Method for identifying species or hybrids of pufferfish

Abstract

To provide methods for discriminating species or hybrids of Takifugu fishes.SOLUTION: Provided is a method for discriminating species or hybrids of Takifugu fishes, comprising detecting at least one single nucleotide polymorphism (SNP) that differs between species of Takifugu fishes in the nuclear DNA derived from a sample, and discriminating the species or hybrids of the Takifugu fishes to which the sample belongs based on the detected SNP genotype.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for distinguishing between species or hybrids of fish of the genus Pufferfish. [Background technology]

[0002] The Food Sanitation Act stipulates that edible species and edible parts of pufferfish of the genus Takifugu are regulated, and pufferfish of unknown species must be eliminated. However, hybrids resulting from intraspecific variation and interspecific hybridization are often found within Takifugu, and it is often difficult to distinguish the species or hybrid from their appearance. Furthermore, when identifying the species or hybrid causing food poisoning caused by eating pufferfish, it is necessary to identify the species or hybrid from leftover food or vomit, rather than relying on appearance.

[0003] Previously, methods for identifying species of pufferfish using mitochondrial DNA (mtDNA) sequence information have been reported (Non-Patent Documents 1 and 2). However, because mtDNA is maternally inherited and hybrids only have the mtDNA of their maternal species, there was a problem in that hybrids could not be identified. Furthermore, because interspecies penetration of mtDNA has occurred between pufferfish species through hybridization during the evolutionary process, there was a problem in that identification methods based on mtDNA would result in a certain probability of misidentification.

[0004] A method for distinguishing species or hybrids of fish of the genus Takifugu using information on polymorphisms in bisexually inherited nuclear DNA has also been reported (Non-Patent Documents 3 and 4). This method utilizes the amplified fragment length polymorphism (AFLP) method, but has the problem of complicated experimental procedures and time-consuming analysis. Furthermore, the AFLP method involves cleaving genomic DNA with a restriction enzyme to fragments, adding specific adapters to the fragments, using these as templates for PCR amplification, and amplifying them using primers with arbitrary bases added to the 3' ends to obtain information on polymorphisms in base sequences. However, if the genomic DNA used is fragmented in advance, the reproducibility of the experiment is reduced. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hsieh, YW et al. "Identification of toxin and fish species in cooked fish liver implicated in food poisoning" Journal of Food Science, 67(3), 948-952, 2002 [Non-patent document 2] Sadako Fukiya and Yukio Kazuta, "Identification of Pufferfish Species by PCR-RFLP and PCR-SSCP Methods," Bulletin of the Yamaguchi Prefectural Environmental Health Center, No. 53, 49-51, 2010 [Non-patent document 3] Takahashi, H. et al. "Asymmetric hybridization and introgression between sibling species of the pufferfish Takifugu that have transgenic explosive speciation" Marine Biology, 164(4), 90, 2017 [Non-patent document 4] Tatsuno, R. et al. "Tetrodotoxin distribution in natural hybrids between the pufferfish species Takifugu rubripes and Takifugu porphyreus" Fisheries Science, 85(1), 247-245, 2019 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides A method for accurately, simply, and quickly distinguishing between a specific species of Takifugu fish, a Takifugu fish other than the specific species, or a hybrid between the specific species of Takifugu fish and a Takifugu fish other than the specific species, and The present invention relates to providing a method for accurately, simply, and quickly identifying species or hybrids of fish of the genus Pufferfish. [Means for solving the problem]

[0007] As a result of various investigations to solve the above problems, the present inventors have found multiple species-specific single nucleotide polymorphisms (SNPs) in the nuclear DNA of pufferfish. Furthermore, the present inventors have discovered that by using these SNPs as indicators, it is possible to easily and quickly distinguish between species or hybrids of pufferfish with high accuracy, and have completed the present invention.

[0008] That is, the present invention provides the following [1] to [9]. [1] A method for distinguishing between species or hybrids of pufferfish, Detecting at least one single nucleotide polymorphism (SNP) that differs between species of pufferfish in nuclear DNA from the sample; and Identifying the species or hybrid of the pufferfish genus to which the sample belongs based on the genotype of the detected SNP; A method comprising: [2] The method of [1], wherein the SNP is at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 1 to 33 and 166 to 168. [3] The SNP is at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 1 to 3, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 4 to 6, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 7 to 9, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 10 to 12, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 13 to 15 and 166, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 16 to 18 and 167. the discrimination method according to [1] or [2], wherein the SNP is at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 19 to 21, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 22 to 24, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 25 to 27 and 168, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 28 to 30, and at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 31 to 33. [4] The discrimination method of [2] or [3], wherein if the genotype of the detected SNP is (ii) in Table 1 below, the species of the sample is discriminated to be the species (i) in Table 1 below; if the genotype of the detected SNP is (iii) in Table 1 below, the species of the sample is discriminated to be a species of Takifugu species other than (i) in Table 1 below; and if the genotype of the detected SNP is (iv) in Table 1 below, the species of the sample is discriminated to be a hybrid of the species (i) in Table 1 below and a species of Takifugu species other than (i).

[0009] [Table 1]

[0010] [5] The method of any one of [1] to [4], wherein the species of the Takifugu species is at least one species selected from the group consisting of red-eyed pufferfish, spotted pufferfish, red-spotted pufferfish, common pufferfish, grass pufferfish, giant pufferfish, stingray pufferfish, Japanese pufferfish, Japanese pufferfish, striped pufferfish, Japanese tiger pufferfish, and crow pufferfish. [6] A probe for distinguishing species or hybrids of fish of the genus Pufferfish, capable of detecting an SNP at the 151st base position of the base sequence shown in any of SEQ ID NOS: 1 to 33 and 166 to 168. [7] A primer set for distinguishing species or hybrids of pufferfish, capable of amplifying a region containing an SNP at the 151st base position of the base sequence shown in any of SEQ ID NOs: 1 to 33 and 166 to 168. [8] A kit for identifying species or hybrids of pufferfish, comprising the probe of [6] and the primer set of [7]. [9] A marker for distinguishing species or hybrids of fish of the genus Pufferfish, comprising a polynucleotide consisting of a partial base sequence of 15 to 100 bases long containing an SNP at the 151st base position of a base sequence shown in any of SEQ ID NOs: 1 to 33 and 166 to 168, or a complementary strand thereof, the 151st base of the base sequence shown in SEQ ID NO: 1 is T or C, the 151st base of the base sequence shown in SEQ ID NO: 2 is C or T, the 151st base of the base sequence shown in SEQ ID NO: 3 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 4 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 5 is T or C, the 151st base of the base sequence shown in SEQ ID NO: 6 is T or G, the 151st base of the base sequence shown in SEQ ID NO: 7 is G or T, the 151st base of the base sequence shown in SEQ ID NO: 8 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 9 is T or C, the 151st base of the base sequence shown in SEQ ID NO: 10 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 11 is G or C; the 151st base of the base sequence shown in SEQ ID NO: 12 is A or C, the 151st base of the base sequence shown in SEQ ID NO: 13 is C or T, the 151st base of the base sequence shown in SEQ ID NO: 14 is C or T, the 151st base of the base sequence shown in SEQ ID NO: 15 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 16 is G or C; the 151st base of the base sequence shown in SEQ ID NO: 17 is T or C, the 151st base of the base sequence shown in SEQ ID NO: 18 is G or A; the 151st base of the base sequence shown in SEQ ID NO: 19 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 20 is C or G, the 151st base of the base sequence shown in SEQ ID NO: 21 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 22 is T or C, the 151st base of the base sequence shown in SEQ ID NO: 23 is C or A, the 151st base of the base sequence shown in SEQ ID NO: 24 is G or C; the 151st base of the base sequence shown in SEQ ID NO: 25 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 26 is A or T, the 151st base of the base sequence shown in SEQ ID NO: 27 is C or G, the 151st base of the base sequence shown in SEQ ID NO: 28 is T or G, the 151st base of the base sequence shown in SEQ ID NO: 29 is C or G, the 151st base of the base sequence shown in SEQ ID NO: 30 is T or C, the 151st base of the base sequence shown in SEQ ID NO: 31 is A or T, the 151st base of the base sequence shown in SEQ ID NO: 32 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 33 is G or A, the 151st base of the base sequence represented by SEQ ID NO: 166 is C or T; the 151st base of the base sequence shown in SEQ ID NO: 167 is G or T; The 151st base of the base sequence represented by SEQ ID NO: 168 is A or T; A marker for identifying species or hybrids of the genus Pufferfish. [Effects of the Invention]

[0011] According to the method of the present invention, A specific species of Takifugu fish, a Takifugu fish other than the specific species, or a hybrid between the specific species of Takifugu fish and the specific species of Takifugu fish other than the specific species, This method allows for easy, rapid identification of species or hybrids of the genus Pufferfish with high accuracy, regardless of their appearance. The SNPs used for such identification are: A specific species of Takifugu fish, a Takifugu fish other than the specific species, or a hybrid between the specific species of Takifugu fish and the specific species of Takifugu fish other than the specific species, The present invention is also useful as an indicator for distinguishing species or hybrids of fish of the genus Puffer. A specific species of Takifugu fish, a Takifugu fish other than the specific species, or a hybrid between the specific species of Takifugu fish and the specific species of Takifugu fish other than the specific species, Provided are a probe, a primer set, and a kit for identifying species or hybrids of fish of the genus Pufferfish. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of detecting the genotype of the SNP at the 151st base position of the base sequence shown in SEQ ID NO: 4 for nuclear DNA derived from samples collected from the spotted pufferfish, the Japanese pufferfish, and hybrids between them. [Figure 2] FIG. 1 shows the results of detecting the genotype of the SNP at the 151st base position of the base sequence shown in SEQ ID NO: 5 for nuclear DNA derived from samples collected from the spotted pufferfish, the Japanese pufferfish, and hybrids between them. [Figure 3]FIG. 1 shows the results of detecting the genotype of the SNP at the 151st base position of the base sequence shown in SEQ ID NO: 6 for nuclear DNA derived from samples collected from the spotted pufferfish, the Japanese pufferfish, and hybrids between them. [Figure 4] FIG. 1 shows the results of detecting the genotype of the SNP at the 151st base position of the base sequence shown in SEQ ID NO: 28 for nuclear DNA derived from samples collected from the spotted pufferfish, the Japanese pufferfish, and hybrids between them. [Figure 5] FIG. 1 shows the results of detecting the genotype of the SNP at the 151st base position of the base sequence shown in SEQ ID NO: 29 for nuclear DNA derived from samples collected from the spotted pufferfish, the Japanese pufferfish, and hybrids between them. [Figure 6] FIG. 1 shows the results of detecting the genotype of the SNP at the 151st base position of the base sequence shown in SEQ ID NO: 30 for nuclear DNA derived from samples collected from the spotted pufferfish, the Japanese pufferfish, and hybrids between them. [Figure 7] FIG. 1 shows the results of detecting the genotype of the SNP at the 151st base position of the base sequence shown in SEQ ID NO: 1 for nuclear DNA derived from samples collected from 11 species of pufferfish. DETAILED DESCRIPTION OF THE INVENTION

[0013] In this specification, the abbreviations for base sequences (nucleotide sequences), nucleic acids, etc. are written using symbols commonly used in the relevant field, such as the IUPAC-IUB rules (IUPAC-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138:9-37, 1984), "Guidelines for the preparation of specifications, etc., including base sequences or amino acid sequences" (edited by the Japan Patent Office), etc. In this specification, "deoxyribonucleic acid (DNA)" includes not only double-stranded DNA but also the single-stranded DNAs, that is, the sense strand and the antisense strand, which constitute it.

[0014] As used herein, "nucleotide," "oligonucleotide," and "polynucleotide" are synonymous with nucleic acid and include both DNA and RNA. Said DNA includes cDNA, genomic DNA, and synthetic DNA. Said RNA includes total RNA, mRNA, rRNA, and synthetic RNA. Furthermore, "nucleotide," "oligonucleotide," and "polynucleotide" may be double-stranded or single-stranded, and when a "nucleotide" (or "oligonucleotide," "polynucleotide") having a certain sequence is mentioned, it also comprehensively means a "nucleotide" (or "oligonucleotide," "polynucleotide") having a complementary sequence, unless otherwise specified.

[0015] As used herein, a "corresponding position" in a nucleotide sequence can be determined by aligning a target sequence with a reference sequence (e.g., the nucleotide sequences represented by SEQ ID NOS: 1 to 33 and 166 to 168) to maximize homology. Alignment of nucleotide sequences can be performed using known algorithms, and the procedures are well known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J.D. et al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Alternatively, Clustal W2 or Clustal omega, which are revised versions of Clustal W, can also be used. Clustal W, Clustal W2, and Clustal omega are available, for example, on the websites of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]) and the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]), operated by the National Institute of Genetics. The position of the target sequence aligned to any position in the reference sequence by the above-mentioned alignment is considered to be the "position corresponding to" that position.

[0016] As used herein, the term "single nucleotide polymorphism (SNP)" refers to a DNA polymorphism in which the base sequence at a specific site on a genomic DNA base sequence differs between individuals, with a frequency of 1% or more in a population, and in particular refers to a polymorphism resulting from the substitution of one base with another. Hereinafter, the genotype of an SNP may be expressed in the order of allele 1 and allele 2. For example, when both alleles are the same, such as AA, the genotype is homozygous, and when both alleles are different, such as AT, the genotype is heterozygous. As used herein, "SNP" includes allele 1, allele 2, and their complementary strands.

[0017] As used herein, "fish of the genus Takifugu" refers to fish belonging to the genus Takifugu in the family Tetraodontiformes. Examples of "species belonging to the genus Takifugu" include, but are not limited to, the standard Japanese names Takifugu chrysops, Takifugu stictonotus, Takifugu pardalis, Takifugu flavipterus, Takifugu alboplumbeus, Takifugu porphyreus, Takifugu exascurus, Takifugu vermicularis, Takifugu xanthopterus, Takifugu snyderi, Takifugu rubripes, and Takifugu chinensis. Of these, the tiger pufferfish and the crow are considered to be very closely related species that are difficult to distinguish genetically, and the present invention does not distinguish between the two. As used herein, the term "hybrid" refers to an individual resulting from the crossbreeding of one species with another, where species refers to the above-mentioned species belonging to the genus Pufferfish.

[0018] The method of the present invention for distinguishing species or hybrids of Takifugu rubripes comprises detecting at least one SNP that differs between species of Takifugu rubripes in nuclear DNA derived from a sample, and distinguishing the species or hybrid of Takifugu rubripes to which the sample belongs based on the genotype of the detected SNP.

[0019] Examples of samples used in the present invention include biological samples collected from pufferfish, such as fins (caudal, dorsal, pectoral, pelvic, and anal fins), blood, muscle, and internal organs (stomach, heart, kidneys, liver, ovaries, and testes). Other examples of samples include food products containing pufferfish, food residue, and vomit, which may contain parts of pufferfish. Nuclear DNA can be extracted from samples using known methods. Examples of nuclear DNA extraction methods include the phenol method, the CTAB method, and the alkaline SDS method. Nuclear DNA may be used as a crude extract or, if necessary, purified using known methods. Commercially available reagents and kits for nuclear DNA extraction may also be used.

[0020] In the present invention, "detecting SNPs" refers to detecting the genotype of a SNP site, i.e., SNP typing. The at least one SNP that differs between species of pufferfish of the present invention is preferably at least one SNP selected from a group consisting of 36 SNPs located at the 151st base position of the base sequences shown in SEQ ID NOS: 1-33 and 166-168. Here, the base sequences shown in SEQ ID NOS: 1-33 and 166-168 are partial base sequences in the pufferfish genome assembly (FUGU5, https: / / www.ncbi.nlm.nih.gov / assembly / GCF_000180615.1 / ) in the NCBI database. Thus, in pufferfish, each SNP is located at a specific base position of a chromosome number or scaffold number, as described below. In species or hybrids of pufferfish of the genus pufferfish other than pufferfish, each SNP is located at a specific base position of the chromosome number or scaffold number, or at a base position corresponding thereto. The term "scaffold" refers to a sequence compiled as a result of constructing a genome sequence using a large number of fragmentary sequences obtained by genome analysis.

[0021] The base sequence shown in SEQ ID NO: 1 is the base sequence of positions 7,007,985 to 7,008,285 on chromosome 2 of FUGU5. The genotype of the SNP at base position 151 (7,008,135) of this base sequence is TT in red eyed pufferfish, CC in species of Takifugu other than red eyed pufferfish, and TC in hybrids of red eyed pufferfish and Takifugu other than red eyed pufferfish. Therefore, the SNP serves as an indicator for determining that a sample is derived from red eyed pufferfish if the genotype is TT, that a sample is derived from Takifugu other than red eyed pufferfish if the genotype is CC, and that a sample is derived from a hybrid of red eyed pufferfish and Takifugu other than red eyed pufferfish if the genotype is TC.

[0022] The base sequence shown in SEQ ID NO: 2 is the base sequence of positions 9,295,032 to 9,295,332 on chromosome 17 of FUGU5. The genotype of the SNP at base position 151 (position 9,295,182) of this base sequence is CC in red eagle pufferfish, TT in species of Takifugu other than red eagle pufferfish, and CT in hybrids of red eagle pufferfish and Takifugu other than red eagle pufferfish. Therefore, the SNP serves as an indicator that a sample is derived from red eagle pufferfish if the genotype is CC, that a sample is derived from Takifugu other than red eagle pufferfish if the genotype is TT, and that a sample is derived from a hybrid of red eagle pufferfish and Takifugu other than red eagle pufferfish if the genotype is CT. At the 132nd base position of the base sequence shown in SEQ ID NO: 2, there is a polymorphism that is not species-specific for FUGU5 in pufferfish and other species of the genus Pufferfish.

[0023] The base sequence shown in SEQ ID NO: 3 is the base sequence of positions 95,305 to 95,605 of scaffold 269 of FUGU5. The genotype of the SNP at base position 151 (position 95,455) of this base sequence is AA in red eagle pufferfish, GG in species of Takifugu other than red eagle pufferfish, and AG in hybrids of red eagle pufferfish and Takifugu other than red eagle pufferfish. Therefore, the SNP serves as an indicator that a sample is derived from red eagle pufferfish when the genotype is AA, that a sample is derived from Takifugu other than red eagle pufferfish when the genotype is GG, and that a sample is derived from a hybrid of red eagle pufferfish and Takifugu other than red eagle pufferfish when the genotype is AG.

[0024] The base sequence shown in SEQ ID NO: 4 is the base sequence of positions 5,580,494 to 5,580,794 on chromosome 2 of FUGU5. The genotype of the SNP at base position 151 (position 5,580,644) of this base sequence is AA in pufferfish, GG in species of Takifugu other than Pufferfish, and AG in hybrids of Pufferfish and Takifugu other than Pufferfish. Therefore, the SNP serves as an indicator that a sample is derived from a pufferfish if the genotype is AA, a sample is derived from a Takifugu other than Pufferfish if the genotype is GG, and a sample is derived from a hybrid of Pufferfish and Takifugu other than Pufferfish if the genotype is AG.

[0025] The base sequence shown in SEQ ID NO: 5 is the base sequence of positions 4,651,817 to 4,652,117 on chromosome 7 of FUGU5. The genotype of the SNP at base position 151 (position 4,651,967) of this base sequence is TT in pufferfish, CC in species of Takifugu other than Pufferfish, and TC in hybrids of Pufferfish and Takifugu other than Pufferfish. Therefore, the SNP serves as an indicator that a sample is derived from a pufferfish if the genotype is TT, a sample is derived from a Takifugu other than Pufferfish if the genotype is CC, and a sample is derived from a hybrid of Pufferfish and Takifugu other than Pufferfish if the genotype is TC.

[0026] The base sequence shown in SEQ ID NO: 6 is the base sequence of positions 145,328 to 145,628 of scaffold 305 of FUGU5. The genotype of the SNP at base position 151 (position 145,478) of this base sequence is TT in pufferfish, GG in species of Takifugu other than Pufferfish, and TG in hybrids of Pufferfish and Takifugu other than Pufferfish. Therefore, the SNP serves as an indicator that a sample is derived from Pufferfish if the genotype is TT, a sample is derived from Takifugu other than Pufferfish if the genotype is GG, and a sample is derived from a hybrid of Pufferfish and Takifugu other than Pufferfish if the genotype is TG.

[0027] The base sequence shown in SEQ ID NO: 7 is the base sequence of positions 1,532,717 to 1,533,017 on chromosome 8 of FUGU5. The genotype of the SNP at base position 151 (position 1,532,867) of this base sequence is GG in pufferfish, TT in species of Takifugu other than Takifugu, and GT in hybrids of Takifugu and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator that a sample is derived from a Takifugu if the genotype is GG, a sample is derived from a Takifugu other than Takifugu if the genotype is TT, and a sample is derived from a hybrid of Takifugu and Takifugu other than Takifugu if the genotype is GT.

[0028] The base sequence shown in SEQ ID NO: 8 is the base sequence of positions 6,067,356 to 6,067,656 on chromosome 14 of FUGU5. The genotype of the SNP at base position 151 (6,067,506) of this base sequence is AA in pufferfish, GG in species of Takifugu other than Takifugu, and AG in hybrids of Takifugu and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator that a sample is derived from a Takifugu if the genotype is AA, that a sample is derived from a Takifugu other than Takifugu if the genotype is GG, and that a sample is derived from a hybrid of Takifugu and Takifugu other than Takifugu if the genotype is AG.

[0029] The base sequence shown in SEQ ID NO: 9 is the base sequence of positions 3,365,155 to 3,365,455 on chromosome 20 of FUGU5. The genotype of the SNP at base position 151 (position 3,365,305) of this base sequence is TT in pufferfish, CC in species of Takifugu other than Takifugu, and TC in hybrids of Takifugu and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator for determining that a sample is derived from Takifugu if the genotype is TT, that a sample is derived from a species of Takifugu other than Takifugu if the genotype is CC, and that a sample is derived from a hybrid of Takifugu and Takifugu other than Takifugu if the genotype is TC.

[0030] The base sequence shown in SEQ ID NO: 10 is the base sequence of positions 3,263,777 to 3,264,077 on chromosome 7 of FUGU5. The genotype of the SNP at base position 151 (position 3,263,927) of this base sequence is AA in common pufferfish, GG in species of Takifugu other than Takifugu, and AG in hybrids of common pufferfish and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator that a sample is derived from a common pufferfish if the genotype is AA, a sample derived from a species of Takifugu other than Takifugu if the genotype is GG, and a sample derived from a hybrid of common pufferfish and Takifugu other than Takifugu if the genotype is AG. At base positions 110, 171, and 203 of the base sequence shown in SEQ ID NO: 10, there are polymorphisms that are not species-specific for FUGU5 in pufferfish and other species of the genus Pufferfish.

[0031] The base sequence shown in SEQ ID NO: 11 is the base sequence of positions 1,940,951 to 1,941,251 on chromosome 10 of FUGU5. The genotype of the SNP at base position 151 (position 1,941,101) of this base sequence is GG in common pufferfish, CC in species of Takifugu other than Takifugu, and GC in hybrids of common pufferfish and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator for determining whether a sample is derived from a common pufferfish if the genotype is GG, from a species of Takifugu other than Takifugu if the genotype is CC, or from a hybrid of common pufferfish and a species of Takifugu other than Takifugu if the genotype is GC. At base positions 136 and 182 of the base sequence shown in SEQ ID NO: 11, there are polymorphisms that are not species-specific for FUGU5 in pufferfish and species of the genus Pufferfish other than pufferfish.

[0032] The base sequence shown in SEQ ID NO: 12 is the base sequence from positions 115,922 to 116,222 of scaffold 334 of FUGU5. The genotype of the SNP at base position 151 (position 116,072) of this base sequence is AA in common pufferfish, CC in species of Takifugu other than Takifugu, and AC in hybrids of common pufferfish and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator that a sample is derived from a common pufferfish if the genotype is AA, a sample is derived from a species of Takifugu other than Takifugu if the genotype is CC, and a sample is derived from a hybrid of common pufferfish and Takifugu other than Takifugu if the genotype is AC. At base positions 117 and 136 of the base sequence shown in SEQ ID NO: 12, there are polymorphisms that are not species-specific for FUGU5 in pufferfish and species of the genus Pufferfish other than pufferfish.

[0033] The base sequence shown in SEQ ID NO: 13 is the base sequence of positions 6,074,995 to 6,075,295 on chromosome 3 of FUGU5. The genotype of the SNP at base position 151 (6,075,145) of this base sequence is CC in Takifugu, TT in species of Takifugu other than Takifugu, and CT in hybrids of Takifugu and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator that a sample is derived from Takifugu if the genotype is CC, that a sample is derived from Takifugu other than Takifugu if the genotype is TT, and that a sample is derived from a hybrid of Takifugu and Takifugu other than Takifugu if the genotype is CT. At the 169th base position of the base sequence shown in SEQ ID NO: 13, there is a polymorphism that is not species-specific for FUGU5 in tiger pufferfish and other species of the genus Pufferfish.

[0034] The base sequence shown in SEQ ID NO: 14 is the base sequence of positions 5,447,010 to 5,447,310 on chromosome 10 of FUGU5. The genotype of the SNP at base position 151 (position 5,447,160) of this base sequence is CC in T. pufferfish, TT in species of T. pufferfish other than T. pufferfish, and CT in hybrids of T. pufferfish and T. pufferfish other than T. pufferfish. Therefore, the SNP serves as an indicator for determining that a sample is derived from T. pufferfish if the genotype is CC, that a sample is derived from a species of T. pufferfish other than T. pufferfish if the genotype is TT, and that a sample is derived from a hybrid of T. pufferfish and T. pufferfish other than T. pufferfish if the genotype is CT.

[0035] The base sequence shown in SEQ ID NO: 15 is the base sequence of positions 5,146,443 to 5,146,743 on chromosome 20 of FUGU5. The genotype of the SNP at base position 151 (position 5,146,593) of this base sequence is AA in Takifugu, GG in species of Takifugu other than Takifugu, and AG in hybrids of Takifugu and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator that a sample is derived from Takifugu if the genotype is AA, that a sample is derived from Takifugu if the genotype is GG, and that a sample is derived from a hybrid of Takifugu and Takifugu other than Takifugu if the genotype is AG. At the 134th base position of the base sequence shown in SEQ ID NO: 15, there is a polymorphism that is not species-specific for FUGU5 in pufferfish and other species of the genus Pufferfish.

[0036] The base sequence shown in SEQ ID NO: 16 is the base sequence of positions 6,199,440 to 6,199,740 on chromosome 9 of FUGU5. The genotype of the SNP at base position 151 (6,199,590) of this base sequence is GG in pufferfish, CC in species of Takifugu other than Takifugu, and GC in hybrids of Takifugu and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator that a sample is derived from a pufferfish if the genotype is GG, a sample is derived from a species of Takifugu other than Takifugu if the genotype is CC, and a sample is derived from a hybrid of Takifugu and Takifugu other than Takifugu if the genotype is GC. At the 160th and 184th base positions of the base sequence shown in SEQ ID NO: 16, there are polymorphisms that are not species-specific for FUGU5 in pufferfish and other species of the genus Pufferfish.

[0037] The base sequence shown in SEQ ID NO: 17 is the base sequence of positions 229,760 to 230,060 on chromosome 18 of FUGU5. The genotype of the SNP at base position 151 (position 229,910) of this base sequence is TT in pufferfish, CC in species of Takifugu other than Takifugu, and TC in hybrids of Takifugu and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator for determining that a sample is derived from Takifugu if the genotype is TT, that a sample is derived from Takifugu other than Takifugu if the genotype is CC, and that a sample is derived from a hybrid of Takifugu and Takifugu other than Takifugu if the genotype is TC.

[0038] The base sequence shown in SEQ ID NO: 18 is the base sequence of positions 9,633,325 to 9,633,625 on chromosome 21 of FUGU5. The genotype of the SNP at base position 151 (9,633,475) of this base sequence is GG in pufferfish, AA in species of Takifugu other than Takifugu, and GA in hybrids of Takifugu and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator for determining that a sample is derived from Takifugu if the genotype is GG, that a sample is derived from Takifugu other than Takifugu if the genotype is AA, and that a sample is derived from a hybrid of Takifugu and Takifugu other than Takifugu if the genotype is GA. At the 128th base position of the base sequence shown in SEQ ID NO: 18, there is a polymorphism that is not species-specific for FUGU5 in pufferfish and species of the genus Pufferfish other than pufferfish.

[0039] The base sequence shown in SEQ ID NO: 19 is the base sequence of positions 4,108,937 to 4,109,237 on chromosome 4 of FUGU5. The genotype of the SNP at base position 151 (position 4,109,087) of this base sequence is AA in Tsubakifugu, GG in species of Tsubakifugu other than Tsubakifugu, and AG in hybrids of Tsubakifugu and Tsubakifugu other than Tsubakifugu. Therefore, the SNP serves as an indicator that a sample is derived from Tsubakifugu if the genotype is AA, that a sample is derived from Tsubakifugu other than Tsubakifugu if the genotype is GG, and that a sample is derived from a hybrid of Tsubakifugu and Tsubakifugu other than Tsubakifugu if the genotype is AG.

[0040] The base sequence shown in SEQ ID NO: 20 is the base sequence of positions 3,271,371 to 3,271,671 of chromosome 10 of FUGU5. The genotype of the SNP at base position 151 (position 3,271,521) of this base sequence is CC in Tsubakifugu, GG in species of Tsubakifugu other than Tsubakifugu, and CG in hybrids of Tsubakifugu and Tsubakifugu other than Tsubakifugu. Therefore, the SNP serves as an indicator that a sample is derived from Tsubakifugu if the genotype is CC, that a sample is derived from Tsubakifugu other than Tsubakifugu if the genotype is GG, and that a sample is derived from a hybrid of Tsubakifugu and Tsubakifugu other than Tsubakifugu if the genotype is CG. At base positions 121, 131, 133, 172 and 176 of the base sequence shown in SEQ ID NO: 20, there are polymorphisms that are not species-specific for FUGU5 in pufferfish and other species of the genus Pufferfish.

[0041] The base sequence shown in SEQ ID NO: 21 is the base sequence of positions 145,534 to 145,834 of scaffold 305 of FUGU5. The genotype of the SNP at base position 151 (position 145,684) of this base sequence is AA in Tsubakifugu, GG in species of Tsubakifugu other than Tsubakifugu, and AG in hybrids of Tsubakifugu and Tsubakifugu other than Tsubakifugu. Therefore, when the SNP genotype is AA, it can be determined that the sample is derived from Tsubakifugu, when the genotype is GG, it can be determined that the sample is derived from species of Tsubakifugu other than Tsubakifugu, and when the genotype is AG, it can be determined that the sample is derived from a hybrid of Tsubakifugu and species of Tsubakifugu other than Tsubakifugu. At base positions 127, 143, and 191 of the base sequence shown in SEQ ID NO: 21, there are polymorphisms that are not species-specific for FUGU5 in pufferfish and species of the genus Pufferfish other than pufferfish.

[0042] The base sequence shown in SEQ ID NO: 22 is the base sequence of positions 143,948 to 144,248 on chromosome 12 of FUGU5. The genotype of the SNP at base position 151 (position 144,098) of this base sequence is TT in pufferfish, CC in species of Takifugu other than pufferfish, and TC in hybrids of pufferfish and Takifugu other than pufferfish. Therefore, the SNP serves as an indicator that a sample is derived from pufferfish if the genotype is TT, a sample is derived from Takifugu other than pufferfish if the genotype is CC, and a sample is derived from a hybrid of pufferfish and Takifugu other than pufferfish if the genotype is TC. At base positions 145, 163, and 176 of the base sequence shown in SEQ ID NO: 22, there are polymorphisms that are not species-specific for FUGU5 in pufferfish and species of the genus Pufferfish other than pufferfish.

[0043] The base sequence shown in SEQ ID NO: 23 is the base sequence of positions 14,860,695 to 14,860,995 on chromosome 13 of FUGU5. The genotype of the SNP at base position 151 (position 14,860,845) of this base sequence is CC in pufferfish, AA in species of Takifugu other than pufferfish, and CA in hybrids of pufferfish and Takifugu other than pufferfish. Therefore, the SNP serves as an indicator that a sample is derived from pufferfish if the genotype is CC, that a sample is derived from Takifugu other than pufferfish if the genotype is AA, and that a sample is derived from a hybrid of pufferfish and Takifugu other than pufferfish if the genotype is CA. At the 175th base position of the base sequence shown in SEQ ID NO: 23, there is a polymorphism that is not species-specific for FUGU5 in tiger pufferfish and other species of the genus Pufferfish.

[0044] The base sequence shown in SEQ ID NO: 24 is the base sequence of positions 1,390,678 to 1,390,978 on chromosome 18 of FUGU5. The genotype of the SNP at base position 151 (position 1,390,828) of this base sequence is GG in pufferfish, CC in species of Takifugu other than pufferfish, and GC in hybrids of pufferfish and Takifugu other than pufferfish. Therefore, the SNP serves as an indicator that a sample is derived from pufferfish if the genotype is GG, that a sample is derived from Takifugu other than pufferfish if the genotype is CC, and that a sample is derived from a hybrid of pufferfish and Takifugu other than pufferfish if the genotype is GC. At base positions 158, 160, 162, 169, and 172 of the base sequence shown in SEQ ID NO: 24, there are polymorphisms that are not species-specific for FUGU5 in pufferfish and other species of the genus Pufferfish.

[0045] The base sequence shown in SEQ ID NO: 25 is the base sequence of positions 11,361,343 to 11,361,643 on chromosome 1 of FUGU5. The genotype of the SNP at base position 151 (position 11,361,493) of this base sequence is AA in plaicefish, GG in species of Takifugu other than plaicefish, and AG in hybrids of plaicefish and Takifugu other than plaicefish. Therefore, the SNP serves as an indicator that a sample is derived from plaicefish if the genotype is AA, that a sample is derived from Takifugu other than plaicefish if the genotype is GG, and that a sample is derived from a hybrid of plaicefish and Takifugu other than plaicefish if the genotype is AG. At the 146th base position of the base sequence shown in SEQ ID NO: 25, there is a polymorphism that is not species-specific for FUGU5 in pufferfish and species of the genus Pufferfish other than pufferfish.

[0046] The base sequence shown in SEQ ID NO: 26 is the base sequence of positions 7,074,989 to 7,075,289 on chromosome 2 of FUGU5. The genotype of the SNP at base position 151 (position 7,075,139) of this base sequence is AA in plaicefish, TT in species of Takifugu other than plaicefish, and AT in hybrids of plaicefish and Takifugu other than plaicefish. Therefore, the SNP serves as an indicator that a sample is derived from plaicefish if the genotype is AA, that a sample is derived from Takifugu other than plaicefish if the genotype is TT, and that a sample is derived from a hybrid of plaicefish and Takifugu other than plaicefish if the genotype is AT. At the 160th and 168th base positions of the base sequence shown in SEQ ID NO: 26, there are polymorphisms that are not species-specific for FUGU5 in pufferfish and other species of the genus Pufferfish.

[0047] The base sequence shown in SEQ ID NO: 27 is the base sequence of positions 11,451,665 to 11,451,965 on chromosome 19 of FUGU5. The genotype of the SNP at base position 151 (position 11,451,815) of this base sequence is CC in plague doctor fish, GG in species of Takifugu other than plague doctor fish, and CG in hybrids of plague doctor fish and Takifugu other than plague doctor fish. Therefore, the SNP serves as an indicator that a sample is derived from a plague doctor fish if the genotype is CC, a sample is derived from a Takifugu other than plague doctor fish if the genotype is GG, and a sample is derived from a hybrid of a plague doctor fish and Takifugu other than plague doctor fish if the genotype is CG.

[0048] The base sequence shown in SEQ ID NO: 28 is the base sequence of positions 2,006,321 to 2,006,621 of chromosome 4 of FUGU5. The genotype of the SNP at base position 151 (position 2,006,471) of this base sequence is TT in pufferfish, GG in species of Takifugu other than Takifugu, and TG in hybrids of Takifugu and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator for determining that a sample is derived from Takifugu if the genotype is TT, that a sample is derived from Takifugu other than Takifugu if the genotype is GG, and that a sample is derived from a hybrid of Takifugu and Takifugu other than Takifugu if the genotype is TG. At the 164th base position of the base sequence shown in SEQ ID NO: 28, there is a polymorphism that is not species-specific for FUGU5 in pufferfish and other species of the genus Pufferfish.

[0049] The base sequence shown in SEQ ID NO: 29 is the base sequence of positions 41,156 to 41,456 on chromosome 6 of FUGU5. The genotype of the SNP at base position 151 (position 41,306) of this base sequence is CC in pufferfish, GG in species of Takifugu other than Takifugu, and CG in hybrids of Takifugu and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator for determining that a sample is derived from Takifugu if the genotype is CC, that a sample is derived from Takifugu other than Takifugu if the genotype is GG, and that a sample is derived from a hybrid of Takifugu and Takifugu other than Takifugu if the genotype is CG.

[0050] The base sequence shown in SEQ ID NO: 30 is the base sequence of positions 683,629 to 683,929 on chromosome 11 of FUGU5. The genotype of the SNP at base position 151 (683,779) of this base sequence is TT in pufferfish, CC in species of Takifugu other than Takifugu, and TC in hybrids of Takifugu and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator for determining that a sample is derived from Takifugu if the genotype is TT, that a sample is derived from Takifugu other than Takifugu if the genotype is CC, and that a sample is derived from a hybrid of Takifugu and Takifugu other than Takifugu if the genotype is TC. At the 134th base position of the base sequence shown in SEQ ID NO: 30, there is a polymorphism that is not species-specific for FUGU5 in pufferfish and species of the genus Pufferfish other than pufferfish.

[0051] The base sequence shown in SEQ ID NO: 31 is the base sequence of positions 5,621,458 to 5,621,758 on chromosome 8 of FUGU5. The genotype of the SNP at base position 151 (position 5,621,608) of this base sequence is AA for pufferfish or crows, TT for species of fish in the genus Pufferfish other than pufferfish and crows, and AT for hybrids of pufferfish or crows with species of fish in the genus Pufferfish other than pufferfish or crows. Therefore, the SNP serves as an indicator for determining that a sample is derived from a pufferfish or crow if the genotype is AA, a sample is derived from a species of fish in the genus Pufferfish other than pufferfish or crows if the genotype is TT, and a sample is derived from a hybrid of a pufferfish or crow with a species of fish in the genus Pufferfish other than pufferfish or crows if the genotype is AT.

[0052] The base sequence shown in SEQ ID NO: 32 is the base sequence of positions 7,185,177 to 7,185,477 on chromosome 14 of FUGU5. The genotype of the SNP at base position 151 (7,185,327) of this base sequence is AA for pufferfish or crows, GG for species of pufferfish other than pufferfish and crows, and AG for hybrids of pufferfish or crows with species of pufferfish other than pufferfish or crows. Therefore, the SNP serves as an indicator that a sample is derived from a pufferfish or crow if the genotype is AA, a sample is derived from a species of pufferfish other than pufferfish or crows if the genotype is GG, and a sample is derived from a hybrid of pufferfish or crows with species of pufferfish other than pufferfish or crows if the genotype is AG.

[0053] The base sequence shown in SEQ ID NO: 33 is the base sequence of positions 11,043,143 to 11,043,443 on chromosome 19 of FUGU5. The genotype of the SNP at base position 151 (position 11,043,293) of this base sequence is GG for pufferfish or crows, AA for species of Takifugu other than pufferfish and crows, and GA for hybrids of Takifugu or crows with species of Takifugu other than pufferfish or crows. Therefore, the SNP serves as an indicator for determining that a sample is derived from a Takifugu or crow if the genotype is GG, a sample is derived from a species of Takifugu other than pufferfish or crows if the genotype is AA, and a sample is derived from a hybrid of a Takifugu or crow with species of Takifugu other than pufferfish or crows if the genotype is GA. At the 174th base position of the base sequence shown in SEQ ID NO: 33, there is a polymorphism that is not species-specific for FUGU5 in pufferfish and other species of the genus Pufferfish.

[0054] The base sequence represented by SEQ ID NO: 166 is the base sequence of positions 5,823,130 to 5,823,430 on chromosome 17 of FUGU5. The genotype of the SNP at base position 151 (position 5,823,281) of this base sequence is CC in Takifugu, TT in species of Takifugu other than Takifugu, and CT in hybrids of Takifugu and Takifugu other than Takifugu. Therefore, the SNP serves as an indicator for determining that a sample is derived from Takifugu if the genotype is CC, that a sample is derived from Takifugu other than Takifugu if the genotype is TT, and that a sample is derived from a hybrid of Takifugu and Takifugu other than Takifugu if the genotype is CT. At the 137th base position of the base sequence shown in SEQ ID NO: 166, there is a polymorphism that is not species-specific for FUGU5 in pufferfish and other species of the genus Pufferfish.

[0055] The base sequence shown in SEQ ID NO: 167 is the base sequence of positions 18,681,377 to 18,681,677 on chromosome 1 of FUGU5. The genotype of the SNP at base position 151 (position 18,681,528) of this base sequence is GG in pufferfish, TT in species of T. pufferfish other than T. pufferfish, and GT in hybrids between pufferfish and species of T. pufferfish other than T. pufferfish. Therefore, the SNP serves as an indicator that a sample is derived from a pufferfish if the genotype is GG, a sample is derived from species of T. pufferfish other than T. pufferfish if the genotype is TT, and a sample is derived from a hybrid between pufferfish and species of T. pufferfish other than T. pufferfish if the genotype is GT. At base positions 168, 174, 188, and 193 of the base sequence shown in SEQ ID NO: 167, there are polymorphisms that are not species-specific for FUGU5 in pufferfish and other species of the genus Pufferfish.

[0056] The base sequence shown in SEQ ID NO: 168 is the base sequence of positions 486,790 to 487,090 on chromosome 2 of FUGU5. The genotype of the SNP at base position 151 (position 486,941) of this base sequence is AA in plaicefish, TT in species of Takifugu other than plaicefish, and AT in hybrids of plaicefish and Takifugu other than plaicefish. Therefore, the SNP serves as an indicator that a sample is derived from plaicefish if the genotype is AA, that a sample is derived from Takifugu other than plaicefish if the genotype is TT, and that a sample is derived from a hybrid of plaicefish and Takifugu other than plaicefish if the genotype is AT. At base positions 133, 135, and 141 of the base sequence shown in SEQ ID NO: 168, there are polymorphisms that are not species-specific for FUGU5 in pufferfish and other species of the genus Pufferfish.

[0057] The relationship between the genotype of each SNP and the species or hybrid of Takifugu rubripes is shown in Table 2 below. In the method of the present invention, when the genotype of the detected SNP is (ii) in Table 2, the species from which the sample originated can be determined to be the species (i) in Table 2; when the genotype of the detected SNP is (iii) in Table 2, the species from which the sample originated can be determined to be a species of Takifugu rubripes other than (i) in Table 2; and when the genotype of the detected SNP is (iv) in Table 2, the species from which the sample originated can be determined to be a hybrid of the species (i) in Table 2 and a species of Takifugu rubripes other than (i).

[0058] [Table 2]

[0059] In the method of the present invention, only one of the above SNPs may be used as an indicator for distinguishing species or hybrids, or two or more SNPs may be combined and used as an indicator for distinguishing species or hybrids, but from the standpoint of discrimination accuracy, it is preferable to use two or more SNPs in combination as an indicator.Among them, since it is possible to collectively distinguish between red-eyed pufferfish, spotted pufferfish, red-spotted pufferfish, common pufferfish, grass pufferfish, spotted pufferfish, Japanese pufferfish, Japanese tiger pufferfish, and tora pufferfish or crows, as well as hybrids therebetween, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 1 to 3, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 4 to 6, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 7 to 9, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 10 to 12, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 13 to 15 and 166, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 16 to 18 and 167, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 19 to 21, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 22 to 24, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 25 to 27 and 168, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 28 to 30, and at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 31 to 33. It is more preferable to use a combination of at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 1 to 33 and 166 to 168 as an indicator, and it is even more preferable to use all of the 36 SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 1 to 33 and 166 to 168 as an indicator.

[0060] SNP detection can be carried out using known methods, including, but not limited to, PCR-SSCP, PCR-RFLP, PCR-SSO, PCR-ASP, TaqMan PCR, cycling probe, smart amplifier, molecular beacon, invader, LAMP, SNaPshot, direct sequencing, Sniper, MALDI-TOF / MS, microarray, DOL, and TDI.

[0061] In the present invention, it is preferable to use the TaqMan PCR method as a method for detecting SNPs in terms of detection time and detection accuracy. Reagents and kits used in the TaqMan PCR method are commercially available, for example, TaqMan (registered trademark) SNP Genotyping Kit. Assays (Thermo Fisher Scientific) may also be used. Detection of SNPs by the TaqMan PCR method can be carried out as follows: A PCR reaction is carried out using two probes (first probe) that specifically hybridize to a base sequence containing the base at which the target SNP is located on a template DNA, where the base corresponding to the SNP position is complementary to allele 1 of the SNP and is labeled with a fluorescent substance at the 5' end and a quencher (quencher) at the 3' end, and a probe (second probe) where the base corresponding to the SNP position is complementary to allele 2 of the SNP and is labeled with a fluorescent substance different from that of the first probe at the 5' end and a quencher (quencher) at the 3' end, a primer set capable of amplifying a region on the template DNA containing the base sequence to which the probe specifically hybridizes, and PCR reagents such as Taq DNA polymerase. If the template DNA contains only allele 1, the first probe hybridizes to the template DNA during the annealing step, but the presence of a quencher on the probe suppresses fluorescence emission even when irradiated with excitation light. During the extension step, the first probe hybridized to the template DNA is degraded by the 5'→3' exonuclease activity of Taq DNA polymerase, liberating the fluorescent substance, which releases fluorescence and releases the inhibition by the quencher. On the other hand, the second probe contains a mismatch at the SNP position, preventing it from hybridizing to the template DNA and preventing its degradation, suppressing fluorescence emission. Therefore, only the fluorescence of the first probe is detected, and not the fluorescence of the second probe. Conversely, if the template DNA contains only allele 2, the fluorescence of the first probe is not detected, and only the fluorescence of the second probe is detected. Furthermore, if the template DNA contains both alleles 1 and 2, the fluorescence of both the first and second probes is detected. Thus, the genotype of the SNP can be determined based on the type and intensity of the detected fluorescence.

[0062] The PCR conditions are not particularly limited, and the optimal conditions may be determined taking into consideration the amplification size, the base length of the probe and / or primer, the GC content, the Tm value, etc., but examples thereof include the following conditions: 1) Thermal denaturation of double-stranded DNA to single-stranded DNA: Usually, heating is performed at about 93 to 95°C for about 3 seconds to 1 minute. 2) Annealing: Heating is usually performed at about 50 to 60°C for about 10 seconds to 1 minute. 3) DNA extension reaction: Usually heated at about 60 to 74°C for about 10 seconds to 5 minutes. Here, the annealing and the DNA elongation reaction can be carried out simultaneously without being separated. The above reactions 1) to 3) are usually carried out for about 30 to 50 cycles, whereby fluorescence derived from the probe can be detected.

[0063] PCR is preferably performed using a real-time PCR device that integrates a thermal cycler and a spectrofluorometer, such as the StepOne (registered trademark) Real-Time PCR System (Thermo Fisher Scientific), as this allows for rapid and simple detection of SNPs.

[0064] In the present invention, the probe may be any probe capable of detecting an SNP at the 151st base position of a base sequence shown in any one of SEQ ID NOS: 1 to 33 and 166 to 168. The probe is preferably an oligonucleotide that specifically hybridizes to a base sequence containing the 151st base (the base at which the SNP is located) of a base sequence shown in any one of SEQ ID NOS: 1 to 33 and 166 to 168, and that comprises a base sequence in which the base corresponding to the SNP position is complementary to allele 1 or 2 of the SNP. The probe may be appropriately designed based on the base sequences shown in SEQ ID NOS: 1 to 33 and 166 to 168. The base length of the probe is generally about 10 to 50 bases, preferably about 10 to 35 bases, and more preferably about 12 to 30 bases. The probe is preferably labeled at the 5' end with a fluorescent substance such as FITC or VIC and at the 3' end with a quencher such as TAMRA or MGB. Such labeled probes are suitable for use in SNP detection by TaqMan PCR. The phrase "specifically hybridizes to a target site (sequence)" means that there is no significant cross-hybridization with other DNA under normal hybridization conditions, preferably under stringent hybridization conditions (e.g., the conditions described in Joseph Sambrook et al., Molecular Cloning: A Laboratory Manual (3 Vol. Set), Cold Spring Harbor Laboratory, NY, 2001). Preferably, the probe has a base sequence complementary to the base sequence of the region containing the base of the SNP site to be detected, but it does not need to be completely complementary as long as such specific hybridization is possible.

[0065] Examples of such probes include oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 34 to 99 and 169 to 174, oligonucleotides consisting of sequences complementary to these nucleotide sequences, and oligonucleotides consisting of nucleotide sequences in which one or two bases have been deleted, substituted, added, or inserted in the oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 34 to 99 and 169 to 174 or oligonucleotides consisting of sequences complementary to these nucleotide sequences, and which have the same function as a probe as the oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 34 to 99 and 169 to 174 or oligonucleotides consisting of sequences complementary to these nucleotide sequences. Of these, oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 34 to 99 and 169 to 174 are preferred. Table 3 below shows the relationship between the probes consisting of the base sequences shown in SEQ ID NOs: 34 to 99 and 169 to 174 and the SNPs they detect.

[0066] [Table 3]

[0067] In the present invention, the primer may be any primer capable of amplifying a region containing an SNP at the 151st base position of the base sequence shown in any one of SEQ ID NOS: 1 to 33 and 166 to 168. The primer is preferably an oligonucleotide that specifically hybridizes upstream or downstream of a region containing a base sequence to which the probe shown in any one of SEQ ID NOS: 1 to 33 and 166 to 168 hybridizes, and can be used as a forward primer or reverse primer, and can be appropriately designed based on the base sequences shown in SEQ ID NOS: 1 to 33 and 166 to 168. The base length of the primer is typically about 15 to 50 bases, preferably about 15 to 45 bases. The forward primer and reverse primer for amplifying a region containing a certain SNP are collectively referred to as a primer set. The base length of the region amplified by the primer set is typically 50 to 300 bp, preferably 70 to 150 bp. Here, "specifically hybridizing to a target site (sequence)" means, as in the case of a probe, that there is no significant cross-hybridization with other DNA under normal hybridization conditions, preferably under stringent hybridization conditions.

[0068] Examples of such primers include oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 100 to 165 and 175 to 180, oligonucleotides consisting of sequences complementary to these nucleotide sequences, and oligonucleotides consisting of nucleotide sequences in which one or two bases have been deleted, substituted, added, or inserted in the oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 100 to 165 and 175 to 180 or oligonucleotides consisting of sequences complementary to these nucleotide sequences, and which have the same function as a primer as the oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 100 to 165 and 175 to 180 or oligonucleotides consisting of sequences complementary to these nucleotide sequences. Of these, oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 100 to 165 and 175 to 180 are preferred. Table 4 below shows the relationship between primers consisting of the base sequences shown in SEQ ID NOs: 100 to 165 and 175 to 180 and SNPs in the regions they amplify. In Table 4, two primers in the same row make up a primer set.

[0069] [Table 4]

[0070] The probe and primer may be synthesized by a conventional chemical synthesis method such as the phosphoramidite method or the phosphate triester method, or may be synthesized using a commercially available automatic oligonucleotide synthesizer.

[0071] The method for distinguishing species or hybrids of pufferfish of the genus Takifugu of the present invention can be carried out more easily by using a kit. The kit includes a probe and primer set for detecting at least one SNP selected from the group consisting of SNPs at the 151st base of the nucleotide sequences shown in SEQ ID NOS: 1 to 33 and 166 to 168. Details of the primers constituting the probe and primer set are the same as those of the probe and primers described above. The kit of the present invention preferably includes the following (1) to (36): (1) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 34 and 35 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 100 and 101; (2) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 36 and 37 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 102 and 103; (3) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 38 and 39 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 104 and 105; (4) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 40 and 41 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 106 and 107; (5) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 42 and 43 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 108 and 109; (6) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 44 and 45 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 110 and 111; (7) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 46 and 47 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 112 and 113; (8) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 48 and 49 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 114 and 115; (9) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 50 and 51 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 116 and 117; (10) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 52 and 53 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 118 and 119; (11) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 54 and 55 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 120 and 121; (12) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 56 and 57 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 122 and 123; (13) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 58 and 59 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 124 and 125; (14) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 60 and 61 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 126 and 127; (15) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 62 and 63 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 128 and 129; (16) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 64 and 65 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 130 and 131; (17) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 66 and 67 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 132 and 133; (18) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 68 and 69 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 134 and 135; (19) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 70 and 71 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 136 and 137; (20) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 72 and 73 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 138 and 139; (21) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 74 and 75 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 140 and 141; (22) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 76 and 77 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 142 and 143; (23) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 78 and 79 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 144 and 145; (24) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 80 and 81 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 146 and 147; (25) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 82 and 83 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 148 and 149; (26) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 84 and 85 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 150 and 151; (27) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 86 and 87 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 152 and 153; (28) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 88 and 89 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 154 and 155; (29) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 90 and 91 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 156 and 157; (30) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 92 and 93 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 158 and 159; (31) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 94 and 95 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 160 and 161; (32) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 96 and 97 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 162 and 163; (33) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 98 and 99 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 164 and 165; (34) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 169 and 170 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 175 and 176; (35) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 171 and 172 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 177 and 178; and (36) two probes consisting of the nucleotide sequences shown in SEQ ID NOs: 173 and 174 and two primers consisting of the nucleotide sequences shown in SEQ ID NOs: 179 and 180; At least one selected from the group consisting of (1) to (3), more preferably at least one selected from the group consisting of (4) to (6), at least one selected from the group consisting of (7) to (9), at least one selected from the group consisting of (10) to (12), at least one selected from the group consisting of (13) to (15) and (34), at least one selected from the group consisting of (16) to (18) and (35), at least one selected from the group consisting of (19) to (21), at least one selected from the group consisting of (22) to (24), at least one selected from the group consisting of (25) to (27) and (36), at least one selected from the group consisting of (28) to (30), and at least one selected from the group consisting of (31) to (33), and even more preferably all of (1) to (36).

[0072] The kit may optionally contain reagents used for SNP detection (e.g., Taq DNA polymerase, dNTPs, etc.) For ease of implementation, the kit may further contain an appropriate reaction diluent, buffer solution, information for identifying species or hybrids based on the detected genotype (e.g., a list of species or hybrids that can be identified by genotype), etc.

[0073] Thus, the method of the present invention makes it possible to distinguish species or hybrids of fish of the genus Pufferfish easily and quickly with high accuracy. The 36 SNPs used in the method of the present invention are specific to species or hybrids of Takifugu species and are useful for distinguishing species or hybrids. In particular, the SNP at the 151st base of the nucleotide sequence shown in any of SEQ ID NOS: 1 to 3 is useful for distinguishing between red-spotted pufferfish, the SNP at the 151st base of the nucleotide sequence shown in any of SEQ ID NOS: 4 to 6 is useful for distinguishing between spotted pufferfish, the SNP at the 151st base of the nucleotide sequence shown in any of SEQ ID NOS: 7 to 9 is useful for distinguishing between red-spotted pufferfish, the SNP at the 151st base of the nucleotide sequence shown in any of SEQ ID NOS: 10 to 12 is useful for distinguishing between common pufferfish, the SNP at the 151st base of the nucleotide sequence shown in any of SEQ ID NOS: 13 to 15 and 166 is useful for distinguishing between grass pufferfish, and the SNP at the 151st base of the nucleotide sequence shown in any of SEQ ID NOS: 16 to 18 and 167 is useful for distinguishing between grass pufferfish and red-spotted pufferfish. A SNP at the 151st base of the base sequence shown in any of SEQ ID NOs: 19 to 21 is useful for distinguishing between pufferfish and tiger pufferfish, a SNP at the 151st base of the base sequence shown in any of SEQ ID NOs: 22 to 24 is useful for distinguishing between pufferfish and tiger pufferfish, a SNP at the 151st base of the base sequence shown in any of SEQ ID NOs: 25 to 27 and 168 is useful for distinguishing between pufferfish and striped pufferfish, a SNP at the 151st base of the base sequence shown in any of SEQ ID NOs: 28 to 30 is useful for distinguishing between pufferfish and tiger pufferfish, and a SNP at the 151st base of the base sequence shown in any of SEQ ID NOs: 31 to 33 is useful for distinguishing between tiger pufferfish and crow pufferfish. Therefore, a polynucleotide consisting of a partial base sequence containing an SNP at the 151st base position of any of the base sequences shown in SEQ ID NOS: 1 to 33 and 166 to 168, or its complementary strand, can be used as a marker for distinguishing species or hybrids of fish of the genus Pufferfish, where the base of the SNP is the base of allele 1 or 2 of the SNP. That is, in the base sequences of the markers, the 151st base of the base sequence shown in SEQ ID NO: 1 is T or C, the 151st base of the base sequence shown in SEQ ID NO: 2 is C or T, the 151st base of the base sequence shown in SEQ ID NO: 3 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 4 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 5 is T or C, the 151st base of the base sequence shown in SEQ ID NO: 6 is T or G, the 151st base of the base sequence shown in SEQ ID NO: 7 is G or T, the 151st base of the base sequence shown in SEQ ID NO: 8 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 9 is T or C, the 151st base of the base sequence shown in SEQ ID NO: 10 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 11 is G or C, the 151st base of the base sequence shown in SEQ ID NO: 12 is A or C, and the 151st base of the base sequence shown in SEQ ID NO: 13 is A or G. the 151st base of the base sequence shown in SEQ ID NO: 14 is C or T, the 151st base of the base sequence shown in SEQ ID NO: 15 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 16 is G or C, the 151st base of the base sequence shown in SEQ ID NO: 17 is T or C, the 151st base of the base sequence shown in SEQ ID NO: 18 is G or A, the 151st base of the base sequence shown in SEQ ID NO: 19 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 20 is C or G, the 151st base of the base sequence shown in SEQ ID NO: 21 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 22 is T or C, the 151st base of the base sequence shown in SEQ ID NO: 23 is C or A, the 151st base of the base sequence shown in SEQ ID NO: 24 is G or C, and the 151st base of the base sequence shown in SEQ ID NO: 25 is A or G,The 151st base of the base sequence shown in SEQ ID NO: 26 is A or T, the 151st base of the base sequence shown in SEQ ID NO: 27 is C or G, the 151st base of the base sequence shown in SEQ ID NO: 28 is T or G, the 151st base of the base sequence shown in SEQ ID NO: 29 is C or G, the 151st base of the base sequence shown in SEQ ID NO: 30 is T or C, the 151st base of the base sequence shown in SEQ ID NO: 31 is A or T, the 151st base of the base sequence shown in SEQ ID NO: 32 is A or G, the 151st base of the base sequence shown in SEQ ID NO: 33 is G or A, the 151st base of the base sequence shown in SEQ ID NO: 166 is C or T, the 151st base of the base sequence shown in SEQ ID NO: 167 is G or T, and the 151st base of the base sequence shown in SEQ ID NO: 168 is A or T. The base length of such a polynucleotide or its complementary strand may be any length that allows it to be specifically recognized on the genome of each species of pufferfish, and is, for example, preferably 15 to 100 bases, more preferably 20 to 50 bases. [Example]

[0074] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0075] Example 1 Identification of SNPs for distinguishing species or hybrids of Pufferfish We detected SNPs across the genomes of 12 species of the genus Takifugu (Takifugu rubripes), including Takifugu rubripes, Takifugu rubripes, Takifugu rubripes, Takifugu rubripes, and Takifugu rubripes, from Japan and three species of Takifugu (Takifugu rubripes), including Takifugu rubripes, Takifugu rubripes, and Takifugu rubripes. Specifically, we extracted genomic DNA from 8 to 16 individuals of each species using the DNeasy Blood & Tissue Kit (Qiagen). Approximately 10 ng of genomic DNA was digested with EcoRI and BglII restriction enzymes, ligated to adapters, and amplified with primers containing barcode sequences unique to each individual to create a library. The library was run on a next-generation sequencer (HiSeq 2000 or HiSeq 2500, Illumina) using 50-bp single-end sequencing. After removing adapter sequences and low-quality reads from the obtained ddRAD-seq reads, they were mapped to the tiger pufferfish genome sequence (FUGU5). Loci containing SNPs were extracted from the mapped reads, and species-specific SNPs (1,232 loci) were identified from among them. For each specific SNP, 150-bp upstream and 150-bp downstream sequences were obtained from FUGU5. The sequences obtained from FUGU5 were compared with the read sequences to detect non-species-specific polymorphisms in tiger pufferfish or other species of the genus Takifugu. Loci suitable for SNP typing were selected, with at least three candidate loci for each species, as they contained no non-species-specific polymorphisms near each SNP and did not contain many repetitive sequences or non-species-specific polymorphisms in the surrounding sequences. TaqMan Assays were designed for these candidates, and validation experiments were performed until three or four loci were identified for each species for which assays could be designed and for which no false positives or false negatives were detected in samples identified by AFLP. Finally, SNPs for species or hybrid identification were determined. Table 5 lists the species and genotypes corresponding to each SNP, and Table 6 lists the probes and primers used.

[0076] [Table 5]

[0077] [Table 6]

[0078] The genotype of SNP (1) above was TT in red-eyed pufferfish and CC in other Takifugu species, demonstrating species specificity for SNP (1). These results suggest that in hybrids between red-eyed pufferfish and other species of Takifugu species, the genotype of SNP (1) is TC. The same can be said for SNPs (2) to (36) above, based on Table 5 above. Therefore, SNPs (1) to (36) are useful for distinguishing species or hybrids of Takifugu species.

[0079] Example 2 Identification of species or hybrids of Pufferfish genus 1 Genomic DNA was extracted from fin sections of seven individuals of the genus Pufferfish (Pufferfish pufferfish), seven individuals of the Japanese pufferfish (Pufferfish spp.), and five hybrids between them, whose species or hybrids had been identified by AFLP, using the Wizard® Genomic DNA Purification Kit (Promega) according to the kit's instructions. The resulting nucleic acid samples were used as templates for real-time quantitative PCR using the probes and primers listed in Table 6 above, as well as TaqMan® GTXpress® Master Mix (Applied Biosystems®), on a StepOne® Real-Time PCR System (Thermo Fisher Scientific) to determine the genotypes of SNPs (4) to (6) and (28) to (30) listed in Table 5 above. A no-template PCR was used as a negative control. PCR was performed for 40 cycles, each cycle consisting of heat denaturation at 95°C for 3 seconds and annealing and extension at 60°C for 20 seconds.

[0080] The typing results for SNPs (4) to (6) are shown as allele discrimination plots in Figures 1 to 3. When the genotype of SNP (4) was used to distinguish individuals with AA as pufferfish, individuals with GG as pufferfish, and individuals with AG as hybrids of pufferfish and pufferfish, the results matched the species or hybrids previously identified by AFLP. Furthermore, when the genotype of SNP (5) was used to distinguish individuals with TT as pufferfish, individuals with CC as pufferfish, and individuals with TC as hybrids of pufferfish and pufferfish, the results matched the species or hybrids previously identified by AFLP. Furthermore, when the genotype of SNP (6) was used to distinguish individuals with TT as pufferfish, individuals with GG as pufferfish, and individuals with TG as hybrids of pufferfish and pufferfish, the results matched the species or hybrids previously identified by AFLP. The typing results for SNPs (28) to (30) are shown as allele discrimination plots in Figures 4 to 6. When the genotype of SNP (28) was used to distinguish individuals with TT as pufferfish, individuals with GG as pufferfish, and individuals with TG as hybrids of pufferfish and pufferfish, the results were consistent with the species or hybrids previously identified by AFLP. Furthermore, when the genotype of SNP (29) was used to distinguish individuals with CC as pufferfish, individuals with GG as pufferfish, and individuals with CG as hybrids of pufferfish and pufferfish, the results were consistent with the species or hybrids previously identified by AFLP. Furthermore, when the genotype of SNP (30) was used to distinguish individuals with TT as pufferfish, individuals with CC as pufferfish, and individuals with TC as hybrids of pufferfish and pufferfish, the results were consistent with the species or hybrids previously identified by AFLP. As described above, the typing results of the six SNPs accurately distinguished between pufferfish, pufferfish, and their hybrids. These SNPs were shown to be useful as indicators for distinguishing between species or hybrids of fish of the genus Pufferfish.

[0081] Example 3 Identification of species or hybrids of Pufferfish 2 The genotypes of SNPs (16) to (18) and (31) to (33) in Table 5 above were determined in the same manner as in Example 2, except that seven individuals of pufferfish, seven individuals of tiger pufferfish, and five hybrids between them were used.

[0082] As a result, when the genotype of SNP (16) was used to distinguish GG individuals as pufferfish, CC individuals as tiger pufferfish, and GC individuals as hybrids between pufferfish and tiger pufferfish, the species or hybrids were consistent with the species or hybrids previously identified by AFLP. Furthermore, when the genotype of SNP (17) was used to distinguish TT individuals as pufferfish, CC individuals as tiger pufferfish, and TC individuals as hybrids between pufferfish and tiger pufferfish, the species or hybrids were consistent with the species or hybrids previously identified by AFLP. Furthermore, when the genotype of SNP (18) was used to distinguish GG individuals as pufferfish, AA individuals as tiger pufferfish, and GA individuals as hybrids between pufferfish and tiger pufferfish, the species or hybrids were consistent with the species or hybrids previously identified by AFLP. Furthermore, when the genotype of SNP (31) was used to distinguish AA individuals as tiger pufferfish, TT individuals as small-spotted pufferfish, and AT individuals as a hybrid between tiger pufferfish and small-spotted pufferfish, the species or hybrids were consistent with the species or hybrids previously identified by AFLP. Furthermore, when the genotype of SNP (32) was used to distinguish AA individuals as tiger pufferfish, GG individuals as small-spotted pufferfish, and AG individuals as a hybrid between tiger pufferfish and small-spotted pufferfish, the species or hybrids were consistent with the species or hybrids previously identified by AFLP. Furthermore, when the genotype of SNP (33) was used to distinguish GG individuals as tiger pufferfish, AA individuals as small-spotted pufferfish, and GA individuals as a hybrid between tiger pufferfish and small-spotted pufferfish, the species or hybrids were consistent with the species or hybrids previously identified by AFLP. As described above, the typing results of the six SNPs accurately distinguished between pufferfish, tiger pufferfish, and their hybrids, demonstrating that the SNPs are useful as indicators for distinguishing between species or hybrids of fish of the genus Pufferfish.

[0083] Example 4 Identification of species or hybrids of Pufferfish genus 3 The genotypes of SNPs (10) to (12) and (28) to (30) in Table 5 above were determined in the same manner as in Example 2, except that seven individuals of the common pufferfish, seven individuals of the Japanese pufferfish, and five hybrids between them were used.

[0084] As a result, when the genotype of SNP (10) was used to distinguish individuals with AA as common pufferfish, individuals with GG as pufferfish, and individuals with AG as hybrids between common pufferfish and pufferfish, the species or hybrids were consistent with the species or hybrids previously identified by AFLP. Furthermore, when the genotype of SNP (11) was used to distinguish individuals with GG as common pufferfish, individuals with CC as pufferfish, and individuals with GC as hybrids between common pufferfish and pufferfish, the species or hybrids were consistent with the species or hybrids previously identified by AFLP. Furthermore, when the genotype of SNP (12) was used to distinguish individuals with AA as common pufferfish, individuals with CC as pufferfish, and individuals with AC as hybrids between common pufferfish and pufferfish, the species or hybrids were consistent with the species or hybrids previously identified by AFLP. Furthermore, when the genotype of SNP (28) was used to distinguish the TT individual as a Japanese pufferfish, the GG individual as a common pufferfish, and the TG individual as a hybrid between the Japanese pufferfish and a common pufferfish, the species or hybrids were consistent with the species or hybrids previously identified by AFLP. Furthermore, when the genotype of SNP (29) was used to distinguish the CC individual as a Japanese pufferfish, the GG individual as a common pufferfish, and the CG individual as a hybrid between the Japanese pufferfish and a common pufferfish, the species or hybrids were consistent with the species or hybrids previously identified by AFLP. Furthermore, when the genotype of SNP (30) was used to distinguish the TT individual as a Japanese pufferfish, the CC individual as a common pufferfish, and the TC individual as a hybrid between the Japanese pufferfish and a common pufferfish, the species or hybrids were consistent with the species or hybrids previously identified by AFLP. As described above, the typing results of the six SNPs accurately distinguished between the common pufferfish, the Japanese pufferfish, and their hybrids. These SNPs were shown to be useful as indicators for distinguishing between species or hybrids of fish in the genus Pufferfish.

[0085] Example 5 Identification of species or hybrids of Pufferfish genus 4 Since hybrids between the species combinations in Examples 2 to 4 are relatively common in nature, it was possible to obtain samples including the hybrids and identify the species or hybrids using the AFLP method. On the other hand, for species combinations where hybrids are rarely or never found in nature, the specificity of species or hybrid identification by detecting the genotypes of the SNPs in Table 5 above was verified using 11 species of pufferfish. The genotypes of SNPs (1) to (3) in Table 5 above were determined in the same manner as in Example 2, except that four individuals each of red-eyed pufferfish, spotted pufferfish, red-spotted pufferfish, common pufferfish, grass pufferfish, giant pufferfish, stingray pufferfish, and tiger pufferfish were used, as well as an equal mixture of red-eyed pufferfish and tiger pufferfish genomic DNA as a heterozygous control.

[0086] The typing results for SNP (1) are shown as an allele discrimination plot in Figure 7. When the SNP (1) genotype was determined to be TT, the CC individual to be a species of Takifugu other than Takifugu, and the TC sample to be a heterozygous control consisting of an equal mixture of genomic DNA from Takifugu and Takifugu, the results matched the species or hybrids previously identified by AFLP. Furthermore, when the SNP (2) genotype was determined to be CC, the TT individual to be a species of Takifugu other than Takifugu, and the CT sample to be a heterozygous control consisting of an equal mixture of genomic DNA from Takifugu and Takifugu, the results matched the species or hybrids previously identified by AFLP. Furthermore, when individuals with the genotype of SNP (3) AA were identified as red-eyed pufferfish, individuals with GG as pufferfish species other than red-eyed pufferfish, and samples with AG as heterozygous controls consisting of equal amounts of red-eyed pufferfish and pufferfish genomic DNA, they matched the species or hybrids previously identified by the AFLP method. The typing results for the three SNPs above accurately distinguished between red-eyed pufferfish, other species of the genus Pufferfish, and possible hybrids between them. No false negatives or false positives were observed, demonstrating that the SNPs are useful as indicators for distinguishing species or hybrids of Pufferfish, particularly red-eyed pufferfish.

[0087] Example 6 Identification of species or hybrids of Pufferfish genus 5 As in Example 5, the specificity of species or hybrid discrimination by detecting the genotypes of SNPs (4) to (33) in Table 5 above was verified using four individuals each of the following species: red-eyed pufferfish, spotted pufferfish, red-spotted pufferfish, common pufferfish, grass pufferfish, spotted pufferfish, Japanese pufferfish, blue-spotted pufferfish, and tiger pufferfish, as well as an equal mixture of genomic DNA of each species and tiger pufferfish as a heterozygous control.

[0088] When individuals with the genotype of SNP (4) AA were identified as pufferfish, individuals with the genotype GG were identified as pufferfish species other than pufferfish, and samples with the genotype AG were identified as heterozygous controls consisting of equal amounts of pufferfish and pufferfish genomic DNA. Similarly, when the species or hybrids of each individual or sample were identified based on the genotypes of SNPs (5) to (33), they all matched the species or hybrids previously identified by AFLP. As described above, the typing results of 30 SNPs accurately distinguished the species of pufferfish and their possible hybrids. No false negatives or false positives were observed, demonstrating that the SNPs are useful as indicators for distinguishing the species or hybrids of pufferfish.

[0089] Example 7 Identification of species or hybrids of Pufferfish genus 6 As in Example 5, seven individuals of each of the following species were used to verify the specificity of species or hybrid discrimination by detecting the genotypes of SNPs (34) to (36) in Table 5: red-eyed pufferfish, spotted pufferfish, red-spotted pufferfish, common pufferfish, grass pufferfish, spotted pufferfish, Japanese pufferfish, and tiger pufferfish, as well as a mixture of equal amounts of genomic DNA of each species and tiger pufferfish as a heterozygous control.

[0090] The genotype of SNP (34) was used to distinguish individuals with CC from pufferfish, individuals with TT from pufferfish species other than pufferfish, and individuals with CT from heterozygous controls, which were equal mixtures of pufferfish and pufferfish genomic DNA. The results matched the species or hybrids previously identified by AFLP. The SNP typing results accurately distinguished pufferfish, pufferfish species other than pufferfish, and potential hybrids between them. No false negatives or false positives were observed, demonstrating the usefulness of the SNP as an indicator for distinguishing pufferfish species or hybrids, particularly pufferfish. Furthermore, when the genotype of SNP (35) was used to distinguish individuals with GG as pufferfish, individuals with TT as pufferfish species other than pufferfish, and individuals with GT as heterozygous controls consisting of equal amounts of pufferfish and pufferfish genomic DNA, the results matched the species or hybrids previously identified by AFLP. The SNP typing results accurately distinguished pufferfish, pufferfish species other than pufferfish, and potential hybrids between them. No false negatives or false positives were observed, demonstrating the usefulness of the SNP as an indicator for distinguishing pufferfish species or hybrids, particularly pufferfish. Furthermore, when the genotype of SNP (36) was determined to be AA pufferfish, TT pufferfish, or a species of Takifugu other than Takifugu, and AT sample, a heterozygous control consisting of an equal mixture of Takifugu and Takifugu genomic DNA, the results matched the species or hybrids previously identified by AFLP. The SNP typing results accurately distinguished Takifugu, Takifugu species other than Takifugu, and possible hybrids between them. No false negatives or false positives were observed, demonstrating that the SNP is useful as an indicator for distinguishing species or hybrids of Takifugu, particularly Takifugu.

Claims

1. A method for distinguishing between a specific species of Takifugu fish, a Takifugu fish other than the specific species, or a hybrid between the specific species of Takifugu fish and a Takifugu fish other than the specific species, comprising: Detecting at least one SNP selected from the group consisting of single nucleotide polymorphisms (SNPs) at the 151st base position of the base sequences shown in SEQ ID NOs: 1 to 33 and 166 to 168 in nuclear DNA derived from a sample; and Based on the genotype of the detected SNP, determining whether the Takifugu fish from which the sample was derived is the specific species of Takifugu fish, a species other than the specific species of Takifugu fish, or a hybrid between the specific species of Takifugu fish and the species other than the specific species of Takifugu fish; Including, when detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 1 to 3, the specific species is Akame pufferfish, when detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 4 to 6, the specific species is pufferfish, when detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 7 to 9, the specific species is pufferfish; when detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 10 to 12, the specific species is common pufferfish, When detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 13 to 15 and 166, the specific species is pufferfish, When detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 16 to 18 and 167, the specific species is pufferfish, When detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 19 to 21, the specific species is pufferfish, when detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 22 to 24, the specific species is pear pufferfish, When detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 25 to 27 and 168, the specific species is pufferfish, when detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 28 to 30, the specific species is pufferfish; When at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 31 to 33 is detected, the specific species is a pufferfish or a crow. method.

2. The method according to claim 1, wherein at least two groups of SNPs selected from the following 11 groups (i) to (xi) are detected in combination: (i) at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 1 to 3; (ii) at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 4 to 6; (iii) at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 7 to 9; (iv) at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 10 to 12; (v) at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 13 to 15 and 166; (vi) at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 16 to 18 and 167; (vii) at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 19 to 21; (viii) at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 22 to 24; (ix) at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 25 to 27 and 168; (x) at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 28 to 30; and (xi) At least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 31 to 33.

3. A method for distinguishing between species or hybrids of fish of the genus Pufferfish, comprising: For nuclear DNA derived from a sample, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOS: 1 to 3, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOS: 4 to 6, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOS: 7 to 9, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOS: 10 to 12, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOS: 13 to 15 and 166, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOS: 16 to 18 and 167 at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 19 to 21, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 22 to 24, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 25 to 27 and 168, at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 28 to 30, and at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 31 to 33; Identifying the species or hybrid of the pufferfish from which the sample originates based on the genotype of the detected SNP; A method comprising:

4. If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 1 is TT, the fish of the genus Takifugu from which the sample was derived is determined to be a red eagle pufferfish; if it is CC, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than a red eagle pufferfish; and if it is TC, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a red eagle pufferfish and a species of fish of the genus Takifugu other than a red eagle pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 2 is CC, the fish of the genus Takifugu from which the sample was derived is determined to be a red eagle pufferfish; if the genotype is TT, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than a red eagle pufferfish; and if the genotype is CT, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a red eagle pufferfish and a species of fish of the genus Takifugu other than a red eagle pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 3 is AA, the fish of the genus Takifugu from which the sample was derived is determined to be a red eagle pufferfish; if it is GG, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than a red eagle pufferfish; and if it is AG, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a red eagle pufferfish and a species of fish of the genus Takifugu other than a red eagle pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 4 is AA, the fish of the genus Takifugu from which the sample was derived is determined to be a pufferfish; if it is GG, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than Takifugu; and if it is AG, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a pufferfish and a species of fish of the genus Takifugu other than Takifugu; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 5 is TT, the fish of the genus Takifugu from which the sample was derived is determined to be a pufferfish; if it is CC, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than Takifugu; and if it is TC, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a pufferfish and a species of fish of the genus Takifugu other than Takifugu; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 6 is TT, the fish of the genus Takifugu from which the sample was derived is determined to be a spotted pufferfish; if it is GG, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than a spotted pufferfish; and if it is TG, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a spotted pufferfish and a species of fish of the genus Takifugu other than a spotted pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 7 is GG, the fish of the genus Takifugu from which the sample was derived is determined to be a red pufferfish; if it is TT, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than a red pufferfish; and if it is GT, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a red pufferfish and a species of fish of the genus Takifugu other than a red pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 8 is AA, the fish of the genus Takifugu from which the sample was derived is determined to be a red pufferfish; if it is GG, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than a red pufferfish; and if it is AG, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a red pufferfish and a species of fish of the genus Takifugu other than a red pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 9 is TT, the fish of the genus Takifugu from which the sample was derived is determined to be pufferfish, if it is CC, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than pufferfish, and if it is TC, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of pufferfish and a species of fish of the genus Takifugu other than pufferfish, If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 10 is AA, the Takifugu fish from which the sample was derived is determined to be a common pufferfish; if it is GG, the Takifugu fish from which the sample was derived is determined to be a species of Takifugu fish other than a common pufferfish; and if it is AG, the Takifugu fish from which the sample was derived is determined to be a hybrid of a common pufferfish and a species of Takifugu fish other than a common pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 11 is GG, the Takifugu fish from which the sample was derived is determined to be a common pufferfish; if it is CC, the Takifugu fish from which the sample was derived is determined to be a species of Takifugu fish other than a common pufferfish; and if it is GC, the Takifugu fish from which the sample was derived is determined to be a hybrid of a common pufferfish and a species of Takifugu fish other than a common pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 12 is AA, the Takifugu fish from which the sample was derived is determined to be a common pufferfish; if it is CC, the Takifugu fish from which the sample was derived is determined to be a species of Takifugu fish other than a common pufferfish; and if it is AC, the Takifugu fish from which the sample was derived is determined to be a hybrid of a common pufferfish and a species of Takifugu fish other than a common pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 13 is CC, the Takifugu species from which the sample was derived is determined to be a Takifugu species; if the genotype is TT, the Takifugu species from which the sample was derived is determined to be a species of Takifugu species other than a Takifugu species; and if the genotype is CT, the Takifugu species from which the sample was derived is determined to be a hybrid of a Takifugu species and a Takifugu species other than a Takifugu species; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 14 is CC, the Takifugu species from which the sample was derived is determined to be a Takifugu species; if the genotype is TT, the Takifugu species from which the sample was derived is determined to be a species of Takifugu species other than Takifugu species; and if the genotype is CT, the Takifugu species from which the sample was derived is determined to be a hybrid of a Takifugu species and a Takifugu species other than Takifugu species. If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 15 is AA, the Takifugu species from which the sample was derived is determined to be a Takifugu species; if the genotype is GG, the Takifugu species from which the sample was derived is determined to be a species of Takifugu species other than a Takifugu species; and if the genotype is AG, the Takifugu species from which the sample was derived is determined to be a hybrid of a Takifugu species and a Takifugu species other than a Takifugu species; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 16 is GG, the fish of the genus Takifugu from which the sample was derived is determined to be a pufferfish; if it is CC, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than Takifugu; and if it is GC, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a pufferfish and a species of fish of the genus Takifugu other than Takifugu; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 17 is TT, the fish of the genus Takifugu from which the sample was derived is determined to be a pufferfish; if it is CC, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than Takifugu; and if it is TC, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a pufferfish and a species of fish of the genus Takifugu other than Takifugu; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 18 is GG, the fish of the genus Takifugu from which the sample was derived is determined to be a pufferfish; if the genotype is AA, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than Takifugu; and if the genotype is GA, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a pufferfish and a species of fish of the genus Takifugu other than Takifugu; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 19 is AA, the Takifugu species from which the sample was derived is determined to be a Takifugu; if the genotype is GG, the Takifugu species from which the sample was derived is determined to be a species of Takifugu other than Takifugu; and if the genotype is AG, the Takifugu species from which the sample was derived is determined to be a hybrid of a Takifugu and a species of Takifugu other than Takifugu; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 20 is CC, the Takifugu species from which the sample was derived is determined to be a Takifugu; if the genotype is GG, the Takifugu species from which the sample was derived is determined to be a species of Takifugu other than Takifugu; and if the genotype is CG, the Takifugu species from which the sample was derived is determined to be a hybrid of a Takifugu and a species of Takifugu other than Takifugu; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 21 is AA, the Takifugu species from which the sample was derived is determined to be a mushifugu; if it is GG, the Takifugu species from which the sample was derived is determined to be a species of Takifugu other than a mushifugu; and if it is AG, the Takifugu species from which the sample was derived is determined to be a hybrid of a mushifugu and a species of Takifugu other than a mushifugu; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 22 is TT, the fish of the genus Takifugu from which the sample was derived is determined to be pufferfish; if it is CC, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than pufferfish; and if it is TC, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of pufferfish and a species of fish of the genus Takifugu other than pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 23 is CC, the Takifugu species from which the sample was derived is determined to be pufferfish; if the genotype is AA, the Takifugu species from which the sample was derived is determined to be a species of Takifugu other than pufferfish; and if the genotype is CA, the Takifugu species from which the sample was derived is determined to be a hybrid of pufferfish and a species of Takifugu other than pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 24 is GG, the Takifugu species from which the sample was derived is determined to be pufferfish; if it is CC, the Takifugu species from which the sample was derived is determined to be a species of Takifugu other than pufferfish; and if it is GC, the Takifugu species from which the sample was derived is determined to be a hybrid of pufferfish and a species of Takifugu other than pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 25 is AA, the Takifugu species from which the sample was derived is determined to be a striped pufferfish; if the genotype is GG, the Takifugu species from which the sample was derived is determined to be a species of Takifugu other than a striped pufferfish; and if the genotype is AG, the Takifugu species from which the sample was derived is determined to be a hybrid of a striped pufferfish and a species of Takifugu other than a striped pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 26 is AA, the fish of the genus Takifugu from which the sample was derived is determined to be a striped pufferfish; if the genotype is TT, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than a striped pufferfish; and if the genotype is AT, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a striped pufferfish and a species of fish of the genus Takifugu other than a striped pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 27 is CC, the Takifugu species from which the sample was derived is determined to be a striped pufferfish; if the genotype is GG, the Takifugu species from which the sample was derived is determined to be a species of Takifugu other than a striped pufferfish; and if the genotype is CG, the Takifugu species from which the sample was derived is determined to be a hybrid of a striped pufferfish and a species of Takifugu other than a striped pufferfish; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 28 is TT, the fish of the genus Takifugu from which the sample was derived is determined to be Takifugu rubripes; if the genotype is GG, the fish of the genus Takifugu from which the sample was derived is determined to be a species of Takifugu other than Takifugu rubripes; and if the genotype is TG, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of Takifugu rubripes and a species of Takifugu other than Takifugu rubripes; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 29 is CC, the fish of the genus Takifugu from which the sample was derived is determined to be Takifugu rubripes, if it is GG, the fish of the genus Takifugu from which the sample was derived is determined to be a species of Takifugu other than Takifugu rubripes, and if it is CG, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of Takifugu rubripes and a species of Takifugu other than Takifugu rubripes, If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 30 is TT, the fish of the genus Takifugu from which the sample was derived is determined to be Takifugu rubripes; if the genotype is GG, the fish of the genus Takifugu from which the sample was derived is determined to be a species of Takifugu other than Takifugu rubripes; and if the genotype is TG, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of Takifugu rubripes and a species of Takifugu other than Takifugu rubripes; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 31 is AA, the fish of the genus Takifugu from which the sample was derived is determined to be a tiger pufferfish or a crow; if it is TT, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than a tiger pufferfish or a crow; and if it is AT, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a tiger pufferfish or a crow and a species of fish of the genus Takifugu other than a tiger pufferfish or a crow; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 32 is AA, the fish of the genus Takifugu from which the sample was derived is determined to be a tiger pufferfish or a crow; if it is GG, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than a tiger pufferfish or a crow; and if it is AG, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a tiger pufferfish or a crow and a species of fish of the genus Takifugu other than a tiger pufferfish or a crow; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 33 is GG, the fish of the genus Takifugu from which the sample was derived is determined to be a tiger pufferfish or a crow; if it is AA, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than a tiger pufferfish or a crow; and if it is GA, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a tiger pufferfish or a crow and a species of fish of the genus Takifugu other than a tiger pufferfish or a crow; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 166 is CC, the Takifugu species from which the sample was derived is determined to be a Takifugu species; if the genotype is TT, the Takifugu species from which the sample was derived is determined to be a species of Takifugu species other than a Takifugu species; and if the genotype is CT, the Takifugu species from which the sample was derived is determined to be a hybrid of a Takifugu species and a Takifugu species other than a Takifugu species; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 167 is GG, the fish of the genus Takifugu from which the sample was derived is determined to be a pufferfish; if it is TT, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than Takifugu; and if it is GT, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a pufferfish and a species of fish of the genus Takifugu other than Takifugu; If the genotype of the detected SNP at the 151st base position of the base sequence shown in SEQ ID NO: 168 is AA, the fish of the genus Takifugu from which the sample was derived is determined to be a striped pufferfish; if it is TT, the fish of the genus Takifugu from which the sample was derived is determined to be a species of fish of the genus Takifugu other than Takifugu; and if it is AT, the fish of the genus Takifugu from which the sample was derived is determined to be a hybrid of a striped pufferfish and a species of fish of the genus Takifugu other than Takifugu. The method according to any one of claims 1 to 3.

5. A probe for discriminating between a specific species of Takifugu genus fish, a Takifugu genus fish other than the specific species, or a hybrid between the specific species of Takifugu genus fish and the specific species of Takifugu genus fish other than the specific species, which can detect a SNP at the 151st base position of a base sequence shown in any of SEQ ID NOs: 1 to 33 and 166 to 168, when detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 1 to 3, the specific species is Akame pufferfish, when detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 4 to 6, the specific species is pufferfish, when detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 7 to 9, the specific species is pufferfish; when detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 10 to 12, the specific species is common pufferfish, When detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 13 to 15 and 166, the specific species is pufferfish, When detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 16 to 18 and 167, the specific species is pufferfish, When detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 19 to 21, the specific species is pufferfish, when detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 22 to 24, the specific species is pear pufferfish, When detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 25 to 27 and 168, the specific species is pufferfish, when detecting at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 28 to 30, the specific species is pufferfish; When at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 31 to 33 is detected, the specific species is a pufferfish or a crow. probe.

6. A primer set for discriminating between a specific species of Takifugu rubripes, a Takifugu rubripes other than the specific species, or a hybrid between the specific species of Takifugu rubripes and the specific species of Takifugu rubripes other than the specific species, which is capable of amplifying a region containing a SNP at the 151st base position of the base sequence shown in any of SEQ ID NOs: 1 to 33 and 166 to 168, when amplifying a region including at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 1 to 3, the specific species is pufferfish, When amplifying a region including at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 4 to 6, the specific species is pufferfish, when amplifying a region including at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 7 to 9, the specific species is pufferfish, When amplifying a region including at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 10 to 12, the specific species is common pufferfish, When amplifying a region including at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 13 to 15 and 166, the specific species is pufferfish, When amplifying a region including at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 16 to 18 and 167, the specific species is pufferfish, When amplifying a region including at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 19 to 21, the specific species is pufferfish, When amplifying a region including at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 22 to 24, the specific species is pear pufferfish, When amplifying a region containing at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 25 to 27 and 168, the specific species is pufferfish, When amplifying a region including at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 28 to 30, the specific species is pufferfish, When amplifying a region containing at least one SNP selected from the group consisting of SNPs at the 151st base position of the base sequences shown in SEQ ID NOs: 31 to 33, the specific species is a pufferfish or a crow. Primer set.

7. A kit for distinguishing between a specific species of Takifugu fish, a Takifugu fish other than the specific species, or a hybrid between the specific species of Takifugu fish and a Takifugu fish other than the specific species, comprising the probe described in claim 5 and the primer set described in claim 6.

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