Characterization of Eu- and heterochromatin of citrus with a focus on the condensation behavior of 45S rDNA chromatin.

To characterize the properties of eu- and heterochromatic regions in Citrus species, the chromosomal distribution of different histone H3 marks, DNA methylation sites (5mC) and 45S ribosomal DNA sites were determined for C. clementina, C. paradisi, C. sinensis, and for the hybrid Ortanique C. reticulata × C. sinensis. Our data show that in the relatively small genomes of investigated Citrus species (genome size ranges from 378-400 Mbp) the euchromatin is characterized by histone H3 lysine 4 mono-, di- and trimethylation (H3K4me1/ 2/3) and histone H3 lysine 9 trimethylation (H3K9me3). In contrast, histone H3 lysine 9 mono- and dimethylation (H3K9me1/2), histone H3 lysine 27 mono-, di- and trimethylation (H3K27me1/2/3) as well as 5-methylcytosine (5mC) were enriched at certain heterochromatin fractions. Whereas H3K9me1/2 and H3K27me1 were preferentially enriched at the chromomycin A(3)-bright (CMA(+)) heterochromatin, H3K27me2/3 showed a higher accumulation at the DAPI brightly-stained heterochromatin. 5mC signals were associated with most of the CMA(+) areas as well as with the DAPI strongly-stained heterochromatin fraction. Therefore, extensive methylation of DNA as well as of H3K9me1/2 and H3K27me1/2/3, and depletion of H3K4me1/2/3 and H3K9me3 appear to be specific features of heterochromatin in Citrus. Transcriptionally active decondensed 45S rDNA sites were found DNA hypomethylated, while the silenced condensed sites were strongly 5mC methylated. Although the number of chromosomal 45S rDNA sites differed between the species, the number of transcriptionally active rDNA sites remains constant.