Comparative genomics of the fungal kingdom

Comparative genomics of the fungal kingdom: a view from the
chytrids

Jason Stajich
University of California, Berkeley

Comparative Genomics

• Tools for studying evolution at level of genomic blueprints.

• Identifying shared, unique, loss and gains of genes.

• Signatures of adaptation

• Identify genes that are under positive directional selection - changing faster at the
amino acid level than expected given neutral rate

• Identiﬁcation of gene families that expand or contract by unexpected amounts

• Contrasting genome organization and evolution of genomic clusters of genes

Fantastic Fungi

• Evolution of modern fungal forms and lifestyles

• Evolution of Multicellularity - independent transitions in Metazoa and Plants.

• Reversions to unicellularity

• Evolution of development; early genes involved in fruiting body development

• Plants and Fungi have cell walls; animals lack cell walls; what were fungal ancestor’s
cell walls like? Fungal-animal ancestor?

• What genes were in the ancestral fungus? Which genes have newly evolved and are
contribute to new morphologies or life stages?

Fantastic opportunities in fungal comparative genomics

• More than 65 available genomes - dozens more in pipeline at sequencing centers
• http://fungalgenomes.org/wiki/Fungal_Genome_Links
• 1(2) Chytrid, 2 Zygomycetes, 8 (12) Basidiomycetes, 3-4 Taphrinomycotina,
• ~30 (+15 strains Coccidoidioides, 3 strains of Histoplasma) Pezizomycotina
• ~22(+20-100 strains S. cerevisiae & S. paradoxus) Saccharomycotina

• Broad Institute & Fungal Genome Initiative, Joint Genome Institute, Stanford Genome
Technology Center, Sanger Centre, Génolevures project & CNRS, BC Genome
Sequencing Center, others.

• US genome sequencing funding: NSF, DOE, NIH

Genome annotation
• Train ab initio gene predictors

• Build good models from protein to genome alignments of take set of curated genes.
Build full-length models from cDNA or assembled ESTs

• Trains on exon-intron, intron length, exon length, and codon/nt biases

• Reﬁne parameters using iterative manner with some gene models held out to assess
improvements

• Generate and combine Annotations

• Take ab initio, homology based, and EST tracks

• Combine into consensus gene models

• GLEAN or Jigsaw (GAZE also)

• Assess performance of different datasets, leave out some models if necessary

Combined predictions
perform better
scaffold_5
1219k 1220k 1221k
% gc
58%

17%
GLEAN
BDEN_JAM81_00470 BDEN_JAM81_00471
probability 0.765437 probability 0.981985
SNAP genes
lenx_scaffold_5-snap.460 lenx_scaffold_5-snap.461

Twinscan genes
TS.scaffold_5.413

Genewise genes
dhan_DEHA0E17479g__scaffold_5__1216332__1226931
egos_AGR101C__scaffold_5__1216332__1226940
klac_KLLA0F11957g__scaffold_5__1216332__1226931
ctro_CTRT_03542__scaffold_5__1216332
lelo_LELT_03523__scaffold_5__1216332

AUGUSTUS genes
scaffold_5-augustus-g372.t1

PASA EST genes
Model.asmbl_4025
Model.asmbl_4026

• Consensus tree of 42 fungal
genomes based on many
thousands of orthologous genes

• Not perfect, but automated
reconstruction can be powerful tool

• Conﬂicts in topology can identify
genes with interesting history

Fitzpatrick DA, Logue ME, Stajich JE, Butler G.
BMC Genomics 2006

Complex fungal genes

• Modern fungi have complex gene structures. How complex were
gene structures in the fungal ancestor?

• Many introns are present in fungal genes

• Intron poor Saccharomyces, U.maydis, and S.pombe are derived

• Evolution of introns in fungi has seen many losses, few gains

Fungal intron size and frequency evolution

500
Hemiascomycota
C. glabrata
Median intron length (bp)

400

300
K. lactis
U. maydis
B.dendrobatidis
Y. lipolytica
200

Euascomycota
Basidiomycota
S.cerevisiae

Zygomycota
100
C. cinerea P. chrysosporium
R. oryzae
C. neoformans
S. pombe
0
0 1 2 3 4 5 6 7
Stajich JE, Dietrich FS, and Roy SW.
Mean number of introns per kb of coding sequence Genome Biology In revision

Podospora anserina (359)
Euascomycota
Chaetomium globosum (463)
Neurospora crassa (336)
Magnaporthe grisea (368)
Fusarium graminearum (372)
Aspergillus fumigatus (481)
Aspergillus terreus (474)
Aspergillus nidulans (469)
Stagonospora nodorum (403)
Hemiascomycota Ashbya gossypii (7)
Kluyveromyces lactis (6)
Saccharomyces cerevisiae (7)
Dikarya
Candida glabrata (6)
Debaryomyces hansenii (5)
Yarrowia lipolytica (30)
Schizosaccharomyces pombe (214)
Basidiomycota
Coprinopsis cinerea (1621)
Opisthokont
Phanerochaete chrysosporium (1615)
Cryptococcus neoformans (1578)
Ustilago maydis (86)
Zygomycota
Rhizopus oryzae (947)
Vertebrates
Homo sapiens (2737)
Mus musculus (2656)
Takifugu rubripes (2685)
Plants Stajich JE, Dietrich FS, and Roy SW.
Arabidopsis thaliana (2290)
Genome Biology In revision
0.1

Intron loss predominates in fungal lineages

Saccharomycetes
P. chrysosporium

Sordariomycetes

Eurotiomycetes
C. neoformans
A

S. nodorum
Vertebrates

Y. lipolytica
A. thaliana

C. cinerea
U. maydis

S. pombe
R. oryzae
5.51 6.62 2.28 0.21 3.80 3.89 3.90 0.52 0.88 1.16 0.97 0.07 0.02

4.03
1.20
0.07

3.59
2.36

2.77
3.59

3.59

3.87

4.98

Stajich JE, Dietrich FS, and Roy SW.
Genome Biology In revision

Intron loss in C. neoformans through mRNA intermediete

C
A
C. gattii, strain WM276

JEC21

BT-100

BT-157
WM276

BT-63
R265

H99

35-23
2462
C. gattii, strain R265
C. neoformans var. neoformans, strain JEC21

C. neoformans var. grubii, strain H99
1.0

B
1kb 5 kb
2 kb 3 kb 4 kb 6 kb

1 2 3 4 5 6 78 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 78 9-19 20 21 22

Stajich JE, Dietrich FS. Euk Cell 2006

Intron gain is rare

• Two studies looked at intron loss and gain in 4 closely related C. neoformans (Sharpton et
al, submitted; Stajich and Dietrich 2006) and found little or no intron gain.

• Nielsen et al, Plos Biology 2004 found moderate amount of intron gain among
Pezizomycota

• Intron gain IS happening in lineages but among sampled closely related genomes there are
few examples of intron gains...

• ... and little convincing evidence of the molecular mechanism of this gain (duplication, self-
splicing, de-novo intron creation)

• More work needed to understand dynamics and mechanisms of gene structure change

B. dendrobatidis genomics

• Amphibian pathogen killing frogs
worldwide

• Chytrid fungus with motile zoospore
and zoosporangia stage

• Genome sequencing of 2 strains

• JEL423 (Joyce Longcore; Panama) motile
and JAM81 (Jess Morgan; Sierras, zoospore
California)

• 24 Mb genome; ~8,000 genes

• Tiling genomic microarray and exon
array in development (Eisen lab)

zoosporangia

B. dendrobatidis genomics

• Amphibian pathogen killing frogs
worldwide

• Chytrid fungus with motile zoospore
and zoosporangia stage

• Genome sequencing of 2 strains

• JEL423 (Joyce Longcore; Panama) motile
C. neoformans ~7,000
and JAM81 (Jess Morgan; Sierras, zoospore
C. cinereus ~10,000
California)
U. maydis ~7,000
S. cerevisiae ~6,000
• 24 Mb genome; ~8,000 genes
A. fumigatus ~10,000
• Tiling genomic microarray and exon
array in development (Eisen lab)

zoosporangia

Gene structure evolution:
B.dendrobatidis genes are intron rich
B.dendrobatidis
BDEN_JAM81_01417

U.maydis
UM03290.1

P.chrysosporium
GLEAN_01130

S.pombe
SPAC644.14c

N.crassa
NCU02741.1

S. cerevisiae
YER095W
Strand exchange protein, forms a helical filament with DNA that searches for homology; involved in the recombinational repair of double-strand
breaks in DNA during vegetative growth and meiosis; homolog of Dmc1p and bacterial RecA protein

Phylogenetic proﬁling

• Classify a genes as to which phylogenetic clades it shares homologs with.

• Can be simply a similarity search (BLAST) to representatives genomes.

• Summarize the number of shared genes by different patterns

• Using Chytrid genes to identify genes present in ancestor, shared with animal
outgroup.

• Find genes lost at different part of tree

• By comparing all genes in lineages back to Chytrid can identify potential gene gains

Phylogenetic proﬁle of B.dendrobatidis genes
Fungi
Basidiomycota

122 262

63 606 556 123
4685 3732
119 395
168

Zygomycota Ascomycota
Animal Plant
1550 (19.2%) Chytrid speciﬁc genes
8068 B. dendrobatidis genes

Phylogenetic proﬁle of B.dendrobatidis genes
Fungi
Basidiomycota

1.5% 3.3%

.7% 7.5% 6.9% 1.5%
58% 46%
1.5% 4.9%
2%

Zygomycota Ascomycota
Animal Plant
1550 (19.2%) Chytrid speciﬁc genes

Fungal cell wall

Latgé JP

Evolution of cell walls

• Fungal cell wall are made of

• Chitin, Beta-glucans, Mannin,
other sugars

• Animals lack cell walls

• Plants have rigid cell walls

• Can learn about opisthokont ancestor
from learning about the ancestral
fungus

Baldauf SL. Science 2003

Evolution of cell wall: 1,3 Beta-glucan synthesis
Genes C Z B A
✘ ✔ ✔ ✔
1,3-beta-D-glucan synthase (FKS1)
1,6 β-glucan 1,3 β-glucan

✘ ✘ ✘ ✔
Cell surface reg kinase (HKR1)

✘ ✘ ✔ ✔
Regulator (SMI1)

✘ ✔ ✔ ✔
1,3-beta-glucanase (EXG1)

✔ ✔ ✔ ✔
Glucosidase (GTB1)

✘ ✘ ✘ ✔
1,6-beta-glucan biosynthesis (KNH1)

✔ ✔ ✔ ✔
glucosyltransferase (KRE5)

✘ ✘ ✔ ✔
Glucosidase activity (KRE6)

✘ ✘ ✔ ✔
Glucosidase activity (SKN1)

✔ ✔ ✔ ✔
uridylyltransferase (UGP1)

Flagella in fungi

• Loss of ﬂagella was a one or a few
events

• Find shared genes in animal and
Chytrid genomes but missing fungi

• Many of these genes are even
shared with cillia & ﬂagellar genes
with Chlamydomonas.

• Microarray expression data
differences between zoospore and
sporangia

• Flagella Dynein 64x up regulated in
zoospores.

Hypothesis for new cell wall genes and transition to
terrestrial life

• Cell wall of ancestral fungus adapted for aquatic fungus which had ﬂagella.

• Loss of ﬂagella as part of adaptation to terrestrial life.

• Additional gene family duplication and specialization.

• Chitin synthase expansions

• FKS1 1,3-Beta-glucan pathway evolution

• Substrate for complex multicellular evolution and morphological elaboration.

Collaboration

• Erica Rosenblum, Michael Eisen, John Taylor; University of California, Berkeley

• Igor Grigoriev, Alan Kuo; DOE Joint Genome Institute

• Christina Cuomo, Antonis Rokas; Broad Institute of MIT and Harvard

• Tim James; Uppsala University

• http://fungal.genome.duke.edu - genome browser and annotations

• http://fungalgenomes.org

• Blog & Wiki for Genome data

• Coming soon: Genome Browser and comparative resources

Comparative genomics of the fungal kingdom

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Comparative genomics of the fungal kingdom