SignificanceThe “centromere paradox” refers to rapidly evolving and highly diverse centromere DNA sequences even in closely related eukaryotes. However, factors contributing to this rapid divergence are largely unknown. Here, we identified large regional, LTR retrotransposon-rich centromeres in a group of human fungal pathogens belonging to the Cryptococcus species complex. We provide evidence that loss-of-functional RNAi machinery and possibly cytosine DNA methylation trigger instability of the genome by activation of centromeric retrotransposons presumably suppressed by RNAi. We propose that RNAi, together with cytosine DNA methylation, serves as a critical determinant that maintains repetitive transposon-rich centromere structures. This study explores the direct link between RNAi and centromere structure evolution.

Supplementary table S1. Coordinates of centromeres and their GC content in M. sympodialis.Coordinates and length of Mtw1-enriched regions in comparison with that of the core centromeres in M. sympodialis. Chromosome numberCore centromere

Centromeres of Candida albicans form on unique and different DNA sequences but a closely related species, Candida tropicalis, possesses homogenized inverted repeat (HIR)-associated centromeres. To investigate the mechanism of centromere type transition, we improved the fragmented genome assembly and constructed a chromosome-level genome assembly of C. tropicalis by employing PacBio sequencing, chromosome conformation capture sequencing (3C-seq), chromoblot, and genetic analysis of engineered aneuploid strains. Further, we analyzed the 3D genome organization using 3C-seq data, which revealed spatial proximity among the centromeres as well as telomeres of seven chromosomes in C. tropicalis. Intriguingly, we observed evidence of inter-centromeric translocations in the common ancestor of C. albicans and C. tropicalis. Identification of putative centromeres in closely related Candida sojae, Candida viswanathii and Candida parapsilosis indicates loss of ancestral HIR-associated centromeres and establishment of evolutionary new centromeres (ENCs) in C. albicans. We propose that spatial proximity of the homologous centromere DNA sequences facilitated karyotype rearrangements and centromere type transitions in human pathogenic yeasts of the CUG-Ser1 clade.

Centromeres are chromosomal regions that serve as platforms for kinetochore assembly and spindle attachments, ensuring accurate chromosome segregation during cell division. Despite functional conservation, centromere DNA sequences are diverse and often repetitive, making them challenging to assemble and identify. Here, we describe centromeres in an oomycete Phytophthora sojae by combining long-read sequencing-based genome assembly and chromatin immunoprecipitation for the centromeric histone CENP-A followed by high-throughput sequencing (ChIP-seq). P. sojae centromeres cluster at a single focus at different life stages and during nuclear division. We report an improved genome assembly of the P. sojae reference strain, which enabled identification of 15 enriched CENP-A binding regions as putative centromeres. By focusing on a subset of these regions, we demonstrate that centromeres in P. sojae are regional, spanning 211 to 356 kb. Most of these regions are transposon-rich, poorly transcribed, and lack the histone modification H3K4me2 but are embedded within regions with the heterochromatin marks H3K9me3 and H3K27me3. Strikingly, we discovered a Copia-like transposon (CoLT) that is highly enriched in the CENP-A chromatin. Similar clustered elements are also found in oomycete relatives of P. sojae, and may be applied as a criterion for prediction of oomycete centromeres. This work reveals a divergence of centromere features in oomycetes as compared to other organisms in the Stramenopila-Alveolata-Rhizaria (SAR) supergroup including diatoms and Plasmodium falciparum that have relatively short and simple regional centromeres. Identification of P. sojae centromeres in turn also advances the genome assembly.

26Centromeres are chromosomal regions that serve as platforms for kinetochore assembly 27 and spindle attachments, ensuring accurate chromosome segregation during cell division. 28Despite functional conservation, centromere DNA sequences are diverse and often repetitive, 29 making them challenging to assemble and identify. Here, we describe centromeres in an 30 oomycete Phytophthora sojae by combining long-read sequencing-based genome assembly and 31 chromatin immunoprecipitation for the centromeric histone CENP-A followed by high-throughput 32 sequencing (ChIP-seq). P. sojae centromeres cluster at a single focus at different life stages and 33 during nuclear division. We report an improved genome assembly of the P. sojae reference strain, 34 which enabled identification of 15 enriched CENP-A binding regions as putative centromeres. 35By focusing on a subset of these regions, we demonstrate that centromeres in P. sojae are 36 regional, spanning 211 to 356 kb. Most of these regions are transposon-rich, poorly transcribed, 37 and lack the histone modification H3K4me2 but are embedded within regions with the 38 heterochromatin marks H3K9me3 and H3K27me3. Strikingly, we discovered a Copia-like 39 transposon (CoLT) that is highly enriched in the CENP-A chromatin. Similar clustered elements 40 are also found in oomycete relatives of P. sojae, and may be applied as a criterion for prediction 41 of oomycete centromeres. This work reveals a divergence of centromere features in oomycetes 42 as compared to other organisms in the Stramenopila-Alveolata-Rhizaria (SAR) supergroup 43 including diatoms and Plasmodium falciparum that have relatively short and simple regional 44 centromeres. Identification of P. sojae centromeres in turn also augments the genome assembly. 45 46 Significance Statement 49Oomycetes are fungal-like microorganisms that belong to the stramenopiles within the 50 Stramenopila-Alveolata-Rhizaria (SAR) supergroup. The Phytophthora oomycetes are infamous 51 as plant killers, threatening crop production worldwide. Because of the highly repetitive nature of 52 their genomes, assembly of oomycete genomes presents challenges that impede identification of 53 centromeres, which are chromosomal sites mediating faithful chromosome segregation. We 54 report long-read sequencing-based genome assembly of the Phytophthora sojae reference strain, 55 which facilitated the discovery of centromeres. P. sojae harbors large regional centromeres 56 enriched for a Copia-like transposon that is also found in discrete clusters in other oomycetes. 57This study provides insight into the oomycete genome organization, and broadens our knowledge 58 of the centromere structure, function and evolution in eukaryotes. 59 60 Accurate segregation of chromosomes during mitosis and meiosis is critical for the 61 development and reproduction of all eukaryotic organisms. Centromeres are specialized regions 62 of chromosomes that mediate kinetochore formation, spindle attachment, and sister chromatid 63 segregation during cell division (1, 2). T...