Transgene-specific PCR and RT-PCR assays for the potato late blight R gene RB

We have developed transgene-specific PCR and RT-PCR assays for the potato late blight resistance gene RB.  Our assays utilize two approaches toward primer design, allowing discrimination between the RB transgene and both the endogenous RB gene and numerous RB paralogues.  First, a reverse primer was designed to take advantage of an 18 bp indel present in the RB transgene but absent in rb susceptibility alleles. This primer enhanced specificity for the transgene, but could not fully differentiate between the transgene and RB paralogues.  Second, a forward primer was designed using the principles of Mismatch Amplification Mutation Assay (MAMA) PCR.  RB was cloned using Long Range (LR)-PCR, resulting in a transgene containing three SNPs relative to the endogenous RB.  The forward primer was designed such that a transgene-specific SNP was at its extreme 3’ end and an intentional mismatch was incorporated in the penultimate position.  Together, the indel reverse primer and the MAMA forward primer provide an assay that is highly specific for the RB transgene, being capable of distinguishing the transgene from all RB endogenous gene copies and from all RB paralogues in a diverse collection of wild and cultivated potato genotypes.  The assay has been successfully multiplexed with an internal control and is useful for both PCR and RT-PCR applications.  Currently, we are exploring a double MAMA primer approach that may be of general application for LR-PCR generated transgenes.