Microsatellite analysis of Ceratocystis fimbriata

Abstract

Ceratocystis fimbriata is the type species for the genus Ceratocystis and was first described as the causal agent of black rot in sweet potatoes. However, evidence from DNA sequence data suggests that C. fimbriata is in fact a species complex (C. fimbriata sensu lato) consisting of many morphologically similar cryptic species. Species in this complex are pathogens of important root and fruit crops and trees in the forestry industry world-wide. Population studies on some of these species have mainly relied on microsatellite markers. However, nothing is known regarding the microsatellite structure within Ceratocystis species or any species in the order Microascales in which Ceratocystis resides. The need for a more robust identification tool is also required to differentiate between species in this complex. The first chapter of this thesis provides a review of the literature on microsatellite markers, particularly in fungi. It also discusses the history of microsatellites, mechanisms of microsatellite evolution and functional importance in selected fungal examples. In addition, isolation methodologies are compared and contrasted to newly developed techniques that include bioinformatic searches of genome sequences. Opportunities to use and develop microsatellite markers in Ceratocystis species is also discussed with an emphasis on the possibilities that more microsatellites markers would provide. Microsatellites are abundant in eukaryotic genomes, and fungi are no exception. Analyses of microsatellite content in eukaryotic and fungal genomes have shown that fungi contain fewer microsatellites and that each organism shows preference for particular motifs. In Chapter 2 of this thesis, the abundance and distribution of microsatellites in the recently sequenced C. fimbriata genome is investigated. Comparisons to other fungi and eukaryotes show that C. fimbriata follows the general pattern of microsatellite structure, however it is unique in its preference for certain motifs. The C. fimbriata sensu lato species complex contains morphologically indistinct species. Microsatellite markers previously developed for a population study could differentiate between some of the cryptic species based on their geographic location and host-specificity. In Chapter 3 a subset of microsatellite markers identified in gene regions in Chapter 2 are used to develop a diagnostic test to differentiate between species in the complex. Microsatellite markers that are polymorphic between species but monomorphic within species were selected for this purpose. However, not all species could be distinguished using this diagnostic test. This thesis is presented as a series of chapters in which Chapters 3 2 and 3 are in manuscript format. Consequently each chapter represents an independent article and repetition between these chapters has been unavoidable.