JP7788097B2 - Male-specific sequences for genetic sexing of bluefin tuna - Google Patents

Description

The present invention relates to a male-specific base sequence used for genetic sex determination of bluefin tuna.

As the world's largest tuna consumer, Japan has international responsibilities regarding the management and sustainable use of tuna resources. Planning resource management measures requires a variety of biological information obtained from catches and resource survey samples, and along with data such as body length, age, and species, sex data is one of the most important pieces of information.

Currently, sexing of tuna is performed using classical methods such as visual inspection of gonads or histological observation of sections. However, these methods have the following drawbacks: 1) they cannot be used to sex juveniles and young individuals whose gonads are not yet fully developed, or eviscerated individuals distributed in the market; 2) the testing is laborious and time-consuming; and 3) it is difficult to sex individuals alive. As a result, for example, sex data remains missing from the majority of bluefin tuna (Thunnus orientalis) resource survey samples, and the influence of sex on the temporal and spatial movements, distribution, and growth characteristics of bluefin tuna life cycles remains unknown. Furthermore, in recent years, tuna farming has been shifting from wild-caught yellowtail to artificially hatched fish. However, because it is difficult to sex captive-reared individuals alive, it is also impossible to determine whether the parent fish population being cultivated has a sex ratio suitable for spawning. For these reasons, there is a need for a simple, highly accurate method for sexing tuna.

The inventors have previously developed a method for genetically determining the sex of tuna species by detecting sex-specific DNA polymorphisms in the genome (Patent Documents 1 and 2).

Japanese Patent Application Laid-Open No. 2019-088234 Japanese Patent Application Laid-Open No. 2020-184917

Methods for genetically sexing tuna species by detecting sex-specific DNA polymorphisms in the genome require the construction of a specific system for detecting the DNA polymorphisms, which requires expensive equipment such as PCR or real-time PCR devices, as well as complicated procedures such as electrophoresis. The objective of the present invention is to provide sex-specific DNA and a sexing method that enables sexing of bluefin tuna even at sampling sites such as research vessels, fishing ports, and aquaculture farms, where such methods are difficult or impossible to implement.

The inventors conducted various studies to solve the above-mentioned problems, and as a result, for the first time discovered a male-specific DNA of approximately 230 kb in the bluefin tuna genome. They also discovered that by using this DNA, it is possible to quickly and easily determine the genetic sex of bluefin tuna with high accuracy, and to develop a highly versatile sex determination method, leading to the completion of the present invention.

That is, the present invention provides the following [1] to [10].
[1] A method for determining the sex of bluefin tuna, comprising detecting male-specific DNA in a sample collected from a bluefin tuna specimen,
The method, wherein the DNA is at least one type of DNA selected from the group consisting of the following (a) to (d):
(a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a partial nucleotide sequence thereof;
(b) DNA consisting of a base sequence having 90% or more identity with the base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity with a partial base sequence of the base sequence represented by SEQ ID NO: 1;
(c) DNA consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 1, or a base sequence in which one or several bases are deleted, substituted or added in a partial base sequence of the base sequence represented by SEQ ID NO: 1;
(d) DNA that hybridizes under stringent conditions with the base sequence represented by SEQ ID NO: 1 or a complementary sequence of a partial base sequence thereof.
[2] The method described in [1], further comprising determining the sex of the subject based on the results of the detection.
[3] The method according to [1] or [2], wherein the DNA is a DNA consisting of the base sequence represented by SEQ ID NO: 1 or a partial base sequence thereof.
[4] The method according to any one of [1] to [3], wherein the detection is carried out by the LAMP method.
[5] Male-specific DNA of bluefin tuna, which is at least one type of DNA selected from the group consisting of the following (a) to (d):
(a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a partial nucleotide sequence thereof;
(b) DNA consisting of a base sequence having 90% or more identity with the base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity with a partial base sequence of the base sequence represented by SEQ ID NO: 1;
(c) DNA consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 1, or a base sequence in which one or several bases are deleted, substituted or added in a partial base sequence of the base sequence represented by SEQ ID NO: 1;
(d) DNA that hybridizes under stringent conditions with the base sequence represented by SEQ ID NO: 1 or a complementary sequence of a partial base sequence thereof.
[6] A marker for determining the sex of bluefin tuna, comprising the DNA described in [5].
[7] Use of the DNA according to [5] for determining the sex of bluefin tuna.
[8] A primer for determining the sex of bluefin tuna, which can specifically amplify the DNA described in [5].
[9] A probe for determining the sex of bluefin tuna, which can specifically hybridize to the DNA according to [5].
[10] A kit for determining the sex of bluefin tuna, comprising the primer according to [8] and/or the probe according to [9].

The present invention provides male-specific DNA for bluefin tuna, a method for genetically sexing bluefin tuna that detects said DNA, and primers, probes, and kits that can be used in said method. By using said DNA as an indicator for sexing, it is possible to develop a highly versatile sexing method without compromising the accuracy of sexing, compared to conventional techniques that use sex-specific DNA polymorphisms as an indicator. In addition to sexing using PCR and other methods, it also becomes possible to develop sexing using the LAMP method, which has traditionally been difficult to develop because it requires high specificity for the target sequence. The LAMP method is highly specific, quick, and simple, and is suitable for implementation at sampling sites with limited experimental resources, such as research vessels, fishing ports, and aquaculture farms.

This figure shows the results of PCR performed to determine the sex of 14 Pacific bluefin tuna, both male and female, and agarose gel electrophoresis of the amplified products.

Abbreviations used in this specification for base sequences (nucleotide sequences), nucleic acids, etc. are written using symbols commonly used in the relevant field, such as the IUPAC-IUB standard (IUPAC-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138:9-37, 1984) and the "Guidelines for the Preparation of Specifications Including Base or Amino Acid Sequences" (edited by the Japan Patent Office). In this specification, "deoxyribonucleic acid (DNA)" encompasses not only double-stranded DNA, but also the single-stranded DNAs that make up the double-stranded DNA, known as the sense strand and antisense strand.

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 unless otherwise specified, when referring to a "nucleotide" (or "oligonucleotide," "polynucleotide") having a certain sequence, this also comprehensively refers to a "nucleotide" (or "oligonucleotide," "polynucleotide") having a complementary sequence.

In this specification, the term "gene" includes double-stranded DNA including genomic DNA, as well as single-stranded DNA (positive strand) including cDNA, single-stranded DNA (complementary strand) having a sequence complementary to the positive strand, and fragments of these, and refers to DNA in which some biological information is contained within the sequence information of the bases that make up the DNA. Furthermore, in this specification, "gene" refers to regulatory regions, coding regions, exons, and introns without distinction, unless otherwise specified.

In this specification, "bluefin tuna" refers to Pacific bluefin tuna (Thunnus orientalis). Bluefin tuna includes both wild-caught and farmed fish. Hereinafter, Pacific bluefin tuna may be referred to simply as bluefin tuna. Bluefin tuna have 24 chromosome sets, with a genome size of approximately 800 million base pairs.

As shown in the Examples below, the inventors have discovered that in the genomic DNA of bluefin tuna, DNA consisting of the 232,647-base sequence represented by SEQ ID NO: 1 is present only in males. They have also discovered that by detecting this male-specific DNA, the genetic sex of bluefin tuna can be determined quickly, easily, and with high accuracy. Therefore, this male-specific DNA is useful as an indicator (marker) for determining the genetic sex of bluefin tuna. Here, "genetic sex" refers to sex determined by genetics, and will hereinafter be referred to simply as "sex" or "gender."

The sex-distinguishing marker for bluefin tuna of the present invention (hereinafter referred to as the marker of the present invention) comprises at least one type of DNA selected from the group consisting of the following (a) to (d):
(a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a partial nucleotide sequence thereof;
(b) DNA consisting of a base sequence having 90% or more identity with the base sequence represented by SEQ ID NO: 1 or a base sequence having 90% or more identity with a partial base sequence of the base sequence represented by SEQ ID NO: 1;
(c) DNA consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 1, or a base sequence in which one or several bases are deleted, substituted or added in a partial base sequence of the base sequence represented by SEQ ID NO: 1;
(d) DNA that hybridizes under stringent conditions with the base sequence represented by SEQ ID NO: 1 or a complementary sequence of a partial base sequence thereof.
The DNAs (a) to (d) are male-specific DNAs. The DNAs (b) to (d) include the variant of (a) insofar as they function as sex-discriminating markers for bluefin tuna. Such variants include naturally occurring allelic variants and non-naturally occurring variants that can be generated using mutagenesis techniques well known in the art.

Here, "male-specific DNA" means that its presence is associated with the male gender, and when this DNA is detected in a sample derived from a subject, the subject can be determined to be male.

The partial base sequence of the base sequence represented by SEQ ID NO: 1 may be any base sequence consisting of consecutive bases in the base sequence represented by SEQ ID NO: 1. Of the base sequence represented by SEQ ID NO: 1, it is preferably a base sequence of 50 or more consecutive bases, more preferably a base sequence of 50 to 100,000 consecutive bases, even more preferably a base sequence of 50 to 50,000 consecutive bases, even more preferably a base sequence of 50 to 10,000 consecutive bases, even more preferably a base sequence of 50 to 5,000 consecutive bases, even more preferably a base sequence of 50 to 3,000 consecutive bases, even more preferably a base sequence of 50 to 1,500 consecutive bases, even more preferably a base sequence of 50 to 1,000 consecutive bases, even more preferably a base sequence of 50 to 500 consecutive bases, and even more preferably a base sequence of 100 to 500 consecutive bases. Examples of partial base sequences include the base sequences of positions 9390 to 9818, 16389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654, which are regions predicted to be genes in the base sequence represented by SEQ ID NO: 1, and partial base sequences thereof. Of these, the gene consisting of the base sequence from positions 32614 to 64628 of SEQ ID NO: 1 has a high base sequence identity with the salt1st6 gene encoding estrogen sulfotransferase in medaka (Oryzias latipes), and since expression of estrogen sulfotransferase has actually been confirmed in southern bluefin tuna, a species closely related to bluefin tuna, it is believed that this gene also functions in bluefin tuna. Generally, gene regions are more highly conserved than non-gene regions, and exon regions are more highly conserved than intron regions. Therefore, the partial base sequence of the base sequence represented by SEQ ID NO: 1 is preferably the base sequence of positions 32614 to 64628 of SEQ ID NO: 1, or a partial base sequence thereof, and more preferably the base sequences of positions 32614 to 32800, 34058 to 34186, 46409 to 46506, 46707 to 46833, 61282 to 61376, 61471 to 61651, or 64519 to 64628 of SEQ ID NO: 1, which are exon regions, or partial base sequences thereof.

A nucleotide sequence having 90% or greater identity to the nucleotide sequence represented by SEQ ID NO: 1 or a partial nucleotide sequence having 90% or greater identity to the nucleotide sequence represented by SEQ ID NO: 1 preferably has 93% or greater identity, more preferably 95% or greater, even more preferably 97% or greater, even more preferably 98% or greater, and even more preferably 99% or greater identity. The identity of nucleotide sequences can be determined using the BLAST algorithm by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 1993, 90:5873-5877). Based on this BLAST algorithm, programs known as BLASTN and BLASTX have been developed (J. Mol. Biol., 1990, 215, pp. 403-410). Alternatively, the homology analysis (Search homology) program in the genetic information processing software Genetyx may be used. The specific techniques for these analysis methods are publicly known (see www.ncbi.nlm.nih.gov).

Unless otherwise defined herein, "one or several" when used in reference to the deletion, substitution, or addition of bases in a base sequence may mean, for example, 1 to 120, preferably 1 to 90, more preferably 1 to 60, even more preferably 1 to 30, even more preferably 1 to 10, even more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1. Furthermore, in this specification, "addition" of a base includes the addition of one or several bases to one end and both ends of a sequence.

As used herein, stringent conditions refer to conditions under which highly identical base sequences hybridize with each other, while less identical base sequences do not. "Stringent conditions" can be varied appropriately depending on the desired level of identity. The more stringent the conditions, the more likely it is that only highly identical sequences will hybridize. Examples of stringent conditions include those described in "Molecular Cloning: A Laboratory Manual" (Second Edition, J. Sambrook et al., 1989). Examples include conditions in which a solution containing 6x SSC (1x SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5x Denhardt's, and 100 mg/mL herring sperm DNA is incubated at 65°C for 8 to 16 hours with the probe for hybridization. Washing conditions are preferably 1x SSC at 60°C or higher, and more preferably 1x SSC at 73°C or higher.

The marker of the present invention may be one type of DNA selected from the group consisting of (a) to (d), or a combination of two or more types of DNA. The marker of the present invention is preferably DNA (a), more preferably DNA consisting of the nucleotide sequence of positions 32614 to 64628 of SEQ ID NO: 1 or a partial nucleotide sequence thereof, and even more preferably DNA consisting of the nucleotide sequences of positions 32614 to 32800, 34058 to 34186, 46409 to 46506, 46707 to 46833, 61282 to 61376, 61471 to 61651, and 64519 to 64628 of SEQ ID NO: 1, or a partial nucleotide sequence thereof.

The method for determining the sex of bluefin tuna of the present invention (hereinafter referred to as the method of the present invention) involves detecting the above-mentioned marker of the present invention in a sample collected from a bluefin tuna specimen. Here, "detecting a marker" means clarifying the presence or absence of the marker in a sample. Because the marker of the present invention is male-specific, it is possible to determine whether a bluefin tuna specimen is male or female based on the detection results of the marker. Specifically, if the marker of the present invention is detected, the specimen can be determined to be male, and if it is not detected, the specimen can be determined to be female.

The bluefin tuna specimen used in the method of the present invention is an individual bluefin tuna whose sex is to be confirmed. It may be a wild-caught fish or a farmed fish, and there are no restrictions on its life stage. The sample collected from the specimen is not particularly limited as long as it contains genomic DNA, and examples include cells, tissues, fins, muscles, internal organs, and surface mucus. Genomic DNA can be extracted from the sample using a known method. Examples of methods for extracting genomic DNA include the phenol method, the CTAB method, and the alkaline SDS method. Genomic DNA may be purified using a known method, if necessary. Reagents and kits for genomic DNA extraction are commercially available, and these may also be used. The method of the present invention may further include collecting a sample from the bluefin tuna specimen. The method of the present invention may also further include extracting genomic DNA from the sample collected from the bluefin tuna specimen.

The markers of the present invention can be detected using nucleic acid detection methods commonly used in the field. Examples of such detection methods include PCR, real-time PCR (e.g., TaqMan® PCR), LAMP (Loop-mediated Isothermal Amplification), ICAN® (Isothermal and Chimeric Primer-Initiated Amplification of Nucleic Acids), RCA (Rolling Circle Amplification), LCR (Ligase Chain Reaction), SDA (Strand Displacement Amplification), HRM (High Resolution Melting), Southern blot hybridization, dot blot hybridization, slot blot hybridization, DNA chips, DNA microarrays, and direct sequencing, all of which are methods for detecting nucleic acids or their amplification products. Reagents and kits for nucleic acid detection are commercially available and may be used. Nucleic acid detection methods are not limited to these, and other known methods may also be used. These methods may be used alone, or two or more may be combined.

When detecting a marker of the present invention using PCR, genomic DNA derived from a sample is used as a template, and a primer pair designed to specifically amplify all or part of a region of the marker of the present invention is used to amplify that region by PCR, and the amplification product is then detected. If an amplification product of the desired size is detected, the subject can be identified as male; if an amplification product is not detected or the amplification product is not of the desired size, the subject can be identified as female.

Primers used in PCR can be appropriately designed depending on the target region, taking into account factors such as GC content, base bias, Tm value, and secondary structure. For example, primer design software such as Primer3 (https://primer3.ut.ee) can be used. To enhance PCR specificity, it is preferable to confirm that the designed primers and the region amplified by the primers are specific to the target region. Specificity can be confirmed, for example, by searching using the BLAST method described above.
The base length of the primer is usually 15 to 50 bases, preferably 15 to 35 bases. The primer may have one to several, preferably one to five, and more preferably one to three mismatches with the base sequence of the template DNA, as long as the primer can specifically amplify the target region.
The amplification size is preferably about 50 to 3000 bp, more preferably about 50 to 1500 bp, and even more preferably about 50 to 500 bp.

Specific examples of primers used in PCR include oligonucleotides consisting of a partial sequence of the marker of the present invention or their complementary strands. The "complementary strand" is not limited to a completely complementary sequence, as long as it specifically recognizes the marker of the present invention, and may be a sequence having a sequence identity of preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more.
More specific examples of primers include, but are not limited to, primers consisting of the nucleotide sequence represented by any one of SEQ ID NOs: 2 to 5. Primer pairs include, for example, a primer pair comprising a primer consisting of the nucleotide sequence represented by SEQ ID NO: 2 and a primer consisting of the nucleotide sequence represented by SEQ ID NO: 3, and a primer pair comprising a primer consisting of the nucleotide sequence represented by SEQ ID NO: 4 and a primer consisting of the nucleotide sequence represented by SEQ ID NO: 5.

The PCR reaction conditions are not particularly limited, and the optimum conditions may be determined depending on the primer pair used, the length of the amplified region, etc., but examples thereof include the following conditions.
1) Thermal denaturation of double-stranded DNA to single-stranded DNA: Heating is usually performed at about 94 to 98°C for about 10 seconds to 1 minute.
2) Annealing: Heating is usually performed at about 50 to 65°C for about 10 seconds to 1 minute.
3) DNA extension reaction: Usually, the reaction mixture is heated at about 68 to 72°C for about 10 seconds to 3 minutes.
Here, the annealing and the DNA elongation reaction can be carried out simultaneously without being separated.
The target region can be amplified to a detectable level by generally carrying out the above reactions 1) to 3) for about 25 to 40 cycles.
The above series of operations can be carried out using a thermal cycler generally used for PCR.

PCR amplification products can be detected using known means capable of specifically recognizing amplification products. For example, the markers of the present invention can be detected by electrophoresing the resulting amplification products and staining them with reagents such as ethidium bromide or SYBR® Green I, which intercalate into double-stranded DNA and emit fluorescence, and then detecting the fluorescence using a UV device, fluorescent plate reader, or the like. Alternatively, dNTPs incorporated during the amplification reaction can be labeled with radioisotopes, fluorescent substances, luminescent substances, or other labels, and then detected. Amplification products incorporating labeled dNTPs can be observed using any method known in the art for detecting the above-mentioned labels. For example, when a radioisotope is used as the label, radioactivity can be measured using, for example, a liquid scintillation counter or a gamma counter. When a fluorescent label is used as the label, the fluorescence can be detected using a UV device, fluorescent plate reader, or the like.

In addition, to demonstrate that the absence of PCR amplification products is not due to a PCR failure, a gene or base sequence that is present in both males and females may be simultaneously amplified as a control during the PCR amplification reaction. Examples of such genes include the mitochondrial COI gene and ND4 gene.

The markers of the present invention can also be detected by real-time PCR, which monitors and analyzes the amount of amplified product in real time. The PCR reaction conditions are as described above. Real-time PCR can be performed using a dedicated real-time PCR device that combines a thermal cycler and a spectrofluorometer.

When detecting a marker of the present invention using TaqMan PCR, genomic DNA derived from a sample is used as a template. A primer pair designed to target and amplify all or a portion of a region of the marker of the present invention is used, along with a probe designed to specifically hybridize within the amplified region, the probe being labeled with a fluorescent substance at the 5' end and a quencher (quenching substance) at the 3' end. The region is then amplified by PCR using Taq DNA polymerase, and the fluorescence emitted during amplification is then detected. If fluorescence is detected, the subject can be identified as male; if fluorescence is not detected, the subject can be identified as female.

The primers used in TaqMan PCR, the amplification size, and the PCR reaction conditions are the same as those in the PCR described above.
The probes used in TaqMan PCR may be designed appropriately depending on the target region. To enhance the specificity of TaqMan PCR, it is preferable to confirm that the designed probe is specific to the target region. Whether or not the probe is specific can be confirmed, for example, by a search using the above-mentioned BLAST.
The probe typically has a base length of 15 to 50 bases, preferably 15 to 35 bases. The primer may have one to several, preferably one to five, and more preferably one to three mismatches with the base sequence of the template DNA, as long as it can specifically hybridize with the target region. The probe is labeled at its 5' end with a fluorescent substance such as FITC or VIC, and at its 3' end with a quencher (quenching substance) such as TAMRA.

Specific examples of probes used in TaqMan PCR include oligonucleotides consisting of a partial sequence of a marker of the present invention, or those having the above-mentioned labels at both ends of their complementary strands. The "complementary strand" is not limited to a completely complementary sequence, as long as it specifically recognizes the marker of the present invention; it may be a sequence that has a sequence identity of preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more.

The fluorescence emitted by TaqMan PCR can be detected using known means such as a UV device or a fluorescent plate reader, and preferably can be detected in real time using a device dedicated to real-time PCR.

When detecting a marker of the present invention using the LAMP method, genomic DNA derived from a sample is used as a template, and all or part of a region of the marker of the present invention is amplified using a primer set designed to specifically amplify this region as a target, and the amplified product is then detected. If an amplified product is detected, the subject can be identified as male, and if an amplified product is not detected, the subject can be identified as female.

The LAMP method uses a primer set consisting of four or six primers to amplify a target sequence at a constant temperature using a strand displacement reaction mediated by a strand-displacing DNA polymerase (International Publication No. WO 2000/028082). In the LAMP method, three regions, F3c, F2c, and F1c, are defined from the 3' end of one strand of the target DNA region, and three regions, B3, B3, and B1, are defined from the 5' end. The regions complementary to these regions are designated F3, F2, F1, B3c, B2c, and B1c, respectively. Based on the base sequences of these regions, four types of primers are designed: FIP, F3 primer, BIP, and B3 primer. FIP consists of a base sequence containing the base sequence of the F1c region on the 5' end and the base sequence of the F2 region on the 3' end. The F3 primer consists of the base sequence of the F3 region. BIP consists of a base sequence containing the base sequence of the B1c region on the 5'-end and the base sequence of the B2 region on the 3'-end. The B3 primer consists of the base sequence of the B3 region. In the LAMP method, six types of primers may be used, including the four types of primers described above and two types of primers, loop primer F and loop primer B. Loop primer F consists of a base sequence complementary to the region between the F1 region and the F2 region, and loop primer B consists of a base sequence complementary to the region between the B1 region and the B2 region. The use of loop primers can increase the number of starting points for nucleic acid amplification and promote the amplification reaction. These primers may be designed appropriately taking into account the Tm value, terminal stability, GC content, secondary structure, distance between primers, etc. The distance between primers is preferably about 100 to 500 bases from the outside of the F2 region to the outside of the B2 region (the amplified region of the LAMP method), more preferably about 120 to 160 bases; the distance from the 5' end of the F2 region to the 5' end of the F1 region (loop-forming region) is preferably 40 to 60 bases; and the distance between the F2 region and the F3 region is preferably 0 to 60 bases. Such primers can be designed using primer design support software such as PrimerExplorer (http://primerexplorer.jp).

Specific examples of primers used in the LAMP method include, but are not limited to, primers consisting of the nucleotide sequence represented by any one of SEQ ID NOs: 8 to 37. Primer sets include, for example, a primer set containing an F3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 8, a B3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 9, an FIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 10, and a BIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 11; a primer set containing an F3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 12, a B3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 13, an FIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 14, and a BIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 15; a primer set containing an F3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 16, a B3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 17, an FIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 18, and a BIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 19; a primer set containing an F3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 20, a B3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 21, an FIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 22, and a BIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 23; and an F3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 24. a primer set comprising an F3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 24, a B3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 25, an FIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 26, and a BIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 27; a primer set comprising an F3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 24, a B3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 25, an FIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 26, a BIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 27, a loop primer F consisting of the nucleotide sequence represented by SEQ ID NO: 36, and a loop primer B consisting of the nucleotide sequence represented by SEQ ID NO: 37; a primer set comprising an F3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 28, a B3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 29, an FIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 30, and a BIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 31; and a primer set comprising an F3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 32, a B3 primer consisting of the nucleotide sequence represented by SEQ ID NO: 33, an FIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 34, and a BIP primer consisting of the nucleotide sequence represented by SEQ ID NO: 35.

In the LAMP method, template DNA, a primer set, strand-displacing DNA polymerase, a substrate, etc. are mixed and kept at a constant temperature of approximately 60-65°C, allowing the nucleic acid amplification reaction to proceed. Because the amplification reaction proceeds isothermally, the reaction can be carried out using a device that can maintain a constant temperature. Examples of such devices include an incubator, thermal cycler, and real-time PCR device.

To detect amplification products using the LAMP method, known means capable of specifically recognizing amplification products can be used. For example, reagents such as ethidium bromide or SYBR Green I, which intercalate into double-stranded DNA and emit fluorescence, can be added to the reaction solution. After the amplification reaction, the reaction tube can be held over a UV device to detect the fluorescence, allowing easy visual confirmation of whether amplification has occurred. Alternatively, pyrophosphate ions released from dNTPs during the extension reaction combine with magnesium ions in the reaction solution to form magnesium pyrophosphate, causing the reaction solution to become cloudy. Therefore, the presence or absence of amplification can be easily confirmed by visually checking the turbidity of the reaction solution in the reaction tube or by using a simple measuring device.

When detecting a marker of the present invention using Southern blotting, for example, genomic DNA derived from a sample, digested with an appropriate restriction enzyme, is transferred onto a membrane according to standard methods, and then a probe labeled with a radioisotope, fluorescent substance, luminescent substance, enzyme, biotin, or the like is hybridized to the genomic DNA. The marker of the present invention can be detected by detecting a signal derived from the label from the formed double strand of the labeled probe and genomic DNA. Preferably, the probe is capable of specifically hybridizing to all or part of a marker of the present invention and detecting the marker. The probe's base length is preferably 100 to 5,000 bases, more preferably 500 to 1,000 bases. As long as the probe can specifically hybridize to a marker of the present invention, it may have one to several, preferably one to five, and even more preferably one to three mismatches with the base sequence of the template DNA.

Specific examples of probes that can be used include polynucleotides, oligonucleotides, or their complementary strands that comprise the entire or partial sequence of a marker of the present invention. The "complementary strand" is not limited to a completely complementary sequence, as long as it specifically recognizes the marker of the present invention, and may be a sequence that has preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity.

The above primers or probes can be DNA or RNA, and can be synthetic or natural. The primers or probes can be synthesized by conventional chemical synthesis methods such as the phosphoramidite method or the phosphate triester method, or can be synthesized using a commercially available automated oligonucleotide synthesizer. Alternatively, they can be prepared from bluefin tuna genomic DNA using standard methods.

In Patent Document 1, a sex-specific DNA polymorphism, a specific base at a specific position in a genomic DNA sequence common to both males and females, is used as a sex-determining marker, so it is necessary to detect this specific base, which places constraints on the construction of a sex-determining method. In contrast, the marker of the present invention is male-specific DNA and is approximately 230 kb in length, allowing for much greater freedom in the construction of a sex-determining method compared to Patent Document 1, making it highly versatile.

According to the method of the present invention, by detecting male-specific DNA, it is possible to quickly and easily determine the sex of bluefin tuna without compromising the accuracy of sex determination. While various techniques can be used to detect this DNA, the LAMP method can be performed using simple equipment. Furthermore, since it uses four or six primers to recognize six regions, amplification specificity is extremely high. Furthermore, the amplification efficiency is high, and the target region can be amplified to a detectable level in a reaction of approximately 15 minutes to one hour, making it an excellent method for rapidity. Due to its high amplification efficiency, the amount of amplified product is large, and the presence or absence of amplification can be easily confirmed using fluorescence or turbidity. Furthermore, the entire process can be completed within the reaction tube, reducing the possibility of contamination or mix-up of the reaction solution. Thus, the LAMP method is quick and easy to use, making it possible to determine the sex of bluefin tuna even in facilities where it was previously impossible or difficult to perform due to equipment limitations. Therefore, the LAMP method is preferable for detecting the male-specific DNA of the present invention.

The method of the present invention may be performed alone or in combination with other sex determination methods. Other sex determination methods include visual inspection of the gonads, observation of histological sections, and detection of sex-specific DNA polymorphisms.

The method of the present invention can be carried out more easily by using a reagent kit for detecting the marker of the present invention. The kit includes a sexing primer and/or a sexing probe. Examples of such kits include a kit containing a sexing primer pair for use in PCR, a kit containing a sexing primer pair and a sexing probe for use in TaqMan PCR, etc., a kit containing a sexing primer set for use in the LAMP method, and a kit containing a sexing probe for use in Southern blot hybridization.

The kit of the present invention may optionally contain a labeling agent, reagents necessary for PCR (e.g., DNA polymerase, dNTPs, etc.), reagents necessary for TaqMan PCR (e.g., Taq DNA polymerase, dNTPs, etc.), and reagents necessary for LAMP (e.g., strand-displacing DNA polymerase, dNTPs, etc.). Furthermore, the kit may contain appropriate reaction diluents, buffers, restriction enzymes, etc., for the convenience of performing the measurement.

The present invention will now be described in more detail using examples, but the present invention is not limited to these examples in any way.

Example 1: Identification of Sex-Specific Nucleotide Sequences in Pacific Bluefin Tuna. PacBio long-read sequencing was performed using one male Pacific bluefin tuna (the gonads had been determined and the sex had been determined using the method described in Patent Document 2). Genome assembly (contig creation using HGAP4, sequence correction using Pilon, and contig linking using LINKS) was performed using the obtained nucleotide sequence data, resulting in the acquisition of a male-specific DNA fragment (SEQ ID NO: 1, sequence name: M175, approximately 230 kb) that is not present in females. The female genome sequence has been registered in the DNA Data Bank of Japan (DDBJ) under Accession Nos. DDBJ: BKCK01000001 to BKCK01000444.

Using protein data (amino acid sequences) from a public database (Ensemble), gene prediction (Exonarate) for this region was performed, resulting in the prediction of 12 genes, as shown in Table 1 below.

In Table 1, the start and end positions each represent the base positions in SEQ ID NO: 1. Of the 12 genes listed above, all but g3 were highly identical to genes associated with transposable elements. Meanwhile, g3 was highly identical to the salt1st6 gene encoding estrogen sulfotransferase in medaka (Oryzias latipes), and was named the salt1st6y gene. Because M175 is male-specific, it is considered useful as an indicator for sex determination. However, given that gene regions generally have higher sequence conservation than non-gene regions, the salt1st6y gene region of M175 was used as an indicator in Examples 2 to 6 below.

Example 2 Gender discrimination analysis 1
Among the base sequences of M175, the base sequence of the sult1st6y gene was targeted, and multiple primer pairs were designed to amplify this by PCR, and a sex discrimination analysis of bluefin tuna specimens was performed. In PCR amplification using the sult1_LP2F primer (SEQ ID NO: 2) and the sult1_LP2R primer (SEQ ID NO: 3), if amplification of a 1352 bp DNA fragment was observed, the genetic sex of the specimen could be determined to be male; if amplification of this DNA fragment was not observed, the genetic sex of the specimen could be determined to be female. At this time, a non-gender-specific 486 bp DNA fragment was also amplified, allowing the success or failure of the PCR to be confirmed. Furthermore, in PCR amplification using the sult1Y_F1 primer (SEQ ID NO: 4) and the sult1Y_R1 primer (SEQ ID NO: 5), if amplification of a 77 bp DNA fragment was observed, the genetic sex of the specimen could be determined to be male; if amplification of this DNA fragment was not observed, the genetic sex of the specimen could be determined to be female. To prevent false negatives, which could occur if a 77-bp DNA fragment is not amplified from a male individual due to human error, resulting in the individual being mistakenly identified as a female, the ND4_F primer (SEQ ID NO: 6) and ND4_R primer (SEQ ID NO: 7) were also prepared for amplifying the ND4 gene of mitochondrial DNA (an internal positive control). PCR amplification using these primers amplified a 268-bp DNA fragment. The sequences of each primer are shown in Table 2. In Table 2, 5'pos indicates the base position of the 5'-end of the primer in the base sequence of SEQ ID NO: 1.

The specific test method was as follows: Muscles were collected using scissors or other tools from each of 12 individuals, consisting of six males and six females (sexed by gonads), consisting of four wild-caught female Pacific bluefin tuna (lanes 1 to 4 in Figure 1 ), two farmed female Pacific bluefin tuna (lanes 5 and 6 in Figure 1 ), three farmed male Pacific bluefin tuna (lanes 7, 11, and 12 in Figure 1 ), and three wild-caught male Pacific bluefin tuna (lanes 8 to 10 in Figure 1 ). The muscles were then treated with protease in a TNES/Urea 6M solution (10 mM Tris-HCl, 125 mM NaCl, 10 mM EDTA, 0.5% SDS, 6M Urea), and genomic DNA was prepared using an automated DNA extraction device (Maxwell RSC system, Maxwell RSC Blood DNA kit, manufactured by Promega Corporation). Using the genomic DNA (final concentration 0.5 ng/μL) as a template, a PCR reaction (35 cycles of 98°C for 10 seconds, 60°C for 30 seconds, and 68°C for 1 minute) was carried out using a PrimeSTAR GXL kit (manufactured by Takara Bio Inc.) and sult1_LP2F primer (final concentration 0.5 μM) and sult1_LP2R primer (final concentration 0.5 μM) with a Proflex PCR system (manufactured by Applied Biosystems). The resulting PCR amplified product was applied to a 2% agarose gel and electrophoresed at 100 V for 15 minutes.
Genomic DNA was also prepared from two farmed Pacific bluefin tuna (one female (lane 13 in Figure 1 ) and one male (lane 14 in Figure 1 ) (sexed by gonads) using the above method. PCR reactions (35 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 15 seconds) were performed using the genomic DNA (final concentration 0.5 ng/μL) as a template and a PrimeSTAR GXL kit (manufactured by Takara Bio Inc.) with sult1Y_F1 primer (final concentration 0.4 μM), sult1Y_R1 primer (final concentration 0.4 μM), and ND4_F primer (final concentration 0.05 μM), and ND4_R primer (final concentration 0.05 μM) using a Proflex PCR system (manufactured by Applied Biosystems). The resulting PCR amplification products were applied to a 2% agarose gel and electrophoresed at 100 V for 15 minutes.

The results are shown in Figure 1. Lanes 1 to 12 in Figure 1 show the results of PCR amplification using the sult1_LP2F and sult1_LP2R primers. The 1,352 bp DNA fragment was not observed in female individuals (lanes 1 to 6) but was specifically observed in male individuals (lanes 7 to 12). A non-gender-specific 486 bp DNA fragment was observed in all lanes, demonstrating that PCR was performed successfully. Lanes 13 and 14 in Figure 1 show the results of PCR amplification using the sult1Y_F1 and sult1Y_R1 primers. A 77 bp DNA fragment was not observed in female individuals (lane 13) but was specifically observed in male individuals (lane 14). A 268 bp amplification product derived from the ND4 gene was observed in all lanes, demonstrating that PCR was performed successfully. The sex of all tested individuals could be accurately determined, demonstrating that by using the newly discovered M175 as an indicator, it is possible to determine the genetic sex of bluefin tuna specimens with high accuracy.

Example 3 Gender discrimination analysis 2
The sex determination analysis of bluefin tuna was carried out using the LAMP method. In the analysis using the LAMP method, which targets male-specific DNA, if an amplification product is detected, the sex of the subject can be determined to be male, and if an amplification product is not detected, the sex of the subject can be determined to be female.
The specific test method is as follows. Genomic DNA was prepared from two farmed Pacific bluefin tuna, one male and one female (the gonads had been determined and the sex had been determined using the method described in Patent Document 2), using the same method as in Example 2. Using the genomic DNA (final concentration 2 ng/μL) as a template, reactions (63°C for 50 minutes, 95°C for 5 minutes) were carried out using a Proflex PCR system (Applied Biosystems) with Loopamp DNA Amplification Reagent D and a Fluorescence and Visual Detection Reagent (manufactured by Eiken Scientific Co., Ltd.) and one of the primer sets (i) to (vii) shown in Table 3 below (F3 and B3 primers at a final concentration of 0.2 μM, FIP and BIP primers at a final concentration of 1.6 μM) as a template. A reaction without DNA was also carried out as a negative control (NTC). The resulting amplified products were held in the reaction tubes over a blue LED transilluminator (LEDB-SBOXHP, Optocode Corporation) and the fluorescence was visually confirmed. In Table 3, 5'pos indicates the base position of the 5'-terminal base of the primer in the base sequence of SEQ ID NO: 1. Since FIP and BIP are primers synthesized from two sequences in opposite directions, no positional information is shown.

The results are shown in Table 4. In Table 4, a "+" indicates that green fluorescence was observed, and a "-" indicates that no fluorescence was observed. Regardless of which of the seven primer sets was used, fluorescence associated with the amplification of the target DNA fragment was observed in male individuals, but no fluorescence was observed in female individuals. Therefore, the LAMP method was able to accurately determine the sex of bluefin tuna.

Example 4 Gender discrimination analysis 3
Of the primer sets shown in Table 3, primer sets (iii) and (iv), which contain exon sequences that are more conserved than intron sequences in the primer region, were used to conduct sexing analysis using the LAMP method. Genomic DNA was prepared from 15 individuals, including six male and seven female wild Pacific bluefin tuna (sexed using the method of Patent Document 2) other than those used in Example 3, and one male and one female farmed Pacific bluefin tuna used in Example 3, using the same method as in Example 2. Using the genomic DNA (final concentration 1.5-3 ng/μL) as a template, sexing analysis of bluefin tuna specimens was conducted using the same method as in Example 3, except for using primer set (iii) or (iv). A reaction without DNA was also performed as a negative control (NTC).

The results are shown in Table 5. In Table 5, a "+" indicates that green fluorescence was observed, a "-" indicates that no fluorescence was observed, and individual numbers 14 and 15 represent the individuals used in Example 3. Regardless of which primer set was used, fluorescence associated with the amplification of the target DNA fragment was observed in all male individuals, while no fluorescence was observed in female individuals. Therefore, the LAMP method was able to accurately determine the sex of bluefin tuna. Furthermore, when primer set (iii) was used, the fluorescence intensity was higher than when primer set (iv) was used, and sex determination ability was higher.

Example 5 Gender discrimination analysis 4
Of the primer sets shown in Table 3, primer set (iii) with higher sex discrimination ability was used to conduct sex discrimination analysis using the LAMP method. Genomic DNA was prepared from 23 individuals, including 10 male and 11 female wild Pacific bluefin tuna (sexed using the gonads and the methods of Patent Documents 1 and 2) other than those used in Examples 3 and 4, and one male and one female farmed Pacific bluefin tuna used in Example 3, using the same method as in Example 2. Sex discrimination analysis of bluefin tuna specimens was conducted using the same method as in Example 3, except that the genomic DNA (final concentration 0.8 to 2 ng/μL) was used as a template and primer set (iii). A reaction without DNA was also performed as a negative control (NTC).

The results are shown in Table 6. In Table 6, a "+" indicates that green fluorescence was observed, a "-" indicates that no fluorescence was observed, and individual numbers 23 and 24 represent the individuals used in Example 3. Fluorescence associated with the amplification of the target DNA fragment was observed in all male individuals, while no fluorescence was observed in female individuals. Therefore, the sex of bluefin tuna could be accurately determined using the LAMP method.

Example 6 Gender discrimination analysis 5
Of the primer sets shown in Table 3, for primer set (v), loop primer F (5-LF: CATCCCAGAATTGGATTTGTGAGT, SEQ ID NO: 36) and loop primer B (5-LB: AAAAAGTGCCAAAACTACGGAGC, SEQ ID NO: 37) were further designed, and sexing analysis by the LAMP method was performed with and without the use of loop primers. Specifically, genomic DNA was prepared from six individuals, including two males and two females of wild Pacific bluefin tuna (sexed using the gonads and the method of Patent Document 2) other than those used in Examples 3 to 5, and one male and one female of the farmed Pacific bluefin tuna used in Example 3, using the same method as in Example 2. The genomic DNA (final concentration 0.2 to 2 ng/μL) was used as a template, and primer set (v) was used, with the exception that the reaction time was 50 minutes when no loop primers were used, and 20 pmol of each loop primer was added to the reaction system and the reaction time was 30 minutes, using the same method as in Example 3. A reaction without DNA was also performed as a negative control (NTC).

The results are shown in Table 7. In Table 7, a "+" indicates that green fluorescence was observed, and a "-" indicates that no fluorescence was observed. Fluorescence associated with the amplification of the target DNA fragment was observed in all male individuals, while no fluorescence was observed in female individuals. Therefore, the LAMP method was able to accurately determine the sex of bluefin tuna. Furthermore, sex determination was possible with a reaction time of 30 minutes.

The results of Examples 2 to 6 demonstrate that the sex of bluefin tuna can be determined with high accuracy using the LAMP method. Because the LAMP reaction proceeds isothermally, all that is required is a device that can maintain a constant temperature, and unlike PCR, no expensive equipment is required. The amplification reaction also takes a short time. Furthermore, after the amplification reaction, the presence or absence of amplification can be easily confirmed by simply holding the reaction tube directly over a UV device and checking for fluorescence. In this way, the LAMP method is quick and easy to use, and enables highly accurate sex determination even in situations with limited experimental resources, such as at aquaculture farms.

Example 7 Primers Below are examples of primer designs used in the method for sexing bluefin tuna of the present invention. Table 8 shows primer pairs 1 to 10 for PCR, and Table 9 shows primer sets 11 to 15 for the LAMP method. In the table, 5'pos indicates the base position of the 5'-end of the primer in the base sequence of SEQ ID NO: 1. As FIP and BIP are primers synthesized from two sequences in opposite directions, no position information is shown.

Reference Example 1: Confirmation of M175 Structure The entire M175 sequence is male-specific. On the other hand, when constructing a method for determining the sex of bluefin tuna using a partial base sequence of M175 as an indicator, if the partial base sequence contains a sequence highly identical to female genomic DNA, care must be taken when designing primers or probes. Therefore, assuming the length of the amplification product using the primers is 100 bp, BLAST was used to verify whether any base sequence consisting of 100 or more consecutive bases in M175 is a 100% match with the female genomic sequence. The results are shown in Table 10.

In Table 10, the start and end positions indicate the base positions in SEQ ID NO: 1 of the 5'-terminal and 3'-terminal bases of the base sequence that 100% matches the female genome sequence. As shown in Table 10, M175 has a partial base sequence that matches the female genome sequence. However, those skilled in the art can select DNA consisting of all or an appropriate partial base sequence of M175 as a marker, depending on the characteristics of the method for detecting the sex-determining marker, and carry out the method for sexing bluefin tuna of the present invention. For example, when PCR is used for sexing, it is sufficient to avoid designing a primer pair that targets any one region of the partial base sequence in Table 10. Even if one primer of the primer pair is designed to target any one region of the partial base sequence, sexing is possible if the other primer is designed outside that region. For example, when TaqMan PCR is used for sexing, it is sufficient to avoid designing a primer pair and probe that target any one region of the partial base sequence in Table 10. Sexing is possible if the probe is designed to target a region outside the partial base sequence in Table 10. For example, when using the LAMP method for sex determination, sex determination is possible by designing a primer set that results in differences in the presence or absence of amplification between males and females, such as by avoiding designing a primer set that targets any one region of the partial base sequence. Furthermore, for example, when using Southern blotting hybridization for sex determination, sex determination is possible even if a probe is designed to target any one region of the partial base sequence, by using an appropriate restriction enzyme when digesting the genomic DNA. Thus, by using an appropriate marker detection method, DNA consisting of all or a partial base sequence of M175 can be used as a sex determination marker. It is preferable for the marker to exclude DNA consisting of a base sequence included in any one of the partial base sequences in Table 10, and more preferably DNA consisting of a partial base sequence obtained by excluding the partial base sequence of Table 10 from M175.

Claims (7)

A method for determining the sex of bluefin tuna, comprising detecting male-specific DNA in a sample collected from a bluefin tuna specimen,
The method, wherein the DNA is at least one type of DNA selected from the group consisting of the following (a) to ( c ):
(a) any of the nucleotide sequences of positions 9390 to 9818, 16389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654 of SEQ ID NO: 1; or any of the nucleotide sequences of positions 9390 to 9818, 16 male-specific DNA consisting of a partial base sequence of any one of the base sequences of positions 389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654, which is a contiguous base sequence of 50 to 10,000 bases ;
(b) a nucleotide sequence having 90% or more identity to any of the nucleotide sequences of positions 9390 to 9818, 16389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654 of SEQ ID NO: 1; or a male-specific DNA consisting of a partial base sequence of any one of the base sequences at positions 389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654, which has 90% or more identity to a contiguous base sequence of 50 to 10,000 bases ;
(c) 1 in any of the base sequences of positions 9390 to 9818, 16389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654 of SEQ ID NO: 1 A male-specific DNA comprising a base sequence in which 1 to 5 bases have been deleted, substituted or added, or a partial base sequence of any of the base sequences at positions 9390 to 9818, 16389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654 of SEQ ID NO: 1, in which 1 to 5 bases have been deleted, substituted or added in a base sequence of 50 to 10,000 consecutive bases . The method of claim 1, further comprising determining the sex of the subject based on the results of the detection. (a) is a male-specific DNA consisting of a base sequence of 50 to 10,000 consecutive bases which is a base sequence of positions 32614 to 64628 of SEQ ID NO: 1 or a partial base sequence of the base sequence of positions 32614 to 64628 of SEQ ID NO: 1,
(b) is a male-specific DNA consisting of a base sequence having 90% or more identity with the base sequence of positions 32614 to 64628 of SEQ ID NO: 1 or a partial base sequence of the base sequence of positions 32614 to 64628 of SEQ ID NO: 1, which base sequence has 90% or more identity with a continuous base sequence of 50 to 10,000 bases,
(c) is a male-specific DNA consisting of a base sequence in which 1 to 5 bases are deleted, substituted or added in the base sequence of positions 32614 to 64628 of SEQ ID NO: 1, or a partial base sequence in which 1 to 5 bases are deleted, substituted or added in a base sequence of 50 to 10,000 consecutive bases in the base sequence of positions 32614 to 64628 of SEQ ID NO: 1,
3. The method according to claim 1 or 2. the DNA is a nucleotide sequence of any one of positions 9390 to 9818, 16389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269 , and 207325 to 207654 of SEQ ID NO: 1; The method according to claim 1 or 2, wherein the male-specific DNA is a partial base sequence of any one of the base sequences at positions 603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654, and is composed of a base sequence of 50 to 10,000 consecutive bases . The method according to any one of claims 1 to 4 , wherein the detection is carried out by the LAMP method. Use of at least one type of DNA selected from the group consisting of the following (a) to (c) for sexing of bluefin tuna :
(a) any of the nucleotide sequences of positions 9390 to 9818, 16389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654 of SEQ ID NO: 1, or positions 9390 to 9818 of SEQ ID NO: 1 male-specific DNA consisting of a partial base sequence of any of positions 16389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654, which is a contiguous base sequence of 50 to 10,000 bases;
(b) a nucleotide sequence having 90% or more identity to any of the nucleotide sequences of positions 9390 to 9818, 16389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654 of SEQ ID NO: 1; or a male-specific DNA consisting of a partial base sequence of any one of the base sequences at positions 389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654, which has 90% or more identity to a contiguous base sequence of 50 to 10,000 bases;
(c) a base sequence in which 1 to 5 bases are deleted, substituted or added in any of the base sequences of positions 9390 to 9818, 16389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269 and 207325 to 207654 of SEQ ID NO: 1, or Male-specific DNA consisting of a partial base sequence of any of the base sequences at positions 6389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654, in which 1 to 5 bases are deleted, substituted, or added in a contiguous base sequence of 50 to 10,000 bases . A kit for determining the sex of bluefin tuna, comprising a primer pair capable of specifically amplifying at least one type of DNA selected from the group consisting of the following (a) to (c) and/or a probe capable of specifically hybridizing to said DNA :
(a) any of the nucleotide sequences of positions 9390 to 9818, 16389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654 of SEQ ID NO: 1; or any of the nucleotide sequences of positions 9390 to 9818, 16 male-specific DNA consisting of a partial base sequence of any one of the base sequences of positions 389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654, which is a contiguous base sequence of 50 to 10,000 bases;
(b) a nucleotide sequence having 90% or more identity to any of the nucleotide sequences of positions 9390 to 9818, 16389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654 of SEQ ID NO: 1; or a male-specific DNA consisting of a partial base sequence of any one of the base sequences at positions 389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654, which has 90% or more identity to a contiguous base sequence of 50 to 10,000 bases;
(c) a base sequence in which 1 to 5 bases are deleted, substituted or added in any of the base sequences of positions 9390 to 9818, 16389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269 and 207325 to 207654 of SEQ ID NO: 1, or Male-specific DNA consisting of a partial base sequence of any of the base sequences at positions 6389 to 17603, 32614 to 64628, 76852 to 77963, 82490 to 82919, 118954 to 119841, 168127 to 168456, 176645 to 176974, 186243 to 186572, 195859 to 196188, 200940 to 201269, and 207325 to 207654, in which 1 to 5 bases are deleted, substituted, or added in a contiguous base sequence of 50 to 10,000 bases .