Chromosomal rearrangements are a major driver of eukaryotic genome evolution, affecting speciation, pathogenicity and cancer progression. Changes in chromosome structure are often initiated by mis-repair of double-strand breaks in the DNA. Mis-repair is particularly likely when telomeres are lost or when dispersed repeats misalign during crossing-over. Fungi carry highly polymorphic chromosomal complements showing substantial variation in chromosome length and number. The mechanisms driving chromosome polymorphism in fungi are poorly understood. We aimed to identify mechanisms of chromosomal rearrangements in the fungal wheat pathogen Zymoseptoria tritici. We combined population genomic resequencing and chromosomal segment PCR assays with electrophoretic karyotyping and resequencing of parents and offspring from experimental crosses to show that this pathogen harbors a highly diverse complement of accessory chromosomes that exhibits strong global geographic differentiation in numbers and lengths of chromosomes. Homologous chromosomes carried highly differentiated gene contents due to numerous insertions and deletions. The largest accessory chromosome recently doubled in length through insertions totaling 380 kb. Based on comparative genomics, we identified the precise breakpoint locations of these insertions. Nondisjunction during meiosis led to chromosome losses in progeny of three different crosses. We showed that a new accessory chromosome emerged in two viable offspring through a fusion between sister chromatids. Such chromosome fusion is likely to initiate a breakage-fusion-bridge (BFB) cycle that can rapidly degenerate chromosomal structure. We suggest that the accessory chromosomes of Z. tritici originated mainly from ancient core chromosomes through a degeneration process that included BFB cycles, nondisjunction and mutational decay of duplicated sequences. The rapidly evolving accessory chromosome complement may serve as a cradle for adaptive evolution in this and other fungal pathogens.

Chromosomal rearrangements are a hallmark of genetic differences between species. But changes in chromosome structure can also occur spontaneously within species, within populations, or even within individuals. The causes and consequences of chromosomal rearrangements affecting natural populations are poorly understood. We investigated a class of fungal chromosomes called accessory chromosomes that are not shared among all individuals within a species. Using a fungal pathogen possessing numerous accessory chromosomes as a model, we assessed chromosome diversity based on whole-genome sequencing and a PCR assay of chromosomal segments that included a global collection of isolates. We show that the accessory chromosomes are highly variable in their gene content and that geographic differences correlate with the number and the structure of the chromosomes. We applied the same approach to document chromosomal rearrangements occurring during sexual reproduction. We identified viable offspring carrying a novel chromosome that originated from a large duplication affecting the majority of the chromosome. Our study showed that chromosomal structure can evolve rapidly within a species to generate a highly diverse set of accessory chromosomes. This chromosomal diversity may contribute significantly to the adaptive potential of fungal pathogens.

Funding: This work was supported by an ETH Zurich grant [ETH-03 12; www.ethz.ch ] to DC and BAM and by Swiss National Science Foundation grants to DC [PA00P3_145360; www.snf.ch ] and BAM [31003A_134755; www.snf.ch ]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2013 Croll et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The large set of accessory chromosomes in Z. tritici and its close relatives provides a powerful model system to elucidate the mechanisms underlying fungal chromosomal polymorphisms and the origins of accessory chromosomes. We combined population genomic resequencing and PCR-based chromosome segment genotyping to measure the diversity in chromosomal structure at a global scale. We then performed controlled sexual crosses to trace the fate of accessory chromosomes through meiosis and to identify structural rearrangements in chromosomes among the progeny. We confirmed the findings from our resequencing data with electrophoretic karyotyping that enabled chromosomal separation, isolation and visualization by Southern blotting. Our study provides the most comprehensive view to date of mechanisms underlying chromosomal polymorphisms in evolving fungal populations.

The largest known complement of accessory chromosomes is found in the wheat pathogen Zymoseptoria tritici (syn. Mycosphaerella graminicola [45] ). The eight smallest chromosomes of the reference genome of Z. tritici, ranging in size from 409–773 kb, were identified as accessory chromosomes [46] . The core chromosomes of the reference genome range in size from 1,186–6,089 kb [46] . In contrast to accessory chromosomes found in other pathogenic fungi, Z. tritici accessory chromosomes contain over six hundred annotated genes, however the function of these genes is poorly understood [46] . The fungus shows extensive chromosomal length and number polymorphisms within random mating field populations [30] , [47] . Some of the chromosomal diversity appears to be generated through meiosis because progeny populations exhibited frequent chromosome loss and disomy as a result of nondisjunction of accessory chromosomes [48] . The origin of the accessory chromosomes of Z. tritici is not known, though both horizontal chromosome transfer from an unknown donor and degeneration of core chromosomes have been proposed [46] . Comparative genomics of closely related species suggested that several accessory chromosomes originated prior to the emergence of Z. tritici [49] . Several lines of evidence suggest that accessory chromosomes may be important for virulence, including the finding that genes on accessory chromosomes are under accelerated evolution and that these are more likely to show a protein signature consistent with a role in pathogenicity [46] , [49] .

Some of the most polymorphic chromosomal complements were found in plant pathogenic fungi. Several species carry chromosomes that are not shared among all members of the species [40] . Chromosomes exhibiting a presence/absence polymorphism within a species have been referred to as B, dispensable, supernumerary or accessory chromosomes to differentiate them from the “core” chromosomes that are shared among all members of a species [29] , [32] , [40] . We refer to the chromosomes not shared among all individuals as accessory chromosomes because many of these chromosomes play an adaptive role in pathogen evolution, hence these chromosomes are not truly dispensable [32] . Nor do they fit the classic definition of B chromosomes, because they can carry many coding genes and may be necessary for survival in some environments. One of the best studied fungal accessory chromosomes was found in isolates of the pathogen Nectria haematococca and contains a gene cluster important for virulence on peas [41] , [42] . The tomato pathogen Fusarium oxysporum f. sp. lycopersici contains several accessory chromosomes that carry a series of genes important for virulence [43] . In the rice blast fungus Magnaporthe oryzae and related species, a major effector called AVR-Pita that confers virulence on rice was frequently translocated between subtelomeric regions of different chromosomes including accessory chromosomes [44] . Flanking retrotransposons likely contributed to the extreme mobility of the AVR-Pita gene within and among closely related species.

Fungal chromosomes are generally too small for traditional cytogenetic analyses based on chromosome staining and microscopic examination. But fungi were found to show extensive chromosomal polymorphisms following the invention of pulsed-field gel electrophoresis (PFGE). Application of PFGE revealed that many fungal species exhibit a high variability in chromosome number and size, even among individuals drawn from the same random mating population [28] – [30] . Mechanisms generating the differences in chromosome length and number remained largely elusive, although chromosome breakage and non-allelic homologous recombination among repetitive elements during meiosis were suggested to play a role [28] , [31] . High chromosomal variability in pathogenic fungi may play an important adaptive role [32] . For example, dramatic changes in copy numbers of an arsenite efflux transporter in Cryptococcus neoformans occurred during experimental evolution favoring arsenite tolerance [33] . Chromosomal disomy was associated with increased antifungal drug resistance in several human pathogens including C. neoformans and Candida albicans [34] , [35] . Copy-number variation and aneuploidy were frequently found in clinical and environmental isolates of the same species [36] – [39] .

Chromosomal rearrangements involve deletions, duplications, inversions and translocations within and among chromosomes. In most cases, the molecular mechanisms that generated the observed rearrangements are not known, but a common explanation is mis-repair of double-stranded DNA breaks [12] , [13] . Repetitive DNA has been strongly associated with chromosome rearrangements in plant and animal genomes and is thought to promote non-allelic homologous recombination during meiosis due to the misalignment of dispersed repeats [14] – [16] . Telomeres play a major role in maintaining chromosome stability [17] , [18] . Although chromosomes lacking a telomere are particularly susceptible to chromosomal fusion, subtelomeric double-strand breaks may also cause chromosomal fusion [19] . McClintock's classic cytogenetic work on maize in the 1930s and 1940s showed that mis-repair of damaged chromosomal ends could generate cycles of chromosomal degeneration termed breakage-fusion-bridge (BFB) cycles [20] , [21] . BFB cycles begin when a telomere breaks off a chromosome. When the damaged chromosome replicates, its sister chromatids fuse and form a bridge during anaphase, with the two centromeres of the fused sister chromatids pulled into opposite poles of the dividing cell. After the bridge breaks, the resulting daughter cells receive defective chromosomes that lack telomeres and can initiate new BFB cycles. BFB cycles have also been identified in animals [22] , [23] and yeast [24] , [25] . In humans, BFB cycles play a significant role in cancer progression [18] , [26] , [27] .

Chromosomal rearrangements are major drivers of genome evolution. Dobzhansky [1] realized that chromosomal polymorphism would “supply the raw materials for evolution”, providing some of the earliest support for Darwin's theory of evolution. Since Dobzhansky's work on Drosophila, cytogenetic studies have revealed a large number of chromosomal rearrangements in the genomes of plant and animal species [2] , including humans [3] . Chromosomal rearrangements were shown to contribute to sex chromosome differentiation [4] , [5] , reproductive isolation [6] , speciation [7] – [10] and complex adaptive phenotypes [11] .

Results

Global populations are highly differentiated for presence or absence of chromosomal segments Z. tritici is distributed globally and exhibits high genetic diversity for neutral markers [50]–[52] as well as high phenotypic diversity for quantitative traits, including virulence and thermal adaptation [51]–[53]. To assess the composition and frequency of accessory chromosomes across global populations, we designed 57 PCR assays covering all 8 known accessory chromosomes found in the reference strain IPO323 (chromosomes 14–21; [46]). Amplicons ranging in size from 400–600 bp were targeted to coding regions and primer sites were chosen in conserved regions of each gene (Table S1). The genes comprised in the PCR assay were evenly distributed along the accessory chromosomes and were located mostly in GC-rich regions interspersed by regions of higher repeat content (Figure 1C and 1D). Gene density varies along accessory chromosomes and the PCR assays covered the entire range of known gene locations for each chromosome (Figure 1E). The function of most genes included in the PCR assay is unknown and only 7 out of 57 genes were characterized by gene ontology (Figure 1F; [46]). As a control we designed 15 additional PCR assays covering core chromosomes 10 and 13. Known microsatellite loci were included in each PCR as a positive control. In total, we surveyed 98 isolates sampled from a global collection of four wheat fields at 72 evenly spaced chromosome positions (Table S2): Oregon, United States (n = 19), Israel (n = 23), Australia (n = 30) and Switzerland (n = 26). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Global survey of diversity in accessory chromosomes of Zymoseptoria tritici. A) Presence or absence of chromosomal segments assayed by PCR in a global collection of four field populations located in Australia, Israel, United States and Switzerland (total n = 98). Green bars represent the number of chromosomal segments found within the populations. Core chromosomes 10 and 13 were included for comparison with the accessory chromosomes 14–21. B) Population differentiation based on the presence of chromosomal segments calculated by Wright's F ST . C) The physical location of each gene used for the PCR assays is shown on schematics drawn for each accessory chromosome. The variation of GC-content along the chromosomes is shown in red. D) Content of short direct repeats assessed in 20 kb segments. E) Location of coding regions according to the reference genome [46]. The location of probes used for Southern hybridizations to CHEF gels are indicated in red. F) Gene ontology terms for genes comprised in the PCR assay. Only genes on accessory chromosomes 14 and 16 were described by gene ontology terms [46]. https://doi.org/10.1371/journal.pgen.1003567.g001 The PCR assays on the core chromosomes 10 and 13 showed that 10 chromosomal segments were present in all 98 isolates (Figure 1A). Three chromosomal segments were missing in 1–3 isolates distributed at random across the populations. One segment on each chromosome was missing in a large fraction of the isolates, but was at approximately the same frequency across all populations (Figure 1A). None of the isolates was missing an entire core chromosome. In contrast, chromosomal segments on accessory chromosomes showed large frequency variations among populations and different accessory chromosomes showed different patterns of segmental presence/absence (Figure 1A). With the exception of chromosome 18, all accessory chromosomes were found at a frequency higher than 50% in the four field populations. Chromosome 16 was present at the highest frequency with several chromosomal segments being fixed within populations. Individual accessory chromosomes showed substantial differences compared to the chromosomes of the Dutch reference strain IPO323. Central chromosomal segments located on chromosome 14 were almost entirely missing in isolates from Australia, the United States and Israel. Swiss isolates showed a central chromosomal segment at approximately half the frequency as segments closer to the telomeric ends of the chromosome. The haplotypic diversity for the presence or absence of individual chromosomal segments was substantial among isolates (Figure S1). Nearly every isolate showed a unique combination of presence or absence of individual accessory chromosome segments. We assessed the population differentiation for presence or absence of chromosomal segments among populations using Wright's F ST statistic. Frequencies of several accessory chromosome segments were strongly differentiated among populations. The central segments of chromosome 14 showed F ST ranging from 0.15–0.55 (Figure 1B). High levels of differentiation were also found for the second segment of chromosome 15 and the first segment of chromosome 17. Chromosome 18 showed elevated levels of differentiation across the chromosome, largely because this chromosome was almost entirely missing from the Australian and USA populations (Figure 1A). In contrast, previous data on neutral genetic markers on core chromosomes showed little differentiation among these and other populations [50].

Population resequencing revealed variation in gene content among homologous chromosomes We found substantial karyotypic diversity in accessory chromosomes among isolates from Switzerland (Figure 2). In order to obtain a fine-scale map of structural variation in accessory chromosomes among isolates, we performed Illumina resequencing on 9 of the Swiss isolates (mapping coverage 10–23×; Table 1). We identified genomic divergence between the reference isolate IPO323 and the resequenced isolates by mapping all sequence reads to the finished reference genome. To avoid spurious read mapping in repetitive regions of the chromosomes, we restricted our comparison to the coding regions of the accessory chromosomes. Furthermore, we considered exons of multi-exon genes separately to avoid biases introduced by gene length. In summary, we mapped reads to 1763 exons corresponding to 654 unique genes. The average exon length on accessory chromosomes is 314 bp compared to 517 bp on core chromosomes. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Electrophoretic karyotype diversity in accessory chromosomes among eight resequenced Swiss Zymoseptoria tritici isolates compared to the reference isolate IPO323. Chromosomal bands were separated by CHEF gel electrophoresis. The size marker is Saccharomyces cerevisiae chromosomes (Sc). https://doi.org/10.1371/journal.pgen.1003567.g002 PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Illumina resequencing and assembly statistics of Zymoseptoria tritici isolates from the Swiss population. https://doi.org/10.1371/journal.pgen.1003567.t001 The read depth from the resequencing data of 9 Swiss isolates mapped against the reference genome did not suggest any disomic chromosomes (i.e. doubled read depth for a particular chromosome). However, the different isolates varied greatly in gene content on accessory chromosomes. Four isolates (3C4, 3D1, 3F5 and 1A5; Figure 3) showed a nearly complete set of coding sequences compared to the reference genome, with a substantial number of coding sequences present on all 8 accessory chromosomes. Isolate 3D7 contained the smallest complement of accessory chromosome genes, as only four chromosomes showed a substantial proportion of coding sequences to be present. The read mapping to coding regions indicated that accessory chromosomes 14, 19 and 21 likely differ in length among homologous chromosomes (Figure 3). Chromosome 16 was found in all isolates except one. However, chromosome 16 likely differs substantially among isolates due to a large number of deletions compared to the chromosome 16 of the reference genome. Nearly all surveyed 20 kb segments along chromosome 16 showed missing genes in at least some of the resequenced isolates. The strongest variation in coding sequence complements was found among variants of chromosome 14. Isolate 3D1 lacked 149 out of 292 coding sequences, while smaller segments of missing coding sequences were found in six isolates (9G4C, 3B8, 3C4, 3F5, 1E4 and 1A5). The number of missing coding sequences ranged from 18–45 among these six isolates. At one end of chromosome 19, isolate 3B8 showed 46 missing coding sequences out of 220 coding sequences. Similarly, isolates 1A5 and 1E4 showed 31 missing coding sequences out of 155 coding sequences on chromosome 21. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Genome resequencing of nine Zymoseptoria tritici isolates from a Swiss population. Illumina sequencing reads were mapped to annotated coding regions of the reference genome IPO323. The height of the bars represents the number of coding regions found in 20 kb segments in the reference genome. Horizontal rows show the nine different resequenced isolates. For each resequenced isolate black segments of the bars indicate the sum of present coding sequences per 20 kb segments. White segments of the bars indicate absent coding sequences. https://doi.org/10.1371/journal.pgen.1003567.g003

Distorted segregation of chromosomes in sexual crosses A major contribution to polymorphisms in accessory chromosomes may arise through meiotic recombination [48]. We performed controlled crosses involving three pairs of isolates from the Swiss population and analyzed 48 progeny from each cross. We applied the same PCR assays targeting 15 chromosomal segments on two core chromosomes and 57 chromosomal segments on the accessory chromosomes. Chromosomal segments on core chromosomes that were missing in either of the two parents were found to be segregating in approximately equal proportions in all three progeny sets (Figure 8). Patterns of segregation were different for several accessory chromosomes. In Cross 1 (9B8B×9G4C), we found a loss of chromosome 16 in one offspring despite the fact that both parental isolates were carrying a near full-length chromosome 16 (Figure 8E). In Cross 2 (1A5×1E4), we found that 8 progeny were missing all chromosome 14 segments, although both parental isolates carried the corresponding chromosome segments (Figure 8C). Similarly, chromosomes 16, 18, 20 and 21 were entirely missing from one offspring though both parents carried these chromosomes. Cross 3 (1A5×3D7) showed the strongest segregation distortions. Parental isolate 3D7 was missing four accessory chromosomes (Figure 8A; chromosomes 14, 15, 18 and 21). Two of these four chromosomes (15 and 21) were inherited in significantly higher proportion than expected under random segregation (X2 test, p<0.0007 multiple comparisons corrected, Figure 8B). Interestingly, in Cross 1 the parental strains similarly differed in their presence of chromosomes 15 and 21, however we did not detect any significant segregation distortion in this cross (Figure 8E). Furthermore, two progeny of Cross 3 lost accessory chromosomes 17, 19 and 20 entirely, although both parental strains carried these chromosomes. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 8. Variability in accessory chromosomes among progeny of three crosses between Zymoseptoria tritici isolates. A) Progeny of a cross between isolates 3D7 and 1A5 (Cross 3). Chromosomal segments of core chromosomes 10 and 13 and accessory chromosomes 14–21 were assayed by PCR. The two top rows indicate the two parental genotypes. The green bars show the number of individual chromosomal segments among the 48 progeny. B), D) and F) Test for random segregation of chromosomal segments that are present in only one of the two parental isolates. The −log 10 transformed p values were corrected for non-independence and the horizontal bar represents the Bonferroni-corrected significance threshold (p<0.0007). C) Chromosomal segment numbers among 48 progeny from a cross between isolates 1E4 and 1A5 (Cross 2). E) Chromosomal segment numbers among 48 progeny from a cross between isolates 9B8B and 9G4C (Cross 1). https://doi.org/10.1371/journal.pgen.1003567.g008

Meiosis generates novel electrophoretic karyotype profiles We randomly selected 24 and 34 offspring from Cross 1 and Cross 2, respectively, in order to identify changes in electrophoretic karyotype profiles among progeny. Progeny of both crosses showed substantial karyotypic diversity. Through hybridization with chromosome-specific probes, we found that parental isolates of Cross 2 showed length variation for chromosome 19 of approximately 0.3 Mb (data not shown). Chromosomes 15 and 21 showed nearly identical chromosome lengths among the parental isolates. Progeny of Cross 2 segregated the two length variants of chromosome 19 in approximately equal proportions (data not shown). Larger chromosomes (1.0–3.0 Mb) of parents and progeny of Cross 2 showed similarly diverse electrophoretic karyotypes (Figure 9A). In Cross 2, we identified two progeny (A2.2 and A66.2) out of 34 tested with a chromosomal band estimated to be around 0.9 Mb. However, neither of the two parents were found to have a chromosomal band in the range of 0.7–1.2 Mb, as shown by different PFGE gels optimized to separate either the smallest (<1.0 Mb) or medium-sized chromosomal bands (1.0–3.0 Mb) (Figures 9A and B). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 9. Pulsed-field gel electrophoresis of accessory chromosomes in the parental isolates 1E4 and 1A5, as well as their progeny A2.2 and A66.2. The isolate IPO323 was added as a reference. Both progeny showed a new chromosomal band at 0.9 Mb that is absent in either parental isolate and other screened progeny. Sc represents chromosomes of Saccharomyces cerevisiae added as a size marker. A) Pulsed-field gel electrophoresis of medium-sized chromosomes (up to approx. 3 Mb) of parental isolates 1E4 and 1A5 and 7 progeny. Progeny A2.2 and A66.2 showed a new chromosomal band at 0.9 Mb indicated by an asterisk. B) Pulsed-field gel electrophoresis of accessory chromosomes of parental isolates 1E4 and 1A5 and progeny A2.2 and A66.2. Asterisks identify chromosome 17 variants identified by hybridization. C) Southern hybridization of a chromosome 17 specific probe on chromosomes separated by pulsed-field gel electrophoresis of parental isolates, progeny and the reference isolate IPO323 as in B). https://doi.org/10.1371/journal.pgen.1003567.g009