Nested PCR Approach for petB Gene Metabarcoding of Marine Synechococcus Populations.

Area of Science:

• Marine microbiology
• Molecular ecology
• Phytoplankton diversity

Background:

• Marine picocyanobacteria, particularly Synechococcus, are crucial phytoplankton components.
• Traditional 16S rRNA gene sequencing has limitations for differentiating closely related picocyanobacteria.
• The single-copy petB gene offers higher resolution for Synechococcus diversity studies.

Purpose of the Study:

• To design and evaluate new primers and a nested PCR protocol (Ong_2022) for marine Synechococcus metabarcoding using the petB gene.
• To assess the specificity and sensitivity of the Ong_2022 protocol compared to existing methods (Mazard_2012).
• To enable simultaneous assessment of Synechococcus genetic diversity and cellular properties using flow cytometry-sorted samples.

Main Methods:

• Development of novel primers targeting the petB gene.
• Implementation of a nested PCR protocol (Ong_2022) for petB metabarcoding.
• Comparison of Ong_2022 with the Mazard_2012 protocol using filtered seawater and flow cytometry-sorted Synechococcus samples from the Southwest Pacific Ocean.

Main Results:

• The Ong_2022 protocol successfully amplified low-DNA samples from flow cytometry-sorted Synechococcus.
• Ong_2022 captured higher genetic diversity within Synechococcus subcluster 5.1 and reduced incorrectly assigned amplicon sequence variants (ASVs) compared to Mazard_2012.
• Both methods identified dominant subclades (Ia, Ib, IVa, IVb), with Ong_2022 showing nuanced abundance differences, particularly in subtropical samples.

Conclusions:

• The Ong_2022 nested PCR protocol using the petB gene is a sensitive and specific method for marine Synechococcus metabarcoding.
• This approach enhances the characterization of Synechococcus community structure in marine ecosystems.
• The protocol's effectiveness with low-DNA samples facilitates integrated studies linking Synechococcus diversity with cellular functions.