Identification and characterization of Phytophthora hybrids using genotyping-by-sequencing

Since the mid-1990’s, several Phytophthora hybrids have been described in multiple phylogenetic clades. As a result of intensified international plant trade, which might bring previously separated species in contact with each other, an increase in the number of hybridization events is suspected. These hybrids might be more aggressive or have an expanded host range in comparison to the parental species. Early detection of new hybrid species is thus warranted, but is not always successful via traditional PCR amplicon sequencing of individual loci. We have used two-enzyme genotyping-by-sequencing (GBS) to reliably identify and characterize hybrids in a European collection of several hundreds of Phytophthora isolates.
DNA was extracted, digested and GBS-adapters were ligated. After PCR amplification, GBS libraries were quantified, pooled and sequenced using the Illumina HiSeq 3000 technology. Read data was preprocessed using custom scripts and subsequently subjected to the GibPSs pipeline to identify a global set of GBS tags across all samples. Subsequently, per sample the absence/presence profiles were scored together with SNP profiles for each GBS tag. The method was highly reproducible and generated about 15000 to 30000 tags for a non-hybrid species. When isolates share a large number of GBS tags with at least two Phytophthora species, this is indicative of a hybridization event. Using this approach, we detected a previously unidentified hybrid of P. chlamydospora and P. lacustris.
Even when the parental species are not known, a large number of GBS tags in a sample can point to a hybridization event, as hybrids contain tags from each of the parental species. We thus identified a number of potential novel hybrid species, which implies that historic hybridization events might be more common in Phytophthora than previously assumed. Genotyping-by-sequencing can thus be used to reliably identify and characterize Phytophthora hybrids.