Nested PCR Approach for Gene Metabarcoding of Marine Populations.

The molecular diversity of marine picocyanobacterial populations, an important component of phytoplankton communities, is better characterized using high-resolution marker genes than the 16S rRNA gene as they have greater sequence divergence to differentiate between closely related picocyanobacteria groups. Although specific ribosomal primers have been developed, another general disadvantage of bacterial ribosome-based diversity analyses is the variable number of rRNA gene copies. To overcome these issues, the single-copy gene, encoding the cytochrome subunit of the cytochrome complex, has been used as a high-resolution marker gene to characterize diversity. We have designed new primers targeting the gene and proposed a nested PCR method (termed Ong_2022) for metabarcoding of marine populations obtained by flow cytometry cell sorting. We evaluated the specificity and sensitivity of Ong_2022 against the standard amplification protocol (termed Mazard_2012) using filtered seawater samples. The Ong_2022 approach was also tested on flow cytometry-sorted populations. Samples (filtered and sorted) were obtained in the Southwest Pacific Ocean, from subtropical (ST) and subantarctic (SA) water masses. The two PCR approaches using filtered samples recovered the same dominant subclades, Ia, Ib, IVa, and IVb, with small differences in relative abundance across the distinct samples. For example, subclade IVa was dominant in ST samples with the Mazard_2012 approach, while the same samples processed with Ong_2022 showed similar contributions of subclades IVa and Ib to the total community. The Ong_2022 approach generally captured a higher genetic diversity of subcluster 5.1 than the Mazard_2012 approach while having a lower proportion of incorrectly assigned amplicon sequence variants (ASVs). All flow cytometry-sorted samples could be amplified only by our nested approach. The taxonomic diversity obtained with our primers on both sample types was in agreement with the clade distribution observed by previous studies that applied other marker genes or PCR-free metagenomic approaches under similar environmental conditions. The gene has been proposed as a high-resolution marker gene to access the diversity of marine populations. A systematic metabarcoding approach based on the gene would improve the characterization/assessment of the community structure in marine planktonic ecosystems. We have designed and tested specific primers to be applied in a nested PCR protocol (Ong_2022) for metabarcoding the gene. The Ong_2022 protocol can be applied to samples with low DNA content, such as those obtained by flow cytometry cell sorting, allowing the simultaneous assessment of the genetic diversity of populations and cellular properties and activities (e.g., nutrient cell ratios or carbon uptake rates). Our approach will allow future studies using flow cytometry to investigate the link between ecological traits and taxonomic diversity of marine .