MICROSATELLITE MARKERS FOR PHYTOPHTHORA INFESTANS: NEW TOOLS FOR AN OLD PROBLEM
ND PIPE, CJ GLIDDON, RC SHATTOCK and DS SHAW
School of Biological Sciences, University of Wales, Bangor, Gwynedd, LL57 2UW, UK
Background and objectives
World-wide, considerable effort has been made to determine the population structure of the late blight fungus, P. ;infestans, using multi-locus DNA fingerprinting. The level of genetic diversity may be established from phenotypic markers. However, these studies have not allowed the analysis of gene frequencies to identify how variation is generated and maintained. Isozymes and single locus RFLP markers have traditionally been used for population studies but are unlikely to provide the required level of polymorphism.
The choice of microsatellite markers  for the analysis of gene-flow was made on the basis that they should be single locus, codominant, highly polymorphic and detectable by PCR. The difficulty of utilizing microsatellite markers is that knowledge of the flanking sequences either side of the microsatellite repeat is required for their use. We describe here the isolation of microsatellite loci from P. ;infestans, and demonstrate their potential for population analysis.
Materials and methods
Microsatellite loci were cloned from small-insert and microsatellite-enriched, partial genomic DNA libraries from P. ;infestans. The small-insert library was created by digesting genomic DNA with AluI, RsaI and HaeIII and gel-purified fragments (100-600 ;bp) were ligated into pGEM3Z plasmid vector. The enriched library was created by digesting genomic DNA with Tsp509I; oligonucleotide Tsp adaptors were ligated to gel purified fragments (100-600 ;bp). Oligonucleotides (AC)13 and (CT)13 were 5'-end-labelled with Biotin-16-ddUTP using T4 polynucleotide kinase and hybridized to streptavidin-coated magnetic beads. The genomic DNA fragments were hybridized to the biotinylated oligonucleotide magnetic beads at 65°C and, using the Tsp adaptors, hybridizing fragments were bulked up using PCR amplification before cloning into pGEM3Z. Ligated DNA from both libraries was transformed into competent JM109 E. ;coli cells and for each library approximately 8300 colonies were replica-plated onto 41 Hybond N filters. To identify microsatellite-containing clones, colony hybridizations were performed using 32P end-labelled (AC)13 and (CT)13 oligoprobes. A total of 130 positive hybridizing colonies were sequenced and microsatellite primers were designed to allow PCR amplification of five microsatellite loci from genomic DNA.
Results and conclusions
Initial screening of microsatellite loci was performed using 20 isolates of P. ;infestans, representing eight phenotypes, as demonstrated with the multilocus fingerprinting probe, RG57. Unexpectedly, only two microsatellite loci were polymorphic. More than 400 isolates collected locally during 1995 and 1996  were screened for alleles at these two loci. The most polymorphic locus, a (CT)27 repeat, S0820, showed five different alleles at this locus. The remaining three microsatellite loci were monomorphic for this collection of isolates. These preliminary data indicate the suitability of microsatellites as single-locus markers for analysis of population structure in P. ;infestans. Additional data from a larger collection of UK isolates and from a collection of isolates from Mexico, where sexual reproduction is thought to be frequent, will be presented.
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