With the advent of high-throughput DNA sequencing, it is now straightforward and inexpensive to generate high-density small nucleotide polymorphism (SNP) maps. Here we combined high-throughput sequencing with bulk segregant analysis to expedite mutation mapping. The general map location of a mutation can be identified by a single backcross to a strain enriched in SNPs compared to a standard wild-type strain. Bulk segregant analysis simultaneously increases the likelihood of determining the precise nature of the mutation. We present here a high-density SNP map between Neurospora crassa Mauriceville-1-c (FGSC2225) and OR74A (FGSC2489), the strains most typically used by Neurospora researchers to carry out mapping crosses. We further have demonstrated the utility of the Mauriceville sequence and our approach by mapping the mutation responsible for the only existing temperature-sensitive (ts) cell cycle mutation in Neurospora, nuclear division cycle-1 (ndc-1). The single T-to-C point mutation maps to the gene encoding ornithine decarboxylase (ODC), spe-1 (NCU01271), and changes a Phe to a Ser residue within a highly conserved motif next to the catalytic site of the enzyme. By growth on spermidine and complementation with a wild-type spe-1 gene, we showed that the defect in spe-1 is responsible for the ts ndc-1 mutation. Based on our results, we propose changing ndc-1 to spe-1 ndc , which reflects that this mutation results in an ODC with a specific nuclear division defect.Single nucleotide polymorphism (SNP) maps between organisms with different genetic backgrounds are useful for identifying point mutations that result in an observable phenotype. By mating a mutant with a strain of a different genetic background and then selecting progeny with and without a phenotype, one is able to map the underlying mutation(s) by bulk segregant analysis (29). After undergoing recombination, each progeny exhibiting a specific phenotype will have the same genetic background as the mutant parent in the vicinity of the mutation but a mixture of DNA inherited from either parent in regions of the genome that are unlinked to the mutation. By analyzing DNA from a mixture of phenotypically mutant progeny from the cross, it is possible to define the genetic region containing the mutation and even to identify the mutation itself. This is what is meant by "bulk segregant analysis."Most recently, molecular mapping strategies based on restriction fragment length polymorphisms (RFLP) (28), cleaved amplified polymorphic sequences (CAPS) (20, 22), or microarray-based restriction-site-associated DNA mapping (RAD mapping) (1, 23) have been used to map mutations in Neurospora crassa. These methods can be time-consuming and laborious and therefore expensive. In addition, once the mutation is mapped to a specific region of the genome, additional primer pairs for CAPS mapping need to be ordered and additional SNPs identified to more precisely map the mutation. Typically, researchers will then attempt complementation of phenotypes by candidate genes. On...