The NnCenH3 Protein and Centromeric DNA Sequence Profiles of Sacred Lotus (Nelumbo nucifera Gaertn.) Reveal the DNA Structures and Dynamics of Centromeres in Basal Eudicots.

Centromeres on eukaryotic chromosomes consist of large arrays of DNA repeats that undergo very rapid evolution. Nelumbo nucifera Gaertn. (sacred lotus) is a phylogenetic relict and an aquatic perennial basal eudicot. Studies concerning the centromeres of this basal eudicot species could provide ancient evolutionary perspectives. In this study, we characterized the centromeric marker protein NnCenH3 (sacred lotus centromere-specific histone H3 variant), and used a chromatin immunoprecipitation (ChIP)-based technique to recover the NnCenH3 nucleosome-associated sequences of sacred lotus. The properties of the centromere-binding protein and DNA sequences revealed notable divergence between sacred lotus and other flowering plants, including the following factors: (i) an NnCenH3 alternative splicing variant comprising only a partial centromere-targeting domain, (ii) active genes with low transcription levels in the NnCenH3 nucleosomal regions, and (iii) the prevalence of the Ty1/copia class of long terminal repeat (LTR) retrotransposons in the centromeres of sacred lotus chromosomes. In addition, the dynamic natures of the centromeric region showed that some of the centromeric repeat DNA sequences originated from telomeric repeats, and a pair of centromeres on the dicentric chromosome1 was inactive in the metaphase cells of sacred lotus. Our characterization of the properties of centromeric DNA structure within the sacred lotus genome describes a centromeric profile in ancient basal eudicots and might provide evidence of the origins and evolution of centromeres. Furthermore, the identification of centromeric DNA sequences is of great significance for the assembly of the sacred lotus genome. Significance Statement Sacred lotus (Nelumbo nucifera Gaertn.), an aquatic perennial, is a basal eudicot. Understanding centromere structure of basal eudicots will inform perspectives on centromere evolution. Here we analyzed the centromere-binding protein (CENH3) and used ChIP-sequencing of centromeric DNA to demonstrate notable differences between sacred lotus and other flowering plants, our results will also be useful for construction of fine-scale genetic and physical maps in sacred lotus.