PCR-based markers for genetic mapping in Neurospora crassa.

Moshi Kotierk and Myron L. Smith, Carleton University, Ottawa-Carleton Institute of Biology, 1125 Colonel By Drive, Ottawa, Ontario, K1S 5B6, Canada

Fungal Genetics Newsletter 51:26-26

Eighteen PCR-based markers are described for use in mapping mutations in Oak Ridge background strains of Neurospora crassa.  These markers are located on each of the seven linkage groups and in the mitochondrial genome to enable course-scale linkage mapping.  Following mapping to a linkage group, additional markers can be developed in the co-segregating region for fine-scale mapping of mutations.  As with the N. crassa RFLP map (Metzenberg and Grotelueschen, 1993; Nelson and Perkins, 2000), the addition of PCR-based markers by members of the Neurospora community will enhance this marker set for mapping purposes.

Map-based or positional cloning is necessary to locate, identify and characterize genes that are associated with spontaneous or induced mutations.  Methods available for locating mutations in Neurospora crassa involve co-segregation analysis using phenotypic (e.g. auxotrophic) markers.  For example, multiply marked centromere tester strains of N. crassa are available for this purpose through the Fungal Genetics Stock Center (e.g. Perkins, 1972; Metzenberg et al., 1984).  Often, however, co-segregation analysis with a set of phenotypic markers will not provide adequate resolution for the fine-scale linkage mapping necessary to clone a trait of interest and may require subsequent crosses to achieve further resolution.

In this note, PCR-based markers are described for locating mutations generated in Oak Ridge background strains.  The method takes advantage of abundant sequence differences between Oak Ridge and Mauriceville genetic backgrounds, and the recently completed N. crassa genome sequence of the Oak Ridge standard strain 74-OR23-1VA (Galagan et al., 2003).  The PCR-based markers are distributed throughout the seven N. crassa linkage groups and the mitochondrial DNA (Figure 1).  Mapping of a mutation involves four steps.  1) Crossing an Oak Ridge-background mat-a strain bearing the mutation of interest with the Mauriceville-background strain, FGSC# 2225.  2) Locating the mutation to linkage group by identifying PCR-based polymorphism(s) that co-segregate with the mutation. 3) Generation of additional markers in the region of interest to carry out high-resolution mapping of the mutation.  4) Identification of the mutated gene by, for example, complementation with co-segregating open reading frames (ORFs) based on the N. crassa genome sequence.

In Table 1, the primer sequences, location and PCR conditions are described for each of three types of polymorphisms identified.  Amplified Product Length Polymorphisms (APLPs) are evident when PCR products are of different sizes.  Also used is presence/absence of PCR product in the Oak Ridge and Mauriceville strains, respectively. Finally, Restriction Fragment Length Polymorphisms (RFLPs) are used to identify internal sequence differences in PCR products that are the same size.

Once the mutation is located to linkage group, mapping can be refined with additional markers in the co-segregating region using the N. crassa genome database as a reference to design primer pairs.  We found the most efficient way to find PCR-based polymorphisms was to use primers placed within adjacent ORFs to obtain amplification products of non-coding, intergenic sequences.  Polymorphisms in these non-coding regions are present in most cases between the Oak Ridge and Mauriceville strains. 

As an example of the use of these markers, we examined their segregation with respect to a Supercontig 12 PCR-marker. Supercontig 12 was anchored to linkage group I and linkage group VI since it bore two and three markers from each linkage group, respectively.  The segregation pattern for the Supercontig 12 marker indicates that it is located on linkage group I, between our met-6 and arg-13 markers.  These 18 and other markers added by the Neurosporacommunity will provide a valuable resource for the positional cloning of traits of interest.

Acknowledgments.  Funding for this research was provided by a Discovery Grant to MLS from the Natural Sciences and Engineering Research Council of Canada and from the Mississaugas of the New Credit First Nation to MK.  Dr. John Vierula kindly provided primers for the nmt-1 marker.

Table 1:  Details of N. crassa PCR-based polymorphisms, including marker location, primer sequences, PCR conditions and polymorphism type.

¹ Marker region, primer names and sequences are based on nearby mapped loci or the Broad Institute’s NC locus designation (Version 1) for assembly version 3.

² +/- = Presence / absence of a PCR product; RFLP = restriction fragment length polymorphism with given restriction enzyme; APLP = amplified product length polymorphism.  Approximate fragment sizes (bp) are given for each under OR ( Oak Ridge ) and M (Mauriceville).  The expected restriction fragment patterns, based on the genome sequence, were obtained in our Oak Ridge background strain for all but the pyr-1 marker.  For the pyr-1 marker, the Mauriceville background strain had the expected Oak Ridge pattern.

Figure 1:  The position of PCR-based markers on each of the seven linkage groups and the mitochondrial DNA are indicated by arrows.  The nuclear maps are based on Perkins et al., (2001).  The numbers beneath the mitochondrial map indicate Oak Ridge EcoRI fragments as designated in Taylor and Smolich, (1985). The mitochondrial map is not on the same scale as the nuclear maps.  The mitochondrial genes indicated are as in Perkins et al., (2001).

References.

Galagan et al. 2003.  The genome sequence of the filamentous fungus Neurospora crassa. 

Nature 422: 859-868.

Metzenberg, R. L., J. N. Stevens, E. U. Selker and E. Morzycka-Wroblewska.  1984.  A method for finding the genetic map position of cloned DNA fragments.  Neurospora Newsl.  31:  35-39.

Metzenberg, R. L. and  J. Grotelueschen.  1993. Restriction polymorphism maps of Neurospora crassa: update.  Fungal Genet. Newsl.  40: 130-138.

Nelson, M. A. and D. D. Perkins.  2000.   Restriction polymorphism maps of Neurospora crassa:  2000 update.  Fungal Genet. Newsl.  47:  25-39.

Perkins, D. D. 1972.  Special purpose Neurospora stocks.  Neurospora Newsl.  19:  30-32.

Perkins, D. D. , A. Radford and M. S. Sachs.  2001.  The Neurospora Compendium: Chromosomal Loci.  Academic Press, New York .

Taylor, J. W. and B. D. Smolich. 1985. Molecular cloning and physical mapping of the Neurospora crassa 74-OR23-1A mitochondrial genome.  Current Genet.  9:  597-603.

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