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Genetic Analysis of Aspergillus Niger' Genetic analysis of Aspeirgil 7us m'ger~ CENTRALE LANDBOUWCATALOGU S 0000 0417 9137 Promotor: dr. R.F. Hoekstra hoogleraar in de populatie- en kwantitatieve genetica Co-promotor: dr. ir.C.J , Bos universitair hoofddocent ^ .vjoB'-o', !"i ;-v3 f. Föns Debets Genetic analysis of Asp&rgi 7 7us niger~ Proefschrift ter verkrijging van de graad van doctor in de landbouw- en milieuwetenschappen op gezag van de rector magnificus, dr. H.C van der Plas, inhe topenbaa r teverdedige n op dinsdag 4decembe r199 0 des namiddags tevie r uuri nd eaul a vand eLandbouwuniversitei t teWageningen . teùJvZé&lîk ^UO?W,:^ ,CS Stellingen 1. Aspergillus niger heeft acht chromosomen. Dit proefschrift 2. Hetgenoo m van Aspergillus niger issignifican t groterda nda t van Aspergillus nidulans. Dit proefschrift 3. Genetische analyse vanhaploid e segreganten vanheterozygot e diploiden van Aspergillus niger zoals door Lhoas voorgesteld, levert geen betrouwbare informatie omtrent deliggin g vane nd eonderling e afstanden tussen genetische markers. P. Lhoas (1967). Genet. Res. 10:45-61 Dit proefschrift 4. Debewerin g date rgee n coincidentie istusse n overkruisinge n haploidisatie in Aspergillus nidulans lijkt instrij dme t de waargenomen verhoogde frequentie vanmitotisch e overkruisingi n disomen. E. Käfer (1977). Adv.Genet . 19:33-131 5. Deverklarin g dieKund u &Da sgeve n voorhe tontstaa n vand edoo r hen geisoleerde recombinanten van Aspergillus niger isnie td emees t waarschijnlijke. P.N. Kundu andA .Da s(1985) . J.Appl . Bacteriol. 59:1-5 6. Deveronderstellin g vanAssinde r et al. dathe tcam Cge n van Aspergillus nidulans verva nhe tcentromee r ligto pd elinke rar m van chromosoom Ii snie t correct. S.J. Assinder, B.Gidding s andA .Upshal l (1986). Hol.Gen .Genet . 202:382-387. H. Arst (1988). Hol.Gen .Genet . 213:545-547 7. Hetgebrui k vaneenker nig e schimmelprotoplaste n intransformati e experimenten waarbij polyethyleenglycol wordt toegepast zal het ontstaan vanheterokaryotisch e transformanten niet verhinderen. J.R.S. Fincham (1989). Hicrobiol. Rev.53:148-17 0 8. Deconclusi e datdoo r mitotische overkruising in Aspergillus nidulans complementaire produkten ontstaan, isgebaseer d opd e foutieve veronderstelling datheteroal le le recombinatie niet verschilt vanrecombinati e tussen niet-allele mutaties. J.A. Roperan dR.H . Pritchard (1955). Nature 175:639 M. Bandiera, D.Armale oan d6 .Morpurg o (1973).Mol .Gen .Genet . 122:137-148 9. Voor eventuele regelgeving tenaanzie n vaninter-specie s recombinant DNA overdracht isme tnam e voor asexuele schimmels dehuidig e taxonomische indeling niet toereikend. 10. Hetbelan g vand eparasexuel e cyclus bijschimmel s voord e onderzoeker isduidelijk , defuncti e voord eschimme l zelf verdient nader onderzoek. 11. Eenstammenverzame lin g isvoo ree nschimme lgeneticu s alsee ncune t vooree nstratenmaker : heti see ndiepte-investerin g dienie t door iedereen opwaard e wordt geschat maar hetbepaal t wel het eindresultaat. 12. Naast deoproe p 'vrouwen gevraagd voor mannenwerk'ka noo k een stimulans aanmanne n omt ekieze n voor 'vrouwenwerk' bijdragenaa n een verbetering vand epositi e vanvrouwe n opd earbeidsmarkt . postbus5 1 13. Door de politieke omwentelingen in Oost-Europa dreigen grote omleggingen van geld- en afvalstromen te ontstaan ten nadele van de Derde wereldlanden. 14. Het gebruik van titulatuur bij het ondertekenen van ingezonden stukken in de krant aangaande 'common interest' zaken getuigt niet van veel vertrouwen in de aangevoerde argumenten. 15. Dopingjacht geeft records 'eeuwigheidswaarde'. B. de Graaf de Volkskrant 25 augustus 1990 Stellingen behorende b.ij het proefschrift van Fons Debets: 'Genetic analysis of Aspergillus niger'. Wageningen, 4 december 1990. Aan Marita en Lynn Aan moeder CONTENTS 1. Introduction 1 1.1 Aspergillus 1 1.2 Aspergillus niger 1 1.3 Genetic analysis based onmitoti c recombination 3 1.3.1 Parasexual mechanisms 3 1.3.2 Haploidization analysis 5 1.3.3 Mitotic crossing-over 7 1.3.3.1 Recombination models 7 1.3.3.2 Determining gene order 11 1.4 Aim and outline of this study 14 2. Mitotic mapping inlinkag e groupV o f Aspergillus niger based on selection of auxotrophic recombinantsb yNovozy m enrichment. 21 3. Genetic analysis of Aspergillus niger: isolationo f chlorate resistance mutants,thei r use inmitoti cmappin g and evidence for aneight h linkagegroup . 31 4. Genetic analysis of amdStransformant s of Aspergillus niger and their use inchromosom emapping . 39 5. Geneticmap s ofeigh t linkagegroup so f Aspergillus niger based onmitoti c mapping. 49 6. An electrophoretic karyotype of Aspergillus niger. 75 7. Summary and general discussion. 87 Samenvatting 97 Nawoord 101 Curriculum vitae 103 CHAPTER1 Introduction 1.1 Aspergillus Micheli (1729, cf Raper and Fennel 1965) introduced the name Aspergillus forth emould s with a characteristic pattern of conidiophores and sporeheads reminding him,a sa priest ,o fa mo pfo rdistributin g holy- water (Lat. Aspergillium). The fungi that belong to this form-genusar e worldwide indistributio n andar efoun d on almost any type of substrate (foods, textile, leather, decaying vegetation inth efield s etc.)(Rape r and Fennel 1965). Because of their metabolic versatility especially the black Aspergilli arewidel y used inindustr y forth eproductio n oforgani c acids such asgalli c acid, citric acid andgluconi c acid (Lockwood 1975), for the production of industrial enzymes such as amylase, glucoamylase, cellulase, hemicellulase, pectinase, glucose oxidase and catalase (Underkofler 1976)an di nfoo d fermentations practiced inth eorien t (Wood 1977). Some Aspergilli can also produce mycotoxins in food and someca n cause aspergillosis (Edwards andAl-Zubaid y 1977). Since DeBar y (1854,c f Raper and Fennel 1965) reported the direct relationship between the ascomycetous genus Eurotium and the Aspergillus glaucus group, the ascosporic states have been found forman y Aspergilli (Raper and Fennel 1965, Onions et al. 1981). They allar ecleistocarpi c andal lbu ton e (A. heterothallicus) arehomothalli c (Raperan dFenne l 1965). 1.2 Aspergillus niger The black Aspergilli areprobabl y more common than anyothe r group within the genus. Because of the characteristic pigmentation of the conidial heads, members of the group are commonly referred to as 1 'Aspergillus niger'. Indeed, within the Aspergillus niger group Van Tieghem's species A. niger isb y farth emos t abundant member, andth e degree of intragroup (morphological) variation is such that specific identifications often become quite difficult (Raper and Fennel 1965).Th e occurrence ofthi s easily recognizable andselectabl e fungus (onth ebasi s of specific growth on20 % tannin, Rippel 1939) isdocumente d from allpart s of theworld , buti ncontras t toman y other Aspergillus species notmor e than 50%o fth efinding s arewithi n thetropic s (Domsch et al. 1980).Th e characteristic pigment production in Aspergillus niger is profoundly influenced byth epresenc e orabsenc e ofminut e quantities ofcoppe r in the substratum and this phenomenon has been used to estimate copper concentrations insoi l (Mulder 1948). Copper dependent laccase activityha s been documented tob e involved inpigmentatio n notonl y in A. niger, but also in Aspergillus nidulans (Clutterbuck 1972). footcell Figure 1.1. Lifecycle ofA .niger . No sexual stage isknow n inth elif e cycle of A. niger. Though A. japonicus from the A. niger group was reported to have a perfect state (Saitoa japonica Rajendram and Muthappa, Onions et al. 1981) this observation hasno tbee n confirmed. Thegrowt h cycle of A. niger therefore consists ofth esequence : hyphae -- airborne conidiospores -- hyphae(Fig . 1.1). The vegetative mycelium consists of septate hyphae which are branching and partially submerged. The conidial apparatus develops from specialized, enlarged, thick-walled hyphal cells (the foot cells) as conidiophores with conidial heads. Conidia are produced by specialized conidiogenous cells called phialide s that coverth esurfac eo fth evesicle , the swollen endo fth econidiophore . In some species, the phialide sar e borne directly on thevesicl e (uniseriate), whereas in biseriate species the phialide s are borne on intermediate cells or metulae which are attached toth evesicle . A. niger shows mixed uni-an dbiseriat e typeso f arrangement sometimes even in the same conidial head (Raper and Fennel 1965). Conidial heads split during aging anda t maturity thedr yconidi a are easily spread byair . Theconidi a of A. niger VanTieghe m arethough t to be uninucleate (Yuill 1950), though also A. niger isolates with binucleate conidia have been reported (Baracho and Coelho 1978). In submerged cultures with agitation and temperature elevation A. niger may show microcycle sporulation, i.e.immediat e recapitulation of sporogenesis following spore germination (Smith 1977). 1.3 Genetic analysis based onmitoti c recombination 1.3.1 Parasexual recombination infung i Recombination without theinvolvemen t ofth efunga l sexual cyclewa s homokaryotic heterokaryon heterozygous strams Opioid non—recombinant diploid crossing-over recombnant diploid (homozygous for part of a chromosome) <2n) non-disjunction recombinant diploid (homozygous for complete cfromosomete)) recombinant anastomosis somatic haploid (plasmogamy) karyogamy Figure 1.2. Origins of new genotypes by parasexual recombination. For genetic analysis haploid segregants and diploid crossing-over recombinants are relevant (see Figures 1.3 and1.6) . first discovered byPontecorv o et al. (1953b) in A. nidulans
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