Wang, B.; Jia, Y.; Dang, N.; Yu, J.; Bush, S. J.; Gao, S.; He, W.; Wang, S.; Guo, H.; Yang, X.; Ma, W.; Ye, K.

Centromeres play a crucial and conserved role in cell division, although their composition and evolutionary history in green algae, the evolutionary ancestors of land plants, remains largely unknown. In this study, we constructed near telomere-to-telomere (T2T) assemblies for two Trebouxiophyceae species, Chlorella sorokiniana NS4-2 and C. pyrenoidosa DBH, with chromosome numbers of 12 and 13, and genome sizes of 58.11 Mb and 53.41 Mb, respectively. We identified and validated centromere sequences using CENH3 ChIP-seq and found that, similar to humans and higher plants, the centromere CENH3 signals of green algae display a pattern of hypomethylation. Interestingly, the centromeres of both species largely comprised transposable elements (TEs), although their composition showed significant differences. In Chlorella species, centromeres primarily consist of multiple elements, such as LINE/L1, LTR/Copia, LINE/RTEX, and Unknown families. However, in early-evolved species such as Chamydomonas reinhardtii, Coccomyxa subellipsoidea, and Chromochloris zofingiensis, centromere composition has evolved towards optimization with a single-element composition. Through phylogenomic and centromeric compositions analysis, We found that the centromeres of the most recent common ancestor of green algae are composed of two transposable element families, LINE/L1 and LTR/Copia. Moreover, we observed significant differences in the composition and structure of centromeres among chromosomes with strong collinearity within the Chlorella genus, suggesting that centromeres in green algae evolve more rapidly than non-centromeric regions. This study not only provides high-quality genome data for comparative genomics of green algae but gives insight into the composition and evolutionary history of centromeres in early plants, laying an important foundation for further research on their evolution.