In this work package 198 species of Q-arthropods have been divided into two priority groups.
To generate from 5-10 barcode sequences (COI and ITS) for about 100 species of Q arthropods (priority 1), and about 50 closely related species.
To reach this aim we will :
- Establish a prioritized list of all Q arthropods relevant for the EU to be barcoded, plus a list of the related species that could be misidentified.
- Collect all Q arthropods and nominated associated species, each from a variety of localities and/or crop hosts.
- Develop reliable DNA extraction procedures for larvae and adults.
- Develop and test primers for PCR amplification of the two proposed genes (COI & ITS) for all species of Q arthropods and nominated associated species.
- Collect DNA barcode sequence data for the two proposed genes (COI & ITS) for all species of Q arthropods and nominated associated species.
- Test the usefulness of this dataset together with independently generated data in BOLD for accurate identification of the Q arthropods.
- Task 3.1 Sampling of species
- Task 3.2 Selection of barcode genes and regions
- Task 3.3 DNA extractions and generation of protocols for amplification of barcode regions
- Task 3.4 Generation of Barcode sequences
- D 3.1 List of selected Q- arthropods and non-Q congeneric species (Month 6)
- D 3.2 List of barcode regions for selected Q-arthropods (Month 6)
- D 3.3 Three protocols for DNA extraction for selected Q-arthropods (Month 12)
- D 3.4 List of protocols for amplification of 2 barcode regions for all taxonomic groups (Month 18)
- D 3.5 About 3000 Barcode sequences of the Q-arthropods (Month 30)
- D 3.6 Providing diagnostic tool (including any current limitations) (Month 36)
- D 3.7 Detailed contingency plan for WP3 (Month 6)
In this work package 198 species of Q-arthropods have been divided into two priority groups. The lists have been erected based on the economic value of the Q-arthropods, their availability and their habitat, trying to cover both agriculture and forest pests. Many contacts with colleagues and many field trips have been made to get the required specimens.
Several DNA extraction methods that are commonly used with arthropods have been tested. Three of them were selected. These three methods performed better and/or are easy to use by non-trained people. Also, a non-destructive protocol was developed.
Cox-1 region and the ITS-2 region of the rDNA were selected to be barcoded and primers for those regions were developed and tested.
The initial objectives were to generate from 5-10 barcode sequences (COI and ITS) for about 100 species of Q arthropods (priority 1), and about 50 closely related species. For priority group 1, 83 Q-species (79.8%) were sequenced for a total of ca. 2500 sequenced PCR amplicons with an average of 20 COI and 10 ITS sequences per species. For priority group 2, 52 Q-species (54.7%) were sequenced for a total of about 1300 sequences with an average of 16 COI and 8 ITS sequences per species.
20 species were included that are not yet considered quarantine species, though they represent a threat to Europe. This priority 3 list was established during the project. Including these species makes this identification tool more adapted to European needs. For these species, a total of ca. 200 sequences with an average of 7 COI and 3 ITS sequences per species were obtained. Instead of the 50 out group species initially proposed (i.e. species that are congeneric of or could be confused with the Q-arthropods), 128 species were sequenced, producing about 1300 sequences with an average of 7 COI and 4 ITS sequences per species.
To improve the representativeness of the Q-bank database of arthropods (i.e. include Q-arthropod species included on priority 1 and 2 lists but not yet available or increase intraspecific variability for better identification), 334 COI sequences mostly produced by USDA and mined from GenBank have been added to our sequence library.
All sequences were carefully validated before inclusion in the Q-bank database (detection of contamination and pseudo genes). Altogether, about 5300 sequences have been generated during the project and 334 sequences have been mined from Genbank. The database now includes ca. 5600 COI and ITS barcodes for 153 species of Q-arthropods (ca. 77% of the priority 1 and 2 lists), 20 species of arthropods that are considered a serious threat for Europe and 140 out group species, far surpassing the original aim. All specimens have been identified by taxonomists, vouchers in INRA and LNPV Montpellier.