The draft assembly of the radically organized Stylonychia lemnae macronuclear genome

Abstract

Stylonychia lemnae is a classical model single-celled eukaryote, and a quintessential ciliate typified by dimorphic nuclei: A small, germline micronucleus and a massive, vegetative macronucleus. The genome within Stylonychia's macronucleus has a very unusual architecture, comprised variably and highly amplified "nanochromosomes," each usually encoding a single gene with a minimal amount of surrounding noncoding DNA. As only a tiny fraction of the Stylonychia genes has been sequenced, and to promote research using this organism, we sequenced its macronuclear genome. We report the analysis of the 50.2-Mb draft S. lemnae macronuclear genome assembly, containing in excess of 16,000 complete nanochromosomes, assembled as less than 20,000 contigs. We found considerable conservation of fundamental genomic properties between S. lemnae and its close relative, Oxytricha trifallax, including nanochromosomal gene synteny, alternative fragmentation, and copy number. Protein domain searches in Stylonychia revealed two new telomere-binding protein homologs and the presence of linker histones. Among the diverse histone variants of S. lemnae and O. trifallax, we found divergent, coexpressed variants corresponding to four of the five core nucleosomal proteins (H1.2, H2A.6, H2B.4, and H3.7) suggesting that these ciliates may possess specialized nucleosomes involved in genome processing during nuclear differentiation. The assembly of the S. lemnae macronuclear genome demonstrates that largely complete, well-assembled highly fragmented genomes of similar size and complexity may be produced from one library and lane of Illumina HiSeq 2000 shotgun sequencing. The provision of the S. lemnae macronuclear genome sets the stage for future detailed experimental studies of chromatin-mediated, RNA-guided developmental genome rearrangements.

Keywords: alternative fragmentation; chromosome copy number; genome rearrangement; histone variant; macronuclear genome; nanochromosome.

Fig. 1.—

Stylonychia macronuclei. (A–C) Illustrations of successive stages of Stylonychia nuclear division during cellular replication, modified from (Bütschli 1876). Replication bands are responsible for DNA synthesis in spirotrichous ciliates (Gall 1959), including Styonychia, and sweep through the MAC during asexually division (Ammermann 1971). The granular structure of macronuclei, due to nucleolar bodies (Postberg et al. 2006) is also shown. There is currently no indication of a classical spindle in macronuclei (Ammermann 1971). (D) A pair of conjugating S. lemnae cells containing a mixture of old fragmenting macronuclei, new macronuclei, and micronuclei (DAPI staining in red; overlaid on a micrograph of the cells) illustrating the complexity of nuclear organization and development.

Fig. 2.—

Synteny of Stylonychia and Oxytricha multigene nanochromosomes. Fold coverage of reads is indicated for total reads mapped with bwa to nanochromosomes from Stylonychia (library 24) and for the Oxytricha MAC genome Illumina library (Swart et al. 2013). Oxytricha has prominent doublet peaks corresponding to telomeric end coverage biases of the PE reads (this phenomenon is due to the larger fragment sizes of the Oxytricha Illumina library). Alternative fragmentation sites are indicated by upward pointing arrows with the number of reads corresponding to the approximate fragmentation site below. Coordinates (italicized numbers) of the contigs (in bp) are given relative to the current assembly.

Fig. 3.—

Conservation of nanochromosome copy number and length. Nanochromosome copy number was determined for nanochromosomes encoding orthologous proteins in the Terminator 2.0 assembly (see Materials and Methods). As the Oxytricha Illumina library is slightly smaller than that of Stylonychia library 24, we multiplied the Oxytricha reads/bp value by 1.242 (total mapped Stylonychia reads/total Oxytricha mapped reads) to normalize the library sizes.

Fig. 4.—

Histone variants in Stylonchia. Scale bars in expected substitutions per site are provided below each phylogeny and bootstrap percentages for branch points are shown when greater than 80%. Phylogenies were rooted using Saccharomyces cerevisiae histone variants (H2A.1/H2A.2, H2B.1/H2B.2, H3, for 4A–C, respectively) as outgroups. Note that in (C) it is possible that long-branch attraction is causing the most divergent histone H3 variants to cluster. See Materials and Methods for a list of accessions for the Saccharomyces cerevisiae and Tetrahymena thermophila histone variants.

Fig. 5.—

Expression of histone variants in Oxytricha trifallax. Gene expression values are the normalized RNA-seq counts obtained from (Swart et al. 2013) and are given in arbitrary units. “Vegetative” represents a normally fed cell culture. The developmental time course on the x axis starts at 0 h when cells from the complementary mating types of O. trifallax were mixed together (see Swart et al. 2013 for additional details about this experiment).