Recurrent chromosome destabilization through repeat-1 mediated rearrangements in a fungal pathogen 2

28 Genomic instability caused by chromosomal rearrangements has severe consequences for organismal 29 fitness and progression of cancerous cell lines. The triggers of destabilized chromosomes remain poorly 30 understood but are often assumed to be associated with fragile sites. Here, we retrace a runaway 31 chromosomal degeneration process observed in a fungal pathogen using telomere-to-telomere 32 assemblies across an experimental pedigree. We show that the same fragile sites triggered reproducible, 33 large-scale rearrangements through non-allelic recombination. Across the four-generation pedigree, 34 chromosomal rearrangements were accompanied by non-disjunction events and caused aneuploid 35 progeny to carry up to four chromosomal copies. We identify a specific transposable element as the 36 most likely trigger for the repeated chromosomal degeneration. The element is associated with higher 37 virulence of the pathogen and has undergone a burst increasing copy numbers across the genome. 38 Chromosome sequences are also targeted by a genome defense mechanism active on duplicated 39 sequences, which may contribute to decay. Our work identifies the exact sequence triggers initiating 40 chromosome instability and perpetuating degenerative cycles. Dissecting proximate causes leading to 41 run-away chromosomal degeneration expands our understanding of chromosomal evolution beyond 42 cancer lines.


Introduction
we mapped reads for the progeny to the 1A5 parent chromosomes and identified regions of chromosome 120 17 with higher than the mean coverage of the core chromosomes (1-13). We found sharp transitions in CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 15, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 15, 2023. ; https://doi.org/10.1101/2023.07.14.549097 doi: bioRxiv preprint kb windows. Vertical dashed lines indicate the chromosomal breakpoints A-F (see fig. 1   CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 15, 2023.

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Additionally, CpG dinucleotides were frequently targeted. New RIP-like mutations were found after and 4) with each carrying two copies of region 0-B on one chromosome. Additionally, both progenies 218 were generated by a backcross with 1A5. Our results show that in addition to rearrangements and non-219 disjunction, RIP is a mutational mechanism driving degenerative cycles in fungi.

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We recapitulated the dynamics of a highly unusual chromosomal rearrangement. Using split long-reads,

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we identified the exact breakpoints and retraced the degeneration through four rounds of meiosis. We 262 found that the primary degenerative rearrangement was caused by non-allelic recombination between a 263 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 15, 2023. ; https://doi.org/10.1101/2023.07.14.549097 doi: bioRxiv preprint copy of a TE family Styx and a region with microhomology to the TE. The degenerated chromosome 264 was composed of a large duplicated region connected to a single-copy chromosomal segment near the

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 15, 2023.   CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 15, 2023. ; https://doi.org/10.1101/2023.07.14.549097 doi: bioRxiv preprint 17 performed twice and the washing step three times. In the last step, the DNA pellet was resuspended in 357 100 µl of sterile water.  CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 15, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 errors in the uncorrected PacBio reads. We expected the alternate allele at polymorphic sites to be shared 466 by ≥80% of the mapped reads for single-copy regions, shared by ≥40% of the reads in regions with 467 evidence for duplications (based on read coverage), shared by ≥27%, ≥20% and ≥16% of the reads in 468 regions with three, four and five copies (based on read coverage), respectively. We also excluded 469 regions with ≤50% or ≥150% of the expected read coverage for further analyses to reduce erroneous 470 variant calls due to inconsistent read mapping.

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 15, 2023.                   . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 15, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023   (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 15, 2023.  Length of consensus (kb) Length of consensus (kb) divergent and likely inactive copies . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 15, 2023. ; https://doi.org/10.1101/2023.07.14.549097 doi: bioRxiv preprint