Whole genome duplication (WGD) generates new genetic material that can contribute to the evolution of developmental processes and phenotypic diversification. A WGD occurred in an ancestor of arachnopulmonates (spiders, scorpions, and their relatives), which provides an important independent comparison to WGDs in other animal lineages. After WGD, arachnopulmonates retained many duplicated copies (ohnologues) of developmental genes including clusters of homeobox genes, many of which have been inferred to have undergone subfunctionalisation. However, there has been little systematic analysis of gene regulatory sequences and comparison of the expression of ohnologues versus their single-copy orthologues between arachnids. Here we compare the regions of accessible chromatin and gene expression of ohnologues and single-copy genes during three embryonic stages between an arachnopulmonate arachnid, the spider Parasteatoda tepidariorum, and a non-arachnopulmonate arachnid, the harvestman Phalangium opilio. We found that the expression of each spider ohnologue was lower than their single-copy orthologues in the harvestman suggesting subfunctionalisation. However, this was not reflected in a reduction in the number of peaks of accessible chromatin because both spider ohnologues and single-copy genes had more peaks than the orthologous harvestman genes. We also found that the number of peaks of accessible chromatin was higher in the late embryonic stage associated with activation of genes expressed later during embryogenesis in both species. Taken together, our study provides a genome-wide comparison of gene regulatory sequences and embryonic gene expression in arachnids and thus new insights into the impact of the arachnopulmonate WGD.

Abstract DNA damage occurs in all cells and must be repaired to maintain genome integrity. Many DNA lesions are targeted for removal by repair systems that excise the damage, thereby generating a temporary single-strand discontinuity in the chromosome. If DNA repair has not been completed prior to a round of genome duplication, the single-strand discontinuity (nick or gap) can be converted to a double-strand break (DSB) by an oncoming replication fork. Because the genomic location of nucleobase damage is stochastic, investigating the fate of replication machinery (replisome) at DNA repair sites with single-strand discontinuities has been limited. Here we address this issue by expressing Cas9 nickases in Bacillus subtilis to create site-specific single-strand discontinuities in a bacterial chromosome. We find that a nick in either leading or lagging strand arrests DNA replication, while the fate of the replicative helicase is distinct and depends upon the strand nicked. Genetic, biochemical, and single cell analyses indicate that replisome/nick encounters generate a single-end DSB which requires recombinational repair to enable PriA-dependent replication restart. Together this work defines the physiologically relevant pathway used by B. subtilis to reinitiate DNA synthesis following replication fork inactivation at a single-strand discontinuity.

In this study, Hedychium coccineum tetraploid plants and octaploid plants induced by colchicine were used as materials. The ploidy levels were precisely identified by combining root tip squash and flow cytometry analyses, and the differences between plants of different ploidy levels were systematically investigated at cytological, morphological, and molecular levels. The results showed that the highest polyploid induction efficiency was achieved when callus tissues were treated with 0.1 g/L colchicine for 4 days. The fluorescence peak value of the induced plants was twice that of the tetraploids, confirming their octaploid status. Compared with tetraploids, octaploid plants exhibited almost no apparent dormancy period, significantly slower growth, earlier flowering, and notably smaller inflorescences. Morphologically, they showed a dwarf phenotype characterized by narrower and lighter-colored leaves, fewer leaves per shoot, shorter internodes, and wider leaf angles, along with enhanced stress tolerance. Cytological observation revealed that cell area in internode tissues at the bud and seedling stages was generally larger in tetraploids than in octaploids, suggesting a reduction in cell size following genome duplication. Furthermore, transcriptome comparison between tetraploids and octaploids identified HcPCNA1 as a candidate gene closely associated with plant height. Functional validation showed that overexpression of HcPCNA1 in Arabidopsis thaliana significantly increased plant height, whereas silencing of HcPCNA1 in H. coccineum via Virus-induced gene silencing (VIGS) resulted in a distinct dwarf phenotype with smaller leaves. Cytological and molecular evidence together indicate that HcPCNA1 may influence plant height in H. coccineum through its role in promoting cell division and elongation. This finding provides new insights into the molecular mechanisms underlying plant architecture development in polyploid species.

DNA replication fork speed, which controls the rate of genome duplication, has emerged as a key regulator of cellular plasticity. However, its role in neurogenesis remains unexplored. Mini-chromosome maintenance complex (MCMs)-binding protein (MCMBP) functions as a chaperone for newly synthesized MCMs, increasing chromatin coverage to restrain fork speed. We demonstrate that selectively deleting Mcmbp in neural progenitor radial glial cells (RGCs) accelerates fork speed, triggering DNA damage, micronuclei formation, and widespread apoptosis, which ultimately activates p53 and causes microcephaly. Unexpectedly, concurrent deletion of Trp53 and Mcmbp further increases fork speed, leading to extensive RGC detachment from the ventricular zone and acquisition of outer-RGC characteristics. Mechanistically, we find that the MCM complex coordinates DNA and centrosome duplication, thereby mediating RGC attachment. Behavioral analysis reveals that embryonic replication stress induced by accelerated fork speed results in lasting anxiety-like behavior in adult mice. These findings unveil a role for replication fork speed in neurogenesis.

BackgroundNMDA receptors (NMDARs) are widely expressed, ligand-gated ion channels that play key roles in brain development and function. Variants have been identified in the GRIN genes encoding NMDAR subunits that are linked to neurodevelopmental disorders, among other manifestations. Zebrafish are a powerful model to study brain development and function given their rapid development and ease of genetic manipulation. As a result of an ancient genome duplication, zebrafish possess two paralogues for most human NMDAR subunits. To evaluate the degree of conservation between human NMDAR subunits and their respective zebrafish paralogues, we carried out detailed in silico analyses, with an emphasis on key functional elements. To further assess the suitability of zebrafish for modeling NMDAR-associated neurodevelopmental disorders, we analyzed the conservation of positions with identified missense variants.ResultsWe find that the human NMDAR subunits are generally well conserved across zebrafish paralogs. Moreover, variants classified as pathogenic and putatively pathogenic are highly conserved, reflecting the importance of key protein regions to neurotypical receptor function. Positions with putatively benign and benign variants are less conserved. Across NMDAR domains, the transmembrane domain is most highly conserved, followed by the ligand-binding domain, which maintains conservation of amino acids that participate in the binding of ligands. The N-terminal domain is less well conserved but aligned models show high degrees of structural similarity. The C-terminal domain is the most poorly conserved region across zebrafish paralogs, but certain key regions that undergo phosphorylation, palmitoylation, and ubiquitylation as well as protein-binding motifs are better conserved.ConclusionsOur findings highlight a strong conservation of human NMDAR subunits in zebrafish, with some exceptions. The ligand-binding domain, the transmembrane domain forming the ion channel, and the short polypeptide linkers that connect them are highly conserved. The N- and C-terminal domains are less conserved but functional motifs in general are more highly conserved relative to the entire domain. Overall, our findings support the utility of zebrafish as a model for studying neurodevelopment and disease mechanisms and provide a template for rigorously considering the relationship between human and zebrafish paralogues.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-025-12274-6.

Replication timing (RT) is the temporal program of genome duplication that is coordinated with 3D genome organization and gene expression1,2. How and when RT is established during mammalian development are questions that remain unsolved. Four recent studies provided the first insights; however, they reached to contradictory conclusions. Nakatani et al. reported a poorly defined RT in mouse zygotes that is progressively consolidated by the 4-cell stage3; Takahashi et al. and Halliwell, et al. reported a complete RT absence in the zygote with abrupt emergence at later stages4,5; finally, Xu et al.6reported a RT already established in the zygote. Discrepancies might reflect differences in computational methods and thresholds applied to sparse single-cell RT data. More recently, Shetty et al. concluded that RT is established in the zygote, and that transcriptional activation and chromatin accessibility emerge at late replicating regions7. Their conclusions challenge the long-standing relationship between gene expression and early replication1,2,8–10and contradict previous studies3–6. This critique demonstrates that methodological flaws and artifacts, stemming from inadequate sample acquisition and data processing led to unreliable RT inference, rendering the conclusions on RT establishment and its inverse correlation with gene expression erroneous. Moreover, re-analysis of their data, with stage-matched controls from previous studies3,5, demonstrates that the canonical relationship between RT, transcriptional activity, and chromatin accessibility are indeed still standing during the early mammalian embryogenesis.

14,15-Dihydroclerodin, or dihydroclerodin-I (DCLR), is a natural product that can also be synthesized through catalytic hydrogenation of clerodin. In this study, dihydroclerodin, purified from Clerodendrum viscosum leaves, was evaluated for its cell cycle arrest and cytotoxic effects in Allium cepa root tip cells and compared with colchicine-induced antimitotic activity. Treatments with dihydroclerodin at concentrations of 100, 200, and 300 µg mL−1, and colchicine at 150 µg mL−1 (as a positive control), were administered for 2, 4, and 4 + 16 h (4-hour treatment followed by 16 h of recovery). The highest frequency of metaphase cells was observed with dihydroclerodin (300 µg mL−1) and colchicine (150 µg mL−1) at 4 h, with values of 75.09 ± 0.93% and 78.31 ± 0.49%, respectively. Both treatments significantly increased the percentages of aberrant cells, chromosomal aberrations (CA), micronuclei (MN), C-metaphases (C-Met), and polyploid (PP) cells. These findings suggest that dihydroclerodin exhibits strong antimitotic effects, warranting further investigation of its potential in cancer chemotherapy due to its ability to induce cell cycle delay and cytotoxicity.

Polyploidization, followed by genome downsizing, is a recurrent evolutionary cycle that dramatically reshapes genome structure. Newly formed polyploids must quickly adjust their cell division machinery to maintain stable chromosome inheritance, while long-term stabilization involves rediploidization, returning the genome to a diploid-like state. Here, we investigate the origin and early genome evolution of Arabidopsis suecica, a hybrid polyploid derived from A. thaliana and A. arenosa. Leveraging a recent genome assembly for A. suecica along with population-level whole-genome resequencing of all three species, we identify the closest progenitors to A.suecica and use this knowledge to examine genes under positive selection and to assess compensatory dynamics between homeologs carrying loss-of-function mutations. Our findings show that both parental species were diploid, including the paternal A. arenosa progenitor. We identify evidence for de novo adaptation to allopolyploidy within the A.arenosa sub-genome of A. suecica, including genes involved in homolog pairing and recombination. Although relaxed purifying selection is evident, likely due to the genome-wide redundancy, we observe functional compensation between homeologous gene pairs in A.suecica: When one copy loses function, the other maintains it. Together, these findings revise the origin of A. suecica and identify early genome stabilization mechanisms, including evidence for meiotic adaptation and mutational buffering through homeologous gene compensation.

Gene gain and loss drive the diversification of gig immune genes in teleosts: structural and regulatory insights from Atlantic salmon.

Albizia odoratissima is a valuable drought-tolerant native tree species in the dry and hot river valleys of China, which has important ecological and economic values. Exploring its genetic background and phylogenetic direction will be conducive to its further exploitation and use, and promote the process of vegetation restoration in the dry hot river valley region. A genome assembly of approximately 719.88 Mb was achieved at the contig level, featuring a contig N50 of 53.74 Mb. Of this, 98.58% of gene sequences were organized into 13 pseudochromosomes. The A. odoratissima genome contained 96.96% of conserved genes, including 1,538 intact single-copy genes and 42 intact duplicated genes. It had an angiosperm palaeotripling event and the last whole genome duplication event occurred approximately 62.9 million years ago. A. odoratissima shares 8,936 gene families with five other legume species, while 1,420 gene families are unique to A. odoratissima. Under drought stress, photosynthesis was significantly inhibited to reduce water consumption, osmoregulatory substances were significantly increased to alleviate osmotic stress, and flavonoid accumulation was induced to enhance antioxidant capacity through the up-regulation of AoANS (Aod07G019900) gene expression, thereby improving drought tolerance. High-quality reference genomes generated through molecular studies are advancing research into the molecular mechanisms of A. odoratissima.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-025-07523-5.

Functional divergence of BnaWRKY7 homologs drives phytosterol variations in polyploid Brassica napus.

Pepper(Capsicum annuum L.) faces multiple biotic and abiotic stresses throughout the phases of peppers' growth and development, with salinity being a major constraint. Recent research shows that cation proton antiporters (CPAs) are vital for ion balance and enhancing plant resilience during abiotic stress. Although the CPA gene family plays important roles in pepper, its characteristics and biological significance have not been systematically characterized. Analyzing the functions of pepper CPA family genes is of great significance for revealing the molecular mechanism of improving adaptability to adversity. Our investigation revealed 33 distinct CaCPA genes within the pepper genome. Phylogenetic analysis coupled with whole genome duplication studies demonstrated that these CaCPA genes can be classified into three distinct subfamilies (CaCHXs, CaNHXs and CaKEAs). Analysis of the cis-acting elements showed that the highest enrichment was for the adversity stress response element. Available transcriptome expression data showed CaCHXs highly expressed in floral tissues, while CaNHXs and CaKEAs displayed varied expression patterns. RNA-seq and RT-qPCR analyses revealed that CaNHX2 exhibited the most pronounced upregulation under 200 mM NaCl, with expression levels increasing 7-fold after 3h of treatment compared to the 0h control. This suggests its potential importance in salt tolerance. The functional role of CaCHX2 requires further experimental validation. Collectively, these findings shed new light on the CPA gene family, yielding important clues about its evolutionary patterns and functional roles in pepper. This study provides fundamental insights for future efforts aimed at breeding salt-resistant pepper varieties.

The minichromosome maintenance (MCM2-7) protein complexes are central drivers of genome duplication. Distinct protein pools, parental and nascent MCMs, and their precise equilibrium are essential to sustain error-free DNA replication. However, the mechanism responsible for generating these pools and maintaining their equilibrium remains largely unexplored. Here, we identified CRL4DCAF12 as a factor controlling the assembly of nascent MCM complexes. During MCM biogenesis, MCMBP facilitates the assembly and transport of newly synthesized MCM3-7 subcomplexes into the nucleus. Once in the nucleus, the MCM2 subunit must be incorporated into the MCM3-7 subcomplex, while MCMBP needs to be removed. CRL4DCAF12 facilitates the degradation of MCMBP and thereby regulates the assembly of MCM2-7 complexes. The absence of CRL4DCAF12 adversely affects the level of chromatin-bound nascent MCMs, resulting in accelerated replication forks and replication stress. Collectively, our findings uncovered the molecular mechanism underlying nascent MCM production essential to counteract genome instability.

All jawed vertebrates have a highly coordinated innate immune response to viral infections driven by a core set of interferon responsive genes (ISGs), but interspecies variation remains substantial. In this study, we examined the genome-wide regulatory basis for antiviral gene expression responses in Atlantic salmon (Salmo salar), representing a teleost family that underwent a whole genome duplication (WGD) event ~100 Mya. We stimulate fish systemically with polyinosinic:polycytidylic acid (poly I:C), a synthetic viral mimic, and profile transcriptomic and epigenomic responses in the primary hematopoietic and lymphoid tissue. We used ATAC-seq and ChIP-seq (H3K27ac and H3K27me3), combined with mRNA-seq, to comprehensively examine modifications in gene regulation following stimulation. We identified a set of 197 ISGs with regulatory elements showing increased chromatin accessibility and H3K27ac signal in concert with increased gene expression in response to poly I:C. Fifty-four of these genes were conserved ISGs in rainbow trout, zebrafish, and human. Our analysis provides evidence for conserved transcription factors (TFs) driving the interferon response by binding ISG promoters, including IRF8, IRF9, STAT1, and STAT2. Regulatory elements within differentially expressed genes were enriched for predicted binding sites for STAT6, PRDM1, IRF6, JDP2, NR2E1, and BCL6, suggesting a central role for these TFs in the antiviral response. Finally, we demonstrate paralog-specific enrichment of interferon-stimulated response element (ISRE) motifs in poly I:C activated promoters of ISGs retained as duplicates from the salmonid WGD. Overall, this study provides novel insights into the genomic regulatory landscape underlying antiviral immunity in a farmed fish with a complex genome.

Abstract Background and Aims Aging is associated with a natural decline in nephron mass and number, leading to progressive kidney function loss and an increased risk of chronic kidney disease (CKD). With individuals over 65 years old being the fastest-growing demographic in developed nations, understanding the mechanisms driving CKD in this population is of critical clinical relevance. CKD often remains undiagnosed in up to 90% of patients due to the kidney tubule's remarkable compensatory capacity to maintain function despite nephron loss. However, these compensatory mechanisms are poorly understood. Aging in most organs is frequently linked to a loss of stem cell pools or impaired progenitor cell proliferation. In the kidney, we have revealed that a population of renal progenitor cells (RPC), interspersed among tubular cells (TC), are key to regenerating kidney tubules after acute kidney injury. Additionally, TC undergo polyploidization, acquiring extra genome copies, enabling them to sustain kidney function during stress. This is a key mechanism underlying kidney's ability to buffer against TC loss. This study investigates these two adaptive responses during physiological kidney aging. Method To trace RPC (Pax2⁺ cells), we used inducible Pax2/Confetti mice, where RPC are labeled with one of four fluorescent reporter genes. To study polyploid TC, we used inducible Pax8/FUCCI2aR mice, identifying polyploid TC by cell cycle fluorescent proteins and DNA content analysis. Mice aged 2, 5, 12, and 20 months were analyzed, and single-cell RNA sequencing (scRNA-seq) was performed at 2 and 20 months of age. Results Aging was associated with a natural decline in nephron number, progressive kidney function decline, fibrosis, and accumulation of senescent TC, culminating in CKD. In Pax2/Confetti mice, we observed a gradual loss of Pax2⁺ RPC and a decrease in their regenerative capacity starting from 12 months of age. scRNA-seq revealed an age-dependent signature in RPC characterized by DNA damage, TGF-β signaling, senescence markers, and enrichment of stress response pathways, which collectively underlie the impaired regenerative capacity of aged RPC. In Pax8/FUCCI2aR mice, tubular polyploidy progressively increased, accumulating additional chromosome sets as evinced by octaploid TC (8C DNA content), throughout aging. Polyploid TC showed elevated DNA damage and were detected in urine, suggesting ongoing loss. scRNA-seq of polyploid TC showed the increment of hypertrophy- and maladaptive-associated genes in aged mice and expression of fibrotic and senescent genes in polyploid TC expressing DNA damage markers. Additionally, we revealed the polyploid characteristic pathways, which were associated with tubulointerstitial fibrosis, including TGF-β signaling and epithelial-to-mesenchymal transition, TNF-α signaling, DNA damage responses, and mitochondrial dysfunction. These findings highlight the dual role of polyploid TC in acquiring a profibrotic/proinflammatory phenotype to buffer against DNA damage throughout aging, while driving fibrosis and CKD progression. Conclusion Aging-related kidney decline is characterized by: These age-related changes likely compromise the kidney tubule's capacity to compensate for nephron loss, increasing the susceptibility to CKD in the elderly population.

BackgroundMADS-box genes encode transcription factors critical for plant development, particularly floral organogenesis, flowering time regulation, and adaptation to environmental stresses. Among these, the MIKCC-type genes are pivotal regulators in floral developmental processes. Although the evolutionary diversification and functional dynamics of MADS-box genes have been extensively characterized in model plants such as Arabidopsis thaliana and Oryza sativa, their evolutionary relationships and functional profiles in Lavandula angustifolia, an economically significant aromatic plant, remain poorly understood.ResultsGenome-wide analysis identified 173 MADS-box genes in L. angustifolia, categorized into type I (Mα: 26; Mβ: 0; Mγ: 10) and type II (MIKCC: 125; MIKC*: 12) based on phylogenetic comparisons with A. thaliana. The MIKCC subgroup was further subdivided into 12 subclasses, including genes central to the ABCDE model of floral organ specification. Structural analyses revealed distinct conserved motifs and exon-intron configurations specific to each subgroup, indicative of functional divergence. Synteny analysis demonstrated Whole Genome Duplication (WGD) and segmental duplications as major contributors to MIKCC gene family expansion, notably among genes linked to floral organ development. Expression profiling via RNA-seq and quantitative real-time PCR (qPCR) showed type II MADS-box genes exhibited higher expression levels with pronounced tissue-specific and developmental stage-specific expression patterns compared to type I genes. Many type II genes displayed significant associations with floral organogenesis, floral transition, and abiotic stress responses, underscoring their essential roles in reproductive development and environmental adaptability in L. angustifolia.ConclusionsThe identification and comprehensive characterization of 173 MADS-box genes in L. angustifolia highlight the significant expansion of the MIKCC subgroup driven primarily by WGD and segmental duplications. The distinct structural features and specific expression patterns observed provide insights into the functional divergence and complexity of these genes, particularly regarding floral organogenesis and adaptation to environmental stress. This study establishes a robust molecular basis for further functional analysis and genetic improvement of aromatic plants.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-025-12120-9.

Gene and genome duplications expand genetic repertoires and facilitate functional innovation. Segmental or whole-genome duplications generate duplicates with similar and somewhat redundant expression profiles across multiple tissues, while other modes of duplication create genes that show increased divergence, leading to functional innovations. How duplicates diverge in expression across cell types in a single tissue remains elusive. Here, we used high-resolution spatial transcriptomic data from Arabidopsis thaliana, Glycine max, Phalaenopsis aphrodite, Zea mays, and Hordeum vulgare to investigate the evolution of gene expression following gene duplication. We found that genes originating from segmental or whole-genome duplications display increased expression levels, expression breadths, spatial variability, and number of coexpression partners. Duplication mechanisms that preserve cis-regulatory landscapes typically generate paralogs with more preserved expression profiles, but such differences generated by mode of duplication fade or disappear over time. Paralogs originating from large-scale (including whole-genome) duplications display redundant or overlapping expression profiles, indicating functional redundancy or subfunctionalization, while most small-scale duplicates diverge asymmetrically, consistent with neofunctionalization. Expression divergence also depends on gene functions, with dosage-sensitive genes displaying highly preserved expression profiles and genes involved in more specialized processes diverging more rapidly. Our findings offer a spatially resolved view of expression divergence following duplication, elucidating the tempo and mode of gene expression evolution, and helping understand how gene and genome duplications shape cell identities.

Poxviruses have a low overall rate of point mutations but are known to exhibit genomic duplications and deletions that can influence viral evolution. We examine the prevalence of large deletions in clade IIb monkeypox virus (MPXV) genomes during the global 2022 outbreak. We observe thirty-one distinct deletions, ranging from 573 to 21,576 bp among over 2000 MPXV genomes during 2022 − 2023 in the United States (U.S.). Almost all deletions are present in the first 25,000 bp or last 50,000 bp of the MPXV genome, excluding the terminal 500 bp. The large deletions result in extensive predicted gene loss as well as novel predicted gene products. Most unique deletions are observed in one case; however, one 3370 bp deletion mutant predominated in a U.S. state during late 2022 and a different 913 bp deletion may have arisen independently multiple times across several MPXV sub-lineages and multiple countries. The recurrent presence of large deletion mutants provides evidence of a mechanism of poxvirus evolution by genomic deletion and gene loss. While no deletion emerged in a dominant variant during 2022 − 2023, large deletions have the potential to result in viruses in which a therapeutic or diagnostic target is deleted.

As the tree of life becomes increasingly accessible to molecular investigations, describing mechanisms underlying evolutionary convergence and constraint will be crucial to understanding diversification. The lineage including the model yeast Saccharomyces cerevisiae evolved aerobic fermentation in part through an ancient whole genome duplication and retention of glycolytic genes. To evaluate the glycolytic rates across diverse yeasts, we developed and deployed an extracellular acidification rates (ECAR) assay on 299 species that span more than 400 million years of evolution and identified a clade in the genus Saturnispora that convergently evolved aerobic fermentation. Through comparative genomics and transcriptomics, we found that several glycolytic genes had higher expression and novel cis-regulatory elements in aerobically fermenting Saturnispora species. When the transcription factor required for their activation was deleted in Saturnispora dispora, the mutants had reduced glycolytic rates and increased respiration. Intriguingly, many of the upregulated genes are orthologous to duplicated glycolytic genes in S. cerevisiae. These divergent genetic mechanisms suggest that there are strong evolutionary constraints on how some traits like aerobic fermentation can arise convergently.

Divergence and conservation of neuropeptide Y receptors in teleosts with diverse feeding habits.