Whole-Genome Sequencing to Identify Mutants and Polymorphisms in Chlamydomonas reinhardtii

Abstract

Whole-genome sequencing (WGS) provides a new platform for the identification of mutations that produce a mutant phenotype. We used Illumina sequencing to identify the mutational profile of three Chlamydomonas reinhardtii mutant strains. The three strains have more than 38,000 changes from the reference genome. NG6 is aflagellate and maps to 269 kb with only one nonsynonymous change; the V(12)E mutation falls in the FLA8 gene. Evidence that NG6 is a fla8 allele comes from swimming revertants that are either true or pseudorevertants. NG30 is aflagellate and maps to 458 kb that has six nonsynonomous changes. Evidence that NG30 has a causative nonsense allele in IFT80 comes from rescue of the nonswimming phenotype with a fragment bearing only this gene. This gene has been implicated in Jeune asphyxiating thoracic dystrophy. Electron microscopy of ift80-1 (NG30) shows a novel basal body phenotype. A bar or cap is observed over the distal end of the transition zone, which may be an intermediate in preparing the basal body for flagellar assembly. In the acetate-requiring mutant ac17, we failed to find a nonsynonymous change in the 676 kb mapped region, which is incompletely assembled. In these strains, 43% of the changes occur on two of the 17 chromosomes. The excess on chromosome 6 surrounds the mating-type locus, which has numerous rearrangements and suppressed recombination, and the changes extend beyond the mating-type locus. Unexpectedly, chromosome 16 shows an unexplained excess of single nucleotide polymorphisms and indels. Overall, WGS in combination with limited mapping allows fast and accurate identification of point mutations in Chlamydomonas.

Keywords: basal bodies; flagellar assembly; intraflagellar transport (IFT); mapping; mating-type.

Figure 1

Electron microscopy of basal bodies from NG6 (fla8-3) and NG30 (ift80-1). (A, B) Cross-sectional images from NG6 showing closed tubules and no hooked tubules. (C−F) Cross-sectional images of NG30 showing normal transition zones. (G) Longitudinal image of NG6 without a cap at the distal end of the transition zone. (H, I) Longitudinal images of NG30 with cap at the distal end of the transition zone. This cap is not observed in wild-type cells or other mutants.

Figure 2

Distribution of changes across the genome by chromosome. (A) Plots of the number of changes (y-axis) in ift80-1 and ac17 strains relative to the JGI reference genome. The x-axis shows the 17 chromosomes with their proportional length. (B) Expansion of the plot in panel A for the number of changes on chromosome 6 for ift80-1 and ac17. (C) Expansion of the plot in panel for the number of changes on chromosome 16 for ift80-1 and ac17.