Report of Systematic Zoology Lab Practicum, Volume 5: e05; August, 2014


Partial sequence of the nucleus-encoded 28S rRNA gene of Littorina brevicula (Gastropoda: Littorinimorpha: Littorinidae) from Oshoro, Hokkaido, Japan


Mana Nishikata and Rika Endo

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



Material and Methods
A gastropod was obtained in the intertidal zone of Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 28 May 2014 by Mana Nishikata and Rika Endo, identified by Hiroshi Kajihara as Littorina brevicula (Philippi, 1844), and then photographed and fixed in 99% EtOH by Takumi Onishi. DNA was extracted from the EtOH-fixed tissue, 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 has been deposited at the Hokkaido University Museum under the catalogue number ICHU2120420 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      PCR amplification of the mitochondrial cytochrome c oxidase subunit I (COI) gene using LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994) was unsuccessful. A hot start PCR was performed by a thermal cycler, iCycler (Bio-Rad), 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). The 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 sec; and a final elongation at 72°C for 7 min.
      An about 1.2K-bp fragment of 28S rRNA gene was amplified by using the primer pair LSU5 (ACCCGCTGAAYTTAAGCA) and LSU3 (TCCTGAGGGAAACTTCGG) (Littlewood. 1994). The thermal cycling condition was the same for COI exept that the elongation time was set for 3 min.
      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. 2011).

Results
Phylum Mollusca
Class Gastropoda
Order Littorinimorpha
Family Littorinidae
Genus Littorina
Littorina brevicula (Philippi, 1844)
[Japanese name: tamakibi-gai]

(Fig. 1)


Only 223 bases near the 5′ end of the PCR products were reliably determined because the chromatogram in the 3′ region was more or less disturbed. A nucleotide BLAST search (Altschul et al. 1997) at the NCBI website (https://blast.ncbi.nlm.nih.gov/) showed that our sequence from Oshoro was 99% identical with HE590805 (100% in query coverage; E value = 6e-109), a sequence of the same species from Goura, Otsu, Kitaibaraki, Ibaraki (Reid et al 2012).


Fig. 1. Littorina brevicula (Philippi, 1844) (ICHU2120420) from Oshoro, Hokkaido, Japan.


References

Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389–3402.

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.

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

Tamura, K.,Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. 2011. MEGA5: Molecullar Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Phylogenetics and Evolution 24: 1596–1599.



Appendix
A 223-bp partial sequence of the 28S rRNA gene from ICHU2120420 identidied as Littorina brevicula.

CTAGATGGTTCGATTAGTCTTTCGCCCcTATACCCAAGTTTGACGATCGATTTGCACGTCAGAATCGCTGCGGACCTCCACCAGAGTTTCCTCTGGCTTCGTCCTACTCAGGCATAGTTCACCATCTTTCGGGTCCCAACGTGCGCGCTCATGCTCCGCCTCACCGACAACGCGGACGAGACGGGCCGGTGGTGCGCCCCCAACATTTGAGGGATCCCACCTG