Molecular Ecology Resources (2009) 9, 399–401 doi: 10.1111/j.1755-0998.2008.02239.x © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd Blackwell Publishing Ltd PERMANENT GENETIC RESOURCES Development and characterization of polymorphic markers for the sap-stain fungus Ophiostoma quercus J. W. GROBBELAAR,*† I. BARNES,*† M-N. CORTINAS,*† P. BLOOMER,* M. J. WINGFIELD† and B. D. WINGFIELD*† *Department of Genetics, University of Pretoria, Pretoria 0002, South Africa, †Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria 0002, South Africa Abstract Eight polymorphic markers were developed from South African isolates of Ophiostoma quercus. The genome was screened for repeat regions using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol and 20 de novo primer pairs flanking putative microsatellite regions were designed. Eight loci were optimized and their polymorphisms evaluated by sequencing. The repeat and flanking regions were highly polymorphic containing both indels and base-pair substitutions revealing a total of 46 alleles in 14 isolates and an average heterozygosity of 0.68. Substantial sequence variability makes these markers useful for genotyping populations in order to calculate diversity and monitor global movement of O. quercus. Keywords: blue-stain fungi, M-FIASCO, microsatellites, Ophiostoma quercus, polymorphic markers, sequence variation Received 20 March 2008; revision accepted 29 April 2008 Ophiostoma quercus is a wood-inhabiting, heterothallic ascomycete causing sapwood stain. The fungus has been recorded in many countries on hardwood and coniferous trees although for many years, it was treated collectively with the morphologically similar Ophiostoma piceae. The advent of DNA sequence-based phylogenies has led to O. quercus being accepted as a discrete taxon, part of the O. piceae complex (Harrington et al. 2001). However, almost nothing is known regarding its population genetic structure. Microsatellites display high levels of polymorphism and are ideal genetic markers to provide resolution in relatedness studies (Tautz & Renz 1984). The aim of this study was to develop polymorphic microsatellite markers specific for O. quercus in order to describe its population genetic structure and worldwide distribution. A microsatellite-enriched library was made using the fast isolation by amplified fragment length polymorphism of sequences containing repeats (FIASCO) protocol (Zane et al. 2002) with modifications (M-FIASCO) as described by Cortinas et al. (2006). Genomic DNA was pooled from six South African isolates of O. quercus (CMW 2520, CMW 2521, CMW 2534, CMW 3119, CMW 3117 and CMW 3116) to yield a total of 2 μg. Cultures were made from single, ger- minating conidia or ascospores and genomic DNA was extracted using the method of Jacobs et al. (2004). Selections of biotinylated oligo probes representing di- to hexanucleotides in different combinations were used in the enrichment procedure. Enriched fragments were cloned using the TOPO 4 TA Kit (Invitrogen) and 576 colonies were selected and grown in 96-well plates containing 2 mL Luria-Bertani (LB) broth. M13 TOPO vector primers (Invit- rogen) were used for colony PCRs and amplicons were cleaned with 1.25 U of Exonuclease I and 1 U Shrimp Alkaline Phosphatase (Fermentas Life Sciences) to digest excess primers and dNTPs. Cloned products were sequenced using the ABI PRISM BigDye Terminator version 3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems Inc.) and the M13 forward and reverse vector primers. Sequenced products were purified using the Applied Biosystems precipitation method and were separated on an ABI PRISM 3100 auto- mated sequencer (Applied Biosystems). Sequences were manually screened for microsatellite regions using vector nti advance 10 software (Invitrogen). Correspondence: Brenda Wingfield, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, 74 Lunnon Road, Hillcrest, Pretoria 0002, South Africa. Fax: +27 12 4203960; E-mail: brenda.wingfield@fabi.up.ac.za