Development and application of new molecular markers for analysis of genetic diversity in Verticillium dahliae populations

The aim of this study was to develop new polymorphic markers for analysis of genetic diversity in the fungal soilborne plant pathogen Verticillium dahliae. Twelve polymorphic markers (five microsatellites and seven polymorphic sequences) were developed from a genomic library enriched for microsatellites. Screening of polymorphic loci was done using a collection of 25V. dahliae isolates of diverse geographic origins, host sources and vegetative compatibility groups (VCGs). Three methods were used to score alleles: polyacrylamide gel electrophoresis (PAGE), sequencing of PCR-amplified loci, and capillary electrophoresis. The new markers were used to assess genetic differentiation between isolates associated with different host plants. Two collections of isolates were analysed, obtained from artichoke (30 isolates) and potato (20 isolates) from crops grown in rotation located in the same area in eastern-central Spain. The resolution of genetic differentiation between these two collections using the new markers was compared to that provided by other often-used markers (SCARs and VCGs). Sequence analysis of the alleles proved to be the most unambiguous technique for scoring microsatellite data. The relatively high genetic differentiation observed between isolates from different crops (genetic differentiation coefficient, G_ST=0·24) and their high genotypic diversity suggest a divergence between V. dahliae from artichoke and potato. It is hypothesized that evolution of V. dahliae from the local resident population in association with the two host crops has occurred. The new markers are useful for resolving population structure within V. dahliae and may contribute to a better understanding of the population biology of this fungus.