Barcoding of Nematodes

In this work package a base list of 32 nematode species was created for which barcodes needed to be collected

Objectives

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

  • Establish a prioritized list of all phytopathogenic nematodes relevant for the EU to be barcoded.
  • Produce data to be used as a decision basis for the choice of 2-3 nematode barcode genes and regions.
  • Compare and validate reliable DNA extraction procedures.
  • Develop robust primers for PCR amplification of the nematode barcode gene regions.
  • Collect barcode data including intra- and interspecies genetic variation on the nematode barcode regions.

Tasks

  • Task 5.1  Selection of targets
  • Task 5.2  Selection of barcode regions
  • Task 5.3  DNA extractions
  • Task 5.4  Generation of protocols for amplification of barcode regions
  • Task 5.5  Generation of Barcode sequences

Deliverables

  • D 5.1 List of selected nematode Q-organisms (Month 6)
  • D 5.2 List of barcode regions for selected nematode Q-organisms (Month 12)
  • D 5.3 Two protocols for DNA extraction for selected nematodes Q-organisms (Month 12)
  • D 5.4 List of protocols for generic amplification of barcode regions (Month 18)
  • D 5.5 About 1600 Barcode sequences of the selected nematode Q-organisms (Month 36)
  • D 5.6 Detailed contingency plan for WP5 (Month 6)

Results

In this work package a base list of 32 nematode species was created for which barcodes needed to be collected. This list contained all quarantine nematodes as well as a number of close relatives. In addition, 43 nematodes species were nominated which would be sequenced if time permitted. These additional species were composed of further relatives of Q-organisms as well as a number of other agronomically relevant nematode species. Material for most of the species on the base list has been acquired as well as material for a large number of additional species.

Five DNA isolation methods were compared, including both commercial kits as well as published methods, and the best two methods were selected for further use.

Primers were developed for the amplification of six potential barcoding regions: the small subunit (SSU) ribosomal RNA gene, the D1-D2 and D2-D3 regions of the large subunit (LSU) ribosomal RNA gene, the second intragenic spacer region (IGS2) of the ribosomal RNA cassette, a fragment of the RNA polymerase II gene and the mitochondrial cytochrome oxidase c subunit 1 (COI) and subunit 2 (COII) genes. A subset of nematode species was assigned to assess both the inter- and intra-species variation of these potential barcode regions and based on these results the SSU, LSU, COI and COII genes were chosen for sequencing in the remaining nematode species.

A total of 1600 sequences for up to 58 species, distributed over the various priority groups, was promised in the project. For each priority group the required amount of sequences was generated and in some cases well surpassed. Of all generated sequences, 1683 were of a high enough quality for inclusion in the Q-Bank nematodes database, originating from a total of 121 species.