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. 2010 Sep;186(1):67–77. doi: 10.1534/genetics.110.117408

Accidental Amplification and Inactivation of a Methyltransferase Gene Eliminates Cytosine Methylation in Mycosphaerella graminicola

Braham Dhillon *,1, Jessica R Cavaletto †, Karl V Wood ‡, Stephen B Goodwin †,2
PMCID: PMC2940312  PMID: 20610411

Abstract

A de novo search for repetitive elements in the genome sequence of the wheat pathogen Mycosphaerella graminicola identified a family of repeats containing a DNA cytosine methyltransferase sequence (MgDNMT). All 23 MgDNMT sequences identified carried signatures of repeat induced point mutation (RIP). All copies were subtelomeric in location except for one on chromosome 6. Synteny with M. fijiensis implied that the nontelomeric copy on chromosome 6 served as a template for subsequent amplifications. Southern analysis revealed that the MgDNMT sequence also was amplified in 15 additional M. graminicola isolates from various geographical regions. However, this amplification event was specific to M. graminicola; a search for MgDNMT homologs identified only a single, unmutated copy in the genomes of 11 other ascomycetes. A genome-wide methylation assay revealed that M. graminicola lacks cytosine methylation, as expected if its MgDNMT gene is inactivated. Methylation was present in several other species tested, including the closest known relatives of M. graminicola, species S1 and S2. Therefore, the observed changes most likely occurred within the past 10,500 years since the divergence between M. graminicola and S1. Our data indicate that the recent amplification of a single-copy MgDNMT gene made it susceptible to RIP, resulting in complete loss of cytosine methylation in M. graminicola.

MYCOSPHAERELLA graminicola (anamorph: Septoria tritici) is the causal organism of Septoria tritici blotch (STB), which is one of the most important diseases of wheat worldwide (Eyal et al. 1985). In addition to Europe and the United States, infection is very severe in other wheat-growing countries in the Mediterranean, East Africa, and Australia, causing significant reductions in yield and quality. Control of STB is compounded by the development of resistance in M. graminicola to the benzimidazoles (Fisher and Griffin 1984) and strobilurins (Fraaije et al. 2005), two classes of fungicide with different modes of action.

Knowledge about pathogen biology, epidemiology, and other related physiological processes could facilitate the design of better strategies for pathogen control and reduction of disease losses. A key resource to better understand these processes is the availability of a genome sequence. Improvements in sequencing technologies and the relatively small sizes of fungal genomes have facilitated a substantial increase in the number of species sequenced during the last few years.

An important feature found in most sequenced genomes (IHGSC 2001; Galagan et al. 2003) is the presence of transposable elements (TEs), DNA fragments that can move to new locations in the host genome. Autonomous TEs code for proteins for their own movement and also can mobilize similar nonautonomous elements that have lost their protein-coding capacity. Due to their ability to mobilize and increase their copy number, TEs may occupy up to 21% of some fungal genomes (Martin et al. 2008). TEs may be beneficial or detrimental to their host genomes. For example, in Drosophila, telomeres are maintained by HeT-A and TART retrotransposons (Pardue and Debaryshe 2003). Alternatively, insertions of TEs into genes can eliminate gene function or lead to the production of chimeric or antisense transcripts, thereby modifying gene expression (Feschotte 2008). In addition to insertional mutagenesis, TEs can contribute to chromosomal rearrangements by facilitating ectopic recombination (Wessler 2006). To minimize their deleterious effects, host genomes have evolved various strategies to minimize the movement of TEs, one of which involves DNA methylation.

DNA cytosine methylation occurs in many well-studied eukaryotes, with a few model organisms, such as the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, and the nematode Caenorhabditis elegans being the notable exceptions (Colot and Rossignol 1999). DNA methylation is catalyzed by a conserved set of proteins called DNA methyltransferases (DNMTs), which usually add a methyl group to cytosine. DNA methylation in plants, mammals, and the filamentous fungus Neurospora crassa is mostly concentrated on repetitive elements (Rabinowicz et al. 1999) and, at least in some cases, limits their movement. Conversely, genome-wide demethylation in plants has been shown to increase the rate of TE activity (Miura et al. 2001; Singer et al. 2001). Numerous qualitative and quantitative methods are available to measure DNA methylation, either for a specific locus or on a global scale. Liquid chromatography coupled with electrospray ionization tandem mass spectrometry (ESI–MS/MS) has been used to determine the whole-genome methylation status of human cell lines with very high sensitivity (Song et al. 2005).

Another genome-defense mechanism, which is strictly limited to fungi, is repeat induced point mutation (RIP). RIP, discovered in N. crassa, was the first genome-defense system to be described in eukaryotes (Selker et al. 1987; Galagan and Selker 2004). During the sexual phase, RIP introduces C:G to T:A mutations in both copies of duplicated sequences >400 bp (Watters et al. 1999). These transition mutations often introduce stop codons, thus inactivating gene expression. Linked, as well as unlinked, dispersed repetitive sequences, including genes, are equally likely to be inactivated, although their genomic location may influence their susceptibility to RIP. In N. crassa, an ectopic insertion led to the duplication of an otherwise single-copy gene, cya-8 (cytochrome aa3 deficient), which then was targeted and mutated by RIP (Perkins et al. 2007). Up to 30% of C:G pairs in duplicated sequences can be mutated during a single sexual cycle (Cambareri et al. 1989). RIP-mutated sequences are frequently methylated in N. crassa (Galagan et al. 2003).

In N. crassa, one known DNMT, DIM-2 (defective in methylation-2) (Foss et al. 1993), and one putative DNMT, RID (RIP defective) (Freitag et al. 2002), have been characterized genetically. All apparent DNA methylation is lost in dim-2 mutants without causing growth defects (Foss et al. 1993), whereas mutations in the rid gene eliminate RIP completely. RIP has been characterized extensively in N. crassa but no report is available for M. graminicola. Although direct evidence is lacking in M. graminicola, repetitive sequences show characteristics of RIP, such as high ratios of transitions to transversions and low GC content (Galagan and Selker 2004).

Due to its economic importance and genetic tractability, the genome of M. graminicola was sequenced to completion by the Joint Genome Institute (JGI) of the U.S. Department of Energy (http://genome.jgi-psf.org/Mycgr3/Mycgr3.home.html). It is the first genome of a filamentous fungus to be finished according to recently proposed standards (Chain et al. 2009); i.e., all 21 chromosomes except the smallest one have been sequenced completely from telomere to telomere with only two gaps of unclonable DNA. We characterized the repetitive sequences in the finished M. graminicola genome to search specifically for high-copy-number gene families as potential targets of RIP. Our aim was to test whether genes or gene fragments have been amplified in the genome and how this amplification could be influenced by RIP. We found an example where a single-copy DNMT gene had been amplified in the M. graminicola genome. This gene-amplification event was most likely followed by RIP-mediated gene inactivation and a concomitant species-wide loss of methylation in M. graminicola.

MATERIALS AND METHODS

Identification and characterization of repetitive sequences:

Sequences of two closely related fungi, the wheat pathogen M. graminicola and the banana pathogen M. fijiensis, were used in this study. The 21 chromosomes of M. graminicola isolate IPO323 have been sequenced completely (JGI, v.2.0, http://genome.jgi-psf.org/Mycgr3/Mycgr3.home.html) while for M. fijiensis, a 7.1× draft sequence is available (JGI, v.1.0, http://genome.jgi-psf.org/Mycfi1/Mycfi1.home.html).

RECON version 1.05 (Bao and Eddy 2002) was used to identify repetitive elements de novo in the M. graminicola genome. During characterization of these repetitive sequences, a repeat family was identified, designated as “fam1,” members of which contained a protein sequence similar to a DNA methyltransferase (MgDNMT). Fam1 elements were further searched for protein domains generally associated with repetitive elements, using BLASTX (Altschul et al. 1997) against the NCBI nonredundant protein database (nr) at e−5, and for structural features such as terminal inverted repeats (TIR) using “Einverted” (http://emboss.sourceforge.net/apps/release/6.2/emboss/apps/einverted.html) and tandem repeats using tandem repeat finder (TRF) version 4.0 (Benson 1999).

Identification and phylogenetic analysis of Dim2-like genes in other fungal genomes:

Sequences similar to the N. crassa DIM-2 gene were identified and copy number was determined in 10 other fungal species: Botryotinia fuckeliana, Coccidioides immitis, Gibberella zeae, Magnaporthe oryzae, Phaeosphaeria nodorum, Pyrenophora tritici-repentis, Sclerotinia sclerotiorum, Podospora anserina, M. fijiensis, and M. graminicola relative S1. The genome sequences for these species were available at http://www.broad.mit.edu/annotation/fgi/, http://podospora.igmors.u-psud.fr/ and http://genome.jgi-psf.org/Mycfi1/Mycfi1.home.html. DIM-2-like sequence from the M. graminicola relative S1 was provided by E. Stukenbrock. Including the Dim-2 gene from N. crassa and the “RIPed” and “deRIPed” M. gramincola sequences, 13 DIM-2-like sequences were aligned using ClustalX version 2.0 (Thompson et al. 1997) and a neighbor-joining (Saitou and Nei 1987) tree was constructed using a 100-replicate bootstrap analysis.

RIP and deRIP in MgDNMT:

To quantify the transitions induced by RIP, DNA sequences of all fam1 elements were aligned using ClustalX version 2.0 (Thompson et al. 1997) and were edited manually using Jalview version 2.3 (Clamp et al. 2004). DeRIPing was done by comparing the base composition at each nucleotide position of aligned sequences and deducing the original sequence prior to RIP. For this analysis, the only nontelomeric MgDNMT sequence, on chromosome 6, was used as a reference. At each polymorphic base position in the alignment, any thymine (T) residue in the reference sequence was changed to a cytosine (C), if any of the other sequences had a corresponding C. Similarly, an adenine (A) in the reference sequence was changed to a guanine (G) when a “G” was present among other sequences in the alignment. This corrected for RIP on both strands of the DNA sequence. Remaining internal stop codons were corrected manually by inserting transitions that changed the stop to an amino acid, usually glutamine (Q), when compared to the S1 DNMT copy. These stops were assumed to be at sites that were RIPed in all the copies and were no longer polymorphic.

Southern hybridization for estimation of copy number:

Sixteen isolates were used for this analysis. Fifteen isolates of M. graminicola from seven countries on five continents, including Turkey (isolates IPO86013 and IPO86022), Argentina (IPO86068), Uruguay (IPO87016, IPO87019), Ethiopia (IPO88004, IPO88018), The Netherlands (IPO001, IPO235, IPO89011, IPO323), Australia (Paskeville), and the United States (I1A.1, I1A.3, ST2), and one isolate of S. passerinii (P77) were used. These isolates were grown in liquid culture as described previously (Goodwin et al. 2001). Mycelia were collected and lyophilized before DNA extraction (DNeasy Plant Mini Kit; QIAGEN, Valencia, CA). Southern analysis was done using alkaline transfer (Sambrook and Russell 1989), and chemiluminescence was used to detect the DIG-labeled MgDNMT probe (Roche Diagnostics, Indianapolis). The probe was designed from the nontelomeric MgDNMT reference sequence on chromosome 6.

Determination of syntenic regions for MgDNMT:

To determine synteny, 50 kb of sequence flanking the MgDNMT sequence was compared to the homologous sequences in M. fijiensis, P. tritici-repentis, P. nodorum, and S. sclerotiorum. Graphic visualization of genomic comparisons was done using the Artemis Comparison Tool (ACT) Release 8 (Carver et al. 2005). This tool was used to display similarity at the nucleotide level even though at the amino acid level the similarity was much higher.

Analysis of genome-wide cytosine methylation:

Three isolates of M. graminicola from different geographical regions (IPO323, IPO86068, Paskeville), four isolates from two new, undescribed species of Mycosphaerella from wild grasses (species S1, isolates ST04IR-221 and ST04IR-431; and S2, isolates ST04IR-111 and ST04IR-3131), three isolates of M. fijiensis (IPO139a, IPO8837, rCRB2), and one isolate of S. passerinii (P63) were used. DNA was hydrolyzed as described previously (Song et al. 2005). This mixture was analyzed by electrospray ionization tandem mass spectrometry. Briefly, 5 μg of genomic DNA was denatured by heating to 100° for 3 min before chilling on ice, followed by addition of 1/10 vol of 0.1 m ammonium acetate (pH 5.3) and 2 units of nuclease P1 and incubation at 45° for 2 hr. Next, 1/10 vol of 1 m ammonium bicarbonate and 0.002 unit of venom phosphodiesterase I were added and the mixture was incubated at 37° for 2 hr. The final incubation was at 37° for 1 hr after addition of 0.5 unit of alkaline phosphatase.

All ESI analyses were carried out on a Finnigan MAT LCQ Classic mass spectrometer system (ThermoElectron, San Jose, CA). The electrospray needle voltage and the heated capillary voltage were set to 4.0 kV and 10 V, respectively. The capillary temperature was set at 207° and the typical background source pressure was 1.2 × 10−5 torr. The sample flow rate was ∼8 μl per minute. The drying gas was nitrogen. The LCQ was scanned to 1000 amu for these experiments. The sample was dissolved in methanol and water.

The MS/MS results were obtained by selecting the ion of interest (the precursor ion). The precursor ion was then subjected to collision-induced dissociation (CID) resulting in the formation of product ions. Helium was introduced into the system to an estimated pressure of 1 mtorr to improve trapping efficiency and also acted as the collision gas during the CID experiments. The collision energy was set to 40% of the maximum available from the 5-V tickle voltage, with a 2-mass-unit isolation window.

RESULTS

Characterization of fam1 repeats:

Repeat analysis of the finished sequence of the M. graminicola genome (http://genome.jgi-psf.org/Mycgr3/Mycgr3.home.html) with RECON (Bao and Eddy 2002) identified 106 families of repetitive elements. One of these, family 1 (fam1) had a total of 16 elements on seven chromosomes (supporting information, Figure S1) with the longest element, ele9870 at ∼9.7 kb long. All fam1 elements except for one on chromosome 6 were located in subtelomeric regions. No other distinguishing structural features associated with TEs such as direct or inverted repeats were found in the elements of this family.

A search for protein domains in fam1 elements identified a DNA cytosine methyltransferase protein (MgDNMT), which is atypical for repetitive elements. This MgDNMT belongs to a class of proteins represented by DIM-2, a genetically well-characterized protein from N. crassa. After searching the M. graminicola genome with the N. crassa DIM-2 (NcDim-2) sequence, 12 additional copies of the MgDNMT-containing repeats were identified, also at subtelomeric locations. Of the 28 fam1 elements distributed on 17 chromosomes, the MgDNMT region was present in 23 (Table S1). However, all elements lacked the usual protein-coding regions commonly associated with TEs such as transposases, reverse transcriptases, or helicases.

Determining the original MgDNMT region and synteny with other genomes:

To determine how the number of MgDNMT sequences increased in the genome, it was essential to identify the original MgDNMT sequence. One interesting candidate for the original “donor” sequence was the 4.4-kb nontelomeric copy present on chromosome 6. Sequences flanking the element on chromosome 6 were unique, suggesting that this 4.4-kb sequence is the possible source for the MgDNMT region in fam1 elements. This comparison helped delimit the extent of the genomic fragment that had become a part of the fam1 repetitive elements.

A search of the genome of M. fijiensis, a relative of M. graminicola, using the MgDNMT sequence identified a single-copy homolog on scaffold 1. Fifty kilobases of genomic sequence flanking all fam1 elements was used to search the M. fijiensis scaffold 1 sequence to identify possible syntenic regions. The only region in M. graminicola conserved for gene number and order between the two genomes was the nontelomeric region on chromosome 6. Two genes present upstream of the MgDNMT sequence, one similar to a UsgS transmembrane protein and the other similar to a conserved hypothetical protein, were present in M. fijiensis but in reverse orientation (Figure 1). In M. graminicola these genes are ∼0.4 kb apart, whereas in M. fijiensis they are separated by a 4.4-kb repetitive element. Similarly, downstream of the MgDNMT sequence is a C6 zinc finger domain-containing protein that is present at the same location in M. fijiensis (Figure 1). Therefore, the four genes in this region appear to be syntenic between M. graminicola and M. fijiensis but not collinear. All three genes surrounding the MgDNMT sequence are specific to fungi.

Figure 1.—

Figure 1.—

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Synteny between Mycosphaerella graminicola and M. fijiensis in the MgDNMT region. One hundred kilobases of sequence on chromosome 6 from M. graminicola and scaffold 1 from M. fijiensis were compared to test for synteny of gene content and order between the two genomes. Graphs at the top and bottom show the percentage of GC for M. graminicola and M. fijiensis, respectively. Positions and orientations of genes are indicated in the tracks for each region. Syntenic genes are linked by lines indicating similarity at the nucleotide level. The shaded regions in the percentage of GC panels show a sharp decrease in GC content in the MgDNMT region of M. graminicola as compared to M. fijiensis.

The four genes shared between M. graminicola and M. fijiensis were present in P. tritici-repentis, P. nodorum, and S. sclerotiorum but were not syntenic. This synteny between M. fijiensis scaffold 1 and the MgDNMT sequence on M. graminicola chromosome 6 strengthens the possibility that the nontelomeric copy on chromosome 6 was the original template for subsequent amplifications.

Amplification of MgDNMT in M. graminicola:

Only one match to the NcDIM-2 protein was found in the genome sequences of 11 phylogenetically diverse species (Figure 2) compared to the 23 copies identified in M. graminicola. The RIPed copy of the MgDNMT sequence was on a much longer branch reflecting its greater rate of change compared to the deRIPed version. Southern analysis using the chromosome 6 MgDNMT sequence as probe revealed multiple copies in 15 M. graminicola isolates from various geographical regions (Figure 3). Higher intensity of some bands, especially in lanes 1 (IPO86013) and 2 (IPO86022), may represent multiple copies, which were either similar in size or too large to be resolved on the gel (Figure 3). Lanes 3 (IPO86068) and 10 (I1A.1) contained much less DNA and showed either no or very little hybridization. However, an overexposure revealed that these isolates had at least five bands. No hybridization with the MgDNMT sequence was visible in a closely related fungus, the barley pathogen S. passerinii, even after overexposure.

Figure 2.—

Figure 2.—

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Phylogenetic relationships among DIM-2-like genes in Mycosphaerella graminicola and 11 other ascomycete species. A neighbor-joining tree is shown for 9 phylogenetically diverse species, along with RIPed and deRIPed M. graminicola sequence, M. fijiensis, and species S1, using 100 bootstrap replicates. Sequences corresponding to the species names are as follows: CIMG_01762, Coccidioides immitis; PODANSg1750, Podospora anserina; FG10766, Gibberella zeae; BC1G_12419, Botryotinia fuckeliana; MGG_00889, Magnaporthe oryzae; SNOG_03039, Phaeosphaeria nodorum; PTRG_02280, Pyrenophora tritici-repentis; SS1G_07976, Sclerotinia sclerotiorum; and NCU02247, Neurospora crassa. Bootstrap values were ≥85% and are indicated at the nodes.

Figure 3.—

Figure 3.—

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Southern analysis of MgDNMT in Mycosphaerella graminicola. The MgDNMT probe highlights a number of bands in each lane. Lanes 3 and 10 show weak hybridization, but this was mostly due to unequal loading and an overexposure revealed multiple bands. Geographic regions and isolates in order of loading are as follows: Turkey, IPO86013 and IPO86022; Argentina, IPO86068; Uruguay, IPO87016 and IPO87019; Ethiopia, IPO88004 and IPO88018; The Netherlands, IPO001 and IPO235; United States, I1A.1 and ST2; Australia, Paskeville; and United States, I1A.3. Also tested but not shown were IPO89011 (The Netherlands), IPO323 (Netherlands isolate that was sequenced), and S. passerinii P77. A 1-kb ladder (lane M) was used as a size standard. The scale at the bottom shows the MgDNMT region from which the probe was designed. The arrows mark the flanking PstI restriction enzyme sites.

Subtelomeric location of fam1 elements:

Each fam1 element contained sequences similar to one or both of the two longest elements, ele799 or ele9870 (Figure S1). On two pairs of chromosomes, 2 and 17 (Figure 4) and 9 and 21, fam1 elements were duplicated and formed complex repetitive structures at the subtelomeric locations. Comparisons of these regions revealed a distinct arrangement of ele799-like and ele9870-like elements with the MgDNMT region sandwiched between them.

Figure 4.—

Figure 4.—

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Comparison of fam1 subtelomeric repeats on chromosomes 2 and 17 of Mycosphaerella graminicola. Similar sequences on chromosomes 2 and 17 are connected by straight (A) or slanting (B) lines. Locations of ele799- and ele9870-like elements and the MgDNMT sequence are indicated by boxes with dark shading, hatch marks, and horizontal lines, respectively. Telomeres are indicated by circles. Chromosome positions are indicated in kilobases. GC content plots are shown above and below each chromosome.

Pairwise comparisons of 23 MgDNMT sequences pointed at one possible mechanism of MgDNMT sequence amplification in the genome. Two nearly identical sequences (2.4 kb each with 99% identity) were found at the same chromosomal position, adjacent to the telomeric repeats, one each on chromosomes 9 and 21. The two sequences differ by seven transitions and one transversion mutation, of which six caused synonymous amino acid substitutions. Other MgDNMT sequences showed transition mutations when compared to these two. A 99% identity between 2 MgDNMT copies and significant differences from the other 21 copies implies that amplification may have occurred recently, subsequent to mutagenesis by RIP. The most likely explanation is that after accumulating mutations, one sequence could have been copied to a new location via reciprocal translocation between the subtelomeres.

Juxtaposition of an LTR retrotransposon fragment:

Repetitive sequences flanking the MgDNMT region in fam1 elements were devoid of any repeat-associated protein domains or characteristic structural features commonly associated with TEs. To test for other similarities to TEs, these flanking sequences were used to search the repetitive sequence library from M. graminicola. A 220-bp fragment immediately upstream of the MgDNMT sequence was similar to a noncoding region from a long terminal repeat (LTR) retrotransposon family (fam15) (Figure S2). In the LTR retrotransposon sequence, the 220-bp fragment was present immediately upstream of the 3′ LTR. This suggests that at some subtelomeric location, MgDNMT sequence was adjacent to a LTR retrotransposon. This similarity also hints at the possibility that the MgDNMT sequence might have been acquired by a LTR retrotransposon and then moved to a telomeric location.

RIP and deRIP in MgDNMT:

Multiple copies of the MgDNMT sequence in the M. graminicola genome make it a likely candidate for RIP. To determine the extent of RIP, the original chromosome 6 MgDNMT sequence was deRIPed by replacing a T with a C, or an A with a G, at positions showing C/T or A/G polymorphisms, respectively, and compared to the remaining MgDNMT copies. The number of transversion mutations between the MgDNMT copies and the deRIPed version of MgDNMT sequence ranged from 0 to 11 per kilobase of sequence compared (Table 1). Two subsets of sequences were present, one with greater than four transversions per kilobase (15 sequences) and the other with fewer than two transversions per kilobase (7 sequences). The sequences with the higher numbers of transversions included all of the full-length MgDNMT sequences, whereas in the other set all sequences were truncated.

TABLE 1.

Pairwise comparison of MgDNMT sequences to the deRIPed nontelomeric sequence on chromosome 6 of Mycosphaerella graminicola

No. Chromosome Element Length Tia Tva Ti/Tv Ti/kb Tv/kb
1 8 1_24 1710 174 19 9.2 101.8 11.1
2 2 1_08 4419 330 29 11.4 74.7 6.6
3 2 1_09 4419 331 29 11.4 74.9 6.6
4 17 1_11 4422 306 26 11.8 69.2 5.9
5 17 1_12 4416 176 25 7.0 39.9 5.7
6 5 1_14 4419 247 25 9.9 55.9 5.7
7 1 1_02 4422 467 25 18.7 105.6 5.7
8 15 1_10 4422 309 24 12.9 69.9 5.4
9 21 1_16 4422 279 24 11.6 63.1 5.4
10 1 1_01 4422 472 23 20.5 106.7 5.2
11 1 1_03 4076 345 21 16.4 84.6 5.2
12 1 1_04 4078 341 21 16.2 83.6 5.1
13 6 1_18 4419 227 22 10.3 51.4 5.0
14 5 1_15 4244 327 21 15.6 77.0 4.9
15 1 1_06 453 10 2 5.0 22.1 4.4
16 12 1_21 2271 202 3 67.3 88.9 1.3
17 9 1_23 2429 37 3 12.3 15.2 1.2
18 21 1_17 2429 34 2 17.0 14.0 0.8
19 18 1_25 1478 109 1 109.0 73.7 0.7
20 1 1_05 2221 75 1 75.0 33.8 0.5
21 17 1_13 2564 53 1 53.0 20.7 0.4
23 16 1_22 2136 109 0 — 51.0 0.0
24b 6 1_19_20 4331 435 0 — 100.4 0.0
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All sequences were sorted by transversions per kilobase in decreasing order.

a

Ti, transitions; Tv, transversions.

b

The original RIPed copy on chromosome 6.

Transition mutations varied from 14 to 107 per kilobase of sequence analyzed. These transitions resulted in 61 stop codons in the putative coding region of the nontelomeric MgDNMT sequence (Figure S3). All fam1 elements, except two, had stop codons that were caused by C → T and G → A transitions. Because the two remaining fam1 elements were truncated, no functional copy of the MgDNMT sequence appears to be present in the M. graminicola genome. Moreover, no sequences corresponding to MgDNMT were identified in the M. graminicola EST data set. In contrast, the single-copy dim-2 homologs in the genomes of M. fijiensis and the other fungal species analyzed were complete with no evidence of RIP or stop codons.

Searching the genome sequence of species S1, the closest known relative of M. graminicola (Stukenbrock et al. 2007) with the deRIPed MgDNMT copy along with its flanking sequence identified only one copy of the MgDNMT-like sequence. On the basis of comparisons to its putative homolog in S1, the 4.4-kb chromosome 6 donor sequence most likely contains the complete predicted DNMT sequence coding for 1281 amino acids.

After adjusting the nucleotide polymorphisms for RIP, the chromosome 6 MgDNMT sequence still had eight stop codons that were resolved by comparison to the S1 sequence. This final deRIPed MgDNMT sequence showed an improvement in significance value of BLAST searches to the NCBI “nr” database from e−52 to e−175. The expected translation product of the deRIPed MgDNMT sequence shows significant similarity to other fungal DIM-2-like proteins, but the N-terminal and C-terminal ends were of different lengths and did not align (Figure S4).

Recent loss of cytosine methylation in M. graminicola:

N. crassa DIM-2, a MgDNMT homolog, is responsible for all of the known cytosine methylation in that organism (Kouzminova and Selker 2001). To determine whether RIP of the MgDNMT sequence affected DNA methylation of M. graminicola, a genome-wide DNA methylation assay was conducted using ESI–MS/MS.

Under the assay conditions, the presence of a specific ion can be confirmed from its fragmentation products; i.e., loss of the dehydrated deoxyribose sugar residue from 5-methyl deoxycytidine (5mdC) will give rise to 5-methyl cytosine (5mC). This can be detected qualitatively in ESI–MS/MS spectra by the presence of an ion at mass-to-charge ratio (m/z) 126 (after loss of dehydrated deoxyribose), in the MS/MS spectrum of m/z 242 (protonated 5mdC). The control MS/MS spectra of m/z 228, protonated cytidine (dC), for both M. fijiensis (Figure 5A) and M. graminicola (Figure 5B) show a similar fragment ion at m/z 112 indicating a loss of dehydrated deoxyribose. However, differences were observed in the MS/MS spectra of m/z 242. In M. fijiensis (Figure 5C), the fragment ion at m/z 126 (5mC) is clearly visible. However, this fragmentation ion is absent in the MS/MS spectrum of m/z 242 from M. graminicola (Figure 5D). Therefore, as compared to M. fijiensis, cytosine methylation is absent from M. graminicola. The cytosine methylation profile of the closely related barley pathogen S. passerinii was similar to that for M. fijiensis.

Figure 5.—

Figure 5.—

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Comparison of ESI–MS/MS mass spectra of Mycosphaerella fijiensis (A and C) and M. graminicola (B and D) genomic DNA. Product ion mass spectra, m/z 228 and m/z 242 of deoxycytidine (A and B) and 5-methyl deoxycytidine (C and D), and their fragmentation products following loss of dehydrated deoxyribose at m/z 112 and 126, respectively, are shown. At least 10 scans were averaged. Absence of the m/z 126 peak in M. graminicola (D) is indicated by a downward-pointing arrow. The fragment ion at m/z 242 can represent other ions, but the transition from m/z 242 to m/z 126 comes only from 5-methyl cytosine. The fragment ions at m/z 187 and 215 correspond to unknowns that are present in DNA of both species.

Isolates of the recently discovered, unnamed species S1 and S2 from uncultivated grasses in Iran also were assayed for cytosine methylation. Cytosine methylation was present in both S1 and S2, which are thought to have diverged from M. graminicola ∼10,500 and 20,000 years ago, respectively (Stukenbrock et al. 2007). Therefore, loss of cytosine methylation in M. graminicola probably happened after its divergence from S1 within the past 10,500 years.

DISCUSSION

Multiple copies of a DNA methyltransferase (MgDNMT) sequence at subtelomeric positions in the M. graminicola genome, all marred with signatures of RIP, is unprecedented. A DNMT domain is not a component of known repetitive elements but, due to its high copy number, the MgDNMT sequence was recognized de novo as a repetitive sequence by RECON. The species-wide presence of this amplification event in M. graminicola was suggested by the occurrence of multiple copies of MgDNMT sequence in 15 isolates from diverse geographical regions. However, only one copy was present in the genome sequence of the closely related species S1 and 10 other ascomycete fungi, so the expansion seems to be recent and unique to M. graminicola.

RIP protects fungal genomes from the detrimental effects of repetitive elements (Galagan and Selker 2004). The only gene known to be involved in RIP is the RID gene from N. crassa, which also is a putative DNMT (Freitag et al. 2002). However, the exact role that RID plays in RIP is unknown. DIM-2, on the other hand, is not required for RIP (Kouzminova and Selker 2001; Freitag et al. 2002). The genomic sequence of M. graminicola contains a putative ortholog of the rid gene. It is single copy and predicted to encode a protein of 742 amino acids, suggesting that RIP is functional in M. graminicola.

Another feature of RIP-induced transitions is a decrease in GC content of the affected sequences. A corresponding decrease in percentage of GC was notable in the DNMT region in M. graminicola as compared to M. fijiensis (Figure 1). Substituting the nucleotides A/T with G/C at polymorphic sites of aligned sequences increased the percentage of GC content of the deRIPed MgDNMT sequence from 43 to 53. Transitions introduced by RIP inactivated all MgDNMT copies in the genome, leading to a loss of cytosine methylation. The absence of cytosine methylation in M. graminicola was alluded to in a previous analysis based on methylation-sensitive and -insensitive restriction enzymes (Goodwin et al. 2001). However, lack of cytosine methylation is not unique among fungi; many yeasts, such as S. cerevisiae and S. pombe, lack detectable methylation (Colot and Rossignol 1999). Moreover, methylation is not essential in N. crassa, as dim-2 mutants are viable. These mutants show no obvious phenotype under laboratory conditions despite genome-wide demethylation (Kouzminova and Selker 2001). Therefore, DNA methylation is not required in all organisms and the lack of a functional copy of DNMT has no obvious fitness cost in M. graminicola.

In the absence of DNA methylation, histone methylation might be responsible for the silencing of repetitive DNA. In N. crassa, the dim-5 gene is responsible for methylation of histone H3 at the lysine 9 residue (H3K9). Cytosine methylation depends upon prior methylation of H3K9 residues (Tamaru and Selker 2001) and binding of H3K9me by heterochromatin protein 1 (hpo gene) (Freitag et al. 2004). Homologs for both dim-5 and the hpo gene are present in M. graminicola.

Identification of the original MgDNMT sequence was facilitated by synteny with M. fijiensis. However, synteny was nonexistent with other less closely related fungi such as P. nodorum, P. tritici-repentis, and S. sclerotiorum. This is not surprising, because closely related organisms typically share a higher number of similar genomic regions and synteny breaks down more drastically in comparison with distant relatives (Liti and Louis 2005). Comparison of orthologous regions in closely related fungi such as N. crassa, F. graminearum, and M. grisea reveals that syntenic blocks usually consist of small numbers of genes, ranging from 3 to 20 (Xu et al. 2006). Even when gene order has been conserved, the transcriptional orientations of the genes relative to one another often are different, as also was observed between M. graminicola and M. fijiensis.

Repetitive sequences also can lead to loss of conserved synteny, because they promote crossing over at nonhomologous chromosomal sites leading to chromosomal rearrangements. In S. cerevisiae, LTR retroelements were frequently associated with rearrangements (Dunham et al. 2002). Although comprehensive synteny analysis between M. graminicola and M. fijiensis is lacking, the role of TEs in the breakdown of synteny was evident during the comparison of 100 kb of sequence flanking the MgDNMT region between the two species. This region in M. graminicola had 15 predicted genes and no repetitive sequences, whereas in M. fijiensis 66% of the sequence was repetitive with only 4 predicted genes (Figure 1). Moreover, in M. fijiensis, a TE was inserted into the intergenic region of syntenic genes. The extensive presence of TEs in this region in M. fijiensis suggests that their insertion in intergenic regions may lead to loss of synteny between closely related species of Dothideomycetes as well.

Reverse transcriptase/endonuclease proteins, especially those in non-LTR LINE retrotransposons, can bind and transcribe cellular mRNAs and integrate them back into the genome, giving rise to processed retrogenes (Esnault et al. 2000). However, to date there appears to be only one case reported in which a LTR retrotransposon has been shown to carry gene fragments. In maize, one of the first retrotransposons to be identified was Bs1, which later was shown to carry transduced fragments from three genes (Elrouby and Bureau 2001). Although no mechanism for gene capture was proposed, the Bs1 results support the idea that LTR retrotransposons are capable of mobilizing and multiplying single-copy genes within genomes. The 220-bp LTR fragment adjacent to the MgDNMT region could have come from a complete LTR retroelement, which was gradually shortened during subsequent recombination events. Alternatively, this LTR fragment might have been present already at subtelomeric locations and was propagated along with the MgDNMT region.

A more plausible explanation for increase in MgDNMT copy number could be amplification by segmental duplication. One mechanism for segmental duplication is double-strand break (DSB) repair (Bailey et al. 2003). Any sequence in the genome may be used ectopically as a template to initiate DSB repair, leading to the duplication of that region (Rong and Golic 2003). In M. graminicola, the original nontelomeric chromosome 6 MgDNMT sequence could have served as a template for DSB repair at a subtelomeric location. Once copied to the subtelomeric location, the MgDNMT region could have been amplified by ectopic recombination between subtelomeric regions on different chromosomes. Ectopic exchange between subtelomeres in M. graminicola is supported by sequences that are identical between the subtelomeric regions of at least two pairs of chromosomes. These identical regions extend well beyond the MgDNMT sequence. In S. cerevisiae, at least three gene families, β-fructofuranosidase, α-galactosidase, and resistance to toxicity of molasses, have been amplified between chromosome ends through ectopic recombination (Louis et al. 1994). In humans, subtelomeric regions are patchworks of interchromosomal segmental duplications (Linardopoulou et al. 2005) with high plasticity, which may increase gene diversity (Trask et al. 1998), as observed in the subtelomeres of M. graminicola.

Four additional chromosomal ends in M. graminicola (between chromosomes 4 and 18 and 8 and 13) also have similar long repetitive sequences near their telomeres, but do not include MgDNMT sequence. Subtelomeric repetitive sequences have been reported in several organisms, but the reason for these structures is still unknown (Flint et al. 1997). It has been suggested that in the absence of telomerase, large blocks of tandem arrays of subtelomeric repeats may help stabilize the telomeres (McEachern et al. 2000). This mechanism of telomerase-independent, recombination-based telomere maintenance has been demonstrated in S. cerevisiae and Kluyveromyces lactis (Lundblad and Blackburn 1993; McEachern and Blackburn 1996) and also may operate in M. graminicola.

The different isolates of M. graminicola showed a high degree of polymorphism for bands corresponding to the MgDNMT sequence. Size polymorphisms on Southern blots usually are attributed to mutations in restriction enzyme recognition sites or the activity of transposable elements. In M. graminicola, two factors, RIP and high recombination at subtelomeres, may have contributed to these size polymorphisms. However, in all isolates, except for the sequenced M. graminicola isolate IPO323, the chromosomal location of MgDNMT sequences is unknown. Only a subset of total MgDNMT sequences was initially recognized by RECON (Bao and Eddy 2002) and the rest were identified by an iterative search. Improper definition of element boundaries is one reason that RECON (Bao and Eddy 2002) may sometimes fail to cluster similar elements into one family.

In organisms where a RIP-like process is absent, genes with altered functions may be created from transposable element-mediated multiplication of captured genes (McCarrey and Thomas 1987) or gene duplication and/or segmental duplication events. However, in filamentous fungi with an active RIP-like process, duplicated sequences are likely to be mutated by RIP. In N. crassa, only 6 from a predicted 10,082 genes have highly similar duplicates, suggesting that evolution via gene duplication has been virtually arrested (Galagan et al. 2003). Loss of function of the N. crassa cya-8 gene was shown to be a direct consequence of gene duplication followed by RIP (Perkins et al. 2007). Our results support the idea that accidental amplification followed by RIP may be a prevalent mechanism to inactivate single-copy genes, leading to significant effects on the basic biological pathways in fungi. These changes can occur rapidly; amplification and subsequent inactivation of MgDNMT sequences probably occurred after the split of M. graminicola and S1, which was estimated at 10,500 years ago (Stukenbrock et al. 2007), and may have been concomitant with the domestication of wheat as a cultivated crop. Whether loss of methylation influenced the shift in M. graminicola host preference from a wild grass to wheat is not known, but it provides a testable hypothesis for future research.

Acknowledgments

We thank Bruce McDonald for providing DNA samples of S1 and S2, Eva Stukenbrock for BLAST searches against the S1 genome sequence, and Gert Kema for help in obtaining the genomic sequence of M. graminicola. We also thank Michael Freitag and Eric Selker for critically reviewing the manuscript. DNA sequencing of M. graminicola and M. fijiensis was performed at the U.S. Department of Energy's Joint Genome Institute through the Community Sequencing Program (www.jgi.doe.gov/CSP/) and all sequence data are publicly available. This work was supported by the U.S. Department of Agriculture (USDA) Current Research Information System (CRIS) project 3602-22000-015-00D. Names are necessary to report factually on available data. However, the USDA neither guarantees nor warrants the standard of the product, and the use of the name implies no approval of the product to the exclusion of others that also may be suitable.

Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.110.117408/DC1.

References

  1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang et al., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25 3389–3402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bailey, J. A., G. Liu and E. E. Eichler, 2003. An Alu transposition model for the origin and expansion of human segmental duplications. Am. J. Hum. Genet. 73 823–834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bao, Z., and S. R. Eddy, 2002. Automated de novo identification of repeat sequence families in sequenced genomes. Genome Res. 12 1269–1276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benson, G., 1999. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 27 573–580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cambareri, E. B., B. C. Jensen, E. Schabtach and E. U. Selker, 1989. Repeat-induced G-C to A-T mutations in Neurospora. Science 244 1571–1575. [DOI] [PubMed] [Google Scholar]
  6. Carver, T. J., K. M. Rutherford, M. Berriman, M. A. Rajandream, B. G. Barrell et al., 2005. ACT: the Artemis Comparison Tool. Bioinformatics 21 3422–3423. [DOI] [PubMed] [Google Scholar]
  7. Chain, P. S., D. V. Grafham, R. S. Fulton, M. G. Fitzgerald, J. Hostetler et al., 2009. Genomics. Genome project standards in a new era of sequencing. Science 326 236–237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clamp, M., J. Cuff, S. M. Searle and G. J. Barton, 2004. The Jalview Java alignment editor. Bioinformatics 20 426–427. [DOI] [PubMed] [Google Scholar]
  9. Colot, V., and J. L. Rossignol, 1999. Eukaryotic DNA methylation as an evolutionary device. BioEssays 21 402–411. [DOI] [PubMed] [Google Scholar]
  10. Dunham, M. J., H. Badrane, T. Ferea, J. Adams, P. O. Brown et al., 2002. Characteristic genome rearrangements in experimental evolution of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 99 16144–16149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Elrouby, N., and T. E. Bureau, 2001. A novel hybrid open reading frame formed by multiple cellular gene transductions by a plant long terminal repeat retroelement. J. Biol. Chem. 276 41963–41968. [DOI] [PubMed] [Google Scholar]
  12. Esnault, C., J. Maestre and T. Heidmann, 2000. Human LINE retrotransposons generate processed pseudogenes. Nat. Genet. 24 363–367. [DOI] [PubMed] [Google Scholar]
  13. Eyal, Z., A. L. Scharen, M. D. Huffman and J. M. Prescott, 1985. Global insights into virulence frequencies of Mycosphaerella graminicola. Phytopathology 75 1456–1462. [Google Scholar]
  14. Feschotte, C., 2008. Transposable elements and the evolution of regulatory networks. Nat. Rev. Genet. 9 397–405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fisher, N., and M. Griffin, 1984. Benzimidazole (MBC) resistance in Septoria tritici. ISPP Chem. Control Newsl. 5 8–9. [Google Scholar]
  16. Flint, J., G. P. Bates, K. Clark, A. Dorman, D. Willingham et al., 1997. Sequence comparison of human and yeast telomeres identifies structurally distinct subtelomeric domains. Hum. Mol. Genet. 6 1305–1313. [DOI] [PubMed] [Google Scholar]
  17. Foss, H. M., C. J. Roberts, K. M. Claeys and E. U. Selker, 1993. Abnormal chromosome behavior in Neurospora mutants defective in DNA methylation. Science 262 1737–1741. [DOI] [PubMed] [Google Scholar]
  18. Fraaije, B. A., F. J. Burnett, W. S. Clark, J. Motteram and J. A. Lucas, 2005. Resistance development to QoI inhibitors in populations of Mycosphaerella graminicola in the UK, pp. 63–71 in Modern Fungicides & Antifungal Compounds IV, edited by H. W. Dehne, U. Gisi, K. H. Kuck, P. E. Russell and H. Lyr. BCPC, Alton, UK.
  19. Freitag, M., R. L. Williams, G. O. Kothe and E. U. Selker, 2002. A cytosine methyltransferase homologue is essential for repeat-induced point mutation in Neurospora crassa. Proc. Natl. Acad. Sci. USA 99 8802–8807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Freitag, M., P. C. Hickey, T. K. Khlafallah, N. D. Read and E. U. Selker, 2004. HP1 is essential for DNA methylation in Neurospora. Mol. Cell 13 427–434. [DOI] [PubMed] [Google Scholar]
  21. Galagan, J. E., and E. U. Selker, 2004. RIP: the evolutionary cost of genome defense. Trends Genet. 20 417–423. [DOI] [PubMed] [Google Scholar]
  22. Galagan, J. E., S. E. Calvo, K. A. Borkovich, E. U. Selker, N. D. Read et al., 2003. The genome sequence of the filamentous fungus Neurospora crassa. Nature 422 859–868. [DOI] [PubMed] [Google Scholar]
  23. Goodwin, S. B., J. R. Cavaletto, C. Waalwijk and G. H. Kema, 2001. DNA fingerprint probe from Mycosphaerella graminicola identifies an active transposable element. Phytopathology 91 1181–1188. [DOI] [PubMed] [Google Scholar]
  24. IHGSC, 2001. International Human Genome Sequencing Consortium. Nature 409 860–921.11237011 [Google Scholar]
  25. Kouzminova, E., and E. U. Selker, 2001. dim-2 encodes a DNA methyltransferase responsible for all known cytosine methylation in Neurospora. EMBO J. 20 4309–4323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Linardopoulou, E. V., E. M. Williams, Y. Fan, C. Friedman, J. M. Young et al., 2005. Human subtelomeres are hot spots of interchromosomal recombination and segmental duplication. Nature 437 94–100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Liti, G., and E. J. Louis, 2005. Yeast evolution and comparative genomics. Annu. Rev. Microbiol. 59 135–153. [DOI] [PubMed] [Google Scholar]
  28. Louis, E. J., E. S. Naumova, A. Lee, G. Naumov and J. E. Haber, 1994. The chromosome end in yeast: its mosaic nature and influence on recombinational dynamics. Genetics 136 789–802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lundblad, V., and E. H. Blackburn, 1993. An alternative pathway for yeast telomere maintenance rescues est1- senescence. Cell 73 347–360. [DOI] [PubMed] [Google Scholar]
  30. Martin, F., A. Aerts, D. Ahren, A. Brun, E. G. Danchin et al., 2008. The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis. Nature 452 88–92. [DOI] [PubMed] [Google Scholar]
  31. McCarrey, J. R., and K. Thomas, 1987. Human testis-specific PGK gene lacks introns and possesses characteristics of a processed gene. Nature 326 501–505. [DOI] [PubMed] [Google Scholar]
  32. McEachern, M. J., and E. H. Blackburn, 1996. Cap-prevented recombination between terminal telomeric repeat arrays (telomere CPR) maintains telomeres in Kluyveromyces lactis lacking telomerase. Genes Dev. 10 1822–1834. [DOI] [PubMed] [Google Scholar]
  33. McEachern, M. J., A. Krauskopf and E. H. Blackburn, 2000. Telomeres and their control. Annu. Rev. Genet. 34 331–358. [DOI] [PubMed] [Google Scholar]
  34. Miura, A., S. Yonebayashi, K. Watanabe, T. Toyama, H. Shimada et al., 2001. Mobilization of transposons by a mutation abolishing full DNA methylation in Arabidopsis. Nature 411 212–214. [DOI] [PubMed] [Google Scholar]
  35. Pardue, M. L., and P. G. DeBaryshe, 2003. Retrotransposons provide an evolutionarily robust non-telomerase mechanism to maintain telomeres. Annu. Rev. Genet. 37 485–511. [DOI] [PubMed] [Google Scholar]
  36. Perkins, D. D., M. Freitag, V. C. Pollard, L. A. Bailey-Shrode, E. U. Selker et al., 2007. Recurrent locus-specific mutation resulting from a cryptic ectopic insertion in Neurospora. Genetics 175 527–544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rabinowicz, P. D., K. Schutz, N. Dedhia, C. Yordan, L. D. Parnell et al., 1999. Differential methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome. Nat. Genet. 23 305–308. [DOI] [PubMed] [Google Scholar]
  38. Rong, Y. S., and K. G. Golic, 2003. The homologous chromosome is an effective template for the repair of mitotic DNA double-strand breaks in Drosophila. Genetics 165 1831–1842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Saitou, N., and M. Nei, 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4 406–425. [DOI] [PubMed] [Google Scholar]
  40. Sambrook, J., and D. W. Russell, 1989. Molecular Cloning. Cold Spring Harbor Laboratory Press, Plainview, NY.
  41. Selker, E. U., E. B. Cambareri, B. C. Jensen and K. R. Haack, 1987. Rearrangement of duplicated DNA in specialized cells of Neurospora. Cell 51 741–752. [DOI] [PubMed] [Google Scholar]
  42. Singer, T., C. Yordan and R. A. Martienssen, 2001. Robertson's Mutator transposons in A. thaliana are regulated by the chromatin-remodeling gene Decrease in DNA Methylation (DDM1). Genes Dev. 15 591–602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Song, L., S. R. James, L. Kazim and A. R. Karpf, 2005. Specific method for the determination of genomic DNA methylation by liquid chromatography-electrospray ionization tandem mass spectrometry. Anal. Chem. 77 504–510. [DOI] [PubMed] [Google Scholar]
  44. Stukenbrock, E. H., S. Banke, M. Javan-Nikkhah and B. A. McDonald, 2007. Origin and domestication of the fungal wheat pathogen Mycosphaerella graminicola via sympatric speciation. Mol. Biol. Evol. 24 398–411. [DOI] [PubMed] [Google Scholar]
  45. Tamaru, H., and E. U. Selker, 2001. A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature 414 277–283. [DOI] [PubMed] [Google Scholar]
  46. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin and D. G. Higgins, 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25 4876–4882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Trask, B. J., C. Friedman, A. Martin-Gallardo, L. Rowen, C. Akinbami et al., 1998. Members of the olfactory receptor gene family are contained in large blocks of DNA duplicated polymorphically near the ends of human chromosomes. Hum. Mol. Genet. 7 13–26. [DOI] [PubMed] [Google Scholar]
  48. Watters, M. K., T. A. Randall, B. S. Margolin, E. U. Selker and D. R. Stadler, 1999. Action of repeat-induced point mutation on both strands of a duplex and on tandem duplications of various sizes in Neurospora. Genetics 153 705–714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wessler, S. R., 2006. Eukaryotic transposable elements: teaching old genomes new tricks, pp. 138–162 in The Implicit Genome, edited by L. H. Caporale. Oxford University Press, New York.
  50. Xu, J. R., Y. L. Peng, M. B. Dickman and A. Sharon, 2006. The dawn of fungal pathogen genomics. Annu. Rev. Phytopathol. 44 337–366. [DOI] [PubMed] [Google Scholar]

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