Genomic DNA extraction and PCR amplification of cyanobacterial genes

The genomic DNA of the ten Chroococcidiopsis sp. strains was extracted using a PowerWater DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, United States) and quantified using the NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). Amplification of apcE2 was performed by using 12 ng of genomic DNA in 12 μl PCR reaction mixtures containing 6 μl of Phusion High-Fidelity PCR Master Mix with HF Buffer (New England Biolabs, Ipswich, MA, United States) and F-apcE2M and R-apcE2M primers as described (Antonaru et al., 2020 (link)). The resulting amplicons were loaded onto a 1.5% agarose gel containing 0.5 mg ml^–1 ethidium bromide and, after electrophoresis, visualized with a transilluminator.
For psaA2 and psaB2, PCR involved 50 ng of gDNA in 25 μl reactions, using a Q5 High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA, United States). Primers are listed in Supplementary Table 1. Thermocycling conditions were as reported above, except for a 1 min extension time, and a gradient annealing temperature as described below. The results were visualized on a 1.5% agarose gel stained with ROTI GelStain Red (Carl Roth GmbH, Karlsruhe, Germany).