Liu, G.; Bousquet, L.; Mayeur, H.; Manirakiza, E.; Daric, V.; Klopp, C.; Noirot, C.; Lopez-Escardo, D.; Grimsley, N. H.; Yau, S.; Krasovec, M.; Echeverria, M.; PIGANEAU, G.

Marine photosynthetic microbes contribute substantially to global primary production, yet many algal lineages still lack reference genomes with the continuity and annotation quality required for fine-scale structural, regulatory and comparative analyses. Ostreococcus tauri, one of the smallest known free-living photosynthetic eukaryotes, has been a model marine picoeukaryote for over two decades. Despite successive improvements to its historical reference genome, previous assemblies retained hundreds of gaps and incomplete genes, hampering high-resolution genomic analyses. Here, we present O. tauri RCC4221 genome version 2026, a telomere-to-telomere assembly of all 20 chromosomes spanning 13.34 Mb with no gaps. This assembly combines PacBio long-read sequencing, Illumina short-read polishing, correction of unresolved regions guided by independent Nanopore-based assemblies. The updated reference supports a curated annotation comprising 7,683 protein-coding genes, 48 tRNA genes, 3 rRNA operons, 116 medium-sized noncoding RNAs, one signal recognition particle RNA and 138 small nucleolar RNAs. It also improves gene-model integrity and recovers candidate coding loci absent from the 2014 reference. Structural analyses resolved the organization of the two atypical low-GC chromosome 2 and 19 that contain duplicated regions that were collapsed or misrepresented in previous assemblies. Finally, bisulfite sequencing and PacBio SMRT sequencing revealed a dual DNA methylation landscape, with CG-context cytosine methylation concentrated in gene bodies and N6-methyladenosine (m6A) enriched at the start codon. The updated O. tauri 2026 assembly provides a complete and curated reference resource for chromosome biology, comparative genomics, epigenomics and RNA biology in a model marine picoeukaryote.