Molecular marker for identifying sebastes species

Abstract

The invention aims to provide a molecular marker for identifying sebastes species, namely a mitochondrial molecular marker and a sebastes species identification method established based on the molecular marker. The molecular marker comprises a marker for detecting sebastes schlegeli with a sequence of SEQ ID NO: 1, a marker for detecting sebastes koreanus with a sequence of SEQ ID NO: 2, a marker for detecting sebastes pachyus with a sequence of SEQ ID NO: 3, a marker for detecting sebastes soup with a sequence of SEQ ID NO: 4, a marker for detecting sebastes pachyus with a sequence of SEQ ID NO: 5, a marker for detecting sebastes armoricanus with a sequence of SEQ ID NO: 6 and a marker for detecting sebastiscus marmoratus with a sequence of SEQ ID NO: 7. The mitochondrial molecular marker provided by the invention overcomes the defect that the existing eDNA universal primer cannot distinguish sebastes species.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic breeding, and particularly relates to a molecular marker for identifying Sebastes species. BACKGROUND

[0002] Traditional methods for monitoring marine fish species, such as trawl fishing, visual surveys, and purse seine fishing, are costly and time-consuming, severely limiting the scope, scale, and reliability of assessing important fish populations from an ecological and commercial perspective. New methods that can monitor more biodiversity at a higher frequency are urgently needed. Environmental DNA (eDNA) is emerging as a new strategy for monitoring marine ecosystems. eDNA is the free DNA released by organisms into the environment. By isolating, amplifying, and sequencing eDNA, species in a particular environment can be identified, providing a non-invasive, cost-effective alternative to traditional survey methods.

[0003] The eDNA metabarcoding technique uses "universal primers" targeting a broad spectrum of target groups to amplify specific barcode sites, allowing the identification of multiple species in a single detection. Combined with next-generation DNA sequencing technology, this technique can reconstruct the community composition of various ecosystems, including marine ecosystems. The eDNA metabarcoding technique has been widely used in marine fish species diversity surveys, providing a potential alternative to time-consuming and labor-intensive fish species monitoring.

[0004] Sebastes, belonging to the family Scorpaenidae of the order Scorpaeniformes, is the most diverse genus in the Scorpaeniformes order, playing a crucial role in the North Pacific coastal ecosystem and being a valuable resource for commercial and recreational fisheries.

[0005] Sebastes is a diverse genus in the North Pacific, with a large number of overlapping distribution areas. The distinguishing characteristics between species are highly repetitive and overlapping, especially between closely related species. Traditional survey methods cannot effectively distinguish Sebastes species, especially during the early life stages. Although eDNA can be an effective alternative for monitoring Sebastes, existing universal metabarcoding primers for bony fish (such as 12S MiFish primers) cannot achieve species-level identification. This deficiency is due to the rapid radiation and evolution of the genus over the past million years, resulting in highly conserved mitochondrial 12S gene sequences targeted by MiFish primers.

[0006] Therefore, effective monitoring of Sebastes requires new methods such as eDNA, but their application depends on the discovery of molecular marker identification sites with faster evolutionary rates and greater sequence variation. SUMMARY

[0007] The purpose of the present invention is to provide a molecular marker for identifying Scorpionfish species, namely a mitochondrial molecular marker and a Scorpionfish species identification method established based on the molecular marker.

[0008] The present invention first provides a molecular marker for identifying species of the genus Scorpaenidae in the coastal waters of China, wherein the molecular marker comprises the following fragments: 1) Detection of Scorpionfish ( Sebastes schlegelii ), the sequence of which is: GTATCCAGGTCAGTTTCTATCTATGGTGTGCTCTTTTCTAGTACGAAAGGACCGAAAAGAAGAGGCCCCTGCTTTAAGCAAGCCTCACCCCCACCTAGTGAAGACAACTAAAATAGGCAAGAGGGCATACCCCCAATGCCTGAGAGAACGGCATGTTG (SEQ ID NO: 1), 2) Detection of Korean scorpionfish ( Sebastes koreanus ) marker, the sequence of which is: GTATCCAGGTCAGTTTCTATCTATGATGTGCTCTTTTCTAGTACGAAAGGACCGAAAAGAAGAGGCCCCTGCTCTAAGCAAGCCTCACCCCCACCTAGTGAAGACAACTAAAGTAGGCAAGAGGGCATACCCCCAATGCCTGAGAGAACGGCATGTTG (SEQ ID NO: 2), 3) Detection of Thickhead Scorpionfish ( Sebastes pachycephalus ), the sequence is: GTATCCAGGTCAGTTTCTATCTATGGTGTGCTCTTTTCTAGTACGAAAGGACCGAAAAGAAGAGGCCCCTGCTCTAAGCAAGCCTCACCCCCACCTAGTGAAAACAACTAAAATAGGCAAGAGGGCATACCCCCAATGTCTGAGAGAACGGCATGTTG (SEQ IDNO: 3), 4) Detection of Tang's flathead scorpionfish ( Sebastes thompsoni ), the sequence of which is: GTATCCAGGTCAGTTTCTATCTATGGTGTGCTCTTTTCTAGTACGAAAGGACCGAAAAGAAGAGGCCCCTGCTCTAAGCAAGCCTCACCCCCACCTAGTGAAGACAACTAAAGTAGGCAAGAGGGCATACCCCCCGTGCCTGAGAGAACGGCATGTTG (SEQ ID NO: 4), 5) Detection of striped scorpionfish ( Sebastes trivittatus ), its sequence is: GTATCCAGGTCAGTTTCTATCTATGGTGTGCTCTTTTCTAGTACGAAAGGACCGAAAAGAAGAGGCCCCTGCTCTAAGCAAGCCTCACCCCCACCTAGTGAAGACAACTAAAGTAGGCAAGAGGGCATACCCCCACTGCCTGAGAGAACGGCATGTTG (SEQ ID NO: 5), 6) Detection of armored flathead scorpionfish ( Sebastes hubbsi ), the sequence of which is: GTATCCAGGTCAGTTTCTATCTATGGTGTGCTCTTTTTTAGTACGAAAGGACCGTAAAGAAGAGGCCCCTGCTTTAAGCAAGCCTCACCCCCACCTAGTGAAAACAACTAAAGTAGGCAAGAGGGCATACCCCCAATGCTGAGAGAACGGCATGTTG (SEQ ID NO: 6), 7) Detection of brown scorpionfish ( Sebastiscus marmoratus ), its sequence is: GTATCCAGGTCAGTTTCTATCTATGGTGTGCTCTTTTCTAGTACGAAAGGACCGAAAAGAAGAGGCCCCTGCTCTAAGCAAGCCTTACCCCCACCTAATGAAGACAACTAAAATAGGCAAGAGGGCATACCCCAATGCCTGAGAGAACGGCATGTTG (SEQ ID NO: 7).

[0009] The present invention provides a use of the molecular marker, which is the use in preparing a molecular product for identifying species of the genus Scorpaenidae in the coastal waters of China; The present invention also provides a molecular detection product for identifying species of the genus Scorpaenidae in the coastal waters of China, wherein the product comprises primers for detecting the above-mentioned molecular markers; The sequence information of one of the specific primers is as follows: Sebastes-F 5'-GTAATCCAGGTCAGTTTCTAT-3' (SEQ ID NO:8), Sebastes-R 5'-CAACATGCCGTTCTCTCA-3' (SEQ ID NO:9).

[0010] The application provides a method for identifying the diversity of Sebastes species in eDNA, which is used for identifying the diversity of Sebastes species by detecting the genotype distribution of the molecular marker in the eDNA sample. The method comprises the following steps: 1) extracting eDNA in seawater; 2) using primers for PCR amplification; 3) detecting the genotype distribution of the molecular marker by amplicon sequencing of the PCR product; 4) judging the species distribution of Sebastes in the environment according to the genotype distribution of the molecular marker obtained by amplicon sequencing.

[0011] Compared with the prior art, the application has the following advantages: 1) The application is faster than the traditional morphological-based species identification, and can directly use the eDNA method to investigate the diversity of Sebastes species, and has a lower detection cost.

[0012] 2) The mitochondrial molecular marker provided by the application makes up for the defect that the existing eDNA universal primer cannot distinguish the Sebastes species. DETAILED DESCRIPTION

[0013] Figure 1 Figure 4 is a legend of five Sebastes and Sebastiscus albus (Korean flathead flounder is missing, according to Liu Jing et al., Huangbohai Fish Log, Science Press, 2015) Figure 2 Figure 5 is a sequence comparison diagram of the mitochondrial molecular marker in Sebastes and Sebastiscus albus. DETAILED DESCRIPTION

[0014] The technical solutions of the application are further described in combination with the following specific examples.

[0015] In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials, reagents, etc. used can be purchased from biological or chemical reagent companies.

[0016] Example 1: Diversity monitoring analysis of Sebastes fish based on simulated eDNA The target species (Sebastes schlegelii (Basil), Sebastes koreanus (Korean flathead flounder), Sebastes rubrivittatus (thick-headed flathead flounder), Sebastes tobeyanu (Tom's flathead flounder), Sebastes alutus (striped flathead flounder), Sebastes goodei (armored flathead flounder), and Sebastiscus albus (brown flathead flounder) Sebastes schlegelii Sebastes koreanus Sebastes pachycephalus Sebastes thompsoni Sebastes trivittatus Sebastes hubbsi Sebastiscus marmoratus ​​​​​​The complete mitochondrial genome sequences of the above species were analyzed, the mitochondrial gene sequences of the above species were compared, and according to the comparison results, a region containing multiple high-information SNP sites and having conserved sequences on both sides was selected as a target amplification segment, and finally the partial sequence of the 16S rRNA gene was selected as a target sequence, and the specific information is as follows: 1) The marker for detecting Sebastolobus altivelis (S. altivelis) is as follows: Sebastes schlegelii GTATCCAGGTCAGTTTCTATCTATGGTGTGCTCTTTTCTAGTACGAAAGGACCGAAAAGAAGAGGCCCCTGCTTTAAGCAAGCCTCACCCCCACCTAGTGAAGACAACTAAAATAGGCAAGAGGGCATACCCCCAATGCCTGAGAGAACGGCATGTTG (SEQ ID NO: 1), 2) The marker for detecting Sebastolobus altivelis (S. altivelis) is as follows: Sebastes koreanus GTATCCAGGTCAGTTTCTATCTATGATGTGCTCTTTTCTAGTACGAAAGGACCGAAAAGAAGAGGCCCCTGCTCTAAGCAAGCCTCACCCCCACCTAGTGAAGACAACTAAAGTAGGCAAGAGGGCATACCCCCAATGCCTGAGAGAACGGCATGTTG (SEQ ID NO: 2), 3) The marker for detecting Sebastolobus altivelis (S. altivelis) is as follows: Sebastes pachycephalus GTATCCAGGTCAGTTTCTATCTATGGTGTGCTCTTTTCTAGTACGAAAGGACCGAAAAGAAGAGGCCCCTGCTCTAAGCAAGCCTCACCCCCACCTAGTGAAAACAACTAAAATAGGCAAGAGGGCATACCCCCAATGTCTGAGAGAACGGCATGTTG (SEQ ID NO: 3), 4) The marker for detecting Sebastolobus altivelis (S. altivelis) is as follows: Sebastes thompsoni ​​​​GTATCCAGGTCAGTTTCTATCTATGGTGTGCTCTTTTCTAGTACGAAAGGACCGAAAAGAAGAGGCCCCTGCTCTAAGCAAGCCTCACCCCCACCTAGTGAAGACAACTAAAGTAGGCAAGAGGGCATACCCCCCGTGCCTGAGAGAACGGCATGTTG (SEQ ID NO: 4), 5) the marker for detecting Sebastes ruberrimus (Sebastolobus altivelis) with the sequence as follows: Sebastes trivittatus GTATCCAGGTCAGTTTCTATCTATGGTGTGCTCTTTTCTAGTACGAAAGGACCGAAAAGAAGAGGCCCCTGCTCTAAGCAAGCCTCACCCCCACCTAGTGAAGACAACTAAAGTAGGCAAGAGGGCATACCCCCCGTGCCTGAGAGAACGGCATGTTG (SEQ ID NO: 4), 6) the marker for detecting Sebastolobus altivelis (Sebastolobus alpinus) with the sequence as follows: GTATCCAGGTCAGTTTCTATCTATGGTGTGCTCTTTTCTAGTACGAAAGGACCGAAAAGAAGAGGCCCCTGCTCTAAGCAAGCCTCACCCCCACCTAGTGAAGACAACTAAAGTAGGCAAGAGGGCATACCCCCCGTGCCTGAGAGAACGGCATGTTG (SEQ ID NO: 4), Sebastes hubbsi 7) the marker for detecting Sebastolobus altivelis (Sebastolobus alpinus) with the sequence as follows: GTATCCAGGTCAGTTTCTATCTATGGTGTGCTCTTTTCTAGTACGAAAGGACCGAAAAGAAGAGGCCCCTGCTCTAAGCAAGCCTCACCCCCACCTAGTGAAGACAACTAAAGTAGGCAAGAGGGCATACCCCCCGTGCCTGAGAGAACGGCATGTTG (SEQ ID NO: 4), 7) the marker for detecting Sebastolobus altivelis (Sebastolobus alpinus) with the sequence as follows: Sebastiscus marmoratus GTATCCAGGTCAGTTTCTATCTATGGTGTGCTCTTTTCTAGTACGAAAGGACCGAAAAGAAGAGGCCCCTGCTCTAAGCAAGCCTTACCCCCACCTAATGAAGACAACTAAAATAGGCAAGAGGGCATACCCCAATGCCTGAGAGAACGGCATGTTG (SEQ ID NO: 7). The above markers can be used as molecular markers for identifying the species of Sebastosuchus in China, which are specific to the corresponding species.

[0017]

[0018] ​To facilitate subsequent amplicon sequencing, the target amplification fragment length was selected in the range of 100-200 bp, and a pair of universal PCR primers was designed using professional primer design software (Primer5). The designed primer sequences were Sebastes-F 5'-GTAATCCAGGTCAGTTTCTAT-3'-, Sebastes-R 5'-CAACATGCCGTTCTCTCA-3', and the amplification length was about 158 bp.

[0019] Example 2: Application of molecular markers in identifying Sebastes species in China's coastal waters Tissue samples of 7 species were obtained, 6 samples for each species, and a small amount of fin tissue was taken for tissue DNA extraction, with the specific steps as follows: 1) Take 25 mg of muscle tissue and cut into small pieces and place in a 1.5 ml microcentrifuge tube.

[0020] 2) Add 180 μL Buffer ATL to the sample, add 20 μL proteinase K, vortex vigorously to disperse the sample thoroughly, and incubate in a 56°C water bath until the tissue blocks are completely digested, mix 2-3 times during the incubation, and vortex mix again for 15 s after digestion is complete.

[0021] 3) Add 200 μl of Buffer AL to the sample and vortex evenly.

[0022] 4) Add 200 μl of ethanol (96-100%) and vortex evenly.

[0023] 5) Pipette the mixture into a DNeasy Mini spin column placed in a 2 ml collection tube, centrifuge at 6,000xg for 1 min, and discard the filtrate and collection tube.

[0024] 6) Place the spin column in a new 2 ml collection tube, add 500 μl of Buffer AW1, and centrifuge at 6,000xg for 1 min.

[0025] 7) Place the spin column in a new 1.5 ml or 2 ml microcentrifuge tube.

[0026] 8) Add 200 pl of Buffer AE to the center of the spin column and elute the DNA. Incubate at room temperature (15-25°C) for 1 min, and centrifuge at 6,000xg for 1 min.

[0027] PCR amplification was performed on DNA extracted from 42 tissues (six samples from each of seven species). A 25 µL reaction system consisted of: Mix 12.5 µL, ddH₂O 7.5 µL, DNA template 2 µL, and 2 µL each of forward and reverse primers (10 µmol / L). The primer sequences were: Sebastes-F 5'-GTAATCCAGGTCAGTTTCTAT-3'-, Sebastes-R 5'-CAACATGCCGTTCTCTCA-3'. The amplified fragment was a partial 16S gene sequence, approximately 158 bp in length.

[0028] Reaction procedure: The reaction was carried out in a BIOER PCR instrument with a pre-denaturation at 94°C for 5 min, followed by 30 cycles of annealing for 30 s at 54.8°C and extension at 72°C for 30 s, followed by extension at 72°C for 10 min.

[0029] The PCR amplified fragments were sequenced and the sequencing results were compared ( Figure 2 By comparing the target regions of mitochondrial 16S rRNA genes of seven scorpionfish species (corresponding to Sebastes schlegelii Reference sequence positions 1-243), the following nucleotide positions were found to be species-specific: 1) Position 49: Sebastes koreanus is adenine (A), and the other six are guanine (G); 2) Positions 130-132: Sebastes hubbsi is a continuous thymine insertion (TTT), and the other six are thymine-cytosine-thymine (TCT); 3) Position 124: Sebastes thompsoni is guanine (G), Sebastes trivittatus is cytosine (C), and the other five are thymine (T); 4) Position 167: Sebastes pachycephalus is thymine (T), Sebastiscus marmoratus The first is adenine (A), and the other five are cytosine (C).

[0030] By detecting the above-mentioned sites, accurate molecular identification of seven target species of the genus Scorpaenidae was achieved.

[0031] Example 3: Detection of mixed eDNA The extracted eDNA of seven fish species was diluted to 20 ng / uL, and 10 equal amounts of each sample were mixed to form simulated mixed eDNA as analysis samples.

[0032] The mixed eDNA was diluted to 5 ng / uL as template for PCR amplification, and the 25 µL reaction system and reaction program were the same as those for the identification of Sebastidae species based on 16S rRNA gene sequences.

[0033] The purified PCR products were quantified using a Qubit® 3.0 fluorometer. After mixing 24 amplicons with different barcodes in equal amounts, a double-end sequencing library was constructed according to the Illumina genomic DNA library preparation process, and double-end sequencing was performed on the Illumina MiSeq PE 250 platform. After sequencing, the raw data was processed by the following process: low-quality sequences (such as reads with a length of less than 50 bp or a tail quality value of less than 20) were filtered using the Trimmomatic software. The paired reads were merged into complete sequences based on the overlapping relationship of the original reads using the FLASH software. Chimeras were removed by the denovo and reference sequence combined strategy of the Usearch software, and the primer sequences were removed using the Cutadapt tool. Sequence denoising was performed by the dada2 process, and the de-redundant sequences after quality control were defined as amplicon sequence variants (ASV). Based on the self-built 16S gene database of Sebastodes fish in the Yellow Sea and Bohai Sea, the representative sequences of ASV were annotated for species classification.

[0034] The results showed that the molecular marker could successfully detect the specific fragments of Sebastes scholii ( Sebastes schlegelii ), Sebastodes koreanus ( Sebastes koreanus ), Sebastodes crassispinis ( Sebastes pachycephalus ), Sebastodes tomzani ( Sebastes thompsoni ), Sebastodes annularis ( Sebastes trivittatus ), Sebastodes armatus ( Sebastes hubbsi ), Sebastodes inornatus ( Sebastiscus marmoratus ), etc. among the mixed samples, confirming that the screened specific markers and the primer pairs used could be used to monitor the above 7 species of Sebastidae in eDNA.

Claims

1. A molecular marker for identifying species of the genus Scorpaenidae in the coastal waters of China, characterized in that: The molecular marker comprises the following fragments: 1) Detection of Scorpionfish ( Sebastes schlegelii ) marker, the sequence of which is SEQ ID NO: 1, 2) Detection of Korean scorpionfish ( Sebastes koreanus ) marker, the sequence of which is SEQ ID NO: 2, 3) Detection of Thickhead Scorpionfish ( Sebastes pachycephalus ) marker, the sequence of which is SEQ ID NO: 3, 4) Detection of Tang's flathead scorpionfish ( Sebastes thompsoni ) marker, the sequence of which is SEQ ID NO: 4, 5) Detection of striped scorpionfish ( Sebastes trivittatus ) marker, the sequence of which is SEQ ID NO: 5, 6) Detection of armored flathead scorpionfish ( Sebastes hubbsi ) marker, the sequence of which is SEQ ID NO: 6, 7) Detection of brown scorpionfish ( Sebastiscus marmoratus ) marker, whose sequence is SEQ ID NO:

7.

2. Use of the molecular marker according to claim 1 in the preparation of a molecular product for identifying species of the genus Scorpaenidae in the coastal waters of China.

3. The use according to claim 2, characterized in that The product is a PCR amplification sequencing kit.

4. A molecular detection product for identifying species of the genus Scorpaenidae in the coastal waters of China, characterized in that: The product contains primers for detecting the above molecular markers.

5. The molecular detection product according to claim 4, wherein the upstream primer sequence is SEQ ID NO: 8, and the downstream primer sequence is SEQ ID NO:

9.

6. A method for identifying species diversity of the genus Scorpaenidae from eDNA, characterized in that: The method is to identify the species diversity of the genus Scorpaenidae by detecting the genotype distribution of the molecular markers according to claim 1 in the eDNA sample.

7. The method according to claim 6, wherein The method comprises the following steps: 1) Extraction of eDNA from seawater; 2) Perform PCR amplification using primers; 3) Perform amplicon sequencing on the PCR products to detect the genotype distribution of molecular markers; 4) Determine the distribution of Scorpaenidae species in the environment based on the genotype distribution of molecular markers obtained through amplicon sequencing.

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