Centromeres are the specialized regions of the chromosomes that direct faithful chromosome segregation during cell division. Despite their functional conservation, centromeres display features of rapidly evolving DNA and wide evolutionary diversity in size and organization. Previous work found that the noncanonical B-form DNA structures are abundant in the centromeres of several eukaryotic species with a possible implication for centromere specification. Thus far, systematic studies into the organization and function of non–B-form DNA in plants remain scarce. Here, we applied the oat system to investigate the role of non–B-form DNA in centromeres. We conducted chromatin immunoprecipitation sequencing using an antibody to the centromere-specific histone H3 variant (CENH3); this accurately positioned oat centromeres with different ploidy levels and identified a series of centromere-specific sequences including minisatellites and retrotransposons. To define genetic characteristics of oat centromeres, we surveyed the repeat sequences and found that dyad symmetries were abundant in oat centromeres and were predicted to form non–B-DNA structures in vivo. These structures including bent DNA, slipped DNA, Z-DNA, G-quadruplexes, and R-loops were prone to form within CENH3-binding regions. Dynamic conformational changes of predicted non–B-DNA occurred during the evolution from diploid to tetraploid to hexaploid oat. Furthermore, we applied the single-molecule technique of AFM and DNA:RNA immunoprecipitation with deep sequencing to validate R-loop enrichment in oat centromeres. Centromeric retrotransposons exhibited strong associations with R-loop formation. Taken together, our study elucidates the fundamental character of non–B-form DNA in the oat genome and reveals its potential role in centromeres.

Centromeres are the specialized regions of the chromosomes that direct faithful chromosome segregation during cell division. Despite their functional conservation, centromeres display features of rapidly evolving DNA and wide evolutionary diversity in size and organization. Previous work found that the noncanonical B-form DNA structures are abundant in the centromeres of several eukaryotic species with a possible implication for centromere specification. Thus far, systematic studies into the organization and function of non–B-form DNA in plants remain scarce. Here, we applied the oat system to investigate the role of non–B-form DNA in centromeres. We conducted chromatin immunoprecipitation sequencing using an antibody to the centromere-specific histone H3 variant (CENH3); this accurately positioned oat centromeres with different ploidy levels and identified a series of centromere-specific sequences including minisatellites and retrotransposons. To define genetic characteristics of oat centromeres, we surveyed the repeat sequences and found that dyad symmetries were abundant in oat centromeres and were predicted to form non–B-DNA structures in vivo. These structures including bent DNA, slipped DNA, Z-DNA, G-quadruplexes, and R-loops were prone to form within CENH3-binding regions. Dynamic conformational changes of predicted non–B-DNA occurred during the evolution from diploid to tetraploid to hexaploid oat. Furthermore, we applied the single-molecule technique of AFM and DNA:RNA immunoprecipitation with deep sequencing to validate R-loop enrichment in oat centromeres. Centromeric retrotransposons exhibited strong associations with R-loop formation. Taken together, our study elucidates the fundamental character of non–B-form DNA in the oat genome and reveals its potential role in centromeres.

Centromeres are the specialized regions of the chromosomes that direct faithful chromosome segregation during cell division. Despite their functional conservation, centromeres display features of rapidly evolving DNA and wide evolutionary diversity in size and organization. Previous work found that the noncanonical B-form DNA structures are abundant in the centromeres of several eukaryotic species with a possible implication for centromere specification. Thus far, systematic studies into the organization and function of non–B-form DNA in plants remain scarce. Here, we applied the oat system to investigate the role of non–B-form DNA in centromeres. We conducted chromatin immunoprecipitation sequencing using an antibody to the centromere-specific histone H3 variant (CENH3); this accurately positioned oat centromeres with different ploidy levels and identified a series of centromere-specific sequences including minisatellites and retrotransposons. To define genetic characteristics of oat centromeres, we surveyed the repeat sequences and found that dyad symmetries were abundant in oat centromeres and were predicted to form non–B-DNA structures in vivo. These structures including bent DNA, slipped DNA, Z-DNA, G-quadruplexes, and R-loops were prone to form within CENH3-binding regions. Dynamic conformational changes of predicted non–B-DNA occurred during the evolution from diploid to tetraploid to hexaploid oat. Furthermore, we applied the single-molecule technique of AFM and DNA:RNA immunoprecipitation with deep sequencing to validate R-loop enrichment in oat centromeres. Centromeric retrotransposons exhibited strong associations with R-loop formation. Taken together, our study elucidates the fundamental character of non–B-form DNA in the oat genome and reveals its potential role in centromeres.