Report of Systematic Zoology Lab Practicum, Volume 4: e08; August, 2013


28S ribosomal DNA partial sequence of the scallop Chlamys (Azumapecten) farreri akazara (Mollusca: Bivalvia) from Oshoro Bay, Hokkaido, Japan


Takumi Adachi and Wataru Saitou

Division of Biology, Department of Biological Sciences, School of Science, Hokkaido University, Sapporo 060-0810, Japan



Material and Methods
A scallop was obtained in the intertidal zone of Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 6 may 2013 by Takumi Adachi, photographed and identified by Hiroshi Kajihara as Chlamys (Azumapecten) farreri akazara (Kuroda, 1932) by a reference to Hayami (2000), and fixed in 99% EtOH. DNA was extracted from the adductor muscle using the silica method (Boom et al. 1990) with some modifications. Extracted DNA was dissolved in 30 µl of deionized water and has been preserved at –20°C. Remaining morphological voucher specimen was in good condition and has been deposited at the Hokkaido University Museum under the catalogue number ICHU2110622 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      An about 1.2Kb fragment of 28S rRNA gene was amplified by polymerase chain reaction (PCR) using LSU5 (5′-ACCCGCTGAAYTTAAGCA-3′) and LSU3 (5′-TCCTGAGGGAAACTTCGG-3′) (Littlewood et al. 1994). A hot start PCR was performed by a thermal cycler, 2720 Thermal Cycler(Applied Biosystems, in a 20-µl reaction volume containing 1 µl of template total DNA (approximately 10–100 ng) and 19 µl of premix made with 632-µl deionized water, 80-µl Ex Taq Buffer (TaKara Bio), 64-µl dNTP (each 25 mM), 8-µl each primer (each 10 µM), and 0.1-µl TaKara Ex Taq (5 U/µl,TaKara Bio). Thermal cycling condition comprised an initial denaturation at 95°C for 30 sec; 30 cycles of denaturation at 95°C for 30 sec, annealing at 45°C for 30 sec, and elongation at 72°C for 45°C and a final elongation at 72°C for 7 min. Amplification of mitochondrial cytochrome c oxidase subunit I gene (COI) using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994) was unsuccessful.
      The PCR product was purified with the silica method (Boom et al. 1990). Both strands were sequenced with a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) following the manufacturer's protocol, using the same primer set as the initial PCR amplification. Sequencing was performed with ABI Prism 3730 DNA Analyzer (Applied Biosystems). Chromatogram and sequence data were operated with MEGA v5 software (Tamura et al. 2007).

Results
A total of 1093 bp of 28S rRNA partial sequence overlaping at the center was determined from Chlamys (Azumapecten) farreri akazara (see Appendix).

Taxonomy
Phylum Mollusca
Class Bivalvia
Order Pectinida
Family Pectinidae Rafinesque, 1815
Genus Chlamys Röding, 1798
Chlamys (Azumapecten) farreri akazara (Kuroda, 1932)
[Japanese name: akazara-gai]
(Figs 1, 2)


Fig. 1. Chlamys (Azumapecten) farreri akazara (Kuroda, 1932) (ICHU2110622).



Fig. 2. Chlamys (Azumapecten) farreri akazara (Kuroda, 1932) (ICHU2110622.



References

Boom, R., Sol, C. J. A., Salimans, M. M. M., Jansen, C. L., Wertheim-van Dillen, P. M. E., and Van der Noordaa, J. 1990. Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 28: 495–503.

Folmer, O., Black, M., Hoeh, W., Lutz, R., and Vrijenhoek, R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299.

Hayami, I. 2000. Pectinidae. Pp. 897–911. In: Okutani, T. (Ed.) Marine Mollusks in Japan. Tokai University Press, Tokyo.

Littlewood, D. T. 1994. Molecular phylogenetics ofcupped oysters based on partial 28S rRNA gene sequences. Molecular Phylogenetics and Evolution 3: 221–229.

Tamura, K.,Dudley, J., Nei, M. and Kumar, S. 2007. MEGA5: Molecullar Evolutionary Genetics Analysis (MEGA) software version 5.0. Molecular Phylogenetics and Evolution 24: 1596–1599.




Appendix
28S rDNA gene sequence from Chlamys (Azumapecten) farreri akazara (Kuroda, 1932) (ICHU2110622).

TATCACTAAGCGGAGGAAAAGAAACTAACAAGGATTCCCCCAGTAACGGCGAGTGAAGTGGGAATAGCCCAGCACCGAATCCCTCAGCCTTGCGCTGCCGGGACCTGTGGTGTTTGGGACGACCGCTGTCGTGTTGTTCGGGTGCCCAAGTCCTCCCGATCGGGGCCTCTCCCACAGTGGGTGTCAGGCCTTTACCGGCACTCGTCGACACGGCTATGGTCGTCCTCGGAGTCGGGTTGTTTGGGAATGCAGCCCAAAGTTGGTGGTAAGCTCCATCTAAGGCTAAATACTGACACGAGTCCGATAGCGGACAAGTACCGTGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCAATAGTACGTGAAACCGCTTAGAGGCAAACGGGTGGATCCGTAAAGTCGACCCGGGGAATTCATCTTGCTGTCAGCGACGGGCGTGTGGCAGAGATTCCTCACGGGACTCTGCTGGCGTCCGGTTGCCGCCGGCGAGTGCACTTTCCCCGGGTTGAGCGCCACGACCGGTTTTCTGGCGGTCATAAGCTCCGCGAGAAGGTAGCTCGTCTCTTCGGGGTCGAGTGTTATAGCTCGCGGTAGTTGTCTTGCCGGGAGACTGAGGGTCTCCAGCGCCTGTCGGCCCGAACTAGCCTGCGTCCGTTCAACTGGGGTAGACTGCTTGCAGTGTTCTCCGACCGCGAGCGTAGTGTGGGTTCGCAGCCCGGCGGCGCGTTGGGTCAGTGGCGATtCGGTCGGCATTCCACCCGACCCGTCTTGAAACACGGACCAAGGAGTCTAACATGTGCGCGAGTCATGGGGTCTTACGAAACCTAAAGGCGCAATGAAAGTGAAGGCAGCCTCCGGTTTGCCTAGGTAGGATCCTTCCTGTAACGGGGAGGCGCACTACCGGCCCGTCTCGTCCGCATTGCCGGGGAGGCGGAGCAAGAGCGTACACGTTGGGACCCGAAAGATGGTGAACTATGCCTGAGTAGGACGAAGCCAGAGGAAACTCTGGTGGAGGTCCGTAGCGATTCTGACGTGCAAATCGATCGTCAAACCTGGGTATAGGGGCGAAAGACTAATCGAACCATCTAGTAGCTGGTTCCCTCCG