JP2002345498A - Method for distinguishing species of scomber yielded in japan - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for distinguishing many samples of fry, immature fish, or the like, collected in the field by a simple method. SOLUTION: This method for distinguishing the species of Scomber yielded in Japan is characterized in that the species are distinguished by the intervening sequence of rDNA of a specimen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for discriminating a species belonging to the genus Mackerel which is difficult to discriminate from the form.

[0002]

2. Description of the Related Art Discrimination of Japanese mackerel species is extremely important for maintenance of catch statistics and ecological research, especially for larval and juvenile ecological surveys. It is difficult to discriminate both from the form. By the way, research to discriminate species from the nucleotide sequence of genes has been conducted.
The following techniques are known.

[0003] A polynucleotide containing the same partial sequence V contained in the sequences represented by the three types of SEQ ID NOs: the same partial sequence contained in the sequence, and the 5 'end of the Using a set consisting of a polynucleotide containing a complementary sequence V ′ of the partial sequence further 3 ′ from the 3 ′ end, PCR using Leishmania parasite DNA as a template was performed, and the three types of SEQ ID NOs: A method for detecting a Leishmania bianja group including a step of amplifying a partial sequence of a sequence to be performed. "
No. 9). This utilizes characteristics such as that F10 band DNA and F4 band DNA are not amplified or not depending on species when PCR is performed with primers specific to arbitrary or Leishmania parasite.

[0004]

However, the arbitrary primer PCR technique has poor reproducibility, and it has been difficult to apply this technique to Japanese mackerel to easily discriminate multiple samples. Moreover, the Leishmania parasite-specific primer cannot be applied to the discrimination of the species of Mackerel in Japan.

[0005] Accordingly, an object of the present invention is to provide a method capable of distinguishing a large number of samples such as fry and immature fish collected in a field by a simple method.

[0006]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to achieve the above object, and as a result, the rDN of Japanese mackerel spp.
Attention was paid to the intervening sequence of A (ITS1 and ITS2). That is, rDNA present at a large number of sites on the chromosome
Is that all rDNA molecules possessed by an individual have almost the same sequence, because mutations occurring in individual rDNA rapidly disappear or spread throughout in a broad recombination reaction with another rDNA molecule. Further, rDNA is inherited from both parents because it is on a chromosome, and the sequence is homogenized by recombination (crossover) between chromosomes. For this reason, differences between individuals within a species are rare. However, since mutations accumulate in the intervening sequence independently for each species, the difference between species was predicted to be large. And, in fact, although intervening sequences have intra-individual mutations, individual differences are rather rare when a common sequence is determined by the majority rule of mutation sites.
In contrast, the interspecies differences were found to be quite large. From these, it was found that the intervening sequence of rDNA was a region suitable for species discrimination, with rare intra-species variation and large inter-species differences, and completed the present invention.

[0007] That is, the present invention provides a method for discriminating the species of Japanese Mackerel, wherein the species is discriminated from the intervening sequence of rDNA of the test subject. Things. The present invention has the following excellent effects by adopting such a configuration. Because of the large number of genes, it can be easily detected from a DNA sample that has been degraded in a small amount. When an intervening sequence of rDNA is used, each type is independently inherited, so that species discrimination can be performed with higher accuracy than when mitochondrial DNA that cannot completely eliminate the risk of misclassification due to intraspecies mutation is used. Unlike mitochondrial DNA, which is inherited by the maternal line, rDNA is inherited from both parents, so that hybrids can be determined. The morphological discrimination method can be improved by applying the method of the present invention to an individual whose morphological discrimination is extremely difficult. The discrimination target can be expanded to larvae and eggs, thereby enabling species discrimination in the entire life cycle, improving the accuracy of ecological surveys, and enriching data for ABC calculation and fishery situation prediction.

[0008] The present invention also relates to a method for discriminating the species of Mackerel in Japan, wherein 5.8S rDNA of rDNA and 18
It is an object of the present invention to provide a method for discriminating the species of the Mackerel genus in Japan, comprising discriminating the species from the length of a base sequence existing in an intervening sequence between SrDNAs. 5.8SrD of rDNA
By measuring the length of the base sequence of the specific portion in the intervening sequence between NA and 18S rDNA, the species can be distinguished with higher accuracy. The present invention also relates to a method for discriminating the Japanese mackerel genus, wherein a sequence that hybridizes to a site where there is no interspecies mutation is used as a primer,
The present invention provides a method for discriminating the species of Japanese mackerel, which comprises performing R and subjecting the PCR product to electrophoresis to discriminate the species. By taking such a configuration,
It is very easy to distinguish species having excellent sensitivity and stability. The present invention also relates to a method for discriminating the Japanese mackerel genus, comprising the step of:
A method for discriminating the species of Japanese mackerel, characterized by performing PCR using primers that match downstream from the terminus and primers that match upstream from the 3 ′ terminus to search for sites where only interspecies variation is not detected. To provide.
With this configuration, the species can be determined by a simple method without using a restriction enzyme. The present invention also relates to a method for discriminating the species of the genus Mackerel in Japan.
The present invention provides a method for discriminating the species of Japanese mackerel, which discriminates the species of Lasicus. By adopting such a configuration, it is possible to easily discriminate between saba and sesame, which are very similar in form and difficult to distinguish morphologically.

[0009]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a determination method according to the present invention will be described. First, the total DNA of Masabana and Sesame mackerel was roughly extracted, and the 3 'end-intervening sequence 1-5.8S of 18S rDNA was used.
rDNA-intervening sequence 2-28S The base sequence at the 5 'end of rDNA is divided into two overlapping fragments and subjected to PCR amplification. In order to divide into two overlapping fragments, a primer matching from the 5 'end of the overlapping portion to the downstream side and a primer matching from the overlapping 3' end to the upstream side may be used. Such a primer can be obtained from a nucleotide sequence common to the same region of a genus slightly distant from Mackerel. Alternatively, it may be divided into two overlapping fragments by performing a restriction enzyme treatment. The amplified fragment is inserted into a plasmid, cloned, and sequenced. The length of this region is 1225 base pairs for Masaba,
In the case of sesame mackerel, it is 1331 to 1340 base pairs. The number of mutation sites is 100 or more in the intervening sequence 1 region and 20 or more in the intervening sequence 2 region. On the contrary,
In the functional rRNA (rDNA) region, intra-individual mutations
There were only a few insertions and deletions of bases and bases.

In the intervening sequence 1, it was observed that 20 base pairs were continuously inserted / deleted. Generally, about 10
When the base sequence length differs by more than%, discrimination by agarose gel electrophoresis becomes possible. Therefore, PCR primers satisfying the following conditions were designed. A base sequence of 200 bases or less including the above 20 base pairs, a base sequence not only capable of interspecies variation, no large deviation in the base sequence, the same bases are not continuous several or more times, and it is difficult to take a single-stranded higher-order structure, A base sequence having general characteristics that a PCR primer should have, such as being difficult to bind between primers and having no similar sequence in the vicinity.

[0011] Using the above PCR primers, PCR amplification is carried out according to a conventional method. By electrophoresis of the obtained PCR product, more preferably agarose gel electrophoresis, it is possible to easily detect the difference in the length of the above-mentioned nucleotide sequence, thereby easily discriminating the species of the Japanese mackerel genus. can do.

Such a method is particularly effective for discriminating between species of Masababa and Sesame mackerel in which insertions / deletions of 20 base pairs or more have been continuously detected.

When it is difficult to determine the species due to the distortion of the electric field during electrophoresis or the like, if the PCR reaction product is treated with a specific restriction enzyme, a species-specific cleavage pattern appears. Can be used to determine the species. For example, when discriminating the species of Masaba and Sesame mackerel,
Using BglI, only one specific place in Masaba
When cI and Tru9I are used, only one specific portion of sesame mackerel is cut.

[0014]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

Example 1 2 from the 3 'end of 18S rDNA of Masababa and Sesame mackerel
Determination of the base sequence up to the 5 'end of 8S rDNA Muscle sites were collected from each of five individuals of Masaba and Sesame mackerel, and total DNA was roughly extracted by a conventional method. The DNA is 18Sr
DNA-intervening sequence 1 (ITS1) -5.8S rDNA-
Includes the structure of intervening sequence 2 (ITS2) -28S rDNA. With respect to this structure, primers matching several nucleotide sequences from the 3 'end of 5.8S rDNA to 18S rDNA and 28S from the 5' end of 5.8S rDNA
PCR amplification was performed using primers matching several base sequences toward the rDNA. The obtained PCR amplified fragment (corresponding to b and c in FIG. 1) had an overlapping portion in the 5.8S rDNA region. The base sequences of the above two primers are the same as those of carp (U87963: International DNA).
Database, accession number) and Xenopus (XELRNO1: International DNA Database, locus name). The base sequence was determined by inserting the PCR-amplified fragment into the plasmid pUC19. The sequence was determined from the consensus sequence of at least 3 clones. 18SrDNA to ITS1 to 5.8SrDNA to I of 5 crab mackerel and 5 crab mackerel
The nucleotide sequences of TS2-28S rDNA are shown in SEQ ID NOs: 1 to 14. SEQ ID NOs: 1 to 5 are the base sequences of Masaba 1 to 5, SEQ ID NO: 6 are Sesame Saba 1, SEQ ID NOs: 7 to 8 are Sesame Saba 2, SEQ ID NOs: 9 to 11 are Sesame Saba 3, SEQ ID NO: 12.
13 to 13 show the base sequences of sesame mackerel 4 and SEQ ID NO: 14 show the base sequences of sesame mackerel 5 (two kinds of sesame mackerels 2 and 4 and three kinds of sesame mackerel 3 exist). FIG. 2 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14. From FIG. 2, it can be seen that in saba, ITS1 in sesame saba is present at 33 to 14 base pairs from the 3 ′ end of ITS1.
Base pairs are deleted, and there is almost no difference between individuals,
It can be seen that the difference between species is large. In contrast, ITS2
As for, there are many intraspecies variations, but the interspecies differences are smaller than ITS1.

Example 2 Design and PCR Amplification of PCR Primers Containing 20 Base Pairs Deleted in Masaba As shown in Example 1, 20 base pairs in ITS1 existing in sesame are And the nucleotide sequence containing this portion has the largest interspecies difference. Therefore, a base sequence of 200 bases or less including the above 20 base pairs, a base sequence in which no interspecies variation is observed, a base sequence having no large deviation, and having a single-stranded higher-order structure in which the same bases do not continue several times or more. A PCR primer having general characteristics that a PCR primer should have, such as being difficult to bind with each other, having no similar sequence in the vicinity, and the like, was designed. The PCR primer on the forward side is SEQ ID NO: 15 (<223> synthesis),
The reverse side PCR primer is SEQ ID NO: 16 (<223>).
(Synthesis). 428 Masabana individuals identified by form,
PCR amplification was performed on 398 sesame mackerel and 8 unknown plants using the PCR primers under the following conditions.

PCR reaction conditions: The reaction system was 30 μL,
100 ng or less of template DNA, 3 μL of dNTP mix (manufactured by Toyobo Co., Ltd.) (However, 2.4 μL when using Takara Shuzo Co., Ltd.), 0.15 μL of rTaq polymerase (manufactured by Takara Shuzo Co., Ltd.), 10 × rTaq polymerase buffer (Manufactured by Takara Shuzo Co., Ltd.)
Add to make up to μL. The reaction system was dispensed into a microtube for PCR and heated at 95 ° C. for 2 to 5 minutes, and then a cycle of 98 ° C. for 10 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds was repeated 30 times. Hold for a minute. As a result, an amplification product of 204 base pairs was obtained for Masaba and 226 base pairs was obtained for Sesame Mackerel.

Example 3 Detection of PCR product by agarose gel electrophoresis 1 to 3 μL of the reaction solution after the PCR reaction was subjected to 2%
The gel was subjected to agarose gel electrophoresis, stained and detected according to a conventional method.

As a result, among the Japanese genus Mackerel identified in the above manner, it was considered that morphological identification was incorrect for 24 individuals. It was also found that three of them were hybrids.

As for ITS2, there were two types in the sesame species, and one was similar to the saba. When the retention status of these two types in sesame mackerel was examined for 95 individuals, 75 individuals possessed both types. Assuming that these two types are alleles, this is considered paralogous, since there is a significant hetero-excess.
The relatively low intraspecies variability in the mackerel encodes the hypothesis from mitochondrial DNA analysis that it has speciated from a portion of the meta population of Sesame mackerel.

REFERENCE EXAMPLE 1 When it is difficult to determine the species due to the distortion of the electric field during electrophoresis, if the PCR reaction product is treated with the following restriction enzymes, a specific cleavage pattern appears and the species can be identified. Become.
With BglI, 139 base pairs and 65
Cleaved into two base pairs. When SacI is used, it is cut into two at 172 bp and 53 bp only in sesame. With Tru9I, it is cut into two, 164 base pairs and 62 base pairs. In addition, when AsuI or BbvI is used, specific cleavage is expected to occur.

[0022]

According to the method of the present invention, species can be easily discriminated with an inexpensive apparatus except that the PCR apparatus is expensive. In particular, for species discrimination between Masababa and Sesame mackerel, PCR is performed using primers designed using a base sequence common to both species as a scaffold. Compared to,
The risk of erroneous determination (erroneous amplification or erroneous non-amplification) is small. When amplifying regions having different lengths, species discrimination is possible only by direct electrophoresis of the PCR reaction solution. Conventionally, PC
The operation procedure was complicated because the species could not be identified unless the R reaction solution was analyzed by restriction enzyme. However, according to the method of the present invention, it was not necessary to perform restriction enzyme analysis. Species discrimination can be performed.

[Sequence List] SEQUENCE LISTING <110> Method of discriminating species of Japanese Scomber <120> <130> J88542A1 <160> 14 <210> 1 <211> 1355 <212> DNA <213> Scomber japonicus <400> 1 tgaacctgcg gaaggatcat taccgggtcg cgtaccgcct ctcctcgtcg ccggggttga 60 agcgagcgac tgtaacctcc tccacacaag ctgagggcct cccgcaccgg ttctctccgg 120 agaacagggc gggggcccga ggcgcgcacg cggccctcct cccgaggggg gccgtgggca 180 aagaaattca cctcttgctc tggaccacgt tcctggccgg acgggcctct cttccccccc 240 ctactttttc cccagctccg ggcatcacgg tgcggcaggg ccggccggcc tggtgcccgt 300 cgctcgggga tcacacgggg gggggtcggg aacagtctac ccgtccggta cgccgggata 360 ggcccaacaa tcggaacctt aacctcagcg cgttgcggcg gctcggcccc tggccgccct 420 gcgcgtgccc ccgggtaccc aactctcctc cttcctccgg agggaccata gaggggttca 480 atgtctccta ccccgccgtg aaggccgttt gcagcccgtc ggcgggacgg agcgcccggg 540 ggtccggtcc tccccttctc aaaccctttt tttcagcagt cgaactctgg caaaagtgaa 600 aacacaaaac aaactgtgac aactcttagc ggtggatcac tcggctcgtg cgtcgatgaa 660 gaacgcagct agctgcgaga acta atgtga attgcaggac acattgatca tcgacacttc 720 gaacgcacct tgcggccccg ggttcctccc ggggctacgc ctgtctgagg gtcgctttga 780 catcaatcgg gaaataacct ttcccgcggc tggggctgtc gcaggcacct gtttgccttc 840 gtccccctaa gtgcagactg tcgggatgcc cggtgcggtg cgggtcgggc cagcaggctc 900 gcctccgttc tccccgtcgg cctaacatcc ccccccccat ctctcaccct cctttttccc 960 cagcctccgg gccggggaag ggggcgcctc cgtcgggccc gcatgagtcg ggcgcggctg 1020 ccggtggact tctggtctcc gcgctgcccg cgctacgcgt gcgtaagtcc ggcgggggtt 1080 gtcggcgcgt caagcgccgg gggtatcacg gggggtgggg ggggaaggcc gcggggtttg 1140 cggggctgtc gagctctgcc gccggtccgc ttccccgcgc cggggtacct ggctacgacc 1200 caccgagtct ccaggtgagc ctccgctccg gactctcggg cccggaagcg gagccaccga 1260 ccgggggccg cctggcggcc cccatcccat tcgactacga cctcagatca gacgagacaa 1320 cccg 1324 <210> 2 <211> 1355 <212> DNA <213> Scomber japonicus <400> 2 gtgaacctgc ggaaggatca ttaccgggtc gcgtaccgcc tctcctcgtc gccggggttg 60 aagcgagcga ctgtaacctc ctccacacaa gctgagggcc tcccgcaccg gttctctccg 120 gagaacaggg cgggggcccg aggcgcgcac gcggcccccc t cccgagggg ggccgtgggc 180 aaagaaattc acctcttgct ctggaccacg ttcctggccg gacgggcctc tcttcccccc 240 cctacttttt ccccagctcc gggcatcacg gtgcggcagg gccggccggc ctggtgcccg 300 tcgctcgggg atcacacggg ggggggtcgg gaacagtcta cccgtccggt acgccgggat 360 aggcccaaca atcggaacct taacctcagc gcgttgcggc ggctcggccc ctggccgccc 420 tgcgcgtgcc cccgggtacc caactctcct ccttcctccg gagggaccat agaggggttc 480 aatgtctcct accccgccgt gaaggccgtt tgcagcccgt cggcgggacg gagcgcccgg 540 gggtccggtc ctccccttct caaacccttt ttttcagcag tcgaactctg gcaaaagtga 600 aaacacaaaa caaactgtga caactcttag cggtggatca ctcggctcgt gcgtcgatga 660 agaacgcagc tagctgcgag aactaatgtg aattgcagga cacattgatc atcgacactt 720 cgaacgcacc ttgcggcccc gggttcctcc cggggctacg cctgtctgag ggtcgctttg 780 acatcaatcg ggaaataacc tttcccgcgg ctggggctgt cgcaggcacc tgtttgcctt 840 cgtcccccta agtgcagact gtcgggatgc ccggtgcggt gcgggtcggg ccagcaggct 900 cgcctccgtt ctccccgtcg gcctaacatc ccccccccca tctctcaccc tcctttttcc 960 ccagcctccg ggccggggaa gggggcgcct ccgtcgggcc cgcatgagtc gggcgcggct 1020 gccggtggac ttctggtctc cgcgctgccc gcgctacgcg tgcgtaagtc cggcgggggt 1080 tgtcggcgcg tcaagcgccg ggggtatcac ggggggtggg gggggaaggc cgcggggttt 1140 gcggggctgt cgagctctgc cgccggtccg cttccccgcg ccggggtacc tggctacgac 1200 ccaccgagtc tccaggtgag cctccgctcc ggactctcgg gcccggaagc ggagccaccg 1260 accgggggcc gcctggcggc ccccatccca ttcgactacg acctcagatc agacgagaca 1320 acccg 1325 <210> 3 <211> 1355 <212> DNA <213> Scomber japonicus <400> 3 gtgaacctgc ggaaggatca ttaccgggtc gcgtaccgcc tctcctcgtc gccggggttg 60 aagcgagcga ctgtaacctc ctccacacaa gctgagggcc tcccgcaccg gttctctccg 120 gagaacaggg cgggggcccg aggcgcgcac gcggccctcc tcccgagggg ggccgtgggc 180 aaagaaattc acctcttgct ctggaccacg ttcctggccg gacgggcctc tcttcccccc 240 cctacttttt ccccagctcc gggcatcacg gtgcggcagg gccggccggc ctggtgcccg 300 tcgctcgggg atcacacggg ggggggtcgg gaacagtcta cccgtccggt acgccgggat 360 aggcccaaca atcggaacct taacctcagc gcgttgcggc ggctcggccc ctggccgccc 420 tgcgcgtgcc cccgggtacc caactctcct ccttcctccg gagggaccat agaggggttc 480 aatgtctcct ac cccgccgt gaaggccgtt tgcagcccgt tggcgggacg gagcgcccgg 540 gggtccggtc ctccccttct caaacccttt ttttcagcag tcgaactctg gcaaaagtga 600 aaacacaaaa caaactgtga caactcttag cggtggatca ctcggctcgt gcgtcgatga 660 agaacgcagc tagctgcgag aactaatgtg aattgcagga cacattgatc atcgacactt 720 cgaacgcacc ttgcggcccc gggttcctcc cggggctacg cctgtctgag ggtcgctttg 780 acatcaatcg ggaaataacc tttcccgcgg ctggggctgt cgcaggcacc tgtttgcctt 840 cgtcccccta agtgcagact gtcgggatgc ccggtgcggt gcgggtcggg ccagcaggct 900 cgcctccgtt ctccccgtcg gcctaacatc ccccccccca tctctcaccc tcctttttcc 960 ccagcctccg ggccggggaa gggggcgcct ccgtcgggcc cgcatgagtc gggcgcggct 1020 gccggtggac ttctggtctc cgcgctgccc gcgctacgcg tgcgtaagtc cggcgggggt 1080 tgtcggcgcg tcaagcgccg ggggtatcac ggggggtggg gggggaaggc cgcggggttt 1140 gcggggctgt cgagctctgc cgccggtccg cttccccgcg ccggggtacc tggctacgac 1200 ccaccgagtc tccaggtgag cctccgctcc ggactctcgg gcccggaagc ggagccaccg 1260 accgggggcc gcctggcggc ccccatccca ttcgactacg acctcagatc agacgagaca 1320 acccg 1325 < 210> 4 <211> 1355 <212> DNA <213> Scomber japonicus <400> 4 gtgaacctgc ggaaggatca ttaccgggtc gcgtaccgcc tctcctcgtc gccggggttg 60 aagcgagcga ctgtaacctc ctccacacaa gctgagggcc tcccgcaccg gttctctccg 120 gagaacaggg cgggggcccg aggcgcgcac gcggccctcc tcccgagggg ggccgtgggc 180 aaagaaattc acctcttgct ctggaccacg ttcctggccg gacgggcctc tcttcccccc 240 cctacttttt ccccagctcc gggcatcacg gtgcggcagg gccggccggc ctggtgcccg 300 tcgctcgggg atcacacggg ggggggtcgg gaacagtcta cccgtccggt acgccgggat 360 aggcccaaca atcggaacct taacctcagc gcgttgcggc ggctcggccc ctggccgccc 420 tgcgcgtgcc cccgggtacc caactctcct ccttcctccg gagggaccat agaggggttc 480 aatgtctcct accccgccgt gaaggccgtt tgcagcccgt cggcgggacg gagcgcccgg 540 gggtccggtc ctccccttct caaacccttt ttttcagcag tcgaactctg gcaaaagtga 600 aaacacaaaa caaactgtga caactcttag cggtggatca ctcggctcgt gcgtcgatga 660 agaacgcagc tagctgcgag aactaatgtg aattgcagga cacattgatc atcgacactt 720 cgaacgcacc ttgcggcccc gggttcctcc cggggctacg cctgtctgag ggtcgctttg 780 acatcaatcg ggaaataacc tttcccgcgg ctggggctgt cgcaggc acc tgtttgcctt 840 cgtcccccta agtgcagact gtcgggatgc ccggtgcggt gcgggtcggg ccagcaggct 900 cgcctccgtt ctccccgtcg gcctaacatc ccccccccca tctctcaccc tcctttttcc 960 ccagcctccg ggccggggaa gggggcgcct ccgtcgggcc cgcatgagtc gggcgcggct 1020 gccggtggac ttctggtctc cgcgctgccc gcgctacgcg tgcgtaagtc cggcgggggt 1080 tgtcggcgcg tcaagcgccg ggggtatcac ggggggtggg gggggaaggc cgcggggttt 1140 gcggggctgt cgagctctgc cgccggtccg cttccccgcg ccggggtacc tggctacgac 1200 ccaccgagtc tccaggtgag cctccgctcc ggactctcgg gcccggaagc ggagccaccg 1260 accgggggcc gcctggcggc ccccatccca ttcgactacg acctcagatc agacgagaca 1320 acccg 1325 <210> 5 <211> 1355 <212> DNA <213> Scomber japonicus <400> 5 gtgaacctgc ggaaggatca ttaccgggtc gcgtaccgcc tctcctcgtc gccggggttg 60 aagcgagcga ctgtaacctc ctccacacaa gctgagggcc tcccgcaccg gttctctccg 120 gagaacaggg cgggggcccg aggcgcgcac gcggccctcc tcccgagggg ggccgtgggc 180 aaagaaattc acctcttgct ctggaccacg ttcctggccg gacgggcctc tcttcccccc 240 cctacttttt ccccagctcc gggcatcacg gtgcggcagg gccggccggc ctggtgcccg 300 tcgctcgggg atcacacggg ggggggtcgg gaacagtcta cccgtccggt acgccgggat 360 aggcccaaca atcggaacct taacctcagc gcgttgcggc ggctcggccc ctggccgccc 420 tgcgcgtgcc cccgggtacc caactctcct ccttcctccg gagggaccat agaggggttc 480 aatgtctcct accccgccgt gaaggccgtt tgcagcccgt cggcgggacg gagcgcccgg 540 gggtccggtc ctccccttct caaacccttt ttttcagcag tcgaactctg gcaaaagtga 600 aaacacaaaa caaactgtga caactcttag cggtggatca ctcggctcgt gcgtcgatga 660 agaacgcagc tagctgcgag aactaatgtg aattgcagga cacattgatc atcgacactt 720 cgaacgcacc ttgcggcccc gggttcctcc cggggctacg cctgtctgag ggtcgctttg 780 acatcaatcg ggaaataacc tttcccgcgg ctggggctgt cgcaggcacc tgtttgcctt 840 cgtcccccta agtgcagact gtcgggatgc ccggtgcggt gcgggtcggg ccagcaggct 900 cgcctccgtt ctccccgtcg gcctaacatc ccccccccca tctctcaccc tcctttttcc 960 ccagcctccg ggccggggaa gggggcgcct ccgtcgggcc cgcatgagtc gggcgcggct 1020 gccggtggac ttctggtctc cgcgctgccc gcgctacgcg tgcgtaagtc cggcgggggt 1080 tgtcggcgcg tcaagcgccg ggggtatcac ggggggtggg gggggaaggc cgcggggttt 1140 gcggggctgt cgagc tctgc cgccggtccg cttccccgcg ccggggtacc tggctacgac 1200 ccaccgagtc tccaggtgag cctccgctcc ggactctcgg gcccggaagc ggagccaccg 1260 accgggggcc gcctggcggc ccccatccca ttcgactacg acctcagatc agacgagaca 1320 acccg 1325 <210> 6 <211> 1355 <212> DNA <213> Scomber australasicus <400> 6 gtgaacctgc ggaaggatca ttaccgggtc gcgttccgcc tctcctcctc gccggggttg 60 aagcgagcga ctgttgcctc cggaacagcc gaggtccccc cgccccagtt ctcttcgggg 120 aacgggggcg aggcccgagg cgcgcacgcg gaccctcctc gtgggggtcc gcgggcacac 180 aaaccctcct cttggtctgg acctcgtccc gaccggacgg gcctctcttc cccccccctt 240 tcccccggca acgggcatca cggtgcggca gggccggccg tcctggtgcc catcgctcgg 300 gggtcgcacg ggggggggtt gggaaccttc tacccgtccg gtgcgccggg acaggcccgc 360 cactcggaac caaaacctca gcgcgttgcg gcggctcggc ccctggccgc cctgcgcgcg 420 cccccgggta cccaactctc ctccctcctc cggagggaac atagaggggt tcaatgtctc 480 ctgccccgcc gacaaaggcc gtttccccca acccgttggg ggttgccgcc cgtcggcggg 540 acggagcgcc cgggggtcct gtcgtccccc ttctcaaacc ctttttttaa cgttcgagct 600 ctggcaaaag tgaaaacaca aaaacaaact gtgacaactc ttagcggtgg atcactcggc 660 tcgtgcgtcg atgaagaacg cagctagctg cgagaactaa tgtgaattgc aggacacatt 720 gatcatcgac acttcgaacg caccttgcgg ccccgggttc ctcccggggc tacgcctgtc 780 tgagggtcgc tttgacatca atcgggaaat aacttttttc ccgcggctgg ggctgtcgca 840 ggcacctgtt tgccttcgtc cccctaagtg cagactgtcg ggatgcccgg tgcggtgcgg 900 atcgggccag caggctcgcc tccgttctcc ccgtcggcct aacatccccc ccccatctct 960 caccctcctt tttccccttc cggggaaggg ggcgcctccg tcgggcccgc atgagtcggg 1020 cgcggctgcc ggtggacttc tggtctccgc gctgcccgcg ctacgcgtgc gtaagtccgg 1080 cgggggtgtc ggcgcgtcaa gcgccggggg tatcacgggg ggtggggggg aaggccgcgg 1140 ggtttgcggg gctgtcgagc tctgctgccg gtccgcttcc ccgcgccggg gtacctggct 1200 acgacccacc gagtctccag gtgagcctcc gctcaagggc tttcgggccc cggagcggag 1260 ccaccgaccg ggggccgcct ggcggccccc atcccattcg actacgacct cagatcagac 1320 gagacaaccc g 1331 <210> 7 <211> 1355 <212> DNA <213> Scomber australasicus <400> 7 gtgaacctgc ggaaggatca ttaccgggtc gcgttccgcc tctcctcctc gccggggttg 60 aagcgagcga ctgttgcctc cggaacagcc gaggtcc ccc cgccccagtt ctcttctcgg 120 ggaacggggg cgaggcccga ggcgcgcacg cggaccctcc tcgtgggggt ccgcgggcac 180 acaaaccctc ctcttggtct ggacctcgtc ccgaccggac gggcctctct tcccccccct 240 ttcccccggc aacgggcatc acggtgcggc agggccggcc gtcctggtgc ccatcgctcg 300 ggggtcgcac gggggggggt tgggaacctt ctacccgtcc ggtgcgccgg gacaggcccg 360 ccactcggaa ccaaaacctc agcgcgttgc ggcggctcgg cccctggccg ccctgcgcgc 420 gcccccgggt acccaactct cctccctcct ccggagggaa catagagggg ttcaatgtct 480 cctgccccgc cgacaaaggc cgtttccccc aacccgttgg gggttgccgc ccgtcggcgg 540 gacggagcgc ccgggggtcc tgtcgtcccc cttctcaaac ccttttttta acgttcgagc 600 tctggcaaaa gtgaaaacac aaaaacaaac tgtgacaact cttagcggtg gatcactcgg 660 ctcgtgcgtc gatgaagaac gcagctagct gcgagaacta atgtgaattg caggacacat 720 tgatcatcga cacttcgaac gcaccttgcg gccccgggtt cctcccgggg ctacgcctgt 780 ctgagggtcg ctttgacatc aatcgggaaa taactttttc ccgcggctgg ggctgtcgca 840 ggcacctgtt tgccttcgtc cccctaagtg cagactgtcg ggatgcccgg tgcggtgcgg 900 atcgggccag caggctcgcc tccgttctcc ccgtcggcct aacatccccc ccccc atctc 960 tcaccctcct ttttccccgg cctccgtgcc ggggaagggg gcgcctccgt cgggcccgca 1020 tgagtcgggc gcggctgccg gtggacttct ggtctccgcg ctgcccgcgc tacgcgtgcg 1080 taagtccggc gggggtgtcg gcgcgtcaag cgccgggggt atcacggggg gtggggggga 1140 aggccgcggg gtttgcgggg ctgtcgagct ctgctgccgg tccgcttccc cgcgccgggg 1200 tacctggcta cgacccaccg agtctccagg tgagcctccg ctcaagggct ttcgggcccc 1260 ggagcggagc caccgaccgg gggccgcctg gcggccccca tcccattcga ctacgacctc 1320 agatcagacg agacaacccg 1340 <210> 8 <211> 1355 <212> DNA <213> Scomber australasicus <400> 8 gtgaacctgc ggaaggatca ttaccgggtc gcgttccgcc tctcctcctc gccggggttg 60 aagcgagcga ctgttgcctc cggaacagcc gaggtccccc cgccccagtt ctcttctcgg 120 ggaacggggg cgaggcccga ggcgcgcacg cggaccctcc tcgtgggggt ccgcgggcac 180 acaaaccctc ctcttggtct ggacctcgtc ccgaccggac gggcctctct tcccccccct 240 ttcccccggc aacgggcatc acggtgcggc agggccggcc gtcctggtgc ccatcgctcg 300 ggggtcgcac gggggggggt tgggaacctt ctacccgtcc ggtgcgccgg gacaggcccg 360 ccactcggaa ccaaaacctc agcgcgttgc ggcggctcgg cccctggccg ccc tgcgcgc 420 gcccccgggt acccaactct cctccctcct ccggagggaa catagagggg ttcaatgtct 480 cctgccccgc cgacaaaggc cgtttccccc aacccgttgg gggttgccgc ccgtcggcgg 540 gacggagcgc ccgggggtcc tgtcgtcccc cttctcaaac ccttttttta acgttcgagc 600 tctggcaaaa gtgaaaacac aaaaacaaac tgtgacaact cttagcggtg gatcactcgg 660 ctcgtgcgtc gatgaagaac gcagctagct gcgagaacta atgtgaattg caggacacat 720 tgatcatcga cacttcgaac gcaccttgcg gccccgggtt cctcccgggg ctacgcctgt 780 ctgagggtcg ctttgacatc aatcgggaaa taactttttt cccgcggctg gggctgtcgc 840 aggcacctgt ttgccttcgt ccccctaagt gcagactgtc gggatgcccg gtgcggtgcg 900 gatcgggcca gcaggctcgc ctccgttctc cccgtcggcc taacatcccc cccccatctc 960 tcaccctcct ttttcccctt ccggggaagg gggcgcctcc gtcgggcccg catgagtcgg 1020 gcgcggctgc cggtggactt ctggtctccg cgctgcccgc gctacgcgtg cgtaagtccg 1080 gcgggggtgt cggcgcgtca agcgccgggg gtatcacggg gggtgggggg gaaggccgcg 1140 gggtttgcgg ggctgtcgag ctctgctgcc ggtccgcttc cccgcgccgg ggtacctggc 1200 tacgacccac cgagtctcca ggtgagcctc cgctcaaggg ctttcgggcc ccggagcgga 1260 gcc accgacc gggggccgcc tggcggcccc catcccattc gactacgacc tcagatcaga 1320 cgagacaacc cg 1332 <210> 9 <211> 1355 <212> DNA <213> Scomber australasicus <400> 9 gtgaacctgc ggaaggatca ttaccgggtc gcgttccgcc tctcctcctc gccggggttg 60 aagcgagcga ctgttgcctc cggaacagcc gaggtccccc cgccccagtt ctcttccggg 120 gaacgggggc gaggcccgag gcgcgcacgc ggaccctcct cgtgggggtc cgcgggcaca 180 caaaccctcc tcttggtctg gacctcgtcc cgaccggacg ggcctctctt ccccccccct 240 ttcccccggc aacgggcatc acggtgcggc agggccggcc gtcctggtgc ccatcgctcg 300 ggggtcgcac gggggggggt tgggaacctt ctacccgtcc ggtgcgccgg gacaggcccg 360 ccactcggaa ccaaaacctc agcgcgttgc ggcggctcgg cccctggccg ccctgcgcgc 420 gcccccgggt acccaactct cctccctcct ccggagggaa catagagggg ttcaatgtct 480 cctgccccgc cgacaaaggc cgtttccccc aacccgttgg gggttgccgc ccgtcggcgg 540 gacggagcgc ccgggggtcc tgtcgtcccc cttctcaaac ccttttttta acgttggagc 600 tctggcaaaa gtgaaaaaca caaaaacaaa ctgtgacaac tcttagcggt ggatcactcg 660 gctcgtgcgt cgatgaagaa cgcagctagcc tgcgagaact aatgtgaatt gcaggacaca 720 ttgatcatcg aca cttcgaa cgcaccttgc ggccccgggt tcctcccggg gctacgcctg 780 tctgagggtc gctttgacat caatcgggaa ataacttttt cccgcggctg gggctgtcgc 840 aggcacctgt ttgccttcgt ccccctaagt gcagactgtc gggatgcccg gtgcggtgcg 900 gatcgggcca gcaggctcgc ctccgttctc cccgtcggcc taacatcccc cccccatctc 960 tcaccctcct ttttccccgg cctccgtgcc ggggaagggg gcgcctccgt cgggcccgca 1020 tgagtcgggc gcggctgccg gtggacttct ggtctccgcg ctgcccgcgc tacgcgtgcg 1080 taagtccggc gggggtgtcg gcgcgtcaag cgccgggggt atcacggggg gtggggggga 1140 aggccgcggg gtttgcgggg ctgtcgagct ctgctgccgg tccgcttccc cgcgccgggg 1200 tacctggcta cgacccaccg agtctccagg tgagcctccg ctcaagggct ttcgggcccc 1260 gagcggagcc accgaccggg ggccgcctgg cggcccccat cccattcgac tacgacctca 1320 gatcagacga gacaacccg 1340 <210> 10 <211> 1355 <212> DNA <213> Scomber australasicus <400> 10 gtgaacctgc ggaaggatca ttaccgggtc gcgttccgcc tctcctcctc gccggggttg 60 aagcgagcga ctgttgcctc cggaacagcc gaggtccccc cgccccagtt ctcttccggg 120 gaacgggggc gaggcccgag gcgcgcacgc ggaccctcct cgtgggggtc cgcgggcaca 180 caaaccctcc tcttggtctg gacctcgtcc cgaccggacg ggcctctctt ccccccccct 240 ttcccccggc aacgggcatc acggtgcggc agggccggcc gtcctggtgc ccatcgctcg 300 ggggtcgcac gggggggggt tgggaacctt ctacccgtcc ggtgcgccgg gacaggcccg 360 ccactcggaa ccaaaacctc agcgcgttgc ggcggctcgg cccctggccg ccctgcgcgc 420 gcccccgggt acccaactct cctccctcct ccggagggaa catagagggg ttcaatgtct 480 cctgccccgc cgacaaaggc cgtttccccc aacccgttgg gggttgccgc ccgtcggcgg 540 gacggagcgc ccgggggtcc tgtcgtcccc cttctcaaac ccttttttta acgttggagc 600 tctggcaaaa gtgaaaaaca caaaaacaaa ctgtgacaac tcttagcggt ggatcactcg 660 gctcgtgcgt cgatgaagaa cgcagctagc tgcgagaact aatgtgaatt gcaggacaca 720 ttgatcatcg acacttcgaa cgcaccttgc ggccccgggt tcctcccggg gctacgcctg 780 tctgagggtc gctttgacat caatcgggaa ataacttttt tcccgcggct ggggctgtcg 840 caggcacctg tttgccttcg tccccctaag tgcagactgt cgggatgccc ggtgcggtgc 900 ggatcgggcc agcaggctcg cctccgttct ccccgtcggc ctaacatccc cccccccatc 960 tctcaccctc ctttttcccc ttccggggaa gggggcgcct ccgtcgggcc cgcatgagtc 1020 gggcgcggct gccggtggac ttctggtc tc cgcgctgccc gcgctacgcg tgcgtaagtc 1080 cggcgggggt gtcggcgcgt caagcgccgg gggtatcacg gggggtgggg gggaaggccg 1140 cggggtttgc ggggctgtcg agctctgctg ccggtccgct tccccgcgcc ggggtacctg 1200 gctacgaccc accgagtctc caggtgagcc tccgctcaag ggctttcggg ccccggagcg 1260 gagccaccga ccgggggccg cctggcggcc cccatcccat tcgactacga cctcagatca 1320 gacgagacaa cccg 1334 <210> 11 <211> 1355 <212> DNA <213 > Scomber australasicus <400> 11 ctcgtgcgtc gatgaagaac gcagctagct gcgagaacta atgtgaattg caggacacat 60 tgatcatcga cacttcgaac gcaccttgcg gccccgggag gaacccgggg ctacgcctgt 120 ctgagggtcg ctttgacatc aatcgggaaa taactttttt cccgcggctg gggctgtcgc 180 aggcacctgt ttgccttcgt ccccctaagt gcagactgtc gggatgcccg gtgcggtgcg 240 gatcgggcca gcaggctcgc ctccgttctc cccgtcggcc taacatcccc cccccatctc 300 tcaccctcct ttttcccctt ccggggaagg gggcgcctcc gtcgggcccg catgagtcgg 360 gcgcggctgc cggtggactt ctggtctccg cgctgcccgc gctacgcgtg cgtaagtccg 420 gcgggggtgt cggcgcgtca agcgccgggg gtatcacggg gggtgggggg gaaggccgcg 480 gggtttgcgg ggctgtcgag ctctgctg ggtccgcttc cccgcgccgg ggtacctggc 540 tacgacccac cgagtctcca ggtgagcctc cgctcaaggg ctttcgggcc ccggagcgga 600 gccaccgacc gggggccgcc tggcggcccc catcccattc gactacgacc tcagatcaga 660 cgagacaacc cg 672 <210> 12 <211> 1355 <212> DNA <213> Scomber australasicus <400> 12 gtgaacctgc ggaaggatca ttaccgggtc gcgttccgcc tctcctcctc gccggggttg 60 aagcgagcga ctgttgcctc cggaacagcc gaggtccccc cgccccagtt ctcttcgggg 120 aacgggggcg aggcccgagg cgcgcacgcg gaccctcctc gtgggggtcc gcgggcacac 180 aaaccctcct cttggtctgg acctcgtccc gaccggacgg gcctctcttc cccccccttt 240 cccccggcaa cgggcatcac ggtgcggcag ggccggccgt cctggtgccc atcgctcggg 300 ggtcgcacgg gggggggttg ggaaccttct acccgtccgg tgcgccggga caggcccgcc 360 actcggaacc aaaacctcag cgcgttgcgg cggctcggcc cctggccgcc ctgcgcgcgc 420 ccccgggtac ccaactctcc tccctcctcc ggagggaaca tagaggggtt caatgtctcc 480 tgccccgccg acaaaggccg tttcccccaa cccgttgggg gttgccgccc gtcggcggga 540 cggagcgccc gggggtcctg tcgtccccct tctcaaaccc tttttttaac gttcgagctc 600 tggcaaaagt gaaaaacaca aaaacaaact gtgacaactc ttagcggtgg atcactcggc 660 tcgtgcgtcg atgaagaacg cagctagctg cgagaactaa tgtgaattgc aggacacatt 720 gatcatcgac acttcgaacg caccttgcgg ccccgggttc ctcccggggc tacgcctgtc 780 tgagggtcgc tttgacatca atcgggaaat aacttttttc ccgcggctgg ggctgtcgca 840 ggcacctgtt tgccttcgtc cccctaagtg cagactgtcg ggatgcccgg tgcggtgcgg 900 atcgggccag caggctcgcc tccgttctcc ccgtcggcct aacatccccc cccccatctc 960 tcaccctcct ttttccccgg cctccgtgcc ggggaagggg gcgcctccgt cgggcccgca 1020 tgagtcgggc gcggctgccg gtggacttct ggtctccgcg ctgcccgcgc tacgcgtgcg 1080 taagtccggc gggggtgtcg gcgcgtcaag cgccgggggt atcacggggg gtggggggga 1140 aggccgcggg gtttgcgggg ctgtcgagct ctgctgccgg tccgcttccc cgcgccgggg 1200 tacctggcta cgacccaccg agtctccagg tgagcctccg ctcaagggct ttcgggcccc 1260 ggagcggagc caccgaccgg gggccgcctg gcggccccca tcccattcga ctacgacctc 1320 agatcagacg agacaacccg 1340 <210> 13 <211> 1355 <212> DNA <213> Scomber australasicus <400> 13 gtgaacctgc ggaaggatca ttaccgggtc gcgttccgcc tctcctcctc gccggggttg 60 aagcgagcga ctgttgcctc cggaacagcc gaggtcc ccc cgccccagtt ctcttcgggg 120 aacgggggcg aggcccgagg cgcgcacgcg gaccctcctc gtgggggtcc gcgggcacac 180 aaaccctcct cttggtctgg acctcgtccc gaccggacgg gcctctcttc cccccccttt 240 cccccggcaa cgggcatcac ggtgcggcag ggccggccgt cctggtgccc atcgctcggg 300 ggtcgcacgg gggggggttg ggaaccttct acccgtccgg tgcgccggga caggcccgcc 360 actcggaacc aaaacctcag cgcgttgcgg cggctcggcc cctggccgcc ctgcgcgcgc 420 ccccgggtac ccaactctcc tccctcctcc ggagggaaca tagaggggtt caatgtctcc 480 tgccccgccg acaaaggccg tttcccccaa cccgttgggg gttgccgccc gtcggcggga 540 cggagcgccc gggggtcctg tcgtccccct tctcaaaccc tttttttaac gttcgagctc 600 tggcaaaagt gaaaaacaca aaaacaaact gtgacaactc ttagcggtgg atcactcggc 660 tcgtgcgtcg atgaagaacg cagctagctg cgagaactaa tgtgaattgc aggacacatt 720 gatcatcgac acttcgaacg caccttgcgg ccccgggttc ctcccggggc tacgcctgtc 780 tgagggtcgc tttgacatca atcgggaaat aacttttttc ccgcggctgg ggctgtcgca 840 ggcacctgtt tgccttcgtc cccctaagtg cagactgtcg ggatgcccgg tgcggtgcgg 900 atcgggccag caggctcgcc tccgttctcc ccgtcggcct aacatccccc cccca tctct 960 caccctcctt tttccccttc cggggaaggg ggcgcctccg tcgggcccgc atgagtcggg 1020 cgcggctgcc ggtggacttc tggtctccgc gctgcccgcg ctacgcgtgc gtaagtccgg 1080 cgggggtgtc ggcgcgtcaa gcgccggggg tatcacgggg ggtggggggg aaggccgcgg 1140 ggtttgcggg gctgtcgagc tctgctgccg gtccgcttcc ccgcgccggg gtacctggct 1200 acgacccacc gagtctccag gtgagcctcc gctcaagggc tttcgggccc cggagcggag 1260 ccaccgaccg ggggccgcct ggcggccccc atcccattcg actacgacct cagatcagac 1320 gagacaaccc g 1331 <210> 14 <211> 1355 <212> DNA <213> Scomber australasicus <400> 14 tgaacctgcg gaaggatcat taccgggtcg cgttccgcct ctcctcctcg ccggggttga 60 agcgagcgac tgttgcctcc ggaacagccg aggtcccccc gccccagttc tcttctcggg 120 gaacgggtgc gaggcccgag gcgcgcacgc ggaccctcct cgtgggggtc cgcgggcaca 180 caaaccctcc tcttggtctg gacctcgtcc cgaccggacg ggcctctctt ccccccccct 240 ttcccccggc aacgggcatc acggtgcggc agggccggcc gtcctggtgc ccatcgctcg 300 ggggtcgcac gggggggggt tgggaacctt ctacccgtcc ggtgcgccgg gacaggcccg 360 ccactcggaa ccaaaacctc agcgcgttgc ggcggctcgg cccctggccg ccctgcgcc 420 gcccccgggt acccaactct cctcctcctc cggagggaac atagaggggt tcaatgtctc 480 ctgccccgcc gacaaaggcc gtttccccca acccgttggg ggttgccgcc cgtcggcggg 540 acggagcgcc cgggggtcct gtcgtccccc ttctcaaacc ctttttttaa cgttggagct 600 ctggcaaaag tgaaaacaca aaaacaaact gtgacaactc ttagcggtgg atcactcggc 660 tcgtgcgtcg atgaagaacg cagctagctg cgagaactaa tgtgaattgc aggacacatt 720 gatcatcgac acttcgaacg caccttgcgg ccccgggttc ctcccggggc tacgcctgtc 780 tgagggtcgc tttgacatca atcgggaaat aacttttttc ccgcggctgg ggctgtcgca 840 ggcacctgtt tgccttcgtc cccctaagtg cagactgtcg ggatgcccgg tgcggtgcgg 900 atcgggccag caggctcgcc tccgttctcc ccgtcggcct aacatccccc ccccatctct 960 caccctcctt tttccccggc ctccgtgccg gggaaggggg cgcctccgtc gggcccgcat 1020 gagtcgggcg cggctgccgg tggacttctg gtctccgcgc tgcccgcgct acgcgtgcgt 1080 aagtccggcg ggggtgtcgg cgcgtcaagc gccgggggta tcacgggggg tgggggggaa 1140 ggccgcgggg tttgcggggc tgtcgagctc tgctgccggt ccgcttcccc gcgccggggt 1200 acctggctac gacccaccga gtctccaggt gagcctccgc tcaagggctt tcgggccccg 1260 gagcggagcc accgaccggg ggccgcctgg cggcccccat cccattcgac tacgacctca 1320 gatcagacga gacaacccg 1339 <210> 15 <211> 18 <212> DNA <213> artificial <220> <400> 15 ccgggtaccc aactctcc 18 <210> 16 <211> 22 <212> DNA <213> artificial <220> <400> 16 caccgctaag agttgtcaca gt 22

[Brief description of the drawings]

FIG. 1 schematically shows the steps of an embodiment of the present invention.

FIG. 2 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 3 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 4 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 5 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 6 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 7 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 8 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 9 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 10 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 11 is an alignment of SEQ ID NOS: 1 to 14.

FIG. 12 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 13 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 14 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 15 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 16 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 17 shows an alignment of SEQ ID NOS: 1 to 14.

FIG. 18 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 19 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 20 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

FIG. 21 is an alignment of SEQ ID NOS: 1 to 14.

FIG. 22 is an alignment of SEQ ID NOS: 1 to 14.

FIG. 23 is an alignment of SEQ ID NOS: 1 to 14.

FIG. 24 is an alignment of SEQ ID NO: 1 to SEQ ID NO: 14.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 AA11 CA01 CA09 HA12 4B063 QA01 QA18 QQ02 QQ42 QR08 QR42 QR55 QR62 QS25 QS34 QS36 QX02

Claims (5)

[Claims] 1. A method for discriminating the species of a Mackerel genus in Japan, comprising discriminating the species from an intervening sequence of rDNA of a test subject. 2. The rDNA of 5.8S rDNA and 18S
2. The method according to claim 1, wherein the species is determined from the length of the nucleotide sequence present in the intervening sequence between the rDNAs. 3. PCR is performed using a sequence that hybridizes to a site where no interspecies variation is observed as a primer.
3. The method of claim 1 or 2, wherein the species is identified by subjecting the product to electrophoresis. 4. Performing PCR using 5.8S rDNA of rDNA using a primer that matches downstream from the 5 ′ end and a primer that matches upstream from the 3 ′ end,
The method of claim 3, wherein a site in which only interspecies variation is not detected is searched. 5. The method for determining the species of the genus Mackerel according to any one of claims 1 to 4, wherein the method is for determining the species of a mackerel (Scomber japonicus) and a sesame mackerel (Scomber australasicus).