JP7399451B2 - Identification method and kit for rosaceans - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act (1) Publisher: Society of Applied Ecological Engineering Fukuoka Secretariat, Publication name: Society of Applied Ecological Engineering Fukuoka 2018, Program, Publication date: December 4, 2018

TECHNICAL FIELD The present invention relates to a method and a kit for identifying rosaceans.

Rhodeus ocellatus kurumeus is a small pure freshwater fish that belongs to the family Cyprinidae, subfamily Cyprinidae. The Japanese rose tanago is distributed in the plains of western Japan, and lives in agricultural canals and reservoirs. Currently, the Japanese rose tanago is on the brink of extinction, as it is listed as endangered in the Ministry of the Environment's Red List 2019. The biggest reason for this is hybridization due to crossbreeding with Rhodeus ocellatus ocellatus, a closely related subspecies of the alien species.

Nippon Baratanago, Tyrikbaratanago, and their hybrids are morphologically very similar and difficult to distinguish. Therefore, discrimination is performed using DNA information such as mitochondrial DNA (mtDNA) (for example, see Non-Patent Document 1) and microsatellite DNA (for example, see Non-Patent Document 2).

Tsukasa Abe et al., “Native Population of Japanese Rose Tanago in the Sanyo Region”, Journal of Ichthyology, Vol. 60, No. 1, pp. 49-55, 2013. Shirai Y et al., “Isolation and characterization of new microsatellite markers for rose bitterlings, Rhodeus ocellatus.”, Mol Ecol Resour., Vol. 9, Issue 3, pp. 1031-1033, 2009.

However, although analysis using mtDNA is relatively low in cost and labor, it is only possible to analyze the maternal genotype, and it is not possible to determine whether an individual's genotype is homozygous or heterozygous. Furthermore, although an analysis method using microsatellite DNA can determine the genotype of an individual, it is costly and technically difficult. Monitoring the invasion of the Japanese rose tanager is an important issue in environmental surveys, etc., but currently there is a method to easily and reliably identify which type of rose tanago is the Japanese rose tanago, the Japanese rose tanager, or their hybrids while keeping costs down. There is no way to do it.

The present invention has been made in view of the above-mentioned circumstances, and provides a simple and reliable new method and kit for identifying rosaceans.

That is, the present invention includes the following aspects.
(1) A method for determining which type of Japanese rose tanago is a Japanese rose tanago, a Japanese rose tanago, and a hybrid thereof, comprising:
A nuclear DNA amplification step in which an amplification product is obtained by PCR using the nuclear DNA of the target rosefish as a template and the four types of primers shown below;
1) Sense primer A having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 1;
2) Antisense primer B having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 2;
3) Sense primer C having a polynucleotide consisting of a sequence complementary to a part of the nucleotide sequence of the 18S rRNA gene of Rose tanago;
4) Antisense primer D having a polynucleotide consisting of a sequence complementary to a part of the nucleotide sequence of the 5.8S rRNA gene of Rose tanago;
If the amplification product X amplified by the sense primer A and the antisense primer D is detected, it is determined that it is a Japanese rose tanago,
If the amplification product Y amplified by the sense primer C and the antisense primer B is detected, it is determined that the plant is a Japanese grasshopper;
If the amplified product
How to identify rosaceans, including.
(2) The method for distinguishing rosacea according to (1), wherein the sense primer C has a polynucleotide consisting of the base sequence shown by SEQ ID NO: 3.
(3) The method for identifying rosacea according to (1) or (2), wherein the antisense primer D has a polynucleotide consisting of the base sequence shown by SEQ ID NO: 4.
(4) Before the nuclear DNA amplification step or after the discrimination step,
A mitochondrial DNA amplification step in which an amplified product is obtained by PCR using the mitochondrial DNA of the target rosefish as a template and the two types of primer sets shown below;
5) A primer set consisting of a sense primer E and an antisense primer F for amplifying the transcript of the cytochrome b gene of Japanese Rose Tanago;
6) A primer set consisting of a sense primer G and an antisense primer H for amplifying the transcript of the cytochrome b gene of the Japanese grasshopper;
When the amplification product W amplified by the sense primer E and the antisense primer F is detected, the genotype of the mitochondrial DNA is determined to be Japanese Rose Tanago,
If the amplification product V amplified by the sense primer G and the antisense primer H is detected, a mitochondrial DNA genotype determination step of determining that the genotype of the mitochondrial DNA is that of the Japanese white-breasted grasshopper;
The method for determining rosacea according to any one of (1) to (3), further comprising:
(5) In (4), the sense primer E has a polynucleotide consisting of the base sequence shown by SEQ ID NO: 5, and the antisense primer F has a polynucleotide consisting of the base sequence shown by SEQ ID NO: 6. How to identify the listed rosaceans.
(6) The sense primer G has a polynucleotide consisting of the base sequence shown by SEQ ID NO: 7, and the antisense primer H has a polynucleotide consisting of the base sequence shown by SEQ ID NO: 8, (4) or (5) The method for determining rosacea species as described in (5).

(7) A kit for discrimination of rosefish, comprising a primer set including the four types of primers shown below.
1) Sense primer A having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 1;
2) Antisense primer B having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 2;
3) Sense primer C having a polynucleotide consisting of a sequence complementary to a part of the nucleotide sequence of the 18S rRNA gene of Rose tanago;
4) Antisense primer D having a polynucleotide consisting of a sequence complementary to a part of the nucleotide sequence of the 5.8S rRNA gene of Rose tanago
(8) The kit for discrimination of rosefish according to (7), wherein the sense primer C has a polynucleotide consisting of the base sequence shown by SEQ ID NO: 3.
(9) The kit for identifying rosacea according to (7) or (8), wherein the antisense primer D has a polynucleotide consisting of the base sequence shown by SEQ ID NO: 4.
(10) The kit for discriminating rosacea according to any one of (7) to (9), further comprising the following two types of primer sets.
5) A primer set consisting of a sense primer E and an antisense primer F for amplifying the transcript of the cytochrome b gene of Japanese Rose Tanago;
6) Primer set (11) consisting of a sense primer G and an antisense primer H for amplifying the transcript of the cytochrome b gene of the Japanese white-breasted tanager. (10), wherein the antisense primer F has a polynucleotide consisting of the base sequence shown by SEQ ID NO: 6.
(12) The sense primer G has a polynucleotide consisting of the base sequence shown by SEQ ID NO: 7, and the antisense primer H has a polynucleotide consisting of the base sequence shown by SEQ ID NO: 8, (10) or (11) The kit for identifying rosaceans as described in (11).

According to the above-mentioned method and kit for identifying rosanthus, it is possible to easily and reliably determine which varietal among the Japanese rosanthus, the Japanese rosanthus, and their hybrids.

FIG. 2 is a diagram showing the results of genotyping of mitochondrial DNA using species-specific primers in Example 1. FIG. 2 is a diagram showing the results of genotyping of nuclear DNA using species-specific primers in Example 1. 2 is a graph showing the distribution ratio of genotypes in each region of Rose tanago individuals in Example 1. FIG. 2 is a schematic diagram comparing the procedures of the method for identifying rosaceans according to the present embodiment and the conventional method for identifying rosaceans (sequence analysis method of mitochondrial DNA or nuclear DNA, and microsatellite DNA analysis method).

Hereinafter, details of a method and a kit for identifying rosehoppers according to an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described.

<How to identify rosefish>
The method for identifying Rosatanago of this embodiment (hereinafter, may be simply abbreviated as "discrimination method of this embodiment") is to determine which variety of Rosetanago is Japanese Rosetanago, Japanese Rosetanago, and their hybrid species. This is the way to do it. The discrimination method of this embodiment includes a nuclear DNA amplification step and a discrimination step.
In the nuclear DNA amplification step, an amplification product is obtained by a PCR method using the nuclear DNA of the target rosefish as a template and the four types of primers shown below.
1) Sense primer A having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 1;
2) Antisense primer B having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 2;
3) Sense primer C having a polynucleotide consisting of a sequence complementary to a part of the nucleotide sequence of the 18S rRNA gene of Rose tanago;
4) Antisense primer D having a polynucleotide consisting of a sequence complementary to a part of the nucleotide sequence of the 5.8S rRNA gene of Rose tanago

In the discrimination step, when the amplification product X amplified by the sense primer A and the antisense primer D is detected, it is discriminated that it is a Japanese Rose Tanago.
When the amplification product Y amplified by the sense primer C and the antisense primer B is detected, it is determined that the plant is a Japanese grasshopper.
When the amplification product X and the amplification product Y are detected, it is determined that the plant is a hybrid of the Japanese rose tanago and the Japanese rose tanago.

The inventors focused on the internal transcribed spacer (internal transcribed spacer 1; ITS1) region of the ribosomal RNA gene in the nuclear DNA of the Japanese rose tanago and the Japanese rose tanago, and as a result of analyzing the base sequences of each subspecies, they found that each The present invention was completed by discovering that each subspecies has a specific mutation and designing primers specific to each subspecies.
As shown in the Examples described below, the discrimination method of this embodiment uses a primer set containing primers specific to each subspecies to determine whether it is a Japanese Rose Tanago, a Japanese Rose Tanago, or a hybrid thereof. It is possible to easily and reliably determine whether the

[Nuclear DNA amplification process]
In the nuclear DNA amplification step, an amplification product is obtained by a PCR method using the nuclear DNA of the target rosefish as a template and the four types of primers shown below.
1) Sense primer A having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 1;
2) Antisense primer B having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 2;
3) Sense primer C having a polynucleotide consisting of a sequence complementary to a part of the nucleotide sequence of the 18S rRNA gene of Rose tanago;
4) Antisense primer D having a polynucleotide consisting of a sequence complementary to a part of the nucleotide sequence of the 5.8S rRNA gene of Rose tanago

In order to amplify the nuclear DNA of a target rosacea, first, the nuclear DNA of the rosacea is prepared and used as a sample. The sample only needs to contain nuclear DNA, and the entire DNA of a rosefish may also be used as a sample. Alternatively, the nuclear DNA of a rosefish may be extracted and used as a sample.

The sample may be prepared from tissue of a living roseate. Alternatively, samples can also be prepared from tissues of carcasses of rosefish. Alternatively, a sample may be prepared from the feces of a rosefish or a tissue peeled off from a rosefish. A known method can be used to extract DNA from a sample of rosefish. Specifically, DNA is extracted from a sample of rosefish using the phenol method or a commercially available nucleic acid extraction kit (manufactured by QIAGEN, Takara Bio, etc.).

Next, the ITS1 region in the nuclear DNA is amplified using the prepared sample as a template. Specifically, a DNA fragment containing the ITS1 region in nuclear DNA can be amplified by DNA amplification using the above four types of primers. More specifically, the sense primer A and the antisense primer D amplify a DNA fragment (amplification product X described below) containing the ITS1 region having a mutation specific to the Japanese Rose Tanago. Using the sense primer C and the antisense primer B, a DNA fragment (amplification product Y to be described later) containing the ITS1 region having a mutation specific to P. aeruginosa is amplified. The sense primer C and the antisense primer D amplify the full length or part of the 18S rRNA gene--ITS1 region-5.8S rRNA gene common to the Japanese rose tanago and the Japanese rose tanager.

The length of the primer used in the amplification step is not particularly limited as long as each target region can be amplified, but it is 15 bases or more and 40 bases or less, and may be 15 bases or more and 35 bases or less; The number may be greater than or equal to 30 bases, or may be greater than or equal to 15 bases and less than or equal to 25 bases.

Sense primer A is not particularly limited as long as it can amplify the ITS1 region having a mutation specific to Japanese Rose Tanago, but for example, it consists of the base sequence shown by SEQ ID NO: 1 (5'-TTTTCCACCCCAGTG-3') Examples include sense primers having polynucleotides.

Antisense primer B is not particularly limited as long as it is capable of amplifying the ITS1 region having a mutation specific to T. argentina; Examples include antisense primers having a polynucleotide.

Sense primer C is not particularly limited as long as it can amplify all or part of the 18S rRNA gene, which is a common sequence in rosefish species. Sense primer C is a sequence that can hybridize to the 18S rRNA gene, and has a sequence that is complementary to a portion of the base sequence of the 18S rRNA gene. For example, a sense primer having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 3 (5'-TCCGTAGGTGAACCTGCGGAAG-3') can be mentioned.

Antisense primer D is not particularly limited as long as it can amplify all or part of the 5.8S rRNA gene, which is a common sequence in rosefish species. Antisense primer D is a sequence that can hybridize to the 5.8S rRNA gene and has a sequence that is complementary to a portion of the base sequence of 5.8S rRNA. For example, an antisense primer having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 4 (5'-GTCCTGCAATTCACATTAGTTC-3') can be mentioned.

The length of the amplified product amplified by PCR is determined by the selected primers. The length of the amplified product can be 10 base pairs or more and 1000 base pairs or less, and preferably 100 base pairs or more and 700 base pairs or less for ease of electrophoresis and observation.

The method for amplifying DNA is not limited to the general PCR method, and known methods such as real-time PCR method, PCR-SSP (Sequence Specific Primers) method, and PCR-SSCP (Single Strand Conformation Polymorphism) method may be used. thing I can do it.

[Discrimination process]
In the discrimination step, by detecting the amplification product obtained in the nuclear DNA amplification step, it is determined which of the Japanese rose tanago, the Japanese rose tanago, and their hybrid species.
Specifically, when the amplification product X amplified by the sense primer A and the antisense primer D is detected, it is determined that the plant is a Japanese Rose Tanago.
When the amplification product Y amplified by the sense primer C and the antisense primer B is detected, it is determined that the plant is a Japanese grasshopper.
When the amplification product X and the amplification product Y are detected, it is determined that the plant is a hybrid of the Japanese rose tanago and the Japanese rose tanago.
In addition, in the detection of the above amplification product, a DNA fragment consisting of the full length or a part of the 18S rRNA gene - ITS1 region - 5.8S rRNA gene (hereinafter sometimes referred to as "amplification product Z") common to the Japanese rose tanago and the Japanese rose tanago is also detected.

The amplification product obtained in the nuclear DNA amplification step may be detected by observing the amplified gene fragment by, for example, agarose gel electrophoresis, polyacrylamide gel electrophoresis, or the like. Alternatively, a real-time PCR method may be used in which a DNA amplification reaction is performed in the presence of an intercalator dye such as SYBR green and the fluorescence of the intercalator dye is detected.

The discrimination method of the present embodiment can further include a step of specifying the maternal genotype of the target Rose Tanago. Specifically, the discrimination method of the present embodiment may further include a mitochondrial DNA amplification step and a mitochondrial DNA genotype determination step described below before the nuclear DNA amplification step or after the discrimination step. .

[Mitochondrial DNA amplification process]
In the mitochondrial amplification step, an amplification product is obtained by PCR using the mitochondrial DNA of the target rosefish as a template and the two types of primer sets shown below.
5) A primer set consisting of a sense primer E and an antisense primer F for amplifying the transcript of the cytochrome b gene of Japanese Rose Tanago;
6) Primer set consisting of sense primer G and antisense primer H for amplifying the transcription product of the cytochrome b gene of the white-spotted grasshopper

In order to amplify the mitochondrial DNA of a target rosefish, first, the mitochondrial DNA of the rosefish is prepared and used as a sample. The sample only needs to contain mitochondrial DNA, and the entire DNA of a rosefish may also be used as a sample. Alternatively, the mitochondrial DNA of rosefish may be extracted and used as a sample.

The sample may be prepared from tissue of a living roseate. Alternatively, samples can also be prepared from tissues of carcasses of rosefish. Alternatively, a sample may be prepared from the feces of a rosefish or a tissue peeled off from a rosefish. Examples of the method for extracting DNA from a sample of rosefish include a method similar to the method exemplified in the above-mentioned "nuclear DNA amplification step".

Next, the cytochrome b gene of mitochondrial DNA is amplified using the prepared sample as a template. Specifically, a DNA fragment containing the cytochrome b gene of mitochondrial DNA can be amplified by DNA amplification using the above two types of primer sets. More specifically, the sense primer E and the antisense primer F amplify a DNA fragment (amplification product W described below) containing the cytochrome b gene having a mutation specific to the Japanese Rose Tanago. Using the sense primer G and the antisense primer H, a DNA fragment (amplification product V to be described later) containing the cytochrome b gene having a mutation specific to P. aeruginosa is amplified.

The length of the primer used in the amplification step is not particularly limited as long as it can amplify the cytochrome b gene of mitochondrial DNA, but it is 15 bases to 40 bases, may be 15 bases to 35 bases, and 15 bases to 40 bases. It may be more than or equal to a base and less than or equal to 30 bases, or may be more than or equal to 15 bases and less than or equal to 25 bases.

The sense primer E and the antisense primer F are not particularly limited as long as they can amplify the cytochrome b gene having a mutation specific to the Japanese Rose Tanago. Sense primer E and antisense primer F are sequences that can hybridize to the cytochrome b gene having a mutation specific to the Japanese roseate, and are part of the base sequence of the cytochrome b gene having a mutation specific to the Japanese roseate. It has a complementary sequence to the part. For example, a sense primer having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 5 (5'-ACCCTATATAGGAGACACCC-3') and a polynucleotide consisting of the base sequence shown by SEQ ID NO: 6 (5'-CTTAATGTGTGGCGGGGTA-3') are used. Examples include antisense primers that have

The sense primer G and the antisense primer H are not particularly limited as long as they can amplify the cytochrome b gene having a mutation specific to P. aeruginosa. Sense primer E and antisense primer F are sequences capable of hybridizing to the cytochrome b gene having a mutation specific to the Japanese white-breasted rosefish, and are part of the nucleotide sequence of the cytochrome b gene that has a mutation specific to the black-and-white grasshopper. It has a complementary sequence to the part. For example, a sense primer having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 7 (5'-GGTTTATTTCTGGCTATGCAC-3') and a polynucleotide consisting of the base sequence shown by SEQ ID NO: 8 (5'-GTTTCCTTATATAAATAGGACCCA-3') are used. Examples include antisense primers that have

The length of the amplified product amplified by PCR is determined by the selected primers. The length of the amplified product can be 10 base pairs or more and 1000 base pairs or less, and preferably 100 base pairs or more and 700 base pairs or less for ease of electrophoresis and observation.

Examples of the method for amplifying DNA include the same methods as those exemplified in the "nuclear DNA amplification step" described above.

[Mitochondrial DNA genotype determination process]
In the mitochondrial DNA genotype determination step, the genotype of the mitochondrial DNA of the target Rose Tanago, that is, the maternal genotype, is determined by detecting the amplification product obtained in the mitochondrial DNA amplification step.
Specifically, when the amplification product W amplified by the sense primer E and the antisense primer F is detected, it is determined that the mitochondrial DNA genotype is Japanese Rose Tanago.
When the amplification product V amplified by the sense primer G and the antisense primer H is detected, it is determined that the plant is a Japanese grasshopper.

Examples of the method for detecting the amplification product obtained in the mitochondrial DNA amplification step include methods similar to those exemplified in the "discrimination step" described above.

The discrimination method of this embodiment includes, in addition to the above-mentioned nuclear DNA amplification step and the above-mentioned discrimination step, a mitochondrial DNA amplification step and a mitochondrial DNA genotyping step, as shown in the implementation section described later. Not only can it be determined which genotype is the Japanese rose tanago, the Japanese rose tanago, or a heterozygous hybrid, but it is also possible to determine whether the haplotype is the Japanese rose tanago or the Japanese rose tanago from the mitochondrial DNA genotype. . As a result, as shown in the Examples described below, it is possible to accurately grasp the genetic invasion rate of the Japanese rosefish that inhabits a specific region.

<Rosacana species identification kit>
The kit for discrimination of rosacea according to the present embodiment (hereinafter may simply be abbreviated as "discrimination kit of the present embodiment") includes a primer set including the following four types of primers.
1) Sense primer A having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 1;
2) Antisense primer B having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 2;
3) Sense primer C having a polynucleotide consisting of a sequence complementary to a part of the nucleotide sequence of the 18S rRNA gene of Rose tanago;
4) Antisense primer D having a polynucleotide consisting of a sequence complementary to a part of the nucleotide sequence of the 5.8S rRNA gene of Rose tanago

By using the primer set described above, the identification kit of the present embodiment can easily and reliably determine which type of Rosa tanago is a Japanese rose tanago, a Japanese rose tanager, or a hybrid thereof.

In the discrimination kit of the present embodiment, the sense primer A, antisense primer B, sense primer C, and antisense primer D may be the same as those exemplified in the above-mentioned "Method for discriminating roseola species."

The discrimination kit of this embodiment can further include two types of primer sets shown below.
5) A primer set consisting of a sense primer E and an antisense primer F for amplifying the transcript of the cytochrome b gene of Japanese Rose Tanago;
6) Primer set consisting of sense primer G and antisense primer H for amplifying the transcription product of the cytochrome b gene of the white-spotted grasshopper

The discrimination kit of this embodiment further includes the two types of primer sets described above, so that it is possible to specify the maternal genotype of the target Rose tanago.

Sense primer E, antisense primer F, sense primer G, and antisense primer H may be the same as those exemplified in the above-mentioned "Method for distinguishing roseate species."

The discrimination kit of this embodiment may further include a probe.

As the probe, a TaqMan (registered trademark) probe can also be used. Fluorescent substances that can be used include FAM, Yakima Yellow (registered trademark), fluorescein, fluorescein isothiocyanate (FITC), VIC (registered trademark), and the like. Further, examples of quenching substances that can be used include carboxytetramethylrhodamine (TAMRA) and the like.

When a DNA amplification reaction is performed in the presence of the above probe, the probe anneals to the DNA fragment to be amplified. The probe is then degraded by the 5'→3' exonuclease activity of DNA polymerase. As a result, the quencher and fluorescent substance, which were present in close proximity on the probe molecule, separate, and the fluorescent substance begins to emit fluorescence. By quantifying this fluorescence, the progress of the DNA amplification reaction can be quantitatively measured.

The length of the probe in the kit of this embodiment is particularly limited if it can anneal to the template DNA before the sense primer and antisense primer (if it has a higher melting temperature (Tm) than the sense primer and antisense primer). For example, it may be from 15 bases to 40 bases, from 15 bases to 35 bases, from 15 bases to 30 bases, from 15 bases to 25 bases. good.

By using the kit of the present embodiment, the above-mentioned "Method for Discriminating Rosetanago" can be carried out by real-time PCR method.

EXAMPLES Hereinafter, the present invention will be explained with reference to examples, but the present invention is not limited to the following examples.

[Example 1]
1. Sample collection Samples of Nippon Rose Tanago, Tyrik Rose Tanago, and their hybrids were collected from five rivers in Kyushu (Rokkaku River, Chikugo River, Kikuchi River) using hand nets or casting nets from 2017 to 2018. , Zuibaiji River, and Kashima River), as well as the Isumi River in eastern Honshu. These sampling locations were selected based on previous studies. A portion of the caudal fin was cut from each captured individual, and the cut tissue was immediately fixed with 99% ethanol. All captured individuals were then immediately released at each sampling location. Total DNA was isolated from the fixed tissue using DNeasy Blood & Tissue Kit (manufactured by Qiagen).

2. Genotyping using species-specific primers in mitochondrial DNA The reference base sequences of the mitochondrial cytochrome b genes of the Japanese rose tanago and the Japanese rose tanager were obtained from the DNA Data Bank of Japan (DDBJ). The accession numbers of Nippon Rose Tanago are AB109000, AB109010, AB366504-AB366507, AB769511-AB769515. The accession numbers of Tairikbaratanago are AB109009, AB366512-AB366513, AB620133, AB769516-AB769519, and KF410776. These nucleotide sequences were analyzed using MEGA7 software (Reference 1: “Kumar S et al., “MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets.”, Mol Biol Evol., Vol. 33, Issue 7, pp. 1870- 1874, 2016.) to identify mutations specific to each subspecies (interspecies mutations) in the mitochondrial cytochrome b gene.

Based on the identified interspecies variation, we designed the subspecies-specific primer set shown below. The base sequences of each primer are shown in Table 1. In Table 1, "forward primer Rok_cytb_F" and "reverse primer Rok_cytb_R" are primer sets for obtaining an amplification product of the mitochondrial cytochrome b gene of Japanese rosefish; primer Roo_cytb_R” is Tyrik This is a primer set for obtaining the amplification product of the mitochondrial cytochrome b gene of rosefish.

PCR was performed using the primer set shown in Table 1 and the total DNA isolated from each tissue obtained in "1." as a template. Specifically, 5 μL of KOD One PCR Master Mix -Blue- (manufactured by Toyobo), 0.5 μL of each primer (concentration relative to the total volume of the mixture is 10 μM (μmol/L)), and 2.5 μL of ultrapure water. A 10 μL reaction mixture containing 0.5 μL of template DNA was prepared, and PCR was performed. The PCR conditions were: 98°C for 30 seconds, followed by 30 cycles of the following: 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 15 seconds. After the cycle was completed, the temperature was maintained at 72°C for 2 minutes. In order to separate the amplification products amplified by PCR, a reaction solution containing 2 μL of each PCR product was electrophoresed at 100 V for 25 minutes using a 10 mass% agarose gel containing ethidium bromide. The molecular weight of the amplified product was determined using a 1 kb Plus DNA ladder (NEW ENGLAND Biolabs Inc.).

First, using the Japanese baratanago (sample No. 1 collected from the Rokkaku River) and the Japanese baratanago (sample No. 25 collected from the Isumi River) whose nucleotide sequences are known, amplification products from the two subspecies were extracted. We verified the differences in the patterns. The results are shown in Figure 1. In FIG. 1, "Fragment W" is an amplification product specific to the Japanese rosacea, and "Fragment V" is an amplification product specific to the Japanese rosacea.
From FIG. 1, it was confirmed that amplification products specific to two subspecies of the cytochrome b gene were obtained. The amplification product specific to Nippon Rosa tanago was 348 base pairs (bp), and the amplification product specific to Japanese rose tanager was 193 bp.

Next, genotyping of the mtDNA was performed on the other 34 samples by PCR to identify the mtDNA genotype.

3. Genotyping using species-specific primers in nuclear DNA Next, ribosomal RNA in the nuclear DNA of the Japanese baratanago (sample No. 1 collected from the Rokkaku River) and the Japanese baratanago (sample No. 31 collected from the Isumi River) was determined. The sequence of the internal transcribed spacer 1 (ITS1) region of the gene was analyzed. “Kanno K et al., “Morphological, distributional, and genetic characteristics of Cottus pollux in the Kyushu Island, Japan: indication of fluvial and amphidromous life histories within a single lineage”, Ichthyological Research, Vol. 65, Issue 4, pp. 462-470, 2018. (Reference 2), a forward primer (18SrRNAF; 5'-TCCGTAGGTGAACCTGCGGAAG-3' (SEQ ID NO: 3)) having a sequence complementary to a part of the nucleotide sequence of the 18SrRNA gene described in PCR was performed using a reverse primer (5.8SrRNAR; 5'-GTCCTGCAATTCACATTAGTTC-3' (SEQ ID NO: 4)) having a sequence complementary to a part of the nucleotide sequence of the 8SrRNA gene, and a 344bp ITS1 region amplification product was obtained. I got it. Specifically, 5 μL of EmeraldAmp (registered trademark) PCR Master Mix (manufactured by TaKaRa), 0.5 μL of each primer (concentration relative to the total volume of the mixture is 10 μM (μmol/L)), and 3.5 μL of ultrapure water. A 10 μL reaction mixture containing 0.5 μL of template DNA was prepared, and PCR was performed. The PCR conditions were as follows: 98°C for 30 seconds, followed by 30 cycles: 98°C for 15 seconds, 50°C for 15 seconds, 72°C for 15 seconds. After the cycle was completed, the temperature was maintained at 72°C for 2 minutes.

In order to confirm the amplification products amplified by PCR, a reaction solution containing 2 μL of each PCR product was electrophoresed at 100 V for 25 minutes using a 1% by mass agarose gel containing ethidium bromide.

The obtained amplification product was purified using ExoSAP-IT PCR Product Cleanup (manufactured by Thermo Fisher). The obtained purified product was then mixed with primers and sequenced using FASMAC DNA Sequencing Service (FASMAC). The primers used were the above 18SrRNAF, the above 5.8SrRNA, the forward primer for amplification of the ITS1 region common to Japanese rose grass (Roo_ITS1_F2; 5'-CGCCTTGGGTTTTAATGTGCC-3' (SEQ ID NO: 9)), and the reverse primer for amplification of the ITS1 region of Japanese rose grass (Roo_ITS1_R2; '-GAACGACACAGACAACGGGGTGGG-3' (SEQ ID NO: 10)). The newly obtained nucleotide sequence was registered in DDBJ (accession numbers: LC485325-LC485326). The obtained nucleotide sequences were analyzed using MEGA7 software (see Reference 1 above), and mutations specific to each subspecies (interspecific mutations) in the ITS1 region were identified.

Based on the identified interspecies variation, we designed the subspecies-specific primer set shown below. Table 2 shows the base sequence of each primer. In Table 2, "forward primer Rok_ITS1_shortF" is a forward primer for obtaining an amplification product of the ITS1 region of Japanese rose togonago, and "reverse primer Rok_ITS1_shortR" is a reverse primer for obtaining an amplification product of the ITS1 region of Japanese rose tanago. be.

PCR was performed using the four types of primers shown below in order to distinguish between Japanese rose tanago and Japanese rose tanago based on nuclear DNA.
1) Rok_ITS1_shortF (SEQ ID NO: 1)
2) Roo_ITS1_shortR (SEQ ID NO: 2)
3) 18SrRNAF (SEQ ID NO: 3)
4) 5.8SrRNAR (SEQ ID NO: 4)

With these primers, the common amplification product (UNIF) amplified by 18S rRNAF and 5.8S rRNAR (approximately 460 bp, 464 bp in Nippon rose tanago and 456 bp in Tyrik rose tanago), as well as subspecies-specific amplification products that are markers of the ITS1 region. Amplification products (134 bp of the amplified product (ROKF) specific to the Japanese rose tortoiseshell amplified by Rok_ITS1_shortF and 5.8S rRNAR, and 336 bp of the amplification product (ROOF) specific to the Japanese rose tortoise amplified by 18S rRNAF and Roo_ITS1_shortR) can be obtained. .

PCR was performed using the same method as in "2. Genotyping using species-specific primers in mitochondrial DNA" above, except for the PCR cycle. PCR cycles were performed after incubation at 98°C for 30 seconds, followed by 30 cycles of: 98°C for 10 seconds, 50°C for 15 seconds, and 72°C for 15 seconds. After the cycle was completed, the temperature was maintained at 72°C for 2 minutes. In order to separate the amplification products amplified by PCR, a reaction solution containing 2 μL of each PCR product was electrophoresed at 100 V for 25 minutes using a 10 mass% agarose gel containing ethidium bromide. The molecular weight of the amplified product was determined using a 1 kb Plus DNA ladder (NEW ENGLAND Biolabs Inc.).

First, using the Japanese baratanago (sample No. 1 collected from the Rokkaku River) and the Japanese baratanago (sample No. 31 collected from the Isumi River) whose nucleotide sequences are known, amplification products from the two subspecies were extracted. We verified the differences in the patterns. The results are shown in Figure 2.
From FIG. 2, UNIF and ROKF were detected in Nippon Baratanago, while UNIF and ROOF were detected in Tairikbaratanago. This confirmed that in addition to common amplification products, amplification products specific to each subspecies were detected. Three types of amplification products, UNIF, ROKF, and ROOF, were detected in the hybrids of Nippon Rose Tanago and Tyrik Rose Tanago.
From the above, it was suggested that by performing PCR analysis using this primer set, it is possible to easily distinguish between Japanese rose tanago, Japanese rose tanago, and hybrid individuals.

Next, the other 34 samples were subjected to genotyping by PCR of the above-mentioned nuclear DNA, and it was determined whether they were Japanese rose tanago, Japanese rose tanago, or a hybrid thereof.

4. Discrimination of Rosetanago We summarized the analysis results of "2. Genotyping using species-specific primers in mitochondrial DNA" and "3. Genotyping using species-specific primers in nuclear DNA" above, and determined the genotype of each individual. was classified into the following six types. The results are shown in Figure 3. In Figure 3, "st.1", "st.2", "st.3", "st.4", "st.5", and "st.6" are Rokkaku River, Chikugo River, Kikuchi River, respectively. Samples collected from Zuibaiji River, Kashima River, and Isumi River are shown.
(1) Maternal genotype is Japanese Rose Tanago (mat-Rok), nuclear DNA genotype is Japanese Rose Tanago (homo-Rok)
(2) Maternal genotype is Japanese Rose Tanago (mat-Rok), nuclear DNA genotype is hybrid (hetero)
(3) The maternal genotype is Japanese Rose Tanago (mat-Rok), and the nuclear DNA genotype is Tyrik Baratanago (homo-Roo).
(4) Maternal genotype is Tyrikbaratanago (mat-Roo), nuclear DNA genotype is Nipponbaratanago (homo-Rok)
(5) The maternal genotype is mat-Roo, and the nuclear DNA genotype is a hybrid (hetero).
(6) Maternal genotype is Tairikbaratanago (mat-Roo), nuclear DNA genotype is Tairikbaratanago (homo-Roo)

From Figure 3, all the individuals collected in the Rokkaku River, Chikugo River, and Kikuchi River (st. 1 to 3) were classified as Japanese Rose Tanago (classification (1) above: mat-Rok, homo-Rok). All the individuals collected from Isumi River (st. 6) were classified as Tairikbaratanago (classification (6) above: mat-Roo, homo-Roo). Subspecies and hybrid species were found in the individuals collected from Zuibaiji River and Kashima River (st. 4 and 5). The individuals collected from the Zuibaiji River were divided into 4 individuals (classification (6) above: mat-Roo, homo-Roo) and 2 hybrids (classification (5) above: mat-Roo, hetero). Classified. Among the individuals collected in the Kashima River, 1 individual was a Tairikbaranago (classification (6) above: mat-Roo, homo-Roo), and 5 were hybrids (classification (2) above: mat-Rok, hetero; classification above). (3): mat-Rok, homo-Roo; classified into the above classification (5): mat-Roo, hetero).

The method for identifying Roseate species using the above primer set requires less effort (3 steps and 3.5 hours) than conventional DNA sequence analysis (9 steps and 8.5 hours) and microsatellite DNA analysis (6 steps and 6 hours). You can easily identify rosaceans based on the time (see Figure 4). This is because the present method for identifying rosetans consists only of an amplification step by PCR and a DNA separation step using agarose gel. Furthermore, this discrimination method omits the step of amplification check and does not require the use of reagents normally used for post-amplification checks, so analysis can be performed at a lower cost than conventional methods.

The present discrimination method can detect hybrid species and Japanese grasshoppers with higher sensitivity than conventional methods of analyzing only mitochondrial DNA. When the individuals collected from the Kashima River (st. 5) in Figure 3 were classified using the conventional mitochondrial DNA analysis method, two out of six individuals were detected as having haplotypes, and the genetics of the Tairikubaratanago were detected. The target penetration rate is 33%. However, with this discrimination method, we can not only detect that the haplotype is Tairikbaratanago from these two individuals, but also detect the heterozygous type of four individuals by using nuclear DNA analysis. It can be deduced that the genetic invasion rate of Rose Tanago is 100%.

According to the method and kit for identifying rosaceans of this embodiment, it is possible to easily and reliably identify rosaceans. The method and kit for identifying rosaceans of this embodiment can be used for environmental surveys and ecological research.

Claims (2)

A method for determining which type of rose tanago is a Japanese rose tanago, a Japanese rose tanago, and a hybrid thereof, comprising:
A nuclear DNA amplification step in which an amplification product is obtained by PCR using the nuclear DNA of the target rosefish as a template and the four types of primers shown in 1) to 4) below;
A discrimination step of determining which of the Japanese rose tanago, the Japanese rose tanago, and their hybrids is determined by electrophoresing and detecting the amplified product obtained in the nuclear DNA amplification step,
1) Sense primer A having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 1;
2) Antisense primer B having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 2;
3) Sense primer C having a polynucleotide consisting of the nucleotide sequence shown by SEQ ID NO: 3, which is complementary to a part of the nucleotide sequence of the 18S rRNA gene of Rose tanago;
4) Antisense primer D having a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4, which is complementary to a part of the base sequence of the 5.8S rRNA gene of Rose tanago;
In the discrimination step, if the amplification product X amplified by the sense primer A and the antisense primer D is detected, it is determined that it is a Japanese Rose Tanago,
If the amplification product Y amplified by the sense primer C and the antisense primer B is detected, it is determined that the plant is a Japanese grasshopper;
If the amplification product
Furthermore, before the nuclear DNA amplification step or after the discrimination step,
A mitochondrial DNA amplification step in which an amplified product is obtained by PCR using the mitochondrial DNA of the target rosefish as a template and the two types of primer sets shown below;
A mitochondrial DNA genotype determination step of determining the genotype of the mitochondrial DNA of the target rosetanago by electrophoresing and detecting the amplification product obtained in the mitochondrial DNA amplification step,
5) A primer set consisting of a sense primer E and an antisense primer F for amplifying the transcript of the cytochrome b gene of Japanese Rose Tanago;
6) A primer set consisting of a sense primer G and an antisense primer H for amplifying the transcript of the cytochrome b gene of the Japanese grasshopper;
The sense primer E has a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5, and the antisense primer F has a polynucleotide consisting of the base sequence shown in SEQ ID NO: 6,
The sense primer G has a polynucleotide consisting of the base sequence shown in SEQ ID NO: 7, and the antisense primer H has a polynucleotide consisting of the base sequence shown in SEQ ID NO: 8,
In the step of determining the genotype of the mitochondrial DNA, if the amplification product W amplified by the sense primer E and the antisense primer F is detected, the genotype of the mitochondrial DNA is determined to be Japanese Rose Tanago,
A method for discriminating Rosetanago, which determines that the mitochondrial DNA genotype is a Japanese Rosetanago when an amplification product V amplified by the sense primer G and the antisense primer H is detected. A kit for discrimination of rosefish, comprising a primer set including four types of primers shown in 1) to 4) below , and two types of primer sets shown in 5) and 6) .
1) Sense primer A having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 1;
2) Antisense primer B having a polynucleotide consisting of the base sequence shown by SEQ ID NO: 2;
3) Sense primer C having a polynucleotide consisting of the nucleotide sequence shown by SEQ ID NO: 3, which is complementary to a part of the nucleotide sequence of the 18S rRNA gene of Rose tanago;
4) Antisense primer D having a polynucleotide consisting of the nucleotide sequence shown by SEQ ID NO: 4, which is complementary to a part of the nucleotide sequence of the 5.8S rRNA gene of Rose tanago.
5) A primer set consisting of a sense primer E and an antisense primer F for amplifying the transcript of the cytochrome b gene of Japanese Rose Tanago;
6) Primer set consisting of sense primer G and antisense primer H for amplifying the transcription product of the cytochrome b gene of the white-spotted grasshopper
The sense primer E has a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5, and the antisense primer F has a polynucleotide consisting of the base sequence shown in SEQ ID NO: 6,
The sense primer G has a polynucleotide consisting of the base sequence shown in SEQ ID NO: 7, and the antisense primer H has a polynucleotide consisting of the base sequence shown in SEQ ID NO: 8.

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