Distribution of SNPs/indels along the 17 <i>Chlamydomonas</i> chromosomes compared to the reference genome (v5.3).

<p>(A) Distribution of SNPs/indels in four wild-type strains (CC-124, CC-125, isoloP, and isoloM). (B) Distribution of SNPS/indels along the length of each chromosome in the CC-124 strain in Mb. The number of SNPs/indels is summed over each 100 kb interval. (C) Distribution in 14 strains used in this study, with the exception of <i>fla18</i> and S1C5. (D) Distribution in strains in part C after the subtraction of SNPs/indels found in the CC-124 strain. The <i>uni1</i> strain is the most divergent. (E) Distribution in 14 strains after the subtraction of SNPs/indels found in the CC-125 strain.</p

Strains used to generate a <i>Chlamydomonas</i> SNP/indel library.

a<p>PE, pair-end reads; SE, single-end reads.</p>b<p>isoloM and isoloP were generated by 10 rounds of backcrosses of the wild-type strain CC124.</p>c<p>The <i>fla18</i> strain sequenced was generated by a cross between <i>fla18</i> and S1C5 (CC-1952).</p>d<p>pers. comm., personal communication.</p

Pellicle phenotype after cell mating.

<p>(A) Pellicle formation after overnight mating between <i>plus</i> and <i>minus</i> gametes. Sheets of dark green pellicles are observed in matings between wild-type gametes (CC-125×CC-124), wild-type mated with either the untransformed (<i>imp3</i>×CC-124) or transformed <i>imp3</i> mutant strain (<i>imp3; HA-PP2A3</i>×CC-124), and between <i>imp3; HA-PP2A3</i> gametes. These pellicles are resistant to vortex or pipette disruption. In contrast, only small clumps of pellicles are found in the mating between <i>imp3</i> cells and they are easily disrupted by vortexing or pipetting. (B) Thick, dark green, multilayer pellicle found in mating between wild-type and <i>imp3</i> gametes. (C) Single layer pellicle that is light in color is found in matings homozygous for the <i>imp3</i> mutation. (D) Magnification view of cells in (C). Scale bar, 0.1 mm.</p

HA-PP2A3 localizes to the basal body region in gametes.

<p>(A) Immunofluorescence with the anti-HA antibody (green, first column) in unmated gametes of wild-type (CC-125), <i>imp3</i>, or <i>imp3</i> transformed with wild-type and mutant forms of <i>HA-PP2A3</i>. Immunofluorescence with the anti-acetylated α-tubulin (red, second column) identifies the <i>Chlamydomonas</i> flagella. Merged images of both staining are shown in the third column. Scale bar, 10 µm. (B) Immunofluorescence of the HA-PP2A3 location in wild-type dikaryons (CC-125×CC-124), in dikaryons between wild-type and <i>imp3</i> gametes transformed with wild-type or <i>V₃₁₀T HA-PP2A3</i>. (C) Percentages of cells in each strain that showed the basal body localization of the HA-PP2A3 signal. Blue columns represent results from the unmated gametes. Red columns represent data from dikaryons identified by their four flagella. For each point, over 50 cells were collected.</p

Distribution of SNPs on all 17 chromosomes in wild-type strains.

<p>Distribution of SNPs on all 17 chromosomes in wild-type strains.</p

Distribution of changes found in the <i>imp3</i> mutant strain.

<p>Distribution of changes found in the <i>imp3</i> mutant strain.</p

The <i>pf7</i> and <i>pf8</i> mutants show splaying phenotype in isolated axonemes similar to splaying in N-DRC mutants.

<p>(A) Fixed axonemes were stained with antibodies against LF5 (green) and acetylated α-tubulin (magenta). (B) Axonemes were scored as intact (blue), sliding (yellow), or splaying (green). At least 100 axonemes were score for each strain.</p

Whole genome sequencing of mutant strains used in this study.

<p>* Changes are defined as being within coding regions or at intron-exon boundaries and have a Phred quality score ≥100.</p><p>Whole genome sequencing of mutant strains used in this study.</p

Sequence changes found in individual mutant strains.

<p>Sequence changes found in individual mutant strains.</p

The localization of polyglutamylated tubulin is affected by both <i>tpg1</i> and <i>pf7</i>.

<p>(A) Two micrograms of flagellar protein were used in immunoblots. α-tubulin is included as a loading control. (B) The nucleoflagellar apparatus (NFAP) from various strains was stained with polyglutamylated tubulin antibody (green) and acetylated α-tubulin (magenta). (C) The length of polyglutamylated tubulin (green) and flagella (indicated by acetylated α-tubulin, magenta). *** indicates p<0.001 by the t-test between the lengths of polyglutamylated tubulin in <i>tpg1</i> and in <i>pf7</i>; <i>tpg1</i>.</p