Cloning and Characterization of Disease Resistance Gene Analogs from Poplar (Populus tremula) Chromosome 1

The majority of verified plant disease resistance genes (R-genes) isolated to date are of the NBS-LRR class, encoding proteins with a predicted nucleotide-binding site (NBS) and a leucine-rich repeat (LRR) region. The conservation between different NBS-LRR R-genes opens the avenue for the use of PCR-based strategies in isolating and cloning other R-gene family members or analogs using degenerate primers for these conserved regions in plants. However, the screening of target sequences among the numerous amplified resistance gene analogs (RGAs) can be very laborious and time-consuming. The amplification of RGAs from specific chromosomes could greatly reduce the number of RGAs to be screened and, consequently, speed up the identification of target RGAs. We have developed a technique that combines chromosome microdissection and homologous sequence amplification in order to acquire RGAs from single chromosomes in forest tree poplars (Populus tremula). A number of RGAs were amplified with the DNA fragments of P. tremula chromosome 1 as template, using consensus-degenerate hybrid oligonucleotide primers based on conserved motifs of the NBS domain. Here, we report the sequence characterization and diversity analysis of these RGAs and their relationships with the NBS sequences of known R-genes from other plant species. These data permit insights into the origin, diversification, and evolution of NBS-LRR R-genes in perennial species such as poplar.