Rediscovery by Whole Genome Sequencing: Classical Mutations and Genome Polymorphisms inNeurospora crassa

Abstract

Classical forward genetics has been foundational to modern biology, and has been the paradigm for characterizing the role of genes in shaping phenotypes for decades. In recent years, reverse genetics has been used to identify the functions of genes, via the intentional introduction of variation and subsequent evaluation in physiological, molecular, and even population contexts. These approaches are complementary and whole genome analysis serves as a bridge between the two. We report in this article the whole genome sequencing of eighteen classical mutant strains of Neurospora crassa and the putative identification of the mutations associated with corresponding mutant phenotypes. Although some strains carry multiple unique nonsynonymous, nonsense, or frameshift mutations, the combined power of limiting the scope of the search based on genetic markers and of using a comparative analysis among the eighteen genomes provides strong support for the association between mutation and phenotype. For ten of the mutants, the mutant phenotype is recapitulated in classical or gene deletion mutants in Neurospora or other filamentous fungi. From thirteen to 137 nonsense mutations are present in each strain and indel sizes are shown to be highly skewed in gene coding sequence. Significant additional genetic variation was found in the eighteen mutant strains, and this variability defines multiple alleles of many genes. These alleles may be useful in further genetic and molecular analysis of known and yet-to-be-discovered functions and they invite new interpretations of molecular and genetic interactions in classical mutant strains.

Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus

“…These uncharacterized MRB mutants were cryogenically stored, awaiting a faster and more economical strategy for identification. Whole-genome sequencing has been used to identify mutant alleles in multiple model organisms (27)(28)(29), including several fungi (30)(31)(32)(33)(34)(35). Here we describe our success in utilizing next-generation sequencing of mutants using Illumina and Ion Torrent platforms to quickly and effectively identify the genetic basis of 12 of the remaining MRB mutants.…”

Meiotic silencing by unpaired DNA is expressed more strongly in the early than the late perithecia of crosses involving most wild-isolated Neurospora crassa strains and in self-crosses of N. tetrasperma

“…However, in N. tetrasperma the ''sex chromosomes'' that bear the mat idiomorphs differ by many small polymorphisms (Ellison et al, 2011), which might lower meiotic silencing activity, say, of the sad-1 gene. Genome re-sequencing projects in Neurospora have revealed a tremendous degree of genomic variation among N. crassa strains (McCluskey et al, 2011), and it is believed that N. crassa strains largely outcross in natural populations. In this scenario, a ''homozygous'' cross such as one between an OR-derived tester with an OR strain, and its associated strong meiotic silencing, might be the exception rather than the norm.…”

Molecular analysis of intragenic recombination at the tryptophan synthetase locus in Neurospora crassa

“…One strain (FGSC 861) has sequence characteristics which confirm that it is unique among this group of strains. None of the 18 strains subject to whole genome analysis (McCluskey et al 2011) had any polymorphisms at the trp-3 locus emphasizing the importance of this gene and the value of well characterized mutant strains with defined genetic lesions. The value of these, and related, materials will increase as more information is available about classical mutations.…”