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Eukaryotic genome replication depends on thousands of DNA replication origins (ORIs). A major challenge is to learn ORI biology in multicellular organisms in the context of growing organs to understand their developmental plasticity. We have identified a set of ORIs of Arabidopsis thaliana and their chromatin landscape at two stages of post-embryonic development. ORIs associate with multiple chromatin signatures including transcription start sites (TSS) but also proximal and distal regulatory regions and heterochromatin, where ORIs colocalize with retrotransposons. In addition, quantitative analysis of ORI activity led us to conclude that strong ORIs have high GC content and clusters of GGN trinucleotides. Development primarily influences ORI firing strength rather than ORI location. ORIs that preferentially fire at early developmental stages colocalize with GC-rich heterochromatin, but at later stages with transcribed genes, perhaps as a consequence of changes in chromatin features associated with developmental processes. Our study provides the set of ORIs active in an organism at the post-embryo stage that should allow us to study ORI biology in response to development, environment, and mutations with a quantitative approach. In a wider scope, the computational strategies developed here can be transferred to other eukaryotic systems., This research was supported by Secretaría de Estado de Investigación, Desarrollo e Innovación, grants BFU2012-34821, BFU2013-50098-EXP, and BFU2015-68396-R to C.G., BIO2016-79043-P to U.B. and AGL2013-43244-R to J.M.C., as well as by institutional grants from Fundacion Ramon Areces and Banco de Santander to the CBMSO.

Full genome replication in eukaryotes depends on the function of thousands of DNA replication origins (ORIs) that constitute the originome. The identification of ORIs in cultured cells has served to define their basic DNA and chromatin features. However, a major challenge is to learn the biology of ORIs in adult organisms to understand their developmental plasticity. Here, we have determined the originome and chromatin landscape in Arabidopsis thaliana at two stages of vegetative development. We found that ORIs associate with multiple chromatin signatures including the most frequent at TSS but also at proximal and distal gene regulatory regions or repressed Polycomb (PcG) domains. In constitutive heterochromatin, a high fraction of ORIs colocalize with GC-rich retrotransposons. Quantitative analysis of ORI activity led us to conclude that strong ORIs possess high scores of local GC content and clusters of GGN trinucleotides that may form G quadruplexes and other G-rich structures. We also found that development primarily influences ORI firing strength rather than ORI location. Moreover, ORIs that preferentially fire at early vegetative stages colocalize with GC-rich heterochromatin whereas those at later stages associate with transcribed genes. Our study provides the first originome of an adult organism, which is a developmentally and genetically tractable, thus opening new avenues of studying the biology of ORIs in response to developmental cues, transcriptional programs, environmental challenges and mutant backgrounds.