Classification of isolates into vegetative compatibility groups (VCGs) using nitrate-non-utilizing (nit) mutants has been widely used for the characterization of Verticillium dahliae populations. However, certain methodological limitations prevent its application on a large scale. Furthermore, systematic investigations into the genetics underlying complementation tests between nit mutants of fungal isolates (i.e. heterokaryon formation) are lacking for Verticillium species. In this work, a diverse collection of 27 V. dahliae isolates – including representatives of all VCGs, both mating types, and heterokaryon self-incompatible isolates – was employed for the development and optimization of (i) a protocol for the rapid generation of nit mutants of V. dahliae isolates using UV-irradiation and (ii) a reproducible high-throughput procedure for complementation tests between nit mutants in liquid cultures using 96-well microplates. The genetic analysis of selected heterokaryons demonstrated that the frequently encountered ‘weak’ cross-reactions between VCGs and their subgroups can be actually heterokaryotic, implying the absence of strict genetic barriers between VCGs. In conclusion, we provide in this work an optimized method for the high-throughput VCG assignment of V. dahliae populations and a genetic analysis of heterokaryons that may have serious implications for the interpretation of VCG classification data. These advancements in the available methodology and the genetic background of vegetative compatibility grouping may contribute to a better understanding of the population biology of V. dahliae and possibly other mitosporic fung
