北海道水産試験場研究報告第86号

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1 Sci.Rep.Hokkaido Fish.Res.Inst. Identification of oysters in Okushiri Island by mitochondrial 16 S rrna analysis TAKUMA KAWASAKI 1,YOHEI SHIMIZU 1,MUTSUMU IWASA 2,SHINYA YOSHIDA 3 and YASUHIRO KUWAHARA 4 1 Mariculture Fisheries Research Institute, Hokkaido Research Organization, Muroran, Hokkaido Okushiri Fishery Technical Guide Office, Okushiri, Hokkaido Oshima Fishery Technical Guide Office, Hakodate, Hokkaido Abashiri Fisheries Research Institute, Hokkaido Research Organization, Abashiri, Hokkaido , Japan Oysters around Okushiri Island are difficult to classify based on appearance. Therefore, we had identified their species using DNA analysis. After obtaining the results, we explored the relationship between species and habitat. Oysters were collected by scuba divers at 1 9 m depth in the sea at 7 different areas around Okushiri Island from November 2011 to the next November. It was difficult to identify the 297 oysters based on their morphological characters. Therefore, we analyzed the nucleotide sequences of mtdna 16S rrna genes. Polymerase chain reaction with a template of total DNA from muscle tissues of oysters successfully amplified DNA fragments of 464 bp in the 16S rrna gene, which were identified as Crassostrea gigas, C. nippona, Ostrea circumpicta, andsaccostrea sp. All of the areas had at least two different species, and species matched their characteristic shell size and depth of habitation, but these were insufficient for certain classification. Therefore, the DNA fingerprinting technique were shown to be beneficial for the classification of oysters. PCR DNA Crassostera nippona Seki, C. gigas Thumberg, DNA c 1 O Foighil et al., S RNA O Foighil et al., 1995 DNA ITS 2000 DNA 2008 DNA 16S RNA A Tel: Fax: E mail: kawasaki takuma@hro.or.jp

2 A G 297 Table1 DNA 30 Number of oyster samples per collecting point and depth SDS Proteinase 2008 DNA Wizard Genomic DNA Purification Kit Promega DNA DNA Nanodorop PCR 30 DNA DNA 16S RNA PCR PCR 16Sar:5 CGCCTGTT TATCAAAAACAT 3 16Sbr:5 CG GTCTGAACT CAGATCACG 3 Kessing et al., 1989 PCR 10µL 10 Ex Taq Buffer 1µl 2.5mM dntp Mixture 0.8µl 5µM 0.5µl TaKaRa Ex Taq 0.05µl DNA ng PCR Veriti PCR PCR ExoSAP IT dntps 30 PCR PCR BigDye Terminator v3.1 Cycle Sequencing Kit PCR PCR Agencort CleanSEQ kit PCR PCR Applied Biosystems 3130xl NCBI BLAST Kaminuma et al., S RNA DDBJ DNA Ostrea circumpicta Pilsbry, ClastalX Thompson et al., 1997 MEGA4 Tamura et al., S RNA Saccostrea sp Liu et al., Saccostrea kegaki Torigoe & Inaba, 1981 S. echinata Quoy & Gaimard, 1835 Fig.1 A F 4 C E 3 B D G 2 B E F A C G D 1 Table2 4 7

3 Tree diagram based on a partial sequence 464 bp of the 16S ribosomal RNA gene of Ostreidae major and the Okushiri oysters. Symbols and numbers in parentheses indicate the Genebank accession number. The numbers in the branched graph shows the match percentage from 1000 bootstrap extractions. Table 2. Results of classification by DNA fingerprinting DNA 3 Fig Fig.3 Fig.4 3 DNA mm mm mm mm mm mm mm mm Fig.5 3m 1 9m 3m 3m 7 9m Fig.6

4 Shells of oysters from station F. Left: Ostrea circumpicta, Center:Crassostrea nippona, Right: Crassostrea gigas. Characteristic shell shape arrow of Saccostrea sp.

5 遺伝子解析による奥尻島周辺のカキ類の種判別 Fig.4 Shells of Rock Oysters Crassostrea nippona from each station. １４１

6 Frequency distribution of shell height. Frequency distribution of collecting depth.

7 DNA 16S RNA DNA DNA Ostrea Ostrea denselamellosa Lischke, S RNA 1 Saccostrea kegaki Torigoe & Inaba, 1981 S. echinata Quoy & Gaimard, m 110mm DNA X pls DNA LAGUNA No pl. I XIII 1 9 Kaminuma E, Mashima J, Kodama Y, Gojobori T, Ogasawara O, Okubo K, Takagi T, Nakamura Y. DDBJ launches a new archive database with analytical tools for next generation sequence data. Nucleic Acids Research, 2010; 38; Database issue D33 D38 Kessing B, Croom H, Martin A, McIntosh C, McMillian WO, Palumbi SP. The simple fool s guide to PCR. University of Hawaii, Honolulu, USA No Liu J, Li Q, Kong L, Yu H, Zheng X. Identifying the true oysters Bivalvia: Ostreidae with mitochondrial phylogeny and distance based DNA barcoding. Mol. Ecol. Resour. 2011; 5:

8 O Foighil D, Gaffney PM, Hilbish TJ. Difference in mitochondrial 16 S ribosomal gene sequences allow discrimination among American Crassostrea virginica Gmelin and Asian C. gigas Thunberg, C. ariakensis Wakiya oyster species. J. Exp. Mar. Biol.Ecol., 1995; 192: O Foighil D, Gaffney PM, Wilbur AE, Hilbish TJ. Mitocondrial cytochrome oxidase gene sequence s support an Asian origin for the Portuguese oyster Crassostrea angulata. Mar.Biol., 1998; 131: Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis MEGA software version 4.0. Molecular Biology and Evolution 2007; 24: URL: pref.hiroshima.lg.jp/uploaded/attachment/41678.pdf Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The Clustal_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 1997; :

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