Induced mutation and somatic recombination as tools for genetic analysis and breeding of imperfect fungi

<p/>Many fungi which are important in Agriculture as plant pathogens or in Biotechnology as producers of organic acids, antibiotics or enzymes, are imperfect fungi. These fungi do not have a sexual stage, which implies that they lack a meiotic recombination mechanism.<p/>However, many imperfect fungi have effective recombination mechanisms operating-during mitotic divisions. The first step in somatic recombination is the fusion of somatic cells. In nature this occurs by hyphal fusion followed by exchange of nuclei, which results in a heterokaryotic mycelium, if the partners are genetically different. Owing to fusion of somatic nuclei (karyogamy), which occurs at a very low frequency, heterozygous diploid nuclei may then arise. Heterozygous diploid strains can be isolated and maintained by transfer of conidia. During division of somatic nuclei (mitosis) recombination of genes can occur by mitotic crossing-over and by loss of chromosomes leading to haploid recombinants.<p/>In the laboratory heterozygous diploid strains can also be obtained via protoplast fusion.<p/>This study concerns the biotechnologically important fungus Aspergillus niger and the phytopathogenic fungus Colletotrichum lindemuthianum (the causal organism of bean anthracnose), whereas studies on fundamental aspects or on the development of procedures were carried out with the genetically well known fungus Aspergillus nidulans. The latter has both a sexual stage and well studied processes of somatic recombination. We used it as a model for studies on mutation induction, heterokaryosis and protoplast fusion.<p/>The chapter on induction and isolation of mutants (Chapter 2) presents studies on UV-survival curves for conidiospores (2.2), the frequency of mutants (2.3) and mutant enrichment procedures (2.4).<p/>The interpretation of the shape of survival curves was discussed. Processes generating an initial shoulder in logsurvival curves have quite different effects: A multi-hit process leads to a much more pronounced initial shoulder than a multi-target process does. In the experimental part is was argued that the initial shoulders in logS curves of haploid uninucleate A.nidulans conidiospores (single target cells) probably are the result of an inherent repair capacity which becomes saturated at a certain UV dose. An increase in target number (number of genomes in the spore) results in an increase of the logS-intercept due to a larger shoulder. At the same time, however, the repair capacity may have increased and complementation of lethal lesions may occur. In general such increases of the logS-intercept lead to overestimation of the target number.<p/>As in practice often high mutagen doses are applied so that mutants are isolated at low survival, the relationship between mutant frequency and survival was analyzed to see whether such high mutagen doses are necessary at all. High doses produce chromosome rearrangements and unnoticed mutations which disturb the genetic background. For several types of mutation it was shown that the highest yield of mutants is found at low mutagen dose (i.e. at about 20-50 % survival). The frequency of mutants among the survivors increases with the dose of mutagen, but levels off and even decreases at higher doses. It was also found, contrary to what is suggested in litterature, that often no simple linear relationship exists between frequency of mutants and the logarithm of the dose or of the surviving fraction. In conclusion our experiments show that mutants can be well isolated at low doses of mutagen. So taking into account the risk of disturbance of the genetic background by unnoticed mutations and chromosomal rearrangments, mutation induction should be done at a survival level of at least 70%.<p/>To compensate for the relatively low frequency of mutants among the survivors, we require procedures for the selection of mutants or for the elimination of the non-mutant cells. The effectiveness of filtration enrichment procedures was demonstrated for different types of mutations. Next, the procedures have been applied to establish a collection of A.niger strains providing genetic markers for genetic analysis and breeding. The first results are reported in Chapter 4.<p/>In Chapter 3 studies on different aspects of somatic recombination are presented: heterokaryosis in C.lindemuthianum, isolation of protoplasts from condidiospores, protoplast fusion and karyogamy in A.nidulans and protoplast fusion in C.lindemuthianum.<p/>To see whether somatic recombination plays a role in the evolution of physiological races of Colletotrichum lindemuthianum the possibilities for heterokaryosis and somatic karyogamy in this fungus were studied (3.2). Although this fungus fulfils some essential requirements for such experiments, the phenomenon of cross-feeding hampered these studies. Between different strains hyphal fusion was observed, but no definite prove for heterokaryosis could be given. Somatic karyogamy could not be demonstrated.<p/>As protoplast fusion seemed a promising alternative for inducing heterokaryosis, methods for isolation (3.3) and fusion (3.4) of conidial protoplasts were first developed with A.nidulans. It was demonstrated that by these methods also in C.lindemuthianum heterokaryons can be formed and maintained. Fusion of protoplasts from conidiospores of strains with different auxotrophic markers resulted in well growing heterokaryons. So at least heterokaryosis may play a role in the development of genetic variation of this phytopathogenic fungus.<p/>In the fusion experiments with A.nidulans protoplasts (3.4) we found that only few protoplasts fused. The system was used to estimate the frequency of somatic karyogamy, which we found to be much higher than the frequency of heterozygous diploid conidia on a heterokaryon. These results confirm that only a small portion of a . balanced heterokaryon consists of heterokaryotic hyphae. This was already indicated by an experiment in which hyphal tips were analyzed.<p/>The conclusion is that a heterokaryon is a dynamic system based on hyphal fusions and segregation of homokaryotic hyphae of the parental types.<p/>In Chapter 4 the start of a program for genetic analysis in A.niger is described. The first results in establishing a collection of mutants providing genetic markers are presented (4.2). Nearly a hundred different mutants are now available, located on at least 30 different genes.<p/>Genetic analysis by haploidization revealed that the 11 loci analyzed belong to at least five linkage groups. In general haploidization was induced by benomyl, but for estimating the frequency of spontaneous haploidization we screened for spontaneous light coloured (fawn) segregants.<p/>The isolation of (homozygous diploid or haploid) recessive recombinants was succesfully done by the enrichment procedure as used for the isolation of mutants. So, for genetic analysis and gene mapping there is no need for special terminal selection markers. In this way genetic analysis and mapping of genes on the chromosomes is possible. The process of mitotic crossing-over proved to be 20-30 less frequent than spontaneous haploidization.<p/>Starting from a reference strain, mutant strains should be induced by as few rounds of mutagenic treatment as possible. Instead, combinations of markers have to be made by recombination. In this way well characterized master strains can be obtained in an isogenic background. These can be used for genetic analysis of other A.niger strains.