Best practices for methylome characterization in novel species: a case study in the microalgae Microchloropsis.

Microalgae remain an important feedstock in the circular bioeconomy. The discovery of new species combined with advanced biotechnology drives optimization of performance predicated on deep knowledge of algal genomics and phenotype. Understanding the contribution of epigenetic processes to algal function provides insight and better approaches for achieving production goals. Here, we provide a methodological framework for investigating epigenetic modifications in new species, including analysis of state-of-the-art techniques, and best practices for discerning novel modifications, focusing on variants of DNA methylation. Further, we demonstrate that specific forms of DNA methylation are overlooked by traditional epigenetic analysis strategies. Using high-throughput, lower cost techniques, we provide several pieces of evidence demonstrating Microchloropsis gaditana and M. salina (formerly Nannochloropsis), two candidate feedstock species, lack the most ubiquitous forms of eukaryotic DNA methylation (5mC and 5hmC) and instead employ N6-adenine methylation (6mA), commonly found in bacteria, in their genomes. Interestingly, transcriptionally diverse physiological conditions do not elicit differential 6mA methylation, suggesting the presence of 6mA may provide stability and protection of the genome. These collective discoveries illuminate not only an exciting avenue for improving feedstock genetic drift, stability, and culture health for bioproduction but also an ideal model species to study other epigenetic processes in microalgae.