The Genome of The Filamentous Fungus Ashbya Gossypii: Annotation and Evolutionary Implications

The genome of the filamentous fungus Ashbya
gossypii: annotation and evolutionary

implications
Sophie Brachat, Fred Dietrich, Sylvia Voegeli, Tom Gaffney, and Peter Philippsen
Abstract
The 9.2 Mb genome of the filamentous fungus Ashbya gossypii consists of seven
chromosomes carrying 4718 protein coding genes, 194 tRNA genes, at least 60
small RNA genes, and 40-50 copies of rRNA genes. With respect to both, the size
and the number of genes, this presently represents the smallest known genome of a
free-living eukaryote. Over 95% of the A. gossypii open reading frames encode
proteins with homology to Saccharomyces cerevisiae proteins. In addition, 90% of
A. gossypii genes show both, homology and a particular pattern of synteny (con-
servation of gene order), with the genome of budding yeast. Gene orders in the
two species are not strictly co-linear because individual clusters of A. gossypii
genes are always syntenic to two distinct S. cerevisiae chromosomal regions but
frequently homologous genes are missing in either of the two regions. These gene
clusters of ancient synteny (CLAS) were found to cover over 90% of both ge-
nomes. Specifically, 95% of the S. cerevisiae genes can be paired in duplicate
blocks that match the gene order of single A. gossypii gene groups. The almost
complete coverage of both genomes by clusters of ancient synteny provides com-
pelling evidence that both species originate from a common ancestor and that the
evolution of S. cerevisiae included a whole genome duplication subsequently fol-
lowed by random deletions of one gene copy in 90% of the duplicated genes. The
alignment of both genomes revealed a complete list of the 10% still remaining du-
plicated genes (twin genes) in today’s genome of S. cerevisiae. The analysis of
this comprehensive set of ancient twin genes in S. cerevisiae suggests that their
evolution is asynchronous. Finally, interpretation of the synteny pattern between
the sixteen S. cerevisiae centromere regions and the homologous gene regions in
A. gossypii suggests that the common ancestor of the two species most likely car-
ried eight chromosomes. The postulated reduction to seven chromosomes in the A.
gossypii lineage very likely marked a key event in the development of this fila-
mentous yeast as a novel species.
Topics in Current Genetics, Vol. 15

P. Sunnerhagen, J. Piškur (Eds.): Comparative Genomics
DOI 10.1007/4735_114 / Published online: 18 November 2005
© Springer-Verlag Berlin Heidelberg 2005
198 Sophie Brachat, Fred Dietrich, Sylvia Voegeli, Tom Gaffney, and Peter Philippsen
Fig. 1. Light microscopy images of S. cerevisiae (A) and A. gossypii (B). Each picture is
provided with a non-exhaustive list of phenotypic differences between the two fungi.
1 Introduction
Eighteen years ago we initiated molecular genetics with A. gossypii in order to

complement our studies with S. cerevisiae. We were intrigued by the fact that this
hemiascomycete exclusively grows like a filamentous fungus (Fig. 1) though phy-
logenetic analyses placed A. gossypii next to Saccharomyces and Kluyveromyces
species as well as the phytopathogenic fungi Erymothecium, Holleya, and Nema-
tospora (Prillinger et al. 1997). Staining of nuclei in A. gossypii hyphae shows that
the filamentous growth of this ascomycete has little in common with pseudohy-
phal growth of S. cerevisiae or Candida albicans (Whiteway and Oberholzer
2004), and very much resembles growth of multinucleated and branching hyphae
of Aspergillus or Neurospora (Philippsen et al. 2005). However, unlike higher as-
comycetes, A. gossypii lacks complex developmental programmes for conidiation.
During the first phase of our A. gossypii studies, we developed tools which al-
lowed targeted deletions of genes and complementation studies with self-
replicating plasmids (Wright and Philippsen 1991; Steiner et al. 1995; Wendland
et al. 2000). In addition, we discovered that the gene order in one third of the ge-
nomic clones was syntenic to the gene order of their homologues in S. cerevisiae,
and that the A. gossypii genome most likely lacked duplications, and that it was
significantly smaller than the genome of S. cerevisiae (Steiner and Philippsen
1994; Mohr 1995; Altmann-Jöhl and Philippsen 1996; Pöhlmann 1996; Wendland
et al. 1999). Based on these results and our several years experience with the S.
cerevisiae genome project, we decided in 1996 to aim at the complete genome se-
quence of A. gossypii. We wanted to gain the necessary information basis for
functional characterization of all genes involved in cellular pathways controlling
The genome of the filamentous fungus Ashbya gossypii 199
growth, nuclear division, and long-range organelle migration. In addition, we en-

visaged that the A. gossypii gene order, once established, would serve as a Rosetta
stone to gain essential insights into the evolution of the S. cerevisiae genome.
The complete sequence was finished early 2002. Completion of its annotation,
and the synteny map of the A. gossypii and the S. cerevisiae genome took an addi-
tional two years. The majority of the work was performed by a small team of sci-
entists working at the Biozentrum at the University of Basel and at Syngenta Bio-
technology Inc. in Research Triangle Park, with essential help in the finishing
phase from the Genome Center at Duke University in Durham. The genome was
sequenced by combining three strategies: end-sequencing of 12000 chromosome-
sorted plasmid and 1000 BAC clones, shotgun-sequencing of 32000 sheared ge-
nomic DNA fragments, and extensive gap filling by 12000 primer walking and
PCR guided sequencing. The sequence assembly was performed in 8 individual
batches (seven chromosomes and the mitochondrial DNA). This strategy led to the
availability of close to 90% of the sequence three years after the launch of the pro-
ject, and provided ample information for functional studies of A. gossypii genes
(Ayad-Durieux et al. 2000; Wendland and Philippsen 2000, 2001; Alberti-Segui et
al. 2001; Knechtle et al. 2003; Bauer et al. 2004). The remaining time was used to
close sequence gaps, to annotate all genes, to complete the synteny map with S.
cerevisiae, and to determine the endpoints of over 300 inversions and transloca-
tions in both genomes for the final publication (Dietrich et al. 2004).
This review describes first the sequence analysis and major results from its an-
notation. The second part focuses on evolutionary implication for both genomes as
revealed from close inspection of the clusters of ancient synteny. This part also in-
cludes results of an attempt to reconstruct the S. cerevisiae genome from 126 du-
plication blocks. The third part briefly summarizes different fates of twin genes
with respect to sequence conservation and divergence. Finally, in the fourth part,
we try to address whether the common ancestor of both organisms carried seven
chromosomes like A. gossypii and gained one chromosome in the S. cerevisiae
lineage prior to the whole genome duplication or whether it carried eight chromo-
somes with subsequent loss of one chromosome in the A. gossypii lineage.
2 Sequence analysis and annotation of the A. gossypii

genome
2.1 General features of the genome sequence
The A. gossypii genome was sequenced and assembled with a 4.2-fold sequence
coverage (Phrap quality > 20) and an estimated accuracy of 99.8%. Three short
regions from 300 bp to 1400 bp could not be sequenced most likely due to a very
high GC-content. Genomic features of the seven chromosomes are summarized in
Table 1. The nuclear genome has a total size of 9.2Mb including rDNA. This is
much smaller than the estimated sizes for other filamentous fungal genomes that
Table 1. A. gossypii genome features
GenBank Accession Size ORFs Intron Coding Coding

Number (Kb) containing DNA (Kb) DNA (%)
genes a
ChrI AE016814 692 381 17 545 78.75
ChrII AE016815 868 462 19 693 79.85
ChrIII AE016816 907 497 26 728 80.26
ChrIV AE016817 1467 819 35 1150 78.38
ChrV AE016818 1519 800 40 1209 79.57
ChrVI AE016819 1813 982 55 1448 79.87
ChrVII b AE016820 1476 777 25 1187 80.41
Genome 8741 4718 217 6970 79.52
a
This number does not include tRNA genes containing introns, of which there are a total of
50. There are five protein-coding genes that encode two introns, so the total number of
introns is 222.
b
The ribosomal DNA Repeat is 8197bp with about 50 copies present on chromosome VII
(Wendland, et al., 1999). Thus the total size of chromosome VII is approximately 1900 kb -
consistent with the observation that this chromosome corresponds to the slowest migrating
band in pulsed field gels (Wendland et al. 1999).
are in the range of 20-50Mb (Osiewacz and Ridder 1991; Kupfer et al. 1997;
Chavez et al. 2001; Galagan et al. 2003). Surprisingly, it is also 30% smaller than
the S. cerevisiae genome (13Mb including rDNA). The small size of the A. gos-
sypii genome is in part due to the almost complete lack of gene duplications (dis-
cussed below) and a very high genetic information density. On average, the A.
gossypii genome carries one protein-coding gene per 1.86 kb. In S. cerevisiae, the
average genetic density is one gene per 2.1 kb and in Schizosaccharomyces pombe
one gene per 2.5 kb (Goffeau et al. 1996; Wood et al. 2002). The average inter-
ORF region in A. gossypii is over 200 bp shorter than in S. cerevisiae indicating
that sequences controlling transcription in A. gossypii are probably shorter than in
S. cerevisiae. The average ORF size is only slightly shorter in A. gossypii com-
pared with S. cerevisiae. The paucity of introns (see Table 1) and the lack of
transposable elements also contribute to the small size of the A. gossypii genome.
Another notable difference is the average GC content which is much higher than
that of S. cerevisiae (52% vs. 38%), a fact that could reflect the different tempera-
tures in their respective habitats (Ashby and Nowell 1926).
2.2 Annotation of the assembled DNA sequences
An essential component of any sequencing effort is the conversion of the DNA

sequence data into useful information. The identification of genomic features such
as protein or RNA coding genes, telomeres, and centromeres and their labelling on
the genome sequence is the first step in this conversion and is referred to as struc-
tural annotation. Once structural annotation is available, functional information for
the discovered genes can be inferred from sequence and domain database searches
and finally from experimental results.
The annotation strategy was designed to make use of the high degree of se-
quence and gene order conservation between the A. gossypii and the S. cerevisiae
genome. As automatic annotation remains prone to errors, our method implied
both an automatic phase and an extensive manual revision. First, all ORFs sharing
homology to S. cerevisiae proteins as available from SGD (Cherry et al. 1998;
Saccharomyces Genome Database, http://www.yeastgenome.org) were automati-
cally extracted and annotated. Based on homology and synteny, other genomic
features, such as tRNA genes, snRNA genes, telomeres and centromeres, were
manually identified and annotated. A careful manual recheck of all annotations
was then conducted in order to disregard ORFs with very low homology to S. cer-
evisiae, which lacked synteny, to confirm low homology ORFs, which showed
synteny, to annotate overlooked syntenic ORFs with low homology to S. cere-
visiae, and to annotate introns. In a second step, we identified overlooked protein-
coding genes in both genomes by comparing translated DNA. This resulted in the
discovery of 46 novel ORFs and the identification of 72 putative annotation errors
in the S. cerevisiae genome (Brachat et al. 2003). A sequence search conducted on
available fungal databases including unfinished genomes and on GenBank, al-
lowed us to identify A. gossypii ORFs that do not have a homologue in S. cere-
visiae but in other species. For this annotation step, we did not apply any size re-
striction for A. gossypii ORFs. Finally, we annotated the remaining A. gossypii
ORFs larger than 150 codons. Systematic A. gossypii gene names were given fol-
lowing the nomenclature procedure used for budding yeast: three letters describing
the organism (A-Ashbya), the chromosome (A-G), and the chromosome arm (L,
R), followed by the gene number counting outwards from the centromere and the
DNA strand (W, C). In contrast to S. cerevisiae, all centromeres have the same
orientation with respect to the conserved DNA elements CDEI, CDEII, and
CDEIII (Hieter et al. 1985). For example, AAL001W is the first protein coding
gene to the left of the centromere on chromosome I and is coded by the Watson
strand. To facilitate functional comparisons we use for syntenic ORFs, in addition
to the systematic name, the common yeast gene name with a prefix Ag, e.g.,
ScCDC14 and AgCDC14. The manual evaluation of start and end points of
synteny was performed by two individuals; inconsistent results were rechecked.
The final annotations of the seven chromosomes and the mitochondrial DNA were
submitted to GenBank (Accession numbers: AE016814 to AE016821). These data
and additional information can be accessed via the Ashbya Genome Database
(Hermida et al. 2005; http://agd.unibas.ch). For a representative section of the an-
notation table for all A. gossypii genes see Table 2.
2.3 Protein coding genes
We have identified 4718 ORFs in the nuclear genome of A. gossypii. When com-
pared with the 6000 ORFs found in S. cerevisiae (Goffeau et al. 1996) and the
4824 ORFs of the fission yeast S. pombe (Wood et al. 2002), A. gossypii contains
Table 2. Features of the A. gossypii genome annotation implemented as spreadsheet.

Footnote to Table 2 overleaf.

This section includes information for 25 protein-coding genes and three tRNA gene. Four
A. gossypii genes are syntenic homologues to two S. cerevisiae genes (twins). Data is auto-
matically extracted from the annotation files and processed manually in Excel®. The “Fea-
ture” row describes the nature of each genomic element: CDS stands for Coding DNA
Strand and refers to protein-coding genes. “Start” and “End” rows describe the gene coor-
dinates on the chromosomes. Inter-ORF sizes are provided to highlight short or long non-
coding genomic regions and are useful to detect potential gene overlaps (yellow) or inter-
ORF regions longer than the average 500 bp (purple). The annotated overlap of 195 bp for
AEL031C and AEL030W was investigated by the 5’ race method, and it was found that the
fourth ATG of AEL030W is the start codon which generates an inter-ORF size of 276 bp.
Systematic and common names for S. cerevisiae homologues are provided. These cells are
filled using colours corresponding to each of the sixteen S. cerevisiae chromosomes and al-
lowing a rapid estimation of ancient synteny . The “synteny” rows depict the A. gossypii
gene categories: syntenic homologue (SH), non-syntenic homologue (NSH) or no homo-
logue in baker’s yeast (NOHBY).
the smallest set of protein coding genes presently described for a free-living eu-
karyote. This suggests that only slightly more than 4500 proteins are required for
an eukaryotic life style which is similar to the 4300 protein coding genes reported
for the prokaryotes E. coli and B. subtilis (Blattner et al. 1997; Kunst et al. 1997)
and less than the 5000 genes predicted for Pseudomonas (Stover et al. 2000),
Streptomyces (Bentley et al. 2002) and Mycobacterium (Cole et al. 1998). The A.
gossypii ORF sizes range from 25 codons for ADR103C, which encodes the
homologue of the S. cerevisiae ribosomal proteins RPL41A and RPL41B, to 4899
codons for AGR074C, which encodes the homologue of the S. cerevisiae protein
Mdn1p. We annotated 217 (4.6%) intron-containing ORFs. Five genes were found
to contain two introns (AFL082W, AEL145W, ADR193W, AFR579W,
AEL262C-A). Similar to S. cerevisiae, introns are small (115bp on average, rang-
ing from 25bp to 667bp). Most introns are located close to the 5’ end of ORFs and
24 introns interrupt the start codon. Interestingly, intron positions within ORFs are
often conserved in the two genomes. These findings were used to identify poten-
tially overlooked introns in the S. cerevisiae genome (Brachat et al. 2003). We
found that 66 A. gossypii ORFs carry an intron, which is not present in the S. cere-
visiae homologue and that for 46 intron-containing S. cerevisiae ORFs the A. gos-
sypii homologues are intron-free. Like in S. cerevisiae, intron-containing A. gos-
sypii genes often belong to particular functional categories. For example, 47
encode ribosomal protein genes, 20 encode proteins involved in cellular traffick-
ing and protein sorting, and 13 are homologues of S. cerevisiae genes encoding
cytoskeleton proteins.
2.4 Sequence conservation of proteins
Our annotation procedure detected, for 95% of the A. gossypii ORFs, homologues
in the budding yeast. The conservation, at the protein level, between these homo-
logues is very variable ranging from 18.1% (homologues of Prm2) and 100%
(homologues of histone H3) identical amino acids, with an average of 53.7%. In
Fig. 2. Identity (at the protein level) between A. gossypii and S. cerevisiae syntenic homo-
logues is independent from their genomic location. Percent identity between A. gossypii and
S. cerevisiae syntenic homologues was evaluated by pairwise comparison of the sequences
using Gap (GCG®) and plotted along the A. gossypii chromosome I. Each line represents a
single A. gossypii gene. The identity level varies between genes independently from their
relative location on the chromosome. The four genes with highest and lowest identity to
their S. cerevisiae syntenic homologues are mentioned. Random distribution of homology
levels along the chromosomes was also observed for the remaining six chromosomes.
order to estimate whether particular regions of the A. gossypii genome are more
susceptible to sequence evolution than others, we plotted levels of identity be-
tween homologues along the A. gossypii chromosomes. Figure 2 shows this plot
for chromosome I. Highly conserved protein coding genes alternate with less con-
served genes in a seemingly random pattern. This indicates that the natural selec-
tion unit is a single gene rather than a chromosomal region. We used functional
Gene Ontology categories (Ashburner et al. 2000) to classify the most similar
(>90% identity) and least similar (< 30%) proteins between the two species. This
analysis confirmed that, as generally observed in eukaryotes, ribosomal proteins,
histones and proteasomal subunits are among the most conserved orthologues. It
also revealed that meiotic proteins are very poorly conserved between the two
fungi. Interestingly, five S. cerevisiae proteins among the 200 most conserved
(>90% similarity) are so far of unknown function (YBR025C, YDR339C,
YGR086C, YGR173W, YPL225W). This implies a critical cellular role for the
encoded proteins. Consistent with this view, the proportion of essential genes,
based on the yeast gene deletion project (Giaever et al. 2002), is nearly twice as
large in the highly conserved gene group (45%) compared to the average of all S.
cerevisiae genes having a homologue in A. gossypii (23%).
2.5 Species-specific proteins
The combination of homology and synteny was used to classify A. gossypii genes
into three categories: Category 1 (syntenic homologue, SH) includes all genes that
are homologous to and in synteny to an S. cerevisiae gene (90% of all A. gossypii
genes). Category 2 (non-syntenic homologues, NSH) includes genes with homol-
ogy to genes in S. cerevisiae but lack synteny (5% of all A. gossypii genes). Cate-
gory 3 (NOHBYs: No Homologue in Baker’s Yeast) includes the remaining
genes, those with no homology in S. cerevisiae (5% of all A. gossypii genes). We
mapped the three gene categories along A. gossypii chromosomes. The three gene
types are, for the most part, evenly distributed across the genome. Gene category
distribution is thus independent from the genomic location. Importantly, this dem-
onstrates that neither NOHBYs nor non-syntenic homologues cluster at specific
regions of the genome but that they are interspersed within syntenic regions, im-
plying an independent evolution of individual genes within these two categories.
The annotation of NOHBYs that can be referred to as “A. gossypii-specific
genes” was challenging and their authenticity can be questioned. However, 170 of
the 262 annotated NOHBYs have homologues in other organisms or encode pro-
teins with known functional domains, confirming that they do not represent anno-
tation artifacts. According to this domain analysis, NOHBYs encode proteins of
diverse functions from enzymes to membrane proteins. The remaining 92 NO-
HBYs encode putative proteins longer than 150 amino acids (34 longer than 250
amino acids). The annotated NOHBYs represent only 5% of all A. gossypii pro-
tein-coding genes, which points to a surprisingly small difference in gene sets be-
tween the two fungi. In contrast to the low number of NOHBYs, we found many
more (1417) S. cerevisiae genes lacking a homologue in A. gossypii. This group
contains both, real genes and also close to 700 short ORFs, which very likely do
not encode proteins (Wood et al. 2001; Brachat et al. 2003).
2.6 RNA-encoding genes
We have identified a total of 194 tRNA-encoding genes in the A. gossypii nuclear

genome, fewer than the 275 tRNA genes of S. cerevisiae, and 50 of the tRNA
genes have introns. The relative abundance of iso-acceptor tRNAs in S. cerevisiae
is very similar in A. gossypii. Similarly as found for protein coding genes, most
tRNA genes map at syntenic locations compared with gene orders in S. cerevisiae.
Interestingly, they often map at the ends of synteny regions, which mark break
points of genome rearrangements (see below). In A. gossypii, tRNA genes repre-
sent the only type of interspersed repeated sequences and, thus, are most likely
sites at which genome rearrangements are initiated (Dietrich et al. 2004). The ri-
bosomal RNA genes are clustered at a single locus on chromosome VII and has
been previously described (Wendland et al. 1999). It is composed of approxi-
mately 50 tandem gene copies (8197bp) coding for the precursor for 18S, 5.8S,
and 26S ribosomal RNA and the same number of gene copies, on the opposite
strand, coding for the 5S ribosomal RNA. The same arrangement of ribosomal
RNA genes is present in S. cerevisiae chromosome XII with conserved proteins
coding genes on both sides of the rDNA clusters. We have so far identified 68
genes encoding snRNAs and snoRNAs most of which at syntenic positions with S.
cerevisiae small RNA genes including four snoRNA genes nested within introns
of protein-coding genes (AgSNR18, AgSNR24, AgSNR54, and
AgSNR39/SNR59) and four clusters of snoRNA genes arranged in a polycistronic
manner like their S. cerevisiae homologues. The total number of small RNAs in A.
gossypii is presently lower than in S. cerevisiae where 84 small RNA products are
known (Saccharomyces Genome Database; A Database for Small Nucleolar
RNAs (snoRNAs) from the Yeast Saccharomyces cerevisiae,
http://www.bio.umass.edu/biochem/rna-sequence/Yeast_snoRNA_Database). The
discrepancy might result from the small size of these small RNA products and
from limited homology between species both rendering their detection difficult.
We assume that additional small RNA genes remain to be discovered in A. gos-
sypii.
2.7 Transposable elements
Strikingly, we could not detect any complete transposable element in the A. gos-
sypii genome. However, one ORF (AGL178W) on chromosome VII has homol-
ogy to the reverse transcriptase gene of the S. cerevisiae transposable element
Ty3. Despite this transcriptase-like gene no remnants of Ty elements could be de-
tected. The lack of bona fide transposable elements is however intriguing because
to our knowledge A. gossypii is the first reported eukaryote lacking mobile ele-
ments.
2.8 Centromeres and telomeres
Centromeres were located based on homology and synteny to S. cerevisiae. The

three elements CDEI, CDEII, and CDEIII (Centromere DNA Element) (Panzeri et
al. 1985; Hieter et al. 1985) could be identified. However, the A. gossypii CDEII is
approximately twice as long as the CDEII of S. cerevisiae and, therefore, resem-
bles more the K. lactis centromeres (Heus et al. 1990). The telomeres of A. gos-
sypii have also been identified and carry the tandemly repeated 24-mer
CGCTGAGAGACCCATACACCACAC. A non-telomeric copy of this repeat is
present in the putative AgTLC1 gene, the A. gossypii homologue of the S. cere-
visiae RNA template component of the telomerase, which is found at a syntenic
position on chromosome I. The A. gossypii telomeres differ to some extent from
the S. cerevisiae telomeres as they have multiple perfect copies of this 24 base pair
repeat. In contrast, the S. cerevisiae repeat unit is composed of the fairly similar
17 base pair template (CACCACACCCACACACA)n but the telomeres contain
mostly degenerate copies of the template (Singer and Gottschling 1994; Cohn et
al. 1998).
3 Evolutionary implications of the A. gossypii genome

sequences
3.1 Possible origins of duplicated gene segments in S. cerevisiae
When the genome sequence of S. cerevisiae was completed two groups proposed a
duplication of the ancestral genome during evolution. Philippsen et al. (1997) dis-
cussed such event as one possible explanation to account for the duplication of six
long gene clusters, representing two thirds of chromosome XIV, with gene clus-
ters on other chromosomes. The duplicated genes within these 55 kb to 130 kb
long regions were interspersed with single copy genes but displayed in most cases
conserved gene orientation and order, a genomic feature termed “relaxed
synteny”. Using the information from the yeast genome consortium Wolfe and
Shields (1997) found that duplicate gene pairs represent 13% of all S. cerevisiae
genes and that two or more of these gene pairs define so-called “duplicate blocks”.
They estimated that duplicate blocks cover 50% of the yeast genome and proposed
that the S. cerevisiae genome is actually the result of a whole-genome duplication
event followed by frequent loss of one copy of the duplicated gene pairs. Accord-
ing to these two proposals the today’s S. cerevisiae genome represents a mosaic of
the ancestral genome as schematically shown in Figure 3A. Explicitly, genes that
were adjacent on a single chromosomal segment in the ancestral genome are now
found alternating between two different chromosomal regions, and the original
Fig. 3. Proposed fate of ancient duplications and reconstruction of ancient synteny. (A)
Schematic representation of the origin of duplicate gene blocks in today’s S. cerevisae ge-
nome by long-range duplications in the ancestral genome or by a single whole genome du-
plication event. The recognition of duplicate gene blocks is not possible in genome regions,
which lack duplicate genes. In addition, reconstruction of the ancient gene order between
duplicate genes is not possible. (B) Schematic illustration of a typical cluster of ancient
synteny (CLAS) between A. gossypii and S. cerevisiae (CLAS). The gene order in A. gos-
sypii is used as Rosetta stone to reconstruct the ancient gene order. In fact, identification of
duplicate blocks in the S. cerevisiae genome no longer depends on the presence of duplicate
genes. All A. gossypii gene regions aligning with homologues of two syntenic yeast chro-
mosomes highlight duplicated S. cerevisiae gene regions and allow reconstruction of an-
cient gene orders.
order of genes between the still recognizable duplicated genes cannot be recon-
structed without additional information. As an alternative hypothesis, Dujon and
colleagues suggested that individual chromosomal segments were duplicated at
different times (Llorente et al. 2000b; Fischer et al. 2001). This theory was con-
sidered less likely due to the absence of long triplicated regions in the S. cere-
visiae genome, but based on the available S. cerevisiae genome information, it
was a viable alternative explanation for the origin of duplicated segments (Piskur
2001). To find convincing evidence for one of the contradicting hypotheses and to
be able to reconstruct the ancient gene order, it was necessary to align S. cere-
visiae genes with homologous genes of a completely sequenced non-duplicated
genome of a hemiascomycete as outlined in Figure 3B. Only a close to complete
alignment of such genome with two gene segments of S. cerevisiae would provide
compelling evidence in favour of the genome duplication hypothesis. The pres-
ence, in such synteny map, of long regions aligning only with single S. cerevisiae
gene segments would favour the segmental duplication hypothesis.
3.2 Proof for an ancient whole-genome duplication in S. cerevisiae
A search for gene duplications in the complete A. gossypii gene set uncovered
only a few tandemly duplicated genes but no gene cluster duplications. This
search also revealed that 96% (see below) of the A. gossypii genome display
synteny to the S. cerevisiae genome as indicated by several hundred clusters of
ancient synteny like the one shown in Figure 3B with homology relations of single
genes or groups of genes alternating between two S. cerevisiae regions. We did
not find A. gossypii genomic segments, which were syntenic with more than two
regions of the S. cerevisiae genome. This result can only be explained by a whole
genome duplication in the S. cerevisiae lineage and does not support the segmen-
tal duplication hypothesis (Dietrich et al. 2004). The same conclusion was drawn
from the analysis of genomic sequences of Kluyveromyces waltii, which carries a
non-duplicated yeast genome (Kellis et al. 2004). We named the blocks of synteny
between one A. gossypii and two S. cerevisiae gene groups “clusters of ancient
synteny” (CLAS). The position and coding information of almost all gene dele-
tions following the whole genome duplication can be inferred in each CLAS from
the gaps in one chromosome segment and the gene still present in the other chro-
mosome segment, respectively. On a whole genome scale this allows reconstruc-
tion of ancient gene orders prior to the genome duplication (Dietrich et al. 2004).
Boundaries of a CLAS mark breaks of synteny either in one or in both S. cere-
visiae chromosome segments. Such breaks define endpoints of viable transloca-
tions or inversions in the evolutionary past of both genomes, which is discussed in
more detail in Chapter 3.6. The longest CLAS comprises 56 A. gossypii genes on
chromosome III (ACR095 to ACR148, including two snRNA genes) syntenic with
S. cerevisiae chromosomes XV (YOR212 to YOR245) and XVI (YPL127 to
YPL158). This CLAS includes 10 duplicate gene pairs (twin genes). Neither of
these three regions was rearranged by a translocation or inversion event since both
Fig. 4 (previous pages). Duplicate block map of the S. cerevisiae genome. Duplication
blocks along the 16 S. cerevisiae chromosomes were reconstructed based on ancient
synteny to the A. gossypii genome. Each block involves two chromosomes and each dupli-
cate region is labelled as A and B. Blocks are depicted as boxes, divided in two coloured
areas representing the two chromosomes. Blocks were drawn to scale and alternate above
or below the chromosome lines for clarity. 95% of the S. cerevisiae protein-coding genes
were found within anciently duplicated regions. Originally, 132 blocks were generated, but
a visual inspection of boundaries led to the reassignment of six short blocks (21, 37, 67, 81,
92, 111) to adjacent blocks. A few short blocks appear more than two times (1B, 43B, 71B,
72B, 89B, 95B, 97B, 114B, 124B, 130B) most likely due to the mapping of gene family
members. In this diagram only translocations are seen which occurred after the genome du-
plication; inversions remain undetected.
species diverged from a common ancester over hundred million years ago. There
are longer regions in A. gossypii chromosomes which did not undergo a rear-
rangement whereas the syntenic S. cerevisiae regions rearranged after the duplica-
tion. The longest region comprises 182 genes (including six snRNA and two
tRNA genes) in chromosome VI (AFL185 to AFL013).
3.3 NOHBY’s and non-syntenic homologues in clusters of ancient

synteny
In order to quantify the proportion of the A. gossypii genome that map within
CLASs, we classified NOHBYs and non-syntenic homologues (NSHs) according
to their location within or outside CLASs using the Ashbya Genome Database.
For example, NOHBY AAR003W occurs within a CLAS whereas the NSH
AAL120W maps at a break of synteny. We found that a total of 178 NOHBYs and
71 non-syntenic homologues map within CLASs. Thus, CLASs comprise 4276
syntenic homologues, 71 non-syntenic homologues and 178 NOHBYs of all 4718
A. gossypii protein-coding genes. In other words, 4525 of the A. gossypii protein-
coding genes (96%) map in clusters of ancient synteny.
3.4 Update of duplicate gene blocks in S. cerevisiae
The almost complete coverage of the A. gossypii genome in clusters of ancient

synteny raised the question which fraction of the S. cerevisiae genes belong to du-
plicated regions. We used ancient synteny information to reconstruct updated S.
cerevisiae duplicate blocks and evaluated their coverage of the yeast genome.
Technically, S. cerevisiae duplicate regions were first derived from each CLAS
and the inferred duplicate loci were re-ordered along the S. cerevisiae chromo-
somes to generate a novel duplicate block map (Fig. 4).
We found that the S. cerevisiae genome can be re-constructed in 126 duplicate
blocks. These blocks cover over 85% of the total genomic DNA (including the ri-
bosomal DNA repeats and the centromeres, but excluding the ORF-free telomeric
regions). We determined that 5969 S. cerevisiae ORFs are included within dupli-
cated regions, representing over 95% of all protein coding genes. This is almost
twice the number of genes previously estimated to participate in duplicated re-
gions of the yeast genome. Interestingly, 13 duplicate blocks lost all “relics” of
their ancient duplication since they lack duplicate genes (remaining twin genes).
Furthermore, additional 28 blocks contain only a single twin pair. Thus, only the
complete synteny map between A. gossypii and S. cerevisiae allowed the identifi-
cation of most likely all duplicate blocks.
3.5 Loss of S. cerevisiae genes after the genome duplication
The synteny map implies that 90% of the gene duplicates lost one copy after the
duplication. This corresponds to approximately 4000 viable gene losses since the
genome duplication event, which happened over 100 million years ago (Piskur
2001). Different mechanisms of gene loss can be envisaged: progressive loss of
function by accumulation of missense mutations or one-step loss of function by a
nonsense mutation, a frame-shift mutation or a deletion event (involving one or
more genes). One rare example for a relic of a duplicate gene was found in a
CLAS involving genes from chromosome II and IV. The sequence between
YBR060C and YBR061C has homology to the ORF YDR037W but the putative
twin ORF on chromosome II is altered by six frame-shifts. The ancient synteny
with A. gossypii suggests that YDR037W is a member of the duplication block 18,
which involves YBR058C to YBR065C and YDR037W to YDR045C (Fig. 4).
Therefore, the YBR061C/YBR060C inter-ORF region most likely still carries the
highly mutated twin copy of YDR037W. We re-sequenced that region in the S.
cerevisiae strain that was used for the yeast genome project and confirmed the
published sequence. Most S. cerevisiae inter-ORF sequences are too short to con-
tain highly degenerate copies for the majority of lost twins. Hence, loss of most
genes of duplicate pairs most likely occurred via single or multiple gene deletions
and is probably still ongoing.
3.6 Synteny breaks as marker of genome rearrangements
The large extent of synteny between A. gossypii and S. cerevisiae provided a

unique opportunity to evaluate the degree of genomic rearrangements that shaped
both genomes after the speciation event (Fig. 5A). As a matter of fact, breaks of
synteny reflect chromosomal rearrangements that took place during evolution of
the two genomes. As depicted in Figure 5B, a synteny break affecting only one of
two S. cerevisiae chromosomes, is indicative of a translocation or inversion event
that occurred in the S. cerevisiae lineage after the genome duplication (Period c,
Fig. 5A). On the other hand, double breaks of ancient synteny can affect both S.
cerevisiae duplicate segments, as shown in Figure 5C, and point to a translocation
or inversion event that took place in the A. gossypii lineage or in the S. cerevisiae
lineage prior to the doubling event (Periods a or b, Fig. 5A).
Fig. 5. Two types of synteny breaks define pre- and post-duplication rearrangements. (A)
Evolution of A. gossypii and S. cerevisiae. Period a refers to the time between the speci-
ation event and today. Periods b and c refer to pre- and post-duplication periods in the S.
cerevisiae lineage. A dashed circle indicates the genome duplication event. (B) Single
break of synteny (white arrow). Genomic rearrangements that occurred in phase c lead in
two clusters of ancient synteny to reciprocal single synteny switches. One of these switches,
from the blue to the orange chromosome, is shown. The other reciprocal switch can be
searched for in the CLAS map (Dietrich et al. 2004). (C) Double break of synteny (black
arrows). Genomic rearrangements in periods a and b lead in two clusters of ancient synteny
to double synteny switches. One of these double synteny switches in the CLAS map is
shown. The reciprocal double synteny switch is sometimes difficult to find in the CLAS
map because subsequent rearrangements can involve previously used sites of rearrange-
ments (Dietrich et al. 2004).
We determined the numbers of double and single synteny breaks on a whole

genome scale (Dietrich et al. 2004). The map of all clusters of ancient synteny
highlighted 328 double and 168 single breaks of synteny, suggesting 164 translo-
cations/inversions during periods a and b and only 82 during period c (one ge-
nome rearrangement creates two break points). However, the number of genome
rearrangements in period c is most likely higher because several single breaks of-
synteny are sometimes masked by double breaks and because some sites of single
breaks may represent an evolutionary hot spot for rearrangements, e.g., sites carry-
ing a transposable element (Dietrich et al. 2004; Fischer et al. 2000). Indeed, we
found that 58 synteny break points carry, in the S. cerevisiae genome, long termi-
nal repeats of TY elements, which, in principle, can initiate genome rearrange-
ments with over two hundred sites in S. cerevisiae chromosomes carrying these
sequence repeats (Goffeau et al. 1996). A more realistic estimate taking into ac-
count repeated use of rearrangement sites in S. cerevisiae due to the presence of
transposable elements predicts about 180 viable translocations or inversions in the
S. cerevisiae lineage, 60 in period b and 120 in period c (Dietrich et al. 2004).
The map of reciprocal S. cerevisiae duplicate regions described in Chapter 3.4
revealed the presence of 126 duplication blocks (Fig. 4). Since inversions remain
undetected in this figure, these blocks were presumably formed by successive re-
ciprocal translocation events after the genome doubling and their number directly
correlates with the number of these rearrangements. At the time of the genome
duplication, the then sixteen chromosomes formed eight long duplicated regions.
As each reciprocal translocation generates two additional duplicated regions, the
126 identified duplication blocks could have originated from some 60 independent
translocations if the majority of the translocation endpoints were only used once.
As the genome duplication took place approximately 100 millions years ago
(Wolfe and Shields 1997; Piskur 2001), viable genome rearrangements occurred at
very low frequency. This implies that those events are either rare or rarely main-
tained in the population due to detrimental effects for the organism. Our results
corroborate an estimate of the number of post-duplication translocations by a
simulation based on 55 blocks covering 50% of the yeast genome (Seoighe and
Wolfe 1998). The authors estimated that between 70 and 100 events resulted in the
55 duplications blocks they had identified and that a total of 150 to 200 paired re-
gions should be identifiable in the yeast genome from comparison to pre-
duplication species.
4 Gene pairs (twins) originating from the genome

duplication
4.1 Identification of twin ORFs
The ancient synteny map of both genomes allowed to identify 496 pairs of S. cer-
evisiae ORFs and their non-duplicated syntenic homologues in A. gossypii (see
examples for the identification of four twin ORFs in Table 2). Each of these ORF
pairs corresponds to duplicates that resulted from the whole genome duplication
and which remained duplicated during evolution. This number of twin ORFs is
significantly higher than the 406 pairs previously proposed (Wolfe and Shields
1997; Seoighe and Wolfe 1999). 367 pairs are shared between the two datasets.
From the remaining 43 ORF pairs suggested by Wolfe and colleagues, 23 pairs
correspond to ORFs lacking a homologue in A. gossypii. They are most likely real
twin ORFs, which lost the syntenic A. gossypii homologue during evolution. In-
deed, each of the 23 pairs can be assigned to syntenic positions in the yeast seg-
ments of clusters of ancient synteny, only that the A. gossypii gene segment lacks
the homologue. In total, 129 novel pairs of S. cerevisiae twin ORFs were identi-
fied which represents a substantial amount of previously overlooked redundancy
in the S. cerevisiae genome.
The earlier identification of genes duplicated in S. cerevisiae was based on an
“all versus all” comparison of the S. cerevisiae protein-coding genes. To be
counted as a duplicate pair, the respective sequences had to fulfil several criteria:
Significant protein sequence homology (BLASTp E-value below 10-18), less than
50 Kb distance between adjacent duplicate pairs of the same block, a minimum of
three adjacent duplicate genes to confirm association of two chromosomal seg-
ments, and conserved gene orders and orientation in duplicate blocks. This under-
standably limited the identification of duplicate ORF pairs. Use of the ancient
synteny map permitted detection of twins sharing homology below the previously
used threshold. This is in agreement with the average amino acid identity between
the 129 newly discovered twins, which is 10% lower compared to the original set
of twins.
4.2 Genetic complexity caused by twin genes
Due to the absence of twin genes in A. gossypii, gene redundancy in this organism
is restricted to a small number of tandem repeats and gene families. This simpli-
fies for example the functional characterization of novel ORFs by reverse genetics
because single gene knockouts show more often a phenotype compared to S. cere-
visiae, making A. gossypii a promising fungal model organism. For example, the
A. gossypii genome encodes only five homologues of CDK-regulating cyclins
compared to nine in S. cerevisiae. Early cell cycle studies in budding yeast have
shown that pairs of cyclins are functionally redundant (Futcher 1996) suggesting
that the A. gossypii cell cycle may be regulated by simpler control system and that
single deletions of cyclin genes may already affect this control system. Indeed, de-
letion of single B-type cyclin genes in A. gossypii results in strong phenotypes (K
Hungerbühler and A Gladfelter, personal communication), whereas the deletion of
three B-type cyclins is needed to lead to a defective cell cycle in budding yeast
(Richardson et al. 1989; Futcher 1996). Additional examples of lack of functional
redundancy in A. gossypii include ribosomal proteins, myosins, RAS GTPases,
and guanine nucleotide exchange factors (GEFs) for small GTPases.
The majority of twin gene products have at least partially overlapping activities
and functional analysis of twin genes will be complicated by this redundancy. The
Yeast Gene Deletion Project provided phenotypic analysis for 451 pairs of twins
(Giaever et al. 2002). For 413 pairs both twins are dispensable for cell survival
and for 38 pairs one of the twins was found to be essential, the other not. This in-
dicates that only 4% of the twins, compared to 15% for the whole genome, are ac-
tually essential, confirming a very high degree of functional redundancy of the

duplicates. Furthermore, for none of the pairs both twin copies are essential for
cell viability. The 38 pairs, for which only one of the twins is essential, might rep-
resent cases in which an essential gene duplicated and one copy functionally di-
verged due to mutations or due to significant decrease in the expression level,
which prevents functional complementation. One example is the novel twin pair
YDL239C and YNL225C with only 21% identical amino acids. YDL239Cp has
been recently described as a component of the spindle pole body critical during
meiosis (Nickas and Neiman 2002) but non-essential for mitosis. YNL225Cp has
an essential function at the spindle pole body during mitosis (Brachat et al. 1998)
and is also essential for meiosis. Table 3 lists 21 twin pairs for which only one of
the two copies was functionally characterized and this knowledge is important to
uncover the function of the other twin copy. Remarkably, for 40 novel twin pairs
both copies are functionally uncharacterized. Only pairwise deletions of these twin
genes might result in a detectable phenotype and may thus lead to the elucidation
of previously unknown gene functions. Examples for such twin pairs were discov-
ered when we screened the 497 twin genes for increased expression during meio-
sis taking advantage on microarray-based genome transcription profiling data
(Primig et al. 2000). We found eight twin pairs for which both twin genes show
increased expression levels during meiosis suggesting a redundant role in this
process. For some of these twins, single deletion strain show no meiotic phenotype
and we expect that double knockouts will affect meiosis.
4.3 Sequence divergence of twin genes
The complete inventory of S. cerevisiae twin genes is not only an important source
of information on functional redundancy but also shows the whole spectrum of se-
quence evolution in twin genes. Gene duplication has long been recognized as an
important mechanism for the creation of new gene functions. However, not all
gene duplications result in the appearance of new functions as the new duplicates
might remain redundant or one of the duplicates might be inactivated either by de-
letion or by accumulation of point mutations. Based on the extended inventory of
S. cerevisiae twin genes, we re-investigated the evolution of their sequences. Im-
portantly, the known syntenic A. gossypii homologues for each twin pair provided
an essential reference for the degree of sequence divergence in the absence of a
second gene copy. We performed a systematic sequence comparison of the twin
gene products and their unique A. gossypii counterparts, creating a similarity tree
for each of the “gene product sets”. The comparison of these trees to the species
tree allows the distinction of three types of protein set phylogenies: i) all the pro-
teins in the set are equally distant to each other; ii) S. cerevisiae duplicates are
closer to each other than to their A. gossypii counterparts; iii) the A. gossypii pro-
tein is more related to at least one of the twins than the twins are to each other. We
defined the twin gene with the highest similarity to the A. gossypii homologue as
twin 1. Figure 6 summarizes the results of the triple sequence comparisons. In 151
Fig. 6. Sequence divergence among S. cerevisiae twins and their A. gossypii homologues.
(A) Triple sequence comparisons showing very similar levels of sequence divergence. 151
groups of twins plus the syntenic A. gossypii showed less than 5% similarity difference
(344 when the threshold was relaxed to 10%). (B) Groups of proteins in which the twins
were more diverged from the A. gossypii reference protein than from each other. 115 be-
long to this class when the threshold was set to 5%. (C) Groups of proteins in which twin 1
was more similar to A. gossypii reference protein than to its duplicate copy. These total 107
cases can be divided in two groups: 96 cases for which twin 2 is equally or more distant to
the A. gossypii protein than to twin 1 suggesting a divergence as described by the classical
model (Fig. 7). In the remaining 11 cases, both twins are closer to their A. gossypii homo-
logue than to each other supporting the subfunctionalization model (Fig. 7).
cases the pairwise similarities are very close to each other (at the most 5% similar-
ity difference) indicating similar selection pressures for the three genes. For 115
triple comparisons the twins are more closely related to each other than either of
them to the A. gossypii homologue (5% similarity difference threshold). In these
cases the A. gossypii gene may have been exposed to less selection pressure than-
the yeast counterparts resulting in a faster sequence divergence between the A.
gossypii gene and both yeast twin genes than between the twin genes. Interest-
ingly, 107 twin 1 proteins are more related to the A. gossypii homologue than to
the respective twin 2 (5% similarity difference threshold) indicating a strong se-
quence divergence among the twins.
Fig. 7. Current models for the evolutionary divergence of duplicate genes. (A) The classical
model suggests that one duplicate retains the ancestral characteristics whereas the second
copy is allowed to freely diverge to acquire new properties. (B) In the subfunctionalization
model, the ancestral functions are split among the two duplicates that can specialize.
It is generally believed that the presence of duplicates allows for diversification

and adoption of new functions. The classical model proposes that one copy retains
the original sequence and function while the second copy is allowed to diverge
(Ohno 1970). An interesting alternative was put forward as the “subfunctionaliza-
tion” model. In this model, both duplicates evolve and the ancestral function is-
subdivided between the two duplicates (Force et al. 1999). Both models are sum-
marized in Figure 7. The 107 divergent pairs of twin genes shown in Figure 6
provide important data for discussion of these models. For only eleven triple com-
parisons, we found that both twins are more closely related to the A. gossypii ref-
erence protein than the twins to each other (5% similarity difference threshold) as
would be predicted by the sub-functionalization model. For the large majority of
cases (96 out of 107), the A. gossypii reference protein is at least as distantly re-
lated to twin 2 than twin 1 is to twin 2. These results clearly favour the classical
Ohno model for duplicate divergence.
This genome-scale analysis of gene duplicates together with pre-duplication
reference genes provides strong evidence that different S. cerevisiae twin pairs,
which arose at the same time, experienced diverse evolutionary fates and fre-
quently evolved in an asynchronous manner. A similar analysis, based on the
comparison of 38 duplicate S. cerevisiae gene pairs with homologues in other as-
comycetes, already suggested this asynchrony (Langkjar et al. 2003).
5 Evolution of chromosome number in A. gossypii
Another evolutionary aspect that can be analyzed by the comparison of the A. gos-
sypii and S. cerevisiae genomes concerns the evolution of the chromosome num-
bers in the two species. S. cerevisiae and closely related species bear sixteen
chromosomes, implying that prior to the duplication event the ancestral genome
carried eight chromosomes. The presence of only seven chromosomes in A. gos-
sypii raises the question of how many chromosomes were present in the common
ancestor of S. cerevisiae and A. gossypii. To address this question, we aligned all
Fig. 8. Comparison of centromeric regions between S. cerevisiae and A. gossypii. (A) Evo-
lutionary twin centromeres of S. cerevisiae were identified for fourteen out of sixteen
chromosomes using synteny to A. gossypii. Syntenic sets of chromosomes are depicted in
groups of three. The insert (B-1) schematically shows the alignment of the remaining two
yeast chromosomes with two syntenic A. gossypii regions both lacking a centromere
(pseudo-centromeric regions). (B-2) Pseudo-centromeric regions depicted in more detail,
schematically showing the gene organization. Circles represent centromeres, genes are de-
picted by boxes and homologous genes are connected by dashed lines.
centromeric regions from S. cerevisiae to the A. gossypii genome. The seven A.

gossypii centromere region align pairwise with fourteen centromere regions in S.
cerevisiae as depicted in Figure 8A. The centromere regions of S. cerevisiae
chromosomes X and XII did not align with any centromeric region in A. gossypii.
But centromere-adjacent genes from the left arms of yeast chromosomes X and
XII perfectly align with A. gossypii genes on chromosome I, and centromere-
adjacent genes from the right arms of chromosomes X and XII align with A. gos-
sypii genes on chromosome III (Fig. 8 B1 and B2). The pattern of ancient synteny
of these alignments is reminiscent of a reciprocal translocation in the A. gossypii
lineage. However, no relics of centromere sequences (pseudo-centromere) are pre-
sent at the presumptive translocation sites in A. gossypii. Either, centromere se-
quences were never present in these regions, or they diverged beyond recognition.
The observed gene order and centromere alignment can be explained by two
models depicted in Figures 9 and 10, respectively. As a first possibility, eight
Fig. 9. Evolutionary model for an ancestral genome with eight chromosomes. One of the
eight chromosomes was lost in the A. gossypii lineage by a centromere break, followed by
the fusion of the two chromosome arms to the telomeric ends of remaining, intact chromo-
somes. The resulting genome consists of only seven chromosomes two of which with in-
creased length. Asterisks label the positions of the two A. gossypii pseudo-centromeric re-
gions.
chromosomes could have been present in the common ancestor and one chromo-
some was lost in the A. gossypii lineage. The breaking of a chromosome at its cen-
tromere would have resulted in the formation of two centromere-free chromoso-
mal pieces. These could have been fused in a non-homologous manner to the ends
of intact chromosomes (Fig. 9). As a second possibility, the common ancestor may
have carried only seven chromosomes. As outlined in Figure 10, a centromere du-
plication event could have generated a dicentric chromosome in the S. cerevisiae
lineage, causing chromosome breaks between the centromeres. Such breaks can be
healed by addition of telomere sequences, thus, creating two smaller chromosomes
as previously shown with artificially generated dicentric yeast chromosomes
(Haber et al. 1984; Jäger and Philippsen 1989a). However, the current pattern of
ancient synteny across the A. gossypii pseudo-centromere would only be observed
if the position of centromere appearance in the S. cerevisiae lineage coincided
with the position of a perfect reciprocal translocation in the A. gossypii lineage.
This model is less likely as it implies two independent and different events at two
syntenic loci in both genomes.
The analysis of the karyotype of other ascomycetes revealed a chromosome
number ranging from six to sixteen (Jäger and Philippsen 1989b; Lankjaer et al.
2000). Species that diverged prior to genome duplication tend to have six or seven
chromosomes. This suggests that alteration of the chromosome number was recur-
rent in the history of ascomycetes. The fact that many species that diverged earlier
than S. cerevisiae and A. gossypii carry less than eight chromosomes could argue
for a seven-chromosome ancestor. Although our comparative analysis of the two
genomes favours the eight-chromosome hypothesis, further evidence will be
needed to distinguish between the two possibilities, e.g., by investigating syntenic
centromere and pseudo-centromere regions in other hemiascomycetes.
Fig. 10. Evolutionary model for an ancestral genome with seven chromosomes. Two inde-
pendent events lead to the generation of the A. gossypii and S. cerevisiae ancestors respec-
tively. In the S. cerevisiae lineage, a centromere duplication event, followed by a chromo-
some break gave rise to an organism with eight chromosomes that subsequently underwent
a whole-genome duplication. In the A. gossypii lineage, a reciprocal translocation occurred
(crossing lines) very close to the location of the centromere acquisition in the S. cerevisiae
lineage.
6 Material and methods
6.1 Sequence and assembly quality
The quality of individual sequence reads was evaluated using Phred quality scores
and the overall quality of the sequence assembly was evaluated using Phrap qual-
ity scores (Ewing and Green 1998; Ewing et al. 1998). When potential frame shift
or stop codons were detected in protein coding genes, these sequences were in-
spected and edited or re-sequenced. Based on the Phrap scores >20 (Ewing and
Green 1998; Ewing et al. 1998) and on our examination of the genomic sequence
data, we estimate that the overall sequence coverage is approximately 4.2-fold and
the average accuracy of the sequence data is 99.8%, with less than 4.5% of the ge-
nome covered by a single sequence read.
6.2 Annotation
Open reading frames (ORFs) larger than 150bp were extracted and searched
against the S. cerevisiae protein dataset as available from SGD (Cherry et al.
1998; Saccharomyces Genome Database) using tFASTA (Pearson and Lipman
1988; Pearson 1994). ORFs with identity to S. cerevisiae proteins ranging from
less than 20% to 100% were retained and automatically annotated. ORFs without
similarity but larger than 450bp were also automatically annotated. Originally, the
most upstream ATG was annotated as start codon but we later reported several ad-
justments to NCBI after experimental determination of several transcription start
sites (S. Voegeli, unpublished). Initial analysis revealed that the intron splice rules
for A. gossypii appear to be identical to those of S. cerevisiae (Rymond and Ros-
bash 1992; Davis et al. 2000). These splice rules where then used to identify and
annotate putative intron containing genes. Annotations were subsequently manu-
ally checked. tRNAs, snRNAs, centromeres, and telomeres were annotated by
homology to S. cerevisiae. The tRNA annotation was compared to tRNA-scan-SE
results (Lowe 1997) but this did not lead to the identification of additional tRNA
genes. As snRNA homology is in general weaker than that observed for tRNA, all
A. gossypii genomic regions syntenic to the 72 snRNA positions in the S. cere-
visiae genome were manually inspected for missed syntenic snRNAs. Inter-ORF
regions were screened against a S. cerevisiae translated genome dataset using
TBlastx and led us to the identification of overlooked protein-coding genes in both
A. gossypii and S. cerevisiae (see Chapter 2). Resulting inter-ORF regions were
then searched against other databases including C. albicans (Stanford Genome
Technology Center), Génolevure (Llorente et al. 2000b), N. crassa (Neurospora
Sequencing Project; Whitehead Institute/MIT Center for Genome Research;
Galagan et al. 2003) and S. pombe (Wood et al. 2002). This allowed the annotation
of A. gossypii genes without homologue in S. cerevisiae (NOHBYs) but having
homologues in other species. Finally, remaining ORFs longer than 150 codons and
non-overlapping other features were annotated as NOHBYs. Gene names were
given following the S. cerevisiae nomenclature from the yeast genome consor-
tium. Chromosome sequences were prepared for submission to GenBank using
sequin (Benson et al. 2003). A minimal set of BAC and plasmid clones covering
the genome sequence was extracted from the assembly information and clone ends
were automatically annotated. Sequence quality information was derived from the
assembly and used to extract regions of the genomes with less than 90% sequence
confidence. These “low quality regions” were also automatically annotated. Genes
having homologues in S. cerevisiae were manually assessed for synteny by two
individuals and the label “syntenic homologue to S. cerevisiae” or “non-syntenic
homologue to S. cerevisiae” was automatically added to the note descriptor of
each gene. When more than one S. cerevisiae homologue was found for a single A.
gossypii gene, the gene with the highest homology is always mentioned first in the
description.
6.3 Data analysis
The data were converted from the GenBank format to the A. gossypii genome
spreadsheet (Table 2) using the Readseq program (Readseq,
http://iubio.bio.indiana.edu/soft/molbio/readseq/), which produces GFF (general
feature format) tables, and manually edited in Excel®. Additional analyses of the
data were done using BLAST (Altschul et al. 1990), Fasta (Pearson 1994), and the
GCG® Wisconsin Package® (Accelerys). Gene conservation levels were obtained
from systematic pairwise sequence alignments of homologous genes using Gap
(GCG® Wisconsin Package® (Accelerys)). The map of the distribution of A. gos-
sypii gene categories was created using GeneSpring®. Ancient synteny informa-
tion was directly derived from the A. gossypii genome spreadsheet and manually
drawn to produce the CLAS map. Gene viability information was taken from the
Yeast Deletion Project data (Giaever et al. 2002) and functional information for
the yeast genes were obtained from SGD (Cherry et al. 1998; Saccharomyces Ge-
nome Database) and the Gene Ontology Consortium (Ashburner et al. 2000). Se-
quence data was organized and maintained within local GCG® sequence data-
bases. Annotation information and all data mining results were organized and
maintained within a local FileMakerPro® database.
6.4 Creation of the map of Duplicate Blocks
The synteny information from the A. gossypii genome spreadsheet was used to as-
sociate each pair of S. cerevisiae chromosomal regions homologous and syntenic
to single A. gossypii loci. S. cerevisiae genes were then reordered along the S. cer-
evisiae chromosomes. Duplication blocks were identified as stretches of the ge-
nome with paired regions involving similar chromosomes. Blocks were numbered
starting from the left arm of chromosome I. First and last gene members of a block
were used to label the block edges. Block edges were then mapped on the com-
plete set of S. cerevisiae features to recover genes absent in A. gossypii. Duplicate
block break points were manually inspected for the presence of transposable ele-
ments and LTRs. The duplicate block map was partially generated using
GeneSpring®.
Acknowledgments
We thank A Lerch, K Gates, A Flavier, S Choi, R Wing, S Steiner, C Mohr, R

Pöhlmann, Ph Luedi, Y Bauer, A Binder, K Gaudenz, S Goff, J Hoheisel, M Jac-
quot, P Knechtle, M Primig, C Rebischung, H-P Schmitz, J Wendland, and the
Syngenta (formerly Novartis) sequencing facility at Research Triangle Park for
their assistance with this project and for valuable discussions. We also acknowl-
edge the help of Iza Kaminski in drawing ancient synteny maps. This work was
supported by major funding obtained from the University of Basel and Syngenta
Biotechnology Inc. (formerly Novartis Agribusiness Biotechnology Research Inc).

We also acknowledge support from the Duke University’s Young Investigator
Start-Up Fund.
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The Genome of The Filamentous Fungus Ashbya Gossypii: Annotation and Evolutionary Implications

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The genome of the filamentous fungus Ashbya

gossypii: annotation and evolutionary

Topics in Current Genetics, Vol. 15

Eighteen years ago we initiated molecular genetics with A. gossypii in order to

growth, nuclear division, and long-range organelle migration. In addition, we en-

2 Sequence analysis and annotation of the A. gossypii

2.1 General features of the genome sequence

Table 1. A. gossypii genome features

GenBank Accession Size ORFs Intron Coding Coding

2.2 Annotation of the assembled DNA sequences

An essential component of any sequencing effort is the conversion of the DNA

2.3 Protein coding genes

Table 2. Features of the A. gossypii genome annotation implemented as spreadsheet.

Footnote to Table 2 overleaf.

2.4 Sequence conservation of proteins

2.5 Species-specific proteins

2.6 RNA-encoding genes

We have identified a total of 194 tRNA-encoding genes in the A. gossypii nuclear

2.7 Transposable elements

2.8 Centromeres and telomeres

Centromeres were located based on homology and synteny to S. cerevisiae. The

3 Evolutionary implications of the A. gossypii genome

3.1 Possible origins of duplicated gene segments in S. cerevisiae

3.2 Proof for an ancient whole-genome duplication in S. cerevisiae

3.3 NOHBY’s and non-syntenic homologues in clusters of ancient

3.4 Update of duplicate gene blocks in S. cerevisiae

The almost complete coverage of the A. gossypii genome in clusters of ancient

3.5 Loss of S. cerevisiae genes after the genome duplication

3.6 Synteny breaks as marker of genome rearrangements

The large extent of synteny between A. gossypii and S. cerevisiae provided a

We determined the numbers of double and single synteny breaks on a whole

4 Gene pairs (twins) originating from the genome

4.1 Identification of twin ORFs

4.2 Genetic complexity caused by twin genes

tually essential, confirming a very high degree of functional redundancy of the

4.3 Sequence divergence of twin genes

It is generally believed that the presence of duplicates allows for diversification

5 Evolution of chromosome number in A. gossypii

centromeric regions from S. cerevisiae to the A. gossypii genome. The seven A.

6 Material and methods

6.1 Sequence and assembly quality

6.3 Data analysis

6.4 Creation of the map of Duplicate Blocks

We thank A Lerch, K Gates, A Flavier, S Choi, R Wing, S Steiner, C Mohr, R

Biotechnology Inc. (formerly Novartis Agribusiness Biotechnology Research Inc).

Alberti-Segui C, Dietrich F, Altmann-Johl R, Hoepfner D, Philippsen P (2001) Cytoplas-

Brachat S, Dietrich FS, Voegeli S, Zhang Z, Stuart L, Lerch A, Gates K, Gaffney T,

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