Meiosis leads to pervasive copy-number variation and distorted inheritance of accessory chromosomes of the wheat pathogen Zymoseptoria tritici

Meiosis leads to pervasive copy-number variation and distorted inheritance of accessory... Meiosis is one of the most conserved molecular processes in eukaryotes. The fidelity of pairing and segregation of homologous chromosomes has a major impact on the proper transmission of genetic information. Aberrant chromosomal transmission can have major phenotypic consequences, yet the mechanisms are poorly understood. Fungi are excellent models to investigate processes of chromosomal transmission, because many species have highly polymorphic genomes that include accessory chromosomes. Inheritance of accessory chromosomes is often unstable and chromosomal losses have little impact on fitness. We analyzed chromosomal inheritance in 477 progeny coming from two crosses of the fungal wheat pathogen Zymoseptoria tritici.For this, we developed a high-throughput screening method based on restriction site-associated DNA sequencing that generated dense coverage of genetic markers along each chromosome. We identified rare instances of chromosomal duplications (disomy) in core chromosomes. Accessory chromosomes showed high overall frequencies of disomy. Chromosomal rearrangements were found exclusively on accessory chromosomes and were more frequent than disomy. Accessory chromosomes present in only one of the parents in an analyzed cross were inherited at significantly higher rates than the expected 1:1 segregation ratio. Both the chromosome and the parental background had significant impacts on the rates of disomy, losses, rearrangements, and distorted inheritance. We found that chromosomes with higher sequence similarity and lower repeat content were inherited more faithfully. The large number of rearranged progeny chromosomes identified in this species will enable detailed analyses of the mechanisms underlying chromosomal rearrangement. Key words: fungi, meiosis, chromosome rearrangements, progeny sequencing. Introduction essential for the faithful segregation of chromosomes Sexual reproduction requires chromosomes to undergo mei- (Naranjo 2012), chromosomes can pair along their entire osis, whereby homologous chromosomes pair, recombine, length or in a segment-specific manner where only some and finally separate and migrate to opposite poles of the mei- regions align (Roeder 1997). This suggests that the length otic cell. Meiosis is a highly conserved process initiated by the and degree of sequence similarity can affect homolog identi- pairing of homologous chromosomes that first recognize one fication and pairing. After pairing, recombination produces another and then establish recombination-dependent links crossovers that physically link homologs, mediate proper seg- between homologs to form the synaptonemal complex regation, and thereby preserves chromosomal integrity (reviewed in Roeder 1997). This is followed by two divisions, (Mather 1938; Baker et al. 1976; Hassold and Hunt 2001). first to separate homologous chromosomes and then to sep- Recombination between misaligned repetitive sequences can arate sister chromatids. While accurate pairing of homologs is generate length variation among the daughter chromosomes The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non- commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 1416 Genome Biol. Evol. 10(6):1416–1429. doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Meiosis Leads to Pervasive Copy-Number Variation and Distorted Inheritance GBE (Montgomery et al. 1991). After pairing and recombination, transmission of chromosomes through meiosis because of segregation occurs via centromeres that bind to chromosome their extreme chromosomal plasticity, the ubiquity of sexual proteins and mediate accurate segregation to the opposite reproduction, and their experimental tractability. poles of the cell. The fungal wheat pathogen Zymoseptoria tritici provides a Aberrant transmission of chromosomes from one genera- striking example of genome plasticity. The bipartite genome tion to the next, including partial and whole chromosome consists of 13 core and up to eight accessory chromosomes duplications or losses, are caused largely by erroneous pairing that exhibit significant length polymorphism within and during meiosis. Such duplication and loss events can affect a among field populations (Goodwin et al. 2011; Croll and large number of genes and alter gene expression across the McDonald 2012). Chromosomal rearrangements played an genome (Harewood and Fraser 2014). The most dramatic important role in adaptation to different host genotypes copy-number variation is aneuploidy. Unequal sets of chro- (Hartmann et al. 2017). The accessory chromosomes are mosomes result from nondisjunction and are the leading ge- highly unstable through meiosis and were shown to undergo netic cause of miscarriages in humans (Hassold and Hunt rearrangements, segregation distortion, and nondisjunction 2001). Atypical phenotypes associated with aneuploid states (Wittenberg et al. 2009; Croll et al. 2013). Z. tritici reproduces are caused by gene dosage imbalances that can cause severe sexually when hyphae originating from two haploid spores of defects (Torres et al. 2008). In general, aneuploidy and chro- opposite mating type fuse to produce a transient diploid stage mosomal rearrangements are associated with lower fitness that undergoes two rounds of meiosis followed by one round (Torres et al. 2008), but in rare circumstances, errors during of mitosis to produce eight ascospores in an ascus. The path- meiosis can provide adaptive genetic variation. For example, ogen tolerates aneuploidy, so chromosomal rearrangements in the human pathogenic fungi Cryptococcus neoformans and generated through this process in both the core and accessory Candida albicans, specific aneuploidies contribute to drug re- genomes can remain viable (Wittenberg et al. 2009; Croll sistance (Selmecki et al. 2006, 2008; Sionov et al. 2010; et al. 2013; Schotanus et al. 2015). Hence, this species is an Ngamskulrungroj et al. 2012). Adaptive aneuploidy is fre- ideal model to analyze patterns of aberrant chromosomal quently associated with response to stressful environments transmission. (Chen et al. 2012). The dosage imbalance and altered stoichi- In this study, we analyze the mechanisms that affect the ometry due to additional copies of genes on a duplicated fidelity of chromosomal inheritance through meiosis, includ- chromosome may not be beneficial under normal conditions, ing identification of chromosomal rearrangements, losses, but can become beneficial under stress (Pavelka, Rancati, and and duplications. For this, we screened hundreds of progeny Li 2010; Pavelka, Rancati, Zhu, et al. 2010). In pathogenic genotypes generated from two independent crosses and de- fungi, aneuploidy often occurs for only a restricted number termined the rate of aneuploidy, patterns of rearrangement of chromosomes, however the mechanisms determining the and distortions in transmission rates. Finally, we investigated rate of aneuploidy generation and its maintenance are poorly whether factors such as length similarity, synteny, recombina- understood. tion rate, and repetitive element content affected the fidelity Aneuploidy also plays an important role in several plant of chromosomal inheritance. pathogenic fungi. Several important plant pathogens have highly dynamic genomes with chromosomes that show sig- Materials and Methods nificant length and number polymorphisms within the Generation of Sexual Crosses species. This chromosomal plasticity is often restricted to a Two crosses were performed between four parental Z. tritici well-defined set of accessory chromosomes. This bipartite ge- isolates collected from two Swiss wheat fields separated by nome structure, characterized by an accessory genome region 10 km. Isolate ST99CH3D1 was crossed with isolate that is rapidly diversifying and a core genome region that ST99CH3D7 (hereafter abbreviated 3D1 and 3D7) and isolate remains conserved, can be associated with the trajectory of ST99CH1A5 was crossed with isolate ST99CH1E4 (abbrevi- pathogen evolution (Croll and McDonald 2012; Dong et al. ated 1A5 and 1E4), producing 359 and 341 haploid asco- 2015). The accessory region is often rich in transposable ele- spore progeny, respectively. The genomes of all four ments that drive chromosomal rearrangements, deletions, parental isolates were sequenced using Illumina technology and duplications (Zhang et al. 2011). Accessory chromosomes (Torriani et al. 2011) and are available under the NCBI SRA are not shared among all members of a species, therefore accession numbers SRS383146 (3D1), SRS383147 (3D7), these chromosomes can contribute significantly to polymor- SRS383142 (1A5), and SRS383143 (1E4). The parental iso- phism within a species. Importantly, many plant pathogens lates were already genetically characterized and have been have been shown to harbor pathogenicity loci on accessory phenotyped for virulence and many other traits (Zhan et al. chromosomes (Mo ¨ ller and Stukenbrock 2017). In contrast, 2005; Croll et al. 2013). Full sib families were produced by the core regions encode essential functions required for sur- coinfecting wheat leaves with asexual conidia from the pa- vival and reproduction. Plant pathogenic fungi provide partic- rental strains of opposite mating types using the crossing ularly powerful models to investigate factors affecting the Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 1417 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Fouche et al. GBE protocol described by Kema et al. (1996). Briefly, spores of a et al. 2014). Only one randomly selected progeny per clonal pair of parents were sprayed onto wheat plants and incu- group was kept for further analyses, reducing the number of bated outdoors for 40–60 days until well-developed symp- progeny to 263 in the 3D13D7cross andto261 in the toms including pseudothecia were observed. Ascospores 1A51E4 cross. were isolated from pseudothecia over a period of several days by placing the infected wheat leaves on wet filter paper Determining Chromosome Number and Length inside Petri dishes. Leaves were covered with upside down Polymorphisms Based on Coverage Petri dish lids that were previously filled with water agar. Restriction sites cut by PstI were identified in silico using the This setup allowed us to capture ascospores that were verti- EMBOSS restrict program (http://www.bioinformatics.nl/ cally ejected from mature pseudothecia. Released ascospores cgi-bin/emboss/restrict; last accessed September 2016). were left to germinate on the water agar to enable inspection Thereafter, the coverage of RADseq reads mapping to for potential contaminants and to ensure that only progeny the restriction sites was determined using the BEDtools resulting from single ascospores were selected. Germinating v. 2.25.0 intersectBed and coverageBed commands ascospores were transferred to individual culture plates for (Quinlan and Hall 2010). Reads were counted if the map- clonal propagation. The mycelium produced by each success- ping quality score was20. The coverage of the se- fully germinated ascospore was used for DNA extraction and quenced parent genomes was determined following the plant infection experiments. Offspring mycelium was pro- same procedure. Progeny with a median read coverage duced in YSB (yeast sucrose broth) liquid media for 6–7 days of<20 were excluded from further analyses to avoid prior to DNA extraction. biases introduced by low-coverage data, resulting in fewer isolates being included in this analysis than in pre- vious studies (Lendenmann et al. 2014, 2016; Stewart Reference Alignment Using Restriction Site-Associated et al. 2018). We retained 249 progeny in the 3D13D7 DNA Sequencing cross and 228 isolates in the 1A51E4 cross. We used We used Restriction Site-Associated DNA Sequencing normalized read counts to detect chromosomal anoma- (RADseq) (Baird et al. 2008) for large-scale sequence gen- lies, where those with a normalized coverage close to zero otyping as described previously (Croll et al. 2015). Briefly, (<0.3) were classified as missing, those with a normalized the RADseq protocol (Etteretal. 2011) was applied to Z. coverage close to one (>¼0.7 and<1.3) were classified as tritici by using the PstI restriction enzyme to digest 1.3 lg present and those with a normalized coverage close to of DNA extracted with the DNAeasy plant mini kit two (>¼1.7) were classified as disomic (fig. 1A). Partially (QIAGEN Inc., Basel, Switzerland) for each offspring. deleted and partially duplicated chromosomes were iden- After digestion and adapter annealing, the pooled DNA tified based on a normalized coverage ratio of>¼0.3 was sequenced on an Illumina HiSeq2000 using a paired- and<0.7 or>1.3 and<1.7, respectively. Deviations end 100-bp library. Pools contained 132 progeny, six from Mendelian inheritance for accessory chromosomes different Illumina TruSeq compatible P2 adapters and 22 present in only one of the parents were determined using P1 adapters with unique barcodes. Progeny DNA with the a chi-squared (v )test. same P2 adapter were distinguishable by using the unique barcodes ligated to the P1 adapters. Distinguishing between Homozygous and Heterozygous Illumina reads were quality trimmed using Trimmomatic v. Disomy 0.30 (Bolger et al. 2014) and separated into distinct sets for each progeny based on the P1 adapter using FASTX toolkit v SNP calling was performed using Freebayes (Version 0.13 (http://hannonlab.cshl.edu/fastx_toolkit/; last accessed 1.0.2_1 1.1.0) (Garrison and Marth 2012)using the bam- March 2015). Reads were aligned to the gapless telomere to files of each isolate mapped to the IPO reference genome. telomere IPO323 reference genome (assembly version MG2, We used the parameters no-indels, no-mnps, no-complex, Septemeber 2008) (Goodwin et al. 2011) with the short-read and ploidy 2. Then we filtered for sites that differed be- aligner version of bowtie 2.1.0 (Langmead and Salzberg 2012) tween the parents (maf 0.2) and considered only these using the default parameters for sensitive end-to-end align- regions to determine whether disomic chromosomes orig- ment (-D 15; -R 2; L- 22; -I S, 1, 1.15). The same parameters inated from one or both parents. We also filtered for from trimming and reference assembly were used to align the depth (minDP 30) and quality (minQ 30). The VCF tools - four parental genome sequences (Croll et al. 2013)to the ref- het function was used to determine the number of homo- erence genome (IPO323). RADseq aligned reads are available zygous sites and the total number of sites. We determined under the NCBI BioProject accession numbers PRJNA256988 the ratio of homozygous sites to the total number of sites and PRJNA256991. Potential clones were identified as geno- and defined those with a ratio>0.6 as homozygous while types sharing>90% identity based on single nucleotide poly- those with a ratio<0.4 were defined as heterozygous. All morphism (SNP) analyses as previously described (Lendenmann other cases were considered to be ambiguous. 1418 Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Meiosis Leads to Pervasive Copy-Number Variation and Distorted Inheritance GBE Normalized coverage <0.3 >= 0.3 & <0.7 >= 0.7 & < 1.3 >= 1.3 & < 1.7 >=1.7 B Loss Loss Loss Partial Normal Partial Duplication Deletion Duplication PstI cut sites Mapped reads Median genome coverage Disomic Missing Normal chromosome chromosome chromosome PstI cut sites Mapped reads Median genome coverage Partially deleted Partially duplicated chromosome chromosome FIG.1.—Procedure to detect chromosomal anomalies. (A) Reads mapped to the PstI restriction sites were used to analyze coverage across the genome. Sequencing data were generated by restriction site-associated DNA sequencing (RADseq). The normalized coverage represents the coverage of each chromosome normalized to the median coverage of all chromosomes of the same progeny. The normalized coverage distribution of progeny from cross 3D13D7 is shown with the cutoffs used to detect a whole chromosome loss (ratio< 0.3), partial deletion (ratio 0.3–0.7), normal transmission (0.7–1.3), partial duplication (1.3–1.7), and whole chromosome duplication (>1.7). (B) Schematic overview of read coverage expected for complete chromosome losses and duplications (in blue). Partial deletions and duplications are shown in green. of TEs on a chromosome was compared with the likelihood of Chromosome Instability and Recombination Rate, being inherited with high fidelity. We also compared the fre- Chromosome Length, Synteny and Transposable Element quency of disomic chromosomes with the frequency of rear- Content of the Parent Chromosomes rangements for all chromosomes in both crosses. We correlated chromosome instability with the percentage length difference in homologs among the parents and recom- bination rates based on the recombination rates reported in Analyses of Progeny Phenotypes Croll et al. (2015). We also correlated synteny and the fidelity with which chromosomes were inherited using the NUCmer Clonally propagated mycelium from each germinated asco- pipeline from MUMmer (version 3.23) software (Kurtz et al. spore was previously used to infect wheat plants in the frame- 2004) to determine the sequence similarity between two ho- work of a QTL mapping study (Stewart et al. 2018). Progeny mologous chromosomes. The minimum cluster length was set from both crosses were phenotyped for percentage of leaf to 50 and we used the –mum option to anchor matches that area covered by lesions (PLACL), pycnidia density (pycnidia/ 2 2 were unique in both the reference and query sequence. The cm leaf area), pycnidia size (mm ), and pycnidia melanization transposable elements (TEs) in the parent genomes were an- on seedlings of the wheat cultivars Runal and Titlis in a previ- notated using RepeatMasker (http://www.repeatmasker.org; ously described glasshouse-based assay (Stewart and last accessed May 2017) and the TE library compiled for Z. tritici McDonald 2014). Gray values were previously shown to be and its sister species (Grandaubert et al. 2015). The percentage a good measure for melanization (Lendenmann et al. 2014). Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 1419 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Frequency Coverage Coverage Chromosome Chromosome Fouche et al. GBE Replication of the infection assays was made possible by in- Patterns of Chromosome Transmission in the Two Crosses oculating replicate wheat plants with a fixed concentration of Analyzing normalized read coverage among progeny revealed blastospores from each progeny mycelium. The assay was high rates of chromosome losses in both crosses. In cross repeated three times over three consecutive weeks, resulting 3D13D7, accessory chromosomes 16, 17, 19, and 20 in three biological replicates and six total replicates per were present in both parents but were missing in 1.6% (4/ progeny-cultivar pair. Automated image analysis of the sec- 249), 4.4% (11/249), 0.4% (1/249), and 1.2% (3/249) of the ond leaf was performed at 23 dpi as previously described progeny, respectively (fig. 2A). In the 1E4 1A5 cross, acces- (Stewart and McDonald 2014). Progeny were also pheno- sory chromosomes 14, 15, 16, 18, 19, 20, and 21 were pre- typed for temperature sensitivity, growth morphology, and sent in both parents but were absent in 7.5% (17/228), 2.2% fungicide sensitivity (Lendenmann et al. 2015, 2016). (5/228), 4.8% (11/228), 6.1% (14/228), 2.2% (5/228), 1.8% Phenotypes were compared in normal progeny and progeny (4/228), and 4.4% (10/228) of the progeny, respectively with “abnormal” (partially deleted, partially duplicated, diso- (fig. 2B). We found no progeny lacking a core chromosome mic, or absent) chromosomes to determine if particular chro- in either of the crosses. mosome genotypes were associated with outlier virulence, We also identified numerous instances of disomy in prog- fungicide resistance, temperature sensitivity, or growth rate eny accessory chromosomes. In cross 3D13D7, chromo- phenotypes. These analyses were performed in R version somes 17, 19, and 20 were present in two copies in 1.6% 3.4.0. (4/249), 0.8% (2/249), and 0.8% (2/249) of the progeny, respectively (fig. 2A). Interestingly, 2.4% (6/249) of the prog- eny were disomic for a core chromosome, with 1.6% (4/249) Results of the progeny disomic for chromosome 5 and 0.8% (2/249) Mapping RADseq Reads to the Reference Genome disomic for chromosome 13. No disomic core chromosomes The chromosome state (absent, present, or duplicated) was were identifiedincross 1E41A5 (fig. 2B), but 1.3% (3/228) determined for each chromosome of the four haploid paren- of the progeny were disomic for chromosome 14, 0.9% (2/ tal isolates (3D1, 3D7, 1A5, 1E4) and 477 progeny, using 228) were disomic for chromosome 18 and chromosomes 16, RADseq reads generated for each progeny mapped to the 19, 20, and 21 were each disomic in 0.4% (1/228) of the IPO323 reference genome. The 3D1 and 1A5 parents had progeny. all 21 chromosomes, while the 3D7 and 1E4 parents were Chromosomal inheritance that differed from the expected missing four and one accessory chromosomes, respectively 1:1 ratio was observed for several chromosomes that were (Croll et al. 2013). None of the four parental strains carried present in only one of the two parents of a cross. In the additional chromosomes beyond the 21 chromosomes iden- 3D13D7 cross, chromosomes 14, 15, 18, and 21 were ab- tifiedinIPO323 (Plissonneau et al. 2018). We selected the sent in the 3D7 parent, hence we expected these chromo- parental isolate from each cross that carried all 21 chromo- somes to be absent in half of the progeny. Instead, somes (3D1 and 1A5) as a reference. We mapped whole- chromosomes 14, 15, 18, and 21 were absent in only genome sequencing data of the two selected parents against 22.5% (56/249), 25.7% (64/249), 30.1% (75/249), and the IPO323 reference genome and identified regions missing 26.9% (67/249) of the progeny, respectively (fig. 2A). The in the parental genomes. Missing regions were not expected inheritance of these chromosomes are significant departures to show coverage in any of the progeny chromosomes and from the canonical Mendelian ratio (chromosome 14: 2 2 were excluded from further analyses. RADseq loci genotyped v ¼37.7, P< 0.001, chromosome 15: v ¼29.4, P< 0.001, in progeny showed an even distribution across all 21 chromo- chromosome 18: v ¼19.7, P< 0.001, and chromosome 21: somes, with no apparent differences between core (1–13) v ¼26.6, P< 0.001). We also tested whether chromosomes and accessory chromosomes (14–21; supplementary fig. 1, 14, 15, 18, and 21 occurred independently from one another Supplementary Material online). Similarly, RADseq loci in progeny. We found that progeny lacking one or four chro- showed homogeneous read coverage across the genome mosomes did not deviate significantly from expectations (v for progeny with high and low overall sequence coverage in ¼ 0.01, P¼ 0.9; v ¼ 0.82, P¼ 0.3, respectively). However, both crosses (supplementary fig. 2A–D, Supplementary we found that progeny having all four chromosomes occurred Material online). For each progeny, we calculated the cover- much more frequently than expected (v ¼ 622.65, age for each chromosome and compared this to the median P< 0.001), while having one or two of the four chromosomes coverage of all chromosomes for that isolate (fig. 1). The nor- also occurred more frequently than expected (v ¼ 14.7, malized coverage per chromosome was close to 1 for the P< 0.001; v ¼ 37.76, P< 0.001, respectively). In the large majority of the chromosomes (supplementary fig. 3, 1E41A5 cross, chromosome 17 was missing in 53.5% Supplementary Material online). The mean normalized cover- (112/228) of the progeny and did not exhibit distorted inher- age ratio was 0.96 and 0.95 for the progeny from cross itance (v ¼0.56, P¼ 0.3) (fig. 2B). Disomy was also found for 3D13D7 and cross 1A51E4, respectively. several accessory chromosomes that were present in only one 1420 Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Meiosis Leads to Pervasive Copy-Number Variation and Distorted Inheritance GBE Cross 3D1x3D7 Chromosomal state 150 Duplicated Loss Normal Partial Deletion Partial Duplication Expected number of chromosomes in progeny [only one parent carried a copy] 1 2 3456789 10 11 12 13 14 15 16 17 18 19 20 21 Chromosome Cross 1E4x1A5 Chromosomal state Duplicated Loss Normal Partial Deletion Partial Duplication 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Chromosome FIG.2.—Summary of the total chromosome anomalies in the progeny of two crosses. Normal, disomic, lost, and rearranged (partially duplicated or deleted) chromosomes are shown separately for cross 3D13D7 and 1E41A5. Dotted lines show the expected number of progeny for chromosomes that were present in only one of the two parental isolates. of the parents. In cross 3D13D7, additional copies of chro- originating from both parents and the other three cases mosome 14 and 18 were identified in 0.4% (1/249) and were ambiguous. As indicated earlier, chromosome 17 could 0.8% (2/249) of the progeny, respectively (fig. 2A). In cross only have originated from one of the parents. 1E41A5, chromosome 17 was disomic in 0.9% (2/228) of the progeny (fig. 2B). Meiosis Generates Novel Chromosome Length Disomic chromosomes can either be heterozygous, carry- Polymorphism ing one of each parental chromosomal copy, or homozygous if the disomy arose from a single parental chromosome In order to identify partially deleted or duplicated chromo- (fig. 3). To distinguish these scenarios, we analyzed disomic somes in the progeny, we investigated chromosomes which progeny chromosomes and restricted the analyses to cases had a normalized coverage between 0.3 and 0.7, and be- where both parents were carrying a chromosomal copy. In tween 1.3 and 1.7 (fig. 1). In cross 3D13D7 (fig. 2A), partial the 3D13D7 cross, 59% (10/17 cases) of the disomic isolates deletions were identified for chromosomes 14 (0.4% of off- were heterozygous, with a chromosome originating from spring, 1/249) and 15 (0.4%, 1/249). Partial duplications were each parent and 29% (5/17 cases) of the disomic isolates detected for chromosomes 14 (0.4%, 1/249), 16 (0.8%, were homozygous, with both chromosomes originating 2/249), 19 (2.4%, 2/249), and 21 (0.4%, 1/249). We also from one parent (fig. 4A). In the case of chromosomes 14 identified one isolate which may have a partially duplicated and 18, the chromosomes could only originate from one par- core chromosome 10. In cross 1E41A5 (fig. 2B), partial ent. In cross 1E41A5, 5 of the 11 disomic isolates were duplications were detected in the progeny for chromosomes homozygous, three disomic isolates had chromosomes 14 (0.9%, 2/228), 15 (0.9%, 2/228), 16 (0.9%, 2/228), Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 1421 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Number of progeny Number of progeny Fouche et al. GBE Mitosis Meiosis I Meiosis II B Meiosis I Meiosis II Mitosis Meiosis I Meiosis I I Mitosis FIG.3.—Schematic overview of how chromosomal nondisjunction can result in chromosome loss or disomy. (A) During canonical meiosis, the haploid nuclei from the two parents fuse resulting in a single diploid nucleus. Parental chromosomes are shown with distinct colors. Chromosomes go through meiosis I and II, followed by mitosis, resulting in eight haploid ascospores. Chromosome loss or disomy can occur as a result of homologous chromosomes failing to segregate during meiosis I (B), resulting in heterozygous disomy with one chromosome originating from each of the parents. The alternative is the failure of sister chromatid segregation during meiosis II (C), generating homozygous disomic progeny with both copies of the chromosome originating from the same parent. 17 (1.8%, 4/228), 19 (0.4%, 1/228), and 20 (1.8%, 4/228). duplication of chromosome 10 while isolate 137.2 had partial Partial losses were identified for chromosomes 14 (0.4%, 1/ duplications of chromosomes 16, 19, and 21. In cross 228), 15 (1.3%, 3/228), 16 (3.5%, 8/228), 17 (2.6%, 6/228), 1E41A5, isolate B23.1 was disomic for chromosome 20 and 21 (3.1%, 7/228). and had partial deletions of chromosomes 17 and 21. This We identified some progeny with multiple chromosomal isolate also had a partially duplicated chromosome 14. Isolate anomalies, however these associations did not deviate signif- B24.2 also had partial deletions of chromosomes 17 and 21. icantly from a random expectation. In cross 3D13D7, isolate Isolate C44.2 had partially deleted chromosomes 16 and 21. 89.1 was disomic for chromosome 13 and had a large, partial Isolate B50.1 was disomic for chromosome 17 and had a 1422 Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Meiosis Leads to Pervasive Copy-Number Variation and Distorted Inheritance GBE Cross 3D1x3D7 Cross 1E4x1A5 1.00 1.0 ● ● ● ● ● 0.8 0.75 Disomic state ● Ambiguous 0.6 ● Heterozygous 0.50 ● Homozygous ● 0.4 0.25 ● ● ● 0.2 5 131417 181920 14 16 17 18 19 20 21 Chromosome Chromosome FIG.4.—Identification of heterozygous and homozygous disomic chromosomes in cross 3D13D7 and cross 1E41A5. Single nucleotide polymor- phism (SNP) loci were screened on progeny chromosomes that showed evidence for disomy. SNPs were genotyped as either homozygous, containing only one of the parental alleles, or heterozygous if both parental alleles were found. The ratio represents the number of homozygous SNPs compared with the total number of genotyped SNPs. Individual dots represent each of the disomic progeny chromosomes identified in the two crosses. Due to uncertainties in SNP calling, we used cut-offs to assign progeny chromosomal states. Chromosomes with a ratio<0.4 were assigned as heterozygous disomic, likely resulting from nondisjunction at meiosis II, >0.6 as homozygous disomic, likely resulting from nondisjunction at meiosis I, and ratios between 0.4 and 0.6 were assigned as ambiguous. partially deleted chromosome 21. Isolate A57.1 was disomic for chromosome 14 and had a partially duplicated chromo- some 16. In cross 3D13D7, we found twelve progeny isolates with partial deletions and duplications. Seven of these partial aneu- ploidies affected chromosomal segments near the telomeric Chromosomal state Loss ends (supplementary fig. 4, Supplementary Material online). Duplicated Isolate 89.1 had a normalized coverage ratio for chromosome Partial Duplication Central 10 of 1.63 suggesting a partial duplication. However the cov- Partial Duplication Telomeric erage along the chromosome was homogeneous, with no Partial Deletion Central apparent duplicated chromosomal regions when compared Partial Deletion Telomeric with the parent chromosomes (supplementary fig. 5, Ambiguous Duplication Supplementary Material online). We considered such cases Ambiguous Deletion as ambiguous duplications. In cross 1A51E4, we found 40 partial deletions and duplications, of which 19 were ambigu- ous and 15 occurred in chromosomal segments near the telo- meric ends (fig. 5). 14 15 16 17 18 19 20 21 Correlation of Chromosomal Features with the Fidelity of Chromosome Transmission FIG.5.—Identification of partial chromosome losses or duplica- During meiosis, chromosomes pair prior to recombination and tions in cross 1E41A5. A summary of all the chromosome number therefore length similarity could play a role in homolog iden- and length polymorphisms in the progeny of cross 1E41A5, as well tification and enable chromosomes to pair and recombine. as the location where the length polymorphism occurred. Most of the However, we found no correlation between the length simi- rearrangements were ambiguous (19), 15 were located toward the larity of the parent chromosomes and the fidelity with which ends of chromosomes and 6 rearrangements occurred in the central chromosomes were inherited (fig. 6A). In general accessory region of the chromosomes. Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 1423 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Ratio Ratio Number of progeny Fouche et al. GBE Chromosomal state Chromosome type ● Duplication Accessory Loss ● Core Cross 3D1 x 3D1 Cross 1E4 x 1A5 ● ● 2 ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ●● ●● ● ● ●● ● ● ● ● ● 05 10 0 5 10 15 Percentage length difference Percentage length difference ● ● 2 ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ●●● ● ● ● ● ●● ● ● 60 80 100 120 40 60 80 100 Recombination rate (cM/Mb) Recombination rate (cM/Mb) ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ●● ● ● 0 ● ● ●●● ● ● ●● ● ● ● ● 65 70 75 80 85 60 70 80 Percentage sequence similarity Percentage sequence similarity ● ● 2 ● ● ● ● ●● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ●● ● ● 15 20 25 30 10 20 30 Percentage repetitive sequences Percentage repetitive sequences FIG.6.—Correlations between chromosome length similarity, recombination rate, percent sequence similarity, fraction of repetitive sequences and the inheritance of chromosomes. Complete and partial chromosome losses and duplications were correlated with length similarity (A), recombination rate (B), sequence similarity (C), and repeat content (D) of the parental chromosomes. Correlations are shown separately for crosses 3D13D7 and 1E41A5. chromosomes were more unstable than core chromosomes. between the recombination rate and chromosome transmis- Interesting exceptions were a disomic core chromosome 13 sion fidelity (fig. 6B). However, in cross 1A51E4, most of the (length difference 5% between the parents) and a disomic chromosome losses and disomies occurred in accessory chro- core chromosome 5 (length difference of 8.4% between the mosomes with a low recombination rate (fig. 6B). Next, we parents). The rate of disomy for these core chromosomes was analyzed sequence similarities between parental chromo- 1.6% (4/249 progeny). We found no significant correlation somes and correlated this with the chromosome transmission 1424 Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Percentage Percentage Percentage Percentage Percentage Percentage Percentage Percentage Meiosis Leads to Pervasive Copy-Number Variation and Distorted Inheritance GBE 14 15 16 17 18 19 20 21 17 Cross 3D1x3D7 ● ● ● ● ● ● ● ● ● Chromosome state Chromosome state Abnormal ● ● ● ● ● ● ● ● ● Duplicated Loss Normal ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Normal 300 300 Partial Deletion ● Partial Duplication ● ● ● ● ● ● ● ● ● ● ● ● ● 100 100 14 15 16 17 18 19 20 21 21 Cross 1E4x1A5 Chromosome state Chromosome state ● ● Abnormal Duplicated Normal Loss Normal Partial Deletion 80 80 Partial Duplication ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 60 60 ● ● ● ● ● ● ● ● ● FIG.7.—Association between accessory chromosomes and phenotypes. Accessory chromosome states, normal or abnormal (duplicated, lost, partially duplicated, or partially lost), were compared with virulence traits using a two-sample t-test (multiple testing correction threshold of P< 0.002). (A)In the progeny of 3D13D7, isolates with a normal chromosome 17 had a significantly higher pycnidia count on the wheat cultivar Runal that isolates with a duplicated or lost chromosome (P¼ 0.0019). (B)In cross 1E41A5, isolates with a lost or partially deleted chromosome 21 had a higher percent leaf area covered by lesions (PLACL) on Titlis than isolates with a normal chromosome 21 (P¼ 0.000024). fidelity. For this, we compared whole chromosome sequences on two wheat cultivars (Runal and Titlis) using data from a and calculated the percentage of syntenic regions between previous study (Stewart and McDonald 2014; Stewart et al. homologous chromosomes. The accessory chromosomes in 2018). Progeny from cross 3D13D7witha normal chromo- the parents for both crosses had a much lower synteny than some 17 had a higher pycnidia count on the cultivar Runal the core chromosomes and had substantially lower transmis- than isolates with an abnormal chromosome 17 (P¼ 0.0019; sion fidelity (fig. 6C). Accessory chromosomes had overall a fig. 7A, supplementary fig. 6, Supplementary Material online). higher content of repetitive elements, which was similarly Isolates missing chromosome 17 had a lower pycnidia count correlated with lower transmission fidelity (fig. 6D). than isolates that were disomic for chromosome 17. On cul- tivar Titlis, progeny from cross 3D13D7 with a normal chro- mosome 18 had significantly darker pycnidia (P¼ 0.0018; Association between Accessory Chromosomes and supplementary fig. 7, Supplementary Material online). Phenotypic Traits Progeny with an abnormal chromosome 19 had a marginally We analyzed whether the chromosome states in progeny higher percent leaf area covered by lesions (PLACL; were correlated with variation in phenotypic traits. For this, P¼ 0.0024; supplementary fig. 7, Supplementary Material we considered first only two chromosome states: normal online). For progeny from cross 1E41A5, we found a corre- (haploid) or abnormal (any loss, duplication, or rearrange- lation of the PLACL produced on Titlis with chromosome 21 ments). We tested for an association with phenotypic traits (P¼ 0.00002; fig. 7B; supplementary fig. 8, Supplementary using two-tailed t-tests (multiple testing significance threshold Material online). Isolates with a partially deleted or lost chro- at P< 0.002). We first tested for associations with virulence mosome 21 had a higher PLACL. For progeny of cross Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 1425 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 PLACL Pycnidia Count PLACL Pycnidia Count Fouche et al. GBE FIG.8.—Correlation between number of disomic progeny and chromosomal rearrangements. Circles and triangles represent accessory chromosomes and core chromosomes, respectively. Chromosomes from cross 3D13D7 are represented in blue, and chromosomes from cross 1E41A5 are in red. 1E41A5, we found that isolates with an abnormal chromo- chromosomal transmission through meiosis. We found exten- some 20 showed higher PLACL on Runal. We found no sig- sive chromosome number and length variation among the nificant correlations for phenotypes related to growth, progeny in both crosses. The rates of disomy and rearrange- fungicide resistance, or temperature sensitivity. ments differed greatly between chromosomes and crosses. Nearly all aberrant chromosomal transmission events affected accessory chromosomes with the rare exception of core chro- Correlation between Disomy and Chromosomal mosome disomies. Several accessory chromosomes showed Rearrangements strongly distorted chromosomal inheritance. Chromosome number polymorphism in Z. tritici has previ- We analyzed whether rates of disomy were correlated with ously been linked to errors occurring during meiosis rates of rearrangements. Nondisjunction results in the loss of a (Wittenberg et al. 2009; Croll et al. 2013). In our study, we chromosome in one progeny and a chromosome gain in the generated a substantially more dense marker coverage using corresponding twin spore from the same ascus. Core chro- the Illumina-based sequencing technique RADseq and were mosomes generally showed only very rare cases of disomy or able to screen more isolates (477 isolates compared with 144 rearrangements (fig. 8). Accessory chromosome 14 was more and 216 isolates, respectively; Wittenberg et al. 2009; Croll frequently disomic and rearranged in progeny from cross et al. 2013). Because RADseq generated a high coverage of 1A41E5. Chromosome 15 underwent partial duplications 100-bp sequences at defined restriction sites, we could pre- and deletions, but we found no evidence for nondisjunction. cisely map sequences to chromosomal positions without hav- Chromosome 16 was both frequently rearranged (4.4%, 10/ ing to rely on genetic map constructions. Physical marker 228) and disomic (0.4% 1/228) among the progeny in positions are particularly important for analyzing accessory 1E41A5. In cross 3D13D7, chromosome 17 was disomic chromosomes of Z. tritici because of their very low rates of in 1.6% (4/249) of the progeny, while in cross 1E41A5 recombination (Croll et al. 2015). In contrast to previous stud- chromosome 17 was more rarely disomic (0.9%, 2/228). ies, our use of RADseq markers allowed us to directly detect Chromosome 17 showed even stronger differences in rear- duplicated chromosomal segments by analyzing variations in rangements among crosses, with 4.4%, (10/228) in cross sequencing coverage. 1E41A5versus0.0% incross 3D13D7. Chromosome 19 Our analyses revealed that all eight accessory chromo- was both more likely to undergo rearrangements and to be somes underwent chromosome loss during meiosis. The inherited as a disomic chromosome in cross 3D13D7. In rate of chromosomal loss depended on the chromosome contrast, chromosome 21 was both more likely to be rear- and varied between the crosses. This confirms the findings ranged and to be inherited in a disomic state in cross of Croll et al. (2013) except that a loss of chromosome 15 had 1E41A5. not previously been detected. We found that 5 progeny (2.1%) had lost this chromosome. No isolate was found lack- Discussion ing a core chromosome despite screening 477 progeny. This We used RADseq data generated for several hundred progeny indicates that all 13 core chromosomes are likely encoding from two crosses of Z. tritici to identify aberrations in essential functions for the growth and survival of the fungus. 1426 Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Meiosis Leads to Pervasive Copy-Number Variation and Distorted Inheritance GBE Chromosome loss most likely occurred as a result of errors We found that an average of 5.9% of the progeny isolates during chromosome segregation, specifically nondisjunction were disomic for one or more chromosomes. This number is of sister chromatids during either meiosis I or II. In accordance similar to what was found for Saccharomyces cerevisiae, with previous studies, we found that the loss of accessory where 8% of the lab strains were estimated to be aneuploid chromosomes during meiosis is common. In natural popula- (Hughes et al. 2000). Disomy is generated when chromo- tions, this may lead to the complete loss of an accessory chro- somes undergo nondisjunction during meiosis, resulting in mosome in the absence of counteracting mechanisms that one daughter cell with two copies of a chromosome and maintain these chromosomes. one daughter cell with no copies of that chromosome Wittenberg et al. (2009) proposed that distorted segrega- (fig. 3). Therefore, for each disomic offspring, we expect a tion of accessory chromosomes could serve as a mechanism corresponding offspring that is missing the same chromo- to prevent their complete loss from a population. some. As expected, we found that chromosomal loss was Chromosomes present in only one parent are expected to often accompanied by disomy. However, contrary to expect- segregate into 50% of the daughter cells. However, we found ations, there was no symmetry in the loss and disomy rates. that in cross 3D13D7 chromosomes 14, 15, 18, and 21 For example, despite finding many progeny lacking chromo- from parent 3D1 were significantly overrepresented in the some 15, no isolate disomic for chromosome 15 was recov- progeny. The transmission advantage resulting from unequal ered. The rates of nondisjunction also differed between segregation is referred to as “meiotic drive” and is frequently chromosomes and between crosses, suggesting that the associated with accessory or B chromosomes (Jones 1991). In loss or disomy of specific chromosomes may be counter se- our study, distorted inheritance was not universal, for exam- lected. In addition, chromosomes differed in their composition ple chromosome 17 in cross 1E41A5 segregated normally. of repetitive elements. Repetitive elements are likely to play an The distorted inheritance pattern in cross 3D13D7 could be important role by influencing the likelihood of faithful disjunc- explained if parent 3D1 already had disomic accessory chro- tion. We also found that nondisjunction was happening dur- mosomes. But our coverage analysis did not detect disomic ing both meiosis I and II. We found heterozygous disomic chromosomes in any of the parents. It is possible that a small chromosomes, which were created as a result of nondisjunc- fraction of the clonal cell pool of a parental mycelium might tion in meiosis I. Heterozygous disomic chromosomes were have harbored disomic chromosomes, but this is not likely to most frequent in cross 3D13D7. In cross 1E41A5, homo- explain the observed rates of disomic accessory chromo- zygous disomy resulting from nondisjunction in meiosis II oc- somes. The overrepresentation of progeny carrying a specific curred more frequently. Aneuploidy can play an important accessory chromosome could be due to selection favoring role in the adaptive evolution of fungal pathogens. In human progeny carrying this chromosome. Such viability selection pathogens, aneuploidy is often associated with drug resis- could not be tested in this experiment because we were un- tance (Hu et al. 2008; Selmecki et al. 2010). Over 50% of able to generate full tetrad sets of offspring and quantify the fluconazole-resistant strains isolated from patients had genotype-specific survival rates. However, if loci located on whole or partial chromosome duplications (Selmecki et al. accessory chromosomes encoded strongly deleterious variants 2006). Correlations between disomic states and phenotypic for growth on culture media, quantitative trait mapping stud- traits in Z. tritici suggests that selection could also be affecting ies performed on the same progeny sets would most likely rates of disomy, albeit with less drastic impacts than in human have identified QTLs linked to accessory chromosomes. pathogens selected for drug resistance. However, no such evidence was found (Lendenmann et al. Aneuploidy typically causes a dosage imbalance, which 2014, 2016). could explain why accessory chromosome aneuploidies are Additional explanations for the observed distortion in in- tolerated more frequently than core chromosome aberra- heritance may include a meiotic drive mechanism such as se- tions. Alternatively, gene expression or dosage compensation lective spore killing. The distortion could also be linked to could have evolved on frequently disomic chromosomes, “sticky” centromeres similar to those found in rye B chromo- which may explain the tolerance for additional copies of cer- somes where the transmission at higher than Mendelian fre- tain chromosomes, but not others (Torres et al. 2008). quencies was explained by the presence of particular Chromosomes that have a higher rate of disomy could have centromeres that ensure that B chromosomes migrate to shorter or nonfunctional telomeres. Telomere defects were the generative pole that will be transmitted to the next gen- found to explain mitotic instability in human mammary epi- eration of plants (Banaei-Moghaddam et al. 2012). In or- thelial cells (Pampalona et al. 2010). Chromosomes with der to distinguish among the possible mechanisms leading shorter telomeres are more likely to undergo nondisjunction. to distorted inheritance, all meiotic products from individ- Furthermore, chromosomes with higher degrees of synteny ual tetrads would have to be analyzed. However, experi- are more likely to pair correctly, resulting in fewer nondisjunc- mental limitations in the generation of large numbers of tion events. We found indications that sequence similarity in individual tetrads prevented us from making more de- the parent chromosomes indeed leads to higher fidelity of tailed investigations. chromosomal inheritance. Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 1427 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Fouche et al. GBE Homologous chromosomes of Z. tritici segregate signifi- accessory chromosomes may be maintained in the species cant structural variation in populations, differing in repeat pool by a selection-drift balance. and gene content, chromosomal length, and recombination Most chromosome rearrangements are thought to be del- rate, as well as telomere and centromere composition (Croll eterious and therefore counter-selected. The ability of Z. tritici et al. 2013, 2015; Schotanus et al. 2015; Plissonneau et al. to tolerate a large number of disomies and chromosomal 2016). Synteny breakpoints are commonly associated with rearrangements makes this species an excellent model for repetitive sequences or transposable element clusters that detailed analyses of rearrangements and nondisjunction can misalign during recombination, thereby generating length events. Despite the fact that the meiotic machinery is highly polymorphism. Such a mechanism was thought to generate a conserved, the strength of selection against erroneous chro- novel chromosome 17 in the progeny of cross 1A51E4 (Croll mosomal transmission can differ widely among species. et al. 2013). In our study, we found no correlation between Relaxed selection on chromosomal transmission can lead to length similarity and recombination rate of the parent chro- highly polymorphic chromosomal sets observed in some eu- mosomes, and the fidelity of chromosome inheritance. karyotic pathogens. Determining the trade-offs involved in However, chromosomes with higher synteny between the maintaining chromosomal integrity and generating chromo- parents and fewer repeats were transmitted more faithfully. somal polymorphism will elucidate how selection operates to Selection favoring the presence or absence of specific ac- maintain the fidelity of meiotic processes. cessory chromosomes would require that accessory chromo- somes directly or indirectly influence phenotypic traits. Supplementary Material However, accessory chromosomes carry few genes and Supplementary data are available at Genome Biology and none are thought to perform a specific function during the Evolution online. life cycle of the fungus (Goodwin et al. 2011). Interestingly, we found a correlation between the presence of chromo- somes 15, 18, and 21 and higher levels of virulence in cross Acknowledgments 3D13D7 (Stewart et al. 2018). In addition, we found a cor- relation between the presence of a normal chromosome 17 We thank Marcello Zala for providing access to progeny col- and an abnormal chromosome 19 and higher levels of pyc- lections and helpful discussions. C.P. was supported by an nidia and PLACL, respectively. In a separate study, whole- INRA Young Scientist grant. S.F. and B.A.M. are supported chromosome deletion mutants of a different Z. tritici strain by the Swiss National Science Foundation (grant were generated by blocking b-tubulin assembly during mitosis 31003A_155955). D.C. is supported by the Swiss National using carbendazim (Habig et al. 2017). A comparison of iso- Science Foundation (grant 31003A_173265). genic lines lacking individual accessory chromosomes showed that the loss of chromosomes 14, 16, 18, 19, and 21 resulted Literature Cited in increased virulence on the wheat cultivar Runal. This finding is in opposition to our own study that showed that the pres- Baird NA, et al. 2008. 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The pri- mary target organ of Cryptococcus gattii is different from that Associate editor:Laura Rose Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 1429 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genome Biology and Evolution Oxford University Press

Meiosis leads to pervasive copy-number variation and distorted inheritance of accessory chromosomes of the wheat pathogen Zymoseptoria tritici

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
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1759-6653
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1759-6653
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10.1093/gbe/evy100
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Abstract

Meiosis is one of the most conserved molecular processes in eukaryotes. The fidelity of pairing and segregation of homologous chromosomes has a major impact on the proper transmission of genetic information. Aberrant chromosomal transmission can have major phenotypic consequences, yet the mechanisms are poorly understood. Fungi are excellent models to investigate processes of chromosomal transmission, because many species have highly polymorphic genomes that include accessory chromosomes. Inheritance of accessory chromosomes is often unstable and chromosomal losses have little impact on fitness. We analyzed chromosomal inheritance in 477 progeny coming from two crosses of the fungal wheat pathogen Zymoseptoria tritici.For this, we developed a high-throughput screening method based on restriction site-associated DNA sequencing that generated dense coverage of genetic markers along each chromosome. We identified rare instances of chromosomal duplications (disomy) in core chromosomes. Accessory chromosomes showed high overall frequencies of disomy. Chromosomal rearrangements were found exclusively on accessory chromosomes and were more frequent than disomy. Accessory chromosomes present in only one of the parents in an analyzed cross were inherited at significantly higher rates than the expected 1:1 segregation ratio. Both the chromosome and the parental background had significant impacts on the rates of disomy, losses, rearrangements, and distorted inheritance. We found that chromosomes with higher sequence similarity and lower repeat content were inherited more faithfully. The large number of rearranged progeny chromosomes identified in this species will enable detailed analyses of the mechanisms underlying chromosomal rearrangement. Key words: fungi, meiosis, chromosome rearrangements, progeny sequencing. Introduction essential for the faithful segregation of chromosomes Sexual reproduction requires chromosomes to undergo mei- (Naranjo 2012), chromosomes can pair along their entire osis, whereby homologous chromosomes pair, recombine, length or in a segment-specific manner where only some and finally separate and migrate to opposite poles of the mei- regions align (Roeder 1997). This suggests that the length otic cell. Meiosis is a highly conserved process initiated by the and degree of sequence similarity can affect homolog identi- pairing of homologous chromosomes that first recognize one fication and pairing. After pairing, recombination produces another and then establish recombination-dependent links crossovers that physically link homologs, mediate proper seg- between homologs to form the synaptonemal complex regation, and thereby preserves chromosomal integrity (reviewed in Roeder 1997). This is followed by two divisions, (Mather 1938; Baker et al. 1976; Hassold and Hunt 2001). first to separate homologous chromosomes and then to sep- Recombination between misaligned repetitive sequences can arate sister chromatids. While accurate pairing of homologs is generate length variation among the daughter chromosomes The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non- commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 1416 Genome Biol. Evol. 10(6):1416–1429. doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Meiosis Leads to Pervasive Copy-Number Variation and Distorted Inheritance GBE (Montgomery et al. 1991). After pairing and recombination, transmission of chromosomes through meiosis because of segregation occurs via centromeres that bind to chromosome their extreme chromosomal plasticity, the ubiquity of sexual proteins and mediate accurate segregation to the opposite reproduction, and their experimental tractability. poles of the cell. The fungal wheat pathogen Zymoseptoria tritici provides a Aberrant transmission of chromosomes from one genera- striking example of genome plasticity. The bipartite genome tion to the next, including partial and whole chromosome consists of 13 core and up to eight accessory chromosomes duplications or losses, are caused largely by erroneous pairing that exhibit significant length polymorphism within and during meiosis. Such duplication and loss events can affect a among field populations (Goodwin et al. 2011; Croll and large number of genes and alter gene expression across the McDonald 2012). Chromosomal rearrangements played an genome (Harewood and Fraser 2014). The most dramatic important role in adaptation to different host genotypes copy-number variation is aneuploidy. Unequal sets of chro- (Hartmann et al. 2017). The accessory chromosomes are mosomes result from nondisjunction and are the leading ge- highly unstable through meiosis and were shown to undergo netic cause of miscarriages in humans (Hassold and Hunt rearrangements, segregation distortion, and nondisjunction 2001). Atypical phenotypes associated with aneuploid states (Wittenberg et al. 2009; Croll et al. 2013). Z. tritici reproduces are caused by gene dosage imbalances that can cause severe sexually when hyphae originating from two haploid spores of defects (Torres et al. 2008). In general, aneuploidy and chro- opposite mating type fuse to produce a transient diploid stage mosomal rearrangements are associated with lower fitness that undergoes two rounds of meiosis followed by one round (Torres et al. 2008), but in rare circumstances, errors during of mitosis to produce eight ascospores in an ascus. The path- meiosis can provide adaptive genetic variation. For example, ogen tolerates aneuploidy, so chromosomal rearrangements in the human pathogenic fungi Cryptococcus neoformans and generated through this process in both the core and accessory Candida albicans, specific aneuploidies contribute to drug re- genomes can remain viable (Wittenberg et al. 2009; Croll sistance (Selmecki et al. 2006, 2008; Sionov et al. 2010; et al. 2013; Schotanus et al. 2015). Hence, this species is an Ngamskulrungroj et al. 2012). Adaptive aneuploidy is fre- ideal model to analyze patterns of aberrant chromosomal quently associated with response to stressful environments transmission. (Chen et al. 2012). The dosage imbalance and altered stoichi- In this study, we analyze the mechanisms that affect the ometry due to additional copies of genes on a duplicated fidelity of chromosomal inheritance through meiosis, includ- chromosome may not be beneficial under normal conditions, ing identification of chromosomal rearrangements, losses, but can become beneficial under stress (Pavelka, Rancati, and and duplications. For this, we screened hundreds of progeny Li 2010; Pavelka, Rancati, Zhu, et al. 2010). In pathogenic genotypes generated from two independent crosses and de- fungi, aneuploidy often occurs for only a restricted number termined the rate of aneuploidy, patterns of rearrangement of chromosomes, however the mechanisms determining the and distortions in transmission rates. Finally, we investigated rate of aneuploidy generation and its maintenance are poorly whether factors such as length similarity, synteny, recombina- understood. tion rate, and repetitive element content affected the fidelity Aneuploidy also plays an important role in several plant of chromosomal inheritance. pathogenic fungi. Several important plant pathogens have highly dynamic genomes with chromosomes that show sig- Materials and Methods nificant length and number polymorphisms within the Generation of Sexual Crosses species. This chromosomal plasticity is often restricted to a Two crosses were performed between four parental Z. tritici well-defined set of accessory chromosomes. This bipartite ge- isolates collected from two Swiss wheat fields separated by nome structure, characterized by an accessory genome region 10 km. Isolate ST99CH3D1 was crossed with isolate that is rapidly diversifying and a core genome region that ST99CH3D7 (hereafter abbreviated 3D1 and 3D7) and isolate remains conserved, can be associated with the trajectory of ST99CH1A5 was crossed with isolate ST99CH1E4 (abbrevi- pathogen evolution (Croll and McDonald 2012; Dong et al. ated 1A5 and 1E4), producing 359 and 341 haploid asco- 2015). The accessory region is often rich in transposable ele- spore progeny, respectively. The genomes of all four ments that drive chromosomal rearrangements, deletions, parental isolates were sequenced using Illumina technology and duplications (Zhang et al. 2011). Accessory chromosomes (Torriani et al. 2011) and are available under the NCBI SRA are not shared among all members of a species, therefore accession numbers SRS383146 (3D1), SRS383147 (3D7), these chromosomes can contribute significantly to polymor- SRS383142 (1A5), and SRS383143 (1E4). The parental iso- phism within a species. Importantly, many plant pathogens lates were already genetically characterized and have been have been shown to harbor pathogenicity loci on accessory phenotyped for virulence and many other traits (Zhan et al. chromosomes (Mo ¨ ller and Stukenbrock 2017). In contrast, 2005; Croll et al. 2013). Full sib families were produced by the core regions encode essential functions required for sur- coinfecting wheat leaves with asexual conidia from the pa- vival and reproduction. Plant pathogenic fungi provide partic- rental strains of opposite mating types using the crossing ularly powerful models to investigate factors affecting the Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 1417 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Fouche et al. GBE protocol described by Kema et al. (1996). Briefly, spores of a et al. 2014). Only one randomly selected progeny per clonal pair of parents were sprayed onto wheat plants and incu- group was kept for further analyses, reducing the number of bated outdoors for 40–60 days until well-developed symp- progeny to 263 in the 3D13D7cross andto261 in the toms including pseudothecia were observed. Ascospores 1A51E4 cross. were isolated from pseudothecia over a period of several days by placing the infected wheat leaves on wet filter paper Determining Chromosome Number and Length inside Petri dishes. Leaves were covered with upside down Polymorphisms Based on Coverage Petri dish lids that were previously filled with water agar. Restriction sites cut by PstI were identified in silico using the This setup allowed us to capture ascospores that were verti- EMBOSS restrict program (http://www.bioinformatics.nl/ cally ejected from mature pseudothecia. Released ascospores cgi-bin/emboss/restrict; last accessed September 2016). were left to germinate on the water agar to enable inspection Thereafter, the coverage of RADseq reads mapping to for potential contaminants and to ensure that only progeny the restriction sites was determined using the BEDtools resulting from single ascospores were selected. Germinating v. 2.25.0 intersectBed and coverageBed commands ascospores were transferred to individual culture plates for (Quinlan and Hall 2010). Reads were counted if the map- clonal propagation. The mycelium produced by each success- ping quality score was20. The coverage of the se- fully germinated ascospore was used for DNA extraction and quenced parent genomes was determined following the plant infection experiments. Offspring mycelium was pro- same procedure. Progeny with a median read coverage duced in YSB (yeast sucrose broth) liquid media for 6–7 days of<20 were excluded from further analyses to avoid prior to DNA extraction. biases introduced by low-coverage data, resulting in fewer isolates being included in this analysis than in pre- vious studies (Lendenmann et al. 2014, 2016; Stewart Reference Alignment Using Restriction Site-Associated et al. 2018). We retained 249 progeny in the 3D13D7 DNA Sequencing cross and 228 isolates in the 1A51E4 cross. We used We used Restriction Site-Associated DNA Sequencing normalized read counts to detect chromosomal anoma- (RADseq) (Baird et al. 2008) for large-scale sequence gen- lies, where those with a normalized coverage close to zero otyping as described previously (Croll et al. 2015). Briefly, (<0.3) were classified as missing, those with a normalized the RADseq protocol (Etteretal. 2011) was applied to Z. coverage close to one (>¼0.7 and<1.3) were classified as tritici by using the PstI restriction enzyme to digest 1.3 lg present and those with a normalized coverage close to of DNA extracted with the DNAeasy plant mini kit two (>¼1.7) were classified as disomic (fig. 1A). Partially (QIAGEN Inc., Basel, Switzerland) for each offspring. deleted and partially duplicated chromosomes were iden- After digestion and adapter annealing, the pooled DNA tified based on a normalized coverage ratio of>¼0.3 was sequenced on an Illumina HiSeq2000 using a paired- and<0.7 or>1.3 and<1.7, respectively. Deviations end 100-bp library. Pools contained 132 progeny, six from Mendelian inheritance for accessory chromosomes different Illumina TruSeq compatible P2 adapters and 22 present in only one of the parents were determined using P1 adapters with unique barcodes. Progeny DNA with the a chi-squared (v )test. same P2 adapter were distinguishable by using the unique barcodes ligated to the P1 adapters. Distinguishing between Homozygous and Heterozygous Illumina reads were quality trimmed using Trimmomatic v. Disomy 0.30 (Bolger et al. 2014) and separated into distinct sets for each progeny based on the P1 adapter using FASTX toolkit v SNP calling was performed using Freebayes (Version 0.13 (http://hannonlab.cshl.edu/fastx_toolkit/; last accessed 1.0.2_1 1.1.0) (Garrison and Marth 2012)using the bam- March 2015). Reads were aligned to the gapless telomere to files of each isolate mapped to the IPO reference genome. telomere IPO323 reference genome (assembly version MG2, We used the parameters no-indels, no-mnps, no-complex, Septemeber 2008) (Goodwin et al. 2011) with the short-read and ploidy 2. Then we filtered for sites that differed be- aligner version of bowtie 2.1.0 (Langmead and Salzberg 2012) tween the parents (maf 0.2) and considered only these using the default parameters for sensitive end-to-end align- regions to determine whether disomic chromosomes orig- ment (-D 15; -R 2; L- 22; -I S, 1, 1.15). The same parameters inated from one or both parents. We also filtered for from trimming and reference assembly were used to align the depth (minDP 30) and quality (minQ 30). The VCF tools - four parental genome sequences (Croll et al. 2013)to the ref- het function was used to determine the number of homo- erence genome (IPO323). RADseq aligned reads are available zygous sites and the total number of sites. We determined under the NCBI BioProject accession numbers PRJNA256988 the ratio of homozygous sites to the total number of sites and PRJNA256991. Potential clones were identified as geno- and defined those with a ratio>0.6 as homozygous while types sharing>90% identity based on single nucleotide poly- those with a ratio<0.4 were defined as heterozygous. All morphism (SNP) analyses as previously described (Lendenmann other cases were considered to be ambiguous. 1418 Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Meiosis Leads to Pervasive Copy-Number Variation and Distorted Inheritance GBE Normalized coverage <0.3 >= 0.3 & <0.7 >= 0.7 & < 1.3 >= 1.3 & < 1.7 >=1.7 B Loss Loss Loss Partial Normal Partial Duplication Deletion Duplication PstI cut sites Mapped reads Median genome coverage Disomic Missing Normal chromosome chromosome chromosome PstI cut sites Mapped reads Median genome coverage Partially deleted Partially duplicated chromosome chromosome FIG.1.—Procedure to detect chromosomal anomalies. (A) Reads mapped to the PstI restriction sites were used to analyze coverage across the genome. Sequencing data were generated by restriction site-associated DNA sequencing (RADseq). The normalized coverage represents the coverage of each chromosome normalized to the median coverage of all chromosomes of the same progeny. The normalized coverage distribution of progeny from cross 3D13D7 is shown with the cutoffs used to detect a whole chromosome loss (ratio< 0.3), partial deletion (ratio 0.3–0.7), normal transmission (0.7–1.3), partial duplication (1.3–1.7), and whole chromosome duplication (>1.7). (B) Schematic overview of read coverage expected for complete chromosome losses and duplications (in blue). Partial deletions and duplications are shown in green. of TEs on a chromosome was compared with the likelihood of Chromosome Instability and Recombination Rate, being inherited with high fidelity. We also compared the fre- Chromosome Length, Synteny and Transposable Element quency of disomic chromosomes with the frequency of rear- Content of the Parent Chromosomes rangements for all chromosomes in both crosses. We correlated chromosome instability with the percentage length difference in homologs among the parents and recom- bination rates based on the recombination rates reported in Analyses of Progeny Phenotypes Croll et al. (2015). We also correlated synteny and the fidelity with which chromosomes were inherited using the NUCmer Clonally propagated mycelium from each germinated asco- pipeline from MUMmer (version 3.23) software (Kurtz et al. spore was previously used to infect wheat plants in the frame- 2004) to determine the sequence similarity between two ho- work of a QTL mapping study (Stewart et al. 2018). Progeny mologous chromosomes. The minimum cluster length was set from both crosses were phenotyped for percentage of leaf to 50 and we used the –mum option to anchor matches that area covered by lesions (PLACL), pycnidia density (pycnidia/ 2 2 were unique in both the reference and query sequence. The cm leaf area), pycnidia size (mm ), and pycnidia melanization transposable elements (TEs) in the parent genomes were an- on seedlings of the wheat cultivars Runal and Titlis in a previ- notated using RepeatMasker (http://www.repeatmasker.org; ously described glasshouse-based assay (Stewart and last accessed May 2017) and the TE library compiled for Z. tritici McDonald 2014). Gray values were previously shown to be and its sister species (Grandaubert et al. 2015). The percentage a good measure for melanization (Lendenmann et al. 2014). Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 1419 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Frequency Coverage Coverage Chromosome Chromosome Fouche et al. GBE Replication of the infection assays was made possible by in- Patterns of Chromosome Transmission in the Two Crosses oculating replicate wheat plants with a fixed concentration of Analyzing normalized read coverage among progeny revealed blastospores from each progeny mycelium. The assay was high rates of chromosome losses in both crosses. In cross repeated three times over three consecutive weeks, resulting 3D13D7, accessory chromosomes 16, 17, 19, and 20 in three biological replicates and six total replicates per were present in both parents but were missing in 1.6% (4/ progeny-cultivar pair. Automated image analysis of the sec- 249), 4.4% (11/249), 0.4% (1/249), and 1.2% (3/249) of the ond leaf was performed at 23 dpi as previously described progeny, respectively (fig. 2A). In the 1E4 1A5 cross, acces- (Stewart and McDonald 2014). Progeny were also pheno- sory chromosomes 14, 15, 16, 18, 19, 20, and 21 were pre- typed for temperature sensitivity, growth morphology, and sent in both parents but were absent in 7.5% (17/228), 2.2% fungicide sensitivity (Lendenmann et al. 2015, 2016). (5/228), 4.8% (11/228), 6.1% (14/228), 2.2% (5/228), 1.8% Phenotypes were compared in normal progeny and progeny (4/228), and 4.4% (10/228) of the progeny, respectively with “abnormal” (partially deleted, partially duplicated, diso- (fig. 2B). We found no progeny lacking a core chromosome mic, or absent) chromosomes to determine if particular chro- in either of the crosses. mosome genotypes were associated with outlier virulence, We also identified numerous instances of disomy in prog- fungicide resistance, temperature sensitivity, or growth rate eny accessory chromosomes. In cross 3D13D7, chromo- phenotypes. These analyses were performed in R version somes 17, 19, and 20 were present in two copies in 1.6% 3.4.0. (4/249), 0.8% (2/249), and 0.8% (2/249) of the progeny, respectively (fig. 2A). Interestingly, 2.4% (6/249) of the prog- eny were disomic for a core chromosome, with 1.6% (4/249) Results of the progeny disomic for chromosome 5 and 0.8% (2/249) Mapping RADseq Reads to the Reference Genome disomic for chromosome 13. No disomic core chromosomes The chromosome state (absent, present, or duplicated) was were identifiedincross 1E41A5 (fig. 2B), but 1.3% (3/228) determined for each chromosome of the four haploid paren- of the progeny were disomic for chromosome 14, 0.9% (2/ tal isolates (3D1, 3D7, 1A5, 1E4) and 477 progeny, using 228) were disomic for chromosome 18 and chromosomes 16, RADseq reads generated for each progeny mapped to the 19, 20, and 21 were each disomic in 0.4% (1/228) of the IPO323 reference genome. The 3D1 and 1A5 parents had progeny. all 21 chromosomes, while the 3D7 and 1E4 parents were Chromosomal inheritance that differed from the expected missing four and one accessory chromosomes, respectively 1:1 ratio was observed for several chromosomes that were (Croll et al. 2013). None of the four parental strains carried present in only one of the two parents of a cross. In the additional chromosomes beyond the 21 chromosomes iden- 3D13D7 cross, chromosomes 14, 15, 18, and 21 were ab- tifiedinIPO323 (Plissonneau et al. 2018). We selected the sent in the 3D7 parent, hence we expected these chromo- parental isolate from each cross that carried all 21 chromo- somes to be absent in half of the progeny. Instead, somes (3D1 and 1A5) as a reference. We mapped whole- chromosomes 14, 15, 18, and 21 were absent in only genome sequencing data of the two selected parents against 22.5% (56/249), 25.7% (64/249), 30.1% (75/249), and the IPO323 reference genome and identified regions missing 26.9% (67/249) of the progeny, respectively (fig. 2A). The in the parental genomes. Missing regions were not expected inheritance of these chromosomes are significant departures to show coverage in any of the progeny chromosomes and from the canonical Mendelian ratio (chromosome 14: 2 2 were excluded from further analyses. RADseq loci genotyped v ¼37.7, P< 0.001, chromosome 15: v ¼29.4, P< 0.001, in progeny showed an even distribution across all 21 chromo- chromosome 18: v ¼19.7, P< 0.001, and chromosome 21: somes, with no apparent differences between core (1–13) v ¼26.6, P< 0.001). We also tested whether chromosomes and accessory chromosomes (14–21; supplementary fig. 1, 14, 15, 18, and 21 occurred independently from one another Supplementary Material online). Similarly, RADseq loci in progeny. We found that progeny lacking one or four chro- showed homogeneous read coverage across the genome mosomes did not deviate significantly from expectations (v for progeny with high and low overall sequence coverage in ¼ 0.01, P¼ 0.9; v ¼ 0.82, P¼ 0.3, respectively). However, both crosses (supplementary fig. 2A–D, Supplementary we found that progeny having all four chromosomes occurred Material online). For each progeny, we calculated the cover- much more frequently than expected (v ¼ 622.65, age for each chromosome and compared this to the median P< 0.001), while having one or two of the four chromosomes coverage of all chromosomes for that isolate (fig. 1). The nor- also occurred more frequently than expected (v ¼ 14.7, malized coverage per chromosome was close to 1 for the P< 0.001; v ¼ 37.76, P< 0.001, respectively). In the large majority of the chromosomes (supplementary fig. 3, 1E41A5 cross, chromosome 17 was missing in 53.5% Supplementary Material online). The mean normalized cover- (112/228) of the progeny and did not exhibit distorted inher- age ratio was 0.96 and 0.95 for the progeny from cross itance (v ¼0.56, P¼ 0.3) (fig. 2B). Disomy was also found for 3D13D7 and cross 1A51E4, respectively. several accessory chromosomes that were present in only one 1420 Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Meiosis Leads to Pervasive Copy-Number Variation and Distorted Inheritance GBE Cross 3D1x3D7 Chromosomal state 150 Duplicated Loss Normal Partial Deletion Partial Duplication Expected number of chromosomes in progeny [only one parent carried a copy] 1 2 3456789 10 11 12 13 14 15 16 17 18 19 20 21 Chromosome Cross 1E4x1A5 Chromosomal state Duplicated Loss Normal Partial Deletion Partial Duplication 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Chromosome FIG.2.—Summary of the total chromosome anomalies in the progeny of two crosses. Normal, disomic, lost, and rearranged (partially duplicated or deleted) chromosomes are shown separately for cross 3D13D7 and 1E41A5. Dotted lines show the expected number of progeny for chromosomes that were present in only one of the two parental isolates. of the parents. In cross 3D13D7, additional copies of chro- originating from both parents and the other three cases mosome 14 and 18 were identified in 0.4% (1/249) and were ambiguous. As indicated earlier, chromosome 17 could 0.8% (2/249) of the progeny, respectively (fig. 2A). In cross only have originated from one of the parents. 1E41A5, chromosome 17 was disomic in 0.9% (2/228) of the progeny (fig. 2B). Meiosis Generates Novel Chromosome Length Disomic chromosomes can either be heterozygous, carry- Polymorphism ing one of each parental chromosomal copy, or homozygous if the disomy arose from a single parental chromosome In order to identify partially deleted or duplicated chromo- (fig. 3). To distinguish these scenarios, we analyzed disomic somes in the progeny, we investigated chromosomes which progeny chromosomes and restricted the analyses to cases had a normalized coverage between 0.3 and 0.7, and be- where both parents were carrying a chromosomal copy. In tween 1.3 and 1.7 (fig. 1). In cross 3D13D7 (fig. 2A), partial the 3D13D7 cross, 59% (10/17 cases) of the disomic isolates deletions were identified for chromosomes 14 (0.4% of off- were heterozygous, with a chromosome originating from spring, 1/249) and 15 (0.4%, 1/249). Partial duplications were each parent and 29% (5/17 cases) of the disomic isolates detected for chromosomes 14 (0.4%, 1/249), 16 (0.8%, were homozygous, with both chromosomes originating 2/249), 19 (2.4%, 2/249), and 21 (0.4%, 1/249). We also from one parent (fig. 4A). In the case of chromosomes 14 identified one isolate which may have a partially duplicated and 18, the chromosomes could only originate from one par- core chromosome 10. In cross 1E41A5 (fig. 2B), partial ent. In cross 1E41A5, 5 of the 11 disomic isolates were duplications were detected in the progeny for chromosomes homozygous, three disomic isolates had chromosomes 14 (0.9%, 2/228), 15 (0.9%, 2/228), 16 (0.9%, 2/228), Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 1421 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Number of progeny Number of progeny Fouche et al. GBE Mitosis Meiosis I Meiosis II B Meiosis I Meiosis II Mitosis Meiosis I Meiosis I I Mitosis FIG.3.—Schematic overview of how chromosomal nondisjunction can result in chromosome loss or disomy. (A) During canonical meiosis, the haploid nuclei from the two parents fuse resulting in a single diploid nucleus. Parental chromosomes are shown with distinct colors. Chromosomes go through meiosis I and II, followed by mitosis, resulting in eight haploid ascospores. Chromosome loss or disomy can occur as a result of homologous chromosomes failing to segregate during meiosis I (B), resulting in heterozygous disomy with one chromosome originating from each of the parents. The alternative is the failure of sister chromatid segregation during meiosis II (C), generating homozygous disomic progeny with both copies of the chromosome originating from the same parent. 17 (1.8%, 4/228), 19 (0.4%, 1/228), and 20 (1.8%, 4/228). duplication of chromosome 10 while isolate 137.2 had partial Partial losses were identified for chromosomes 14 (0.4%, 1/ duplications of chromosomes 16, 19, and 21. In cross 228), 15 (1.3%, 3/228), 16 (3.5%, 8/228), 17 (2.6%, 6/228), 1E41A5, isolate B23.1 was disomic for chromosome 20 and 21 (3.1%, 7/228). and had partial deletions of chromosomes 17 and 21. This We identified some progeny with multiple chromosomal isolate also had a partially duplicated chromosome 14. Isolate anomalies, however these associations did not deviate signif- B24.2 also had partial deletions of chromosomes 17 and 21. icantly from a random expectation. In cross 3D13D7, isolate Isolate C44.2 had partially deleted chromosomes 16 and 21. 89.1 was disomic for chromosome 13 and had a large, partial Isolate B50.1 was disomic for chromosome 17 and had a 1422 Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Meiosis Leads to Pervasive Copy-Number Variation and Distorted Inheritance GBE Cross 3D1x3D7 Cross 1E4x1A5 1.00 1.0 ● ● ● ● ● 0.8 0.75 Disomic state ● Ambiguous 0.6 ● Heterozygous 0.50 ● Homozygous ● 0.4 0.25 ● ● ● 0.2 5 131417 181920 14 16 17 18 19 20 21 Chromosome Chromosome FIG.4.—Identification of heterozygous and homozygous disomic chromosomes in cross 3D13D7 and cross 1E41A5. Single nucleotide polymor- phism (SNP) loci were screened on progeny chromosomes that showed evidence for disomy. SNPs were genotyped as either homozygous, containing only one of the parental alleles, or heterozygous if both parental alleles were found. The ratio represents the number of homozygous SNPs compared with the total number of genotyped SNPs. Individual dots represent each of the disomic progeny chromosomes identified in the two crosses. Due to uncertainties in SNP calling, we used cut-offs to assign progeny chromosomal states. Chromosomes with a ratio<0.4 were assigned as heterozygous disomic, likely resulting from nondisjunction at meiosis II, >0.6 as homozygous disomic, likely resulting from nondisjunction at meiosis I, and ratios between 0.4 and 0.6 were assigned as ambiguous. partially deleted chromosome 21. Isolate A57.1 was disomic for chromosome 14 and had a partially duplicated chromo- some 16. In cross 3D13D7, we found twelve progeny isolates with partial deletions and duplications. Seven of these partial aneu- ploidies affected chromosomal segments near the telomeric Chromosomal state Loss ends (supplementary fig. 4, Supplementary Material online). Duplicated Isolate 89.1 had a normalized coverage ratio for chromosome Partial Duplication Central 10 of 1.63 suggesting a partial duplication. However the cov- Partial Duplication Telomeric erage along the chromosome was homogeneous, with no Partial Deletion Central apparent duplicated chromosomal regions when compared Partial Deletion Telomeric with the parent chromosomes (supplementary fig. 5, Ambiguous Duplication Supplementary Material online). We considered such cases Ambiguous Deletion as ambiguous duplications. In cross 1A51E4, we found 40 partial deletions and duplications, of which 19 were ambigu- ous and 15 occurred in chromosomal segments near the telo- meric ends (fig. 5). 14 15 16 17 18 19 20 21 Correlation of Chromosomal Features with the Fidelity of Chromosome Transmission FIG.5.—Identification of partial chromosome losses or duplica- During meiosis, chromosomes pair prior to recombination and tions in cross 1E41A5. A summary of all the chromosome number therefore length similarity could play a role in homolog iden- and length polymorphisms in the progeny of cross 1E41A5, as well tification and enable chromosomes to pair and recombine. as the location where the length polymorphism occurred. Most of the However, we found no correlation between the length simi- rearrangements were ambiguous (19), 15 were located toward the larity of the parent chromosomes and the fidelity with which ends of chromosomes and 6 rearrangements occurred in the central chromosomes were inherited (fig. 6A). In general accessory region of the chromosomes. Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 1423 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Ratio Ratio Number of progeny Fouche et al. GBE Chromosomal state Chromosome type ● Duplication Accessory Loss ● Core Cross 3D1 x 3D1 Cross 1E4 x 1A5 ● ● 2 ● ● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ●● ●● ● ● ●● ● ● ● ● ● 05 10 0 5 10 15 Percentage length difference Percentage length difference ● ● 2 ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ●●● ● ● ● ● ●● ● ● 60 80 100 120 40 60 80 100 Recombination rate (cM/Mb) Recombination rate (cM/Mb) ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ●● ● ● 0 ● ● ●●● ● ● ●● ● ● ● ● 65 70 75 80 85 60 70 80 Percentage sequence similarity Percentage sequence similarity ● ● 2 ● ● ● ● ●● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ●● ● ● 15 20 25 30 10 20 30 Percentage repetitive sequences Percentage repetitive sequences FIG.6.—Correlations between chromosome length similarity, recombination rate, percent sequence similarity, fraction of repetitive sequences and the inheritance of chromosomes. Complete and partial chromosome losses and duplications were correlated with length similarity (A), recombination rate (B), sequence similarity (C), and repeat content (D) of the parental chromosomes. Correlations are shown separately for crosses 3D13D7 and 1E41A5. chromosomes were more unstable than core chromosomes. between the recombination rate and chromosome transmis- Interesting exceptions were a disomic core chromosome 13 sion fidelity (fig. 6B). However, in cross 1A51E4, most of the (length difference 5% between the parents) and a disomic chromosome losses and disomies occurred in accessory chro- core chromosome 5 (length difference of 8.4% between the mosomes with a low recombination rate (fig. 6B). Next, we parents). The rate of disomy for these core chromosomes was analyzed sequence similarities between parental chromo- 1.6% (4/249 progeny). We found no significant correlation somes and correlated this with the chromosome transmission 1424 Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Percentage Percentage Percentage Percentage Percentage Percentage Percentage Percentage Meiosis Leads to Pervasive Copy-Number Variation and Distorted Inheritance GBE 14 15 16 17 18 19 20 21 17 Cross 3D1x3D7 ● ● ● ● ● ● ● ● ● Chromosome state Chromosome state Abnormal ● ● ● ● ● ● ● ● ● Duplicated Loss Normal ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Normal 300 300 Partial Deletion ● Partial Duplication ● ● ● ● ● ● ● ● ● ● ● ● ● 100 100 14 15 16 17 18 19 20 21 21 Cross 1E4x1A5 Chromosome state Chromosome state ● ● Abnormal Duplicated Normal Loss Normal Partial Deletion 80 80 Partial Duplication ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 60 60 ● ● ● ● ● ● ● ● ● FIG.7.—Association between accessory chromosomes and phenotypes. Accessory chromosome states, normal or abnormal (duplicated, lost, partially duplicated, or partially lost), were compared with virulence traits using a two-sample t-test (multiple testing correction threshold of P< 0.002). (A)In the progeny of 3D13D7, isolates with a normal chromosome 17 had a significantly higher pycnidia count on the wheat cultivar Runal that isolates with a duplicated or lost chromosome (P¼ 0.0019). (B)In cross 1E41A5, isolates with a lost or partially deleted chromosome 21 had a higher percent leaf area covered by lesions (PLACL) on Titlis than isolates with a normal chromosome 21 (P¼ 0.000024). fidelity. For this, we compared whole chromosome sequences on two wheat cultivars (Runal and Titlis) using data from a and calculated the percentage of syntenic regions between previous study (Stewart and McDonald 2014; Stewart et al. homologous chromosomes. The accessory chromosomes in 2018). Progeny from cross 3D13D7witha normal chromo- the parents for both crosses had a much lower synteny than some 17 had a higher pycnidia count on the cultivar Runal the core chromosomes and had substantially lower transmis- than isolates with an abnormal chromosome 17 (P¼ 0.0019; sion fidelity (fig. 6C). Accessory chromosomes had overall a fig. 7A, supplementary fig. 6, Supplementary Material online). higher content of repetitive elements, which was similarly Isolates missing chromosome 17 had a lower pycnidia count correlated with lower transmission fidelity (fig. 6D). than isolates that were disomic for chromosome 17. On cul- tivar Titlis, progeny from cross 3D13D7 with a normal chro- mosome 18 had significantly darker pycnidia (P¼ 0.0018; Association between Accessory Chromosomes and supplementary fig. 7, Supplementary Material online). Phenotypic Traits Progeny with an abnormal chromosome 19 had a marginally We analyzed whether the chromosome states in progeny higher percent leaf area covered by lesions (PLACL; were correlated with variation in phenotypic traits. For this, P¼ 0.0024; supplementary fig. 7, Supplementary Material we considered first only two chromosome states: normal online). For progeny from cross 1E41A5, we found a corre- (haploid) or abnormal (any loss, duplication, or rearrange- lation of the PLACL produced on Titlis with chromosome 21 ments). We tested for an association with phenotypic traits (P¼ 0.00002; fig. 7B; supplementary fig. 8, Supplementary using two-tailed t-tests (multiple testing significance threshold Material online). Isolates with a partially deleted or lost chro- at P< 0.002). We first tested for associations with virulence mosome 21 had a higher PLACL. For progeny of cross Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 1425 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 PLACL Pycnidia Count PLACL Pycnidia Count Fouche et al. GBE FIG.8.—Correlation between number of disomic progeny and chromosomal rearrangements. Circles and triangles represent accessory chromosomes and core chromosomes, respectively. Chromosomes from cross 3D13D7 are represented in blue, and chromosomes from cross 1E41A5 are in red. 1E41A5, we found that isolates with an abnormal chromo- chromosomal transmission through meiosis. We found exten- some 20 showed higher PLACL on Runal. We found no sig- sive chromosome number and length variation among the nificant correlations for phenotypes related to growth, progeny in both crosses. The rates of disomy and rearrange- fungicide resistance, or temperature sensitivity. ments differed greatly between chromosomes and crosses. Nearly all aberrant chromosomal transmission events affected accessory chromosomes with the rare exception of core chro- Correlation between Disomy and Chromosomal mosome disomies. Several accessory chromosomes showed Rearrangements strongly distorted chromosomal inheritance. Chromosome number polymorphism in Z. tritici has previ- We analyzed whether rates of disomy were correlated with ously been linked to errors occurring during meiosis rates of rearrangements. Nondisjunction results in the loss of a (Wittenberg et al. 2009; Croll et al. 2013). In our study, we chromosome in one progeny and a chromosome gain in the generated a substantially more dense marker coverage using corresponding twin spore from the same ascus. Core chro- the Illumina-based sequencing technique RADseq and were mosomes generally showed only very rare cases of disomy or able to screen more isolates (477 isolates compared with 144 rearrangements (fig. 8). Accessory chromosome 14 was more and 216 isolates, respectively; Wittenberg et al. 2009; Croll frequently disomic and rearranged in progeny from cross et al. 2013). Because RADseq generated a high coverage of 1A41E5. Chromosome 15 underwent partial duplications 100-bp sequences at defined restriction sites, we could pre- and deletions, but we found no evidence for nondisjunction. cisely map sequences to chromosomal positions without hav- Chromosome 16 was both frequently rearranged (4.4%, 10/ ing to rely on genetic map constructions. Physical marker 228) and disomic (0.4% 1/228) among the progeny in positions are particularly important for analyzing accessory 1E41A5. In cross 3D13D7, chromosome 17 was disomic chromosomes of Z. tritici because of their very low rates of in 1.6% (4/249) of the progeny, while in cross 1E41A5 recombination (Croll et al. 2015). In contrast to previous stud- chromosome 17 was more rarely disomic (0.9%, 2/228). ies, our use of RADseq markers allowed us to directly detect Chromosome 17 showed even stronger differences in rear- duplicated chromosomal segments by analyzing variations in rangements among crosses, with 4.4%, (10/228) in cross sequencing coverage. 1E41A5versus0.0% incross 3D13D7. Chromosome 19 Our analyses revealed that all eight accessory chromo- was both more likely to undergo rearrangements and to be somes underwent chromosome loss during meiosis. The inherited as a disomic chromosome in cross 3D13D7. In rate of chromosomal loss depended on the chromosome contrast, chromosome 21 was both more likely to be rear- and varied between the crosses. This confirms the findings ranged and to be inherited in a disomic state in cross of Croll et al. (2013) except that a loss of chromosome 15 had 1E41A5. not previously been detected. We found that 5 progeny (2.1%) had lost this chromosome. No isolate was found lack- Discussion ing a core chromosome despite screening 477 progeny. This We used RADseq data generated for several hundred progeny indicates that all 13 core chromosomes are likely encoding from two crosses of Z. tritici to identify aberrations in essential functions for the growth and survival of the fungus. 1426 Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Meiosis Leads to Pervasive Copy-Number Variation and Distorted Inheritance GBE Chromosome loss most likely occurred as a result of errors We found that an average of 5.9% of the progeny isolates during chromosome segregation, specifically nondisjunction were disomic for one or more chromosomes. This number is of sister chromatids during either meiosis I or II. In accordance similar to what was found for Saccharomyces cerevisiae, with previous studies, we found that the loss of accessory where 8% of the lab strains were estimated to be aneuploid chromosomes during meiosis is common. In natural popula- (Hughes et al. 2000). Disomy is generated when chromo- tions, this may lead to the complete loss of an accessory chro- somes undergo nondisjunction during meiosis, resulting in mosome in the absence of counteracting mechanisms that one daughter cell with two copies of a chromosome and maintain these chromosomes. one daughter cell with no copies of that chromosome Wittenberg et al. (2009) proposed that distorted segrega- (fig. 3). Therefore, for each disomic offspring, we expect a tion of accessory chromosomes could serve as a mechanism corresponding offspring that is missing the same chromo- to prevent their complete loss from a population. some. As expected, we found that chromosomal loss was Chromosomes present in only one parent are expected to often accompanied by disomy. However, contrary to expect- segregate into 50% of the daughter cells. However, we found ations, there was no symmetry in the loss and disomy rates. that in cross 3D13D7 chromosomes 14, 15, 18, and 21 For example, despite finding many progeny lacking chromo- from parent 3D1 were significantly overrepresented in the some 15, no isolate disomic for chromosome 15 was recov- progeny. The transmission advantage resulting from unequal ered. The rates of nondisjunction also differed between segregation is referred to as “meiotic drive” and is frequently chromosomes and between crosses, suggesting that the associated with accessory or B chromosomes (Jones 1991). In loss or disomy of specific chromosomes may be counter se- our study, distorted inheritance was not universal, for exam- lected. In addition, chromosomes differed in their composition ple chromosome 17 in cross 1E41A5 segregated normally. of repetitive elements. Repetitive elements are likely to play an The distorted inheritance pattern in cross 3D13D7 could be important role by influencing the likelihood of faithful disjunc- explained if parent 3D1 already had disomic accessory chro- tion. We also found that nondisjunction was happening dur- mosomes. But our coverage analysis did not detect disomic ing both meiosis I and II. We found heterozygous disomic chromosomes in any of the parents. It is possible that a small chromosomes, which were created as a result of nondisjunc- fraction of the clonal cell pool of a parental mycelium might tion in meiosis I. Heterozygous disomic chromosomes were have harbored disomic chromosomes, but this is not likely to most frequent in cross 3D13D7. In cross 1E41A5, homo- explain the observed rates of disomic accessory chromo- zygous disomy resulting from nondisjunction in meiosis II oc- somes. The overrepresentation of progeny carrying a specific curred more frequently. Aneuploidy can play an important accessory chromosome could be due to selection favoring role in the adaptive evolution of fungal pathogens. In human progeny carrying this chromosome. Such viability selection pathogens, aneuploidy is often associated with drug resis- could not be tested in this experiment because we were un- tance (Hu et al. 2008; Selmecki et al. 2010). Over 50% of able to generate full tetrad sets of offspring and quantify the fluconazole-resistant strains isolated from patients had genotype-specific survival rates. However, if loci located on whole or partial chromosome duplications (Selmecki et al. accessory chromosomes encoded strongly deleterious variants 2006). Correlations between disomic states and phenotypic for growth on culture media, quantitative trait mapping stud- traits in Z. tritici suggests that selection could also be affecting ies performed on the same progeny sets would most likely rates of disomy, albeit with less drastic impacts than in human have identified QTLs linked to accessory chromosomes. pathogens selected for drug resistance. However, no such evidence was found (Lendenmann et al. Aneuploidy typically causes a dosage imbalance, which 2014, 2016). could explain why accessory chromosome aneuploidies are Additional explanations for the observed distortion in in- tolerated more frequently than core chromosome aberra- heritance may include a meiotic drive mechanism such as se- tions. Alternatively, gene expression or dosage compensation lective spore killing. The distortion could also be linked to could have evolved on frequently disomic chromosomes, “sticky” centromeres similar to those found in rye B chromo- which may explain the tolerance for additional copies of cer- somes where the transmission at higher than Mendelian fre- tain chromosomes, but not others (Torres et al. 2008). quencies was explained by the presence of particular Chromosomes that have a higher rate of disomy could have centromeres that ensure that B chromosomes migrate to shorter or nonfunctional telomeres. Telomere defects were the generative pole that will be transmitted to the next gen- found to explain mitotic instability in human mammary epi- eration of plants (Banaei-Moghaddam et al. 2012). In or- thelial cells (Pampalona et al. 2010). Chromosomes with der to distinguish among the possible mechanisms leading shorter telomeres are more likely to undergo nondisjunction. to distorted inheritance, all meiotic products from individ- Furthermore, chromosomes with higher degrees of synteny ual tetrads would have to be analyzed. However, experi- are more likely to pair correctly, resulting in fewer nondisjunc- mental limitations in the generation of large numbers of tion events. We found indications that sequence similarity in individual tetrads prevented us from making more de- the parent chromosomes indeed leads to higher fidelity of tailed investigations. chromosomal inheritance. Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 1427 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Fouche et al. GBE Homologous chromosomes of Z. tritici segregate signifi- accessory chromosomes may be maintained in the species cant structural variation in populations, differing in repeat pool by a selection-drift balance. and gene content, chromosomal length, and recombination Most chromosome rearrangements are thought to be del- rate, as well as telomere and centromere composition (Croll eterious and therefore counter-selected. The ability of Z. tritici et al. 2013, 2015; Schotanus et al. 2015; Plissonneau et al. to tolerate a large number of disomies and chromosomal 2016). Synteny breakpoints are commonly associated with rearrangements makes this species an excellent model for repetitive sequences or transposable element clusters that detailed analyses of rearrangements and nondisjunction can misalign during recombination, thereby generating length events. Despite the fact that the meiotic machinery is highly polymorphism. Such a mechanism was thought to generate a conserved, the strength of selection against erroneous chro- novel chromosome 17 in the progeny of cross 1A51E4 (Croll mosomal transmission can differ widely among species. et al. 2013). In our study, we found no correlation between Relaxed selection on chromosomal transmission can lead to length similarity and recombination rate of the parent chro- highly polymorphic chromosomal sets observed in some eu- mosomes, and the fidelity of chromosome inheritance. karyotic pathogens. Determining the trade-offs involved in However, chromosomes with higher synteny between the maintaining chromosomal integrity and generating chromo- parents and fewer repeats were transmitted more faithfully. somal polymorphism will elucidate how selection operates to Selection favoring the presence or absence of specific ac- maintain the fidelity of meiotic processes. cessory chromosomes would require that accessory chromo- somes directly or indirectly influence phenotypic traits. Supplementary Material However, accessory chromosomes carry few genes and Supplementary data are available at Genome Biology and none are thought to perform a specific function during the Evolution online. life cycle of the fungus (Goodwin et al. 2011). Interestingly, we found a correlation between the presence of chromo- somes 15, 18, and 21 and higher levels of virulence in cross Acknowledgments 3D13D7 (Stewart et al. 2018). In addition, we found a cor- relation between the presence of a normal chromosome 17 We thank Marcello Zala for providing access to progeny col- and an abnormal chromosome 19 and higher levels of pyc- lections and helpful discussions. C.P. was supported by an nidia and PLACL, respectively. In a separate study, whole- INRA Young Scientist grant. S.F. and B.A.M. are supported chromosome deletion mutants of a different Z. tritici strain by the Swiss National Science Foundation (grant were generated by blocking b-tubulin assembly during mitosis 31003A_155955). D.C. is supported by the Swiss National using carbendazim (Habig et al. 2017). A comparison of iso- Science Foundation (grant 31003A_173265). genic lines lacking individual accessory chromosomes showed that the loss of chromosomes 14, 16, 18, 19, and 21 resulted Literature Cited in increased virulence on the wheat cultivar Runal. This finding is in opposition to our own study that showed that the pres- Baird NA, et al. 2008. 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The pri- mary target organ of Cryptococcus gattii is different from that Associate editor:Laura Rose Genome Biol. Evol. 10(6):1416–1429 doi:10.1093/gbe/evy100 Advance Access publication May 29, 2018 1429 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1416/5020729 by Ed 'DeepDyve' Gillespie user on 16 June 2018

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Genome Biology and EvolutionOxford University Press

Published: May 29, 2018

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