Comparative genomics of the fungal kingdom

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

Jason StajichUniversity 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

http://fungalgenomes.org/wiki/Fungal_Genome_Links

http://fungalgenomes.org/wiki/Fungal_Genome_Links

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

1219k 1220k 1221k

scaffold_5scaffold_5

% gc58%

17%

GLEANBDEN_JAM81_00470

probability 0.765437

BDEN_JAM81_00471


SNAP geneslenx_scaffold_5-snap.460 lenx_scaffold_5-snap.461

Twinscan genesTS.scaffold_5.413

Genewise genes

ctro_CTRT_03542__scaffold_5__1216332

dhan_DEHA0E17479g__scaffold_5__1216332__1226931

egos_AGR101C__scaffold_5__1216332__1226940

klac_KLLA0F11957g__scaffold_5__1216332__1226931

lelo_LELT_03523__scaffold_5__1216332

AUGUSTUS genesscaffold_5-augustus-g372.t1

PASA EST genesModel.asmbl_4025

Model.asmbl_4026


1219k 1220k 1221k


% gc58%

17%



BDEN_JAM81_00471




Genewise genes








Model.asmbl_4026


1219k 1220k 1221k


% gc58%

17%



BDEN_JAM81_00471




Genewise genes








Model.asmbl_4026


1219k 1220k 1221k


% gc58%

17%



BDEN_JAM81_00471




Genewise genes








Model.asmbl_4026


1219k 1220k 1221k


% gc58%

17%



BDEN_JAM81_00471




Genewise genes








Model.asmbl_4026

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

• 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

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

C. neoformans

P. chrysosporiumC. cinerea

R. oryzae

C. glabrata

S.c

ere

vis

iae

Y. lipolytica

K. lactis

EuascomycotaBasidiomycota

Hemiascomycota

U. maydis

S. pombe

Zygomycota

B.dendrobatidis

0 1 2 3 4 5 6 7

0

100

200

300

400

500

Media

n intr

on length

(bp)

Mean number of introns per kb of coding sequence

Fungal intron size and frequency evolution

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

Basidiomycota

Hemiascomycota

Euascomycota

Opisthokont

Vertebrates

Plants

Dikarya

Zygomycota

Podospora anserina (359)

Chaetomium globosum (463)

Neurospora crassa (336)

Magnaporthe grisea (368)

Fusarium graminearum (372)

Aspergillus fumigatus (481)

Aspergillus terreus (474)

Aspergillus nidulans (469)

Stagonospora nodorum (403)

Ashbya gossypii (7)

Kluyveromyces lactis (6)

Saccharomyces cerevisiae (7)

Candida glabrata (6)

Debaryomyces hansenii (5)

Yarrowia lipolytica (30)

Schizosaccharomyces pombe (214)

Coprinopsis cinerea (1621)

Phanerochaete chrysosporium (1615)

Cryptococcus neoformans (1578)

Ustilago maydis (86)

Rhizopus oryzae (947)

Homo sapiens (2737)

Mus musculus (2656)

Takifugu rubripes (2685)

Arabidopsis thaliana (2290)0.1


A.

thalia

na

R.

ory

zae

U.

mayd

is

C.

neo

form

an

s

C.

cin

ere

a

P. c

hry

so

sp

oriu

m

S.

po

mb

e

Sordariomycetes

Eurotiomycetes

Y.

lipo

lytica

Saccharomycetes

Vertebrates

5.51 6.62 2.28 0.21 3.80 3.89 3.90 0.52 0.88 1.16 0.07 0.02

3.59

3.59

4.03

0.07

2.36

2.77

3.59

3.87

4.98

A

P. a

nserin

a

N.

cra

ssa

C.

glo

bsu

m

0.861.110.81

0.90

0.95

M.

grisae

0.89

F. g

ram

inearu

m

0.90

0.85

0.89

A.

nid

ula

ns

A.

terr

eu

s

A.

fum

igatu

s

1.13 1.16 1.14

1.16

1.16

1.17

B Sordariomycetes EurotiomycetesS

. n

od

oru

m

0.97

S.

no

do

rum

0.97

1.20

1.20

Intron loss predominates in fungal lineages


Intron loss in C. neoformans through mRNA intermediete

0.1

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

JEC21

WM276

R265

H99

2462

35-23

BT-100

BT-63

BT-157C. gattii, strain WM276

C. gattii, strain R265

C. neoformans var. neoformans, strain JEC21

C. neoformans var. grubii, strain H99

A

B

C

1 2 3 4 5 6 7 8 9-19

1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 18 19 20 21 22

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) and JAM81 (Jess Morgan; Sierras, California)

• 24 Mb genome; ~8,000 genes

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

motilezoospore

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) and JAM81 (Jess Morgan; Sierras, California)

• 24 Mb genome; ~8,000 genes

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

motilezoospore

zoosporangia

C. neoformans ~7,000C. cinereus ~10,000U. maydis ~7,000S. cerevisiae ~6,000A. fumigatus ~10,000

BDEN_JAM81_01417

NCU02741.1

UM03290.1

SPAC644.14c

GLEAN_01130

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

Gene structure evolution: B.dendrobatidis genes are intron rich

B.dendrobatidis

U.maydis

P.chrysosporium

S.pombe

N.crassa

S. cerevisiae

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

Basidiomycota

AscomycotaZygomycota

1550 (19.2%) Chytrid specific genes

606

4685

168395

122

63

119

Fungi

PlantAnimal

262

3732

556 123

Phylogenetic profile of B.dendrobatidis genes

8068 B. dendrobatidis genes

Basidiomycota

AscomycotaZygomycota

1550 (19.2%) Chytrid specific genes

7.5%

58%

2%4.9%

1.5%

.7%

1.5%

Fungi

PlantAnimal

3.3%

46%

6.9% 1.5%

Phylogenetic profile of B.dendrobatidis 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 A1,3-beta-D-glucan synthase (FKS1) ✘ ✔ ✔ ✔

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) ✔ ✔ ✔ ✔

1,3 β-

gluc

an1,

6 β-

gluc

an

Evolution of cell wall: 1,3 Beta-glucan synthesis

Genes C Z B A1,3-beta-D-glucan synthase (FKS1) ✘ ✔ ✔ ✔

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) ✔ ✔ ✔ ✔

1,3 β-

gluc

an1,

6 β-

gluc

an

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

http://fungal.genome.duke.edu

http://fungal.genome.duke.edu

http://fungalgenomes.org

http://fungalgenomes.org

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Comparative genomics of the fungal kingdom