Barcoding of Fungi

In this work package a short list of 19 Q-species were selected for barcoding.

Objectives

To generate barcode sequences from a selected set of genetic regions and for a selected set of relevant/quarantine and related fungi. To reach this aim we will:

  • Establish a prioritized list of all phytopathogenic fungi relevant for the EU to be barcoded.
  • Maintain and expand existing collection(s) with relevant fungal isolates.
  • Produce data to be used as a decision basis for the choice of 3 fungi barcode genes and regions.
  • Evaluate existing or develop new reliable DNA extraction procedures.
  • Develop robust primers for PCR amplification of the fungal barcode gene regions.
  • Collect barcode data including intra- and interspecies genetic variation on the fungal barcode regions.

Tasks

  • Task 2.1  Selection of targets
  • Task 2.2  Selection of barcode regions
  • Task 2.3  DNA extractions
  • Task 2.4  Generation of protocols for amplification of barcode regions
  • Task 2.5  Generation of Barcode sequences

Deliverables

  • D 2.1 List of available fungal Q-organisms and related species (Month 6)
  • D 2.2 List of potential barcode regions for selected fungal Q-organisms – five candidate loci (Month 6), workable list of barcode regions – two loci (Month 15)
  • D 2.3 Three protocols for DNA extraction for selected fungal Q-organisms (Month 12)
  • D 2.4 List of 2 protocols for generic amplification of barcode regions (Month 15)
  • D 2.5 At least 110 barcode sequences of the selected fungal Q-organisms and 132 barcode sequences for the related species (Month 34)
  • D 2.6 Detailed contingency plan for WP2 (Month 6)

Results

In this work package a short list of 19 Q-species were selected for barcoding. For those species several gene regions were screened to identify suitable barcoding loci. Protocols for efficient DNA extraction, generic amplification and sequencing of the selected loci were evaluated. For some species it was difficult to obtain larger numbers of isolates per quarantine species. This work package succeeded to make available to the Q-bank database: 791 sequences for 193 strains from 25 quarantine species; as well as 6107 sequences from 1145 strains of 612 related species. Of these sequences, 360 sequences for 81 species were extracted from studies in peer-reviewed journals (mainly for the related species of the unculturable obligate biotroph genera Melampsora, Puccinia and Thecaphora). For each species of quarantine importance in the Q-bank database, hyperlinks to EPPO and EU Council Directive documents are provided. A field “Diagnostic locus for identification in Q-bank” is present for each quarantine species to help the end-user to determine which locus is needed for identification in the database and polyphasic identifications are possible per genus group. A molecular decision scheme showing the route to an identification starting with DNA isolation and amplification of the internal transcribed spacers (ITS) of the nrRNA operon as primary barcode is provided on the Q-bank fungi website. A link to MycoBank, a database for the taxonomy of fungal names, is also provided for each species.