Pollen mother cell transcriptome sequencing (RNAseq) from Rhynchospora pubera. Holocentromeres in Rhynchospora are associated with genome-wide centromere-specific repeat arrays interspersed among euchromatin

The present study provides through extensive analysis of NGS sequencing of genomic DNA, ChIP-seq, RNAseq and BAC sequencing, a cytogenomic characterization of the first identified centromere-specific DNA sequences from a holocentric species. NGS of genomic DNA was used to identify putative centromeric repeats, while RNAseq allowed us to identify and characterize the CENH3 gene of Rhynchospora pubera, our model species. An antibody recognizing the holocentromeres of that species was then designed. ChIP-seq was used to validate the association of the centromeric repeat candidates, a satellite DNA (Tyba) and a centromeric retrotransposon (CRRh), to the functional centromeric nucleosomes from a holocentric species. Finally, assembly of sequenced BACs containing Tyba centromeric tandem repeat was used to examine the properties of the multiple centromeric units found.