Development of polymorphic microsatellite markers for the genetic characterisation of Knoxdaviesia proteae (Ascomycota: Microascales) using ISSR-PCR and pyrosequencing

Knoxdaviesia proteae is one of the first native ophiostomatoid fungi discovered in South Africa, where it consistently occurs in the infructescences of the iconic Cape Biome plant, Protea repens. Although numerous studies have been undertaken to better understand the ecology of K. proteae, many questions remain to be answered, particularly given its unique niche and association with arthropods for dispersal. We describe the development and distribution of microsatellite markers in K. proteae through Interspersed Simple Sequence Repeat-Polymerase Chain Reaction (ISSR-PCR) enrichment and pyrosequencing. A large proportion of the 31492 sequences obtained from sequencing the enriched genomic DNA were characterised by microsatellites consisting of short tandem repeats and di- and tri-nucleotide motifs. Seventeen percent of these microsatellites contained flanking regions sufficient for primer design. Twenty-three primer pairs were tested, of which 12 amplified and 10 generated polymorphic fragments in K. proteae. Half of these could be transferred to the sister species, K. capensis. The developed markers will be used to investigate the reproductive system, genetic diversity and dispersal strategies of K. proteae.