DEVELOPMENT OF A MOLECULAR BARCODE SYSTEM FOR SOIL NEMATODE IDENTIFICATION

In Edinburgh, the Blaxter Nematode Genetics lab has been awarded a three year grant by the NERC Soil Biodiversity Initiative to develop and apply a molecular barcode identification system for soil nematodes. The workers on the grant are Dr. Artemis Papert and Robin Floyd.

Nematodes are speciose but morphological resolution of their diversity is hindered by a clash between the detail and the wider picture available from morphology. On the one hand, much of the morphology is only discernable at the level of the high power light microscope and very few characters in relation to the known or expected species number are available to systematists for study. On the other hand, rank homoplasy and uncertainty in assigning homology makes the use of even these few characters problematic. Indeed, for any one taxonomic nematologist to become expert in more than one strictly circumscribed group is rare. Beyond this, identification is often impossible without a population to study, with males and females available, and the identification of larval stages is well-nigh impossible.

Molecular barcodes offer a route to identification that promises to be both specific (in that taxa may be identified and placed taxonomically with some certainty) and relatively easy to use. It is based on the premise that some molecular changes will become fixed in the genomes of nematode species at rates equivalent to or slightly greater than the rate of cladogenesis (speciation) and thus will retain a record of these events. Two species can be typed for their DNA, and this DNA fingerprint or barcode used to provide a diagnosis. While it is possible to use anonymous markers for this (eg AFLP) there are problems with subsequent use of the dataset to place novel taxa into preexisting schemas, and with the definition of how much difference corresponds to a distinct taxon. DNA sequence data offers a set of characters that may both barcode the organism, and allow taxonomic and phylogenetic reconstruction. The ideal genetic marker for barcode development would combine ease of measurement with a mix of conserved and rapidly evolving segments. The ribosomal RNA gene cistron meets these criteria: it is a homogenous, multi-copy gene with well tested primer sites for amplification and sequencing. We have developed a set of nematode-specific primers for use with the small subunit gene (18S rDNA). The 5' end of the 18S rDNA contains much of the variability of the whole molecule and is a facile target for single nematode amplification protocols.

Molecular barcoding involves isolation of the nematodes (as individuals or in bulk), amplification of the target gene (as individuals or in bulk), and then processing of the PCR products to assess species content, species anbundance and diversity.

The final funding of the project was ~60% of the original application, so we are currently reconfiguring the program of work to suit this reduced award.