Breaking the Limits - Multichromosomal Structure of an Early Eudicot Pulsatilla Patens Mitogenome Reveals Extensive RNA-editing, Longest Repeats and Chloroplast Derived Regions Among Sequenced Land Plant Mitogenomes

Background: The mitogenomes of vascular plants are one of the most structurally diverse molecules. In the present study we characterize mitogenome of a rare and endangered species Pulsatilla patens. We investigated the gene content and its RNA editing potential, repeats distribution and plastid derived sequences.Results: The mitogenome structure of early divergent eudicot, endangered Pulsatilla patens does not support the master chromosome hypothesis, revealing the presence of three linear chromosomes of total length 986 613 bp. The molecules are shaped by the presence of extremely long, exceeding 87 kbp, repeats and multiple chloroplast derived regions including nearly complete inverted repeat. Since the plastid IR content of Ranunculales is very characteristic, the incorporation into mitogenome could be explained rather by intracellular transfer than mitochondrial HGT. The mitogenome contains an almost complete set of genes known from other vascular plants with exception of rps10 and sdh3, the latter being present but pseudogenised. Analysis of long ORFs enabled the identification of genes which are rarely present in plant mitogenomes, including RNA and DNA polymerases, albeit their presence even at species level is variable. Mitochondrial transcripts of P. patens were edited with a high frequency, exceeding the level known in other analyzed angiosperms, despite strict qualification criteria of editing event’s count and analysis of generally less frequently edited leaf transcriptome. The total number of edited sites was 902 and nad4 was identified as the most edited gene with 65 C to U changes. Non-canonical, reverse U to C editing was not detected. Comparative analyses of mitochondrial genes of three Pulsatilla species revealed a level of variation comparable to chloroplast CDS dataset and much higher infrageneric differentiation than in other known angiosperm genera. The variation found in CDS of mitochondrial genes is comparable to values found among Pulsatilla plastomes. Despite a complicated mitogenome structure, 14 single copy regions not splitted by repeats or MTPT of 329 kbp revealed potential for phylogenetic, phylogeographic and population genetics studies by revealing intra- and interspecific collinearity.Conclusions: This studies provides valuable new information about mitochondrial genome of early divergent eudicots, Pulsatilla patens, revealed multi-chromosomal structure and shed new light on mitogenomics of early eudicots.