2.1. PHASE 1 - DEFINITION OF TARGET TAXA AND ESTABLISHMENT OF
REFERENCE DATABASES
The first step was to identify the main purpose of the toolkit and the
research questions being addressed. In the case of this study, the goal
was to develop a screening tool capable of determining the presence of
bivalve and crustacean larvae in zooplankton samples, with particular
interest in the Irish shellfish fisheries and aquaculture industry.
Thus, a total of 24 species were chosen based on their ecological and
economic importance in the context of Irish coastal waters, including
seventeen bivalve species and decapod species (Table 1 ). To
identify the most suitable genes for molecular species identification, a
literature review was conducted for previously published studies
including genetic markers designed for taxonomic resolution of selected
taxa. In order to identify the final target and off-target species
occurring in the geographic region of interest (Irish marine coastal
waters), a review of organisms of interest and other closely related
species (i.e. same Order) was carried out by consulting the World
Register of Marine Species (WoRMS) and Ireland’s National Biodiversity
Data Centre databases (August 2022).
A dedicated reference DNA database was created by combining publicly
available barcode sequences with new data from locally sourced
organisms. Firstly, based on results obtained from the above review of
target and off-target species, previously published DNA sequence data of
selected genes from all relevant taxa was obtained from the Barcode Of
Life Database (BOLD) and GenBank database. Secondly, to fill any data
gaps and confirm target DNA sequences of local taxa, DNA barcode
sequence data was also obtained from taxonomically identified voucher
specimens (e.g. adult individuals obtained from local markets or
opportunistically). For this latter step, DNA was isolated from 50 mg of
muscle tissue using the Blood and Tissue DNEasy kit (Qiagen), and 658 bp
fragments of the COI gene were amplified and sequenced following the
protocol described in . Finally, the resulting dedicated reference DNA
database was organized in relevant gene-specific alignments created
using the mafft alignment plugin as implemented in Geneious Prime
2020.2.1 (Biomatters
Ltd).