Complete sequences of organelle genomes from the medicinal plant Rhazya stricta (Apocynaceae) and contrasting patterns of mitochondrial genome evolution across asterids

Abstract

Background: Rhazya stricta is native to arid regions in South Asia and the Middle East and is used extensively in folk medicine to treat a wide range of diseases. In addition to generating genomic resources for this medicinally important plant, analyses of the complete plastid and mitochondrial genomes and a nuclear transcriptome from Rhazya provide insights into inter-compartmental transfers between genomes and the patterns of evolution among eight asterid mitochondrial genomes.

Results: The 154,841 bp plastid genome is highly conserved with gene content and order identical to the ancestral organization of angiosperms. The 548,608 bp mitochondrial genome exhibits a number of phenomena including the presence of recombinogenic repeats that generate a multipartite organization, transferred DNA from the plastid and nuclear genomes, and bidirectional DNA transfers between the mitochondrion and the nucleus. The mitochondrial genes sdh3 and rps14 have been transferred to the nucleus and have acquired targeting presequences. In the case of rps14, two copies are present in the nucleus; only one has a mitochondrial targeting presequence and may be functional. Phylogenetic analyses of both nuclear and mitochondrial copies of rps14 across angiosperms suggests Rhazya has experienced a single transfer of this gene to the nucleus, followed by a duplication event. Furthermore, the phylogenetic distribution of gene losses and the high level of sequence divergence in targeting presequences suggest multiple, independent transfers of both sdh3 and rps14 across asterids. Comparative analyses of mitochondrial genomes of eight sequenced asterids indicates a complicated evolutionary history in this large angiosperm clade with considerable diversity in genome organization and size, repeat, gene and intron content, and amount of foreign DNA from the plastid and nuclear genomes.

Conclusions: Organelle genomes of Rhazya stricta provide valuable information for improving the understanding of mitochondrial genome evolution among angiosperms. The genomic data have enabled a rigorous examination of the gene transfer events. Rhazya is unique among the eight sequenced asterids in the types of events that have shaped the evolution of its mitochondrial genome. Furthermore, the organelle genomes of R. stricta provide valuable genomic resources for utilizing this important medicinal plant in biotechnology applications.

Figure 1

Maps of the organelle genomes of Rhazya stricta. The inner and outer circles represent the plastid and mitochondrial genomes, respectively. Genes on the inside and outside of each map are transcribed clockwise and counterclockwise direction, respectively. The thick lines on the plastid map indicate the inverted repeats (IRa and IRb), which separate the genome into large and small singles copy region. Ψ denotes a pseudogene.

Figure 2

Schematic representation of transfers of plastid DNA and transposable elements into the mitochondrial genome of Rhazya stricta. Each green line within the circle shows the regions of the plastid genome that have been inserted into different locations of the mitochondrial genome. Red lines outside of the mitochondrial genome indicate the location of integrated transposable elements (TEs) and asterisks indicate TEs that have inserted into genic regions. Genes indicated as blue and green boxes on the inside and outside of maps are transcribed clockwise and counterclockwise direction, respectively.

Figure 3

Gene transfer of rps14 . A. Schematic diagram of rps14 gene transfer from mitochondrial genome to the nucleus. The mitochondrial rps14 copies are identified in two repeat regions and are co-transcribed with rpl5. Boxes indicate mitochondrial targeting presequence (mTP; blue) and a conserved domain (ribosomal S14; red). The grey box arrow in the nuclear genome represents the non-functional copy of rps14 and the dotted red line indicates a conserved domain. Internal stop codons are indicated with asterisks. B. Nucleotide sequence alignment of the two nuclear, transcript, and mitochondrial copies of rps14 from Rhazya stricta. Shaded red box shows an 8 bp deletion that caused a frameshift. C. Amino acid sequence alignment of two nuclear and one mitochondrial copy of rps14 of Rhazya with seven nuclear-encoded and five mitochondrial copies from other angiosperms (see Additional file 1: Table S13). Blue boxes indicate mitochondrial targeting presequences. Red boxes indicate the conserved domain of ribosomal S14. mt = mitochondrial, n = nuclear.

Figure 4

Maximum likelihood phylogenetic trees for mitochondrial genes of Rhazya transferred to the nucleus. A. Mitochondrial and nuclear rps14 sequences of angiosperms. B. Mitochondrial and nuclear sdh3 sequences of angiosperms. Bold font indicates mitochondrial (blue) and nuclear copies (red) of rps14 and sdh3 in Rhazya stricta. Bootstrap support values >50% are shown at nodes. Red and black lines indicate nuclear and mitochondrial sequences, respectively.

Figure 5

Gene transfer of sdh3 . A. Schematic diagram of sdh3 gene transfer from mitochondrial genome to the nucleus. A transposable element (TE) insertion is shown in red in the mitochondrial DNA. The dashed line outlines a pseudogene due to mutations in the start codon as indicated. Boxes and oval indicate mitochondrial targeting presequence (mTP; blue), heat shock protein (hsp; orange), and a conserved domain (succinate dehydrogenase C; red). B. Nucleotide sequence alignment of the nuclear, transcript, and mitochondrial copies of Rhazya stricta. The shaded red area shows the intron within predicted heat shock protein (hsp22). C. Amino acid sequence alignment of Rhazya nuclear and mitochondrial sdh3 with six nuclear-encoded and three mitochondrial-encoded copies from other angiosperms (see Additional file 1: Table S13). Boxes indicate mitochondrial targeting presequence (blue) and a conserved domain of succinate dehydrogenase C (red), and shaded red boxes indicate the remaining portions of genes into which sdh3 was transferred (heat shock proteins, Rhazya and Gossypium-hsp22, Arabidopsis-hsp70; Iron-sulfur cluster scaffold-like protein, Ledum; hypervariable Bacillus group-specific protein, Strychnos; Pyrimidine (PYR) binding domain of thiamine pyrophosphate (TPP)-dependent enzyme, Lobelia). mt = mitochondrial, n = nuclear.

Figure 6

Genome size, amount of plastid-like and repetitive DNA and transposable elements in eight asterid mitochondrial genomes. A. Genome sizes, the number of bp of repetitive DNA, plastid-derived sequences and transposable elements. See Figure 8 legend for details of how the tree was constructed. B. Average of the percentage of different types of transposable elements of asterids. C. Repeat size and frequency (above), and proportion of repetitive DNA per genome (below).

Figure 7

Distribution of repetitive DNA in Rhazya mitochondrial genome compared to seven other asterids. Black lines within circular maps indicate the positions of the pairs of repeats, with crossed connecting lines denoting reverse repeats. Black boxes on the inner and outer circle indicate the positions of mitochondrial genes.

Figure 8

Phylogenetic distribution of gene/intron content (loss and gain) among eight asterids and two outgroups. The tree was inferred using the maximum likelihood (ML) method with the ‘GTRGAMMA’ evolutionary model under the rapid bootstrap algorithm (1000 replicates). Bootstrap support values > 50% are shown at nodes. The 24 genes include atp[1,4,6,8,9], ccm[B, C, Fc, Fn], cob, cox[1-3], matR, mttB, and nad[1, 2, 3, 4, 4L, 5, 6, 7, 9]. The cox1 introns have been lost in the ancestor of angiosperms.