Some cell biological studies of Neurospora crassa have been limited by the rapid rates of hyphal growth and fusion. In this study, we investigated the causative mutation in the standard C24 allele of for (FGSC #9) and assayed the growth and circadian phenotype of the for strain under different nutritional conditions. We show that the for strain can be maintained as metabolically active single cells for 2 days before its growth advances into branched mycelia. This culturing system offers the potential to advance subcellular dynamic research and to facilitate greater understanding of N. crassa in the early developmental stages.
A significant number of classical genetic Neurospora crassa biochemical mutants remain anonymous, unassociated with a physical genome locus. By utilizing short read next-generation sequencing methods, it is possible to sequence the genomes of mutant strains rapidly and economically for the purpose of identifying genes associated with mutant phenotypes. We have taken this approach to connect genes and mutations to “methionineless” phenotypes in N. crassa.
Many filamentous ascomycete fungi reproduce primarily asexually, with only occasional sexual generations. This can lead to a departure from the 1:1 mating type ratio that is expected in obligate sexual populations. The relaxed selection on sexual traits also can lead to a decrease in the frequency of female fertile strains in field populations, while male fertility does not similarly decrease since male gametes also can serve as asexual spores. Both changes ultimately impact the strength of genetic drift in populations. The frequency of female sterility likely increases with the time since the last generation of sexual reproduction, such that it can be used to estimate the relative frequency of sexual reproduction. Here I provide additional details relevant to Leslie and Klein’s (1996) model of mixed sexual reproduction and vegetative propagation as related to the frequency of female sterility. This includes new or modified equations that allow for simpler calculations of i) two estimates of relative Ne, ii) the expected range of hermaphrodite frequencies during the cycles of mixed reproduction, and iii) the relative frequency of sex. These equations also are included in spreadsheet templates into which researchers can directly enter frequencies computed from their population data to estimate these parameters for their own populations. These resources will make the results of Leslie and Klein (1996) more accessible and should increase the use of this model in evaluating the frequency of sexual reproduction of filamentous fungi.
The split-marker (SM) protocol has become a popular method for production of knockout mutations in fungi. We used Southern hybridization to compare the performance and efficiency of the SM protocol with the more traditional double-crossover intact marker (IM) method for creating deletions of the mating type genes in Fusarium graminearum. Both methods successfully produced knockouts at a rate of between 24 and 75%: the SM method produced mutants more efficiently for larger constructs (>1 kb), but it was similar to IM for a smaller construct that was 865 bp. Both methods also produced strains with additional ectopic integrations at a similar rate of approximately 10%, but on average the SM produced a higher number of independent integrations in those strains. Ectopic integrations produce off-site mutations, and strains with multiple integrations are less desirable since it is more difficult to remove them by backcrossing. Southern hybridizations will be generally superior to PCR to identify strains with fewer ectopic integrations for experimental purposes.
Background A large portion of nuclear DNA is composed of transposable element (TE) sequences, whose transposition is controlled by diverse host defense strategies in order to maintain genomic integrity. One such strategy is the fungal-specific Repeat-Induced Point (RIP) mutation that hyper-mutates repetitive DNA sequences. While RIP is found across Fungi, it has been shown to vary in efficiency. To date, detailed information on the TE landscapes and associated RIP patterns exist only in a few species belonging to highly divergent lineages. Result We investigated 18 nearly gapless genome assemblies of ten Neurospora species, which diverged from a common ancestor about 7 MYA, to determine genome-wide TE distribution and their associated RIP patterns. We showed that the TE contents between 8.7-18.9% covary with genome sizes that range between 37.8-43.9 Mb. Degraded copies of Long Terminal Repeat (LTR) retrotransposons were abundant among the identified TEs, and these are distributed across the genome at varying frequencies. In all investigated genomes, TE sequences had signs of numerous C-to-T substitutions, suggesting that RIP occurred in all species. RIP signatures in all genomes correlated with TE-dense regions. Conclusions Essentially gapless genome assemblies allowed us to identify TEs in Neurospora genomes, and reveal that TEs contribute to genome size variation in this group. Our study suggests that TEs and RIP are highly correlated in Neurospora , and hence, the pattern of interaction is conserved over the investigated evolutionary timescale. We show that RIP signatures can be used to facilitate the identification of TE-rich region in the genome.
