EST analysis of Ostreococcus lucimarinus, the most compact eukaryotic genome, shows an excess of introns in highly expressed genes.

EST Analysis of Ostreococcus lucimarinus, the Most
Compact Eukaryotic Genome, Shows an Excess of Introns
in Highly Expressed Genes
William Lanier1, Ahmed Moustafa1, Debashish Bhattacharya1,2, Josep M. Comeron1,2*
1 Interdisciplinary Program in Genetics, University of Iowa, Iowa, United States of America, 2Department of Biological Sciences and Roy J. Carver Center for Comparative
Genomics, University of Iowa, Iowa, United States of America
Abstract
Background: The genome of the pico-eukaryotic (bacterial-sized) prasinophyte green alga Ostreococcus lucimarinus has one
of the highest gene densities known in eukaryotes, yet it contains many introns. Phylogenetic studies suggest this unusually
compact genome (13.2 Mb) is an evolutionarily derived state among prasinophytes. The presence of introns in the highly
reduced O. lucimarinus genome appears to be in opposition to simple explanations of genome evolution based on
unidirectional tendencies, either neutral or selective. Therefore, patterns of intron retention in this species can potentially
provide insights into the forces governing intron evolution.
Methodology/Principal Findings: Here we studied intron features and levels of expression in O. lucimarinus using
expressed sequence tags (ESTs) to annotate the current genome assembly. ESTs were assembled into unigene clusters that
were mapped back to the O. lucimarinus Build 2.0 assembly using BLAST and the level of gene expression was inferred from
the number of ESTs in each cluster. We find a positive correlation between expression levels and both intron number
(R = +0.0893, p=,0.0005) and intron density (number of introns/kb of CDS; R = +0.0753, p=,0.005).
Conclusions/Significance: In a species with a genome that has been recently subjected to a great reduction of non-coding
DNA, these results imply the existence of selective/functional roles for introns that are principally detectable in highly
expressed genes. In these cases, introns are likely maintained by balancing the selective forces favoring their maintenance
with other mutational and/or selective forces acting on genome size.
Citation: Lanier W, Moustafa A, Bhattacharya D, Comeron JM (2008) EST Analysis of Ostreococcus lucimarinus, the Most Compact Eukaryotic Genome, Shows an
Excess of Introns in Highly Expressed Genes. PLoS ONE 3(5): e2171. doi:10.1371/journal.pone.0002171
Editor: Robert DeSalle, American Museum of Natural History, United States of America
Received January 15, 2008; Accepted March 25, 2008; Published May 14, 2008
Copyright: � 2008 Lanier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the National Science Foundation awarded to JMC (DEB-03-44209) and to DB (EF 04-31117, EF 06-25440). WL
was supported by an Institutional NRSA (T 32 GM98629) from the National Institutes of Health.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: josep-comeron@uiowa.edu
Introduction
The genus Ostreococcus encompasses a group of globally
distributed photosynthetic, unicellular green algae in the anciently
diverged class Prasinophyta [1]. These cells are the smallest known
eukaryotes [2], with cell sizes smaller than 1 mm (Figure 1a).
Among free-living taxa, Ostreococcus species also contain the most
compact (i.e., gene-dense) genomes [3]. For example, in
comparison to the compact genome of Saccharomyces cerevisiae
(12.1 Mbp) that contains ,6000 open reading frames, with one
gene every 2.0 kb, O. tauri and O. lucimarinus have genomes sizes of
12.6 and 13.2 Mbp with a gene occurring every 1.6 and 1.7 kb,
respectively [3–5]. This degree of genome miniaturization is
unparalleled among free-living eukaryotes and has led to the
contraction of intergenic regions to an average of less than 200 bp
in O. tauri, which is the smallest among eukaryotes [6,7]. Yet
surprisingly, the dearth of noncoding DNA is not maintained
across the Ostreococcus genomes because introns are present in an
estimated 20–25% of transcripts, compared to less than 4% of
intron-containing genes in the budding yeast [4,5]. This algal
genome structure is in sharp contrast to the general positive
relationship between intron and intergenic size, and ultimately
genome size, observed across a broad range of eukaryotes [7]. In
fact, Ostreococcus species display the highest ratio of intragenic to
intergenic noncoding DNA detected thus far.
This extreme level of genome compression has lead to both a
downsizing of gene families and in some cases to the creation of
gene fusion proteins [3,4]. These trends are conserved across the
Ostreococcus genomes which are characterized by a high degree of
synteny for a core set of 18 chromosomes. Apart from this
conservation, several chromosomes appear to have unique
characteristics when compared both to other chromosomes and
when compared across Ostreococcus spp. Chromosome 21 appears
to be a unique feature of the O. lucimarinus genome resulting from a
fusion of duplicated regions within Chromosomes 9 and 13 [4].
Chromosome 2, although displaying regions of synteny between
species, has also been characterized as unique for its increased
presence of non-coding DNA (intergenic and intronic) and
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transposable elements. Genes encoded on Chromosome 2 contain
a higher proportion of shorter (40–65 bp) introns lacking canonical
splice site and branch point sequences [3].
Despite these differences both within and between the
Ostreococcus genomes, trends common to both suggest that similar
mechanisms have led to the optimization of cell and genome size
[4]. Thus the elevated presence of introns in these otherwise highly
compact genomes, together with phylogenetic studies suggesting
that this unusual genome structure is an evolutionarily derived
state in prasinophytes [8], provides an unique opportunity to study
evolutionary features associated with shifts in the amount of
noncoding DNA and genome size. Here, we studied the possible
role of natural selection acting on intronic DNA by investigating
the influence of varying levels of expression on intron presence and
length in O. lucimarinus. Our results reveal significant correlations
between the levels of expression and the number of introns present
in assembled EST clusters, strongly suggesting non-neutral
processes. We present and discuss several possible selective
mechanisms acting on intronic DNA focusing on explanations
for intron retention in compact genomes.
Methods
EST reads of O. lucimarinus that are available at the Joint
Genome Institute (http://jgi.doe.gov/) were downloaded and
assembled into unigene clusters using the TGI clustering package
(http://www.tigr.org/software). Single contig clusters were
mapped backed to the genome assembly of this species using
blastn. Individual clusters which mapped to multiple loci and/or
arose from cluster assemblies producing multiple contigs were
omitted to reduce the mapping of clusters potentially containing
EST reads of duplicate genes and closely related members of gene
families.
Assembled clusters were verified as originating from a single
locus by examining the clustered output for congruency within the
directionality of forward and reverse EST reads. Clusters that
contained forward or reverse reads in both the sense and antisense
orientation of an assembled contig were examined to identify
potential over-clustering within overlapping 39 ends of transcripts.
Over-clustering is not unexpected in Ostreococcus spp. due to the
known presence of overlapping genes on opposite DNA strands in
these species [4] . This procedure entailed identifying the presence
of annotated protein models on both the sense and antisense
orientations of the cluster. In cases where over-clustering was
found, ESTs from overlapping loci were manually separated into
sense and antisense bins, re-clustered independently, and tested for
their ability to retrieve .90% of a single protein model prior to
being designated as a unigene cluster. Individual over-clustered
contigs that did not conform to these criteria were removed from
the analysis. In addition, Chromosome 21 was omitted from the
analysis because it may be a recent acquisition representing a
chimera of Chromosomes 9 and 13 [4]. Finally, given the
relatively high levels of expression and intron content of ribosomal
proteins in eukaryotic genomes, the contribution of this class to
Figure 1. Ostreococcus lucimarinus and length distribution of
clustered contigs and open reading frames. The prasinophyte
green algal genus Ostreococcus is the smallest-known free-living
eukaryotes, with an average size of 0.8 mm. (a) Image of Ostreococcus
strain RCC 143 kindly provided by W. Eikrem and J. Throndsen
(University of Oslo) (image also available in Wiki Commons). (b) and (c)
Histograms showing the frequency and length distribution for clustered
contigs (b) and longest open reading frames (c) for the Ostreococcus
lucimarinus EST library.
doi:10.1371/journal.pone.0002171.g001
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overall trends in the data was assessed by querying the assembled
clusters with all available nuclear encoded ribosomal models
(n = 106) using blastx.
Tentative introns were identified as instances where complete
and uninterrupted queries were not recovered with a single high-
scoring pair (HSP). Intron presence was confirmed only in cases
where non-overlapping HSPs, with an identity of 99% or greater,
could be used to recover .97% of the query length. In cases
where blastn was not capable of unambiguously aligning the
terminal edges of exons, absolute intron size was calculated using
the genomic distance between aligned neighboring HSPs.
Adjustments to absolute intron size were performed by centering
HSPs along their respective neighboring exons and stipulating the
single inclusion of each query nucleotide in either one or the other
HSP. Absolute intron size was therefore either decreased or
increased in situations where terminal exon nucleotides were
omitted or accounted for multiple times respectively in the local
alignments. Statistical analyses of expression and intron compo-
sition were done using the JMP statistical software package
(http://www.jmp.com/). Given the unique features of Chromo-
some 2 with respect to increased intron presence, we redid the
analyses excluding this chromosome. Differences in the level of
gene expression for Chromosome 2 were also compared against all
other chromosomes to assess potential differences in gene
expression and their influence on the correlations of expression
and intron presence. Functional annotation of the 265 intron-
containing clusters was performed using Blast2GO (www.blast2go.
de).
Results
To investigate the distribution of introns in O. lucimarinus, ESTs
from this species were downloaded and assembled into unigene
clusters (see Methods). Analysis of the 19,200 raw EST reads
generated 2,050 clusters. The average cluster size was 7.57 6
18.62 ESTs/cluster and there was an upper bound of several
hundred reads and an under-representation of smaller transcripts
(otherwise common to eukaryotic transcriptomes). These results
are typical for size-selected and non-normalized cDNA libraries
(Figures 1b, 1c). The BLAST results generated using nuclear
encoded ribosomal proteins as the query returned 18.9% (20/107)
of the working models within the clustered EST data (Table S1).
As suggested by the distribution of the EST clusters in Figures 1b
and 1c, the ribosomal models identified within the expression data
were biased towards the identification of longer cDNAs with the
shortest ribosomal protein models absent from the data set.
Investigation of potentially over-clustered ESTs due to overlap-
ping transcriptional units on opposite strands that span small
intergenic regions identified 8.9% (148/1647) of these cases (See
Methods). Of these, only 26 clusters (15 are intronless and 11
contain introns) were found to contain either Ostreococcus models or
data present in the NCBI non-redundant (nr) database in an
orientation indicative of paired loci overlapping on opposite
strands. Three of the intronless clusters were discarded from
further analysis due to an extensive amount of overlap and the
inability to properly bin the EST reads as belonging to one of the
working models. Corrections of the starting clusters in this manner
resulted in a final data set of 1,670 clusters for downstream
analysis.
EST-cluster to genome alignments were used to map the 1,670
clusters to single loci at greater than 97% query coverage. The
number of clusters mapped per chromosome was in most cases
proportional to the number of annotated genes present in the
individual chromosome scaffolds for O. lucimarinus Build 2.0 with
an average of 22.4 6 3.4% of annotated genes per chromosome
within our mapped unigene clustered dataset (Table 1). Manual
inspection of these clusters revealed 265 intron-containing clusters
accounting for a total of 457 introns with an average size of 188 bp
(Table 2). Forty-four of these clusters (16.7%) mapped to
chromosome 2 and accounted for 181 (39.6%) of the introns
(Table 1).
Although our limited EST data address only ,22% of the
annotated genes (1,670/7,456 predicted genes), the coverage of
these transcripts is largely unbiased in their chromosomal
distribution. Furthermore, our analyses indicate a similar average
intron size (188 bp) to that described for the complete genome
assembly that utilized both experimental and bioinformatic
methods to calculate this number (187 bp). This suggests the
Table 1. Summary of genes predicted and EST-annotated
(mapped) for O. lucimarinus.
Chr. #
Genes
Predicted
Clusters
mapped
Clusters with
Introns
% Map/
Predicted
1 684 146 19 21.4
2 489 136 46 27.8
3 589 149 19 25.2
4 524 117 14 22.3
5 492 115 17 23.4
6 454 83 12 18.3
7 463 111 10 24.0
8 426 91 15 21.4
9 414 85 13 20.5
10 358 73 17 20.4
11 328 68 10 20.7
12 325 68 10 20.9
13 298 76 7 25.5
14 373 82 14 21.9
15 267 46 7 17.2
16 262 52 11 19.9
17 209 50 10 23.9
18 79 26 1 32.9
19 92 20 4 21.7
20 330 76 9 23.0
Totals 7456 1670 265 22.4
Genes predicted for O. lucimarinus Build 2.0 in relation to the unigene clusters
mapped, mapped clusters with introns, and the ratio of unigene clusters
mapped per genes predicted in the currently genome assembly.
doi:10.1371/journal.pone.0002171.t001
Table 2. Summary statistics of predicted introns in O.
lucimarinus.
Average Intron Size, bp 188
Longest Intron, bp 1773
Shortest Intron, bp 26
Average Intron Number per transcript 1.74
Maximum Introns In Single transcript 13
doi:10.1371/journal.pone.0002171.t002
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EST data utilized here do not deviate significantly from
characteristics of the complete genome and therefore represent
an accurate picture of the overall pattern of intron evolution in O.
lucimarinus. Furthermore, analysis of expression patterns failed to
find a difference in the levels of gene expression for clusters that
mapped to intron-rich Chromosome 2 when compared to all other
chromosomes (Mann-Whitney U-test; Z= 1.24, p,0.2157) sug-
gesting that although different in intron composition, the levels of
relative expression for the genes on Chromosome 2 are
comparable to the remainder of the genome.
Nonparametric Spearman’s rank correlation analysis using
intron number in relation to the number of overlapping sequenced
clones contained within unique gene clusters (used as a proxy for
expression levels) returned a positive correlation between intron
presence and increased expression (R= +0.0893, p,0.0005); an
equivalent result is observed after excluding clusters mapped to
Chromosome 2 (R= +0.0972, p,0.0005; see Table 3). Congruent
with the Spearman’s rank correlation, the comparison of intron-
less and intron-containing genes showed significantly reduced
expression for intron-less genes (Mann-Whitney U-test; Z= 3.95,
p,0.0001; Figure 2a). Note that these results remain consistent if
ribosomal proteins (n = 20) are excluded from the analysis, which
results in a positive correlation between intron presence and
expression levels (R= +0.0970, p,0.0005) and significantly
reduced expression for intron-less genes when compared to
intron-containing genes (Mann-Whitney U-test; Z= 4.18,
p,0.0001). These latter results are not surprising given the limited
contribution of ribosomal proteins to the EST library (Table S1;
see Discussion).
Interestingly, gene expression was positively correlated with
both assembled cluster length (R= 0.4741, p,0.0005) and
corresponding longest open reading frame (R= 0.4771,
p,0.0005) in all analyses. The tendency for EST libraries to be
biased towards longer transcripts has been previously reported [9].
These studies suggest that the direct use of EST data might be
problematic and that it should be normalized for length if
transcript abundance is to be used as a measure of expression
levels. If a standard normalization of expression is applied here
(i.e., Normalized Expression=EST reads/Cluster Length [9]) the
correlation between intron presence and increased expression is
further strengthened (R= +0.1251, p,0.0001). In addition, no
statistical differences were found between intron-containing or
intron-less unigene clusters in terms of either the assembled cluster
lengths or longest open reading frames (Figures 2b, 2c and
Table 4). This distinction is important because it addresses the
possibility of our results being generated from the bias of intron
containing transcripts simply being longer than the intron-less
transcripts.
On the other hand, previous studies conducted on the
expression profiles of other highly compact eukaryotic genomes
have noted that basic processes, such as transcription, are usually
modified with a tendency for overlapping transcripts [10]. This
issue was addressed in our study by examining the potential for
over-clustered contigs using existing protein models and the
directionality of clustered EST reads within individual contigs (See
Figure 2. Levels of gene expression for intron containing and
intronless genes in Ostreococcus lucimarinus. (a) Mean expression
values and +/2 standard errors for clusters lacking introns and all
clusters containing introns. Expression values are in terms of the
number of sequenced clones represented within a single unigene
cluster. Boxplots showing the clustered contig lengths (b) and longest
open reading frames (c) for contigs without introns (red, n = 265),
contigs with introns (green, n = 1405), the combined data set of intron
containing and intronless contigs (blue n= 1670), and for the complete
EST library (yellow, n = 2050).
doi:10.1371/journal.pone.0002171.g002
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Methods). Summary statistics suggest that the effect of over-
clustered loci residing within unique clusters is minimal (Table 4).
Lastly, the trend of intron presence and increased expression
does not appear to be an artifact of longer transcripts simply
containing more introns because a measure of intron density
(number of introns/length ORF) is also positively associated with
expression (R=+0.0753, p,0.005, R=+0.1196, p,0.0001 when
corrected for length), whereas intron number and assembled
cluster length was consistently found to be non-significant in all
analyses. The removal of ribosomal proteins from the analysis
depicts an equivalent trend, with intron density increasing with
expression (R= +0.0833, p,0.005).
Discussion
Attempts to model the evolution of genome size typically involve
a tendency for the mutational process of small insertions and
deletions (indels) to be biased towards deletions (deletion bias) that
progressively eliminate nonessential portions of the genome. This
observation offers a quandary in terms of intron retention in the
reduced genomes of Ostreococcus [11–20]. Yet, unidirectional
differences in deletion bias alone cannot account for the ratios of
genome sizes and retention of non-coding sequence in all instances
because these phenomena have been shown to not always
correlate with observed patterns in genome size. This in turn
suggests that genome sizes evolve out of a combination of
mutational biases, neutral drift, and selection [21–23]. Hence
recent models have focused on the balance between expansion and
contraction of non-coding DNA due to both small and larger
indels (i.e., repetitive elements) while including selective forces that
might vary between non-coding sequences in an attempt to better
characterize these effects in terms of the evolution of genome size
[15,21,24].
Several biological arguments have previously been made to
justify extreme directional shifts in genome size as a byproduct of
selection working towards larger and smaller cell volumes.
Examples of cell size and corresponding shifts in genome size have
been proposed for gamete dispersal, duration of meiotic division,
and responses to CO2 in plants [13]. These explanations however
cannot explain differences between the preferential retention of
particular non-coding sequences (i.e., intergenic vs. intronic) in cases
where genome reduction has occurred as a result of either ‘random’
deletion biases or selective forces driving smaller cell volumes.
The analyses described here were aimed at investigating
features of intron evolution and to provide a potential explanation
for the disproportionate representation of introns in O. lucimarinus
when compared to other non-coding regions of the genome.
Interestingly, we find a positive correlation of intron presence and
increased expression, which suggests a selective advantage (i.e.,
function) for introns in highly expressed genes that is not present
(or present to a smaller degree) in genes with reduced expression.
This finding is not unique to the miniaturized genome of
Ostreococcus because it has previously been reported in yeast. In S.
cerevisiae, a few intron-containing genes produce nearly a third of
the total mRNA transcripts, with a large fraction of these intron-
containing genes corresponding to ribosomal proteins [25].
Nevertheless, Ostreococcus represents a more extreme case in this
respect because its genome is not only more gene dense but also
clearly shows a much higher proportion of genes with introns.
Furthermore, the reported relationships between expression and
intron features are also observed after removing ribosomal
proteins from the analyses in Ostreococcus. Indeed, several functional
categories were represented by intron-containing genes with
diverse roles in metabolism, responses to light, and chloroplast
function (Table S2).
Table 3. Spearman’s rank correlation coefficient analysis of EST-annotated introns in O. lucimarinus.
All chromosomes Excluding Chr. 2 Chr.2 Only Excluding ribosomal proteins
Sequenced Clones1 vs. Intron Number 0.0893 *** 0.0972 *** 0.0132 n.s. 0.0970 ***
Sequenced Clones vs. Intron Number/LORF2 0.0753 ** 0.0839 ** 20.0300 n.s. 0.0833 **
Sequenced Clones vs. Mean Intron Length 0.1008 *** 0.0987 *** 0. 0459 n.s. 0.1019 ***
Sequenced Clones vs. Cluster Length 0. 4741 *** 0.4627 *** 0. 6049 *** 0.4764 ***
Sequenced Clones vs. LORF 0. 4771 *** 0.4687 *** 0. 5757 *** 0.4783 ***
Cluster Length vs. Intron Number 20.0189 n.s. 20.0130 n.s. 20.0608 n.s. 20.0103 n.s.
1Number of sequenced clones within assembled unigene clusters used as a measure of gene expression.
2Length of the open reading frame.
*, p,0.05;
**, p,0.005;
***, p,0.0005;
n.s., p.0.05
doi:10.1371/journal.pone.0002171.t003
Table 4. Summary statistics for cluster lengths and longest
open reading frames (LORFs) for all unigene clusters.
All Data Intronless Intronic
Intronic excluding
Chr. 2
Clusters n= 1405 n = 265 n= 219
Longest, bp 4309 4309 3502 3502
Shortest, bp 344 344 456 456
Mean, bp 1358 1361 1340 1351
Median, bp 1308 1304 1321 1335
s2, bp 455 450 479 495
LORFs
Longest, bp 4230 4230 3192 3192
Mean, bp 1059 1060 1052 1078
Median, bp 1007 1011 1005 1017
Shortest, bp 201 201 288 288
s2, bp 492 497 463 465
doi:10.1371/journal.pone.0002171.t004
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