KR20070107346A - Identifying method of chum salmon species or its originated stocks, polynucleotide probe, dna chip and kit for identifying the same - Google Patents

Abstract

An identifying method of chum salmon species or its originated stocks is provided to discriminate differences of nucleotide sequence between chum salmon species or its originated stocks rapidly and accurately by using probes capable of detecting various mutations. An identifying method of chum salmon species or its originated stocks comprises the steps of: extracting DNA from the blood, cell or tissue of chum salmon; performing PCR(polymerase chain reaction) of the extracted DNA to obtain PCR products; binding the PCR products with polynucleotide probes comprising at least one DNA sequence selected from SEQ ID NO:5 to SEQ ID NO:34 or its complementary DNA sequences; and identifying chum salmon species or its originated stocks by confirming the binding of PCR products with polynucleotide probes, wherein the extracted DNA contains a single nucleotide polymorphism of COIII-ND3-ND4L gene and the polynucleotide probe contains 15-30 consecutive nucleotide sequences.

Description

Identifying Method of Chum Salmon Species or Its Originated Stocks, Polynucleotide Probe, DNA Chip and Kit for Identifying The Same}

1 is a schematic diagram showing the gene sequence on salmon mitochondrial DNA,

2 to 6 are schematic views showing 730 sequential sequences including a single nucleotide polymorphism site of COIII-ND3-ND4L gene in salmon mitochondrial DNA according to a preferred embodiment of the present invention.

7 is an electrophoretic photograph of a PCR product amplified by a PCR method according to an embodiment of the present invention,

8 is a schematic diagram illustrating the structure of a DNA chip including an oligonucleotide probe prepared based on the monobasic polymorphism shown in FIGS. 2 to 6,

9A to 9F are photographs showing the reaction result after combining the oligonucleotide probe and PCR amplification products of various salmon according to the present invention using the DNA chip of FIG. 8.

The present invention relates to a method for discriminating a salmon species or a family, and to a polynucleotide probe, a DNA chip, and a kit for discriminating a salmon species or a family, in particular, each family of salmon (chum salmon) stocked by each species of salmon and the North Pacific coastal countries. To distinguish between simple, quick and accurate. More specifically, polynucleotide probes based on single nucleotide polymrphism present in the mitochondrial DNA COIII-ND3-ND4L region of salmon, and kits and DNA chips containing the same are prepared. will be.

Salmon and fish are cold water species that live in the North Pacific and North Atlantic. Typical salmon species in the North Pacific include salmon (chum salmon, Oncorhynchus keta ), pink salmon ( O. gorbuscha ), red salmon (sockeye salmon, O. nerka ), chinook salmon ( O. tshawytscha ) and silver salmon (coho salmon, O. kisutch ), sea salmon (cherry salmon, O. masou masou ), and the Atlantic Ocean (Atlantic salmon, Salmo salar ). In addition, the typical fish species inhabiting the land of salmon and fish include mountain trout (ishikawae salmon, O. masou ishikawae ), rainbow trout ( O. mykiss ) and cutthroat trout ( O. clarkii ). Most of them are widely used for food and are considered an important fishery resource in the North Pacific and North Atlantic coastal countries. On the other hand, salmon ( O. keta ), a species widely distributed in the North Pacific, the Bering Sea, the Okhotsk Sea and the rivers that enter these seas, has produced large numbers of artificially produced fish from Korea, Japan, Russia, Canada and the United States. By discharging, they increase their salmon resources.

Salmon-based coastal countries are making efforts to efficiently manage and rational use of salmon resources, and actively make scientific and diplomatic efforts to secure sovereignty over salmon resources. However, in order to achieve such a purpose, it is necessary to quickly and accurately discriminate between salmon species and flocks.

Gene sequence on mitochondrial DNA of salmon and fish is shown in FIG. The COIII-ND3-ND4L region, which is the subject region of the present invention, is located on the opposite side of the control region, which has been studied in the prior art, and the 203 base (position 1-203), tRNA- of the 3 'end of the COIII (CO3) gene gly gene 70 base (204-273), ND3 gene 351 base (274-624), tRNA-Arg gene 68 base (625-692), ND4L gene 5 'terminal 52 base (693-744). Until now, genotyping salmon has been limited to regulatory regions, but the present inventors analyzed monobasic polymorphisms distinguishing species and families from different COIII, ND3, and ND4L regions. The invention led to the invention of a DNA chip that quickly and accurately identifies salmon species and herds.

Initially, isoenzyme analysis was attempted to identify salmon (Okazaki, 1981; Winans et al. 1994; Wilmot et al. 1998). This method distinguishes enzyme proteins with alleles on a gel using electrophoresis, which does not have good resolution and isozymes are not sufficiently separated (Lewontin, 1991).

Another method was to classify families using restriction fragment length polymorphisms (RFLPs) (Cronin et al. 1993; Seeb and Crane, 1999). However, this method also has a disadvantage in that resolution is reduced because the entire genome of the comparative organism is cut with restriction enzymes and analyzed by electrophoresis to compare the length differences of the cut DNA fragments between the organisms. Since then, attempts have been made to distinguish salmon species and flocks using differences in minisatellite DNA (Beacham, 1996), but this also has many problems in distinguishing species and flocks consistently and quickly.

On the other hand, as genetic sequencing methods have been developed, methods for identifying salmon species using the differences in nucleotide sequences and revealing their flexibility relationship have been developed. Mitochondrial regulatory region sequences from eight species of salmon and fish were analyzed (Shedlock et. Al., 1992), and ATP6 and ND3 sequences were also analyzed (Domanico and Phillips, 1995). In addition, growth hormone-type 2 was also analyzed to determine when and by what species each species of salmon and fish were produced (McKay et. Al., 1996). In addition, growth hormone type 1 and type 2 introns have been investigated to reveal the relationship between salmon and fish (Oakley and Phillips, 1999). These studies provide differences in sequences that can distinguish between species of salmon and fish.

Recently, it is known that there are many mutations in the control region on salmon mitochondrial DNA (Sato et al. 2001), which analyzes the entire nucleotide sequence of the region and identifies the mutations (single nucleotide polymorphisms, SNPs). On the basis of this, a method of classifying flocks was tried (Abe et al., 2002). This method has high resolution in terms of comparing primary differences in nucleotide sequences as a comparative measure. However, the control regions that were analyzed did not turn out to be large enough to distinguish the various families of salmon.

Therefore, in order to classify salmon families, it is necessary to accumulate sequencing data for genetic markers having high resolution because of more mutations, and there is an urgent need for an analysis method capable of quickly distinguishing nucleotide differences between families. to be.

The present invention has been made in order to solve the above problems, the DNA is extracted from the blood (blood), cells and muscle tissue of the salmon (salmon), the amplified PCR product of any one of SEQ ID NO: 5 to SEQ ID NO: 34 It is to provide a method for analyzing salmon genotype by reacting with a polynucleotide probe containing a sequence identical or complementary to the DNA sequence.

Even in the identification of salmon species, even if differences in sequencing are found, having a standardized way to analyze them quickly and accurately can save a lot of time, human resources, and money. In the present invention, to accurately determine the difference in the sequence of each species according to each species or group, and to produce a polynucleotide probe to be different for each salmon species based on this, using a DNA chip or kit comprising the same The purpose of this study is to provide a method for quickly and accurately analyzing the differences in sequence of different species of salmon.

Another object of the present invention is to provide a probe comprising 15 to 30 contiguous nucleotides including a region having a nucleotide sequence difference between a species and a family, a DNA chip comprising the same, and a kit including the same. The present invention is intended to provide a method for simple, rapid and accurate screening of genotyping for multiple salmon species or family on one slide.

Based on the site of single nucleotide polymorphism (SNPs) of the COIII-ND3-ND4L gene in mitochondrial DNA of various salmon species, we have found an optimal polynucleotide probe that can specifically bind to each species of salmon. I want to make it.

The present invention for achieving the above object comprises the steps of extracting DNA from the blood, cells or tissues of salmon (salmon); Performing a polymerase chain reaction (PCR) on the extracted DNA to obtain a PCR product; Combining the PCR product with a polynucleotide probe comprising a nucleotide sequence identical to or complementary to the DNA sequence of any one of SEQ ID NOs: 5 to 34; And determining the species or species to which the salmon belongs by checking whether the combination is present.

Another embodiment for achieving another object of the present invention is to be used in the method for discriminating salmon species or family as described above, and the same or complementary to the DNA sequence of any one of SEQ ID NO: 5 to SEQ ID NO: 34 ) Polynucleotide probe for discriminating salmon species or family consisting of 15-30 consecutive sequences. Furthermore, the present invention binds to the mononucleotide polymorphism (SNP) region DNA sequence of the COIII-ND3-ND4L gene in salmon mitochondrial DNA, and is identical or complementary to the DNA sequence of any one of SEQ ID NOs: 5 to 34. ) May be a DNA chip comprising a polynucleotide probe for discriminating salmon species or family consisting of 15-30 consecutive nucleotide sequences.

Another embodiment for achieving another object of the present invention may be a DNA chip containing the above-described polynucleotide probe or a kit containing the same. That is, it binds to the mononucleotide polymorphism (SNP) region DNA sequence of the COIII-ND3-ND4L gene in salmon mitochondrial DNA, and is identical or complementary to the DNA sequence of any one of SEQ ID NOs: 5 to 34. A polynucleotide probe for discriminating salmon species or herds, characterized in that it comprises 30 consecutive nucleotide sequences; A kit for discriminating salmon species or herds, comprising a; primer for amplifying the salmon mitochondrial DNA by polymerase chain reaction (PCR).

Hereinafter, with reference to the accompanying drawings, preferred embodiments of the present invention will be described in detail.

In the method for discriminating a salmon species or family according to the present invention, first, DNA is extracted from blood, cells, or tissues of salmon, and PCR products are performed by performing PCR analysis on the extracted DNA. Go through the steps to get it. Since the extraction of DNA from the salmon is for analyzing the extracted DNA to determine the species or family of salmon, it is not particularly limited in which part of the salmon the DNA is extracted or in what way. The extracted DNA preferably includes a single nucleotide polymorphism (SNPs) region of the COIII-ND3-ND4L gene region in salmon mitochondrial DNA. In addition, amplifying the DNA thus extracted by PCR is also used to extend the test sample, and the method of obtaining the amplified product is not particularly limited. It is obvious that other DNA extraction methods or amplification methods known to those skilled in the art also belong to the scope of the present invention.

Subsequently, the most important feature of the present invention is the step of combining the PCR product with a polynucleotide probe comprising a nucleotide sequence identical to or complementary to the DNA sequence of any one of SEQ ID NOs: 5 to 34. Thereafter, the step of determining whether the binding belongs to the species or family belonging to the salmon, but this also may be used to determine whether all of the binding method commonly used in the art.

DNA sequences of SEQ ID NO: 5 to SEQ ID NO: 23 according to the present invention are as shown in Table 1, Table 1-1, and Table 2 below.

Table 1: DNA Sequences for Salmon Species Identification

 DNA sequence for salmon species determination  Probe Name  DNA sequence  Reaction salmon species DNA sequence position  sm05 (SEQ ID NO: 5)  5'-gac gca gaa aag ttg tct ccc ta-3 '  salmon  364-386  sm06 (SEQ ID NO: 6)  5'-ccc ctt ggg gtc tgc ccg tct-3 '  Humpback Salmon  402-422  sm07 (SEQ ID NO: 7)  5'-tcc tag tat caa tga gta taa gtg ac-3 '  Sockeye Salmon  208-233  sm08 (SEQ ID NO: 8)  5'-gat ttg atc cat tag ggt ccg c-3 '  King salmon  395-416  sm09 (SEQ ID NO: 9)  5'-gac cct tta gga tcc gcc cgt-3 '  Shima Salmon  402-418  sm10 (SEQ ID NO: 10)  5'-ctc aat acc ccc gcc cta-3 '  Shima Salmon  517-534  sm11 (SEQ ID NO: 11)  5'-tgc tgt gct tgc cct cc-3 '  Mountain Trout  551-568  sm12 (SEQ ID NO: 12)  5'-gac ccc tta ggg tcc gcc cgc-3 '  Rainbow trout  399-420  sm13 (SEQ ID NO: 13)  5'-att tga ccc cct agg atc cg-3 '  cutthroat trout  396-415  sm14 (SEQ ID NO: 14)  5'-aca aat aac gcc cga cgc a-3 '  Atlantic Salmon  351-369  sm15 (SEQ ID NO: 15)  5'-tag tat taa cgc gta taa gtg-3 '  Coho salmon  211-231  sm16 (SEQ ID NO: 16)  5'-tag tat taa cac gta taa gtg-3 '  All other species  211-231  sm17 (SEQ ID NO: 17)  5'-tct att tac tga tga ggc tc-3 '  Salmon and all other species  181-200

Table 1-1: DNA Sequence for Salmon Species Identification

 DNA sequence for salmon species determination  Probe Name  DNA sequence  Reaction salmon species DNA sequence position  sm24 (SEQ ID NO: 24)  5'-gca gag aag tta tcc cc-3 '  King salmon  367-383  sm25 (SEQ ID NO: 25)  5-atc acc att aca ctg tct gc-3 '  Coho salmon  301-320  sm26 (SEQ ID NO: 26)  5-tag tta caa caa tca tcg ct-3 '  Sockeye Salmon  280-300  sm27 (SEQ ID NO: 27)  5'-act ctt gga cta att tat gag tg-3 '  Sockeye Salmon  571-593  sm28 (SEQ ID NO: 28)  5'-cca ctg ctg tac ttg ccc tcc tt-3 '  Shima Salmon  548-571  sm29 (SEQ ID NO: 29)  5'-cca ctg ctg tgc ttg ccc tcc tt-3 '  Mountain Trout  548-571  sm30 (SEQ ID NO: 30)  5'-atg gac cca ggg agg cct tg-3 '  Atlantic Salmon  591-611  sm31 (SEQ ID NO: 31)  5'-tcc tcc ccc tac cct gag gg-3 '  Humpback Salmon  491-510  sm32 (SEQ ID NO: 32)  5'-ctt gac act cgt ttg gtc ca-3 '  Coho salmon  531-550  sm33 (SEQ ID NO: 33)  5'-cct aac act cgt ctg atc ca-3 '  Shima salmon, mountain fish  531-550  sm34 (SEQ ID NO: 34)  5'-gat caa ctc cac acc ccg ac-3 '  Rainbow trout  511-530

Table 2: DNA Sequences for Salmon Family (Genotype) Identification]

 DNA sequence for determination of salmon family  Probe Name  DNA sequence  Salmon genotype  DNA sequence position  sm18 (SEQ ID NO: 18)  5'-aat cat tac tat tac cat cac att-3 '  C1  291-314  sm19 (SEQ ID NO: 19)  5'-acc cca acc cta aca ctt att t-3 '  A1  523-544  sm20 (SEQ ID NO: 20)  5'-tta cta tca ccc tca cat tgt cc-3 '  D2  296-318  sm21 (SEQ ID NO: 21)  5'-ccc caa ccc tga cac tta tt-3 '  B1, C1, D1, D2  524-543  sm22 (SEQ ID NO: 22)  5'-taa ttt atg aat gaa ccc aag gag-3 '  A1, D1, D2  581-604  sm23 (SEQ ID NO: 23)  5'-taa ttt atg agt gaa ccc aag gag-3 '  B1, C1  581-604

As shown in Table 1, Table 1-1 and Table 2 above, the DNA sequence according to the present invention can be divided into to determine the species of salmon and to determine the salmon family. The DNA sequence includes a single nucleotide polymorphism (SNPs) site of the COIII-ND3-ND4L gene in salmon mitochondrial DNA, and represents a general base sequence of a salmon species or a target product to be known. The location of each DNA sequence is also shown. Therefore, the polynucleotide probe according to the present invention preferably has a base sequence complementary to the DNA sequence so as to bind to the DNA sequence of SEQ ID NO: 5 to SEQ ID NO: 34. In addition, in using a PCR product obtained by amplifying DNA extracted from salmon, it is also possible to use either strand of the PCR product, and thus a target having a nucleotide sequence complementary to the DNA sequence of SEQ ID NO: 5 to SEQ ID NO: 34 In order to react with the PCR product of interest, the probe according to the present invention may have the same nucleotide sequence as the DNA sequence of SEQ ID NO: 5 to SEQ ID NO: 34. The present inventors used the base sequence complementary to the DNA sequence of the PCR product according to each sequence number, and named each probe name corresponding to the sequence number sm5 to sm34.

As shown in Figures 2 to 6, the inventors found a single nucleotide polymorphism (SNPs) site of the COIII-ND3-ND4L gene in mitochondrial DNA of various salmon species, based on this Polynucleotide probes that can specifically bind to each were prepared.

2 to 6 are schematic diagrams showing 730 base sequences sequentially including a single nucleotide polymorphism site of the COIII-ND3-ND4L gene in salmon mitochondrial DNA according to a preferred embodiment of the present invention. That is, Figure 2 is a variety of salmon species, specifically salmon (chum salmon, O. keta), salmon salmon (pink salmon, O. gorbuscha), red salmon (sockeye salmon, O. nerka), salmon salmon (chinook salmon, O. tshawytscha, coho salmon (O. kisutch), shima salmon (cherry salmon, O. masou masou), mountain trout (ishikawae salmon, O. masou ishikawae), rainbow trout (O. mykiss), In the mitochondrial DNA of cutthroat trout (O. clarkii) and Atlantic salmon (Salmo salar) species, DNA sequences 1 to 150 of the COIII-ND3-ND4L gene are shown, and FIG. 3 is 151 of the gene. To 300 DNA sequence, Figure 4 is a DNA sequence of the 301 to 450 of the gene, Figure 5 is a DNA sequence of Nos. 451 to 600 of the gene, Figure 6 is a 601 to 730 of the gene DNA sequences up to 1 time are shown sequentially.

Here, the monobasic polymorphism site refers to a nucleotide sequence site showing a genotype different from a general nucleotide sequence of a salmon (chum salmon) species, as shown in FIGS. That is, the site indicated by ". (Dot)" indicates the same genotype as the general nucleotide sequence of the (chum salmon) species, specific A, C, G or T shown in the atlantic of pink to FIG. Is a site showing monobasic polymorphism (SNP).

2 to 6 are linked diagrams showing genomic DNA portions comprising 730 nucleotide sequences including the mutated bases of the COIII-ND3-ND4L gene in the salmon mitochondrial DNA of the present invention. This linkage is a comparison of 730 sequences in the micochondrial DNA COIII-ND3-ND4L region of Korea, Japan, the United States, and Canada salmon. It shows that there are a number of specific monobasic polymorphic mutations. Although divided into 50 nucleotide sequences due to the limitation of the paper, Figures 2 to 6 mean the drawings in order. Among them, the DNA sequence position portions shown in Tables 1 and 2 above represent regions in which species- or family-specific monobasic polymorphisms that can be used as polynucleotide probes to be used in the microarray method exist.

After numerous studies and efforts, the inventors have confirmed that the DNA sequence corresponding to SEQ ID NO: 5 to SEQ ID NO: 34 in the COIII-ND3-ND4L gene in salmon mitochondrial DNA is optimally suitable for distinguishing each salmon species or family. By using the polynucleotide probe having the same or complementary nucleotide sequence as SEQ ID NO: 5 to SEQ ID NO: 34, it can be seen that the species or crab group of salmon can be distinguished significantly better than any other conventional method. Since these polynucleotide probes are prepared based on the mononucleotide polymorphism (SNP) sites of each species of salmon, they are characterized by binding to the intended salmon species or the DNA of the family, but not to other salmon species or the family.

In the present invention, 'polymorphism' means the occurrence of two or more alternative sequences or alleles in a genetically determined population. The polymorphic marker or site is the location where divergence occurs. Preferred markers have two or more alleles that exhibit a frequency of occurrence of at least 1%, more preferably at least 10% or 20%, in the selected population. The polymorphic site may be a single base pair.

Accordingly, the present invention may be a nucleotide or nucleic acid fragment comprising the same or a complement thereof including one or more polymorphic sites according to the present invention in addition to the above-described nucleotide sequence. In addition, in view of the limited mutations in one salmon individual and the characteristics of the salmon collectively flocked to each family, a more preferred form of the polymorphic region according to the present invention has two or more individual polymorphic sites for discrimination between salmon families. From the same genotype is preferred. In addition, the polymorphic site according to the present invention means that two or more alternative sequences or alleles occur in a genetically determined population, as described above, and is identical to the nucleotide sequence of the polymorphic site shown in FIGS. 2 to 6. Or a base sequence including a DNA sequence capable of complementarily binding thereto.

Based on such polymorphic sites, the polynucleotide probe according to the present invention corresponds to the present invention as long as the polynucleotide probe includes a nucleotide sequence capable of complementarily binding to the DNA sequence of any one of SEQ ID NOs: 5 to 34. do. Preferably, at least 10 and up to 100 consecutive polynucleotides or complements thereof, including base sequences capable of complementarily binding to the DNA sequence, more preferably at least 10 and up to 50 And most preferably 15 or more and 30 or less.

The polynucleotide probe binds to a mononucleotide polymorphism site of the COIII-ND3-ND4L gene region in salmon mitochondrial DNA in binding to a target DNA extracted from salmon or a PCR product amplified from the extracted DNA. It is characterized in that it is made differently depending on the species or family. Thus, the present invention determines the species or family of salmon according to the combination of the probe and the target product, the species and family of salmon that can be analyzed by the probe according to the present invention is shown in Tables 3 and 4 below. .

Table 3: Analytical Salmon Species

 Analytical Salmon Species     Salmon Salmon (chum salmon, O. keta ) Pink salmon, O. gorbuscha Sockeye salmon (sockeye salmon, O. nerka ) Chinook salmon ( O. tshawytscha ) Coho salmon ( O. kisutch ) Cherry salmon ( O. masou masou ) Wild Trout (ishikawae salmon, O. masou ishikawae ) Rainbow trout ( O. mykiss ) cutthroat trout ( O. clarkii ) Atlantic salmon ( Salmo salar )

Table 4: Analyzeable Chum Salmon Family

 Analytical chum salmon family (genotype)   Salmon  Korean and Japanese military (genotype A1)  Canadian, Alaska, Russian Military (Genotype B1)  United States Troops (Genetic C1)  Korean and Japanese military (genotype D1)  Republic of Korea (genetic D2)

In Tables 3 and 4, the analytical salmon species refers to the type of salmon, and the analytical salmon family refers to the salmon family corresponding to the salmon (chum salmon) species among the salmon species. . In particular, the family of salmon (chum salmon) species was divided into A1, B1, C1, D1 and D2 genotypes, these genotypes represent the same meaning as the salmon genotype shown in Table 2 above.

As described above, the polynucleotide probe according to the present invention is a mononucleotide polymorphism site of the COIII-ND3-ND4L gene region in salmon mitochondrial DNA in binding to a target DNA extracted from salmon or a PCR product amplified the extracted DNA. Combined with, characterized in that the combination is made differently depending on the species or family of salmon.

Specifically, the binding is made differently depending on the species or family of salmon, as shown in Table 2, the polynucleotide probe of SEQ ID NO: 5 (this includes a polynucleotide comprising a base sequence complementary to the DNA sequence of SEQ ID NO: 5) Nucleotide probe, which is the same in each sequence number below), specifically binds to the 364-386th DNA sequence of the COIII-ND3-ND4L gene region in the mitochondrial DNA of the salmon (chum salmon, O. keta ) species, and In the case of No. 6, the 402-422th DNA sequence of pink salmon ( O. gorbuscha ) species, and in the case of SEQ ID NO: 7, the 208-233th DNA sequence of the sockeye salmon ( O. nerka ) species In the case of No. 8, the 395-416th DNA sequence of the chinook salmon ( O. tshawytscha ) species, in the case of SEQ ID NO: 9 the 402-418th DNA sequence of the cherry salmon ( O. masou masou ) species, In the case of SEQ ID NO: 10, the salted salmon (cherry salm) on, O. masou masou ) 517-534 DNA sequence, SEQ ID NO: 11 is the 551-568 DNA sequence of the species (ishikawae salmon, O. masou ishikawae ), rainbow trout (SEQ ID NO: 12) rainbow trout, O. mykiss ) 399-420 DNA sequence, SEQ ID NO: 13 is the 396-415 DNA sequence of cutthroat trout ( O. clarkii ) species, Atlantic salmon, 351-369 DNA sequence of the Salmo salar species, coho salmon ( O. kisutch ) for the 211-231 DNA sequence of the species, coho salmon, O for the SEQ ID NO: 16 kisutch ) and other 211-231 DNA sequences of all species, SEQ ID NO: 17 of the salmon (chum salmon, O. keta ) of the 181-200th DNA sequence of all other species, SEQ ID NO: 24 of the salmon 367-383 DNA sequence, SEQ ID NO: 25 is the 301-320 DNA sequence of the coho salmon species; SEQ ID NO: 26 is the 280-300 DNA sequence of the salmon In the case of No. 27, the 571-593th DNA sequence of a red salmon salmon, SEQ ID NO: 28, the 548-571th DNA sequence of a Shima salmon species, and in the case of SEQ ID NO: 29, the 548-571th DNA sequence of a wild salmon species, sequence No. 30 is the 591-611th DNA sequence of the Atlantic salmon species, No. 31 is the 491-510 DNA sequence of the musk salmon species, No. 32 is the 531-550th DNA sequence of the coho salmon species, In the case of SEQ ID NO: 33, the 531-550th DNA sequence of the Shima salmon and wild salmon species, and the case of SEQ ID NO: 34 may be characterized in that it specifically binds to the 511-530 DNA sequence of the rainbow trout species.

Among them, in the case of SEQ ID NO: 16, specifically binding to the DNA sequence of all species other than coho salmon ( O. kisutch ), coho salmon, O of the analysis available salmon species shown in Table 3 All salmon species except kisutch , including salmon (chum salmon, O. keta), pink salmon (O. gorbuscha), sockeye salmon (O. nerka), and salmon (chinook salmon). tshawytscha, cherry salmon (O. masou masou), mountain trout (ishikawae salmon, O. masou ishikawae), rainbow trout (O. mykiss), cutthroat trout (O. clarkii) and Atlantic salmon (atlantic salmon) , Salmo salar). Similarly, the sequence number in the case of salmon 17 (chum salmon, O. keta) that all other species of binding DNA sequence and specifically is salmon (chum salmon from a salmon analyzable species shown in Table 3, O. keta ) , Except salmon salmon (pink salmon, O. gorbuscha), sockeye salmon (O. nerka), salmon (chinook salmon, O. tshawytscha), and salmon (coho salmon, O. kisutch) ), Sea salmon (cherry salmon, O. masou masou), mountain trout (ishikawae salmon, O. masou ishikawae), rainbow trout (rainbow trout, O. mykiss), cutthroat trout (O. clarkii) and Atlantic salmon (atlantic salmon, Salmo salar).

In addition, when the polynucleotide probe according to the present invention binds to a target DNA extracted from salmon or a PCR product obtained by amplifying the extracted DNA, another meaning of the binding being different according to salmon species or family is different. This is to determine the family of salmon (chum salmon, O. keta) species among salmon species. That is, the polynucleotide probe of SEQ ID NO: 18 specifically binds to the 291-314 DNA sequence of the COIII-ND3-ND4L gene region in the mitochondrial DNA of the salmon (chum salmon) belonging to the American family (type C1), and SEQ ID NO: 19 is the 523-544 DNA sequence of the Korean and Japanese family (type A1), the 296-318 DNA sequence of the Korean family (type D2) is SEQ ID NO: 20, Canada, Alaska and 524-543th DNA sequence of Russian family (type B1) and US family (type C1), Korean and Japanese family (type D1), Korean family (type D2), and in case of SEQ ID NO: 22, Korean and Japanese family (A1 type) ), The 581-604th DNA sequence of the Korean and Japanese family (type D1), the Korean family (type D2), and SEQ ID NO: 23 for the Canadian, Alaska, and Russian family (type B1) and the US family (type C1). It is also possible to specifically bind to the -604th DNA sequence.

The present invention uses a DNA sequence of any one of SEQ ID NO: 5 to SEQ ID NO: 34 having a single nucleotide polymorphism site in the various salmon species, the same or complementary to the DNA sequence (complementary) A polynucleotide probe containing a sequence of possible sequences is characterized by binding the salmon's DNA extract (exactly the PCR product) we want to know.

The polynucleotide probe according to the present invention is produced based on the mononucleotide polymorphism (SNP) site of each species of salmon, and binds to the intended salmon species or the DNA of the family, but not to other salmon species or the family. Therefore, the step of binding the polynucleotide probe is preferably performed at least one or more times. Among them, if the repeated extraction of the probe according to the present invention and DNA extracted before the step of confirming the binding repeatedly, the binding process can be more surely secured to more tightly bind the probe and the target DNA product. In addition, repeatedly performing the binding of the extracted DNA and the probe according to the present invention after the step of confirming the binding has the effect of predicting the result of the determination indirectly using a variety of probes. Therefore, the step of binding the polynucleotide probe is preferably performed repeatedly at least one or more times several times (particularly 3 to 4 times).

Another feature of the present invention is the step of extracting DNA from blood, cells or tissues of the salmon (salmon), performing a polymerase chain reaction (PCR) on the extracted DNA to obtain a PCR product, the polymerization The enzyme chain reaction (PCR) may be performed using a polynucleotide consisting of a DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a polynucleotide consisting of a nucleotide sequence complementary to the DNA sequence as a forward primer, or SEQ ID NO: 3 or SEQ ID NO: It may be characterized by using a polynucleotide consisting of a DNA sequence of 4 or a polynucleotide consisting of a nucleotide sequence complementary to the DNA sequence as a reverse primer.

In other words, to amplify DNA extracted from salmon, a specific primer is used. This particular primer matches the mononucleotide polymorphism (SNP) region DNA sequence of the COIII-ND3-ND4L gene in salmon mitochondrial DNA, thereby further amplifying the extracted DNA. To this end, the DNA extracted from the blood, cells or tissues of salmon preferably comprises a monobasic polymorphism (SNP) site of the COIII-ND3-ND4L gene.

The forward primer and the reverse primer used to amplify the DNA extracted in the present invention by PCR are as shown in Table 5 below.

Table 5: Primers for Salmon Species Identification

 Primer base sequence for salmon species identification  primer  Sequence  Sequence position  salmon (SEQ ID NO 1) 5'-tga tat tga cac ttt gta gac gt -3 '  136-158 (SEQ ID NO: 2) 5'-tga tac tga cac ttt gta gac gt -3 '  136-158 (SEQ ID NO 3) 5'-aag ggt ctt gtt ttg gac t -3 '  649-629 (SEQ ID NO: 4) 5'-aag ggt ctt att ttg gac t -3 '  649-629

As shown in Table 5, the primer used in the PCR process of the present invention is also formed to optimally fit the mononucleotide polymorphism (SNP) region DNA sequence of the COIII-ND3-ND4L gene in salmon mitochondrial DNA. It is produced on the basis of the most common sequencing regardless of the various salmon species or families. The inventors of the present invention, after investigating and analyzing DNA sequences and SNPs of various salmon species shown in FIGS. 2 to 6, can use PCR sequences to produce PCR products most effectively. It was confirmed.

Specifically, using the polynucleotide consisting of a DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a polynucleotide consisting of a base sequence complementary to the DNA sequence as a forward primer, COIII-ND3- in the salmon mitochondrial DNA shown in Figure 2 Bind to positions 136-158 of the ND4L gene. The reverse primer may be a polynucleotide consisting of a DNA sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or a polynucleotide consisting of a nucleotide sequence complementary to the DNA sequence. To the position one. Then, regardless of the various salmon species or family, salmon mitochondrial DNA extract will be amplified by the PCR method, thereby increasing the number of PCR products amplified significantly.

In addition, the present invention is a rhodamine (rhodamine), cy3, cy5 or biotin is attached to the forward primer or the reverse primer to confirm the hybridization reaction performed after combining the PCR product and the probe according to the present invention, Checking whether the binding is also possible to determine the salmon species, characterized in that by determining the binding by detecting the fluorescence expressed by the rhodamine, cy3, cy5 or biotin. Specifically, the above confirming step may be characterized by using rhodamine, CY3, CY5 or Syreptavidin-Cyanine in combination with biotin, and conveniently performing genotyping by analyzing the results using a microarray scanner. have.

Another embodiment for achieving another object of the present invention is to be used in the method for discriminating salmon species or family as described above, the same or complementary to the DNA sequence of any one of SEQ ID NO: 5 to SEQ ID NO: 34 ) Is a polynucleotide probe for salmon species or herds, characterized in that consisting of 15-30 consecutive nucleotide sequences. Furthermore, the present invention binds to the mononucleotide polymorphism (SNP) region DNA sequence of the COIII-ND3-ND4L gene in salmon mitochondrial DNA, and is identical or complementary to the DNA sequence of any one of SEQ ID NOs: 5 to 34. ) May be a DNA chip comprising a polynucleotide probe for discriminating salmon species or family consisting of 15-30 consecutive sequences. Here, the polynucleotide probe is preferably in the form of being immobilized on a substrate on the glass.

In the present invention, a plurality of salmon species can be simultaneously identified on one slide using a DNA microarray technique. About 20 probes capable of discriminating salmon species were integrated by setting a number of zones on one glass slide treated with chemical functional groups, and the target gene sequence was obtained by fluorescence label obtained by symmetric or asymmetric PCR. By identifying the hybridization reaction, the correct salmon species can be identified.

As described above, the polynucleotide probe is composed of 15-30 consecutive nucleotide sequences identical to or complementary to the DNA sequence of any one of SEQ ID NOs: 5 to 34. Because it is made on the basis of polymorphism (SNP) sites, it is characterized by binding to the intended salmon species or the DNA of the family, but not to other salmon species or the family. Accordingly, by using the polynucleotide probe having the same or complementary nucleotide sequence as SEQ ID NO: 5 to SEQ ID NO: 34, it is possible to distinguish the species or family of salmon remarkably superior to any other conventional method.

In addition, another embodiment for achieving another object of the present invention may be a kit for determining salmon species or flocks used in the method for discriminating salmon species or flocks as described above.

That is, it binds to the mononucleotide polymorphism (SNP) region DNA sequence of the COIII-ND3-ND4L gene in salmon mitochondrial DNA, and is identical or complementary to the DNA sequence of any one of SEQ ID NOs: 5 to 34. A polynucleotide probe for discriminating salmon species or herds, characterized in that it comprises 30 consecutive nucleotide sequences; Primer for amplifying the salmon mitochondrial DNA by polymerase chain reaction (PCR); may be a kit for determining salmon species or family, in which case the primer is a DNA of SEQ ID NO: 1 or SEQ ID NO: 2 Preferably, the primer is a forward primer consisting of the same or complementary base sequence or a reverse primer consisting of the same or complementary base sequence as the DNA sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

Such a salmon species or family identification kit uses a microarray method using the above-described polynucleotide probe, and discriminates the group-specific monobasic polymorphism according to the hybridization of DNA, thereby analyzing the genotype of salmon without analyzing the nucleotide sequence. It has the utility to quickly determine the family. In addition, it is possible to analyze the exact genotype of a large number of salmon fish species in one experiment, thereby maximizing the applicability of technology development to standardize and automate salmon genotype and herd method. That is, the present invention significantly shortened the time required for analyzing a sample compared to the conventional method, and has an effect of processing a large amount of samples in a short time.

Hereinafter, one preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings. The invention may be better understood by the following examples, which are intended for purposes of illustration of the invention and are not intended to limit the scope of protection defined by the appended claims.

Example 1 Synthesis and Sequence of Primers for Salmon Species Identification

First, primers for amplifying DNA extracted from salmon were synthesized. In the present invention, the primers used for symmetric or asymmetric PCR are as shown in Table 5 above.

Reverse (antisense) primers used for symmetric or asymmetric PCR were prepared by attaching rhodamine, cy3, cy5 to the ends or by biotin to confirm the fluorescence after the hybridization reaction. It was used to make and combine. Primers used in the present invention was synthesized by the German Metabion company.

Example 2 Salmon DNA Extraction and Symmetric or Asymmetric PCR Reactions

1) Salmon DNA was extracted using DNeasy tissue kit from Qiagen.

2) The symmetric PCR reaction was performed in a DNA engine (MJ Research, USA) by the method of the conditions shown in Table 6 below.

3) The asymmetric PCR reaction was performed in the DNA engine (MJ Research, USA) by the method of the conditions shown in Table 7 below.

4) After PCR, add 1ul of gel loading buffer (0.2% orange G, 0.25% xylene cyanol FF, 60% glycerol) to 5ul of PCR product and electrophores on 2% agarose gel containing 1ug / ml ethidium bromide. After checking the PCR band in the image analyzer (HITACHI, Japan) attached UV transilluminator (see Fig. 7). Figure 7 is an electrophoretic picture of the PCR product amplified by the PCR method according to an embodiment of the present invention.

Table 6: Salmon Symmetric PCR Reaction Conditions

 Salmon Symmetric PCR Reaction Conditions  Reaction composition condition  Reaction temperature condition  10X buffer  2.0ul  95 degrees, 5.0 minutes  1 time  2.5mM dNTP mix  1.0ul  10 pmol forward (sense) primer  1.0ul  94 degrees, 15 seconds 53 degrees, 45 seconds 72 degrees, 30 seconds  35 times  10 pmol reverse (antisense) primer  1.0ul  5U Taq  0.2ul  Extracted DNA  1.0ul  Sterile distilled water  13.8ul  72 degrees, 5.0 minutes  1 time  Sum  20.0ul

Table 6: Salmon Asymmetric PCR Reaction Conditions

 Salmon Asymmetric PCR Reaction Conditions  Reaction composition condition  Reaction temperature condition  10X buffer  2.0ul  95 degrees, 5.0 minutes  1 time  2.5mM dNTP mix  1.0ul  10 pmol forward (sense) primer  1.0ul  94 degrees, 15 seconds 53 degrees, 45 seconds 72 degrees, 30 seconds  35 times  10 pmol reverse (antisense) primer  1.0ul  5U Taq  0.2ul  Extracted DNA  1.0ul  Sterile distilled water  13.8ul  72 degrees, 5.0 minutes  1 time  Sum  20.0ul

Example 3 Probe Synthesis and Sequence for the Preparation of Salmon Species DNA Chip

It is to immobilize the probe according to the invention to produce a DNA chip. First, in order to covalently bond the probe according to the present invention on the glass on which the aldehyde functional group is treated, the mino link is modified at the 5 'end of the selected probe, and the spatial interference between the probes integrated on the glass slide during the hybridization reaction. In order to minimize the 10-20 oligos (dT) was attached, and then synthesized by adding the base sequence (complementary DNA sequence) as shown in Table 1, Table 2 above. Synthesis was performed by Metabion, Germany. The base sequence of the produced probe was divided into a total of 10 different genotypes of salmon and fish as shown in Table 3 and a total of 5 salmon family genotypes as shown in Table 4.

Example 4 Preparation of Salmon Species Chip

1) In order to fabricate oligonucleotide chip, the same amount of 3X SSC was mixed and mixed with CSS-100 Silylated Slides (cell associate, USA) at the concentration of 50pmole. The reaction is carried out at room temperature for 16 hours. After the reaction was completed, the slides were washed twice with 0.2% SDS for 5 minutes and then twice with distilled water for 5 minutes.

2) The slides were reacted with sodium borohydride (1.3 g NaBH ₄ 375 ml PBS, 125 ml 100% EtOH) for 5 minutes and then reacted in boiling distilled water for 3 minutes, and then dried in a vacuum centrifuge at 800 rpm for 10 minutes.

3) For multiple reactions on one slide, the samples were separated into a perfusion chamber (BioGrace, USA) and stored in the dark at room temperature until the experiment.

The DNA chip thus produced is exemplarily shown in FIG. 8. FIG. 8 is a schematic diagram illustrating the structure of a DNA chip including an oligonucleotide probe prepared based on the monobasic polymorphism shown in FIGS. 2 to 6.

Example 5 Hybridization Reaction

1) PCR products bound with rhodamine, cy3, cy5 or biotin were mixed with hybridization solution (3X SSC, 0.25% SDS) in a ratio of 1: 9. When biotin was used to generate fluorescence, Syreptavidin-Cyanine was added at a concentration of 1 ug / ml and reacted.

2) The prepared hybridization solution was applied to the chmaber and reacted at 60 degrees for 1 hour.

3) After the first wash for 5 minutes in 1X SSC, 0.1% SDS, the second wash was performed for 3 minutes at 0.1X SSC.

4) The washed slides were dried for 10 minutes at a speed of 800rpm in a centrifuge.

5) Confirmation of the experimental results was performed by using a LuxScan-10K / A (CapitalBio, China) scanner to measure the fluorescence genotype analysis, the results are shown in Figure 9a to 9f.

9A to 9F are photographs showing the reaction result after combining the oligonucleotide probe and PCR amplification products of various salmon according to the present invention using the DNA chip of FIG. 8, wherein FIG. 9A is a salmon (chum) 9b is a multi-pumped salmon, FIG. 9c is a rainbow trout, FIG. 9d is a sima salmon, FIG. 9e is a mountain trout, and FIG. 9f is a result of atlantic salmon. As shown here, in the present invention, since the fluorescent label is different depending on the salmon species or the flocks, it was confirmed that the salmon species or flocks can be determined accordingly.

On the other hand, while the present invention has been shown and described with respect to certain preferred embodiments, the invention is variously modified and modified without departing from the technical features or fields of the invention provided by the claims below It will be apparent to those skilled in the art that such changes can be made.

According to the present invention as described above, the step of extracting DNA from the blood (blood), cells or tissue of the salmon (salmon); Performing a polymerase chain reaction (PCR) on the extracted DNA to obtain a PCR product; Combining the PCR product with a polynucleotide probe comprising a nucleotide sequence identical to or complementary to the DNA sequence of any one of SEQ ID NOs: 5 to 34; And determining the species or species to which the salmon belongs by checking whether the combination is present.

According to the present invention, the genotyping analysis of a plurality of salmon species or family on one slide has the effect that can be examined simply, quickly and accurately. By using the salmon mitochondrial DNA COIII-ND3-ND4L region according to the present invention as a gene marker, it is possible to solve the problem of low resolution, which has been conventionally used to discriminate salmon genotype and belonging family. As in the prior art, using the COIII-ND3-ND4L region, which has been identified as having more monobasic polymorphism according to the present invention than using a mitochondrial DNA control region as a gene marker, is more effective than conventional techniques. By providing advanced forms of genetic analysis, genotypes can be more effectively identified and resolution can be significantly increased.

The inventors have found a single nucleotide polymorphism (SNPs) site of the COIII-ND3-ND4L gene in mitochondrial DNA of various salmon species, and based on this, polynucleotides that can specifically bind to each species of salmon. Probes can now be manufactured. Since these polynucleotide probes are prepared based on the mononucleotide polymorphism (SNP) sites of each species of salmon, they are characterized by binding to the intended salmon species or the DNA of the family, but not to other salmon species or the family.

After numerous studies and efforts, the inventors have confirmed that the DNA sequence corresponding to SEQ ID NO: 5 to SEQ ID NO: 34 in the COIII-ND3-ND4L gene in salmon mitochondrial DNA is optimally suitable for distinguishing each salmon species or family. By using a polynucleotide probe having the same or complementary nucleotide sequence as the DNA sequence of SEQ ID NO: 5 to SEQ ID NO: 34, it can be seen that the species or crab group of salmon is remarkably superior to any other conventional method. Could.

In addition, using the microarray method using the polynucleotide probe described above, the genotype and its family of salmon can be determined without analyzing the nucleotide sequence by discriminating the family specific mononucleotide polymorphism according to the hybridization of DNA. It has applicability to make a quick judgment. In addition, it is possible to analyze the exact genotype of a large number of salmon fish species in one experiment, thereby maximizing the applicability of technology development to standardize and automate salmon genotype and herd method. That is, the present invention significantly shortened the time required for analyzing a sample compared to the conventional method, and has an effect of processing a large amount of samples in a short time.

<110> Korea Ocean Research & Development Institute          Genocheck <120> Identifying Method of Chum Salmon Species or Its Originated          Stocks, Polynucleotide Probe, DNA Chip and Kit for Identifying          The Same <130> 0001 <160> 34 <170> KopatentIn 1.71 <210> 1 <211> 23 <212> DNA <213> salmon <400> 1 tgatattgac actttgtaga cgt 23 <210> 2 <211> 23 <212> DNA <213> salmon <400> 2 tgatactgac actttgtaga cgt 23 <210> 3 <211> 19 <212> DNA <213> salmon <400> 3 aagggtcttg ttttggact 19 <210> 4 <211> 19 <212> DNA <213> salmon <400> 4 aagggtctta ttttggact 19 <210> 5 <211> 23 <212> DNA <213> salmon <400> 5 gacgcagaaa agttgtctcc cta 23 <210> 6 <211> 21 <212> DNA <213> salmon <400> 6 ccccttgggg tctgcccgtc t 21 <210> 7 <211> 26 <212> DNA <213> salmon <400> 7 tcctagtatc aatgagtata agtgac 26 <210> 8 <211> 22 <212> DNA <213> salmon <400> 8 gatttgatcc attagggtcc gc 22 <210> 9 <211> 21 <212> DNA <213> salmon <400> 9 gaccctttag gatccgcccg t 21 <210> 10 <211> 18 <212> DNA <213> salmon <400> 10 ctcaataccc ccgcccta 18 <210> 11 <211> 17 <212> DNA <213> salmon <400> 11 tgctgtgctt gccctcc 17 <210> 12 <211> 21 <212> DNA <213> salmon <400> 12 gaccccttag ggtccgcccg c 21 <210> 13 <211> 20 <212> DNA <213> salmon <400> 13 atttgacccc ctaggatccg 20 <210> 14 <211> 19 <212> DNA <213> salmon <400> 14 acaaataacg cccgacgca 19 <210> 15 <211> 21 <212> DNA <213> salmon <400> 15 tagtattaac gcgtataagt g 21 <210> 16 <211> 21 <212> DNA <213> salmon <400> 16 tagtattaac acgtataagt g 21 <210> 17 <211> 20 <212> DNA <213> salmon <400> 17 tctatttact gatgaggctc 20 <210> 18 <211> 24 <212> DNA <213> salmon <400> 18 aatcattact attaccatca catt 24 <210> 19 <211> 22 <212> DNA <213> salmon <400> 19 accccaaccc taacacttat tt 22 <210> 20 <211> 23 <212> DNA <213> salmon <400> 20 ttactatcac cctcacattg tcc 23 <210> 21 <211> 20 <212> DNA <213> salmon <400> 21 ccccaaccct gacacttatt 20 <210> 22 <211> 24 <212> DNA <213> salmon <400> 22 taatttatga atgaacccaa ggag 24 <210> 23 <211> 24 <212> DNA <213> salmon <400> 23 taatttatga gtgaacccaa ggag 24 <210> 24 <211> 17 <212> DNA <213> salmon <400> 24 gcagagaagt tatcccc 17 <210> 25 <211> 20 <212> DNA <213> salmon <400> 25 atcaccatta cactgtctgc 20 <210> 26 <211> 20 <212> DNA <213> salmon <400> 26 tagttacaac aatcatcgct 20 <210> 27 <211> 23 <212> DNA <213> salmon <400> 27 actcttggac taatttatga gtg 23 <210> 28 <211> 23 <212> DNA <213> salmon <400> 28 ccactgctgt acttgccctc ctt 23 <210> 29 <211> 23 <212> DNA <213> salmon <400> 29 ccactgctgt gcttgccctc ctt 23 <210> 30 <211> 20 <212> DNA <213> salmon <400> 30 atggacccag ggaggccttg 20 <210> 31 <211> 20 <212> DNA <213> salmon <400> 31 tcctccccct accctgaggg 20 <210> 32 <211> 20 <212> DNA <213> salmon <400> 32 cttgacactc gtttggtcca 20 <210> 33 <211> 20 <212> DNA <213> salmon <400> 33 cctaacactc gtctgatcca 20 <210> 34 <211> 20 <212> DNA <213> salmon <400> 34 gatcaactcc acaccccgac 20

Claims (13)

Extracting DNA from blood, cells or tissues of the salmon; Performing a polymerase chain reaction (PCR) on the extracted DNA to obtain a PCR product; Combining the PCR product with a polynucleotide probe comprising a nucleotide sequence identical to or complementary to the DNA sequence of any one of SEQ ID NOs: 5 to 34; Determining whether the species belong to the species or the family belonging to the salmon; The method of claim 1, wherein the extracted DNA comprises single nucleotide polymorphisms (SNPs) of the COIII-ND3-ND4L gene region in salmon mitochondrial DNA. The method of claim 1, wherein the polynucleotide probe consists of 15-30 consecutive nucleotide sequences. According to claim 1, wherein the polynucleotide probe comprising a nucleotide sequence complementary to any one of SEQ ID NO: 5 to SEQ ID NO: 34 binds to the monobasic polymorphism site of the COIII-ND3-ND4L gene region in salmon mitochondrial DNA By the way, the combining method is characterized in that the salmon species or flocks, characterized in that made differently depending on the salmon species or flocks. The method according to claim 4, wherein the binding is different depending on the salmon species or family, wherein the polynucleotide probe of SEQ ID NO: 5 is a gene of the COIII-ND3-ND4L gene region in the mitochondrial DNA of the salmon (chum salmon, O. keta ) It specifically binds to the 364-386th DNA sequence, and in the case of SEQ ID NO: 6, the 402-422 DNA sequence of the pink salmon ( O. gorbuscha ) species, and in the case of SEQ ID NO: 7, sockeye salmon, O. nerka ) DNA sequence 208-233, SEQ ID NO: 8 for the salmon salmon (chinook salmon, O. tshawytscha ) 395-416 DNA sequence for SEQ ID NO: 9 salmon salmon (cherry salmon, 402-418th DNA sequence of O. masou masou species; cherry salmon, O. masou masou ) 517-534 DNA sequence of SEQ ID NO: 11; , O. masou ishikawae ) 551-568th DNA sequence, and in the case of SEQ ID NO: 12 a rainbow trout (rai nbow trout, O. mykiss ) 399-420 DNA sequence, SEQ ID NO: 13 396-415 DNA sequence of cutthroat trout ( O. clarkii ) species, Atlantic salmon, 351-369 DNA sequence of the Salmo salar species, coho salmon ( O. kisutch ) for the 211-231 DNA sequence of the species, coho salmon, O for the SEQ ID NO: 16 kisutch ) and other 211-231 DNA sequences of all species, SEQ ID NO: 17 of the salmon (chum salmon, O. keta ) of the 181-200th DNA sequence of all other species, SEQ ID NO: 24 of the salmon 367-383 DNA sequence, SEQ ID NO: 25 is the 301-320 DNA sequence of the coho salmon species, SEQ ID NO: 26 is the 280-300 DNA sequence of the sockeye salmon species; Is the 571-593 DNA sequence of SEQ ID NO: 28, the 548-571 DNA sequence of the Shima salmon species, the SEQ ID NO: 29 is the 548-571 DNA sequence of the wild salmon species, SEQ ID NO: 30 is the 591-611 DNA sequence of the Atlantic salmon species, SEQ ID NO: 31 is the 491-510 DNA sequence of the musk salmon species, and SEQ ID NO: 32 is the 531-550 DNA sequence of the salmon salmon species In case of SEQ ID NO: 33, salmon species or family characterized by specifically binding to the 531-550th DNA sequence of Shima salmon and wild trout species, and SEQ ID NO: 34 of 511-530 DNA sequences of rainbow trout species How to Determine The method according to claim 4, wherein the binding is different according to salmon species or family, wherein the polynucleotide probe of SEQ ID NO: 18 COIII-ND3-ND4L in the mitochondrial DNA of salmon (chum salmon) belonging to the American family (type C1) Specifically binds to the 291-314th DNA sequence of the gene region, and in the case of SEQ ID NO: 19, the 523-544 DNA sequence of the Korean and Japanese family (type A1); and in the case of SEQ ID NO: 20, the Korean family (D2) 296-318 DNA sequence of SEQ ID NO: 21 for the Canadian, Alaska and Russian family (type B1) and the American family (type C1), the Korean and Japanese family (type D1), and the Korean family (type D2) 543th DNA sequence, SEQ ID NO: 22 is the Korean and Japanese family (A1 type), Korea and Japan family (D1), Korean family (D2) 581-604 DNA sequence, and SEQ ID NO: 23 Canada Specific to the 581-604th DNA sequence of the US, Alaska, and Russian (Type B1) and US (Type C1) Salmon species or determination method of the flock characterized in that. The method of claim 1, wherein the binding of the polynucleotide probe is performed at least once. The method according to claim 1, wherein the PCR is performed by using a polynucleotide consisting of a DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a polynucleotide consisting of a base sequence complementary to the DNA sequence as a forward primer Or a polynucleotide consisting of a DNA sequence of SEQ ID NO: 3 or SEQ ID NO: 4 or a polynucleotide consisting of a base sequence complementary to the DNA sequence as a reverse primer. The method of claim 8, wherein the forward primer or the reverse primer is attached to the rhodamine (rhodamine), cy3, cy5 or biotin, and confirming the binding is fluorescence expressed by the rhodamine, cy3, cy5 or biotin Determination method of salmon species or flocks characterized in that to determine whether the detection by combining. 15-30 which is the same or complementary to the DNA sequence of any one of SEQ ID NO: 5 to SEQ ID NO: 34, which is used in the method for discriminating salmon species or family according to any one of claims 1 to 9. Salmon species or family group polynucleotide probe, characterized in that consisting of two consecutive nucleotide sequences. Binding to the mononucleotide polymorphism (SNP) region DNA sequence of the COIII-ND3-ND4L gene in salmon mitochondrial DNA, 15-30 identical or complementary to the DNA sequence of any one of SEQ ID NOS: 5-34 Salmon species or family group DNA chip comprising a polynucleotide probe for discriminating salmon species or family group consisting of a sequence. Binding to the mononucleotide polymorphism (SNP) region DNA sequence of the COIII-ND3-ND4L gene in salmon mitochondrial DNA, 15-30 identical or complementary to the DNA sequence of any one of SEQ ID NOS: 5-34 A polynucleotide probe for discriminating salmon species or herds, characterized in that the sequence consists of a sequence; And a primer for amplifying the salmon mitochondrial DNA by polymerase chain reaction (PCR). The method of claim 12, wherein the primer is a forward primer consisting of a base sequence identical or complementary to the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or consisting of a base sequence identical to or complementary to the DNA sequence of SEQ ID NO: 3 or SEQ ID NO: Salmon species or flocks determination kit, characterized in that the reverse primer.

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