Comparative Analysis of Extrachromosomal Circular DNA in Wild and Cultivated Oryza Species

Due to its societal and economic importance as a staple crop for human nutrition, Oryza sativa is one of the most studied crops and has attracted a lot of genomic studies thanks to its small and well-assembled genome. Currently, multiple genomes are available for O. sativa and a growing number also cover wild related species. These genomes provide solid tools to better explore the phylogenetic and molecular relationships between cultivated rice and its wild ancestors and progenitors. Despite the abundance of genomic studies published in the last decades, the extrachromosomal circular DNA (eccDNA) content of these species is still widely unexplored. eccDNA is a unique form of DNA that is characterized by its circular shape and its seeming independence from the chromosomes. In this research work, we evaluated the abundance and diversity of eccDNA in the wild (AA genome) and cultivated species of the Oryza genus. To this aim, we isolated eccDNA from O. rufipogon, O. nivara, O. glaberrima, and O. sativa spp. japonica var Nipponbare, and O.sativa spp japonica var TNG67. We used a dual sequencing strategy with both long reads (Oxford Nanopore) and short reads (Illumina). Our results demonstrate that the presence of mitochondrial eccDNA contributes to the vast majority (over 85%) of the eccDNA content of O. rufipogon and O. nivara. Strikingly, for all the species, over 90% of eccDNA-producing regions were annotated as Transposable Elements. Most TE families detected in the eccDNA were class II retrotransposons, with members of the Gypsy subclasses being by far the most prevalent in every family. Other well-represented categories for most accessions were Copia retrotransposons, Helitrons, and subclass I DNA transposon. Surprisingly we could not find evidence for an active production of eccDNA besides those derived from mitochondrial eccDNA and rDNA. Overall, the distribution of TE families in eccDNA followed the distribution of genomic TE families. These results provide important information about the diversity and abundance of eccDNA in the Oryza species and shed light on the differences in extrachromosomal DNA accumulation between cultivated rice and its wild ancestors.