Report of Systematic Zoology Lab Practicum, August, 2011


28S ribosomal RNA partial sequence of Terebellidae sp. (Annelida: Polychaeta)


Shinichiro Honma

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



Material and Methods
A polychaete was obtained among holdfast of brown algae at Oshoro Bay, Hokkaido, Japan, about 43°12′N, 140°51′E, on 6 June 2011 by Laurynas Vanagas, photographed and identified by Hiroshi Kajihara as Terebellida sp, and fixed in 99% EtOH. DNA was extracted from one of the peristomial tentacles 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 ICHU22090151 (contact: Dr. Hiroshi Kajihara, kazi@mail.sci.hokudai.ac.jp).
      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.
      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, 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). 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.
      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 v4 software (Tamura et al. 2007).

Results
A total of 1045 bp of 28S rRNA partial sequence overlaping at the center was determined from Terebellidae sp. (see Appendix).

Taxonomy
Phylum Annelida
Class Polychaeta
Order Telebellida
Terebellidae gen. et sp.
(Fig. 1)



Fig. 1. Terebellidae sp. (ICHU22090151), lateral view.



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.

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. MEGA4: Molecullar Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Phylogenetics and Evolution 24: 1596–1599.




Appendix
28S rDNA gene sequence from Terebellidae sp. (ICHU22090151).

CTCAGAGAGGGTGACAGGCCTGTAcGGCGTCGCGgACTACCGTGCCAAAAGGCGTCCCTAGAGTCGCGTtgTTTGGGATTGCAAcGCAAAGTAGGTGGTAGACTCCATCCAAGGCTAAATACTGTCAcGAGTCCGATAGCGGACAAGTACCGTGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCAACAGTACGTGAAACCGTTTAGAGGCAATCGGATGGGACCGCAAATAGTCCGACCGGGCGGGCCCGTGTTTTTCGGAACACGGGTTCCATCGGTTCTCGGTTGGgcGAATGCCACGACCGGTTcGGCGGTGGCGTAAAATCCTCGCATTAGGTAGGTCGTCGGTGGCCCTCGGGTCCCGGCTTCCGTTTGGGTGTGAGCGAAGATGGCCGCACTGCCGGACCGAGG