Resistance gene analogs (RGA) are potential gene fragments that contain
structural motifs, such as the NBS and LRR regions, that are common to most
cloned R genes. Recovery of RGAs using degenerate
primer PCR was first described for cultivated potato, and has become a popular
method for recovering RGAs from many dicot and monocot plant species. In this study, we are
applying the degenerate primer PCR approach to the disease resistant, wild
potato species, Solanum bulbocastanum. We
aimed to compare total genomic DNA versus pooled BAC DNA as template for RGA
recovery in S. bulbocastanum. From total genomic
DNA, we observed an excess of PCR fragments originating from highly repetitive
genomic regions. Normalization during BAC library construction enriches for euchromatic, gene-rich regions, potentially minimizing the
highly repetitive sequences available for amplification. We compared total
genomic DNA versus pooled BAC DNA RGA reactions for the recovery of R gene like
fragments and for the overall amplification of repetitive elements using DNA
hybridization. We further evaluated the pooled BAC strategy by estimating the
frequency of fragments originating from BAC vector and E. coli DNA. Finally, we
determined the minimum genomic equivalents needed to produce a consistent
banding pattern by varying the number of BAC clones of each pool.