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...