THE DM3 REGION IN LETTUCE CONTAINS A COMPLEX MULTIGENE FAMILY OF RESISTANCE GENE CANDIDATES

¹ Department of Vegetable Crops, University of California, Davis, CA 95616 USA

² DuPont Agricultural Products, Biotechnology Group, Delaware Technology park, Newark, DE 19714 USA

The majority of genes conferring resistance to lettuce downy mildew (Dm genes) map to three major clusters in lettuce (Lactuca sativa). The major resistance gene cluster contains at least ten Dm genes, including Dm3, which provide resistance against the fungal pathogen Bremia lactucae. We used a combination of map-based cloning and candidate gene techniques to clone Dm3 and to analyze the physical organization of the region.

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. Primers designed based on conserved motifs in the nucleotide binding site (NBS) of known plant disease resistance gene amplified candidate sequences from Dm3-localized BAC clones. Further analysis revealed that these BACs contain a large and diverse multigene family (the RGC2 family). These resistance gene candidates encode a predicted NBS and more than 40 irregular leucine-rich repeats (LRRs). 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. Genomic sequencing determined that these genes span 7-14 kb. RT-PCR analysis confirmed intron and exon splice sites and indicated numerous members of the family are transcribed. 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.