Insights into an Extensively Fragmented Eukaryotic Genome: De Novo Genome Sequencing of the Multinuclear Ciliate Uroleptopsis citrina

Abstract

Ciliated protists are a large group of single-celled eukaryotes with separate germline and somatic nuclei in each cell. The somatic genome is developed from the zygotic nucleus through a series of chromosomal rearrangements, including fragmentation, DNA elimination, de novo telomere addition, and DNA amplification. This unique feature makes them perfect models for research in genome biology and evolution. However, genomic research of ciliates has been limited to a few species, owing to problems with DNA contamination and obstacles in cultivation. Here, we introduce a method combining telomere-primer PCR amplification and high-throughput sequencing, which can reduce DNA contamination and obtain genomic data efficiently. Based on this method, we report a draft somatic genome of a multimacronuclear ciliate, Uroleptopsis citrina. 1) The telomeric sequence in U. citrina is confirmed to be C4A4C4A4C4 by directly blunt-end cloning. 2) Genomic analysis of the resulting chromosomes shows a "one-gene one-chromosome" pattern, with a small number of multiple-gene chromosomes. 3) Amino acid usage is analyzed, and reassignment of stop codons is confirmed. 4) Chromosomal analysis shows an obvious asymmetrical GC skew and high bias between A and T in the subtelomeric regions of the sense-strand, with the detection of an 11-bp high AT motif region in the 3' subtelomeric region. 5) The subtelomeric sequence also has an obvious 40 nt strand oscillation of nucleotide ratio. 6) In the 5' subtelomeric region of the coding strand, the distribution of potential TATA-box regions is illustrated, which accumulate between 30 and 50 nt. This work provides a valuable reference for genomic research and furthers our understanding of the dynamic nature of unicellular eukaryotic genomes.

Fig. 1.

—Outline of the classes in the phylum Ciliophora. Species involved in our research are highlighted.

Fig. 2.

—Total genomic DNA (A) and telomere-primer PCR amplified DNA (B) of Uroleptopsis citrina. (A) and (B) Marker unit: bp. (C) Living cell of U. citrina under DIC microscope. Scale bar: 40 μm.

Fig. 3.

—Whole assembly circle plot. About 31,754 contigs are separated into two groups based on the presence/absence of a homologous genes. Contigs are joined together and arranged by length (clockwise, from long to short). Homologous genes are linked by black lines within the circle. The outermost layer shows sequencing depth (calculated as TPM value). Middle layer shows distribution of 2/1/0-telomere contigs.

Fig. 4.

—Accumulated contig length distribution based on GC content of different data sets from Uroleptopsis citrina. Telo2: Contigs with telomeres on both 5′- and 3′-ends. Telo1: Contigs with one telomere on the 5′- or 3′-end. Telo0: Contigs without telomeres. All contigs: Combination of all contigs.

Fig. 5.

—(A) Amino acids usage of Uroleptopsis citrina, Saccharomyces cerevisiae, Oxytricha trifallax, Paramecium tetraurelia, and Tetrahymena thermophile. Codons TAA and TAG were calculated individually. (B) Spearman correlation coefficient of amino acid usage between each two species.

Fig. 6.

—Sliding-window analysis for Shannon entropy (H) and nucleotide distribution of complete chromosomes.

Fig. 7.

—(A) A over T skew, (B) G over C skew, (C) AG over CT skew for the first 200 nt of sense- and antisense-strand of coding contigs in Uroleptopsis citrina. Sense-strand refers to the 5′ subtelomeric region of coding strands, reverse to the 5′ region of antisense-strands, which is the reverse complementary strand of 3′ subtelomeric region of coding strands.

Fig. 8.

—Distribution of potential TATA-box regions. The X-axis is the distance from the proximal end of telomere. The Y-axis is the number of putative TATA-box regions, which observed as pure-AT. Both 5′ and 3′ subtelomeric regions are involved and analyzed.