Telomere-to-telomere Characterization of Rdna Chromosome in the Myxomycete Didymium Iridis

Abstract Background 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. Results 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. Conclusions 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.