| Abstract Detail
Conference Wide Syverson, Ryan [1], Bradeen, James [1]. Fine Mapping of Resistance Gene Regions in Solanum bulbocastanum using a Novel MAMA PCR-based Mapping Approach. 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 penultimate mismatch, the specificity at the 3’ most nucleotide site allows the PCR primer to specifically anneal to the target 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, including 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 Solanum 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 a standard PCR primer, was designed to target 10 different SNPs identified in this region. At each site, 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.
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Related Links:
Potato Pathology & Genomics Program at University of Minnesota
1 - University of Minnesota, Plant Pathology, 495 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN, 55108, USA
Keywords:
mapping
PCR markers
SNP
fine mapping.
Session: Poster-80
Location: Ballroom CD/Monona Terrace
Date: Tuesday, July 25th, 2006
Time: 8:00 AM
Abstract ID:53 |
