Fine Mapping of Resistance Gene Regions in Solanum bulbocastanum using a Novel MAMA PCR-based Mapping
Approach.
Submitted by
Department of Plant Pathology
612-624-3437 •
syve0039@umn.edu
Mismatch
amplification mutation assay (MAMA) is a PCR-based technique allowing
differentiation of sequences based on single nucleotide polymorphisms (SNPs).
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. Previously, we adopted a MAMA approach to
develop transgene-specific PCR and RT-PCR assays for the late blight resistance
gene RB.
Here, we report efforts to adapt MAMA-PCR for efficient fine mapping
applications. First we addressed technical considerations of MAMA PCR, testing optimal
annealing temperatures and nucleotide composition at the penultimate position
(i.e. transition vs. transversion)). As
a model system, we used two BAC clones originating from different haplotypes of
the diploid S. bulbocastanum genotype
PT29. These BACs, associated with RB, were fully sequenced and share an
overlapping region of approximately 31 kb.
A series of MAMA PCR primers, each paired with standard PCR primers,
were designed to target 10 different SNPs identified in this region. At each SNP, MAMA primers were designed incorporating
each possible nucleotide at the penultimate position. All primer pairs were singly tested over a
range of annealing temperatures (51°C-61°C) using S. bulbocastanum genotype PT29 genomic DNA, and BAC DNA from both
haplotypes as template. Each reaction
included positive control primer pairs designed from the RNA Polymerase II gene and the pBELOBAC11 vector, in addition to the
MAMA primer pair. We are now demonstrating
the utility of this approach for fine mapping by examining recombination
frequencies within this region using a segregating F1 S. bulbocastanum mapping population. Our fine mapping data will be integrated with
AFLP data generated from the same population, yielding a whole-genome linkage
map.