Comparative genomics of Chlamydomonas

Abstract

Despite its role as a reference organism in the plant sciences, the green alga Chlamydomonas reinhardtii entirely lacks genomic resources from closely related species. We present highly contiguous and well-annotated genome assemblies for three unicellular C. reinhardtii relatives: Chlamydomonas incerta, Chlamydomonas schloesseri, and the more distantly related Edaphochlamys debaryana. The three Chlamydomonas genomes are highly syntenous with similar gene contents, although the 129.2 Mb C. incerta and 130.2 Mb C. schloesseri assemblies are more repeat-rich than the 111.1 Mb C. reinhardtii genome. We identify the major centromeric repeat in C. reinhardtii as a LINE transposable element homologous to Zepp (the centromeric repeat in Coccomyxa subellipsoidea) and infer that centromere locations and structure are likely conserved in C. incerta and C. schloesseri. We report extensive rearrangements, but limited gene turnover, between the minus mating type loci of these Chlamydomonas species. We produce an eight-species core-Reinhardtinia whole-genome alignment, which we use to identify several hundred false positive and missing genes in the C. reinhardtii annotation and >260,000 evolutionarily conserved elements in the C. reinhardtii genome. In summary, these resources will enable comparative genomics analyses for C. reinhardtii, significantly extending the analytical toolkit for this emerging model system.

Figure 1

Images of unicellular species. A, Chlamydomonas reinhardtii. B, C. incerta SAG 7.73. C, C. schloesseri SAG 2486 (=CCAP 11/173). D, E. debaryana SAG 11.73 (=CCAP 11/70). Scale bars, 20 µm. All images kindly provided by Thomas Pröschold.

Figure 2

ML phylogeny of 15 Volvocales species and three outgroups. The phylogeny was inferred using the LG+F+R6 model and a concatenated protein alignment of 1,624 chlorophyte BUSCO genes. All ultrafast bootstrap values ≥99%. Species in bold have gene model annotations and were included in the OrthoFinder-based phylogenies (Supplemental Figure S3, B and C). Phylogeny was rooted on the three Sphaeropleales species (highlighted in pink).

Figure 3

Circos plot (Krzywinski et al., 2009) representation of synteny blocks shared between C. reinhardtii and its close relatives. Circos plots between C. reinhardtii and C. incerta (A) and C. reinhardtii and C. schloesseri (B). Chlamydomonas reinhardtii chromosomes are represented as colored segments and split across the left and right Circos plots, and C. incerta/C. schloesseri contigs as gray segments. Contigs are arranged and orientated relative to C. reinhardtii chromosomes, and adjacent contigs with no signature of rearrangement relative to C. reinhardtii are plotted without gaps. Dark gray bands highlight putative C. reinhardtii centromeres and asterisks represent rDNA. Note that colors representing specific chromosomes differ between (A) and (B).

Figure 4

Phylogenetic relationship and centromeric clustering of Zepp-like elements. A, ML phylogeny of chlorophyte L1 LINE elements inferred using the LG+F+R6 model and alignment of endonuclease and reverse transcriptase protein domains. Bootstrap values ≤70% are shown. Phylogeny is rooted on plant L1 elements. Species are provided by the element name suffix, as follows: CR/cRei = C. reinhardtii; VC = V. carteri; cInc = C. incerta; cSch = C. schloesseri; eDeb = E. debaryana eud = Eudorina sp. 2016-703-Eu-15. B, Density (0%–100%) of ZeppL-1_cRei in 50 kb windows across C. reinhardtii chromosomes. Dark bands represent putative centromeres, x-axis ticks represent 100-kb increments and y-axis ticks represent 20% increments. Plot produced using karyoploteR (Gel and Serra, 2017). Note that ZeppL-1_cRei is a synonym of the Repbase element L1-1_CR (see Supplemental Data Set S1).

Figure 5

Upset plot (Lex et al., 2014) representing the intersection of orthogroups between six core-Reinhardtinia species. Numbers above bars represent the number of orthogroups shared by a given intersection of species.

Figure 6

Synteny representation across the C. reinhardtii MT^– haplotype and inferred MT^– haplotypes in C. incerta and C. schloesseri. Shown is the synteny between the C. reinhardtii genes across the MT^– haplotype and flanking autosomal sequence and (A) inferred C. incerta MT^– haplotype and flanking sequence genes (contig C0033), or (B) inferred C. schloesseri MT^– haplotype and flanking sequence genes (contig C0045). The T, R, and C domains of the C. reinhardtii MT^– are highlighted. Genes with inverted orientations are shown in blue. Note that for C. schloesseri, the region syntenous to the C. reinhardtii MT is entirely on contig C0045, but C0105 was appended to C0045 to show the genes syntenous with the most telomere-proximal region of C. reinhardtii chromosome 6.

Figure 7

Putatively neutral divergence and alignability across the core-Reinhardtinia. A, Estimates of putatively neutral divergence under the GTR model, based on the topology of Figure 2 and 1,552,562 C. reinhardtii 4D sites extracted from the Cactus WGA. B, A representation of the C. reinhardtii genome by site class, and the number of aligned sites per C. reinhardtii site class for each other species in the Cactus WGA.

Figure 8

Coding potential analyses. A, Boxplot of PhyloCSF scores for control and test set genes. B, Boxplot of the ratio of genetic diversity at 0D and 4D sites (π_0D/4D) for control and test set of genes. Gray dashed line represents 95th percentile of control gene values. C, Boxplot of codon adaptation, as quantified by I_TE for control and test set genes. Gray dashed line represents fifth percentile of control gene values. D, Density plot of Kozak scores, quantified as the per gene agreement of the start codon sequence context to that of the C. reinhardtii Kozak consensus sequence. Low CP (i.e. low coding potential), the 250 test set genes that failed all three coding potential analyses; control, the opposite half of the control set to that used to produce the Kozak consensus sequence (see the “Materials and methods” section); random, 10,000 sequences generated based on an average GC content of 64.1%

Figure 9

Intron lengths and overlap with CEs. A, Intron length distributions for five model organisms (C. ele = C. elegans, D. mel = D. melanogaster, N. cra = Neurospora crassa, A. thal = A. thaliana, E. sil = Ectocarpus siliculosus). The brown alga E. siliculosus is included as an example of an atypical distribution similar to that seen in the core-Reinhardtinia. B, Intron length distributions for six core-Reinhardtinia species (C. rei = C. reinhardtii, C. inc = C. incerta, C. sch = C. schloesseri, E. deb = Edaphochlamys debaryana, G. pec = G. pectorale, V. car = V. carteri). C, Correlation between mean intron length per bin and the proportion of sites overlapped by CEs. Introns were ordered by length and separated into 50 bins, so that each bin contained the same number of introns.