Sordaria_macrospora_mutant_sequencing_pro23_pro44

Forward genetics approaches, i.e. the identification of affected genes in mutants with distinct phenotypes, are among the most powerful methods to study biological processes. However, procedures to identify the causative mutations are usually laborious and time-consuming, and require previous knowledge of genetic and/or physical markers or resources like genomic or cDNA libraries for complementation. Here, we present an alternative, rapid method to identify mutated genes in developmental mutants of the filamentous fungus Sordaria macrospora through Illumina/Solexa whole genome-sequencing of mutant strains. We sequenced pooled DNA from 40 single spore isolates per strain for four mutants and the wild type. The sequenced mutant strains have the genotypes pro23/fus and pro44/fus, pro44 and r2, i.e. two carry the spore color mutation fus and one of the sterility-causing mutations pro23 or pro44. Sequencing was done with Illumina/Solexa 50-76 nt mate-pair runs. We obtained one lane of mate-pair sequences (Illumina/Solexa GAII) each for the wt and pro23/fus, and two lanes mate-pair sequences and one lane single reads for pro44/fus as well as one Illumina/Solexa HiSeq lane for pro44 and two lanes for r2. Reads were mapped to the Sordaria macrospora genome sequence (Nowrousian et al. 2010, PLoS Genet. e1000891, acc. numbers CABT01000001-CABT01004783) with BWA (Li and Durbin 2009, Bioinf 25:1754-1760). We were able to identify the causative mutations for three genes through bioinformatics analysis.