Long transposon-rich centromeres in an oomycete reveal divergence of centromere features in Stramenopila-Alveolata-Rhizaria lineages

Abstract

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.

Repetitive Elements Contribute to the Diversity and Evolution of Centromeres in the Fungal Genus Verticillium

“…In most filamentous fungi and oomycetes, AT- and repeat-rich centromeres are embedded in heterochromatin that is characterized by methylated DNA and by particular histone modifications (H3K9me3 and H3K27me3) ( 13 , 15 , 16 , 25 , 30 , 46 ). We recently determined chromatin states in the genome of V. dahliae strain JR2 and revealed that repetitive sequences outside the LS regions display characteristics of heterochromatin ( 36 ).…”

“…H3K4me2 borders most centromeres in Z. tritici ( 27 ) and is associated with centromeres in Schizosaccharomyces pombe and some animals and plants ( 63 – 66 ). H3K4me2 has not been observed at centromeres in most fungi, including V. dahliae , and in the oomycete plant pathogen Phytophthora sojae ( 30 ). Changes in heterochromatin in N. crassa lead to altered CenH3 positioning ( 25 ), suggesting that heterochromatin is similarly required for centromere maintenance and function in V. dahliae .…”

“…Centromeres are often enriched for a variety of different retrotransposons and other repetitive elements ( 15 , 16 , 25 , 29 , 30 , 68 – 70 ). We similarly observed that centromeres in all Verticillium species are repeat rich.…”

“…Knowledge on centromeres has been impaired by their repetitive nature, which hampers their assembly and subsequent analyses ( 15 , 28 ). However, recent advances in long-read sequencing technologies enable studies of the constitution and evolution of centromeres ( 11 , 16 , 29 – 31 ). By using long-read sequencing technologies in combination with optical mapping, we previously generated gapless genome assemblies of two strains of the fungal plant pathogen Verticillium dahliae ( 32 ).…”

<strong>SMARTdenov</strong><strong>o: A <em>de novo</em> Assembler Using Long Noisy Reads</strong>

“…SMARTdenovo has been available since 2015 on github, but its performance not only remains comparable with current assemblers, and but also has several advantages as described. Furthermore, given its excellent performance for use on corrected long sequence reads, it continues to be widely used in for genome assembly projects today [33][34][35][36][37][38][39][40][41][42].…”