Sequence and
polymerase chain reaction-restriction fragment length polymorphism analysis
of the large subunit rRNA gene of bivalve: Simple and widely applicable
technique for multiple species identification of bivalve larva
M. Hosoi, S. Hosoi-Tanabe, H. Sawada, M.
Ueno, H. Toyohara, I. Hayashi-2004
Fisheries
Science, 70(4):
629-637
Abstract:
One of the biggest and long-standing
difficulties in investigation of larval ecology in the field is
species-level identification. In the present study, we developed polymerase
chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis
based on the large subunit (LSU) rRNA gene (rDNA) D1/D2/D3 region for
identification of multiple species of bivalve larvae using 14 species of
bivalve collected from Maizuru Bay. The LSU rDNA D1/D2/D3 region of all
analyzed species could be amplified by PCR using bvD1f/bvD3r primers, and
RFLP analysis by HaeIII digestion on the PCR products showed
species-specific fragment patterns. Furthermore, this analysis applied to
single bivalve larvae in Maizuru Bay revealed efficient amplification of the
target region and the species-specific pattern from 80% of the larvae, 75%
of which showed a pattern that matched a certain pattern of the adult
bivalves. In addition, the analysis of inter- and intraspecies variation of
the LSU rDNA D1/D2/D3 region using the sequence data of the genus Crassostrea
from the DDBJ database showed high applicability of this RFLP analysis on
closely related species. Because of the wide applicability and technical
simplicity, this method can become the standard for the identification of
bivalve larvae species once the sequence data of the LSU rDNA D1/D2/D3
region of many bivalve species have been accumulated.
(Division of Applied Biosciences, Graduate School of
Agriculture, Kyoto University, Kyoto 606-8502, Japan, Tel:
81-75-753-6446. Fax: 81-75-753-6446. Email of H. Toyohara: toyohara@kais.kyoto-u.ac.jp)