Complete Sequence, Multichromosomal Architecture and Transcriptome Analysis of the Solanum tuberosum Mitochondrial Genome

Abstract

Mitochondrial genomes (mitogenomes) in higher plants can induce cytoplasmic male sterility and be somehow involved in nuclear-cytoplasmic interactions affecting plant growth and agronomic performance. They are larger and more complex than in other eukaryotes, due to their recombinogenic nature. For most plants, the mitochondrial DNA (mtDNA) can be represented as a single circular chromosome, the so-called master molecule, which includes repeated sequences that recombine frequently, generating sub-genomic molecules in various proportions. Based on the relevance of the potato crop worldwide, herewith we report the complete mtDNA sequence of two S. tuberosum cultivars, namely Cicero and Désirée, and a comprehensive study of its expression, based on high-coverage RNA sequencing data. We found that the potato mitogenome has a multi-partite architecture, divided in at least three independent molecules that according to our data should behave as autonomous chromosomes. Inter-cultivar variability was null, while comparative analyses with other species of the Solanaceae family allowed the investigation of the evolutionary history of their mitogenomes. The RNA-seq data revealed peculiarities in transcriptional and post-transcriptional processing of mRNAs. These included co-transcription of genes with open reading frames that are probably expressed, methylation of an rRNA at a position that should impact translation efficiency and extensive RNA editing, with a high proportion of partial editing implying frequent mis-targeting by the editing machinery.

Keywords: RNA editing; Solanaceae family; comparative genomics; mitochondria; mtDNA; multichromosomal structure; potato; repeated sequences.

Figure 1

S. tuberosum mitochondrial genome assembly. (A) The 6 unitigs output by HGAP for the Cicero mitogenome. Colored blocks on the unitigs show repeats. Labeled ticks give primers positions, with those in black in forward orientation and those in light red in reverse orientation. (B) The 3 contigs obtained after PCR validation. Colored blocks on the contigs show repeats. The larger contig could not be circularized.

Figure 2

Organization of the potato mtDNA. Molecules 1, 2 and 3 are represented in green, light blue and red respectively. Gene sequences are shown below the sequence line, with protein genes in dark blue and rRNA and tRNA genes in red. Repeated sequences larger than 100 bp are shown above the sequence as orange arrows. Green bars indicate sequences of plastidial origin. Bent green and red lines indicate 5′ and 3′ transcript boundaries, respectively. Grey horizontal arrows represent major transcripts. Green horizontal arrows indicate consensus promoters found upstream of transcripts. Thin vertical lines indicate editing sites.

Figure 3

Methylation of the 18S rRNA. RNA-seq data revealed in about half of the reads an A-to-T mismatch at position 960 of 18S rRNA (position 968 in domain 31 of E. coli 16S rRNA). Such mismatch is diagnostic of mis-incorporation during cDNA synthesis because of m¹A methylation. (A) Comparison of the corresponding domains of the E. coli 16S rRNA (which is methylated at positions 966 and 967, m²G and m⁵C respectively) and of the potato 18S rRNA. Sequence differences are in lower case. (B) In the 3D structure of the bacterial ribosome, these nucleotides are close to the anticodon of the tRNA at position P (dark blue nucleotides in the tRNA structure pairing with the mRNA), with base 966 stacking with the first anticodon nucleotide.

Figure 4

Representative examples of variable mitochondrial protein sequences in Solanaceae species. The primary structures of S. tuberosum ATP6 (A) and COX2 (B) proteins were compared with those of other Solanaceae species available in GenBank. (A) Red and green bars indicate non-conserved or conserved aa sequences in ATP6, respectively. (B) Cultivated (S. lycopersicum) and wild (S. pennellii) tomato have additional 194 aa at the C-terminus of the protein compared with potato and other Solanaceae species. The numbers above red and green bars indicate the start and end positions of potato mitochondrial proteins.

Figure 5

Phylogenetic tree of Solanaceae species. Phylogram of the best maximum-likelihood (ML) tree as determined using the RAxML software from the concatemer of the coding sequences of 15 protein-coding genes. Numbers associated with branches are ML bootstrap support values. Vitis vinifera and Ipomea nil were used as outgroups.