Henk J. Schouten*, Alexander H.J. Wittenberg*, Andrzej Kilian#,
*Plant Research International, PO Box 16, 6700 AA Wageningen, The Netherlands;
#Diversity Arrays Technology Pty Limited, CAMBIA Bdg, GPO Box 3200, Canberra, ACT 2601, Australia
DArT™ (Diversity Arrays Technology) is a novel technique for analysis of DNA polymorphism. DArT is based on hybridisation and uses micro-arrays as a platform. This makes gel-based separation and reading of bands redundant, therewith increasing the throughput and reducing the costs per data point. DArT does not require DNA sequence information. This enables applications in practically any species. We validated this technology, using Arabidopsis thaliana as a model. We digested genomic DNA of A. thaliana Landsberg erecta (Ler) using three restriction enzymes. After attachment of adapters we amplified a specific set of restriction fragments. A subset of these fragments was individualised by means of cloning, and spotted on micro-array slides. We then hybridised the arrays simultaneously with restriction fragments amplified from Ler and labelled with Cy3 and restriction fragments amplified from A. thaliana Colombia (Col) and labelled with Cy5. Spotted fragments that showed a high hybridisation signal for Ler but a low signal for Col were putative marker fragments. These fragments were sequenced. Using BLAST, these marker sequences from Ler were compared with the published Col DNA sequence. The comparison allowed us to validate nearly all of the candidate markers and determine the molecular basis of their polymorphism. We made a genetic linkage map containing the DArT markers, using JoinMap. This genetic linkage map showed a perfect collinearity with the sequence map of A. thaliana. This underlines the quality of the DArT markers.
Contact: henk.schouten @ wur.nl, Alexander.wittenberg @ wur.nl, a.kilian @ cambia.org.au