The complete mitochondrial genome of a medicinal fungus, Tolypocladium ophioglossoides

Abstract As part of a genome sequencing project for Tolypocladium ophioglossoides (T. ophioglossoides), a complete mitochondrial (mt) genome was assembled as a single circular dsDNA of 35,159 bp. Conserved genes including the large and small rRNA subunits, 25 tRNA and 15 protein-coding genes, were identified. In addition, 4 non-conserved open reading frames (ncORFs) in the intergenic and intronic regions were also identified. Transcription analyses using RNA-seq validated the expression of most conserved genes and ncORFs. Seven introns (groups I and II) were found within conserved genes, accounting for 21% of the mitogenome. All structural genes are located on one strand and are apparently transcribed in one direction. The complete mt genomes of T. ophioglossoides would be useful for future investigation of genetics, evolution and clinical identification of Tolypocladium species.

Tolypocladium ophioglossoides is a species of fungus in the family Ophiocordycipitaceae. It is parasitic on fruit bodies of the truffle-like Elaphomyces, ectomycorrhizal fungi closely related to Aspergillus and Penicillium (Landvik et al. 1996). It is distributed throughout many parts of the Northern Hemisphere (Mains 1957). This fungus has been used as a traditional rare and precious medicine in China for centuries. In this study, we report the complete mitochondrial genome sequence of T. ophioglossoides to provide useful genetic information for the future research in biology and medicine.
T. ophioglossoides was provided by Key Laboratory of Microbial Biochemistry and Metabolism Engineering of Zhejiang Province. Genomic DNA was prepared using CTAB method as described previously (Sun et al. 2013). One short sequencing library (ca. 200 bp) and three mate-pair librarys (ca. 3, 8, 10 kb) were constructed and sequenced using ABI PGM platform. Genome assembly and annotation were performed by CLC Genomies Workbench and Mfannot tool with default settings. One contig representing the mitochondrial DNA was identified on the basis of extensive sequence similarity to known fungal mitochondrial genomes. The complete mitochondrial genome of T. ophioglossoides was deposited in GenBank database with the accession number KX455872.The complete mitochondrial genome sequence of T. ophioglossoides is 35,159 bp in length and AT content of 72.47%. It consists of 15 core mitochondrial protein-coding genes, 25 tRNA genes, 2 rRNA genes (small and large subunit rRNA) and 2 unique and separate open-reading frames (ORFs) encoding proteins without apparent homology to any known proteins. Protein-encoding genes include ATP-synthase subunits 6, 8, and 9 (atp6, atp8, and atp9), cytochrome oxidase subunits (cox1, cox2, and cox3), apocytochrome b (cob), and NADH dehydrogenase subunits (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6) whose products involved in mitochondrial electron transfer, oxidative phosphorylation and mitochondrial protein synthesis. All 44 protein-and RNAencoding genes are located on the same strands and are transcribed in one direction. No Tandem Repeat Finder (TRF) hits were found on T. ophioglossoides used by Dfam. The gene order of T. ophioglossoides was identical with that of the known representatives of Metarhizium anisopliae mitochondrial genomes.
The set of 25 tRNA genes code for all 19 standard amino acids. Seventeen tRNA genes are adjacent to rnl, 4 tRNA genes approach to rns, and 4 tRNA genes are located around three protein-coding genes (cob, cox1, and cox3). Seven introns invade three genes including rnl (two), cob (one), cox1 (four). All these introns belong to group I introns. Intronic proteins include ribosomal protein S3 and GIY endonucleases.
Phylogenetic analysis based on whole mitogenome sequences confirms T. ophioglossoides as a member of the fungal order Hypocreales. T. ophioglossoides is clustered together with M. anisopliae and M. chamydosporia within the family Clavicipitaceae according to our phylogenetic analysis (Figure 1), with consistent taxonomic status according to phylogenetic analysis of nuclear genes of Hypocreales (Zhang et al. 2016).

Disclosure statement
This study was supported by the National Science Foundation of China under Grant (31370064). The authors have declared that no competing interests exist.

Funding
This study was supported by the National Science Foundation of China under Grant [31370064].