A new oomycete metabarcoding method using the rps10 gene

Authors: Foster, Zachary; ALBORNOZ, F - Oregon State University; FIELAND, V - Oregon State University; Larsen, Meredith; JONES, F - Oregon State University; TYLER, B - Oregon State University; NGUYEN, H - Agriculture And Agri-Food Canada; BURGESS, T - Murdoch University; RIDDELL, C - Forest Research; VOGLMAYR, HERMANN - University Of Vienna; Martin, Frank; Grunwald, Niklaus - Nik

Submitted to: Phytobiomes Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/10/2022
Publication Date: 9/13/2022
Citation: Foster, Z.S., Albornoz, F.E., Fieland, V.J., Larsen, M.M., Jones, F.A., Tyler, B.M., Nguyen, H.D., Burgess, T., Riddell, C., Voglmayr, H., Martin, F.N., Grunwald, N.J. 2022. A new oomycete metabarcoding method using the rps10 gene. Phytobiomes Journal. 6(3):214-226. https://doi.org/10.1094/PBIOMES-02-22-0009-R.
DOI: https://doi.org/10.1094/PBIOMES-02-22-0009-R

Interpretive Summary: Oomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause diseases in plants and animals. Improved methods are needed to rapidly characterize the diversity of oomycete species found in a community. We have identified the mitochondrial 40S ribosomal protein S10 gene (rps10) as being useful for oomycete community sequencing. We evaluated its utility relative to the current gold standard, the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a community we synthesized in the laboratory community. The amplified rps10 region has a higher taxonomic resolution than ITS1, allowing for greater discrimination of closely related species. We present a new website with a searchable rps10 reference database for species identification and all protocols needed for oomycete community sequencing. The rps10 barcode and methods described herein provide an effective tool for characterizing oomycetes using environmental DNA sequencing.

Technical Abstract: Oomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause diseases in plants and animals. Improved methods are needed for rapid and accurate characterization of oomycete communities using DNA metabarcoding. We have identified the mitochondrial 40S ribosomal protein S10 gene (rps10) as a locus useful for oomycete metabarcoding and provide primers predicted to amplify all oomycetes based on available reference sequences from a wide range of taxa. We evaluated its utility relative to the current gold standard, the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a mock community using Illumina MiSeq. Both methods yielded amplified sequence variants (ASVs) with sequences matching 21 of the 24 species in the mock community and matching all 24 when allowing for a 1 bp difference. Taxonomic classifications of the ASVs generated by a commonly used method included 23 members of the mock community for rps10 and 17 for ITS1. Sequencing the environmental samples suggests the proposed rps10 locus results in substantially less amplification of non-target organisms (e.g., plants and fungi) than the ITS1 method. The amplified rps10 region also has higher taxonomic resolution than ITS1, allowing for greater discrimination of closely related species. We present a new website with a searchable rps10 reference database for species identification and all protocols needed for oomycete metabarcoding. The rps10 barcode and methods described herein provide an effective tool for metabarcoding oomycetes using short-read sequencing.