The genome of rye, Secale cereale, is distinguished by large repetitive regions including subtelomeric heterochromatin and retrotransposon-dominant centromeres, which contrast with the satellite-repeat-based centromeres in most characterized plant genome assemblies. This study aims to decode the architecture and evolution of these elusive regions through high-resolution genome assembly, with a focus on centromere dynamics and chromatin regulation.

Using PacBio HiFi and Nanopore sequencing, we generate a chromosome-scale assembly encompassing three complete centromeres and resolving subtelomeric heterochromatin. We identify terminal tandem repeat arrays as key determinants in establishing specialized chromatin environments linked to retrotransposon deposition. Notably, rye centromeres exhibit an unconventional epigenetic signature depleted of conventional activation and repression marks but displaying unique DNA hypomethylation patterns. This retrotransposon-enriched landscape promotes both the integration of young LTR retrotransposons and the recruitment of CENH3. Cross-species CENH3 ChIP-seq analyses reveal that Cereba retrotransposons are associated with enhanced CENH3 loading in cultivated and wild rye lineages, particularly through their conserved protease and integrase domains, suggesting a potential positive feedback loop for centromere evolution.

Our findings establish retrotransposons as autonomous organizers of centromere chromatin and identity in rye, challenging the paradigm of satellite-dependent centromere specification. The dual role of retrotransposons in maintaining CENH3 recruitment while facilitating genomic innovation provides a mechanistic basis for centromere plasticity. This work advances functional genomics of Triticeae crops and opens new avenues for centromere engineering to manipulate meiotic stability and chromosome transmission in crop breeding.