Mitochondrial haplotype analysis as a tool for differentiating isolates of Verticillium dahliae

The ability to monitor mitochondrial background in Verticillium dahliae may provide an additional tool for population studies and monitoring clonal populations. Published mitochondrial genome sequences of V. dahliae (DQ351941) were used to design primers for amplification of spacer regions for assessment of mitochondrial haplotype differences among isolates. Five regions were examined (5,229 bp, or 19% of the total genome size) for 30 isolates representing a range in vegetative compatibility group (VCG), host, and geographic origin. Observed differences among isolates were due to single nucleotide polymorphisms, different numbers of bases in specific homopolymeric regions, and copies of subrepeated sequences. When the differences observed for each locus were totaled there were 28 total groupings; when the results of each locus for individual isolates were combined there were 15 mitochondrial haplotypes. Some of the observed groupings correlated with VCG. For example, five VCG-1A and VCG-1B isolates from California, Spain, and Greece had identical haplotypes; however, this was not observed for VCG-2 or VCG-4 isolates. While some VCG-2 isolates also were identical and fell into a single haplotype, five haplotypes were found for this group (five other haplotypes were observed for other isolates that had not been characterized to VCG but grouped with VCG-2 isolates in the phylogenetic analysis). Likewise, five VCG-4 isolates fell into four mitochondrial haplotypes, one of which was identical to the largest VCG-2 grouping. A heterokaryon self-incompatible isolate that was reported in the literature to cluster with VCG-2 isolates by amplified fragment length polymorphism analysis was identical with VCG-1 isolates for four of the five loci, but was intermediate between VCG-1 and VCG-2 in the haplotype analysis. Phylogenetic analysis with these regions revealed the mitochondrial background of VCG-1 and VCG2-B to be monophyletic but VCG-2A and VCG-4 could not be separated. The results obtained indicate that there is variation in mitochondrial haplotypes and this type of analysis may be a useful for characterization of isolates. While data from all five regions was used for the haplotype separation in this study, depending on the VCG or the level of variability observed within a population it is possible to use fewer loci.