Telomere-to-telomere characterization of rDNA chromosome in the myxomycete Didymium iridis

<h4>Background</h4>The ribosomal DNA (rDNA) of the myxomycete Didymium iridis is located on a linear, multi-copy, non-Mendelian chromosome. Efforts to determine the complete sequence by short-read sequencing technologies have been prevented by the presence of highly repetitive regions. Here we use high coverage (~10,000 x) long-read Oxford Nanopore Technology to determine the rDNA chromosome sequence in haploid amoebae from telomere-to-telomere.<h4>Results</h4>The 20 kb rDNA chromosome, which is present at ~ 132 copies per haploid genome, is capped by regular TTAGGG telomeric repeats at both ends and carries an 11.3 kb pre-rRNA transcription unit coding for the small and large subunit rRNAs. The rRNA genes are further interrupted by autocatalytic group I introns, one of which encodes a homing endonuclease and two catalytic RNA domains with different functions in RNA processing. RNA mapping analyses from amoeba, microcyst, flagellate, and plasmodium stages, based on Illumina short-read sequencing, support the presence of a mature intron homing endonuclease mRNA both in haploid and diploid life stages in D. iridis. The non-transcribed sequence region upstream of the transcription unit contains several direct repeat arrays, including a highly complex upstream promoter region likely to be involved in pre-rRNA transcription regulation. Adjacent to the upstream telomere, a 4.2 kb palindromic region with potential for cruciform structure formation is found. Here, two putative replication origin candidates are located.<h4>Conclusions</h4>High coverage Oxford Nanopore Technology sequencing results in excellent resolution of complex sequence repeat feature in the D. iridis rDNA chromosome. The rRNA genes are interrupted by complex group I introns and RNA sequencing supports intron autocatalytic processing in haploid and diploid life stages. This study provides new insights into structural arrangements of nuclear rDNA in eukaryotic microorganisms.