Chromosome Research

The germline-restricted chromosome (GRC) of the zebra finch Taeniopygia guttata represents a well-established model of programmed DNA elimination in vertebrates. Although the DNA composition of the GRC, as well as elimination processes during spermatogenesis and early embryogenesis, have been characterised previously, little is known about the cytogenetic features underlying its unusual behaviour, including its stable transmission through the maternal germline. Here, we provide a detailed characterisation of the zebra finch GRC at the diplotene stage of female meiosis, when chromosomes are actively transcribed and acquire the form of giant lampbrushes. We identified a transcriptionally repressed region on the GRC, which we term the belt. Microdissection and sequencing of the belt revealed that it is predominantly composed of a tandem repeat derived from the dph6 gene, robo1 gene fragments, and ERVs. Notably, the terminally located functional centromere of the GRC lacks typical zebra finch centromeric satellites and, conversely, consists of the newly identified GRC-specific tandem repeats Tgut16-201 and Tgut17-167. The canonical centromeric repeat Tgut716 was observed in the GRC belts. Moreover, belts, like the terminal GRC centromere, were associated with coilin-containing nuclear bodies, which serve as markers of centromeric regions on zebra finch lampbrush chromosomes. Together, our findings provide evidence for the presence of one functional and one putative centromeric region on the zebra finch GRC, suggesting their role in non-Mendelian inheritance of the GRC. Author summaryGermline-restricted chromosomes (GRCs) are unusual chromosomes that are retained in germ cells but eliminated from somatic cells during early development. They have evolved independently in several groups of organisms, but are particularly notable in passerine birds, a large monophyletic vertebrate clade ([~]6,700 species) in which GRCs have persisted for at least 44 million years. Passerine GRCs are normally transmitted to the next generation through the maternal germ cell, however, the mechanisms ensuring their inheritance remain unknown. To address this question, we examined the structure of the zebra finch GRC during female meiosis. We found that the GRC differs from all other chromosomes in possessing two distinct centromeric regions: a functional terminal centromere and an extended heterochromatic region exhibiting centromeric properties. These unusual features suggest a mechanism by which the GRC may achieve its preferential transmission through the female germline. Our findings substantially advance the understanding of the zebra finch GRC and the general biology of passerine GRCs. By revealing chromosome features that may underlie their non-Mendelian inheritance, this work provides new insights into the evolution and behaviour of GRCs and other selfish chromosomes that bias their own transmission.

The genus Rhynchospora Vahl (beak-sedges) comprises approximately 381 accepted species with a worldwide distribution, all of which possess holocentric chromosomes, where centromeric activity is distributed almost along the entire chromosome. Despite the recent advances, the mechanisms governing the dynamics of meiotic recombination in holocentric plants remain poorly understood. Here, we developed haplotype-specific oligo-FISH probes for chromosomes 1, 2, and 3 based on a haplotype-phased genome assembly of Rhynchospora breviuscula (n = 5), enabling homolog-specific chromosome painting. Each probe set was labelled with a distinct fluorophore and hybridised in situ to metaphase chromosomes of the reference plant and seven F1 individuals derived from self-crossed reference plants. This approach allowed the unambiguous discrimination of homologous haplotypes and the indirect visualisation of crossover (CO) events in recombined chromosomes. We observed that recombination events were predominantly located in terminal chromosomal regions, consistent across individuals. These results corroborate previous findings from single-cell recombination mapping and provide independent cytological validation of the recombination landscape in this species. Our study establishes haplotype-specific chromosome painting as a robust tool for high-resolution mapping of meiotic recombination in holocentric plants across generations. Furthermore, these probes provided a foundation for future investigations into inverted meiosis, a mechanism characterized by an alternative pattern of chromosome segregation in holocentric species.

Centromeres ensure accurate chromosome segregation and are typically confined to a single, localised region in monocentric chromosomes. In contrast, holocentric chromosomes exhibit kinetochore activity distributed along the chromosome length. Although holocentricity is widespread in Cyperaceae, the composition and organisation of these centromeres, as well as their evolutionary diversification, remain poorly understood. Here, we investigated centromere organisation in representatives of the subfamilies Mapanioideae (Hypolytrum schraderianum Nees) and Cyperoideae (Cladium mariscus (L.) Pohl) by combining genome assemblies, repeatome characterisation (RepeatExplorer), fluorescence in situ hybridisation (FISH), and immunolocalisation. Comparative synteny analyses incorporating the genomes of Rhynchospora breviuscula (n = 5) and Carex littledalei (n = 29) identified conserved blocks, eventually expanding almost whole chromosomes of H. schraderianum (n = 30) and Cl. mariscus (n = 39), despite divergent chromosome numbers and deep evolutionary distances within Cyperaceae. Mobile elements showed very low abundances and were uniformly dispersed, with Ty1/Copia Angela being the most abundant in both species. In Cl. mariscus, holocentromeres showed an extended distribution of centromere- and kinetochore-associated proteins along the chromosomes, largely colocalised with two satellite DNA repeats that form dispersed clusters. In contrast, H. schraderianum also displayed kinetochore signals along chromatids, but the most abundant satellite DNA family was enriched in distal and interstitial chromosomal regions rather than interspersed along the chromatids. Together, these results reveal different genomic architectures underlying holocentric organisation in phylogenetically distinct Cyperaceae lineages, suggesting that holocentromeres in this family have diversified with variation in centromere organisation in regard to its association with repetitive DNA.

Long-read sequencing and assembly of human genomes have revealed extreme variation in centromeric alpha-satellite array sizes across chromosomes and individuals. However, assessing the impact of centromeric array size on centromere function remains challenging. In this study, using quantitative FISH and microscopy assays, we established a framework for the systematic functional evaluation of naturally occurring variations in centromeric array size during mitosis. By comparing 8 pairs of homologous chromosomes exhibiting 1.24 - 4.6 -fold variation in centromeric array size, we uncovered size-based differences in function. We found that between a pair of homologous chromosomes, the chromosome harboring the smaller centromeric array is more prone to chromosome missegregation. Centromere size-based differences in chromosome missegregation rates cannot be explained by differential enrichment of molecular factors like centromeric histone CENP-A, CENP-B, or most kinetochore proteins. However, the smaller centromere out of a homologous pair consistently exhibits increased cohesion fatigue, suggesting the involvement of the cohesin complex in size-based differences in centromere cohesion. Consistent with our hypothesis, complete deprotection of the cohesin complex by depletion of shugoshin-1, removes the size-based bias in centromere cohesion. These results suggest that while CENP-A-enriched core centromeres are essential for kinetochore assembly, variation in overall centromeric array size has a significant impact on centromere performance and the fidelity of chromosome segregation during mitosis.

Human nucleoli are multivalent, involving contributions from up to ten NOR-bearing acrocentric chromosome p-arms. Precision mega-base scale chromosome engineering defines the requirements for this major genome organisational event. NOR deletions reveal that p-arm nucleolar association is rDNA independent. Deletion of all NOR-distal or proximal sequences individually have only a marginal effect on nucleolar association. Finally, deletion of an entire p-arm, while leaving centromere function intact, destroys the nucleolar association potential of that acrocentric. We propose that formation of multivalent nucleoli is not a nucleolar fusion event per se; rather it is driven by the surrounding chromosomal context of NORs.

Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells.

Sexual reproduction in flowering plants relies on double fertilization, a process marked by two fusion events between the male and female gametes that lead to seed formation. Because this process unfolds within the embryo sac embedded deep inside the ovule, direct observation remains technically demanding, especially in maize, where the large size of female reproductive organs presents additional obstacles. The described method enables high-resolution visualization of cellular events unfolding during maize double fertilization. The approach integrates optimized fixation, clearing and confocal imaging of embryo sacs from ears pollinated with fluorescent pollen marker lines. Precise timing of embryo sac fixation is critical, allowing capture of key events such as pollen peri-germ cell membrane break-down or gamete karyogamy. The protocol provides detailed guidance for ovule dissection, fixation, preparation and renewal of the clearing solution and confocal imaging of embryo sacs. This method offers unprecedented access to the cellular events of double fertilization in maize, establishing a robust framework for studying reproductive processes and supporting future discoveries in plant reproduction.

During meiosis, accurate chromosome segregation requires significant condensation and compaction. These processes are mediated by condensins, cohesins, and histone tail modifications. We identified that MET-2, a histone methyltransferase that catalyzes the dimethylation of histone H3 lysine 9 (H3K9me2), differentially impacts chromosome size in the male vs. female C. elegans germline. In met-2 null worms, autosomes during spermatogenesis are significantly larger than wild type, while chromosome size during oogenesis is unaffected. X-univalent size in males is also unaffected by loss of MET-2, indicating MET-2 differentially regulates autosomal and X-chromosome compaction in male spermatogenesis. Autosome size is not changed when males harbor a catalytically deficient MET-2 (met-2CD) or have mutations preventing germline histone H3K9 methylation (H3K9R). In addition, met-2 males, in contrast to met-2CD or H3K9R males, have more active RNA pol II in later stages of meiosis. These data suggest MET-2 plays a noncatalytic role in mediating chromosome structure and transcription. In met-2 male germ lines, genes on the X chromosome, which is typically enriched in H3K9me2, are significantly more likely to be upregulated than genes on autosomes, even though X-univalent size is unchanged. These results suggest that MET-2 plays a sex-specific role that is not limited to its enzymatic activity.

The three-dimensional architecture of genomes plays major roles in biological processes such as gene expression and DNA replication. The architecture of genomes has evolved substantially with distinct 3D genome shapes being identified in different lineages. The factors driving the evolution of genome architectures have primarily been assessed in animals and plants, yet large parts of the tree of life remain poorly explored. Fungi offer excellent models to assess the evolution of 3D genome architecture in a phylogenetic context given rapid genome size changes and chromosomal sequence turnover. Here, we analyzed chromosome conformation data (Hi-C) of 55 fungal species with completely assembled genomes. We identified ten species with Rabl, one species with chromosome territories and ten with a novel, intermediate chromosomal architecture, where centromeres and telomeres are at opposites in the nucleus (Rabl-like) but with a distinct 3D organization. This "bean" shape likely evolved several times independently. The discovery of a genome with a chromosome territories conformation was unexpected, as this was thought to be associated with condensin II subunits in the animal kingdom. We investigated whether 3D conformations correlated with genome size and repeat content using phylogenetic independent contrasts, however we found no genomic feature to be significantly associated with changes in genome architecture. Overall, we report the first large-scale comparison of 3D genome architecture in the fungal kingdom and identify a novel "bean" configuration. SignificanceThree-dimensional genome architecture strongly influences gene regulation, yet little is known about 3D genome architecture in an organismal group that has adapted to nearly all ecosystems on our planet, Fungi. We reconstructed 3D genome architectures from 55 fungal species covering three different phyla and demonstrate that most species do not conform to the existing definitions of 3D architectures. We identified the first case of Chromosome Territories in the Fungal Kingdom and a previously undescribed organization that we label "bean-shaped", and show that some fungal species do not conform to the canonical 3D-architecture categories of the animal and plant kingdoms. The diversity of genome architectures observed in the study could reflect the diverse gene regulatory mechanisms known from Fungi and marks the beginning of mapping out 3D genome organizations in this diverse clade. Further research in this area will uncover the diverse strategies employed by Fungi in light of their rapid adaptation.

Recent technological advances allow for the large-scale acquisition of genetic and morphological data: high-throughput sequencing has transformed the field of genomics while synchrotron X-ray microtomography enables rapid, noninvasive 3D imaging. However, integrating these approaches for the same specimens is challenging because X-rays can fragment DNA, and DNA extraction damages internal morphology, particularly relevant for small bodied organisms, such as insects. We systematically tested multiple extraction protocols and irradiation conditions across three model insect species. We irradiated more than 1,000 specimens under varying conditions and tested DNA quality through DNA barcoding and UCE sequencing. Our results demonstrate that high-quality DNA and high-resolution tomograms can be obtained from the same individuals, provided that the parameters are carefully optimized and rapid SR-CT scanning precedes DNA extraction. In this respect, our findings establish practical guidelines for combining genomics and phenomics, paving the way for comprehensive integrative digitization of biodiversity.

The haplochromine cichlid fishes of Lake Malawi form one of the most dramatic examples of recent rapid radiation in vertebrates. Here we describe nine new diploid telomere-to-telomere (T2T) genome sequences generated using ultra-long ONT reads, which include 78 ungapped chromosomes. We provide accurate annotations of transposable elements and tandem repeats, identify rDNA cluster regions and putative centromeres, and confirm previously reported large chromosomal inversions. The putative centromeres are primarily composed of satellite tandem arrays of previously reported 237 bp repeats, but notably on most chromosomes these are organised in a novel structure in which four blocks of satellites in alternating orientation are separated by an inverted pair of ~15 kb sequences we term 'centroids', which have similarity to a non-autonomous DNA transposable element and containing potential CENP-B binding boxes. The methylation dip region indicating the likely active centromere always lies between the centroids, whose separation is almost always around 200 kb (interquartile range 151-221kb). A structurally equivalent but non-homologous organisation is seen in the distantly related Etroplus cichlid genera from South Asia. By comparing these structures across chromosomes and species, we suggest how they may have evolved, and potentially how they could contribute to the rampant sympatric speciation seen in these species, based on meiotic drive and chromosome missegregation.

We present a chromosome-level genome assembly of a female great grey owl (Strix nebulosa lapponica). The assembly comprises two pseudo-haplotypes of 1554 Mb and 1242 Mb, with 83.2% and 91.4% scaffolded into 40 autosomal chromosomes, in addition to the W and Z sex chromosomes both placed in hap1. Assembly completeness is high (BUSCO 99.2% and 94.8%), with 18,493 and 17,279 annotated protein-coding genes for hap1 and hap2, respectively. This genome establishes a reference for investigating genetic variation and chromosome evolution in great grey owls. Compared with the previous S. nebulosa assembly, this assembly includes both sex chromosomes, separates regions that were previously collapsed, and resolves 82 chromosomes total. While larger chromosomes show broadly conserved synteny across owl assemblies, the recovery of additional conserved microchromosome-associated genes suggests that ONT reads improved resolution of the smallest chromosomes relative to HiFi-based assemblies.

Centromere architecture can determine where transposable elements persist, yet its effects on retrotransposon turnover remain poorly understood. Here we test a chromosome-level "nowhere-to-hide" model, in which holocentric chromosomes, owing to distributed centromere activity and reduced chromatin compartmentalization, provide fewer stable repeat-rich refugia than monocentric chromosomes. We combined genome-wide LTR retrotransposon annotation, spatial modelling and FISH across 40 holocentric plant species and 31 closely related monocentric relatives from Poales, Cuscuta, and Melanthiaceae. Overall LTR retrotransposon abundance and Ty1-copia/Ty3-gypsy composition were explained mainly by lineage history and chromosome size, rather than by holocentricity itself. By contrast, element persistence and removal showed dependence on centromere architecture. Intact LTR retrotransposons were younger in holocentric genomes, and holocentric chromosomes lacked the chromosome-size-dependent spatial clustering of element age observed in monocentrics. Solo-LTR profiles further revealed weaker spatial clustering of removal signatures in holocentric chromosomes, consistent with a more homogeneous chromosome-wide landscape of ectopic recombination. Epigenomic analyses of a matched Luzula-Juncus pair indicated that young elements can occur in centromeric chromatin, whereas solo LTRs are associated with more euchromatic contexts. These results support the nowhere-to-hide model, showing that centromere architecture does not shape LTR retrotransposons accumulation, but their persistence and removal efficiency.

Degenerate sex chromosomes (e.g., the Y or W) remain among the most difficult regions of eukaryotic genomes to assemble because they are highly repetitive and structurally complex. While many recent lepidopteran genome assemblies contain W chromosome scaffolds, the accuracy and consistency of these assemblies remain uncertain, due to lack of replication within species. However, the silkworm moth Bombyx mori is an exception, with numerous independent W chromosome assemblies currently available. We compared six independent long-read W chromosome assemblies, which proved to be highly inconsistent in structure, even among nominally identical genotypes. In contrast, autosomes and the Z chromosome were highly concordant among these assemblies, indicating that current assemblies remain unreliable for resolving W chromosome structure. Additionally, we analyzed repetitive DNA content across the genome. First, we combined assembly- and read-based repeat-discovery methods to generate a comprehensive and curated Bombyx repeat library, which we make publicly available. Assessing repeat content and diversity, we find that the W chromosome is comprised almost entirely of repetitive DNA but that the richness and divergence of W repeats are substantially reduced compared to the remainder of the genome. This reduced diversity, initially inferred from assemblies, is confirmed by direct analysis of PacBio HiFi sequencing reads partitioned by chromosome. We also demonstrate that the B. mori p50ma genome assembly (the current NCBI RefSeq assembly) carries a W chromosome and mitochondrial genome introgressed from B. mandarina. This discovery provided an opportunity to investigate patterns of divergence between closely related W haplotypes, revealing substantially more rapid turnover of repeat content on the W than elsewhere in the genome. Together, our results show that current W chromosome assemblies, although structurally flawed, nevertheless capture robust biological patterns of repeat diversity and support the hypothesis that rapid repeat turnover, rather than frequent chromosome replacement, may underlie the apparent lack of W chromosome homology across Lepidoptera.

Multicellularity has evolved multiple times across the eukaryotic tree of life, including among protist lineages. Because transposable elements (TEs) strongly influence genome architecture and gene regulation, understanding their potential impact on genome structure and their relationship with gene expression may provide insight into the evolution of multicellularity. Here, we generated a new genome assembly for the facultatively multicellular amoeba Acrasis kona and performed comparative analyses of TE composition, TE diversity, and TE-density organization across diverse protist lineages. Comparative analyses included unicellular and multicellular representatives from across the tree of eukaryotes, (Heterolobosea, Filasterea, Cristidiscoidea, and Chlorophyceae), including Naegleria spp., Tetramitus jugosus, Capsaspora owczarzaki, Pigoraptor spp., Fonticula alba, Parvularia atlantis, Volvox carteri, and Chlamydomonas reinhardtii. To examine relationships between TEs and gene regulation, we integrated transcriptomic datasets from A. kona, Capsaspora owczarzaki, and Volvox carteri with genome-wide TE-density analyses of differentially expressed genes. TE abundance and composition varied substantially among lineages, with species that exhibit more complex developmental or cellular organization generally containing higher TE proportions than closely related unicellular taxa. Patterns of TE-density organization near up-regulated, down-regulated, and non-differentially expressed genes also differed among systems, ranging from strong TE depletion in A. kona to weaker or cell-type-specific patterns in Capsaspora and Volvox. Together, these findings suggest that transposable elements are associated with multicellularity across diverse protist lineages, although the specific roles they play appear to be complex, lineage-specific, and not yet fully understood.

Molecular motors travelling along DNA introduce positive supercoils that present as barriers to replication leading to genome instability. To counter these, bacterial cells express DNA gyrase, a topoisomerase that introduces negative supercoils. While much is known about DNA gyrase from genetic and in vitro biochemical studies, the spatiotemporal dynamics of this enzyme remain a mystery. Only recently have we been able to observe the in vivo spatiotemporal dynamics down to single molecule level using advanced super-resolution microscopy techniques. We used Slimfield microscopy, a cutting-edge molecule microscopy technique to address the gap in our knowledge. We analysed a dual fluorescently labelled Escherichia coli strain expressing the replisome marker DnaN-mCherry along with mYPet-GyrB as the enzyme marker. We performed sequential Slimfield microscopy of the labelled proteins from the same strain and analysed in vivo GyrB dynamics in live E. coli cells in relation to the replisome. We find that the majority of replisomes are associated with GyrB. Inhibition of gyrase activity reduces the proportion of replisomes associated with GyrB. Interestingly, GyrB behaviour is distinct from that observed for GyrA in a previous study. Our results reveal the previously unknown dynamics of GyrB inside living bacterial cells highlighting the advantages of in vivo single molecule investigations. Our findings also demonstrate the importance of analysing all subunits of a functional enzyme complex to gain comprehensive understanding of its in vivo mechanisms. This study demonstrates the utility of single-molecule super-resolved microscopy as a valuable underpinning technology to understand in vivo behaviour of biomedically important molecules. Our insights will help impact discovery and development of novel antibiotics that interfere with gyrase function, thus contributing to tackling the growing problem of antimicrobial resistance.

The Saccharomyces cerevisiae Pch2 protein is a conserved meiotic AAA+ ATPase whose activity must be tightly regulated to ensure proper chromosome dynamics during meiotic prophase I. Its function relies on remodeling the HORMA-domain protein Hop1, promoting conformational transitions that are essential for chromosome axis organization, checkpoint signaling, and recombination control. Here, we identify threonine 428 (T428), located within a conserved threonine-glutamine (TQ) putative phosphorylation motif, as a critical regulatory residue of Pch2. We found that, in zip1{Delta} cells, the meiotic recombination checkpoint response is partially or completely abolished in the pch2-T428A and pch2-T428D mutants, respectively. Both mutations alter Pch2 subcellular localization, leading to its increased nuclear accumulation; however, forced nuclear exclusion of Pch2-T428A, but not Pch2-T428D, restores the zip1{Delta} meiotic block, indicating an additional effect of the T428D substitution on checkpoint function beyond subcellular distribution. Analysis in synapsis-proficient strains reveals that this residue also plays a critical role in coordinating Hop1 chromosomal enrichment with Mek1 activation along the synaptonemal complex. In contrast to pch2{Delta} or the ATPase-defective pch2-E399Q mutant, introduction of a negative charge at the 428 position uncouples Hop1 accumulation from its phosphorylation, preventing Mek1 activation despite robust Hop1 association with meiotic chromosomes. These findings support emerging models in which Pch2 regulates Hop1 to control not only its chromosomal abundance, but also the maintenance of sufficient levels of Hop1 in a phosphorylation-competent conformation, thereby ensuring proper checkpoint signaling and faithful meiotic progression.

Sperm heteromorphy, the production of multiple sperm morphs with distinct functions, has evolved repeatedly in animals but its developmental and molecular basis remains poorly understood. In the Drosophila obscura group, males produce fertilising eusperm and non-fertilising parasperm, yet when and how these lineages diverge is unclear. Here, we applied single-cell RNA sequencing to whole testes of D. pseudoobscura to resolve cell-type composition, developmental trajectories, and transcriptional differences between sperm morphs. Analysis of [~]6,500 cells across two replicates identified germline and somatic cell populations and reconstructed spermatogenesis from stem cells to elongated spermatids. We identified a bifurcation in the germline trajectory at the early spermatocyte stage, corresponding to eusperm and parasperm lineages, which was maintained through spermiogenesis. Euspermatocytes, destined to generate longer eusperm, exhibited higher transcriptional activity than paraspermatocytes. Differentially expressed genes included structural sperm tail components, chromatin condensation proteins, post-meiotically transcribed genes and gene duplications resulting in paralogues with reciprocal expression patterns. We also identified markers of somatic cyst cells, including head and tail cyst cells, but found no evidence of morph-specific specialisation within the cyst cell lineage. The transcriptional activities of the distinct germline trajectories corresponded with differential expression of orthologues of critical transcriptional regulators known in D. melanogaster. The transcriptional activator TGIF was enriched in euspermatocytes, while the transcriptional repressor kmg was upregulated in paraspermatocytes. GFP-tagged Kmg showed higher abundance and greater chromatin localisation in paraspermatocytes. These findings demonstrate that sperm morph identity is first apparent in early primary spermatocytes and is largely germline intrinsic. We propose that differential transcriptional provisioning and developmental timing underpin sperm length, providing a framework for understanding the evolution and regulation of sperm heteromorphy.

Meiosis is a fundamental process in producing both sperm and eggs, yet recombination landscapes often exhibit sexual differences, known as heterochiasmy. Since meiotic proteins are generally expressed in both sexes, the molecular mechanism driving heterochiasmy remains elusive. The -kleisin subunit gene of meiotic cohesin, Rec8, is expressed bisexually in mammals, while its putative teleost ortholog, rec8a, is expressed in a female-biased manner, presumably due to the presence of its paralog originating from the teleost-specific whole-genome duplication (TGD). Here, we elucidated the evolutionary history and expression dynamics of -kleisin genes across teleost lineages. Through comprehensive phylogenetic and synteny analyses, we revealed that major teleost lineages retain two copies of rec8 and rad21, with rec8 loci experiencing drastic chromosomal rearrangements immediately after the TGD. Using in situ hybridization and single-cell transcriptome data in medaka and zebrafish, we demonstrated a conserved sexually biased expression pattern: rec8a is predominantly female-biased, whereas rec8b exhibits male-biased expression during gametogenesis. Furthermore, comparative epigenetic analyses revealed that the conserved sexually biased expression is driven by lineage-specific cis-regulatory elements, rather than conserved ones. Motif analyses imply that regulatory rewiring by transcription factors, including foxl2l in particular, might have played a crucial role in the establishment and maintenance of this paralog divergence. Our findings highlight how whole-genome duplication and subsequent genomic and epigenetic rewiring subdivided the bisexual function of rec8, offering insights into sexually distinct meiotic regulation. HighlightsO_LITeleosts possess a unique -kleisin repertoire originating from the TGD. C_LIO_LITeleost rec8 paralogs exhibit conserved sex-biased expression during meiosis. C_LIO_LIDrastic genomic rearrangements after the duplication rewired the teleost rec8 loci. C_LIO_LIThe conserved expression pattern is governed by lineage-specific CREs. C_LIO_LIThose CREs harbor similar types of TFBSs such as Fox-family TFs. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/731870v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@c8c84dorg.highwire.dtl.DTLVardef@1d65668org.highwire.dtl.DTLVardef@c2d732org.highwire.dtl.DTLVardef@1be54a2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background and ObjectivesThe linear relation between human metaphase chromosome length and DNA content has never been rigorously reconciled with modern Hi-C models of mitotic chromatin folding. We tested whether the relation implies a quantitative unit of mitotic chromosome organization. MethodsWe pooled metaphase lengths for 24 human chromosomes from five cytogenetic studies of cultured peripheral lymphocytes and regressed length against base-pair content from GRCh38 and T2T-CHM13 v2.0. Pre-specified analyses comprised ordinary least-squares and power-law fits, arm-level decomposition, and reconciliation with the Gibcus 2018 helical loop-array model. Orthogonal validation used Rao 2014 Hi-C boundary counts and Pope 2014 Repli-Seq. ResultsLength scales linearly with DNA content as L (m) = 0.0329 x Mb + 0.043 (R-squared = 0.998, power-law exponent 0.98 +/-0.01), with a cross-karyotype compaction density of 33.4 +/-1.0 nm/Mb. T2T-CHM13 reanalysis identifies satellite over-condensation; the arm-level residual correlates with p-arm fraction (r = 0.65, p = 5.6e-4). We propose an operational Metaphase Chromatin Unit (MCU) as a quantitative scaling unit (not a discrete structural quantum, as quantization tests are negative): 1 MCU = 7.6 Mb DNA = 0.25 m axial length, numerically corresponding to one Gibcus late-metaphase helical turn; the human haploid genome scales to 406 MCUs. The pairwise megabase-shift slope of 32.75 nm/Mb (R-squared = 0.996) and the approximately 6 Mb Abbe optical detection threshold correctly classify 9 of 9 microdeletion syndromes as karyotype-visible versus FISH-required. ConclusionsThe MCU provides a unified quantitative unit for human mitotic chromosome organization, integrating cytogenetic, Hi-C, polymer-biophysical, and clinical scales.