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Aspergillus DNA barcoding – progress so far

Aspergillus DNA barcoding – progress so far. János Varga, Jos Houbraken, Martin Meijer, Pedro W. Crous & Robert A. Samson. Problems with using cox1 or other mtDNA genes as barcodes in Aspergilli 1. Low intraspecific variation: not

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Aspergillus DNA barcoding – progress so far

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  1. AspergillusDNA barcoding – progress so far János Varga, Jos Houbraken, Martin Meijer, Pedro W. Crous & Robert A. Samson

  2. 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.) Centraalbureau voor Schimmelcultures

  3. g e n e c o b m t D N A t y p e 1 : s t a r t e x o n 1 e x o n 2 i n t r o n 1 m t D N A t y p e 4 : s t a r t 1 g e n e c o x e x o n B e x o n C i n t r o n B m t D N A t y p e 1 : i n t r o n B i n t r o n C e x o n B e x o n C e x o n D m t D N A t y p e 4 : Altered intron content in cob and cox1 genes of A. japonicus mtDNA types 1 & 4 Centraalbureau voor Schimmelcultures

  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 Aspergilluscox1 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 Centraalbureau voor Schimmelcultures

  5. Intron movement in the cox1 gene after protoplast fusion ofA. niger and A. tubingensis e : e x o n e 1 e 2 i 1 1 a : A . n i g e r i : i n t r o n 2 b : A . t u b i n g e n s i s e 1 e 2 e 3 i 3 e 4 i 1 i 2 i n v a s i o n o f i n t r o n s f r o m 2 b m t D N A ( e n d o n u c l e a s e c l e a v a g e ) e 1 e 2 i 1 h o m i n g o f 2 b m t D N A i n t r o n s t o 1 a D S B R e 1.1 i 3 i 1 i 2 e1.2 e2.1 e2.2 Centraalbureau voor Schimmelcultures

  6. 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 Centraalbureau voor Schimmelcultures

  7. Trees of section Fumigati based on mt cytb and -tubulin sequences Wang et al. JCM 38: 1352, 2000 Wang et al. JCM 38: 1352, 2000 Centraalbureau voor Schimmelcultures

  8. 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 Centraalbureau voor Schimmelcultures

  9. Case study: the Aspergillus niger species complex (Al-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 Centraalbureau voor Schimmelcultures

  10. A. ellipticus A. brasiliensis A. vadensis NJ tree based on -tubulinsequences A. tubingensis 8/8 species can be distinguished A. piperis A. foetidus A. costaricaensis A. lacticoffeatus A. niger 0.1

  11. A. ellipticus A. brasiliensis NJ tree based on calmodulinsequences A. niger A. lacticoffeatus A. vadensis 8/8 species can be distinguished A. costaricaensis A. piperis A. foetidus A. tubingensis 0.1

  12. A. ellipticus A. brasiliensis A. lacticoffeatus NJ tree based on IGSsequences A. niger 8/8 species can be distinguished (high intraspecific variability) A. vadensis A. piperis A. tubingensis A. costaricaensis A. foetidus 0.1

  13. A. ellipticus A. brasiliensis A. niger, A. lacticoffeatus NJ tree based on ITSsequences 4/8 species can be distinguished A. tubingensis, A. foetidus, A. piperis, A. vadensis A. costaricaensis 0.01

  14. Several other gene sequences are able to identify at least 3-4 species of the A. niger species complex • 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)

  15. A. ellipticus A. niger A. brasiliensis A. niger A. vadensis A. niger A. niger A. tubingensis A. lacticoffeatus A. tubingensis A. costaricaensis A. tubingensis A. niger NJ tree based on cox1 sequences A. niger A. niger, A. tubingensis 2(?)/8 species can be distinguished; inter- and intraspecific variability overlaps A. foetidus A. tubingensis A. foetidus A. niger A. niger A. niger A. niger, A. tubingensis, A.brasiliensis 0.01

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