Chromatids of mitotic chromosomes were suggested to coil into a helix in early cytological studies and this assumption was recently supported by chromosome conformation capture (3C) sequencing. Still, direct differential visualization of a condensed chromatin fibre confirming the helical model was lacking. Here, we combined Hi-C analysis of purified metaphase chromosomes, biopolymer modelling and spatial structured illumination microscopy of large fluorescently labeled chromosome segments to reveal the chromonema - a helically-wound, 400 nm thick chromatin thread forming barley mitotic chromatids. Chromatin from adjacent turns of the helix intermingles due to the stochastic positioning of chromatin loops inside the chromonema. Helical turn size varies along chromosome length, correlating with chromatin density. Constraints on the observable dimensions of sister chromatid exchanges further supports the helical chromonema model.

The invention of chromosome conformation capture (3C) techniques, in particular the key method Hi-C providing genome-wide information about chromatin contacts, revolutionized the way we study the three-dimensional organization of the nuclear genome and how it affects transcription, replication, and DNA repair. Because the frequency of chromatin contacts between pairs of genomic segments predictably relates to the distance in the linear genome, the information obtained by Hi-C has also proved useful for scaffolding genomic sequences. Here, we review recent improvements in experimental procedures of Hi-C and its various derivatives, such as Micro-C, HiChIP, and Capture Hi-C. We assess the advantages and limitations of the techniques, and present examples of their use in recent plant studies. We also report on progress in the development of computational tools used in assembling genome sequences.

Centromeres are essential for chromosome movement. In independent taxa, species with holocentric chromosomes exist. In contrast to monocentric species, where no obvious dispersion of centromeres occurs during interphase, the organization of holocentromeres differs between condensed and decondensed chromosomes. During interphase, centromeres are dispersed into a large number of CENH3-positive nucleosome clusters in a number of holocentric species. With the onset of chromosome condensation, the centromeric nucleosomes join and form line-like holocentromeres. Using polymer simulations, we propose a mechanism relying on the interaction between centromeric nucleosomes and structural maintenance of chromosomes (SMC) proteins. Different sets of molecular dynamic simulations were evaluated by testing four parameters: (i) the concentration of Loop Extruders (LEs) corresponding to SMCs, (ii) the distribution and number of centromeric nucleosomes, (iii) the effect of centromeric nucleosomes on interacting LEs and (iv) the assembly of kinetochores bound to centromeric nucleosomes. We observed the formation of a line-like holocentromere, due to the aggregation of the centromeric nucleosomes when the chromosome was compacted into loops. A groove-like holocentromere structure formed after a kinetochore complex was simulated along the centromeric line. Similar mechanisms may also organize a monocentric chromosome constriction, and its regulation may cause different centromere types during evolution.

The synaptonemal complex (SC) forms between homologous chromosomes during meiosis. In Arabidopsis thaliana, its central region (CR) is composed of the transverse filament protein ZYP1 and the central element proteins SCEP1 and SCEP2. Here we identify SCEP3 as a CR protein that is evolutionarily conserved across plant species. SCEP3 spatiotemporally overlaps with other CR proteins and localizes to the SC CR. The loss of SCEP3 prevents SC assembly, abolishes crossover (CO) assurance and interference, and eliminates sex-specific differences in CO rates (heterochiasmy) through increased CO in females. SCEP3 is required for a subset of COs in SC-deficient mutants, such as zyp1. Although SCEP3 physically interacts with ZYP1, it loads independently of other CR proteins. We propose that SCEP3 may associate with certain recombination intermediates, stabilizing them and/or recruiting additional factors, such as ZYP1, to a subset of these intermediates, thereby promoting and interlinking SC assembly and CO formation.

SUMMARY: Efficient chromatin condensation is required to transport chromosomes during mitosis and meiosis, forming daughter cells. While it is well accepted that these processes follow fundamental rules, there has been a controversial debate for more than 140 years on whether the higher‐order chromatin organization in chromosomes is evolutionarily conserved. Here, we summarize historical and recent investigations based on classical and modern methods. In particular, classical light microscopy observations based on living, fixed, and treated chromosomes covering a wide range of plant and animal species, and even in single‐cell eukaryotes suggest that the chromatids of large chromosomes are formed by a coiled chromatin thread, named the chromonema. More recently, these findings were confirmed by electron and super‐resolution microscopy, oligo‐FISH, molecular interaction data, and polymer simulation. Altogether, we describe common and divergent features of coiled chromonemata in different species. We hypothesize that chromonema coiling in large chromosomes is a fundamental feature established early during the evolution of eukaryotes to handle increasing genome sizes.

Centromeres are essential for chromosome segregation, yet their organisation and evolution remain poorly understood in holocentric species, where kinetochore activity is distributed along entire chromosomes ^1,2 . While monocentric centromeres are often structured by megabase-sized satellite arrays ^3–5 , the role of repetitive DNA in holocentric systems remains enigmatic. Here, we analyse the dynamics of centromeric Tyba satellite DNA repeats and transposable elements across a chromosome-scale pangenome comprising 56 long-read haplotype assemblies from 20 Rhynchospora species ^6,7 , a plant genus with repeat-based holocentromeres ^8,9 . We identify over 4.6 million monomers of the Tyba satellite repeat, arranged into 43,400 discrete arrays that span all chromosomes. CENH3 ChIP-seq reveals that, unexpectedly, the same Tyba satellite defines holocentromere across the entire genus, demonstrating deep conservation of centromeric DNA over over 40 million years despite extensive karyotype evolution and centromere array turnover. We show that Tyba arrays function as modular centromeric units whose number and spacing, but not size, scale with chromosome length. Tyba sequence diversity recapitulates species phylogeny, while higher-order repeat formation and antagonism with transposable elements shape array turnover. A novel synteny-aware algorithm reveals rapid gain, loss, and rearrangement of arrays across homologous chromosomes. Using cytogenetics and polymer simulations, we demonstrate that inter-array spacing governs chromatin loop length and chromatid thickness, linking repeat-based holocentromere organisation directly to chromosome mechanics. Our findings uncover a scalable, modular logic for holocentromere function and establish a framework for understanding the plasticity of repeat-based centromere evolution and genome architecture in eukaryotes.