aspergillus dna barcoding – progress so far jános varga, jos houbraken, martin meijer, pedro w....
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Altered intron content in cob and cox1 genes of A. japonicus mtDNA types 1 & 4TRANSCRIPT
AspergillusAspergillus DNA DNA barcoding – progress so barcoding – progress so
farfar
János Varga, Jos János Varga, Jos Houbraken, Martin Houbraken, Martin Meijer, Pedro W. Crous Meijer, Pedro W. Crous & Robert A. Samson& Robert A. Samson
Problems with using cox1 or other mtDNA genes as barcodes
in Aspergilli 1. Low intraspecific
variation: not Black Aspergilli
exhibit high levels of intraspecific variability not only in intron content, but also in exonic sequences (Hamari et al. 2003, Juhász et al. 2003, pers. comm.)
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mtDNA type 1:
mtDNA type 4:
intron BexonB exonC
intronB intron CexonB exonDexonC
cox1 gene
mtDNA type 1:start
genecob
mtDNA type 4:start
intron 1exon1 exon2
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Altered intron content in Altered intron content in cobcob and and cox1cox1 genes of genes of A. japonicusA. japonicus
mtDNA types 1 & 4mtDNA types 1 & 4
Problems with using cox1 or other mtDNA genes as barcodes
in Aspergilli 2. Lack of recombination: not
Inter- and intraspecific recombination detected in several cases among Aspergillus cox1 sequences even without selection pressure
Hamari et al. 2003 (A. japonicus) Juhász et al. 2003 (A. niger, A. tubingensis) Tóth et al. 1998 (A. niger) Juhász Á. pers. comm. (A. carbonarius) Varga & Croft 1995, etc. (A. nidulans, A.
quadrilineatus)Not only intron jumps, but exonic sequences
also undergo changes
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A. niger 1a:
A. tubingensis 2b:e2e1 i1 i2 e3 i3 e4
e1.2e1.1 i1 i2 e2.1 i3 e2.2
e: exoni : intron
e1 e2i1
e1 e2i1
invasion of introns from 2b mtDNA(endonuclease cleavage)
DSBR homing of 2b mtDNA introns to 1a
Intron movement in the cox1 gene after protoplast fusion of A. niger
and A. tubingensis
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Problems with using cox1 or other mtDNA genes as barcodes
in Aspergilli 3. Phylogenies based on nuclear and
mitochondrial gene sequences are frequently incongruent
Geiser et al. 1996: mt and nc rRNA genes
Wang et al. 2000: mt cytb genes
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Wang et al. JCM 38: 1352, 2000
Trees of section
Fumigati based on mt cytb and -
tubulin sequences
Wang et al. JCM 38: 1352, 2000
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Problems with using cox1 or other mtDNA genes as barcodes
in Aspergilli 4. Easily accessible: not
Cox1 sequences are available for 6 Aspergillus sp. (3 black Aspergilli, A. nidulans, A. fumigatus and A. oryzae)
Newly designed primers do not work for all Aspergilli (more to be designed and tested)
The cox1 gene of several Aspergillus species carry numerous introns which could make further work tedious
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Case study: theCase study: the Aspergillus Aspergillus nigerniger species complex species complex (Al-(Al-
Musallam, 1981; Samson et al. 2004)Musallam, 1981; Samson et al. 2004) Includes 8 species: A. niger A. tubingensis A. foetidus A. piperis A. brasiliensis A. vadensis A. costaricensis A. lacticoffeatus
ITS, -tubulin, calmodulin, IGS and cox1 sequences were determined for 60-70 isolates
A. niger
A. lacticoffeatus
A. brasiliensisA. ellipticus
A. tubingensis
A. foetidus
A. piperis
A. costaricaensis
A. vadensis
0.1
NJ tree NJ tree based based on on --
tubulintubulin sequensequen
cesces8/8 species 8/8 species
can be can be distinguisheddistinguished
A. niger
A. lacticoffeatus
A. brasiliensis
A. ellipticus
A. tubingensis
A. foetidusA. piperis
A. costaricaensisA. vadensis
0.1
NJ tree NJ tree based on based on calmodulicalmoduli
nn sequencesequence
ss 8/8 species 8/8 species can be can be
distinguisheddistinguished
A. niger
A. lacticoffeatus
A. brasiliensis
A. ellipticus
A. tubingensis
A. foetidus
A. piperis
A. costaricaensis
A. vadensis
0.1
NJ tree NJ tree based based on IGSon IGS sequensequen
cesces
8/8 species 8/8 species can be can be
distinguished distinguished (high (high
intraspecific intraspecific variability)variability)
A. niger, A. lacticoffeatus
A. brasiliensisA. ellipticus
A. tubingensis, A. foetidus, A. piperis, A. vadensis
A. costaricaensis
0.01
NJ tree NJ tree based based on ITSon ITS sequensequen
cesces4/8 species 4/8 species
can be can be distinguisheddistinguished
Several other gene sequences Several other gene sequences are able to identify at least 3-4 are able to identify at least 3-4 species of the species of the A. nigerA. niger species species
complexcomplex Pyruvate kinase, pectin lyase,
polygalacturonase, arabinoxylan-arabinofuranohydrolase, etc. (J. Visser)
Translation intitiation factor 2, pyruvate carboxylase, 70 kD heat shock protein, chaperonin complex component (TCP-1), ATPase (D. Geiser)
Translation elongation factor 1-α, RNA polymerase, actin (S. Peterson)
A. nigerA. niger
A. lacticoffeatus
A. brasiliensisA. brasiliensis
A. ellipticus
A. tubingensisA. tubingensis
A. foetidusA. foetidus
A. costaricaensis
A. vadensis
A. nigerA. nigerA. nigerA. niger
A. nigerA. niger
A. nigerA. niger
A. nigerA. niger
A. tubingensisA. tubingensis
A. tubingensisA. tubingensis
A. foetidusA. foetidusA. tubingensisA. tubingensis
A. nigerA. niger
A. nigerA. niger, A. tubingensisA. tubingensis, A.A. brasiliensisbrasiliensis
A. nigerA. niger
A. nigerA. niger
A. nigerA. niger, A. tubingensisA. tubingensis
0.01
NJ tree NJ tree based based
on on cox1 cox1 sequensequen
cesces2(?)/8 species 2(?)/8 species
can be can be distinguished; distinguished;
inter- and inter- and intraspecific intraspecific variability variability overlapsoverlaps