Vol. 68, No 2/2021 277–286 https://doi.org/10.18388/abp.2020_5555 Regular paper Analysis of the complete mitochondrial genome sequence of the resurrection plant Haberlea rhodopensis Vesselin Baev1,2*, Zdravka Ivanova1*✉, Galina Yahubyan1,2, Valentina Toneva1,2, Elena Apostolova1,2, Georgi Minkov1,2 and Ivan Minkov1,3 1Institute of Molecular Biology and Biotechnology, Plovdiv, Bulgaria; 2Department of Plant Physiology and Molecular Biology, University of Plov- div, Plovdiv, Bulgaria; 3Centre of Plant Systems Biology and Biotechnology, Plovdiv, Bulgaria Haberlea rhodopensis is a paleolithic tertiary relict spe- genome molecules proved to be difficult to assemble be- cies that belongs to the unique group of resurrection cause of their variable structure (Bi et al., 2016). plants sharing remarkable tolerance to desiccation. Mitochondria are essential organelles in plants, often When exposed to severe drought stress, this species called the energy factory of the cell. They possess their shows an ability to maintain structural integrity of its own genome which is often used for comparative and deactivated photosynthetic apparatus, which easily re- evolutionary studies. A recent study highlights mt genes activates upon rehydration. In addition to its homoi- as significant markers for resolving relationships among ochlorophyllous nature, the resurrection capability of genera, families, and higher rank taxa across angio- H. rhodopensis is of particular importance to the global sperms. It was observed that the low substitution rates climate change mitigation. In this study, we sequenced, of mt genes in comparison to cp genes make them very assembled, and analyzed the mitochondrial (mt) genome useful in the reconstruction of ancient phylogenetic rela- of H. rhodopensis for the first time. The master circle has tionships (Qiu et al., 2010). Phylogenetic trees represent a typical circular structure of 484 138 bp in length with the true relationship between conserved core genome a 44.1% GC content in total. The mt genome of H. rho- sequences and are used to resolve taxonomic grouping dopensis contains 59 genes in total, including 35 pro- among different species. Most mt genomes are extremely tein-coding, 21 tRNAs, and 3 rRNAs genes. 7 tandem large and complex when compared to those of animals repeats and 85 simple sequence repeats (SSRs) are dis- and fungi, which generally show a stable and conserva- tributed throughout the mt genome. The alignment of tive mode of evolution (Li et al., 2009). Land plants’ 20 plant mt genomes confirms the phylogenetic position mitochondrial genomes exhibit some features, such as a of H. rhodopensis in the Lamiales order. Our comprehen- significant size expansion, frequent gene loss and gene sive analysis of the complete mt genome of H. rhodo- transfer to the nucleus, RNA editing, genomic rearrange- pensis is a significant addition to the limited database of ment, and replacement of some tRNAs by their cp coun- organelle genomes of resurrection species. Comparative terparts (Knoop, 2004), that highlight their evolutionary and phylogenetic analysis provides valuable information dynamics. Mitochondrial genomes vary significantly in for a better understanding of mitochondrial molecular structure, size, and gene order (Bi et al., 2016). Much evolution in plants. of these variations occur even between members of the same family or genera (Alverson et al., 2010). These main Keywords: Haberlea rhodopensis, resurrection plants, mitochondrial features contrast with animal mtDNAs which are struc- genome, genome assembly turally conserved, relatively small in size, and have very Received: 28 November, 2020; revised: 09 March, 2021; accepted: fast nucleotide substitution rates (Ballard et al., 2004). 26 March, 2021; available on-line: 12 May, 2021 Previous studies revealed that the nucleotide substitu- tion rates between animals and plants differ due to the ✉e-mail: [email protected] fact that the plant and animal kingdoms diverged about Acknowledgements of Financial Support: This project has re- ceived funding from the European Union’s Horizon 2020 research 1000 million years ago and their patterns of evolution and innovation programme under grant agreement No 739582 have become different. Also, researchers observed sig- (Project PlantaSYST). nificantly different rates in nucleotide substitution rates Availability of data and material: Complete mtDNA of Haberlea rho- among plants. Comparison between plant mitochondrial dopensis is deposited in the NCBI GeneBank database under acces- sion number MH757117. (mt), chloroplast (cp) and nuclear (n) DNA sequences *Joint first authors revealed slower substitution rates in the mitochondrial Abbreviations: cp, chloroplast; CV, Composition Vector; mt ge- DNA, which is maybe due to a lower mutation rate. In nome, plant mitochondrial genome; SSRs, simple sequence re- contrast to mammalian mtDNA, where mitochondrial peats; WGS, whole-genome sequencing sequences evolve 5 times faster than nDNA, mtDNA sequences in angiosperms evolve at least 5 times more INTRODUCTION slowly than nDNA (Wolfe et al., 1987). Plant mt genomes contain various repeat sequenc- Over the last several years, many plant genomes have es, such as tandem repeat sequences, simple repeat se- been sequenced and assembled by the next-generation quences, and large repeats (Alverson et al., 2010, 2011). sequencing (NGS) technologies. Due to their small size, In many studies it was observed that mt repeats contain conserved gene order, and content, chloroplast (cp) ge- valuable genetic information and are regarded as com- nomes were sequenced more frequently than mitochon- ponents in intramolecular recombination (Chang et al., drial genomes. In comparison to the cp genomes, the mt 2013). Methylated sites are linked to tandem repeats in the maize mt genome (Clifton, 2004). Simple sequence 278 V. Baev and others 2021 repeats play an important role in the evolution of plant GEN), according to the manufacturer’s instructions. The mt genomes and are responsible for structural variations quality and quantity of DNA were checked with an Ep- and size variability of plant mt genomes (Chang et al., och microplate spectrophotometer and an agarose gel. 2013). Some genes with large repeats may have multiple Library preparation and sequencing by HiSeqX Illumina copies. Recombination across large direct repeats may technology were performed at Macrogen (Seoul, South divide mt genomes into pairs of subgenomic molecules, Korea) by Illumina standard protocol. The isolated DNA whereas inverted repeats may generate isomeric circles was used to generate reads with a 150 bp paired-end (Handa, 2003; Clifton, 2004; Chang et al., 2013). data library and insert size of 350 bp. DNA sequencing The gene content of the plant mt genomes is high- is generated in a total of 2 × 366909885 reads. ly variable. The number of protein-coding genes in the mt genomes varies from 3 to 67, whereas the number Genome assembly and annotation of tRNA genes varies from 0 to 27 (Adams & Palmer, De novo assembly of the H. rhodopensis mt genome 2003). In the course of evolution, many mt genes orig- (mtDNA) was performed by applying NOVOPlasty inally found in plant mt genomes have been lost dur- (https://github.com/ndierckx/NOVOPlasty), а sole de ing transfer to the nucleus (Lei et al., 2013). For exam- novo assembler. A seed-and-extend algorithm was used ple, the sdh2, rps9, rps11, and rps16 genes were lost in which assembles organelle genomes from whole-genome the evolution of plant mt genomes. The protein-coding sequencing (WGS) data, starting from a related or dis- genes: rps12, sdh3, and sdh4 were lost in monocots, while tant single seed sequence with kmer 39 (Dierckxsens rps2 was lost in dicots (Zhang et al., 2012). et RNA editing, a post-transcriptional process of chang- al., 2016). According to the manual of this assembler, ing nucleotide sequence of any RNA molecule, chal- we used the total DNA sequencing reads (including lenged the basic concept of molecular biology that the nuclear, mt, and cp reads) instead of mapping them to primary RNA sequence reflects the sequence of DNA the reference genome and filtering mitochondrial reads. from which it is transcribed. The changes encompass in- The mtDNA nucleotide sequence of the cox1 gene from sertions and deletions of uridine residues, and a conver- the closely related species B. hygrometrica (JN107812) was sion of a cytidine to uridine within the RNA molecule. used as a seed sequence in the process of genome as- RNA editing affects the transcripts of protein-coding sembly. This was subsequently elongated, resulting in genes, non-coding transcribed regions, structural RNAs one contig and a successfully circularized genome. and intron sequences, and serves as a buffer for less Annotation of the H. rhodopensis mt genome was preferred mutations in the coding sequences (Covello & achieved using MITOFY (https://vcru.wisc.edu/cgi-bin/ Gray, 1989). mitofy/mitofy.cgi) with manual start and stop codon correction and validation via comparing to the mt genes In plant organelles, RNA editing sites were found in of previously annotated genomes. The mt gene nomen- the coding regions of mRNA, introns, and non-translat- clature was based on that of published land plant mt ed regions. The majority of post-transcriptional modifi- genomes available in the NCBI database. Transfer RNA cations include U-to-C, A-to-I, and the RNA-editing of genes (tRNA) were identified by MITOFY and validated C-to-U was identified in most of the angiosperms (Gray by the tRNAscan-SE program (http://lowelab.ucsc.edu/ et al., 1999). The process of site editing can generate an initiation or termination codon, but in most cases it gen- tRNAscan-SE/) with default settings (Schattner et al., erates an internal codon with functional relevance (Han- 2005). The mt genome circular representation was gen- da, 2003). Mt and cp RNA editing in plants is essential erated by OrganelllarGenomeDraw (OGDRAW) (Lohse for the normal functioning of their translation and respi- et al., 2013), and the complete mtDNA sequence was ration activity, and is beneficial for understanding gene deposited in the NCBI GeneBank database under acces- expression.