Chloroplast genome sequencing analysis of Heterosigma akashiwo CCMP452 (West Atlantic) and NIES293 (West Pacific) strains

Abstract

Background: Heterokont algae form a monophyletic group within the stramenopile branch of the tree of life. These organisms display wide morphological diversity, ranging from minute unicells to massive, bladed forms. Surprisingly, chloroplast genome sequences are available only for diatoms, representing two (Coscinodiscophyceae and Bacillariophyceae) of approximately 18 classes of algae that comprise this taxonomic cluster. A universal challenge to chloroplast genome sequencing studies is the retrieval of highly purified DNA in quantities sufficient for analytical processing. To circumvent this problem, we have developed a simplified method for sequencing chloroplast genomes, using fosmids selected from a total cellular DNA library. The technique has been used to sequence chloroplast DNA of two Heterosigma akashiwo strains. This raphidophyte has served as a model system for studies of stramenopile chloroplast biogenesis and evolution.

Results: H. akashiwo strain CCMP452 (West Atlantic) chloroplast DNA is 160,149 bp in size with a 21,822-bp inverted repeat, whereas NIES293 (West Pacific) chloroplast DNA is 159,370 bp in size and has an inverted repeat of 21,665 bp. The fosmid cloning technique reveals that both strains contain an isomeric chloroplast DNA population resulting from an inversion of their single copy domains. Both strains contain multiple small inverted and tandem repeats, non-randomly distributed within the genomes. Although both CCMP452 and NIES293 chloroplast DNAs contains 197 genes, multiple nucleotide polymorphisms are present in both coding and intergenic regions. Several protein-coding genes contain large, in-frame inserts relative to orthologous genes in other plastids. These inserts are maintained in mRNA products. Two genes of interest in H. akashiwo, not previously reported in any chloroplast genome, include tyrC, a tyrosine recombinase, which we hypothesize may be a result of a lateral gene transfer event, and an unidentified 456 amino acid protein, which we hypothesize serves as a G-protein-coupled receptor. The H. akashiwo chloroplast genomes share little synteny with other algal chloroplast genomes sequenced to date.

Conclusion: The fosmid cloning technique eliminates chloroplast isolation, does not require chloroplast DNA purification, and reduces sequencing processing time. Application of this method has provided new insights into chloroplast genome architecture, gene content and evolution within the stramenopile cluster.

Figure 1

Fosmid cloning technique. High molecular weight, total DNA is subject to pulse-field electrophoresis to recover sheared DNA of 45 to 50 kb. This DNA is used to generate a fosmid library which is selectively screened for cpDNA-containing clones, which are then sequenced, annotated and assembled.

Figure 2

H. akashiwo CCMP452 (A) and NIES293 (B) genome maps. Outer rim: genes on plus and minus strand, color coded according to function (see legend); Second ring: small inverted (red) and tandem (blue) repeats; Third ring: sequence comparison to the other H. akashiwo genome, including SNPs (blue), small insertions (green), deletions (red) and regions of extremely poor alignment (orange); Fourth ring: Location and size of fosmid clones color coded according to their orientation: supports depicted isoform (green), supports alternate isoform (pink), uninformative (black); Fifth ring: location of inverted repeats, large and small single copy domains. Red bar depicts location of 8 kb region inverted in CCMP452 relative to NIES293; inner circle: GC content.

Figure 3

Isomeric cpDNA populations. Single copy regions are flipped resulting from a recombination event. Arrows show positions of sequence in large and small single copy regions.

Figure 4

Comparison of H. akashiwo CCMP452, H. akashiwo NIES293, Chaetosphaeridium globosum and Prototheca wickerhamii recombinases. Gray shading indicates residues completely conserved among the four proteins. Stars indicated conserved residues important in catalytic function. Overline and underline are box I and box II respectively.

Figure 5

Synteny among stramenopile and red-lineage chloroplast genomes. H. akashiwo vs (A) Odontella sinensis and (B) Porphyra purpurea; Thalassiosira pseudonana vs (C) Odontella sinensis and (D) Porphyra purpurea.