Outcrossing in the homothallic oomycete,Pythium ultimum,detected with molecular markers

The oomycete Pythium ultimum is homothallic, thus a single isolate completes the sexual stage in pure culture. It has been generally assumed that homothallic oomycetes are predominantly inbreeding. In P. ultimum, antheridia occasionally develop from hyphae not directly connected to the oogonium and appear to participate in fertilization, suggesting a possible mechanism for outcrossing. We have used molecular markers to confirm that outcrossing can occur between isolates of P. ultimum. Genetic markers based on randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLP) were used to distinguish isolates in a collection of P. ultimum. Two isolates displaying a high level of polymorphism were mixed and placed on media which allows the development of the sexual stage. RAPD markers were used to screen single oospore progeny to identify potential hybrids between the two parental isolates. Subsequent self-fertilization of one putative F₁ yielded a F₂ population which demonstrated segregation and independent assortment of RAPD and RFLP markers. A similar strategy was used to show that an isolate which is incapable of producing oospores in pure culture can outcross when mixed with a homothallic isolate. These results suggest that other homothallic oomycetes may be capable of outcrossing, and sexual reproduction may, therefore, play an important role in the generation of variation in homothallic oomycetes.