JP2011045276A - Method for discriminating species of bigeye tuna of genus thunnus by pcr and intraspecific phylogeny - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a method for discriminating an intraspecific phylogeny, which simultaneously detects the presence or absence of an amplification band specific to bigeye tuna type β and the presence or absence of an amplification band specific to bigeye tuna type α and uses a multiplex PCR having higher accuracy. <P>SOLUTION: DNA sequences in many regions of mitochondria genomes of bigeye tuna are newly determined and a single base substitution that has a sufficiently high frequency of appearance in bigeye tuna type α group and a negligible low frequency of appearance in a bigeye tuna type β group is found in an ND1 region. Further a concrete set of primers using the single base substitution are provided. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention belongs to the field of a method for discriminating tuna bigeye species and two species within the species. Further, the present invention belongs to the field of a method for discriminating two lines in the species based on the base sequence difference in the two lines of the mitochondrial DNA (mtDNA) of the subject. Furthermore, the present invention belongs to the field of a method for discriminating two systems in the species rapidly and with high accuracy by performing multiplex PCR using nucleotide sequence differences at a plurality of sites.

  In recent years, overfishing of fishery resources has become a problem due to various factors such as global population growth and changes in food demand trends. In order to achieve sustainable use of fishery resources, it is necessary to prevent overfishing and to manage fishery resources effectively. To that end, it is indispensable to accurately grasp fishing areas. On the other hand, in recent years, fraudulent production of food has become a social problem, but transshipment of fish at sea and false reporting of fishery areas have become a problem, and in fact, the bigeye caught in the Atlantic is fake as being from the Indian Ocean. An example of being landed in Japan has been reported. This will lead to the depletion of bigeye fishery resources in the future and may have a negative impact on the bigeye stock assessment. Therefore, the necessity of discriminating the species and the catching sea area of the bigeye fish landing product has been recognized, and the development of a simple, quick and accurate discrimination method for this purpose is awaited.

  The bigeye tuna (English name: bigeye tuna, scientific name: Thunnus obesus) is distributed in the temperate and tropical oceans of the world and has a life span of about 10-15 years. Egg laying is an anniversary / separating buoyant egg. For edible use, sashimi and sushi are generally used.

  There are two strains of bigeye (α and β types) genetically differentiated within the species (see Non-patent Document 1), which are PCR of the ATPase6-COIII gene region (ATCO region) of mitochondrial DNA (mtDNA) -Discrimination is possible by RFLP analysis (see Non-Patent Document 2 and Non-Patent Document 3). The frequency of appearance of these two lines is clearly different between sea areas (for example, in the Atlantic Ocean, α-type is present in a proportion of about 70 to 80% in the population, whereas in India and the Pacific, it is almost β-type and α The type is rarely seen (see Fig. 1), and it is possible to determine the origin using this distinct gene frequency difference. That is, the production area can be determined by extracting a certain number of individual samples from the bigeye fish landing product to be examined and determining the genotype ratio (α type: β type).

  As described above, the PCR-RFLP method has been conventionally used for the discrimination between the two systems, but since the method is complicated, it is not always satisfactory in terms of time, labor, and cost until results are obtained. I didn't want to go. Therefore, the present inventors previously examined the applicability of multiplex PCR (Multiplex PCR; MP-PCR) for the purpose of simplifying and speeding up the determination of the location of the bigeye (Non-Patent Document 4). However, because this method discriminated based on the presence or absence of an amplification band specific for β-type in two strains, a mutation (single base substitution due to genetic variation within the β-type population) occurred at the site where the PCR primer was set. It became clear that there was a misjudgment.

Genetic divergence between Atlantic and Indo-Pacific stocks of bigeye tuna (Thunnus obesus) and admixture around South Africa.Chow S, Okamoto H, Miyabe N, Hiramatsu K, Barut N. Mol Ecol. 2000 Feb; 9 (2): 221- 7. Mitochondrial DNA sequence variation within and between tuna Thunnus species and its application to species identification.H. Takeyama, S. Chow, H. Tsuzuki, T. Matunaga, Journal of Fish Biology Volume 58 Issue 6, Pages 1646-1657 Published Online: 19 Apr 2005 Fish species identification manual for Tuna species April 27, 2005 (partially revised on December 14, 2006) National Agriculture, Forestry and Fisheries Consumption Technology Center National Fisheries Research Center Attempts to speed up the identification of Japanese hornets using multiplex PCR Kenji Nohara (Mizukense Toyomizuken), Atsuko Senba (Mizukense Toyomizuken), Nobuaki Suzuki (Suizou Water Research Institute), Mizuken Se Central Water Research Institute), Okamoto Hiroaki (Mizuken Se Ensuisuiken) Abstracts of Annual Meeting of the Fisheries Science of Japan Volume: 2008 Page: 244 Special Issue: Spring

  In view of the above background, the present researchers have developed a more accurate discrimination method that can simultaneously detect the presence or absence of an amplification band specific for α-type as well as the presence or absence of an amplification band specific for the β-type of bigeye Tried. In order to achieve this goal, a single base substitution with a sufficiently high frequency of occurrence in the α-type population (very few genetic variations) and a low enough occurrence frequency in the β-type population to be negligible Must be found on the genome sequence.

  The present inventors newly determined DNA sequences for a large number of regions on the mitochondrial genome of the bigeye, the frequency of occurrence within the α-type population is sufficiently high, and the frequency of occurrence within the β-type population is negligible The lowest possible single base substitution was found in the ND1 region. The entire mitochondrial DNA of bluefin tuna (Pacific) (Thunnus orientalis) has been determined (GenBank: AB185022.1), and the newly determined bigeye α-type ND1 region is about 94% identical. It is.

  Bluefin Tuna (Pacific) (PNBT), Atlantic Bluefin Tuna (ANBT), Southern Bluefin Tuna (SBT), Bigeye α Type (BETa), Bigeye β Type (BETb), Yellowfin (YFT), Binaga (ALB), Cochinaga (LTT) ), The result of aligning the alignment of the ND1 region is shown in FIG. In the figure, based on the PNBT sequence, “.” Is used when the bases are the same, and A, T, G, C, R (= A or G), Y (= C or T when the bases are different. ), K (= G or T). The number in [] at the right end represents the relative position of the base in each sequence, and the number in () on the left side represents the number of sequences of each species used for alignment. As is clear from the figure, the two C (cytosine) surrounded by the gray empty square were T (thymine) in the α form of the bigeye.

  Therefore, at least one of the two base sequences located in the ND1 region of the mitochondrial DNA in the sample is examined, and if it is T (thymine), it is determined that the sample is derived from the bigeye α type I can do it. In addition, when it is already known that the specimen is a bigeye-derived specimen, at least one of the two base sequences located in the ND1 region of the mitochondrial DNA in the specimen is examined, and this is T (thymine) If it is, it can be determined that the sample is derived from the bigeye α type, and if it is C (cytosine), it can be determined that the sample is derived from the bigeye β type.

  As a method for examining whether or not at least one of the two base sequences located in the ND1 region of the mitochondrial DNA in the sample is T (thymine), in addition to the base sequencing method (sequencing), It is convenient to determine whether an oligonucleotide having a complementary sequence anneals to the site, or whether an extension reaction proceeds from the annealed oligonucleotide (primer). As the method, a PCR method, a 5 'nuclease assay, etc. are convenient.

  In addition to determining whether it is a bigeye α type by the method of the present invention, for example, a method for judging whether it is a tuna genus, a method for judging whether it is a bigeye, whether it is a bigeye β type A method for determining whether or not can be used in combination. As a method of using these methods in combination, multiplex PCR, multiplex 5 'nuclease assay and the like are excellent in terms of rapidity, simplicity and accuracy.

It is a figure which shows the geographical distribution of a bigeye type 2 genotype (alpha type and beta type). Bluefin Tuna (Pacific) (PNBT), Atlantic Bluefin Tuna (ANBT), Southern Bluefin Tuna (SBT), Bigeye α Type (BETa), Bigeye β Type (BETb), Yellowfin (YFT), Binaga (ALB), Cochinaga (LTT) ), An alignment of the sequences of the ND1 region. It is a figure which shows the amplification pattern at the time of performing multiplex PCR using the α-type and β-type specimen of the bigeye.

[Definition]
Bigeye Specimen: In this specification, the term “beepy specimen” refers not only to a specimen from a bigeye, but also a case where it is presumed to be a specimen from a bigeye or a specimen that is not considered to be a bigeye Shall be included. However, if a bigeye specimen is collected from a bigeye individual under the control of a reliable subject, there is no doubt that it is actually a bigeye-derived specimen.

[Experimental materials and methods]
<Bigeye specimen>
For the bigeye specimen, a muscle piece fixed in frozen storage or 70% ethanol was used.

<Primer design>
The primer was purchased from Bex Co., Ltd., an oligonucleotide synthesis company, and desalted. The Tm values of the primers are L8562 (52.3 ° C.), H9432 (50.3 ° C.), L8772 (45.5 ° C.), L8859 (53.7 ° C.), ND1L (45.5 ° C.), and ND1H (49.6 ° C.), respectively.
Table 1 shows the primer sequences used in the present invention.

  The primer set I (L8562 and H9432) is for determining whether or not it is a genus Tuna (genus Thunnus). Primer set II (L8772 and H9432) is for judging whether it is a tuna hornet. Primer set III (L8859 and H9432) is for determining whether it is the β type of bigeye. Primer set IV (ND1L and ND1H) is for determining whether or not it is α type of bigeye. Primer sets I, II, and III are primers designed based on the known sequence of the ATCO region, and fragments of 915, 706, and 615 base pairs (bp), respectively (amplicons I, II, and (Sometimes called III). Primer set IV is a primer designed based on the newly sequenced ND1 region sequence and amplifies a 330 base pair fragment (sometimes referred to as amplicon IV).

<Extraction of mitochondrial DNA>
A small amount of muscle fragment was cut out from the ethanol-fixed specimen and fresh sample, and crude total DNA was extracted using Gentra Puregene Tissue Kit (QIAGEN Cat. No. 158667), a commercially available DNA extraction kit, according to the attached manual. The recovered DNA was washed with 70% ethanol, dried, and dissolved in 100 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The dissolved DNA was stored refrigerated (4 ° C.) until it was subjected to PCR reaction.

<PCR reaction and electrophoresis conditions>
1) Reaction liquid composition
PCR reaction was performed using PCR Thermal Cycler Dice (TaKaRa) or PCR Thermal Cycler SP (TaKaRa). The primer used was a mixed primer of L8562: L8772: L8859: H9432: ND1L: ND1H = 15: 5: 1: 21: 2: 2 and Taq has no calibration activity. DNA polymerase (ABI) or TaKaRa Taq DNA polymerase (TaKaRa) was used. The total volume of PCR reaction solution is 10 μl, DW 4.95 μl, 10 × PCR buffer (contains 1.5 mM MgCl2) 1.0 μl, 2.5 mM each dNTP 1.0 μl, mix primer 2.0 μl, Taq DNA polymerase 0.25 U, DNA 1.0 μl Performed with composition.
2) PCR reaction cycle
The PCR cycle consists of heat denaturation at 94 ° C for 2 minutes, heat denaturation at 94 ° C for 30 seconds, annealing at 52 ° C for 30 seconds, and extension at 72 ° C for 1 minute 26 times, and then maintaining 72 ° C for 5 minutes. went.
3) Electrophoretic conditions
PCR products were run for 40 minutes at 100 V constant voltage in 0.5 × TBE buffer containing ethidium bromide using 2% agarose gel (GIBCO, BRL). As a molecular marker, a 100 bp ladder marker (manufactured by Bex) with 100 base pairs was used.

[Results and discussion]
FIG. 3 shows the amplification pattern when multiplex PCR is performed using α-type and β-type specimens of the bigeye. The three lanes indicated by A in FIG. 3 are the results of electrophoresis when multiplex PCR was performed using mitochondrial DNA derived from a bigeye α-type specimen as a template. As expected, bands of 915, 706, and 330 base pairs with primer sets I, II, and IV were observed. On the other hand, the three lanes indicated by B are the results for the bigeye β type. Again, as expected, bands of size 915, 706, and 615 base pairs with primer sets I, II, and III were observed. As a result of base sequence analysis of the amplified fragment, it was confirmed that it was an amplification product at that position (data not shown).

  Although it is clear from FIG. 3 that the amplification of the target band by multiplex PCR is performed with good reproducibility, the present inventors performed multiplex PCR on a total of more than 1,000 bigeye specimens, and discriminates by this method. The accuracy of was verified. The results are shown in Table 2.

  When multiplex PCR was performed on a total of 1109 bigeye samples, 427 samples showed A pattern (primer sets I, II, and IV amplify), 630 samples showed B pattern (primer sets I, II, and III were Amplify). For 52 samples, other amplification patterns were shown and could not be distinguished.

  All 427 specimens showing the A pattern were verified using the conventional PCR-RFLP method (see Non-Patent Document 2 and Non-Patent Document 3), and all 427 specimens were found to be Atlantic Atlantic wasp type (AT_a type). It was confirmed that. In addition, all 630 specimens showing B pattern were verified using PCR-RFLP method. As a result, 148 specimens were Atlantic bee β-type (AT_b type) and 482 specimens were from Indian / Pacific bee beta-type (Ind -Pac b type). Furthermore, the nucleotide sequence of the ND1 region was determined for all 52 samples showing other amplification patterns. One sample was Atlantic wasp α-type, 11 samples were Atlantic bee β-type, and 40 samples were Indian / Pacific wasps. It was found to be β type. Samples that showed an indistinguishable amplification pattern had a single base substitution (genetic variation within the population) at any primer binding site. That is, when the bigeye specimen showing A pattern or B pattern is determined to be α-type or β-type, respectively, the misjudgment rate is 0%, and the omission of discrimination is 0.23% for α-type (= 1/428) The β type was 7.17% (= (11 + 40) / (159 + 522)).

  When the same 1109 bigeye specimens were judged only by the presence or absence of the band of primer set IV, the specimens judged to be α-type or β-type were 436 specimens or 673 specimens, respectively (other band pattern (Breakdown data not shown). The misjudgment rate was 2.06% or 0.15% for α-type or β-type, respectively, and the omission of discrimination was 0.23% for α-type and 1.32% for β-type.

  From the above, in both cases of multiplex PCR using primer sets I, II, III, and IV, and PCR using only primer set IV, the α and β forms of bees are highly reliable. It became clear that can be discriminated.

  According to the present invention, it is possible to provide a quick, simple, accurate, and inexpensive method for discriminating α-type and β-type of bigeye, and primers and primer sets used for carrying out the method Alternatively, a kit can be provided. Further, it is possible to provide a method for determining the production location of the bigeye sample population by calculating the ratio (gene frequency) of α type and β type in the bigeye sample population from the results obtained by implementing the method. it can. Furthermore, a primer, a primer set, or a kit for performing the production area discrimination method can be provided.

PNBT Bluefin Tuna (Pacific) (Thunnus orientalis)
ANBT Atlantic bluefin tuna (Thunnus thynnus)
SBT southern bluefin tuna (Thunnus maccoyii)
BETa Bigeye α type (Thunnus obesus)
BETb Bigeye β type (Thunnus obesus)
YFT yellowfin (Thunnus albacares)
ALB albacore (Thunnus alalunga)
LTT Cochinaga (Thunnus tonggol)
ND1L ND1L primer
ND1H ND1H primer
L8562 L8562 Primer
L8772 L8772 primer
L8859 L8859 primer
H9432 H9432 Primer

Claims (7)

Mitochondrial DNA from bigeye specimens, and
An oligonucleotide having the base sequence set forth in SEQ ID NO: 9 (L8562 primer),
An oligonucleotide having the base sequence set forth in SEQ ID NO: 10 (H9432 primer),
An oligonucleotide having the base sequence set forth in SEQ ID NO: 11 (L8772 primer),
An oligonucleotide having the base sequence set forth in SEQ ID NO: 12 (L8859 primer),
An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 13 (ND1L primer), and
An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 14 (ND1H primer),
Co-exist to perform PCR,
Amplified fragment (amplicon I) with L8562 primer and H9432 primer,
Amplified fragment (amplicon II) with L8772 primer and H9432 primer,
Amplified fragment (amplicon III) with L8859 primer and H9432 primer, and
Amplified fragment (amplicon IV) with ND1L primer and ND1H primer,
By examining the presence or absence of
A method for discriminating whether the bigeye sample is derived from an α-type or β-type individual. Collect multiple bigeye specimens from a population of bigeye,
Mitochondrial DNA from each bigeye specimen, and
An oligonucleotide having the base sequence set forth in SEQ ID NO: 9 (L8562 primer),
An oligonucleotide having the base sequence set forth in SEQ ID NO: 10 (H9432 primer),
An oligonucleotide having the base sequence set forth in SEQ ID NO: 11 (L8772 primer),
An oligonucleotide having the base sequence set forth in SEQ ID NO: 12 (L8859 primer),
An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 13 (ND1L primer), and
An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 14 (ND1H primer),
Co-exist to perform PCR,
Amplified fragment (amplicon I) with L8562 primer and H9432 primer,
Amplified fragment (amplicon II) with L8772 primer and H9432 primer,
Amplified fragment (amplicon III) with L8859 primer and H9432 primer, and
Amplified fragment (amplicon IV) with ND1L primer and ND1H primer,
By examining the presence or absence of
Determine whether each bigeye specimen is derived from α-type or β-type individual,
Find the ratio of α-type individuals and β-type individuals in the population of the bigeye,
By comparing with the known ratios by production area,
A method of determining the production area of the bigeye group. An oligonucleotide having the base sequence set forth in SEQ ID NO: 9 (L8562 primer),
An oligonucleotide having the base sequence set forth in SEQ ID NO: 10 (H9432 primer),
An oligonucleotide having the base sequence set forth in SEQ ID NO: 11 (L8772 primer),
An oligonucleotide having the base sequence set forth in SEQ ID NO: 12 (L8859 primer),
An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 13 (ND1L primer), and
An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 14 (ND1H primer),
A set of primers for discriminating whether the bigeye specimen is derived from an individual of α type or β type. An oligonucleotide having the base sequence set forth in SEQ ID NO: 9 (L8562 primer),
An oligonucleotide having the base sequence set forth in SEQ ID NO: 10 (H9432 primer),
An oligonucleotide having the base sequence set forth in SEQ ID NO: 11 (L8772 primer),
An oligonucleotide having the base sequence set forth in SEQ ID NO: 12 (L8859 primer),
An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 13 (ND1L primer), and
An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 14 (ND1H primer),
A kit for discriminating the origin of a group of bigeye bees.   Regarding the ND1 region of mitochondrial DNA derived from a bigeye sample, when the base corresponding to position 441 in SEQ ID NO: 3 and SEQ ID NO: 4 is T (thymine), the bigeye sample is a sample derived from an α-type individual How to judge.   Regarding the ND1 region of mitochondrial DNA derived from a bigeye sample, when the base corresponding to position 436 of SEQ ID NO: 3 and SEQ ID NO: 4 is T (thymine), the bigeye sample is a sample derived from an α-type individual How to judge. Collect multiple bigeye specimens from a population of bigeye,
Whether the base corresponding to either positions 436 or 441 of SEQ ID NO: 3 and SEQ ID NO: 4 is T (thymine) or C (cytosine) for the ND1 region of mitochondrial DNA derived from each bigeye specimen By examining
Determine whether each bigeye specimen is derived from α-type or β-type individual,
Find the ratio of α-type individuals and β-type individuals in the population of the bigeye,
By comparing with the known ratios by production area,
A method of determining the production area of the bigeye group.