Development and use of microsatellite markers to study diversity, reproduction and population genetic structure of the cereal pathogen <i>Ramularia collo-cygni</i>

Ramularia collo-cygni (Rcc) is a major pathogen of barley that causes economically serious yield losses. Disease epidemics during the growing season are mainly propagated by asexual air-borne spores of Rcc, but it is thought that Rcc undergoes sexual reproduction during its life cycle and may also disperse by means of sexual ascospores. To obtain population genetic information from which to infer the extent of sexual reproduction and local genotype dispersal in Rcc, and by implication the pathogen's ability to adapt to fungicides and resistant cultivars, we developed ten polymorphic microsatellite markers, for which primers are presented. We used these markers to analyse the population genetic structure of this cereal pathogen in two geographically distant populations from the Czech Republic (n =30) and the United Kingdom (n =60) that had been sampled in a spatially explicit manner. Genetic diversity at the microsatellite loci was substantial, Ht =0.392 and Ht =0.411 in the Czech and UK populations respectively, and the populations were moderately differentiated at these loci (Θ =0.111, P <0.01). In both populations the multilocus genotypic diversity was very high (one clonal pair per population, resulting in >96% unique genotypes in each of the populations) and there was a lack of linkage disequilibrium among loci, strongly suggesting that sexual reproduction is an important component of the life cycle of Rcc. In an analysis of spatial genetic structure, kinship coefficients in all distance classes were very low (-0.0533 to 0.0142 in the Czech and -0.0268 to 0.0042 in the Scottish population) and non-significant (P >0.05) indicating lack of subpopulation structuring at the field scale and implying extensive dissemination of spores. These results suggest that Rcc possesses a high evolutionary potential for developing resistance to fungicides and overcoming host resistance genes, and argue for the development of an integrated disease management system that does not rely solely on fungicide applications.