RESISTANCE GENE CANDIDATES IDENTIFIED BY PCR WITH DEGENERATE OLIGONUCLEOTIDE PRIMERS MAP TO CLUSTERS OF RESISTANCE GENES IN LETTUCE

The recent cloning of genes for resistance against diverse pathogens f rom a variety of plants has revealed that many share conserved sequenc e motifs, This provides the possibility of isolating numerous addition al resistance genes by polymerase chain reaction (PCR) with degenerate oligonucleotide primers. We amplified resistance gene candidates (RGC s) from lettuce with multiple combinations of primers with low degener acy designed from motifs in the nucleotide binding sites (NBSs) of RPS 2 of Arabidopsis thaliana and N of tobacco. Genomic DNA, cDNA, and bac terial artificial chromosome (BAC) clones were successfully used as te mplates. Four families of sequences were identified that had the same similarity to each other as to resistance genes from other species. Th e relationship of the amplified products to resistance genes was evalu ated by several sequence and genetic criteria. The amplified products contained open reading frames with additional sequences characteristic of NBSs, Hybridization of RGCs to genomic DNA and to BAC clones revea led large numbers of related sequences. Genetic analysis demonstrated the existence of clustered multigene families for each of the four RGC sequences. This parallels classical genetic data on clustering of dis ease resistance genes. Two of the four families mapped to known cluste rs of resistance genes; these two families were therefore studied in g reater detail. Additional evidence that these RGCs could be resistance genes was gained by the identification of leucine-rich repeat (LRR) r egions in sequences adjoining the NBS similar to those in RPM1 and RPS 2 of A. thaliana, Fluorescent in situ hybridization confirmed the clus tered genomic distribution of these sequences, The use of PCR with deg enerate oligonucleotide primers is therefore an efficient method to id entify numerous RGCs in plants.