A New Oomycete Metabarcoding Method Using the rps10 Gene

Abstract

Oomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause plant and animal diseases. 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 for oomycete metabarcoding and provide primers predicted to amplify all oomycetes based on a wide range of available reference sequences. We evaluated its utility relative to the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a mock community using Illumina MiSeq. Amplified sequence variants (ASVs) and operational taxonomic units (OTUs) were identified per community. Observed sequences and predicted taxonomy of ASVs and OTUs were compared to the known composition of the mock community. Both rps10 and ITS yielded 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 ASVs included 23 members of the mock community for rps10 and 17 for ITS1. Sequencing results for the environmental samples suggest that the rps10 locus results in substantially less amplification of non-target organisms 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.

An open-access T-BAS phylogeny for emerging Phytophthora species

“…Recently, it has also been proposed that the rps10 mitochondrial locus be used to barcode Phytophthora species [ 98 ]. Although both CoxI and rps10 can differentiate many Phytophthora species, it is important to include nuclear barcodes as well [ 63 ].…”

“…urerae and P . betacei , more whole genome sequence data and whole mitogenome data [ 98 ] will be needed to confidently understand the evolutionary relationships among 1c clade species. This clade is economically relevant and important in evolution studies and studies on the origins of the P .…”

Oomycete communities are influenced by land use and disease status in Christmas tree production in Southern Québec, Canada

“…However, the presence and abundance of these species still need to be confirmed either with direct isolation, species-specific molecular tools, or new metabarcodes, such as the rps10 gene (Foster et al, 2022), enabling the resolution of these clusters at the species level.…”

Oomycete compositon in Proteaceae orchards and natural stands on three continents

“…A nested PCR approach was used with primary Phytophthora-speci c primers 18h2f and 5.8RBis in the rst round, and nested primers ITS6 and 5.8S-IR in the second round. Oomycete-speci c primers targeting the 40S ribosomal protein S10 (RPS10) gene region (Foster et al 2022) were ampli ed. We used a nested approach using PRV9-F and PRV9-R in the rst round (Martin and Coffey 2012) and oomycete-speci c primers RPS10-F and RPS10-R in the second round (http://oomycetedb.cgrb.oregonstate.edu/).…”