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Originally published as Genetics Published Articles Ahead of Print on July 29, 2007.

Genetics, Vol. 177, 1163-1171, October 2007, Copyright © 2007
doi:10.1534/genetics.107.078147

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

Zachary A. Lewis, Anthony L. Shiver, Nicholas Stiffler, Michael R. Miller, Eric A. Johnson and Eric U. Selker1

Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229

1 Corresponding author: Institute of Molecular Biology, 1370 Franklin Blvd., Eugene, OR 97403-1229.
E-mail: selker{at}molbio.uoregon.edu

Manuscript received June 25, 2007. Accepted for publication July 24, 2007.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
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).

STUDY of filamentous fungi, such as Neurospora crassa, has provided important insights into fundamental biological processes (DAVIS and PERKINS 2002). Neurospora was originally popularized by Dodge in the 1930s and Neurospora researchers now enjoy a rich repertoire of biochemical and genetic techniques developed for this organism. The available genome sequences of Neurospora and other filamentous fungi further facilitate genetic and evolutionary analyses of Neurospora and related organisms (GALAGAN et al. 2003, 2005). As with other model organisms, the ability to create and identify new mutants continues to drive discovery in Neurospora. An extensive collection of Neurospora mutant strains that display morphological or biochemical defects exists and many of the genes responsible for these phenotypes have been mapped genetically and/or physically (PERKINS et al. 2001). Classically, the first step in identifying a new mutant gene has involved finding genetic linkage to a previously mapped gene. To facilitate this process, strains that contain markers on multiple chromosomes were developed (PERKINS 1990, 1991; PERKINS et al. 2001). One can often determine linkage to a specific chromosome by scoring a single cross with these strains. In most cases, however, additional crosses must be carried out to determine the precise location of the mutation to allow for map-based cloning. As a result, using phenotypic markers for map-based identification of new mutants remains a time-consuming endeavor, even with these special mapping strains.

This mapping difficulty has been partially circumvented by the use of DNA sequence polymorphisms as markers for linkage mapping, which can be done with progeny from a single cross. Polymorphisms are plentiful between the standard wild-type strain of N. crassa, Oak Ridge, and other strains collected from the wild, such as Mauriceville. 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. For example, each marker must be assayed individually by Southern hybridization (for RFLP mapping) or by PCR. An increase in map density can be achieved only by increasing the number of probes or primer pairs. Therefore, as map density increases, the scale and cost of the experiment increases. More significantly, detection of these markers is limited to experiments that use the Oak Ridge and Mauriceville strains. To perform an experiment with another strain, one must identify a new set of markers to be assayed in the new strain. This limits genetic analysis of additional wild-type strains. Here, we report a mapping method for detecting restriction site polymorphisms using N. crassa microarrays based on the recently described restriction-site-associated DNA (RAD) mapping method (MILLER et al. 2007a,b). This method circumvents some of the problems associated with the mapping methods currently in use. We describe a set of RAD markers that represent EcoRI restriction site polymorphisms between Oak Ridge and Mauriceville strains. Furthermore, we demonstrate the efficacy of this technique by using bulk segregant analysis (MICHELMORE et al. 1991) to detect RAD markers that are tightly linked to a mutation defining a gene, defective in methylation-7 (dim-7).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Strains and growth conditions:
Culture conditions for vegetative growth and sexual crosses have been described previously (DAVIS and DE SERRES 1970). The dim-7 mutation was isolated following UV mutagenesis of strain N2977 [his-3RIP::barm; {Delta}inl am132 ; hphm(EC) a]. Detailed information about the construction of this strain will be published elsewhere. To construct the barm allele, the bar gene and a fragment of the his-3 gene were targeted to the his-3 locus. The strain was crossed to introduce repeat-induced point mutations within the duplicated his-3 sequence. A strain that displayed silencing of the bar gene was then used for this study (Z. A. LEWIS, K. K. ADHVARYU and E. U. SELKER, unpublished data). The silenced hphm allele has been described previously (IRELAN and SELKER 1997). The original isolate was backcrossed to strain N3311 [his-3RIP::barm sad-1; {Delta}inl am132;hphm(EC) A] to obtain a homokaryon and to confirm that the mutant phenotype was caused by a mutation in a single gene. To isolate recombinant progeny, a homokaryotic dim-7 strain, N3312 [his-3RIP::barm; {Delta}inl am132 dim-7; hphm(EC) a], was then crossed to strain N32 (Mauriceville A; FGSC 2225; cross AX1). For DNA isolation, cultures were grown at 32° for 48 hr with shaking in 20- x 150-mm glass tubes containing 5 ml of liquid medium (1x Vogel's salts, 1.5% sucrose, amino acid supplements) (VOGEL 1956).

DNA isolation and analysis:
DNA isolation and Southern blotting was performed as described previously (LUO et al. 1995). For traditional RFLP mapping, cosmids from the Orbach–Sachs cosmid library (ORBACH 1994) linearized with NotI restriction endonuclease (New England Biolabs), or DNA fragments generated by PCR, were radioactively labeled by random-primed DNA synthesis according to the manufacturer's instructions (Rediprime II DNA labeling system, GE Healthcare) in the presence of CTP[{alpha}-32P] (Amersham Biosciences). The map1 probe was amplified from genomic DNA using primers map1 fp (5'-TCGGTGGCATGGTCTTTGAGG-3') and map1 rp (5'-TTTGCTGGCGTTGTCTGCTCA-3). The map2 probe was amplified from genomic DNA using primers map2 fp (5'-AGCATATTGCATGGTATTTGA-3') and map2 rp (5'-CCGCTGCCAGATGTCGGAGAC-3'). The inl probe was generated using a SacI–BglII fragment from the plasmid pRATT09 (generously provided by Robert Pratt and Rodolfo Aramayo, Texas A&M University). The name and location of RFLP probes are listed in Table 1.


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  		TABLE 1 RFLP mapping probes

 
Microarrays:
N. crassa oligo arrays containing 10,990 70-mer oligonuculeotide probes were generated by the Neurospora Genome Project (KASUGA et al. 2005; DUNLAP et al. 2007; TIAN et al. 2007) and were obtained from the Fungal Genetics Stock Center (FGSC) (MCCLUSKEY 2003). A detailed description of the N. crassa microarrays can be found at http://www.yale.edu/twonsend/index.html. The slides were processed as described (FGSC, http://www.fgsc.net/ncrassa.html). Briefly, DNA was crosslinked by exposure to 600 mJ UV light in a UV crosslinker (Stratagene, La Jolla, CA). The slides were prehybridized at 42° in prehybridization buffer 5x SSC (MANIATIS et al. 1982), 0.1% sodium lauryl sulfate (SDS), and 0.1 mg/ml bovine serum albumin) for 1 hr and then washed three times in 0.1x SSC. To decrease autofluorescence, slides were incubated for 30 min at 42° in blocking buffer (2x SSC, 0.05% SDS, 0.25% sodium borohydride) and then washed three times in 0.1x SSC (RAGHAVACHARI et al. 2003). Slides were dried by centrifugation for 5 min at 800 rpm in a Beckman Allegra 25R centrifuge and used within 24 hr.

Isolation of RAD tags:
Isolation of RAD tags was perfomed as described (MILLER et al. 2007b). Samples of DNA (2 µg) isolated from strain N32 (Mauriceville) or strain N3312 (dim-7) were digested with EcoRI restriction endonuclease (New England Biolabs, Beverly, MA). The digested DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with ethanol and then resuspended in 9.75 µl of a reaction mix containing 1x T4 DNA ligase buffer and 1 µM of biotinylated EcoRI linker (generated by annealing primers 5'-biotin-TTCGACGCTCGCATCTGGACAGG-3' and 5'-phosphate-AATTCCTGTCCAGATGCGAGCGTC-3'; Integrated DNA Technologies). The mixture was incubated at 55° for 2 min and then at room temperature for 10 min. Next, 0.25 µl of T4 DNA ligase (200,000 units/ml; New England Biolabs) was added to each tube and the reaction was incubated at 16° for 2 hr. An aliquot (1 µl) of the reaction was fractionated by electrophoresis on a 0.8% agarose gel to verify successful ligation (evident when ligated linkers display slower mobility than linkers alone). The biotinylated DNA was then purified by extraction with phenol/chloroform/isoamyl alcohol and ethanol precipitation and resuspended in 500 µl of "low TE" buffer (10 mM Tris–HCl, 0.1 mM EDTA, pH 7.5). DNA was sheared by two rounds of sonication [10 pulses with 2 min on ice between each round of sonication (duty cycle 80, output 1.2) using a Branson sonicator 450 to yield an average DNA fragment size of ~1.5 kb]. The DNA was concentrated by ethanol precipitation and resuspended in 10 µl low TE buffer. Samples (1 µl) were assayed by gel electrophoresis to confirm that the digested DNA was sheared to the correct size. Next, the volume was increased to 100 µl by addition of low TE buffer. The biotinylated RAD tags were purified with streptavidin-coated magnetic beads (Dynabeads M280 streptavidin, Invitrogen, San Diego) according to the manufacturer's instructions. Briefly, a 50-µl volume of the bead suspension was equilibrated by washing three times with 2x binding and washing buffer (2x BW buffer, 10 mM Tris–Hcl, pH 7.5, 1 mM EDTA, 2.0 M NaCl). A total of 100 µl of 2X BW buffer was added to each 100-µl solution containing RAD tags. The solution was then added to the equilibrated beads and incubated for 15 min at room temperature with occasional mixing. The beads were washed three times with 1x BW solution and once with 1x low TE buffer. Following washing, the RAD tags were released from the beads. To accomplish this, the supernatant was removed and the beads were suspended in 100 µl 1x EcoRI buffer containing 5 units of EcoRI. After incubating the beads at 37° for 1 hr (mixing the bead suspension approximately every 15 min), the supernatant containing the RAD tags was transferred to a new tube. The RAD tags were purified by extraction with phenol/chloroform/isoamyl alcohol and ethanol precipitated. Next, the tags were suspended in 10 µl low TE buffer and 1 µl of each sample was examined on a gel to confirm that comparable concentrations of RAD markers had been isolated from each tube. A typical yield was 200–500 ng of RAD tags. Rather than amplify the tags by PCR, as in the original procedure, ~200 ng of purified RAD tags was labeled and hybridized to microarrays (see below). To perform bulk segregant analysis, RAD tags were isolated from pools of genomic DNA generated from Dim+ or Dim– strains in the manner described above.

Microarray hybridization:
Following purification of RAD tags, the tags from each strain (or pool) were differentially labeled by random-primed DNA synthesis with a Bioprime plus array CGH labeling kit (Invitrogen) using the procedure described previously (MILLER et al. 2007b). Briefly, 50-µl reactions containing 200 ng of purified RAD tags, 1.5 nmol of a Cy3- or Cy5-conjugated dCTP, random primers, 1x –dCTP reaction buffer, and 5 units of DNA polymerase Klenow fragment were incubated at 37° for 3–6 hr. Unincorporated dyes were removed with the Bioprime plus array purification module according to the manufacturer's instructions (Invitrogen). The differentially labeled samples were combined and dried in a Speedvac. The dried samples were resuspended in 42 µl of hybridization buffer (50% formamide, 5x SSC, 1% SDS, 1 µg/µl calf thymus DNA), boiled for 2 min, and hybridized to processed microarrays for ~18 hr at 42°. Hybridizations were carried out under 24- x 60-mm m-Series LifterSlips (Erie Scientific) in submerged hybridization chambers (Corning).

Data analysis:
Microarrays were scanned using a Genepix 4000a scanner (Axon). Fluorescence intensity data were analyzed using Genepix pro software (Axon). The data were median normalized (Genepix software). To determine the number of RAD markers that could be detected in the parental strains (the dim-7 Oak Ridge strain and the wild-type Mauriceville strain), two independent experiments were performed. In the first experiment, RAD tags isolated from the wild-type Mauriceville strain were labeled with Cy3, and RAD tags from the dim-7 (Oak Ridge) strain were labeled with Cy5. In the second experiment, the dyes were swapped. Spots were scored as reliable RAD markers if the median-of-ratios value indicated greater than twofold enrichment in either strain in both experiments. A Pearson's correlation coefficient was calculated for all spots that showed a log2 value ≥1 or ≤ –1 in either experiment. For bulk segregant analysis, the RAD tags isolated from pooled DNA showing the wild-type phenotype were labeled with Cy3 and the RAD tags isolated from pooled DNA showing the mutant phenotype were labeled with Cy5. Two independent experiments were performed. Spots that showed a twofold or greater enrichment in both parental experiments, in addition to both bulk segregant experiments, were scored as linked RAD markers. In all cases, enrichment values were displayed as the log2 (median of ratios). Relative enrichment values were determined by dividing the average log2 (median of ratios) value from the two bulk segregant experiments by the average log2 (median of ratios) value from the two parental hybridizations (the value from the second parental experiment was multiplied by –1 before averaging).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
We sought to develop a method to map mutations in N. crassa that would be both efficient and cost effective. To this end, we modified the RAD mapping procedure developed by MILLER et. al. (2007a,b) and demonstrated its utility by mapping the mutation in a new defective in methylation (dim) strain. The modified mapping procedure is illustrated in Figure 1. Briefly, DNA from two polymorphic strains was first digested with a restriction enzyme, EcoRI in this study. Following digestion of the DNA, biotinylated linkers containing EcoRI cohesive ends were ligated to the digested DNA. The DNA was sheared by sonication, yielding fragments of ~1.5 kb. The RAD tags were then affinity purified by incubation with streptavidin beads followed by release of the tags by digestion with EcoRI. Each pool of RAD tags was differentially labeled with either Cy3 or Cy5 dyes and hybridized to a N. crassa microarray. RAD tags that were common to both samples (associated with restriction sites that are separated by < ~1.5 kb) yielded yellow spots on the microarray whereas RAD tags that were unique to either strain (i.e., associated with restriction site polymorphisms) yielded red or green spots on the microarray, hereafter referred to as RAD markers.


Figure 1
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  		FIGURE 1.— Schematic of the RAD mapping procedure. Genomic DNA of two polymorphic strains is digested with a restriction enzyme. In this study, we used the dim-7 (N3312) strain, which is predominantly an Oak Ridge-type strain, and the polymorphic Mauriceville (N32) wild-type strain. Biotinylated linkers are then ligated to the restriction sites, the DNA is sheared, and the RAD tags are purified using streptavidin beads. The purified tags from each strain are differentially labeled with Cy3 or Cy5 and then hybridized to a microarray. Shared restriction sites generate yellow spots, whereas strain-specific restriction sites generate red or green spots.

 
Initially, we wanted to determine the number and genomic distribution of RAD markers that were detectable using the N. crassa oligonucleotide microarrays available to the community through the Fungal Genetics Stock Center (MCCLUSKEY 2003; KASUGA et al. 2005; DUNLAP et al. 2007; TIAN et al. 2007). We therefore performed a competitive hybridization with EcoRI–RAD tags purified from the previously unmapped dim-7 mutant (strain N3312), which was isolated in a strain containing predominantly Oak Ridge DNA and the commonly used polymorphic strain, Mauriceville (strain N32). (Isolation and characterization of dim-7 will be discussed in the future [Z. A. LEWIS, A. L. SHIVER, N. STIFFLER, M. R. MILLER, E. A. JOHNSON and E. U. SELKER, unpublished data.]) Hybridizations were performed for two experimental replicates and yielded highly reproducible data (Pearson's correlation coefficient, r = –0.87; Figure 2). The dyes were swapped in the second experiment to eliminate any dye bias. A microarray probe was scored as an informative RAD marker when the Cy5/Cy3 ratio indicated a twofold or greater enrichment of the RAD tag in either the dim-7 (Oak Ridge) or the Mauriceville strain in both experiments (see MATERIALS AND METHODS). Analysis of the microarray data revealed that 776 EcoRI–RAD markers were detected using this approach and that these markers were distributed across all seven N. crassa linkage groups (LGs) (Figure 3; supplemental Table 1 at http://www.genetics.org/supplemental/). On average, this is one marker every ~50,000 bp. Of the 776 markers, 487 were enriched in the Oak Ridge strain and 289 were enriched in the Mauriceville strain. The greater number of markers identified from the Oak Ridge strain is not surprising because a subset of the microarray probes are presumably complementary to polymorphic sequences from the Mauriceville strain, resulting in poor hybridization of the Mauriceville DNA to the array, which is based on the DNA sequence of the Oak Ridge strain (GALAGAN et al. 2003). In addition, since the Oak Ridge strain used in the experiment (dim-7) lacks all DNA methylation, some of the shared EcoRI sites may be methylated in the Mauriceville strain and unmethylated in the Oak Ridge strain. Since EcoRI is sensitive to cytosine methylation, these sites would yield Oak Ridge-specific RAD tags in our experiment.


Figure 2
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  		FIGURE 2.— RAD tags are reproducibly detected. The log2 (N3312 dim-7/N32 Mauriceville) values from the first parental experiment are plotted on the y-axis and the log2 (N32 Mauriceville/N3312 dim-7) values from the second experiment are plotted on the x-axis. The negative slope is a result of reciprocal ratios produced in the dye swap experiment. The Pearson's correlation coefficient (r = –0.87) was calculated for all spots that showed a log2 value ≤ –1 or ≥1.

 

Figure 3
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  		FIGURE 3.— Distribution of EcoRI–RAD markers across Neurospora chromosomes. Spots that showed a twofold or more enrichment in the dim-7 (Oak Ridge) or Mauriceville strains in two independent experiments were scored as informative RAD markers. The vertically arranged rectangles represent the seven N. crassa LGs. For each LG, dim-7-specific RAD markers are depicted as solid triangles on the right and Mauriceville-specific RAD markers are depicted as solid triangles on the left. Horizontal lines along the LG separate consecutive contigs. To generate a representation of EcoRI–RAD marker distribution, sequence files were assembled for each LG. Contig order was based on the physical map data available at the Neurospora gene list e-Compendium (http://www.bioinf.leeds.ac.uk/~gen6ar/newgenelist/genes/physical_maps.htm). Contigs that cannot be given an orientation (e.g., a "+" or "–" strand) are shaded. Contigs that cannot be assigned to a specific LG are labeled "unmapped" at the bottom right. Contigs that can be placed on an LG but not ordered within the LG are shown beneath LGs V, VI, and VII. The position of each RAD marker was determined by aligning the microarray probe sequence with the assembled sequence files using BLAST (ALTSCHUL et al. 1990). A Perl script was written to parse the BLAST data and draw an image for each LG.

 
The distribution of the RAD markers identified was not uniform throughout the genome. Certain regions appeared to have a higher density of markers while other regions were devoid of markers (e.g., compare the center of LG VII with the arms of LG VII). The pedigree of the dim-7 strain is complex and some Mauriceville DNA may be present in this strain. However, the dim-7 parent strains were crossed to an Oak Ridge strain three times before mutagenesis to ensure a predominantly Oak Ridge background. Moreover, dim-7 and its parent strains were found to be Oak Ridge compatible in heterokaryon tests (data not shown), suggesting a predominantly Oak Ridge background. The regions that are devoid of markers are likely regions of the dim-7 genome that contain residual Mauriceville DNA. Despite this, the marker density appeared sufficient to map the dim-7 mutation to a relatively small region. We therefore crossed dim-7 to the Mauriceville strain to generate recombinant progeny (cross AX1), isolated 59 progeny, and scored them for DNA methylation by Southern hybridization (data not shown). DNA from the Dim+ and Dim– strains was then pooled (31 and 28 strains, respectively), and RAD tags were isolated from each pool, differentially labeled, and used in a competitive microarray hybridization (see MATERIALS AND METHODS). To identify RAD markers that were linked to the dim-7 mutation, we identified microarray probes that displayed a Cy5/Cy3 ratio, indicating a twofold or greater enrichment in the pooled DNA from either Dim+ or Dim– progeny, reasoning that unlinked markers should be equally represented in both pools. We eliminated probes that exhibited a twofold enrichment in the Dim+ or Dim– pool but were not included in the 776 probes identified in the parental hybridizations. Finally, we generated a relative enrichment value for the remaining markers by dividing the value from the bulk segregant experiment by the value obtained in the parental experiment (see MATERIALS AND METHODS). Tightly linked markers were expected to have high relative enrichment values whereas more distant markers were expected to have low relative enrichment values.

Application of these criteria resulted in the identification of 31candidate dim-7-linked EcoRI–RAD markers (Table 2). Closer inspection of the data revealed that all 31 markers reside on linkage group V, placing dim-7 on this linkage group. Results from replicate experiments are shown in Figure 4A (also see supplemental Tables S2 and S3 at http://www.genetics.org/supplemental/). Again, the data were highly reproducible. In general, relative enrichment values were slightly lower in replicate 2 compared with replicate 1.


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  		TABLE 2 Enrichment of RAD markers in bulk segregant analysis

 

Figure 4
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  		FIGURE 4.— RAD mapping by bulk segregant analysis places dim-7 on LG V . RAD markers that showed a twofold or more enrichment in either the wild-type or the mutant pools are shown as black bars. The height of each bar indicates the relative enrichment of the RAD marker (bulk segregant experiment/parental). The RAD markers that did not show an enrichment of twofold or more in either the wild-type pool or the mutant pool are shown as gray triangles. The asterisk indicates the location of the NCU04209.1 array element. The 0.4-Mb bar indicates the location of dim-7 confirmed by traditional RFLP mapping. The numbers beneath the graph indicate contig numbers corresponding to the Neurospora Genome Sequence Assembly 7 (http://www.broad.mit.edu/annotation/genome/neurospora/Home.html). To generate the representation of the bulk segregant data, a sequence file for LG V was assembled. Contig order was based on the physical map data available at the Neurospora gene list e-Compendium (http://www.bioinf.leeds.ac.uk/~gen6ar/newgenelist/genes/physical_maps.htm). Contigs containing rDNA repeats were not included in the figure. Contigs 46, 88, and 91 cannot be assigned an orientation (± strand). The position of each RAD marker was determined by aligning the microarray probe sequence with the LG V sequence files using BLAST (ALTSCHUL et al. 1990). A Perl script was written to parse the BLAST data and draw the image. (A) The data from individual replicate experiments. (B) The average value from replicates 1 and 2. Only probes that showed a twofold or more enrichment in both replicate experiments are plotted as black bars in B.

 
We examined the relative enrichment values of the 31 RAD markers to identify those markers that were tightly linked to dim-7 (Figure 4B; Table 2). Several markers on contig 7.13 appeared to be tightly linked to the mutation on the basis of the following criteria: (1) The marker had a high relative enrichment value, (2) the adjacent markers had high relative values, and (3) nearly all the potential markers in the region were enriched in one pool or the other (Figure 4B; Table 2). The most notable variation between experiments occurred for markers that were not closely linked to dim-7 on the basis of the criteria outlined above. For example, markers on contigs 11 and 37 were enriched in replicate 1 but not in replicate 2. In contrast, the markers that were closely linked to dim-7 on the basis of these criteria showed the least variability (e.g., all but one marker in the center of contig 7.13 was enriched in both replicate experiments).

We next sought to verify the microarray mapping data using more traditional methods. First, we used PCR to amplify a 2-kb fragment of DNA that included an EcoRI site near the tightly linked Oak Ridge-specific EcoRI–RAD marker, detected by the array element NCU04209.1 (Tables 1 and 2). Next, we performed a Southern analysis of EcoRI-digested Oak Ridge and Mauriceville DNA using this fragment, called map1, as a probe. As expected, we were able to detect an RFLP, indicating that the EcoRI–RAD marker represents a strain-specific restriction site polymorphism (data not shown). To confirm the map location of dim-7, we examined the segregation of the map1 EcoRI RFLP in the recombinant progeny from the AX1 cross. Impressively, all 28 Dim– progeny contained the Oak Ridge-specific RFLP whereas all the 31 Dim+ progeny contained the Mauriceville RFLP, indicating tight linkage to this marker (data not shown).

We next used additional probes to analyze segregation of RFLPs surrounding this area in the AX1 progeny. We generated a second PCR product, called map2, adjacent to another dim-7-linked RAD marker detected by array element NCU04298.1 (Tables 1 and 2). The NCU04298.1 RAD marker displayed a lower relative enrichment value and neighboring RAD markers were not detected in the bulk segregant analysis experiment, suggesting that there was recombination between dim-7 and this marker. Indeed, we were able to identify 10 recombinants (of 59 total recombinant progeny) using map2 as a probe for detecting an EcoRI RFLP. We then used cosmid probes to narrow the region containing dim-7. The genotypes of the select recombinant progeny are shown in Table 3. All probes made between and including cosmid G21H5 and cosmid G25G6 detected RFLPs that showed complete linkage to the dim-7 mutation (Table 3). We conclude that dim-7 is in the 431-kb region flanked by cosmids G12F12 and G12B6, the closest probes giving recombinants. These results demonstrate the effectiveness of the RAD markers for recombination mapping in N. crassa.


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  		TABLE 3 RFLP mapping of the dim-7 mutation

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
We have developed an efficient, cost-effective method for detecting RAD markers across the entire N. crassa genome in a single microarray experiment. To demonstrate the utility of RAD markers for rapid mapping in Neurospora, we used the method for mapping dim-7, a previously unmapped mutation resulting in loss of DNA methylation. Importantly, this technique utilizes available resources (e.g., Neurospora oligonucleotide microarrays) to assay an unprecedented number of markers simultaneously.

The use of microarrays to simultaneously assay a large number of markers provides a significant advantage over other mapping methods. For example, many PCR-based markers (e.g., cleaved amplified polymorphic sequences, or CAPS) require the use of a unique primer set for each marker (KONIECZNY and AUSUBEL 1993). With such methods, an increase in map density corresponds to a significant increase in cost as well as labor. Using RAD mapping, one can approximately double the number of markers simply by hybridizing RAD tags produced with a second restriction enzyme to a second microarray. In principle, the number of markers that one can detect is limited only by the number of restriction enzymes and slides that one is willing to use. Moreover, unlike many molecular mapping techniques, detection of RAD markers is not limited to a single pair of strains. RAD mapping can be used to identify restriction site polymorphisms between any two polymorphic strains without a priori knowledge of the polymorphisms. This will allow Neurospora researchers to easily move beyond the standard Oak Ridge and Mauriceville wild-type strains to examine genetic variation in other natural isolates (TURNER et al. 2001). For example, the high density of RAD markers will facilitate analysis of quantitative trait loci (QTL) that contribute to phenotypic variation. The short generation time of Neurospora allows for easy construction of recombinant inbred lines that display the desired phenotype. One could easily use RAD markers to pinpoint the QTL that contribute to the phenotypic variation associated with the selected trait. It would also be practical to examine the segregation of RAD markers in pools containing DNA from recombinant F1 progeny that display distributional extremes for a trait of interest (TANKSLEY 1993; ABIOLA et al. 2003).

We observed some variability between the replicate bulk segregant experiments. However, the most significant variation between the two replicates occurred for markers that were not closely linked to the dim-7 mutation. This may result from variation in the sonication step between replicate experiments. If the average size of the RAD tags were reduced in the second replicate, a lower enrichment value for some RAD markers would be expected. Despite the subtle variation observed between replicates, the reproducibility of our experiments was sufficient to localize the dim-7 mutation and should be acceptable for most applications. Further replication and statistical analysis of variation could be used as additional criteria for identifying the most closely linked markers. While this is not necessary for identification of single-locus mutations, such analysis would be beneficial for QTL studies.

The use of readily available oligonucleotide arrays (KASUGA et al. 2005; DUNLAP et al. 2007; TIAN et al. 2007) ensures that RAD mapping can be easily employed by the entire Neurospora community. Given the low density of probes on the oligo array (one probe per gene), we generated relatively large tags (~1.5 kb). This modification of the original procedure will mask strain-specific restriction sites that are separated by <1.5 kb. Furthermore, restriction sites that are >1.5 kb away from a probe on the array will not be detected using the available microarray. Although these limitations could be avoided by using a tilling array that contains a higher density of probes accompanied by shearing the DNA to a smaller fragment size, the number of markers detected in our experiments is sufficient for most genotyping applications.

The description of 776 EcoRI–RAD markers represents a significant increase in map density for N. crassa. While the distribution of markers is not uniform (likely due to a small amount Mauriceville DNA in the dim-7 strain), the ability to assay such a large collection of sequence polymorphisms simultaneously allows for high-resolution mapping of a single gene mutation. We were able to rapidly map the dim-7 mutation to a small region using a bulk segregant analysis approach. The use of 59 recombinant progeny was sufficient to immediately identify the markers that showed 100% linkage to the mutation (0/59 ≤1.6 MU). Although map distance and physical distance can vary greatly across the genome (BOWRING and CATCHESIDE 1999), map-based cloning is certainly manageable with this resolution, even in regions that exhibit relatively low recombination rates (such as the region that harbors dim-7).

The use of larger numbers of progeny may further increase the resolution of mapping by bulk segregant analysis, but is not necessary for identification of closely linked RAD markers. In this study, we used RFLP markers to confirm and finely map the location of the dim-7 mutant. This represented the most labor-intensive stage of the mapping process. An increase in the number of progeny would be beneficial at this stage by providing a larger number of recombinants for pinpointing the location of the mutation. In this study, we were able to localize dim-7 to a region of <431 kb. If one were to double the number of progeny, it is likely that this distance would be reduced further.

Neurospora has been the subject of numerous genetic studies over the past half-century. As a result, a large collection of mutant Neurospora strains that display defects in all aspects of the fungal life cycle exists and is readily available to the community (PERKINS et al. 2001; MCCLUSKEY 2003). Many of these mutations have been mapped at low resolution to chromosomes. The 776 putative EcoRI restriction site polymorphisms reported here can be readily adapted as RFLP or CAPS markers for higher-resolution mapping of these mutations.

RAD genotyping is a facile tool that will aid genetic analysis of Neurospora and can be easily applied to other filamentous fungi. Moreover, this technique can be easily applied to a wide range of organisms for which microarrays are available. This technique will promote identification of mutant loci as well as facilitate genetic analysis at the individual and population level.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
This work was supported by grants to E.U.S. from the National Institutes of Health (GM025690-22) and the National Science Foundation (MCB-0121383). Z.A.L. is supported by a postdoctoral fellowship from the American Cancer Society (PF-04-122-01-GMC).


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

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Communicating editor: M. S. SACHS





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