Simple sequence repeat markers distinguish among morphotypes of Sphaeropsis sapinea

Sphaeropsis sapinea is a fungal endophyte of Pinus spp. that can cause dise ase following predisposition of trees by biotic or abiotic stresses. Four m orphotypes of S. sapinea have been described from within the natural range of the fungus, while only one morphotype has been identified on exotic pine s in the Southern Hemisphere. The aim of this study was to develop robust p olymorphic markers that could be used in both taxonomic and population stud ies. Inter-short-sequence-rep eat primers containing micro satellite sequen ces and degenerate anchors at the 5' end were used to target microsatellite -rich areas in an S. sapinea isolate. PCR amplification using an annealing temperature of 49 degreesC resulted in profiles containing 5 to 10 bands. T hese bands were cloned and sequenced, and new short-sequence-repeat (SSR) p rimer pairs were designed that flanked microsatellite-rich regions. Eleven polymorphic SSR markers were tested on 40 isolates of S, sapinea representi ng different morphotypes as well as on 2 isolates of the closely related sp ecies Botryosphaeria obtusa. The putative I morphotype was found to be iden tical to B. obtusa. Otherwise, the markers clearly distinguished the remain ing three morphotypes and, furthermore, showed that the C morphotype was mo re closely related to the A than the B morphotype. The B morphotype was the most genetically diverse, and the isolates could be further divided based on their geographic origins. Sequencing of different alleles from each locu s showed that the most polymorphic markers had mutations within a microsate llite sequence.