Towards Efficient Isolation of R Gene Orthologs from Multiple Genotypes: Optimization of Long Range-PCR

Resistance (R) genes and the proteins they encode are key components of the defense system of plants. The exploration of R gene diversity enables the study of R gene evolution and may facilitate the isolation of new and functional alleles. Most cloned R genes occur in clusters of related sequences. Thus, the development of a tool for reliable recovery of orthologous R gene sequences to the exclusion of paralogous sequences will facilitate R gene diversity analysis. The late blight resistance gene RB is a single functional locus embedded within a cluster of related sequences. Previously, the functional RB allele was cloned from wild potato using a Long Range-PCR (LR-PCR) technique, suggesting this method may be a promising tool for recovery of R gene orthologs in other genotypes. Using the RB gene as a model, we explored the limitations and improved three technical aspects of LR-PCR for multi-genotype applications. We present improved primers for the recovery of the RB locus and have identified efficient DNA extraction procedures and reliable amplification systems. We document that consensus sequences built from three independently generated LR-PCR clones can be up to 100% accurate. Our results show encouraging advances toward successful application of LR-PCR for isolating alleles from orthologous R gene loci.