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Recombination of Genetic Material

Introduction

Genetics is the study of heredity, that is, the storage, translation and transmission of information on the genes, chromosomes and in nuclei. In general terms, genetics is the study of genes. While this is not a chapter on the genetics of Eukaryotic organisms, it does consider the way fungi store, transfer and mix genes. We suggest you refer to a good text on genetics to develop understanding of this important topic.

Plants and animals are basically diploid. That is, the structure or organism we see has two sets of chromosomes in a single nucleus. The vast majority of fungi spend most of their life with haploid nuclei, each with only one set of chromosomes. In haploid organisms each gene is potentially expressed in the phenotype. The masking of genes found in diploid organisms is mostly absent in fungi. LINK

Clonal habit, plasticity of form

Fungi are essentially clonal in nature. Even where the life cycle is short, the fungus produces huge numbers of asexual spores, and maybe hyphal fragments, that maintain the genotype over time. The result is a huge number of identical thalli, each tiny and short-lived, interspersed by vegetative propagules of variable size and longevity.

In long-lived mycelia, the clonal nature of the fungus along with anastomosis may establish the fungus over huge areas where conditions are appropriate (45 ha reported for Armillaria gallica in one forest, with an estimated age of more than 15,000 years). The fungus, may in addition, produce sexual spores that may form individuals that intersperse the older genet. During dikaryotisation, cytoplasm is sometimes also exchanged LINK. The resultant thallus may contain extranuclear genetic factors (extrachromosomal DNA, such as in mitochondria, plasmids and viruses) of both haploids and the community, genetically is more varied.

Fungi may also take on many forms. These forms are either induced by the environment, and called phenotypic plasticity, or they are differences due to the stage of the life cycle, and are referred to as pleomorphism.

Phentypic plasticity is due to variation in genotypic expression. Hyphal growth in a variable or heterogenous environment means that different parts of the thallus are exposed to different environmental stimuli. The response of each part may vary, different parts of the thallus may develop in different ways. These responses also apply to different responses over time. Phenotypic variation is seen in all the thallus, but is most obvious in the vegetative parts. Size of sexual structures vary in only a small way, hence their widespread use in taxonomy.

Pleomorphism is also widespread among fungi. At its simplest, the anamorph and teleomorph are different. Some fungi are only recognised as one or the other form, despite the presence of both forms in the wild. For instance, the sexual state of some Rhizoctonia sp is a short lived and cryptic state. Only in the laboratory can we see Tulasnella, even though the sexual state is possibly widespread. In addition, some rust fungi have several asexual stages. These differ markedly in appearance, and in some species may occur on different hosts. Finally, the obvious state of some plant pathogens is expressed in the field on living plants. The anamorph is maintained in culture and the fungus is understood almost entirely on the basis of its vegetative characteristics. One practical consequence of pleomorphism is the lack of recognition of the connections between asexual and sexual states. Use of sequence information is allowing recognition of the linkages between different forms and states.

Combined with phenotypic plasticity, pleomorphism enables fungi to variably react to their environment, in space and time.

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Diploid State

The diploid exists for very short periods in most fungi. In the widely studied Neurospora, the life cycle may take as little as 2 weeks in culture. The diploid is a very short period, which immediately follows karyogamy. The diploid nucleus immediately undergoes meiosis, and is followed by the formation of 4 haploid nuclei. Each nucleus undergoes mitosis in the ascus, resulting in an ordered array of ascospores in the ascus. LINK

The ordered array of ascospores in Neurospora made it possible to study crossing-over during meiosis, segregation during first and second divisions, reciprocal and non-reciprocal exchange of genetic material, and has been used to map chromosomes and study linkage of genes on the chromosomes. In these experiments, each spore in the experimentally derived ascus was cultured independently to determine its genetic characteristics. A single culture might contain many thousands of ascospores, each arranged independently of all other arrays. Thus by careful analysis, some very useful data has been derived. The early research of fungal genetics was extraordinarily detailed and tedious, but it was important, historically, for the research provided insights into how genes were arranged on chromosomes, and alleles mobilised during sexual divisions. The study of fungi underpins our understanding of all genetics of eukaryotic organisms.

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Nuclear State During Growth

Essentially, in the Ascomycota, Glomeromycota, Zygomycota and zoosporic fungi the majority of the life cycle is haploid. In the Ascomycota, dikaryotisation is followed immediately by the diploid, which then passes through meiosis to form haploid spores. Members of the Zygomycota form diploid zygospores, which pass through meiosis at germination. LINK

In the Basidiomycota, growth of the haploid phase may be extensive, as may the dikaryotic phase. The diploid phase is brief, and spores released from the basidium are haploid. Exceptions do occur, for some mycelia are diploid. LINK

A few yeasts and zoosporic fungi are known to grow during haploid and diploid phases. The cells are morphologically similar, though the diploid may be larger.

The number of nuclei within a compartment varies between the divisions of fungi. In the Mucorales and Glomeromycota, hyphae are aseptate and the compartments, potentially, huge. Members of the mucorales all appear to be short-lived, and so their thallus probably consists of identical haploid nuclei. However, thalli of the Glomeromycota form AM fungi and live for a long time, and the fungi are an ancient group. Diverse communities of nuclei may be housed within a mycelium.

A sexual state has been described for only one species of Glomeromycota, Gigaspora decipiens. Most other species are assumed to be asexual. Mechanisms to ensure removal of untoward mutations are unknown in this group of fungi. Retention of nuclei with different sequences is possible within the multi-nucleate mycelium.

Differences in the ITS region of the DNA from within a spore of Glomus mosseae have been recognised for some time. The ITS probably does not code for a cellular function. Variation among ITS may have been due to differences between nuclei in one multinucleate spore, or within each nucleus. This issue has only recently been clarified for the remainder of the DNA. Genetic variation both within and between nuclei in one spore has been established.

One consequence of variation is that each fungus may form partnerships with many different plants, perhaps because they can carry many different genes for recognition or colonisation of each of the plants. Further, establishment of specific plant partnerships may alter the density of nuclei carryng specific genetic components among the parts of the fungus assocated with that host, but different genetic components with another plant species attached to the same mycelium. Nuclear plasticity within the fungi has many different forms.

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Parasexual Cycle

Another phenomenon, known as the parasexual cycle (see below) LINK, is found in fungi. In brief, some haploid nuclei in the heterokaryon may fuse to form diploid nuclei. These nuclei are rare. Subsequently, chromosomal exchange due to mitotic crossing over may take place during mitotic divisions. The resultant nucleus is unstable, and chromosomes are lost during subsequent mitotic divisions. The haploid state is finally attained and commonly, because of crossing over and random loss of chromosomes, the resultant haploid cell is genetically different from the parent.

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Plant Pathology

Fungal genetics is important in the study of the activity of plant pathogens. Many of the concepts underlying our understanding of plant/pathogen interactions rely on the development of the genetic background to that interaction. For instance, plant breeders have in the past inserted genes for pathogen resistance into crop plants only to find that mutation of a gene in the pathogen enables subsequent infection of the new host genotype. Plants and fungi have a long and interlinked association. LINK

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Recombinant DNA Technology

Many fungi are relatively easy to grow. One group, the yeasts, have been used in gene manipulation because they have a small genome and eukaryotic structure. The genome of bakers yeast Saccharomyces cerevisiae has been thoroughly characterised. Further, because culture of yeasts under industrial conditions is simple, yeasts might be used more extensively for production of important gene products in commercial quantities.

Of considerable importance to recombinant technology was the recognition that most yeast cells contained a huge number of plasmids in the cytoplasm. This provided a mechanism to shuttle genes into the target cell. However, yeasts also have a thick wall which has to be removed before transformation can be attempted.

In principle, the process of transformation follows the following path:

  1. Cell walls are removed by enzymes
  2. Protoplasts are washed and stabilised to promote transformation
  3. DNA transformation is attempted using a vector such as a yeast plasmid, Agrobacterium or ballistic processes
  4. Protoplasts are regenerated on a selective medium that enables transformed cells to regenerate the wall and grow.

While yeasts have been successfully transformed, their value as eukaryotic hosts is uncertain. Many eukaryotic genes have been successfully produced. However, the genome of yeast, and filamentous fungi, is significantly larger than E. coli, making them more complicated. The future of fungi in Recombinant DNA Technology remains to be determined.

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Summary

While inbreeding is apparently common in fungi, the consequences are avoided by selection against deleterious forms in the haploid state. Further, fungi appear to utilise a variety of mechanisms to ensure the genome is shuffled at intervals.

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References

Burnett J (2003) Fungal Populations and Species. OUP.

Clutterbuck AJ, Chapters 11 and 12 of Gow NAR & Gadd GM Eds (1995)The Growing Fungus. Chapman & Hall, pp 239 - 274.

Moore D & Novak Frazer LA 2002 Essential Fungal Genetics. Springer.

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Parasexual Cycle

Many septate fungi are only known from their asexual state (ie Deuteromycota). In some cases, the asexual state has not been linked with a teleomorph, and it has been assumed that the fungi do not have a sexual stage in the wild. Meiosis is important because it is associated with the recombination of genes, mixing of genotypes, if you like. So it might be assumed that either these fungi are so highly fitted to their environment that recombination is unnecessary, or they have other means of reorganising the genome. The discovery of the parasexual cycle was important because it demonstrated that the genome can become reorganised without passing through meiosis.

Sexual and parasexual recombination provide quite different processes to a similar end result. However,

  1. Sexual reproduction is an event precisely controlled by the host genome, whereas the parasexual cycle appears to be uncommon and uncontrolled.
  2. Sexual reproduction involves a second genotype leading to an increase in possible genetic outcomes in the offspring, whereas parasexual recombination is limited to the genetic material already in the thallus, and only one or a few chromosomes. This may lead to a different offspring, but the numbers of possible recombinations is reduced.
  3. Segregation following meiosis leads to controlled separation of chromatids into nuclei, whereas in the parasexual the haploid state is attained following loss of chromosomes.

The parasexual cycle is less efficient than the sexual cycle, and is really only of value when enormous numbers of offspring are placed under selection pressure. The variation attained from parasexual recombination is slight in comparison to a meiotic recombination event.

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