Figure 2

Pair of pseudogenes of OA-DHCα in E. huxleyi CCMP1516. (a) The domain structure of outer arm-dynein heavy chain-α (OA-DHCα) represented by the Chlamydomonas reinhardtii Swiss-Prot ortholog (upper bar), with the most highly conserved DHC structural elements indicated: A1-A6, AAA ATPase domains; N2, N-terminal region; S, Stalk. (a, middle) A complete homolog with all DHC structural elements is encoded on an Illumina paired-end read contig from E. huxleyi strain 92A. Dotted vertical lines mark introns. (a, bottom) Scaffolds 68 and 529 in the CCMP1516 genome assembly share high synteny and homology between each other and the 92A contig but exhibit distinct loss-of-function deletions in the DYHA_CHLRE-homolog. Regions of high nucleotide identity with the 92A contig are indicated. Each scaffold encodes only short segments of the original DHC gene; >95% nucleotide identity over >100 bp sections is indicated between each scaffold and the 92A contig (purple) and between the two scaffolds (blue). Only parts of each scaffold are indicated (numbers indicate nucleotide positions in the JGI assembly), yet the entire Scaffold_529 was identified by JGI as a ‘diploid allele’ of Scaffold_68 (http://genome.jgi.doe.gov and Read et al., 2013). Thin white bars within scaffolds indicate where targeted PCR confirmed scaffold structure, and white bars below scaffolds indicate Illumina paired-end read contigs matching uniquely to one or the other scaffold. (b, c) PCR confirmation of homology break in Scaffold_529. (d) Control PCR amplifying section of C-terminal DHC homology maintained in Scaffold_529. (e) Long-range PCR confirming that the primers used in (b) amplify a large, ≈8.5 kb fragment from RCC1216 genomic DNA (gDNA) and RCC1217 cDNA, corresponding to the major section of DHC homology missing from Scaffold_529 but present in 92A. Only the small fragment is amplified from CCMP1516 gDNA. End sequencing confirmed the products from RCC1216 and RCC1217 were the DHC homologous sections (Supplementary Information). (f) Short-range PCR confirming that the DHC-homologous region found in the 92A scaffold is found in RCC1216 gDNA and RCC1217 cDNA, but not in CCMP1516 gDNA. Samples tested by PCR: random-primed RCC1217 1N cDNA, 1; oligo-dT-primed RCC1217 1N cDNA, 2; RT- RCC1217 1N RNA, 3; RCC1216 2N gDNA, 4; RCC1217 1N gDNA, 5; CCMP1516 gDNA, 6; H2O, 7; RCC1216 2N cDNA.