David D. Perkins

The Cytogenetics of Neurospora

Publisher Summary This chapter concerns genetically significant aspects of Neurospora cytology, and the relation between genes and chromosomes, with special emphasis on chromosome rearrangements. In addition to reviewing the published literature, numerous results have been presented. Many of these are cytological, appearing under various headings. The chapter is also concerned with the morphology and identification of individual chromosomes. New genetic results concerning chromosome rearrangements and general characteristics of aberrations are given and a brief description of each known rearrangement has been provided. The chapter also deals briefly with other topics such as accessory, the genetic control of recombination, and the cytoplasmic genome. Neurospora possesses several favorable features compared to the more conventional organisms that are used in cytogenetic research, and these, in part, compensate for the small size of its chromosomes. All four products of individual meioses survive. Progeny can be obtained either as random meiotic products, as unordered tetrads, or as ordered tetrads whose linear spore arrangement reflects the events of meiosis. The spores show high viability and germination. The vegetative (somatic) part of the life cycle is haploid. Duplications are more readily identified as partial diploids against a haploid background than as partial triploids against a diploid background. Somatic variants can be obtained in pure culture. Any somatic cell can serve as a germ cell. For Aspergillus, only a few rearrangements have been recognized in other eukaryotic microorganisms, where usually they are more difficult to detect than in Neurospora, with its pigmented spores. Failure to recognize an existing rearrangement can lead to spurious conclusions regarding linkage, recombination, interference, preferential segregation, or the presence of synthetic lethal genes.

Map construction in Neurospora crassa.

Publisher Summary In this chapter linkage and centromere data from neurospora crassa have been compiled from all published material, as well as unpublished sources. Tetrad data from gene–centromere and gene–gene intervals have been placed on a uniform basis for mapping by computing map lengths from second-division segregation frequencies and tetratype segregation frequencies, respectively. Maps of the seven linkage groups have been constructed from tetrad data. Seventy-five loci are shown. Confidence limits are indicated for the position of each locus. Two sets of maps are presented, the first is completely uncorrected for multiple crossovers and the second is corrected by means of the mapping function. A few additional genes have been assigned to specific linkage groups on the basis of random isolates. The use of random isolates for mapping is also discussed.

Neurospora from natural populations: Toward the population biology of a haploid eukaryote

Abstract Natural populations of the ascomycete Neurospora have been sampled systematically throughout much of the world, and the haploid strains from colonies in nature have been characterized genetically in the laboratory. Our findings are described in the context of a broader review of wild-collected strains, their uses, and their significance for population genetics. Visible Neurospora colonies found on recently burned vegetation are usually unique in genotype. More than three-fourths are pure strains originating from a single ascospore; the remainder can be purified. Thus, despite the potential for clonal propagation, these colonies provide effective population samples comparable to those collected for higher plants and animals. Over 3900 isolates from burned substrates have been analyzed from over 500 collection sites, mostly from tropical and subtropical regions. These strains have been assigned to five species—four heterothallic species with eight-spored asci and one pseudohomothallic species with four-spored asci. Each species has a unique pattern of distribution, but each overlaps with all the others in one or another part of its range. All of these species are similar in vegetative morphology, with orange or yellow-orange conidia. All have two homologous mating types, but the different species are reproductively isolated from one another. Fertility in crosses with reference strains has provided a reliable and convenient criterion for species classification of heterothallic strains. The species of a newly obtained haploid strain is determined by finding a tester strain with which it is fully fertile and produces predominantly viable ascospores. Viable ascospores are extremely rare for most interspecific combinations, but genes can nevertheless be transferred by matings among all but one of the nonhomothallic species. Abundant but mostly inviable ascopores are produced by some interspecific combinations. Karyotypes, karyogamy, and meiotic chromosome behavior are similar for all the known Neurospora species. There are seven chromosomes and a single terminal nucleolus organizer. This pattern also applies to the five eight-spored homothallic Neurospora lines that were designated by their discoverers as different species on the basis of ascospore morphology. These homothallic lines all lack orange pigment and are devoid of conidia. They were obtained by enrichment from soil samples and would not have been obtained by our collecting methods, which rely on visibility in the field. Examination of wild-collected strains of N. crassa and N. intermedia has revealed a wealth of intraspecific genetic variation. Genetic polymorphism of isozymes in local populations is comparable to that in outbreeding higher animals and plants. DNA restriction fragment length polymorphisms are also abundant, as are differences at vegetative (heterokaryon) incompatibility loci and recessive genes that adversely affect one or more stages of the sexual diplophase. Chromosomally located factors, called Spore killer, act in the sexual phase to produced meiotic drive. The four Spore-killer-sensitive ascospores in every ascus are killed in crosses of sensitive × killer, but all eight ascospores remain viable in crosses of killer × killer and sensitive × sensitive. Mitochondrial genomes of wild strains differ in both length mutations and nucleotide substitutions. Many isolates contain mitochondrial plasmids. A few strains have been found to undergo senescence following insertion of a foreign element into mitochondrial DNA.

Neurospora : a model of model microbes

In the 1940s, studies with Neurospora pioneered the use of microorganisms in genetic analysis and provided the foundations for biochemical genetics and molecular biology. What has happened since this orange mould was used to show that genes control metabolic reactions? How did it come to be the fungal counterpart of Drosophila? We describe its continued use during the heyday of research with Escherichia coli and yeast, and its emergence as a biological model for higher fungi.

STRAINS OF NEUROSPORA COLLECTED FROM NATURE

The fungus Neurospora is genetically and biochemically one of the most studied eukaryotic microorganisms. However, little is known of natural populations, and there has been little systematic collecting or study to provide information on population genetics or ecology. This study was undertaken with the hope that investigations of Neurospora from nature might contribute significantly to evolutionary biology in several ways. It seemed of interest to examine some of the tenets of population genetics using an organism whose life cycle and life style are radically different from those of the diploid organisms conventionally considered. It was hoped specifically to gather information on the role and significance of sexual reproduction, heterokaryosis, and vegetative incompatibility, on genic and chromosomal variability within and between Mendelian populations, on reproductive isolation, and on systematic relationships. The studies reported here show that it is feasible to find and observe Neurospora in the field, and that populations can readily be sampled by obtaining cultures that have originated from single ascospores. The results indicate clearly that Neurospora can contribute information of value to evolutionary biology. At the same time, an evolutionary perspective promises to enrich laboratory investigations of the fungi. Researches on nutritional and regulatory variants, variations in chromosome structure and behavior, heterokaryon compatibility, and the sexual mating types, have been largely concerned in the past with mechanisms, and have given little

Chromosome rearrangements in Neurospora and other filamentous fungi.

Knowledge of fungal chromosome rearrangements comes primarily from N. crassa, but important information has also been obtained from A. nidulans and S. macrospora. Rearrangements have been identified in other Sordaria species and in Cochliobolus, Coprinus, Magnaporthe, Podospora, and Ustilago. In Neurospora, heterozygosity for most chromosome rearrangements is signaled by the appearance of unpigmented deficiency ascospores, with frequencies and ascus types that are characteristic of the type of rearrangement. Summary information is provided on each of 355 rearrangements analyzed in N. crassa. These include 262 reciprocal translocations, 31 insertional translocations, 27 quasiterminal translocations, 6 pericentric inversions, 1 intrachromosomal transposition, and numerous complex or cryptic rearrangements. Breakpoints are distributed more or less randomly among the seven chromosomes. Sixty of the rearrangements have readily detected mutant phenotypes, of which half are allelic with known genes. Constitutive mutations at certain positively regulated loci involve rearrangements having one breakpoint in an upstream regulatory region. Of 11 rearrangements that have one breakpoint in or near the NOR, most appear genetically to be terminal but are in fact physically reciprocal. Partial diploid strains can be obtained as recombinant progeny from crosses heterozygous for insertional or quasiterminal rearrangements. Duplications produced in this way precisely define segments that cover more than two thirds of the genome. Duplication-producing rearrangements have many uses, including precise genetic mapping by duplication coverage and alignment of physical and genetic maps. Typically, fertility is greatly reduced in crosses parented by a duplication strain. The finding that genes within the duplicated segment have undergone RIP mutation in some of the surviving progeny suggests that RIP may be responsible for the infertility. Meiotically generated recessive-lethal segmental deficiencies can be rescued in heterokaryons. New rearrangements are found in 10% or more of strains in which transforming DNA has been stably integrated. Electrophoretic separation of rearranged chromosomal DNAs has found useful applications. Synaptic adjustment occurs in inversion heterozygotes, leading progressively to nonhomologous association of synaptonemal complex lateral elements, transforming loop pairing into linear pairing. Transvection has been demonstrated in Neurospora. Beginnings have been made in constructing effective balancers. Experience has increased our understanding of several phenomena that may complicate analysis. With some rearrangements, nondisjunction of centromeres from reciprocal translocation quadrivalents results in 3:1 segregation and produces asci with four deficiency ascospores that occupy diagnostic positions in linear asci. Three-to-one segregation is most frequent when breakpoints are near centromeres. With some rearrangements, inviable deficiency ascospores become pigmented. Diagnosis must then depend on ascospore viability. In crosses between highly inbred strains, analysis may be handicapped by random ascospore abortion. This is minimized by using noninbred strains as testers.

New markers and map sequences inNeurospora crassa, with a description of mapping by duplication coverage, and of multiple translocation stocks for testing linkage

Thirty previously unmapped markers have been located; 13 are at newly designated loci. Numerous sequences for previously mapped genes have also been determined. A revised map of linkage group I is presented. The order from conventional mapping has been confirmed by testing recessive markers in IL for coverage by duplications. Assignment of new mutants to linkage groups is greatly facilitated by using gene-tagged multiple translocation strains for linkage detection; these “alcoy” tester strains and procedures for using them are described. Recent mapping data of other workers are compiled. Distal markers are now known for all but one of the 14 chromosome arms, but extensive map segments are still devoid of markers.

Different cell types in Neurospora crassa

Neurospora possesses more cell types than are commonly recognized. We have been able to identify 28 morphologically distinct types. Having the cell types clearly defined will be important for genome annotation, describing new mutant phenotypes, and determining sites of gene expression. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol50/iss1/8 Number 50, 2003 17 Different cell types in Neurospora crassa George N. Bistis, David D. Perkins, and Nick D. Read Department of B iology, Drew University, M adison, NJ 07940, Department of B iological Sciences, Stanford University, Stanford, CA 94305-5020, Department of Cell and Molecular Biology, University of Edinburgh, Rutherford Building, Edinburgh EH8 9QU, U.K. Fungal Genet. Newsl. 50:17-19 Neurospora possesses more cell types than are commonly recognized. We have been able to identify 28 morphologically distinct types. Having the cell types clearly defined will be important for genome annotation, describing new mutant phenotypes, and determining sites of gene expression. ____________________________________________________________________________________ Neurospora is a morphologically complex multicellular organism with many more cell types than the unicellular yeast Saccharomyces. Most workers are familiar with mycelia, macroconidia, perithecia, asci, and ascospores, but the diversity of cell types produced by Neurospora may not be fully appreciated. Now that the products of specific genes can be localized using GFP and o ther fluorescent proteins, attention will be focused increasingly on particular cell types that differ in morphology, physiology, or developmental origin. D istinguishing different cell types is also important for genome annotation. For convenience, we need to use the terms ‘cell’ and ‘cell type’ rather loosely to cover both cellular elements such as hyphae and discrete cells such as spores (see discussion by Read, 1994). The basic undifferentiated, totipotent cellular element is the compartmentalized vegetative hypha at the colony periphery (the leader hypha). Certain other cell types are comprised of differentiated hyphae (e.g., fusion hyphae, ascogonia, trichogynes, ascogenous hyphae, asci, paraphyses, and periphyses). At the other extreme are highly differentiated nonhyphal cells such as ascospores, microconidia, and the different wall cells of protoperithecia and perithecia. Twenty-eight morphologically distinct cell types are listed and described below. Designation of protoperithecia and microconidia as vegetative or sexual is arbitrary. Additional types or subtypes will no doubt be revealed.

Main features of vegetative incompatibility in Neurospora.

Main features of vegetative incompatibility in Neurospora. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This mini review is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol35/iss1/26

Neurospora discreta, a new heterothallic species defined by its crossing behavior

Abstract The species is described and named Neurospora discreta sp. nov. because of its stringent reproductive isolation. Isolates collected from burned vegetation at a single site near Kirbyville, Texas, include both mating types (Aanda). Experimental criteria based on cross-fertility were used for assigning species status. Crosses between isolates of opposite mating type are highly fertile, producing abundant eightspored asci. In contrast, when the Kirbyville strains are crossed to sexually compatible speciestester strains representing N. crassa, N. intermedia, N. sitophila , and N. tetrasperma , perithecia are rudimentary and no ascospores are produced. The haploid chromosome number is 7. Chromosomes at pachytene resemble those of other Neurospora species. Biotin is required. Linear growth is slower than for other heterothallic species. When A and a strains from Kirbyville grow toward one another and intersect on crossing medium, there is no barrage. A single homogeneous band of perithecia is formed where they meet, indicating that opposite mating types are vegetatively compatible. The Kirbyville population differs from other heterothallic Neurospora species in ascospore morphology and vegetative traits. Ascospores from Kirbyville parents are larger, and the ribs between confluent parallel grooves are ornamented with dot-like pits. Vegetative cultures from Kirbyville are yellowish rather than orange, and large empty barren protoperithecia or false perithecia are produced abundantly in unfertilized haploid cultures. Isolates from two other N. discreta populations resemble other Neurospora species more closely with respect to these morphological traits but are clearly conspecific with the Kirbyville strains on the basis of fertility in crosses.