Whole-Genome Sequencing ofSordaria macrosporaMutants Identifies Developmental Genes

Abstract

The study of mutants to elucidate gene functions has a long and successful history; however, to discover causative mutations in mutants that were generated by random mutagenesis often takes years of laboratory work and requires previously generated genetic and/or physical markers, or resources like DNA libraries for complementation. Here, we present an alternative method to identify defective genes in developmental mutants of the filamentous fungus Sordaria macrospora through Illumina/Solexa whole-genome sequencing. We sequenced pooled DNA from progeny of crosses of three mutants and the wild type and were able to pinpoint the causative mutations in the mutant strains through bioinformatics analysis. One mutant is a spore color mutant, and the mutated gene encodes a melanin biosynthesis enzyme. The causative mutation is a G to A change in the first base of an intron, leading to a splice defect. The second mutant carries an allelic mutation in the pro41 gene encoding a protein essential for sexual development. In the mutant, we detected a complex pattern of deletion/rearrangements at the pro41 locus. In the third mutant, a point mutation in the stop codon of a transcription factor-encoding gene leads to the production of immature fruiting bodies. For all mutants, transformation with a wild type-copy of the affected gene restored the wild-type phenotype. Our data demonstrate that whole-genome sequencing of mutant strains is a rapid method to identify developmental genes in an organism that can be genetically crossed and where a reference genome sequence is available, even without prior mapping information.

A Fungal Sarcolemmal Membrane-Associated Protein (SLMAP) Homolog Plays a Fundamental Role in Development and Localizes to the Nuclear Envelope, Endoplasmic Reticulum, and Mitochondria

“…Ascospore isolates that carried the hph marker cassette instead of pro45 and that were devoid of the ku70 background were obtained from crosses of primary transformants to a fus spore color mutant (55). Two randomly selected ascospore isolates, N161 (⌬pro45) and R7329 (⌬pro45/fus), were subjected to Southern analysis, confirming the deletion of the pro45 gene (see Fig.…”

RNA Editing During Sexual Development Occurs in Distantly Related Filamentous Ascomycetes

“…For S. macrospora , the wild type strain used for RNA-seq was a derivative of the wild type strain used for the original genome sequencing (Nowrousian et al. 2010); however the genome version (v02) used in our analysis is based on corrections of the original genome sequence based on resequencing of the strain also used for RNA-seq (Nowrousian et al. 2012, Teichert et al.…”

PRO40 Is a Scaffold Protein of the Cell Wall Integrity Pathway, Linking the MAP Kinase Module to the Upstream Activator Protein Kinase C

“…DNA was extracted from 40 sterile progeny as described previously [25]. 40 fertile strains were collected from three crosses of mutants pro30, pro32, and pro34 to fus (Figure S8).…”