Rapid genetic mapping in Neurospora crassa

Jin, Yuan; Allan, Sabrina; Baber, Lauren; Bhattarai, Eric K.; Lamb, Teresa M.; Versaw, Wayne K.

doi:10.1016/j.fgb.2006.09.002

Cited by 15 publications

(21 citation statements)

References 34 publications

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“…Given estimates of the total genetic length of the N. crassa genome ranging from 500 ( Jin et al 2007) to 1000 (Perkins and Barry 1976) MU, the SNP map can be expected to contain several markers linked to any given genetic locus. This number is close to the saturation level for this cDNA library, for the chosen assay ( Figure 2C).…”

Section: Resultsmentioning

confidence: 99%

“…For these reasons, the use of phenotypically neutral polymorphisms is preferred, as these are much more abundant and occur all in a single strain. PCR-based marker sets have been developed (Kotierk and Smith 2004;Jin et al 2007); these are sufficiently dense to establish linkage of a mutant locus to a chromosome arm but not to limit the search to a manageable number of candidate genes. These sets were developed either by trial and error or from a limited amount of sequencing data and thus cannot be easily expanded to include a higher density of confirmed markers in a region of interest.…”

Section: Discussionmentioning

confidence: 99%

“…By combining an accurate SNP detection algorithm (Altshuler et al 2000) with efficient CAPS design we obtained a nearsaturating marker density (given the data set and the assay type), with, on average, one SNP every 7 cM. This is close to the minimum recombination that can be distinguished from perfect linkage using bulked segregant analysis (BSA) ( Jin et al 2007). We then mined the existing library of pSNPs and screened for additional CAPS polymorphisms to increase the local density of the SNP map to a level allowing high-precision mapping.…”

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confidence: 94%

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A High-Density Single Nucleotide Polymorphism Map for Neurospora crassa

Lambreghts¹,

Shi²,

Belden³

et al. 2009

Genetics

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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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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 94%

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A High-Density Single Nucleotide Polymorphism Map for Neurospora crassa

Lambreghts¹,

Shi²,

Belden³

et al. 2009

Genetics

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“…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.…”

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confidence: 99%

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

2011

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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...

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“…A restriction fragment length polymorphism (RFLP) map based on EcoRI digests of DNA from the Oak Ridge and Mauriceville strains is available (Metzenberg et al 1984;Metzenberg and Grotelueschen 1987;Perkins et al 2001). More recently, PCR-based mapping methods have been developed to detect sequence polymorphisms between the Oak Ridge and Mauriceville strains (Kotierk and Smith 2004;Jin et al 2007). While these approaches provide several advantages over phenotypic markers, they have significant limitations.…”

Section: S Tudy Of Filamentous Fungi Such As Neurospora Crassamentioning

confidence: 99%

High-Density Detection of Restriction-Site-Associated DNA Markers for Rapid Mapping of Mutated Loci in Neurospora

et al. 2007

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The wealth of sequence information available for Neurospora crassa and other fungi has greatly facilitated evolutionary and molecular analyses of this group. Although ''reverse'' genetics, in which genes are first identified by their sequence rather than by their mutant phenotypes, serves as a valuable new approach for elucidating biological processes, classical ''forward'' genetic analysis is still extremely useful. Unfortunately, mapping mutations and identifying the corresponding genes has typically been slow and laborious. To facilitate forward genetics in Neurospora, we have adapted microarray-based restriction-site-associated DNA (RAD) mapping for use with N. crassa oligonucleotide microarrays. This technique was used to simultaneously detect an unprecedented number of genomewide restriction site polymorphisms from two N. crassa strains: Mauriceville and Oak Ridge. Furthermore, RAD mapping was used to quickly map a previously unknown gene, defective in methylation-7 (dim-7).

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Rapid genetic mapping in Neurospora crassa

Cited by 15 publications

References 34 publications

A High-Density Single Nucleotide Polymorphism Map for Neurospora crassa

A High-Density Single Nucleotide Polymorphism Map for Neurospora crassa

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

High-Density Detection of Restriction-Site-Associated DNA Markers for Rapid Mapping of Mutated Loci in Neurospora

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