comprises: a step of performing multiple PCR using microsatellite marker; and a step of detecting allele through electrophoresis and determining genotype.

Description

Microsatellite marker and methods for identification of dogs}

The present invention relates to the development of supersatellite markers for gene identification of dogs, and more specifically, to the use of genes in a faster and more economical manner than conventional techniques through multiplex PCR using a microsatellite marker. It relates to the identification and use of paternity.

Dogs are used as dogs as animals familiar to us. The domestic dog industry continues to grow due to the increase in national income, personalization of lifestyle and expansion of digital culture. In 2006, the number of domestic dogs is estimated at 5.3 million, and the population of dogs is expected to reach 3.5 million. In addition, the economic value of the dog industry in 2007 is expected to be at least 1 trillion won. However, in 2008, due to the economic recession and the increase in feed prices, the number of organic dogs has soared nationwide, which is a serious social problem such as the destruction of the natural ecosystem due to the spread of diseases such as rabies and wild dogs. In recent years, 32,600 dogs have been caught in Seoul from 2006 to 2008, and the budget for protecting and managing them is close to 2.25 billion won. Busan has 12,500 heads, and Gyeonggi-do has 31,200 heads of catches in 2008 alone.

In this case, the animal registration system using bio-injectable microchips may be implemented depending on the necessity of introducing a system for integrated management of dogs. However, dogs have a dislike of the owner of the dog due to the insertion of foreign bodies and the possibility of disease of the inserted biological part, and all information can be lost when the microchip is removed. Therefore, in order to establish a systematic tracking history system, There is a need for obtaining unique genetic information and national integrated management.

As a way of confirming this genetic information, countries around the world recognize the value of preservation and utilization of conventional livestock, and various hereds such as mitochondrial DNA, blood proteins, minisatellite, microsatellite, etc. Genetic studies have been carried out using red markers.

Diversity is recognized between individuals according to the number of repetitions of the repeating unit at a specific locus, and if polymerase chain reaction (PCR) is carried out using primers designed in adjacent regions when the number of repetitions has a polymorphism between varieties, Polymorphisms are observed in the length of the PCR product, making it possible to detect DNA polymorphisms.

Among these genetic markers, microsatellite, called supersatellite markers, is a repetitive DNA group that repeats 2 to 6 sequences and is distributed in various genomes over 50,000 to 100,000. It is a non-coding DNA sequence with a very high polymorphism.

The supersatellite markers are also called short tandem repeats (STRs) or simple sequences repeats (SSRs), which are usually composed of short repeat sequences of two to six base pairs, which are specific to the individual's genotype combination of markers. In combination with a number of markers, it is possible to identify not only the breed but also the individual. On the other hand, in order to increase the accuracy and rapidity of the gene identification technique, the investigation should be carried out on a number of supersatellite markers. There was a problem of wasting time and manpower. Therefore, in order to solve this problem, multiple PCR (multiplex-PCR) method of putting primers of several markers in one PCR reaction and performing the reaction at the same time has been proposed as an alternative. For example, using a triplex system that simultaneously performs reactions of three supersatellite markers can reduce the time, reagents, equipment, and manpower to one third. Due to the advantages of the multiplex PCR method, developed countries are accelerating to establish a multiplex PCR method suitable for their own country.

The microsatellite is very useful in the field of molecular breeding of animals and is useful for preventing extinction due to inbreeding, and is also a technology that can be used to select and improve specific traits.

Therefore, multiplex PCR is faster and more efficient than conventional technology.

Development of an individual identification method for dogs using genes as an expedient method is desired.

An object of the present invention to solve the above problems is to provide a dog individual identification method, characterized in that to perform multiple PCR using a primer specific for the dog supersatellite markers.

Another object of the present invention is to provide a primer set specific for dog supersatellite markers.

Still another object of the present invention is to provide a method for identifying individual dogs and identifying paternity by providing a primer set specific to a dog supersatellite marker.

In order to achieve the above object, the present invention is a supersatellite marker, which is a first set consisting of FH3005, FH2537, FH3921, FH3381, FH3116, FH3372, REN62M06, REN277O05, REN51C16, FH3399, FH3027, FH2097, REN204K13, FH5814, FH1014H2834 , One or more combinations of FH2712, REN112C08, FH2998, FH2584, REN197E16, and a third set of FH2054, REN181K04, FH2079, REN01O23, FH2582, FH2790 To provide.

In addition, the present invention is the first set of the FH3005, FH2537, FH3921, FH3381, FH3116, FH3372, REN62M06, REN277O05, REN51C16, FH3399, FH3027, the FH2097, REN204K13, FH1014, FH3058, FH12834, FH12834, FH2834H98 , A second set of REN197E16, and a third set of FH2054, REN181K04, FH2079, REN01O23, FH2582, and FH2790 each provide a supersatellite marker comprising forward and reverse primers.

In another aspect, the present invention is characterized in that the first set, the second set and the third set of the supersatellite, characterized in that it comprises a forward and reverse primer consisting of identification numbers 1 to 22, identification numbers 23 to 42, identification numbers 43 to 54, respectively Provide a marker.

The present invention also provides a superset marker, FH3005, FH2537, FH3921, FH3381, FH3116, FH3372, REN62M06, REN277O05, REN51C16, FH3399, FH3027, FH2097, REN204K13, FH1014, F83430 F2, H259H2 Multiplex PCR amplification using a supersatellite marker combining a second set of FH2998, FH2584, REN197E16, and a third set of FH2054, REN181K04, FH2079, REN01O23, FH2582, FH2790; And it provides a dog identification method comprising the step of determining the genotype by detecting the allele and the size of the product amplified in the amplification step through an electrophoresis device.

In another aspect, the present invention provides a dog individual identification method further comprising the step of arranging the size of the alleles analyzed by the electrophoretic device by individual and breed, and creating a number and frequency distribution of alleles based on the same. .

In addition, the present invention is characterized in that the first set, the second set and the third set of the individual dogs, characterized in that it comprises a forward and reverse primer consisting of identification numbers 1 to 22, identification numbers 23 to 42, identification numbers 43 to 54, respectively Or, it provides a method of identifying individual dogs to identify parents.

As described above, the supersatellite marker and the dog individual identification method using the same according to the present invention have the effect of identifying the individual dog by using amplification after performing multiple PCR using primers specific for the dog supersatellite marker. have.

In addition, the supersatellite marker according to the present invention and the individual identification method of the dog using the same by analyzing multiple alleles that may appear in the dog breed through multiple PCR amplification of 27 supersatellite markers, the individual in a faster, more accurate and economical way It can be used for identification and paternity, to establish the scientific basis of the dog registration system and to supplement the tracking history system.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. First, it should be noted that in the drawings, the same components or parts denote the same reference numerals as much as possible. In describing the present invention, detailed descriptions of related well-known technologies or configurations are omitted in order not to obscure the subject matter of the present invention.

As used herein, the terms "about", "substantially", and the like, are used at, or in close proximity to, numerical values when manufacturing and material tolerances inherent in the meanings indicated are intended to aid the understanding of the invention. Accurate or absolute figures are used to assist in the prevention of unfair use by unscrupulous infringers.

As used herein, "microsatellites" refers to short repeat sequences in the form of mono-, di-, tri- and tetranucleotides distributed throughout the eukaryotes gene. In general, such repeat sequences are present in a tandem repeat of about 10 to 40 times in the chromosome.

As used herein, "microsatellite marker" refers to a DNA polymorphism detection marker using microsatellite.

The present invention is a genetic identification method using a multiplex PCR method for gene identification of a dog, wherein the selected microsatellite marker is reported in a mapviewer database of the National Center for Biotechnology Information (NCBI). It was selected based on the supersomal loci of two dogs, and the detailed selection condition, the allele frequency, the annealing temperature of the primer, the product size, the fluorescent material (Dye), etc. In consideration of the above, it was finally divided into three sets and combined to enable multiplex PCR using 27 supersatellite markers in total.

According to an embodiment of the present invention, a dog identification method includes amplifying alleles corresponding to dog supernatant markers; Determining the genotype of the product amplified in the amplification step by detecting an allele through an electrophoretic apparatus and analyzing the size of the allele; And arranging the sizes of alleles analyzed by the electrophoretic apparatus by individual and variety, and preparing a number and frequency distribution of alleles based on the sizes of alleles.

The supersatellite markers can be divided into three sets, the first set being FH3005 (identification numbers 1 and 2), FH2537 (identification numbers 3 and 4), FH3921 (identification numbers 5 and 6), FH3381 (identification number 7) And 8), FH3116 (Identifiers 9 and 10), FH3372 (Identifiers 11 and 12), REN62M06 (Identifiers 13 and 14), REN277O05 (Identifiers 15 and 16), REN51C16 (Identifiers 17 and 18), FH3399 (SEQ ID NOs: 19 and 20), and amplified by forward and reverse array primers consisting of FH3027 (Identifiers 21 and 22).

In addition, the second set is FH2097 (identification numbers 23 and 24), REN204K13 (identification numbers 25 and 26), FH1014 (identification numbers 27 and 28), FH3058 (identification numbers 29 and 30), FH2834 (identification numbers 31 and 32) , And inverted sequence consisting of FH2712 (ID 33 and 34), REN112C08 (ID 35 and 36), FH2998 (ID 37 and 38), FH2584 (ID 39 and 40), REN197E16 (ID 41 and 42) Amplified with a primer.

In addition, the third set is FH2054 (identification numbers 43 and 44), REN181K04 (identification numbers 45 and 46), FH2079 (identification numbers 47 and 48), REN01O23 (identification numbers 49 and 50), FH2582 (identification numbers 51 and 52) , Amplified with forward and reverse array primers consisting of FH2790 (Identifiers 53 and 54).

Table 1 below shows the combination of each set of supersatellite markers used in the present invention, and shows the chromosome position, primer sequence, amplification product size, fluorescence staining method, and sequence number.

set chromosome
location Supersatellite markers Primer Sequence
(F: forward 5 '→ 3',
R: Reverse 3 '→ 5') Amplification Product Size (bP) Fluorescent material SEQ ID NO: First set 2 FH3005 F ACTCATTTCCAAGGTGATTTG 200-236 Fam One R GTACTCACCGCAAGTGCAAG 2 10 FH2537 F AAAAAGTGTAGAGCTTTCTTCAAA 146-176 Fam 3 R ATTGAGACCCAAGACTGTTAGTG 4 10 FH3921 F CCTTCTTCTTAACACCTCTTCC 364-394 NED 5 R CTCTGTTTGCCAGATGATAACC 6 10 FH3381 F CCCAGAAACTCAACTGATGC 276 ~ 312 Fam 7 R AGCTCTTACACGCATTGAGG 8 12 FH3116 F GAGAAATCCTGTCATGTGCTG 186 ~ 200 VIC 9 R CCTTTTCCCTTCTTTCCTTG 10 19 FH3372 F AGTGCCTTTGAATGTTAATGC 142-162 VIC 11 R ACATCAAAATGGTTACACTTGG 12 26 REN62M06 F AAGTGGAATGGAGTCTGC 243-255 NED 13 R CATGAACCTGTCGTAAGC 14 27 REN277O05 F CCTCCTCTCACTTGTCCTGC 331-338 VIC 15 R AAATGGTGTCTTCAGCTCCG 16 30 REN51C16 F CAGTTCATCCTTCCCCCTCTC 246-264 VIC 17 R GTGCTAGTCTGGCTGTGCTCA 18 38 FH3399 F TCTCTATGCCTGCAGTTTCC 234-282 PET 19 R TTCTGATGCCCTCATAAAGC 20 X FH3027 F GTTTCCTCACATGCAAAAGC 196-234 PET 21 R GCTGGAGGTCAAGGATAAGG 22 2nd set 4 FH2097 F CAATGTCGAATTCCATGGTG 268-300 NED 23 R ATGGAGCAAGATGTGTTTGTG 24 8 REN204K13 F TCGGGATGTTTCTCTTCCAC 246-254 VIC 25 R CTGCTTAAATTCTCCCAGCG 26 9 FH1014 F AGGCTATTAACCCCTGATCG 242-250 Fam 27 R CGATGCCTTACTTAAACAAACC 28 16 FH3058 F GCCTTCCATAGATGAATGAGG 218-234 Fam 29 R CGATGCCTTACTTAAACAAACC 30 18 FH2834 F GCAAGCTTTAAAATACCTTTCC 263-265 Fam 31 R GCCTGAACTGATTGATGACC 32 31 FH2712 F AAGGTAGTCCCACGATCCTC 170-186 PET 33 R GCCTGAACTGATTGATGACC 34 35 REN112C08 F ATGGCCCACCGATACACA 218-236 NED 35 R TCGGGGACATACTTGAACC 36 36 FH2998 F GATTTTGATACCCTGAGAATGC 196-228 PET 37 R CTCACTGGCTCTCACATGC 38 X FH2584 F GTTAGGTTCACAGTGGGCGT 299-317 VIC 39 R ACTCAAAGACCTGGAGGGGT 40 Y REN197E16 F TGGGTGTGAGTCATCCAAGA 140-160 Fam 41 R CGTTACTGTATGCTTAAGCTTTTGA 42

Third set 12 FH2054 F GCCTTATTCATTGCAGTTAGGG 148-180 Fam 43 R ATGCTGAGTTTTGAACTTTCCC 44 23 REN181K04 F ACAAGCCGACTCTAGCGAAA 214-228 Fam 45 R AGATGGGGCCTAACCAAAGT 46 24 FH2079 F CAGCCGAGCACATGGTTT 269 ~ 293 NED 47 R ATTGATTCTGATATGCCCAGC 48 26 REN01O23 F TTCCCTGCAGCCCTTCCTCA 185-203 VIC 49 R TGTGCCTCATTCCTTTTTAT 50 31 FH2582 F TGGAGTGTGTTCCAAGGTCA 342-386 NED 51 R GTTGTTCCCACAAAAGGCAG 52 33 FH2790 F CCAATATTGTTAAGAAGTTCAAGC 204-208 Fam 53 R AGGCCTTCTCTGTCCTCTTG 54

The test animals and DNA samples used in the present invention are six dog breeds, Labrador Retriever, German Shepherd, English Springer Spaniel, Belgian Malinois, Jindo dog. Dog), PoongSan Dog, and 8 heads each, 48 heads were used to isolate genomic DNA from the blood using a genomic DNA extraction kit (Promega, USA).

Multiplex PCR was performed with 6 ul of template genomic DNA (20 ng / ul), 0.3 ul-0.4 ul for each set of fluorescent dyes (10 pmoles), and Hot Start Taq DNA polymerase (2 Unit / ul). 1ul, 10X buffer 4ul, 2.5mM dNTP 3ul was filled with distilled water to the total reaction solution was 25ul. The mixture was denatured at about 95 ° C. for 60 seconds, bound at about 62 ° C. for 75 seconds, using a thermal cycler PTC-0240 (MJ Research, Inc., MA, USA), starting with the first reaction at 95 ° C. for 15 minutes. 5 cycles at about 72 ° C. for 60 seconds, denatured at about 95 ° C. for 60 seconds, bound at about 61 ° C. for 75 seconds, extended at about 72 ° C. for 60 seconds, 5 cycles, at about 95 ° C. for 60 seconds, about 60 seconds After 25 cycles of bonding for 75 seconds at 60 ° C., 60 seconds at 72 ° C., and finally a final stretching reaction at about 65 ° C. for 30 minutes.

The amplified product after PCR was subjected to electrophoresis using ABI-3730 DNA automatic sequencing device (Applied Biosysytems, USA) to classify by size and fluorescent material, and PCR using GeneMapper version 4.0 (Applied Biosystem, USA) Data were collected by classifying the product size and the types of markers.

Finally determined alleles for each microsatellite marker are sorted by analysis group and individual group using the microsatellite toolkit software (Park, 2000) and expected heterozygosity for the entire population. Expected heterozygosity, observed heterozygosity, allele frequency, number of alleles for each locus and number of alleles for each population group were calculated. Expectations, observed heterozygosity rates, and polymorphic information content (PIC) values for each of the six varieties were calculated to express the diversity of allele types for each of the six varieties.

Hereinafter, embodiments of the present invention will be described in detail.

Example  One

Six dog breeds include Labrador Retriever, German Shepherd, English Springer Spaniel, Belgian Malinois, Jindo Dog, and PoongSan Dog. A total of 48 heads were used to isolate genomic DNA from the blood using a genomic DNA extraction kit (Promega, USA) using 8 heads, respectively. Multiple PCR was performed using the forward and reverse alignment primers of identification numbers 1 to 22 of REN277O05, REN51C16, FH3399, and FH3027.

Multiplex PCR was performed with 6 ul of template genomic DNA (20 ng / ul), 0.3 ul-0.4 ul for each set of fluorescent dyes (10 pmoles), and Hot Start Taq DNA polymerase (2 Unit / ul). 1ul, 10X buffer 4ul, 2.5mM dNTP 3ul was filled with distilled water to the total reaction solution was 25ul. The mixture was denatured at about 95 ° C. for 60 seconds, bound at about 62 ° C. for 75 seconds, using a thermal cycler PTC-0240 (MJ Research, Inc., MA, USA), starting with the first reaction at 95 ° C. for 15 minutes. 5 cycles at about 72 ° C. for 60 seconds, denatured at about 95 ° C. for 60 seconds, bound at about 61 ° C. for 75 seconds, extended at about 72 ° C. for 60 seconds, 5 cycles, at about 95 ° C. for 60 seconds, about 60 seconds After 25 cycles of bonding for 75 seconds at 60 ° C., 60 seconds at 72 ° C., and finally a final stretching reaction at about 65 ° C. for 30 minutes.

The amplified product after PCR was subjected to electrophoresis using ABI-3730 DNA automatic sequencing device (Applied Biosysytems, USA) to classify by size and fluorescent material, and PCR using GeneMapper version 4.0 (Applied Biosystem, USA) Data were collected by classifying product size and number of alleles by marker type.

1 and 2 compare the frequency of alleles for each of the dog breeds for FH3381 and FH3399 in the first set of supersatellite markers according to an embodiment of the present invention.

In the drawings, L is a Labrador Retriever, G is German Shepherd, E is English Springer Spaniel, B is Belgian Malinois, and J is Jindo Dog. ), P represents the breed of PoongSan Dog.

In the case of using the supersatellite marker FH3381 in Figure 1, in the case of Labrador retriever (L), the allele frequency is relatively high at 294bP, and the individual identification is possible, and at 290bP, the most allele of German Shepherd (G) is 312bP. Labrador Retriever (L) is the most common. In the case of amplification using FH3399 in FIG. 2, only the allele of German Shepherd (G) is present at 234bP, the Belgian Malinoids (B) at 238bP, and the allele frequency of Poongsan dog (P) at 270bP is most present. Features make it possible to identify each individual.

Example  2

The same procedure as in Example 1 was performed, except that the second-order FH2097, REN204K13, FH1014, FH3058, FH2834, FH2712, REN112C08, FH2998, FH2584, and REN197E16 were used to identify and reverse array primers. Multiple PCR was performed and data were collected by classifying the size of PCR product and the number of alleles by marker type.

3 and 4 compare the frequency of alleles for each of the dog breeds for FH2097 and FH2998 of the second set of supersatellite markers according to an embodiment of the present invention.

In the case of amplification using a supersatellite marker of FH2097 in FIG. 3, Belgian malinois (B) has the most allele frequency at 276 bP, and a gene of allele (J) is present at 300 bP. In the case of amplification using FH2998 in FIG. 4, only alleles of Labrador retriever (L) and Poongsan dog (P) exist at 196bP, and only Labrador retriever (L) and German Shepherd (G) are present at 224bP, and at 228bP Alleles of only Shepard (G) and Jindo dog (J) exist to identify each individual.

Example  3

Example 1, but as a supersatellite marker was subjected to multiple PCR using the forward and reverse array primers of the identification number 43 to 54 of the third set of FH2054, REN181K04, FH2079, REN01O23, FH2582, FH2790 Data were collected by classifying product size and number of alleles by marker type.

5 and 6 compare the frequency of alleles for each of the dog breeds for FH2054 and FH2582 of the third set of supersatellite markers according to an embodiment of the present invention.

In the case of amplification using a supersatellite marker of FH2054 in FIG. 5, a relatively large allele of German Shepherd (G) is present at 168 bP, and alleles of only Jindo dog (J) and Poongsan dog (P) exist at 172 bP. At 176 bP, Jindo allele (J) is present. In the case of amplification using FH2582 in FIG. 6, an allele of only Pungsan Dog (P) is present at 342bP, and an allele of only English Springer Spaniel (E) is present at 354bP, and Jindo Dog (J) and Pungsan Dog (P) at 382bP. Only at 386bP, there are alleles of only Labrador Retriever (L) and Jindo Dog (J), and each individual can be identified.

Table 2 below shows the results of Examples 1 to 3 carried out above, the amplification product size, allele number, and heterozygosity ratio of each breed of dogs.

set Supersatellite
Marker Allele size distribution Number of alleles Total release rate All varieties (L) (G) (E) (B) (J) (P) First set
Example 1 FH3005 200-236 8 4 2 4 3 5 5 0.588 FH2537 146-176 8 3 One 2 2 6 5 0.499 FH3921 364-394 11 5 2 One 3 5 8 0.578 FH3381 276-312 13 7 4 5 3 7 8 0.765 FH3116 186-200 4 2 One One 2 2 2 0.183 FH3372 142-162 6 4 One 3 3 4 5 0.521 REN62M06 243-255 5 One One 3 2 2 4 0.267 REN277O05 331-339 5 3 One 2 4 2 3 0.350 REN51C16 246-264 7 2 3 3 2 4 3 0.506 FH3399 234-282 13 8 3 4 3 8 6 0.725 FH3027 196-234 11 3 2 4 4 6 5 0.664 2nd set
Example 2 FH2097 268-300 9 5 4 3 5 8 4 0.658 REN204K13 246-254 5 2 2 2 2 4 3 0.443 FH1014 242-250 5 One 2 2 4 4 3 0.465 FH3058 218-234 7 3 2 2 3 5 5 0.610 FH2834 263-265 2 One 2 2 2 2 2 0.335 FH2712 170-186 8 3 3 3 4 7 5 0.686 REN112C08 218-236 5 2 2 2 2 4 3 0.440 FH2998 196-228 9 6 5 5 6 6 6 0.803 FH2584 299-317 8 3 One 2 3 8 3 0.560 REN197E16 140-160 6 3 4 2 4 3 4 0.597 Third set
Example 3 FH2054 148-180 9 4 5 4 6 6 6 0.743 REN181K04 214-228 5 3 3 2 4 3 3 0.567 FH2079 269-293 6 4 One 2 3 2 3 0.491 REN01O23 185-203 5 2 One One 2 3 3 0.306 FH2582 342-386 12 5 4 5 3 8 5 0.784 FH2790 204-208 2 2 2 2 2 2 One 0.301

Where (L) is Labrador Retriever, (G) is German Shepherd, (E) is English Springer Spaniel, (B) is Belgian Malinois, ( J) refers to Jindo Dog, and (P) refers to PoongSan Dog.

In addition, alleles of the microsatellite markers of the multiple PCR amplified products were sorted by analysis group and individual group using the microsatellite toolkit software (Park, 2000) and then applied to the entire population. Expected heterozygosity and observed heterozygosity, allele frequency, number of alleles for each locus and number of alleles for each breed group were calculated. Figure 4 shows the expected and observed heterojunction rates and PIC values for the six breeds.

set Supersatellite
Marker Labrador Retriever German Shepherd English Springer Spaniel Ex H Ob H PIC Ex H Ob H PIC Ex H Ob H PIC First set
Example 1 FH3005 0.650 0.500 0.559 0.125 0.125 0.110 0.575 0.250 0.483 FH2537 0.508 0.375 0.427 0.000 0.000 0.000 0.400 0.500 0.305 FH3921 0.767 0.625 0.670 0.525 0.875 0.371 0.000 0.000 0.000 FH3381 0.842 0.750 0.765 0.792 1,000 0.694 0.800 1,000 0.708 FH3116 0.325 0.375 0.258 0.000 0.000 0.000 0.000 0.000 0.000 FH3372 0.517 0.500 0.443 0.000 0.000 0.000 0.592 1,000 0.456 REN62M06 0.000 0.000 0.000 0.000 0.000 0.000 0.708 1,000 0.590 REN277O05 0.433 0.500 0.371 0.000 0.000 0.000 0.500 0.750 0.359 REN51C16 0.400 0.500 0.305 0.608 0.500 0.496 0.608 0.625 0.496 FH3399 0.842 1,000 0.766 0.492 0.625 0.398 0.758 0.875 0.658 FH3027 0.492 0.375 0.398 0.525 0.125 0.371 0.742 0.500 0.645 2nd set
Example 2 FH2097 0.808 0.750 0.717 0.642 0.875 0.547 0.425 0.500 0.354 REN204K13 0.325 0.375 0.258 0.533 0.750 0.375 0.400 0.500 0.305 FH1014 0.000 0.000 0.000 0.458 0.625 0.337 0.233 0.250 0.195 FH3058 0.575 0.375 0.447 0.500 0.750 0.359 0.500 0.750 0.359 FH2834 0.000 0.000 0.000 0.400 0.500 0.305 0.500 0.500 0.359 FH2712 0.658 0.500 0.544 0.658 0.750 0.544 0.425 0.500 0.354 REN112C08 0.400 0.500 0.305 0.400 0.250 0.305 0.525 0.375 0.371 FH2998 0.833 0.750 0.748 0.667 0.875 0.586 0.817 0.875 0.727 FH2584 0.575 0.375 0.447 0.000 0.000 0.000 0.525 0.375 0.371 REN197E16 0.508 0.375 0.427 0.525 0.250 0.458 0.458 0.625 0.337 Third set
Example 3 FH2054 0.642 0.750 0.547 0.725 0.857 0.632 0.733 1,000 0.630 REN181K04 0.508 0.125 0.427 0.714 0.000 0.555 0.233 0.000 0.195 FH2079 0.780 0.286 0.674 0.000 0.000 0.000 0.458 0.625 0.337 REN01O23 0.233 0.250 0.195 0.000 0.000 0.000 0.000 0.000 0.000 FH2582 0.817 0.875 0.727 0.758 1,000 0.646 0.700 1,000 0.595 FH2790 0.233 0.000 0.195 0.527 0.286 0.370 0.458 0.625 0.337

Ex H: Expected Heterozygosity, Ob H: Objective Heterozygosity, PIC: polymorphic information content

set Supersatellite
Marker Belgian Malinois Jindo dog Pungsan Dog Ex H Ob H PIC Ex H Ob H PIC Ex H Ob H PIC First set
Example 1 FH3005 0.750 0.250 0.582 0.708 0.500 0.618 0.717 0.750 0.629 FH2537 0.458 0.625 0.337 0.783 0.625 0.702 0.842 0.875 0.755 FH3921 0.508 0.625 0.427 0.758 0.571 0.657 0.908 0.750 0.835 FH3381 0.442 0.375 0.387 0.817 1,000 0.735 0.900 1,000 0.825 FH3116 0.325 0.375 0.258 0.125 0.125 0.110 0.325 0.125 0.258 FH3372 0.567 0.750 0.468 0.675 0.625 0.570 0.775 0.750 0.682 REN62M06 0.125 0.125 0.110 0.125 0.125 0.110 0.642 0.625 0.547 REN277O05 0.700 0.750 0.605 0.125 0.125 0.110 0.342 0.375 0.294 REN51C16 0.233 0.250 0.195 0.675 0.625 0.570 0.508 0.500 0.427 FH3399 0.592 0.750 0.456 0.850 0.750 0.776 0.817 0.750 0.730 FH3027 0.592 0.375 0.510 0.833 0.625 0.748 0.800 0.250 0.708 2nd set
Example 2 FH2097 0.608 0.500 0.539 0.875 0.750 0.799 0.592 0.375 0.510 REN204K13 0.325 0.375 0.258 0.650 0.750 0.559 0.425 0.500 0.354 FH1014 0.742 0.875 0.636 0.725 0.625 0.622 0.633 0.500 0.511 FH3058 0.575 0.750 0.482 0.742 1,000 0.642 0.767 0.625 0.679 FH2834 0.525 0.875 0.371 0.125 0.125 0.110 0.458 0.625 0.337 FH2712 0.750 0.875 0.644 0.858 0.875 0.779 0.767 0.750 0.679 REN112C08 0.458 0.625 0.337 0.517 0.625 0.443 0.342 0.375 0.294 FH2998 0.833 0.875 0.748 0.825 0.750 0.744 0.842 0.875 0.758 FH2584 0.675 0.500 0.556 0.925 0.500 0.852 0.658 0.250 0.544 REN197E16 0.650 0.875 0.530 0.658 1,000 0.544 0.783 1,000 0.685 Third set
Example 3 FH2054 0.808 1,000 0.723 0.813 0.857 0.719 0.733 0.875 0.654 REN181K04 0.742 0.625 0.645 0.538 0.143 0.427 0.667 0.000 0.555 FH2079 0.575 0.875 0.447 0.440 0.286 0.325 0.692 0.375 0.575 REN01O23 0.525 0.625 0.371 0.538 0.571 0.427 0.542 0.250 0.428 FH2582 0.667 0.333 0.535 0.923 1,000 0.841 0.842 0.750 0.755 FH2790 0.325 0.125 0.258 0.264 0.000 0.215 0.000 0.000 0.000

Ex H: Expected Heterozygosity, Ob H: Objective Heterozygosity, PIC: polymorphic information content

As described above, in the case of analyzing multiple alleles that may appear in dog breeds by multiplying PCR using three sets of 27 supersatellite markers, it can be used for individual identification and paternity identification in a faster, more accurate and economic manner. This can be used for various purposes, such as to establish the scientific basis of the dog registration system and to supplement the tracking history system.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the inventions. It will be clear to those who have knowledge of.

1 and 2 compare the frequency of alleles for each of the dog breeds for FH3381 and FH3399 in the first set of supersatellite markers according to an embodiment of the present invention.

3 and 4 compare the frequency of alleles for each of the dog breeds for FH2097 and FH2998 of the second set of supersatellite markers according to an embodiment of the present invention.

5 and 6 compare the frequency of alleles for each of the dog breeds for FH2054 and FH2582 of the third set of supersatellite markers according to an embodiment of the present invention.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Microsatellite marker and methods for identification of dogs <130> 9P-3505 <160> 54 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FH3005 forward primer <400> 1 actcatttcc aaggtgattt g 21 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH3005 reverse primer <400> 2 gtactcaccg caagtgcaag 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> FH2537 forward primer <400> 3 aaaaagtgta gagctttctt caaa 24 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> FH2537 reverse primer <400> 4 attgagaccc aagactgtta gtg 23 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> FH3921 forward primer <400> 5 ccttcttctt aacacctctt cc 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> FH3921 reverse primer <400> 6 ctctgtttgc cagatgataa cc 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH3381 forward primer <400> 7 cccagaaact caactgatgc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH3381 reverse primer <400> 8 agctcttaca cgcattgagg 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FH3116 forward primer <400> 9 gagaaatcct gtcatgtgct g 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH3116 reverse primer <400> 10 ccttttccct tctttccttg 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FH3372 forward primer <400> 11 agtgcctttg aatgttaatg c 21 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> FH3372 reverse primer <400> 12 acatcaaaat ggttacactt gg 22 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> REN62M06 forward primer <400> 13 aagtggaatg gagtctgc 18 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> REN62M06 reverse primer <400> 14 catgaacctg tcgtaagc 18 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> REN277O05 forward primer <400> 15 cctcctctca cttgtcctgc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> REN277O05 reverse primer <400> 16 aaatggtgtc ttcagctccg 20 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> REN51C16 forward primer <400> 17 cagttcatcc ttccccctct c 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> REN51C16 reverse primer <400> 18 gtgctagtct ggctgtgctc a 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH3399 forward primer <400> 19 tctctatgcc tgcagtttcc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH3399 reverse primer <400> 20 ttctgatgcc ctcataaagc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH3027 forward primer <400> 21 gtttcctcac atgcaaaagc 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH3027 reverse primer <400> 22 gctggaggtc aaggataagg 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH2097 forward primer <400> 23 caatgtcgaa ttccatggtg 20 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FH2097 reverse primer <400> 24 atggagcaag atgtgtttgt g 21 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> REN204K13 forward primer <400> 25 tcgggatgtt tctcttccac 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> REN204K13 reverse primer <400> 26 ctgcttaaat tctcccagcg 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH1014 forward primer <400> 27 aggctattaa cccctgatcg 20 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> FH1014 reverse primer <400> 28 cgatgcctta cttaaacaaa cc 22 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FH3058 forward primer <400> 29 gccttccata gatgaatgag g 21 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> FH3058 reverse primer <400> 30 cgatgcctta cttaaacaaa cc 22 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> FH2834 forward primer <400> 31 gcaagcttta aaataccttt cc 22 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH2834 reverse primer <400> 32 gcctgaactg attgatgacc 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH2712 forward primer <400> 33 aaggtagtcc cacgatcctc 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH2712 reverse primer <400> 34 gcctgaactg attgatgacc 20 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> REN112C08 forward primer <400> 35 atggcccacc gatacaca 18 <210> 36 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> REN112C08 reverse primer <400> 36 tcggggacat acttgaacc 19 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> FH2998 forward primer <400> 37 gattttgata ccctgagaat gc 22 <210> 38 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> FH2998 reverse primer <400> 38 ctcactggct ctcacatgc 19 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH2584 forward primer <400> 39 gttaggttca cagtgggcgt 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH2584 reverse primer <400> 40 actcaaagac ctggaggggt 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> REN197E16 forward primer <400> 41 tgggtgtgag tcatccaaga 20 <210> 42 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> REN197E16 reverse primer <400> 42 cgttactgta tgcttaagct tttga 25 <210> 43 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> FH2054 forward primer <400> 43 gccttattca ttgcagttag gg 22 <210> 44 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> FH2054 reverse primer <400> 44 atgctgagtt ttgaactttc cc 22 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> REN181K04 forward primer <400> 45 acaagccgac tctagcgaaa 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> REN181K04 reverse primer <400> 46 agatggggcc taaccaaagt 20 <210> 47 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> FH2079 forward primer <400> 47 cagccgagca catggttt 18 <210> 48 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FH2079 reverse primer <400> 48 attgattctg atatgcccag c 21 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> REN01O23 forward primer <400> 49 ttccctgcag cccttcctca 20 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> REN01O23 reverse primer <400> 50 tgtgcctcat tcctttttat 20 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH2582 forward primer <400> 51 tggagtgtgt tccaaggtca 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH2582 reverse primer <400> 52 gttgttccca caaaaggcag 20 <210> 53 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> FH2790 forward primer <400> 53 ccaatattgt taagaagttc aagc 24 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FH2790 reverse primer <400> 54 aggccttctc tgtcctcttg 20  

Claims (6)

As a supersatellite marker, First set consisting of FH3005, FH2537, FH3921, FH3381, FH3116, FH3372, REN62M06, REN277O05, REN51C16, FH3399, FH3027, A second set consisting of FH2097, REN204K13, FH1014, FH3058, FH2834, FH2712, REN112C08, FH2998, FH2584, REN197E16, and A supersatellite marker capable of identifying a dog individual by combining one or more of the third set consisting of FH2054, REN181K04, FH2079, REN01O23, FH2582, and FH2790. The method of claim 2, The first set consisting of FH3005, FH2537, FH3921, FH3381, FH3116, FH3372, REN62M06, REN277O05, REN51C16, FH3399, FH3027, A second set consisting of FH2097, REN204K13, FH1014, FH3058, FH2834, FH2712, REN112C08, FH2998, FH2584, REN197E16, and And the third set of FH2054, REN181K04, FH2079, REN01O23, FH2582, and FH2790 comprises forward and reverse primers, respectively. The method of claim 1, Wherein said first set, second set and third set comprise forward and reverse primers comprising identification numbers 1 to 22, identification numbers 23 to 42, and identification numbers 43 to 54, respectively. A supersatellite marker, First set consisting of FH3005, FH2537, FH3921, FH3381, FH3116, FH3372, REN62M06, REN277O05, REN51C16, FH3399, FH3027, A second set consisting of FH2097, REN204K13, FH1014, FH3058, FH2834, FH2712, REN112C08, FH2998, FH2584, REN197E16, Multiplex PCR amplification using a supersatellite marker combining at least one set of the third set consisting of FH2054, REN181K04, FH2079, REN01O23, FH2582, and FH2790; And Dog identification method comprising the step of determining the genotype of the product amplified in the amplification step by detecting an allele through an electrophoresis device and size analysis. The method of claim 3, The individual identification method of the dog further comprises the step of preparing the number and frequency distribution of alleles based on the size of the alleles analyzed by the electrophoretic device by individual and breed. The method of claim 3, The first set, the second set, and the third set include forward and reverse primers consisting of identification numbers 1 to 22, identification numbers 23 to 42, and identification numbers 43 to 54, respectively. How can I identify a dog?

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