CLONING AND CHARACTERIZATION OF DM3 AND RELATED SEQUENCES FROM LETTUCE

¹ Department of Vegetable Crops, University of California, Davis, CA 95616 USA
² Present address: Dupont Agricultural Products - Biotechnology, PO Box 6104, Newark, DE 19714-6104 USA

The major resistance gene cluster in lettuce contains at least ten Dm genes, which provide resistance against the fungal pathogen Bremia lactucae. This cluster has been studied extensively using classical genetic techniques and includes the target of our cloning efforts, the Dm3 specificity. Using a combination of map-based cloning and candidate gene techniques, we have cloned a family of genes from the region containing Dm3 and analyzed the physical organization of this family. A physical map of the Dm3 region was constructed using a combination of a large number of molecular markers, a panel of fast-neutron irradiated mutants, and BAC clones mapped to this region. Sequence analysis of these BACs revealed a large and diverse multigene family (the RGC2 family). The RGC2 family is comprised of more than 24 members, spanning at least 3.5 Mb; most members have been mapped within the Dm3 region. These resistance gene candidates encode a predicted nucleotide binding site (NBS) and more than 40 irregular leucine-rich repeats (LRRs). RGC2 genes encode proteins of over 1800 amino acids. Sequence analysis indicates that the LRR-encoding region shows an alternating pattern of conservation and hypervariability. The patterns of variation support the predicted structure of LRR regions with solvent-exposed hypervariable residues that are potentially involved in binding pathogen-derived ligands. Data indicates that one of the cloned copies is Dm3.