KA SHEN, BC MEYERS, DB CHIN, SJ KIM and RW MICHELMORE

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

Background and objectives
The majority of genes conferring resistance to lettuce downy mildew (Dm genes) map to three major clusters in lettuce. The major resistance gene cluster contains at least 10 Dm genes (including Dm3) that provide resistance against the fungal pathogen Bremia lactucae. We are using a combination of map-based cloning and candidate gene approaches to clone Dm3 and to analyse the physical organization of the region.

We have generated a Bacterial Artificial Chromosome (BAC) library of large genomic clones of lettuce [1]. Molecular markers were used to isolate clones from the Dm3 region. A panel of fast-neutron (FN) irradiated mutants has been generated with deletions spanning the region [2]. BAC clones were arranged into contigs and used in conjunction with our FN mutants and molecular markers to generate a physical map of the region. Several BACs contain markers missing in all deletion mutants.

PCR with degenerate primers was used on these BACs to identify candidates for Dm3. Cloned resistance genes against diverse pathogens from a variety of plants fall into several distinct classes of proteins. Primers were designed based on conserved motifs in the nucleotide binding sites (NBSs) of the largest class which is characterized by the presence of NBSs as well as leucine-rich repeats (LRRs). Hybridization of RGCs to genomic DNA and to BAC clones revealed a large number of related sequences which are clustered in the genome, suggesting that RGCs are members of a multigene family. We are now characterizing the structural and transcriptional complexity of this cluster.

Results and conclusions
The majority of BACs from the Dm3 region contain members of the multigene family, indicating this is a complex region consisting of duplicated RGC sequences. Over 20 members of the family have been identified, 17 of which were mapped to the Dm3 cluster using the deletion-mutant map and markers from the region. Sequence analyses show that lettuce RGC sequences contain an NBS and a large LRR, as well as several introns. The sequences are divergent and show varying degrees of conservation across the open reading frame. Further analyses of this family include phylogenetic and physical mapping comparisons, as well as development of a model for the evolutionary mechanisms by which this resistance gene family has evolved.

RT-PCR analysis indicates that at least six RGC members are transcribed. Comparison of genomic and cDNA sequences confirmed intron and exon splice sites. RGCs are large genes, encoding proteins over 1800 amino acids. The candidate for Dm3 (RGC2b) based on mutant and marker criteria is over 12 kb (including introns) excluding the promoter and comprises an NBS domain plus a long LRR of approximately 45 irregular leucine repeats. To test function, selected RGCs were subcloned and introduced by Agrobacterium-mediated transformation into two genotypes lacking Dm3.

References
1. Frijters ACG, Zhang Z, van Damme M et al., 1997. Theoretical and Applied Genetics 3, 390-399.
2. Okubara PA, Anderson PA, Michelmore RW, 1994. Genetics 137, 867-874.