Multi-genotype allelic mining at the RB late blight resistance locus reveals broad allele distribution and an in planta recombination event

James M. Bradeen, Maria J. Sanchez, and Dimitre S. Mollov

Previously, the broad spectrum late blight resistance gene RB was cloned from the wild potato Solanum bulbocastanum via long range-PCR (LR-PCR). RB is a member of the prominent NBS-LRR class of resistance genes and is one of five related loci within a 50kb region. Only RB has known function. We optimized LR-PCR for isolation of orthologs (true alleles) from additional S. bulbocastanum genotypes, recovering 8-12kb fragments from 17 of 44 tested genotypes (~40% success). Sequence analysis of the intron and LRR confirmed recovery of RB orthologs rather than paralogs. Thus, LR-PCR can be an effective method for multi-genotype allelic mining. Recovered were representatives of two previously reported alleles: RB (functional allele) and rb (non-functional allele). Surprisingly, rb-like alleles predominated in our survey. Also recovered was a new allele, rc-RB. Detailed sequence analysis and recovery of rc-RB from replicate reactions of different individuals from the same population suggest that it is the product of an in planta, inter-locus recombination event. The recovery of rc-RB suggests the RB cluster may be dynamic and inter-locus recombination may be an important mechanism for generation of new alleles. Current efforts focus on transcriptional and functional testing of rc-RB and determination of its distribution in S. bulbocastanum populations.