Transcript Alternative areas to consider

New and old regions for
Barcoding
Teun Boekhout
CBS
Problems with Mitochondrial Genomes
• Taphrinomycotina versus Saccharomycotina
versus Pezizomycotina
• Cryptococcus gattii
Three published scenario’s on
fission yeasts
(1)
(2)
(3)
Saccharomycotina
Pezizomycotina
Saccharomycotina
Pezizomycotina
Taphrinamycotina
Taphrinamycotina
Taphrinamycotina
Saccharomycotina
Pezizomycotina
Most nuclear
genes
No support
Mitochondrial support
Cryptococcus gattii
• 4 monophyletic lineages (phylogenetic species)
• 6 nuclear concordant nuclear gene phylogenies
• Mitochondrial versus nuclear genes incongruent
• [ATP 6 & MtLrRNA] vs [CNLAC, TEF1α, RPB1, RPB2,
ITS, IGS1]
• Mitochondrial loci incongruent
• Mitochondrial recombination
Nuclear AFLP/MLST 4 with varying mitochondria
CBS6998
CBS1622/CBS6992
100
M3
ATP6
haplotype 3
MtLrRNA haplotype 2
M1
ATP6
haplotype 4
MtLrRNA haplotype 2
CBS6290
63
60
48A/56A
64
CBS8273/RV20186T
74
Nucleus
CBS6289
93
Mitochondrion
WM830
ATP6
MtLrRNA
WM276
60
WM176
99
M2
ATP6
haplotype 1
MtLrRNA haplotype 2
M5
ATP6
haplotype 2
MtLrRNA haplotype 1
M4
ATP6
haplotype 2
MtLrRNA haplotype 4
5032738
CBS7229T/RV54130
60
E566
WM179R
CBS919T
64
CBS7748
CBS883T
10
blue = AFLP4
green = AFLP6
purple = AFLP4 like
yellow = AFLP6 like
red = chimera AFLP4/AFLP6
100
a
ATP6
WM714
WM714
H99
Cryptococcus neoformans
125.91
CBS6886
JEC20
RAM2
ICB184
A1MR265
A1MR406
A1M368
CBS1930
CBS6956
AFLP6
CBS8684
WM178
VGII
A1MR409
MLST
6
95 HEC11102
A1MF2866
A1MF2932
A1MR269
A1MR271
CBS7750
WM179 (M5)
(M5)
E566
CBS883 (M4)
84
CBS7748 (M4)
CBS919 (M4)
65 CBS1622 (M3)
AFLP4
CBS6992 (M3)
CBS7229 (M2)
VGI
RV54130 (M2)
MLST4
WM176 (M2)
(M2)
5032738
75
WM276 (M2)
WM830 (M2)
B5748
AFLP7
97 B5742
VGIV
M27055
WM779
MLST7
CBS6290 (M1)
(M1)
56A
93
AFLP4
74 CBS8273 (M1)
(M1)
48A
VGI
CBS6289 (M1)
CBS6998 (M1)
MLST4
RV20186 (M1)
CBS6993
WM161
WM728
AFLP5
CBS5758
94
CBS8755
VGIII
WM726
380C
MLST5
CBS6955
82 CN043
1
CBS6996
98 384C
H99
125.91
CBS6886
JEC20
ICB184
HEC11102
CBS1930
CBS6956
CBS7750
CBS8684
A1MR269
A1MR368
WM178
A1MR406
A1MR409
A1MF2932
A1MR265
A1MR271
A1MF2866
RAM2
(M5) WM179
(M5) E566
100
(M4) CBS919
63
(M4) CBS883
(M4) CBS7748
(M3) CBS1622
(M3) CBS6992
(M2) CBS7229
(M2) RV54130
(M2) WM176
(M2) 5032738
(M2) WM276
(M2) WM830
62
B5748
B5742 WM779
M27055
(M1) CBS6290
(M1)
56A
(M1) CBS8273
48A
(M1)
(M1) CBS6289
(M1) CBS6998
(M1) RV20186
CBS6996
380C
CBS6955
CBS6993
384C
WM161
CBS5758
WM726
CBS8755
CN043
WM728
81
0.1
MtLrRNA
b
The ‘old’ alternative
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rDNA universally present
ITS + D1/D2 LSU rDNA (Kurtzman > 20years)
Basidio + Asco yeasts
Yeast book chapters: both needed
Clinical yeasts (C. glabrata / C. nivariensis / C.
bracarensis)
Large datasets available
Proven useful for ID (Kurtzman, Fell, CBS, et al.)
Luminex technology (ID, M. Diaz, Miami)
Cryptococcus, Trichosporon, Candida, Malassezia
Alternatives 2
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‘Unique’ fungal pathways
E.g. Ergosterol pathway (membranes)
Concatenated ERG genes resolve TOL
ERG7 (lanosterol synthase) fungi/animals
ERG1 (squalene epoxidase) fungi/animals/plants
ERG11 (lanosterol C14 demethylase) fungi/animals
Alternatives 3
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Comparative Genomics
33 Fungal Genomes
4852 KOGs
70 single protein Kogs (paralogs!)
32 gene KOs not viable (S. cerevisae) > essential genes
Reference tree based on 531 KOGs
KOG2671 showed highest correlation (0.97) with 531
KOG tree
• Cophenetic correlation between individual protein NJ
trees > 0.5 > 64 KOGs remaining
• Development phylogenetic signal
64 concatenated proteins
Sac. cerevisiae RM11-1a
* > 80%
*
*
*
*
*
*
*
Sac. cerevisiae S288c
Sac. paradoxus
Sac. mikatae
Sac. kudriavzevii
Sac. bayanus
Sac. castellii
Can. glabrata
*
IA
Ash. gossypii
Kluyveromyces lactis
*
Sac. kluyveri
Can. guilliermondii
*
*
Debaromyces hansenii
*
Can. albicans
Can. lusitaniae
I
Yarrowia lipolytica
*
*
*
*
*
Neurospora crassa
IB1
Magnaporthe grisea
Fusarium graminearum
*
*
*
Chaetomium globosum
*
*
Sclerotinia sclerotiorum
IB
Botritys cinerea
Asp. fumigatus
IB3
Asp. nidulans
Coccidioides immitis
Stagonospora nodorum
*
IC
Schizosaccharomyces pombe
*
*
*
Coprinopsis cinereus
Phanerochaete chrysosporium
*
Cry. neoformans var. neoformans JEC21
II
IIA
III
IIB
IIIA
Cry. neoformans var. grubii H99
Ustilago maydis
Rhizopus oryzae
Caenorhabditis elegans
0.1
IB2
IB4
Development of phylogenetic signal
20
1.10
18
1.00
0.90
16
0.80
14
0.70
12
0.60
10
0.50
8
0.40
6
0.30
4
0.20
2
0.10
0
0.00
1
3
5
7
9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69
Information Storage and Processing
Cellular Processes and Signaling
Poorly characterized
Correlation value
Metabolism
Mainly Information storage and Processing & Cellular processes and signaling
Potential Barcoding candidates
Top 5 orthologues with high discriminatory potential
• KOG2671
• KOG0340
• KOG4089
• KOG0173
• KOG2728
• Etc. Etc.
Putative RNA methylase
ATP-dependent RNA helicase
Predicted mitochondrial ribosomal
protein L23
20S proteasome
Uncharacterized conserved protein
My suggestions
• There may be alternative genes
• D1/D2 + ITS 1+2!
• Panfungal / Paneukaryotic / Single copy / PCRable
• Need to be investigated further for Barcoding (and TOL) potential
• Option: Multilocus Barcoding ?
• Webtool: Different loci per (higher) taxon
• Selection depends on taxonomic resolution