Mitochondrial haplotype determination in the oomycete plant pathogen Phytophthora ramorum

The mitochondrial genome of an isolate of Phytophthora ramorum from Europe (EU) was sequenced and compared to the previously published genome sequence of an isolate from California (NA). The EU mitochondrial genome had the identical gene order and encoded for the same suite of genes as the NA mitochondrial genome, but had 13 single nucleotide polymorphisms (SNPs) and at 39,494 bp was 180 bp longer. This length difference was due to an increase in the size of the spacer region between the nad5 and nad6 genes caused by a chimeric region containing duplication of the spacer sequence and additional sequences from the flanking genes. Recombination between the 1,150 bp-inverted repeats (IR) generated orientational isomers where the gene order was reversed between the IR. A total of seven primer pairs were developed for amplification of regions where the SNPs were located and two other regions where additional SNPs were encountered when a larger number of isolates were examined. Sequence data for a total of 5,743 bp for 40 isolates collected from a range of geographic areas was compared and 28 loci were found to be polymorphic. The combination of these polymorphisms revealed a total of 4 mitochondrial haplotypes; the traditional EU (haplotype I), the traditional NA (haplotype IIa), the third nuclear lineage of the pathogen recovered from a nursery in Washington State (haplotype III) and a new haplotype representing a subgroup of NA isolates from an Oregon forest (haplotype IIb). Phylogenetic analysis using the sequences generated from the haplotype analysis supported a high affinity for haplotypes IIa and IIb, both of which were distinct from haplotype I, with haplotype I basal to these and haplotype III representing the ancestral state.