THE DM3 REGION IN LETTUCE (LACTUCA SATIVA) CONTAINS A COMPLEX MULTIGENE FAMILY OF RESISTANCE GENE CANDIDATES
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
We have generated a Bacterial Artificial Chromosome (BAC) library of large genomic clones of lettuce . 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 . 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
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.