Abstract Background and Aims Three out of four RNA components of ribosomes are encoded by 45S rDNA loci, whose transcripts are processed into 18S, 5.8S and 26S ribosomal RNAs. The loci are organized as long head-to-tail tandem arrays of nearly identical units spanning over several megabases of sequence. Due to this peculiar structure, the number of rRNA genes, their sequence composition and expression status remain unclear, especially in complex polyploid genomes harbouring multiple loci. Here we conducted a complex study to decipher structure and activity of both major and minor rRNA loci in hexaploid bread wheat ( Triticum aestivum ). Methods We employed an original, multi-omics approach, combining chromosome flow sorting and optical mapping with transcriptome and methylome sequencing. Key Results The former two techniques enabled unbiased quantification of rDNA units in particular loci of the wheat genome. Total number of rRNA genes organized in tandem arrays was 4388, with 64.1, 31.4, 3.9 and 0.7% located in short arms of chromosomes 6B, 1B, 5D and 1A, respectively. At the expression level, only 1B and 6B loci contributed to transcription at roughly 2:1 ratio. The 1B:6B ratio varied among five analysed tissues (embryo, coleoptile, root tip, primary leaf, mature leaf), being the highest (2.64:1) in mature leaf and lowest (1.72:1) in coleoptile. Cytosine methylation was considerably higher in CHG contexts in the silenced 5D locus compared to the active 1B and 6B loci. Conclusions A fine genomic organization and tissue-specific expression of rRNA loci were deciphered, for the first time, in a complex polyploid species. We documented various mechanisms of rRNA dosage control, including gene elimination and stable inactivation related to nucleolar subdominance of A and D-genome loci, and a subtle, developmentally regulated silencing of one of the major loci. The results are discussed in the context of wheat evolution and transcription regulation.

Anatomy, transcription dynamics and evolution of wheat ribosomal RNA loci deciphered by a multi-omics approach