Development of microsatellite markers for the pine needle blight pathogen, Dothistroma pini

Dothistroma needle blight of Pinus spp. is a serious disease that can be caused by two distinct fungal species: Dothistroma septosporum and D. pini. Dothistroma septosporum has a broad pine host range, a worldwide distribution and has caused many serious epidemics. In contrast, D. pini has a more limited distribution; known only from Europe and the USA. Unlike the situation for the better-known D. septosporum, the population biology of D. pini has not been intensively studied. Microsatellite markers developed for D. septosporum either do not amplify in D. pini or they are uninformative. The aim of this study was to develop a robust set of microsatellite markers that amplify across different D. pini isolates from a range of geographical locations and different host species. The genome of an isolate from Ukraine was sequenced using Mi-Seq Illumina sequencing. Thirty-two primer pairs were designed to amplify microsatellite regions targeting a mixture of di-, tri-, tetra-, penta- and hexa- nucleotide repeat regions. The results yielded 16 polymorphic markers that amplified across 32 isolates from seven countries and four hosts. Screening of the polymorphic markers on a population of D. pini from France and Slovenia revealed that the Slovenian isolates were clonal but that those from France were genetically more diverse. The markers developed in this study will provide a useful tool with which to study the population structure and genetic diversity of D. pini populations in countries where it is emerging as an important pathogen.