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.