J. Genet., Vol. 75, Number 3, December 1996, pp. 281-286. 9 Indian Academyof Sciences Parasexual recombination in fungi A. JOHN CLUTTERBU CK Institute of Biomedicaland Life Sciences, Molecular Genetics Division, Pontecorvo Building, University of Glasgow, Glasgow G12 8QQ, Scotland, U K Abstract. Parasexual recombination is a valuable tool in the laboratory, particularly for asexual fungi, and a number of developments in methodologyare outlined. In biotechnology, the parasexual cyclehas proved less useful than at one time predicted,but it retains a function in analysis of the products of genetic manipulation, and as a convenientdetection system for environmental chemicals that may disturb mitosis. In nature, recent evidence suggests that parasexual recombinationis rare, in part at least because of the prevalence of heterokaryon incompatibility of many wild fungi. Keywords. Parasexual cycle;recombination; fungi. 1. Introduction As in meiosis, parasexual recombination may occur either by the segregation of whole chromosomes during haploidization, or by mitotic crossing over, the main difference fi'om the sexual (meiotic) process being that the steps of the parasexual cycle are independent of one another, and the frequency of occurrence of each is very much lower. Since its discovery in AspergiUus nidulans (Pontecorvo 1954) and A. niger (Pontecorvo et al. 1953), the parasexual cycle has been demonstrated to operate in at least eight other AspergilIus species (Clutterbuck 1992). The parasexual cycle in general has been reviewed by Roper (1966), and its operation in phytopathogenic fungi by Tinline and McNeil (1969). When Pontecorvo first described the parasexual cycle (Pontecorvo 1956) he immedi- ately recognized its importance in three fields: for mapping markers in both sexual and asexual fungi in the laboratory, for strain improvement in biotechnology, and for generation of biodiversity in the wild. I shall therefore review the current uses and understanding of parasexual recombination under these three headings. 2. Parasexual recombination in the laboratory The value of the parasexual cycle in the laboratory was evident froIT1 the start, even for a sexual species such as A. nidulans. Localization of a new mutation to a chromosome is achieved by observing the segregation of whole chromosomes during haploidization of a diploid, and master strains, bearing markers on all chromosomes, have been constructed for this purpose (McCully and Forbes 1965; Bos et al. 1988). Mitotic crossing over is less commonly used in routine analysis, since it requires prior construction of a strain with a distal selectable marker in coupling with the marker(s) to be mapped. Nevertheless, mitotic recombination has proved invaluable in A. nidulans for mapping centromeres and for confirmation of the order of markers on long chromo- somes. K~ifer (1977) has extended this analysis by employing translocation-bearing 281 282 A. John Clutterbuck strains. While translocations are generally thought of as introducing unwanted compli- cations, such as false linkages, to genetic analysis, their breakpoints may also be treated as valuable markers in otherwise barren chromosomal regions, and in some cases a translocation may reposition a chromosome segment so as to provide a selectable marker on a chromosome previously lacking one. Classical genetic analysis of asexual species relies entirely on the parasexual cycle, and though such a process gives a less detailed picture than meiotic recombination, effective chromosome maps can be built, e.g. for A. niger (Debets et al. 1993). This analysis has been based on long-established techniques, of which perhaps the most important are the use of auxotrophic markers for selection of heterokaryons and diploids, and complementing spore colour markers to simplify recognition of both diploids and their mitotic segregants. Some newer tricks have also been employed; these include the use of chlorate resistance to give selectable nitrate-assimilation mutants at a number ofloci (Debets et al. 1990a), the use of randomly integrated amdS transforming plasmids as genetic markers (Debets et aI. 1990b), and selection for auxotrophic mutants and mitotic segregants by means of the lytic enzyme Novozyme (Debets et al. 1989; Bos et al. 1992). Chlorate-resistant mutants are widely used as a source of selectable auxotrophs, e.g. for Cladosporium (Arnau and 0liver 1993; Arnau et al. 1994), while in Penicillium roqueforti, selective markers were obtained by transformation of parent strains with plasmids bearing different resistance markers (Durand et al. 1993). These authors also employed random amplified polymorphic DNA (RAPD) markers, which, along with restriction fragment length polymorphism (RFLP) markers, are naturally available in most wild populations. While they are suitable for monitoring recombination, DNA polymorphisms cannot be selected, and other markers must be used for forcing heterokaryons and selecting diploids. A. nidulans appears to be unusual in that there is relatively little natural variation for these DNA features, so segregants from an interspecies hybrid between A. niduIans and A. quadrilineatus have been used to identify A. nidula~s chromosomes carrying DNA markers peculiar to each species (Varga and Croft 1994). Another feature of parasexual analysis that has changed over the years is due to the introduction of haploidizing agents. In the early days of parasexual analysis (Pon- tecorvo and K~fer 1958) spontaneous haploids were isolated by simultaneous selection for two markers, either on different chromosomes or on different arms of the same chromosome. Combined selection for such markers as acriflavine resistance or for a spore colour marker, along with the suppressor of adenine, suAadE20, made it unlikely that the selected segregants were due to mitotic crossing over, which produces homozygosity for markers on only one chromosome arm at a time. The disadvantage of this procedure was the necessity for diploids to be heterozygous for suAadE20 and also for a second selectable marker, but homozygous for adE20. The first haploidizing agent regularly used was p-fluorophenylalanine (Lhoas 1961; McCully and Forbes 1965), which had the disadvantage of selecting against phenylalanine auxotrophs and has now been superseded by benlate (Hastie 1970; Upshall et al. 1977). Other agents tried were arsenate (van Arkel 1963), acridine yellow (D. Apirion and J. R. Kinghorn, unpublished), N-glycosyl polifungin (Baler al. 1975) and griseofulvin (Jha and Sinha 1991, 1992). It is difficult to be certain whether the classical parasexual cycle operates in all fungi. It is well known that diploids are unobtainable in Neurospora (Perkins and Barry 1977), Parasexual recombination 283 but in other species recombinants have been recovered from heterokaryons, without the isolation of a diploid. The usual explanation for this is that a transient diploid is formed, but is too unstable for isolation. In Trichoderma pseudokoningii a filtration- enrichment procedure was used to obtain recombinants and this would have actively selected against the diploid (Bagagli et al. 1995), while in Pseudocercosporella, an interspecific diploid was selected, but this was asporogenous and could not be characterized, although clear-cut recombinants for nutritional and isozyme markers were recovered as faster growing, apparently hapIoid sectors (Hocart and McNaughton 1994). In other investigations with Trichoderrna species (Stasz and Harmon 1990), failure to detect diploids has led to the suggestion that recombinants are generated by transfer of genetic information between nuclei in the heterokaryon. In support of this idea, we have evidence that an autonomously replicating plasmid can be transferred between nuclei in an Aspergillus heterokaryon with a fl'equency of approximately 4 x 10- 5 (Aleksenko and Clutterbuck 1995). 3. The parasexual cycle in applied biology Much was written in the 1970s about the possible applications of parasexuality to industrial fungi (e.g. Macdonald et al. 1972; Ball 1973; Holt et al. 1976). However, the extraordinary effectiveness of mutagenesis followed by selection for overproducers has meant, firstly, that recombination methods would have to be very good to improve existing strains, and secondly that production strains are so full of chromosome aberrations as to be impossible to analyse. More recently, molecular-genetic methods involving introduction into the fungus of genes manipulated for overproduction have been the focus of attention of industrial geneticists. Paras exual analysis may then develop a new role: to analyse and recombine the various products of gene manipulation, e.g. for heterologous chymosin production (Bodie et al. 1994). Diploids ofA. l~iger have also been investigated as possible industrial producers of citric acid (Sarangbin et al. 1994). An important feature of molecular genetics is introduction of manipulated DNA into a genome, and this normally requires vegetative recombination to integrate the DNA into a chromosome to enable it to replicate. Once integrated, such plasmids are usually stable in vegetative growth, and through subsequent parasexual cycles (Arnau and Oliver 1993). Recent analysis of transformants with an autonomously replicating plasrnid, which does not require this recombination for its replication, but can be used to detect it, have shown that the process of transformation itself appears to induce recombination (Aleksenko and Clutterbuck 1995). Autonomously replicating plasmids transform