The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

Abstract

Background: Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of beta-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri.

Results: The D. salina organelle genomes are large, circular-mapping molecules with approximately 60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA) sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: approximately 1.5 and approximately 0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA.

Conclusions: These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the development of a viable plastid transformation system for this model alga, and they will complement the forthcoming D. salina nuclear genome sequence, placing D. salina in a group of a select few photosynthetic eukaryotes for which complete genome sequences from all three genetic compartments are available.

Figure 1

Complete mitochondrial genome maps for Dunaliella salina (middle), Chlamydomonas reinhardtii (inner), and Volvox carteri (outer). The mitochondrial genome of D. salina (this study) is 28.3 kb, that of C. reinhardtii (GenBank accession numbers EU306617-EU306623) ranges from 15.8-18.9 kb, depending on the presence of optional introns, and that of V. carteri (GenBank accession numbers EU760701 and GU084821) is ~35 kb. Note that the C. reinhardtii mtDNA is a linear molecule. Arrows within the coding regions denote transcriptional polarities. The small subunit and large subunit rRNA-coding regions are fragmented into modules. Transfer RNA-coding regions are designated by the single-letter abbreviation of the amino acid they specify. Purple asterisks denote the sites of palindromic repeat clusters (see Figure 5 for more details). Rtl codes for a putative reverse-transcriptase-like protein.

Figure 2

Complete plastid genome maps for Dunaliella salina (middle), Chlamydomonas reinhardtii (inner), and Volvox carteri (outer). The D. salina plastid genome (this study) is 269 kb. The C. reinhardtii and V. carteri plastid genomes (GenBank accession numbers FJ423446 and GU084820) are 204.2 kb and ~525 kb, respectively. Arrows within the coding regions denote transcriptional polarities. Transfer RNA-coding regions are designated by the single-letter abbreviation of the amino acid they specify. Introns within intergenic regions are labeled with blue asterisks. Pseudogenes are labeled with a ψ. For all three genomes, the psaA gene is fragmented; the translational order of these fragments is set out using superscript numbers. The portions of the D. salina genome map that are gray (as opposed to black) highlight gene colinearity (not including introns) with either the C. reinhardtii or V. carteri plastid genomes.

Figure 3

Venn diagram comparing the gene repertoires of chlamydomonadalean mitochondrial genomes. Chlamydomonadalean algae are labeled as follows: Ce = Chlamydomonas eugametos; Ch = Chlorogonium elongatum; Ci = Chlamydomonas incerta; Cr = Chlamydomonas reinhardtii; Ds = Dunaliella salina; Ps = Polytomella capuana, Polytomella parva, and Polytomella piriformis (strain SAG 63-10); Vc = Volvox carteri. *Rtl codes for a putative reverse-transcriptase-like protein: in C. reinhardtii and C. incerta this gene is independent of an intron, whereas in V. carteri it is within a group-II intron. Note, the C. eugametos mtDNA contains a duplicate copy of trnM.

Figure 4

Scaling of noncoding DNA content with genome size in completely sequenced organelle DNAs. Chlamydomonadalean algae are labeled as follows: Ce = Chlamydomonas eugametos; Ch = Chlorogonium elongatum; Ci = Chlamydomonas incerta; Cr = Chlamydomonas reinhardtii; Ds = Dunaliella salina; Ps = Polytomella capuana, Polytomella parva, and Polytomella piriformis (strain SAG 63-10); Ts = Trifolium subterraneum; Vc = Volvox carteri.

Figure 5

Consensus sequences and secondary structures of the D. salina mitochondrial palindromic repeat elements. The number of times each element appears in the D. salina mitochondrial genome is shown in red numbers. The locations of these palindromic elements within the mtDNA are depicted on Figure 1 using purple asterisks.