Multilocus Sequencing of Phytophthora and Downy Mildew

Sequences from eight loci were obtained from each sample; primers and annealing temperatures for amplification and sequencing are listed in S2 Table. Methods for obtaining and analyzing the ITS sequence of Phytophthora cyperi will be discussed in a separate manuscript (in preparation). PCR conditions are listed in S3 Table; different amplification conditions were used for nuclear and mitochondrial loci. The FRiz forward ITS primer was initially developed for routine amplification and sequencing of Pythium and Phytophthora but proved to be useful for potentially plant-contaminated downy mildew samples due to its apparent specificity to oomycetes. In both Bremia species, any amplicon containing the ITS2 region produced multiple bands, including some much larger than expected, a known issue with DMPH [68 (link)]; as a result, only the ITS1 was amplified and sequenced from the two samples. The combination of BTubF4 and BTubR3 proved able to amplify the beta tubulin locus without also amplifying host genes, which was particularly problematic for the Phytophthora cyperi sample. The reverse primer SecYtrnC-R was used for some of the downy mildew samples that did not amplify well with SecY-R. The internal primers SecY-F2 and SecY-R2 were developed because P. taxon mugwort contains homopolymers in both the 5' and 3' end of its secY ORF, which rendered downstream sequences difficult to read; the additional primers listed for the cox2-cox1 contig also reflect the presence of homopolymers in many samples, both in-frame and in the cox2-cox1 intergenic spacer. PCR products were cleaned with Exo-SAP-IT (Thermo Fisher Scientific, Waltham MA, USA) according to manufacturer's instructions and Sanger sequencing performed by the UC Davis ^UCDNA Sequencing Facility (Davis, CA, USA). Chromaseq was used to call bases and assemble contigs [69 –72 ]; sequences were uploaded to GenBank (S1 Table).