Analysis of Haplotype Frequency of the Late Blight Resistance Gene, RB, in Solanum bulbocastanum using a Novel MAMA PCR-based Approach.

Ryan Syverson, Ben Millett and James Bradeen

University of Minnesota

Department of Plant Pathology

495 Borlaug Hall/1991 Upper Buford Circle

St. Paul, MN USA

Previously, the broad spectrum late blight resistance gene, RB, was cloned from wild potato (Solanum bulbocastanum), and we reported a Mismatch amplification mutation assay (MAMA) transgene-specific assays. MAMA PCR encompasses an approach to primer design that yields robust single nucleotide polymorphism (SNP) differentiation. Briefly, a SNP-specific PCR primer is designed incorporating the SNP at the ultimate (3’) position and a mismatch at the penultimate position. Despite the mismatch at the penultimate position, the specificity at the 3’ most nucleotide site allows the PCR primer to specifically anneal to a desired sequence, enabling amplification. Here, we report adoption of MAMA-PCR for assessment of haplotype frequency of RB. A series of 18 MAMA PCR primers, each paired with standard PCR primers, were designed to target SNPs identified within RFLP, CAPS and BAC end markers within RB region. Two fully sequenced BAC clones, associated with RB and originating from different haplotypes of the diploid S. bulbocastanum genotype PT29 were used for primer design. In total, 17 of 18 markers encompass a 287kb region of S. bulbocastanum chromosome 8, including the RB gene, with one marker falling outside of this range at an undetermined distance. Segregation analysis in an S. bulbocastanum F1 population was used to validate haplotype specificity of each marker. MAMA primers were used to characterize haplotypes of 60 S. bulbocastanum genotypes including two individuals from each of 30 populations. Conclusions about haplotype frequency will be reported.