Degenerate ITS7 primer enhances oomycete community coverage and PCR sensitivity to Aphanomyces species, economically...

Metagenomic analysis of oomycetes through deep amplicon sequencing has been conducted primarily using the ITS6-ITS7 primer set that targets the ITS1 region. While this primer set shows a perfect match to most oomycete taxa, ITS7 contains 3 mismatches to the corresponding binding site of plant pathogens within the genus Aphanomyces. Polymerase chain reaction (PCR) efficiency differs for taxa with uneven primer matching characteristics, which may explain why previous studies have detected this genus at low abundance. To overcome the impact of these mismatches on PCR sensitivity, the mismatched nucleotides were replaced with degenerate nucleotides. Oomycete communities from 35 soil samples collected from asymptomatic and root rot diseased sites in pea fields across Alberta were analyzed simultaneously using ITS6-ITS7 and ITS6-ITS7-a.e. (modified version of ITS7) primer sets on 1 Illumina MiSeq run. The number of high-quality reads obtained by ITS6-ITS7-a.e. was more than twice that of ITS6-ITS7. The relative abundance of Pythium spp. was reduced and Aphanomyces spp. increased. Aphanomyces cf. cladogamus and Aphanomyces euteiches were the second and third most abundant species, respectively, in the pea rhizosphere using the ITS7-a.e. primer, but were rare using the ITS7 primer. These results indicate that use of ITS7-a.e. provides a more accurate picture of oomycete communities than ITS7 by enhancing PCR sensitivity to Aphanomyces.