Originally published as Genetics Published Articles Ahead of Print on November 17, 2008.

Genetics, Vol. 181, 767-781, February 2009, Copyright © 2009
doi:10.1534/genetics.108.089292

A High-Density Single Nucleotide Polymorphism Map for Neurospora crassa

Randy Lambreghts^*, Mi Shi^*, William J. Belden^*, David deCaprio, Danny Park, Matthew R. Henn, James E. Galagan, Meray Batürkmen^,1, Bruce W. Birren, Matthew S. Sachs^,1, Jay C. Dunlap^* and Jennifer J. Loros^*,,2

^* Department of Genetics and Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts 02142 and Department of Environmental and Biomolecular Systems, Oregon Health & Science University, Beaverton, Oregon 97006

² Corresponding author: Department of Genetics, Dartmouth Medical School, Hanover, NH 03755.
E-mail: jennifer.loros{at}dartmouth.edu

We report the discovery and validation of a set of single nucleotide polymorphisms (SNPs) between the reference Neurospora crassa strain Oak Ridge and the Mauriceville strain (FGSC 2555), of sufficient density to allow fine mapping of most loci. Sequencing of Mauriceville cDNAs and alignment to the completed genomic sequence of the Oak Ridge strain identified 19,087 putative SNPs. Of these, a subset was validated by cleaved amplified polymorphic sequence (CAPS), a simple and robust PCR-based assay that reliably distinguishes between SNP alleles. Experimental confirmation resulted in the development of 250 CAPS markers distributed evenly over the genome. To demonstrate the applicability of this map, we used bulked segregant analysis followed by interval mapping to locate the csp-1 mutation to a narrow region on LGI. Subsequently, we refined mapping resolution to 74 kbp by developing additional markers, resequenced the candidate gene, NCU02713.3, in the mutant background, and phenocopied the mutation by gene replacement in the WT strain. Together, these techniques demonstrate a generally applicable and straightforward approach for the isolation of novel genes from existing mutants. Data on both putative and validated SNPs are deposited in a customized public database at the Broad Institute, which encourages augmentation by community users.

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