MOLECULAR STRATEGIES FOR THE EFFICIENT CHARACTERIZATION OF RESISTANCE GENE MARKER AND ALLELIC DIVERSITY IN SOLANUM SPECIES

Riccardo Aversano^a,b, Maria J. Sanchez^a, Domenico Carputo^b, Luigi Frusciante^b, and James M. Bradeen^a

^aUniversity of Minnesota, Department of Plant Pathology, 495 Borlaug Hall / 1991 Upper Buford Circle, St. Paul, MN 55108 USA

^bUniversity of Naples “Federico II”, Department of Soil, Plant, and Environmental Science, Via Università 100, 80055 Portici (NA) Italy

raversan@unina.it

            Ongoing efforts in our laboratories include the characterization of marker diversity near and allelic diversity at resistance loci in Solanum species.  The broad spectrum late blight resistance gene RB was previously cloned from the Mexican diploid S. bulbocastanum.  Using a reference set of 42 S. bulbocastanum genotypes, here we demonstrate strategies and methods for the characterization of marker diversity near the RB locus, and improved methods for the isolation of RB orthologs (true alleles).

We generated PCR markers of known physical distance from the RB locus from BAC end sequence.  We next adapted EcoTilling for use in Solanum species. EcoTilling is a powerful technique for discovering polymorphisms in natural populations.  Originally developed in self-pollinated species, EcoTilling utilizes a heteroduplex-specific endonuclease to detect single nucleotide polymorphisms.  Most potatoes are outcrossing and highly heterozygous, complicating traditional EcoTilling approaches.  Using our modified protocol and our S. bulbocastanum reference set, we are currently exploring haplotype diversity at the RB locus by examining marker correlations.  We are further exploring the potential for geographic distribution, subspecies classification, and within vs. between population relationships to predict marker status near the RB locus.  Details of our approach and techniques will be presented.

            Like most resistance genes cloned to date, RB resides in a cluster of related sequences.  We have optimized long range-PCR (LR-PCR) for the isolation of RB orthologs (true alleles) to the exclusion of related but non-functional paralogs, and adapted our approach for multi-genotype allelic characterization.  In this study we identified efficient template DNA preparation and LR-PCR amplification conditions.  We further demonstrate sequencing strategies yielding 100% sequence accuracy.  Using a subset of sequences generated from our S. bulbocastanum reference set, we document successful isolation of RB orthologs (true alleles) from previously uncharacterized genotypes.