Efficient Recovery of Resistance Gene Analogs from Solanum bulbocastanum

            Resistance genes that have been cloned from plants have a number of conserved structural motifs, such as the nucleotide binding site (NBS) and leucine rich repeat present in their proteins.  From these motifs, degenerate PCR primers have been developed for amplification of fragments analogous to R genes.  This method of recovering resistance gene analogs (RGAs) was first described in cultivated potato and is now used widely for both dicot and monocot species.  Many studies in various plant species have successfully used common degenerate primer sequences for RGA recovery.  We used previously developed primers for RGA recovery from the disease resistant, wild potato species, Solanum bulbocastanum.  In this study, genomic DNA, pooled BAC DNA, and cDNA, all derived from S. bulbocastanum, were used as templates for degenerate PCR reactions.  The efficiency of these various PCR templates for RGA recovery was compared to determine the best template source for the creation of an RGA library of S. bulbocastanum.  The comparison was based on the total number of resulting unique RGAs recovered, the overall percentage of cloned fragments that were RGAs, the percentage of highly repetitive sequences (HRS) recovered, and the percentage of non-RGA and/or non-HRS sequences recovered.  Degenerate PCR was further applied to related plant species to determine if highly repetitive sequences are recovered.