title: Bulk segregant analysis followed by high-throughput sequencing reveals the Neurospora cell cycle gene, ndc-1, to be allelic with the gene for ornithine decarboxylase, spe-1.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biophysics, 2011 ALS Bldg., Oregon State University, Corvallis, OR 97331-7305, USA.
authors.forename: Kyle R
authors.initials: KR
authors.lastname: Pomraning
authors.forename: Kristina M
authors.initials: KM
authors.lastname: Smith
authors.forename: Michael
authors.initials: M
authors.lastname: Freitag
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D002455:Cell Division / Q000235:genetics; D002874:Chromosome Mapping; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009492:Neurospora crassa / Q000235:genetics*; D009955:Ornithine Decarboxylase / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013095:Spermidine / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D009955:Ornithine Decarboxylase; D013095:Spermidine