Fungal Genetics

JRS Fincham, University of Edinburgh, Edinburgh, UK

The fungal sexual cycle is, in its alternation of haploid and diploid phases, essentially the same as in higher plants and animals, with fusion of haploid gamete nuclei (karyogamy) to give diploidy, and meiosis to restore haploidy. Where most fungi differ from higher eukaryotes is that, meiosis follows immediately after karyogamy, so that the diploid phase is confined to the meiotic cell. In the most studied groups of fungi, Ascomycetes and Basidiomycetes, which are sexually reproducing, there are differences in their modes of formation of haploid spores.

Secondary article
Article Contents
. Relevant Features of Fungal Life Cycles . Heterokaryons . Breeding Systems . Gene Function and Structure . Formal Genetic Analysis Information from Tetrads . Parasexual Analysis in Artificial Diploids . Mitochondrial Inheritance . Cloning and Transformation . Analysis of Whole Genomes

Relevant Features of Fungal Life Cycles

Genetic investigation of fungi has been concentrated mainly on sexually reproducing species of the two major groups Ascomycetes and Basidiomycetes. The fungal sexual cycle is, in its alternation of haploid and diploid phases, essentially the same as in higher plants and animals, with fusion of haploid gamete nuclei (karyogamy) to give diploidy, and meiosis to restore haploidy. Where fungi dier from higher eukaryotes is that, with the exception of the budding yeasts and certain Phycomycetes (with which this article will not be concerned), meiosis follows immediately after karyogamy, so that the diploid phase is conned to the meiotic cell. The dening distinction between Ascomycetes and Basidiomycetes lies in the dierent modes of formation of the haploid spores, the products of meiosis. Whereas in the former group the ascospores are contained within the ascus, formed from the mother cell wall, the basidiospores of the latter are budded from the outside of the mother cell, the basidium. Meiosis gives a tetrad of haploid products. Each basidium bears four basidiospores, and in the ascomycete yeasts each ascus contains four ascospores. But in most Ascomycetes other than yeasts there is a single mitotic nuclear division after meiosis and before spore formation, so the ascus contains eight ascospores, a tetrad of spore pairs. The great majority of fungi are lamentous, growing as branching laments (hyphae), collectively called mycelium, divided by septa into compartments. In Ascomycetes, hyphal compartments usually contain multiple nuclei, a mode of organization more correctly referred to as coenocytic rather than cellular. The yeasts, in contrast, propagate as single uninucleate cells, either by budding or by binary ssion. The well known yeasts are Ascomycetes, but among the Basidiomycetes the smut fungi grow as budding yeasts, in their unmated nonpathogenic haploid phase. A feature possessed by many lamentous Ascomycetes and their asexual relatives is their ability to form abundant asexual spores (conidia) on their mycelium; these are important both for their natural dispersal and for their experimental manipulation. In the lamentous Ascomycetes and Basidiomycetes, there is an interval between sexual fertilization and karyogamy, during which the associated haploid nuclei, still unfused, divide in synchrony in binucleate cells (dikaryons). In the mushroom-like Basidiomycetes (Agaricales) the dikaryon is the main growth phase of the life cycle, proliferating in the soil or other substrate, and, under favourable conditions, emerging to form fruit bodies which bear the basidia, within which karyogamy nally occurs. In the smut fungi (Ustilaginales), it is dikaryotic hyphae that parasitize the host plant. In the lamentous Ascomycetes, the dikaryotic phase is much more abbreviated, being conned to the ascogenous hyphae, a system of short branching laments, stemming generally from a single initial association of nuclei and giving rise to a hundred or so diploid asci in each haploid fruit body, the form of which varies between dierent ascomycete subgroups. The ascomycete yeasts have no dikaryotic phase; karyogamy occurs immediately after sexual cell fusion. In ssion yeast (Schizosaccharomyces pombe) meiosis normally occurs at once, but in budding yeast (Saccharomyces spp.) there is a budding diploid phase of indenite duration, with meiosis and ascospore formation induced only under starvation conditions. The key features of the life cycles of some experimentally important fungal species are summarized in Table 1.

Heterokaryons
Although Ascomycetes and their asexual relatives do not have a free-growing dikaryotic phase, they are in general able to form heterokaryons, that is mycelium with more than one kind of nucleus propagating together in a common cytoplasm. Fusions of hyphae can occur freely, provided that the confronted strains are heterokaryon gueret et al., 1994) can be compatible. Incompatibility (Be brought about either by dierences at the same genetic locus (there are 11 dierent incompatibility loci in
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Table 1 Fungi most used in genetics, and their main characteristics Ascomycetes, yeasts Saccharomyces cerevisiae. Common budding yeast. One mating type locus, two idiomorphs (a/a), with switching. Ascospores in tetrads. Buds either as diploid or haploid; meiosis induced in diploid only under starvation conditions. Schizosaccharomyces pombe. Fission yeast. One mating type locus, two idiomorphs (mat1, mat2), with switching. Ascospores in tetrads. Meiosis follows immediately after karyogamy, so dividing cells are haploid. Ascomycetes, lamentous The ascus is normally the only diploid cell in the life cycle, but a dikaryotic phase (ascogenous hyphae) intervenes between fertilization and karyogamy. All heterothallic species have one mating-type locus, two idiomorphs. Aspergillus nidulans. Green mould. Homothallic. Unordered eight-spored asci. Diploid mycelium can be articially selected. Abundant uninucleate green conidia. Neurospora crassa. Heterothallic. Red bread mould. Ordered eight-spored asci. Ascospore germination activated by heat. Abundant conidia, mostly multinucleate. Sordaria brevicollis. Heterothallic. Ordered eight-spored asci. Ascospore colour mutants available. No conidia. Sordaria micola. As S. brevicollis but homothallic. Podospora anserina. Pseudohomothallic. Four-spored asci with ascospores each containing nuclei of both mating types. Ascospore colour mutants available. No conidia. Ascobolus stercorarius. Cup fungus. Heterothallic. Ascospores in unordered eight-spored asci. Many ascospore colour mutants available. No conidia. Magnaporthe grisea. Rice blast fungus. Rice pathogen. Heterothallic. Unordered eight-spored asci. Abundant conidia. Basidiomycetes Haploid except for the basidium, but with an important dikaryotic phase. Ustilago maydis. Maize smut. Heterothallic with two mating-type loci, one (b) with multiple alternatives (haplotypes or idiomorphs) and the other (a) with only two. Basidiospores in tetrads. Haploid cells prevented from mating grow as saprophytic budding yeast. When mated they give parasitic dikaryotic, still haploid, mycelium, forming abundant black dikaryotic brandspores, which germinate to form basidia, with karyogamy and meiosis. Coprinus cinereus, Schizophyllum commune. Mushroom-like, respectively ink-cap and bracket fruit bodies. Heterothallic with two mating-type loci (A and B), both in multiple alternative versions. Basidiospores in tetrads, germinating to give haploid mycelium, which, on mating, gives the vigorous dikaryotic mycelium that proliferates and forms the fruit bodies.

Neurospora crassa, including the mating-type locus) or by certain combinations of alleles of dierent genes heterogenic incompatibility, best investigated in Podospora anserina. Since there are generally numerous nuclei within each hyphal compartment, ratios of dierent nuclear types within a heterokaryon can vary over a wide range. Heterokaryons can be resolved into their components (homokaryons) by excision of hyphal tips or, more conveniently, by isolation of colonies grown from single conidia where these are available. In Aspergillus spp., which have strictly uninucleate conidia, each such colony will be homokaryotic. In Neurospora, where most conidia have more than one nucleus, screening of numerous colonies or serial reisolation is necessary. We return to the experimental uses of heterokaryons below.

Breeding Systems
The sexually reproducing fungi do not, as a rule, have separated sexes in so far as there are distinct male and female structures, as there often are in Ascomycetes, they
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are borne on the same hermaphroditic mycelium. Selffertilization is, however, very commonly avoided through a system of mating types, whereby only dierent and compatible strains can mate. Species with distinct mating types are called heterothallic and those capable of selffertilization homothallic. Nearly all Basidiomycetes and many, probably most, Ascomycetes are heterothallic (see Table 1). Dierent strains of homothallic species will still form hybrid fruits in mixed culture, but these have to be separated from the selfed fruits for genetic analysis the technique for doing this in Aspergillus nidulans is a wellpractised routine. Most lamentous Ascomycetes have eight-spored asci (the consequence of meiosis plus a postmeiotic mitosis), but some, though closely related to eight-spored heterothallic species, have four-spored asci, with each ascospore yielding a self-fertile culture. Examples are Neurospora tetrasperma and Podospora anserina. These species are basically heterothallic, but contrive to include within each of their four spores two nuclei of dierent mating type, so that each sporeling is eectively internally mated from the start. These systems of pseudohomothallism presumably became established in evolution because the short-term

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advantage of assured ascospore production (the ascospore is the most durable stage in the life cycle) may outweigh the long-term advantages of outbreeding. The same apparent reversion to inbreeding is achieved in the yeasts, both Saccharomyces and Sch. pombe, by systems of mating-type switching, whereby each of the two mating types constantly converts to the other (Schmidt and Gutz, 1994). Nonswitching stocks are used in most genetic studies. The genetic basis of mating type varies between fungal groups and, with analysis of genes at the DNA level, turns out to be unexpectedly complex (Figure 1). Heterothallic Ascomycetes have two mating types, called a/a in Saccharomyces and a/A in Neurospora, determined by what were traditionally termed alleles at a single chromosome locus. Molecular analysis, rst in Saccharomyces and

Neurospora and later in Podospora anserina and several other species (Glass and Nelson, 1994), showed that they were not alleles in the sense of dierent forms of the same gene but quite dierent genes, or nests of genes, that were alternative occupants of the same locus. The term idiotype instead of allele has been proposed for this situation. In the yeast the bizarre system of mating-type switching depends on the permanent presence of unexpressed copies of both idiomorphs at reserve (cassette) loci which can transfer their sequence information to the active mating-type locus through a kind of gene conversion mechanism (Schmidt and Gutz, 1994). In the Basidiomycetes, the most detailed studies of mating type have been made on the agarics Coprinus cinereus, the ink-cap, and Schizophyllum commune, a

a2 a (a) A3 A a1 (b) a W1 W2 mfa1 a1 pra2 (c) a2 a1 A (d) B a2 A2 2

a1 1 1 kb

A1 1 kb

E1 E2 pra1 mfa2 b1 b2 d1 d2 1 kb 1 kb

Figure 1 Some structures of mating-type loci. (a) Saccharomyces cerevisiae. Genes a1 and a1 are necessary for haploid mating functions. The protein products of a1 and a2 form a dimer that represses haploid functions; a2 has no known function. The differently coloured a and a sequences are unrelated and different in length, though at the same chromosome locus. Flanking the expressed mating-type locus there are two (cassette) loci (not shown), with silent copies of a and a respectively, that donate information in the a K a switching process. (b) Neurospora crassa. The two mating types, A and a, are determined by two unrelated alternative segments at the same locus (idiomorphs, A containing three genes and a one). The protein products of genes A1 and a1 cooperate to bring about mating. A1 is also a heterokaryon-incompatibility gene. A2 and A3 provide postfertilization functions in fruit body development. Podospora anserina is very similar. Information from Glass and Nelson (1994). (c) Ustilago maydis. There are two mating-type loci, a and b. The a locus has two alternatives, a1 and a2, responsible for mutual recognition: pra1 encodes a receptor for mfa2-encoded pheromone, and pra2 a receptor for mfa1-encoded pheromone. The b locus has numerous alternative forms, two shown here, each with divergently transcribed genes of the E and W classes. Dikaryon development following cell fusion requires dimerization of E and W protein products, but only E W combinations from different mating types are compatible: e.g. E1 W2 or E2 W1 but not E1 W1 or E2 W2. Information from Ka mper et al. (1994). (d) Coprinus cinereus. Two loci, A and B, each present in the population in many different forms. The A locus (an idealized general representative shown here) contains paired and divergently transcribed HD1 (a1, b1, c1) and HD2 (a2, b2, c2) genes, analogous to the E and W genes of Ustilago, but in multiple copies with substantial sequence differences both within and between mating types. Stable dikaryon formation requires dimerization of an HD1 with an HD2 product, but HD1 and HD2 from the same mating type are mutually incompatible. The relatively widely separate a and b subloci show about 0.1% recombination. The B locus, analogous to Ustilago a, includes pheromones and pheromone receptor genes (respectively green and blue), but in multiple copies. Mutual recognition does not occur between pheromones and receptors encoded in the same mating type. Information from Casselton and Ku es (1994) and OShea et al. (1998).

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bracket fungus (Casselton and Ku es, 1994); and on Ustilago maydis, the maize smut (Ka mper et al., 1994). In each of these species there are two mating-type loci, even more complex than in the Ascomycetes but in quite a dierent way. In the agarics there are multiple alternatives at both loci and, for sexual compatibility, two strains must be dierent at both. The eect of having multiple mating types rather than just two is to make any isolate compatible with the great majority of the rest of the species instead of only half of it. In Ustilago it is only the b locus that has multiple forms; the a locus has only two. The recently elucidated complex structures of matingtype loci of several fungi are illustrated in Figure 1, with a brief outline of what is known about their functions.

Gene Function and Structure

The one geneone enzyme hypothesis, which was arguably the birth of molecular biology, arose from work in the 1940s on auxotrophic mutants of Neurospora crassa. These are mutants, usually induced by mutagenic treatments, that fail to grow on the minimal growth medium that suces for the wild type (containing just sugar, salts and single vitamin D-biotin). For growth they require nutritional supplements, usually a single amino acid, purine or pyrimidine base, or vitamin. Further analysis, involving tests of postulated precursors of the required substance, or the detection of accumulated intermediates in the blocked biosynthetic pathway, and nally direct assay for biosynthetic enzymes, led to the generalization that each auxotroph was decient in a single metabolic step, due to loss of a single enzyme. These studies, as well as clarifying the functions of genes, made a major contribution to the elucidation of biosynthetic pathways. The initial sorting of auxotrophic mutants into dierently defective groups was facilitated by the formation of heterokaryons between pairs of mutants complementation tests. The heterokaryon grows like wild type on minimal medium if each mutant is able to supply the function missing in the other, but not if they share the same deciency. In this way groups of mutants blocked in dierent steps in the same biosynthetic pathway can be distinguished, even though they require the same endproduct. Crosses between mutants showed that members of the same complementation group gave very few wildtype recombinants (none, it was initially thought), indicating that their mutational sites were coincident or at least very close together within the same gene. The one geneone enzyme concept was rened to one geneone polypeptide chain to cover the occasional enzymes composed of dissimilar subunits under the control of dierent genes. Essentially the same kind of analysis has been carried out on a large scale in other fungi, especially S. cerevisiae,
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where complementation tests are made by construction of diploids rather than heterokaryons. In Aspergillus nidulans, where both heterokaryons and diploids are possible, both kinds of complementation test can be made, generally with concordant results. An apparent anomaly, found fairly frequently, is some degree of complementation between mutants decient in the same enzyme protein. In such cases, the mutants can usually still be assigned to the same complementation group since each is usually noncomplementary with many or most other mutants in the group. This phenomenon, called allelic complementation (which would at one time have been regarded as a contradiction), can be accounted for in terms of enzyme structural and/or functional complexity, without compromising the genepolypeptide generalization (Fincham, 1977). Auxotrophic mutants have also been of vital importance in the demonstration that the gene is not indivisible, as was originally thought, but is rather a linear array of individually mutable and separable sites. If sucient meiotic spores are screened numbers in the range ten thousand to a million the great majority of crosses between mutants in the same functional gene yield frequencies of wild-type recombinants far in excess of spontaneous reverse-mutation rates. The wild-type recombinants automatically select themselves on minimal medium against a great preponderance of nongrowing mutants. A number of methods, which cannot be elaborated here (use of anking markers, deletion mapping, conversion polarity), can be used to place the mutational sites in a linear sequence. Auxotrophic mutants are conditional, in the sense that their no-growth phenotypes are seen only under certain nutritional conditions. They played a predominant role in the early days of molecular genetics, but now temperatureconditional mutants are equally or more important. Only a minority of losses of proteins can be compensated for by nutritional supplement, but all proteins can be inactivated by heat, and some by cold also, and all can be altered by mutation in such a way as to make them nonfunctional over a part of their normal temperature range. Temperature-conditional mutants, growing, for example, at 258C but not at 358C, have been used to identify genes with a great range of essential functions. For example, in both S. cerevisiae and Sch. pombe it has been possible to dissect the cell cycle into successive protein-mediated steps through temperature-sensitive mutants (Hartwell, 1978). The identication of essential yeast cell-cycle genes has led to the discovery of homologous genes in animals.

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Formal Genetic Analysis Information from Tetrads

The fact that the products of fungal meiosis are often recoverable as tetrads of spores (or spore pairs), all of which can be separately grown and scored, permits the direct verication of a basic tenet of the chromosome theory, namely that the Mendelian 1:1 segregation of alleles in heterozygotes results from a 2:2 ratio in each and every meiotic tetrad. The demonstration is particularly graphic in several species of Ascomycetes (particularly Sordaria species and Ascobolus stercorarius), in which many mutations aect ascospore colour, which is determined autonomously by the haploid nucleus of the spore itself. Thus a cross between a pale-spored mutant and the wild type shows a 4:4 segregation, 2:2 in terms of spore pairs, in all (or nearly all) asci. The same applies to all gene dierences and not just those aecting spore colour. Moreover, in species (e.g. Sordaria micola and Neurospora spp.) in which the second-division meiotic spindles are end-on to each other in a linear ascus (an ordered tetrad), the products of the two second divisions end up in dierent halves of the ascus, and so it is possible to see whether the segregation of unlike alleles occurred at the rst or the second division. The signicance of this relates to the distance of the gene locus from its chromosome centromere. Sister chromatids remain held together at the
First division

centromere until the second division of meiosis, and so any gene dierence close to a centromere will segregate mostly at the rst division. It is only if crossing-over, which involves two out of four chromatids, occurs between the gene locus and its centromere, that second-division segregation will occur (Figure 2). Thus the frequency of second-division segregation measures the frequency of crossing-over, and hence genetic map distance, in the gene centromere interval, and this permits the inclusion of the centromere along with the gene loci in the linkage map. Tetrad analysis requires a special terminology. Where two allelic dierences, say A/a and B/b, are segregating from the same cross AB ab, there are, if elementary genetic principles hold, only three kinds of unordered tetrad: parental ditypes (P), AB AB ab ab; nonparental ditypes (N) Ab Ab aB aB; and tetratypes (T), AB Ab aB ab. If genes A and B are eectively unlinked, on dierent chromosomes or far apart on the same chromosome, P and N asci will be statistically equal in frequency. P signicantly more frequent than N is evidence for linkage. Without linkage, the frequency of T asci will depend on the distances of A and B from their respective centromeres; if neither shows any second-division segregation, T asci cannot occur. If A and B are far apart on the same chromosome, the frequency of T will approach two-thirds. A nding of P % N and T signicantly less than two-thirds means that A and B are on dierent chromosomes, neither very distant

Second division y

y y y + + y + +

+ +

(a) y y y y + + + y + + (b)
Figure 2 Segregation at (a) the first or (b) second division of meiosis of a pale-ascospore (e.g. yellow, y) mutation depending on whether or not there is crossing-over between the gene locus and the centromere. On the right are shown first- and second-division patterns seen in the asci. Since the orientation of disjoining centromeres is random with respect to the top and base of the ascus, the alternative spore arrangements indicated by the arrows are all equally likely.

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+ + b b + + b b + + b b + + b b + + b b 1 +y +y b+ b+ 2 ++ +y b+ by

from its centromere. This type of analysis is only possible with tetrads, not with randomized meiotic products. With linked loci, T asci are most simply accounted for as single crossovers; N asci, generally rare, must be due to double crossovers, with the second exchange involving the two chromatids not involved in the rst (four-strand doubles) (Figure 3). With three linked marker loci, dening two intervals, four kinds of asci can be distinguished with a crossover in each interval: four-strand doubles, two-strand doubles in which the same two chromatids are involved each time, and two kinds of three-strand doubles, with one chromatid taking part on each occasion, two once each and one not at all. The four kinds are found to be statistically equal in frequency, indicating that the chromatid participation in each crossover is random. Unfortunately for simple theory, but very usefully for the elucidation of mechanisms of recombination, tetrad analysis shows that the basic 2:2 rule of meiotic segregation does not always hold. In Saccharomyces, the extreme case, most allelic dierences segregate 3:1 or 1:3 in several per cent of asci, though, since these exceptions are roughly equal in frequency, the overall 1:1 rule is preserved. Corresponding 6:2 and 2:6 ratios can be seen with ascospore colour dierences in Sordaria spp. and Ascobolus stercorarius, though in these species the frequencies are considerably lower generally of the order of 0.010.1%. To compound the subversion of basic genetic principles, the eight-spored species also show a similarly low frequency, varying from one mutant to another, of 5:3 and 3:5 ratios and an additional class, called aberrant 4:4s, where the overall ratio is the result of the mismatch of two spore pairs, which should each be identical twins. These observations show that segregation of allelic dierences can be delayed until the postmeiotic mitosis, a violation of the doctrine of purity of the meiotic products and a clear indication that postmeiotic chromosomes can be internally hybrid, presumably with mismatching base pairs in the DNA double helix. Postmeiotic segregation can also be shown, though at relatively lower frequency, in the fourspored asci of Saccharomyces by spreading the colonies grown from single ascospores and showing them to be sometimes 50:50 mixtures. In this species only 5:3 and 3:5 asci are found, not aberrant 4:4s. These non-Mendelian tetrad ratios demonstrate gene conversion, that is the unilateral transfer of genetic information (DNA sequence) from one chromatid to another during the rst prophase of meiosis. Postmeiotic segregation is evidence that only single DNA strands are transferred, and the 6:2 and 2:6 ratios are thought to be the consequence of correction of the mismatches so produced. Gene conversion causes recombination, often without crossing-over, but it is nevertheless correlated with crossing-over. There is generally a 3040% chance of a crossover occurring close to each conversion event possibly directly adjacent to it in at least a proportion of cases. This leads to the hypothesis, discussion of which
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y y + + y y + +

y y + + y y + + y y + + 3 ++ ++ by by + + b b y y + +

Figure 3 The explanations in terms of crossing-over (absent, single or double) of (1) parental ditype, (2) tetratype and (3) nonparental ditype tetrads from crosses involving two linked markers. The example shown is the cross between two distinguishable pale-ascospore mutants of Sordaria brevicollis, buff (b) and yellow (y); the double mutant by is white. Double crossovers are infrequent, so the great majority of recombinants occur in tetratype asci. No account is taken here of first- versus second-division segregation, and the order of the spore pairs shown in the asci is arbitrary.

cannot be attempted in this short article, that recombination between chromosomes always involves one-way transfers of DNA strands to form joint structures which can always result in conversion (if the sequence transferred includes a gene dierence) and leads to crossing-over in a proportion of cases (Fogel et al., 1978).

Parasexual Analysis in Artificial Diploids

Genetic recombination and reassortment can also occur during diploid mitosis, albeit at much lower frequency than at meiosis. Thus in Saccharomyces cerevisiae, which is habitually diploid, crossing-over at meiosis between homologous chromosomes, if it occurs between a site of heterozygosity and the centromere, leads to homozygous segregants with 50% frequency (Figure 4; Pontecorvo and Kafer, 1958). This is a rare event, but can be markedly stimulated by irradiation with X-rays or ultraviolet light. X-ray-stimulated mitotic recombination has been used to demonstrate the separability of sites of mutation within the same yeast gene. A heterozygote m1 +/+ m2 gives rise to ++ recombinants with a frequency dependent on X-ray dose. If the mutants m1 and m2 are auxotrophs, the wild-

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+ paba

+ y

1 2 3 4

+ + paba paba

+ + y y

1 2 3 4

+ + paba paba

+ + y y

2 and 4 to same pole paba paba paba y y y + paba paba y y y

Figure 4 Mitotic crossing-over in an Aspergillus nidulans diploid heterozygous for recessive mutations paba (p-aminobenzoic acid requirement) and y (yellow rather than wild-type green conidia), linked in the same chromosome arm, with y the more distant from the centromere. Yellow-conidial sectors of mycelium result from mitotic crossing-over between y and the centromere when the chromatids disjoin 1, 3/2, 4 (50% probability). A yellow sector is also paba-requiring when the crossover is closer to the centromere than paba, but not when it is between the two loci. This example from Pontecorvo and Ka fer (1958).

type recombinants can be easily selected on minimal medium. This provides a method for mapping of sites within genes since, with a given X-ray dose, the recombination frequency is proportional to distance between sites. Mitotic (parasexual) genetic analysis has been most important in Aspergillus nidulans. This species is habitually haploid, but diploids can be articially selected in either of two ways, both dependent on complementation between haploid genomes and the strictly uninucleate nature of the conidia. If one obtains a heterokaryon between two dierent auxotrophs, or multiple auxotrophs, each component possessing the functions that the other lacks, only rare diploid conidia, resulting from fusion of unlike nuclei, will grow to form colonies on minimal medium. The second method depends on complementation of conidial colour mutants. If the two components of the heterokaryon carry colour mutations in dierent genes, yellow and white respectively, the great majority of conidia will be yellow or white, since the colour of each is determined by its own single nucleus. A patch of mycelium bearing conidia of the wild-type green colour signals the formation of a diploid sector, which can be subcultured. Aspergillus diploids, once formed, are fairly stable, but produce occasional sectors showing segregation and recombination of parental traits. These sectors are of two kinds. Most of those that occur spontaneously are haploid, and arise through sequential chromosome loss. After one chromosome is lost, to give a more or less subviable 2n 2 1 aneuploid, further losses leading to haploidy restore vigorous growth. Since chromosomes are lost at random,

and without crossing-over, the haploid derivatives show random reassortment of marker genes associated with dierent chromosome pairs, but complete linkage of markers on the same chromosome. The second class of segregants are diploid, and show segregation of recessive parental markers as a result of mitotic crossing-over with their respective centromeres, a rare event but greatly stimulated by ultraviolet irradiation. The rule is that all originally heterozygous markers distal to a crossover in the same chromosome arm become homozygous together, leaving those between the crossover and the centromere heterozygous (Figure 4). Thus analysis of diploid segregants gives information about the order of genes within chromosome arms, while haploid analysis shows whether genes belong to the same or dierent chromosome pairs. Parasexual genetic analysis can also be applied to a number of asexual species such as Aspergillus niger, and Penicillium spp. In practice it does not seem to have been very much used in these industrially important species, probably because the heavy mutagenesis to which commercial strains have been subjected makes their genetic behaviour complicated and unpredictable. It is also possible to select diploids, and demonstrate mitotic crossing-over in the ssion yeast Sch. pombe and the yeast-like monokaryon of Ustilago maydis, but diploids in Neurospora and other lamentous Ascomycetes seem either not to exist, or to lose chromosomes so rapidly as to be irrecoverable.

Mitochondrial Inheritance
The genetic role of mitochondria, the universal eukaryotic organelles the function of which is to generate energy by respiration, has been most clearly worked out in the budding yeast, Saccharomyces cerevisiae (Wolf, 1995). Like the plastids of green plants, mitochondria in general contain DNA (mtDNA) which, in the case of yeast, is in the form of a closed loop of about 50 kilobase pairs. Saccharomyces spp. are uniquely suitable for the investigation of mitochondrial function because their ability to grow anaerobically, by fermentation, means that losses of mitochondrial function can occur without being lethal. Respiration-decient yeast mutants form small colonies whether oxygen is present or not and (rst studied in Paris) are called petites. They were found to fall into two classes, one, due to mutation in nuclear genes, showing normal Mendelian inheritance, and the other, later shown to be defective in their mtDNA, showing nonMendelian transmission in crosses to wild type or failing to transmit their respiratory deciency at all. The nontransmissible petites, called neutral, occur spontaneously with a frequency of the order of 1%, and turn out to have lost all their mtDNA. Another category, frequent after treatment with the DNA-intercalating
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acridine dyes, are called suppressive petites because their defect is transmitted through crosses, and their mtDNA, which has more or less extensive deletions in its sequence, tends to displace the normal mtDNA and eventually to take over in all descendant cells. Two further kinds of mitochondrial mutants make possible detailed analysis of the deletions, and are the result of point mutations within specic mitochondrial genes. One class (mit 2 ) is respiration-defective with deciencies in single mitochondrially encoded components of the respiration complex, such as cytochrome b and/or one of the components of the cytochrome oxidase and the reduced nicotinamideadenine dinucleotide (NADH) oxidase complexes. The second class of point mutants is resistant to antibiotics such as chloramphenicol, erythromycin and paromomycin. Ribosomes of mitochondria, like those of bacteria, are inhibited by these antibiotics, and the resistant mutants mostly have altered mitochondrial ribosome components (21S or 16S ribosomal RNA). Crosses between strains carrying dierent mitochondrial mutations can yield wild-type (or double mutant) recombinants, and surprisingly, since each yeast cell normally contains about 20 mtDNA genomes, recombinant types are segregated out in pure form in the hybrid diploid cells after a few budding cycles. Presumably the mtDNA population must be reduced to small numbers a population bottleneck soon after mating. Unlike meiotic recombinants, the frequencies of dierent mitochondrial recombinants give little information about distances between mutant sites. Except for the closest linkages, recombination frequencies all tend to approach a maximum of around 25%, presumably as the result of several rounds of random pairwise interactions within the mixed mtDNA population. However, genetic maps can be made using the partial deletions in suppressive petites. The extent of each deletion can be determined physically, by comparison of the circular restriction site map of each partially deleted mtDNA with that of the wild type. The genes retained or lost through each deletion can then be determined by crossing to a set of point mutants, and determining which deletion mutants can contribute the function lost in each point mutant. Or, starting with an antibiotic-resistant mutant, deletion petites can be selected; it can then be determined which segment of the mtDNA must be retained in order for the drug resistance to continue to be transmissible through crosses. Mitochondrial genetics of the strictly aerobic lamentous fungi is much less well developed than that of Saccharomyces, but several cytoplasmically transmitted traits have been identied and attributed to mitochondria. In those Ascomycetes where, unlike yeast, there is a distinction between male and female gametes, the classical test of reciprocal crossing can be used to identify cytoplasmic inheritance. In Neurospora crassa, for example, the slow-growth poky and the stopstart stopper phenotypes are transmitted to most or all progeny through
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the female fruit body initials (protoperithecia), but to few or none through the male fertilizing conidia. These abnormal strains have rearranged mtDNA, with deletions (Griths et al., 1995). The phenomenon of senescence progressive loss of viability after a certain number of transfers has often been observed in lamentous Ascomycetes and has been particularly studied in Podospora anserina and Neurospora spp. It is transmitted through vegetative hyphal fusion as well as through crosses. In Podospora it has been shown to be associated with the proliferation either of cut-down pieces of mtDNA, or of a mitochondrial intron, a portion of a gene transcript normally excised during the processing of messenger RNA, but capable of reverse transcription and occasional replication on its own account (Marbach and Stahl, 1994). In Neurospora spp., senescence is in several cases associated with the presence of mitochondrial plasmids linear or closed-loop DNA elements able to replicate autonomously (Griths et al., 1995). The element kalilo of N. intermedia is a well-investigated example. Both independently replicating introns and plasmids may be considered selsh DNA elements.

Cloning and Transformation

The experimental genetics of fungi has been revolutionized by the development of methods for cloning fragments of DNA and using them to transform cells. Cloned DNA is usually propagated in the bacterium Escherichia coli, using a vector with the capacity for autonomous replication in the bacterium. For E. coli, the DNA vector is commonly a cosmid, a construct with a replication origin derived from a plasmid (usually colE1) and sequences to promote its packaging in lambda bacteriophage particles. For cloning in yeast, one can use yeast articial chromosomes (YACs) but, unless very large DNA fragments have to be cloned, it is generally more convenient to use a vector based upon the yeast 2-mm plasmid, which can replicate in either budding or ssion yeast without integration into the chromosomes. Most useful of all are the so-called shuttle vectors, which incorporate both E. coli plasmid and 2-mm sequences, so that DNA sequences inserted into the shuttle can be propagated in either E. coli or yeast (Beggs, 1978). Cloned DNA can be inserted (transformed) into yeast cells, or into many other fungi, by a number of dierent methods, the rst to be devised involving enzymatic removal of cell walls, suspension of the resulting naked protoplasts (spheroplasts) in some nontoxic osmotic stabilizer such as sorbitol solution, and addition of the DNA with calcium ions and, most usually, polyethylene glycol to cause clumping. Later it was found that intact yeast can be transformed in the presence of high concentrations of lithium. Another increasingly favoured method is electroporation treatment in the presence of

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Fungal Genetics

DNA with a uctuating electric current. Like the lithium method, electroporation works without the need for cell wall removal. If an auxotrophic or temperature-conditional lethal mutant is transformed with a library of cloned DNA sequences, the cells that have picked up the required gene will grow up selectively on minimal medium or at the restrictive temperature. When the repair of the mutational defect is not signalled by growth (as was the case when transformation was used to capture mating-type genes; Glass and Nelson, 1994), the transformants have to be screened by more laborious means. It remains to recover the cloned gene from the transformed cell. This is no problem in yeast transformed using a shuttle vector which can easily be reisolated. But there are no vectors that replicate autonomously in Neurospora, and in this fungus stable transformation depends on integration of the transforming DNA into a chromosome, from which it can be recovered only with diculty. The solution to this problem is sib selection. In the rst demonstration of this method (Vollmer and Yanofsky, 1986), mutants were transformed with 3072 dierent cosmid clones, divided into 32 pools of 96. Pools that succeeded in repairing the mutant were divided into 12 subpools of 8, each of which was tested again. The cosmids in the successful subpool could then be tested individually. Now that so many genes have been cloned and sequenced from so many organisms, it has become possible, using hybridizing DNA probes, to pick out fungal genes that resemble genes from, for example, the mouse. Or cloned mouse genes can be tested for their ability to repair yeast mutants.

conditions under which these genes are necessary remain to be found. The yeast genome project was the rst to be completed in fungi, but will certainly not be the last. Complete DNA sequences for Neurospora crassa, Aspergillus nidulans and probably others are likely to follow within the next decade. Experimental genetics will become increasingly based on scrutiny of the known DNA sequence and precisely targeted DNA manipulations. It will still, however, be interesting to know what fungal genetic systems will do when left to themselves.

References
Beggs JD (1978) Transformation of yeast by a replicating hybrid plasmid. Nature 275: 104109. gueret J, Turcq B and Clave C (1994) Vegetative incompatibility in Be lamentous fungi: het genes begin to talk. Trends in Genetics 10: 441 446. Casselton LA and Ku es U (1994) Mating type genes in Homobasidiomycetes. In: Wessels JGH and Meinhardt F (eds) The Mycota, vol. I, pp. 307321. Berlin: Springer-Verlag. Clayton RA, White O, Ketchum KA and Venter JC (1997) The rst genome from the third domain of life. Nature 387: 459462. Fincham JRS (1977) Allelic complementation reconsidered. Carlsberg Research Communications 42: 421430. Fogel S, Mortimer R, Lusak K and Tavares F (1979) Meiotic gene conversion: a signal of the basic recombination event in yeast. Cold Spring Harbor Symposia in Quantitative Biology 43: 13251341. Glass NL and Nelson MA (1994) Mating-type genes in mycelial Ascomycetes. In: Wessels JGH and Meinhardt F (eds) The Mycota, vol. I, pp. 295306. Berlin: Springer-Verlag. Griths AWF, Collins RA and Nargang FE (1995) Mitochondrial genetics of Neurospora. In: Ku ck U (ed.) The Mycota, vol. II, pp. 93 105. Berlin: Springer-Verlag. Hartwell L (1978) Cell division from a genetic perspective. Journal of Cell Biology 77: 627637. Ka mper J, Bo lker M and Kahmann R (1994) Mating-type genes in Heterobasidiomycetes. In: Wessels JGH and Meinhardt F (eds) The Mycota, vol. I, pp. 351366. Berlin: Springer-Verlag. Marbach K and Stahl U (1994) Senescence of mycelia. In: Wessels JGH and Meinhardt F (eds) The Mycota, vol. I, pp. 195210. Berlin: Springer-Verlag. OShea SF, Chaure PT, Halsall JR et al. (1998) A large pheromone and receptor gene complex determine multiple B mating type specicities in Coprinus cinereus. Genetics 148: 10811090. Pontecorvo G and Kafer E (1958) Genetic analysis by means of mitotic recombination. Advances in Genetics 9: 71104. Schmidt H and Gutz H (1994) The mating type switch in yeasts. In: Wessels JGH and Meinhardt F (eds) The Mycota, vol. I , pp. 283294. Berlin: Springer-Verlag. Vollmer SJ and Yanofsky C (1986) Ecient cloning of genes in Neurospora crassa. Proceedings of the National Academy of Sciences of the USA 83: 48674873. Wolf K (1995) Mitochondrial genetics of yeast. In: Ku ck U (ed.) The Mycota, vol. II, pp. 7591. Berlin: Springer-Verlag.

Analysis of Whole Genomes

Great emphasis is currently being placed on genome projects aimed at the determination of the DNA sequences of entire genomes. At the time of writing, the analysis has been completed for several species of bacteria and a single fungus, the yeast Saccharomyces cerevisiae (Clayton et al., 1997). The yeast project has dened the complete SNA sequence of the 16 chromosomes, totalling about twelve million base pairs and including about 6 000 genes. Many of the genes were known from previous studies, and others could be tentatively identied on the basis of their resemblance to well-characterized genes from other organisms, but many are so far just apparent coding sequences for hitherto unknown proteins. It is now fairly straightforward in yeast to delete or alter any of these newly discovered genes by cloning, in vitro manipulation, and reintroduction into the cell by transformation. One of the major surprises so far is the high proportion of genes whose loss appears to be inconsequential; perhaps the

Further Reading
Bennett JW and Lasure LL (eds) (1985) Gene Manipulations in Fungi. London: Academic Press.

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Fungal Genetics

Bennett JW and Lasure LL (eds) (1991) More Gene Manipulations in Fungi. London: Academic Press. Fincham JRS, Day PR and Radford A (1979) Fungal Genetics, 4th edn. Oxford: Blackwell Scientic Publications. Ku ck U (ed) (1995) The Mycota, vol. II, Genetics and Biotechnology. Berlin: Springer-Verlag.

Stahl U and Tudzynski P (eds) (1992) Molecular Biology of Filamentous Fungi. Weinheim: VCH. Wessels JGH and Meinhardt F (eds) (1994) The Mycota, vol. I, Growth, Dierentiation and Sexuality. Berlin: Springer-Verlag.

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ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net