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Heat-responsive ONSEN long terminal repeats integrate heat shock factor motifs, DNA methylation and natural sequence variation in Arabidopsis.

ONSEN is a heat-activated Ty1/copia retrotransposon in Arabidopsis thaliana controlled by heat shock factors (HSFs) and epigenetic silencing. Heat shock element (HSE)-like sequences in ONSEN long terminal repeats (LTRs) contribute to heat responsiveness, but relationships among sequence architecture, basal DNA methylation and natural variation remain unclear. We combined transcription-factor motif prediction, transposable-element comparisons, methylome and RNA sequencing (RNA-seq) data, and Arabidopsis genome assemblies. In silico disruption of five HSE cores eliminated HSF-family motif compatibility in the selected design and all 5119 exact-guanine-cytosine (GC) alternatives. Across 16 curated Columbia-0 terminal windows, ONSEN contained 33-49 non-redundant HSF motif-coordinate placements per 800 bp window and was strongly enriched relative to 1930 non-ONSEN transposable elements across score thresholds and continuous metrics. Direct comparison with 779 non-ONSEN LTR retrotransposons showed selectively elevated basal CHH methylation (where H = A, C or T) at ONSEN termini. Genome-wide RNA-seq analysis revealed broad heat-responsive gene and transposable-element changes, including strong ONSEN induction, whereas candidate-window analysis distinguished ONSEN from most HSF-rich non-ONSEN outliers. ONSEN-like variants across eight accessions generally retained HSF-compatible motifs while altering predicted DNA binding with one finger-family motif composition. Together, these findings define ONSEN terminal regions as HSF-rich regulatory sequences that retain heat-responsive potential within a methylated chromatin context and identify candidates for functional analysis.

DNA Methylation

Endogenous Retroelement Activation is Implicated in Interferon-α Production and Anti-Cyclic Citrullinated Peptide Autoantibody Generation in Early Rheumatoid Arthritis.

OBJECTIVE: Endogenous retroelements (EREs) stimulate type 1 interferon (IFN-I) production but have not been explored as potential interferonogenic triggers in rheumatoid arthritis (RA). We investigated ERE expression in early RA (eRA), a period in which IFN-I levels are increased. METHODS: ERE expression (long terminal repeat [LTR] 5, long interspersed nuclear element 1 [LINE-1], and short interspersed nuclear element [SINE]) in disease-modifying treatment-na&#xef;ve eRA whole-blood and bulk synovial tissue samples was examined by reverse transcription-polymerase chain reaction and NanoString alongside IFN-&#x3b1; activity. Circulating lymphocyte subsets, including B cell subsets, from patients with eRA and early psoriatic arthritis (ePsA) were flow cytometrically sorted and similarly examined. Existing established RA and osteoarthritis (OA) synovial single-cell sequencing data were reinterrogated to identify repeat elements, and associations were explored. RESULTS: There was significant coexpression of all ERE classes and IFNA in eRA synovial tissue samples (n = 22, P < 0.0001) and significant positive associations between whole-blood LINE-1 expression (n = 56) and circulating IFN-&#x3b1; protein (P = 0.018) and anti-cyclic citrullinated peptide (anti-CCP) titers (P < 0.0001). ERE expression was highest in circulating eRA B cells, particularly na&#xef;ve B cells compared with ePsA, with possible ERE regulation by SAM and HD Domain Containing Deoxynucleoside Triphosphate Triphosphohydrolase 1 transcription (SAMDH1) implicated and associations with IFNA again observed. Finally, in established RA synovium, LTRs, particularly human endogenous retroviral sequence K (HERVK), were most increased in RA compared with OA, in which, for all synovial subsets (monocytes, B cells, T cells, and fibroblasts), ERE expression associated with increased IFN-I signaling (P < 0.001). CONCLUSION: Peripheral blood and synovial ERE expression is examined for the first time in eRA, highlighting both a potential causal relationship between ERE and IFN-I production and an intriguing association with anti-CCP autoantibodies. This suggests EREs may contribute to RA pathophysiology with implications for future novel therapeutic strategies.

Humans

Recent Non-LTR Retrotransposon Activity Predicts Cancer Prevalence in Mammals.

Non-long terminal repeat retrotransposons (nLTRs), including long and short interspersed nuclear elements (L1 and SINEs), are the most abundant and active mobile elements in mammals. NLTRs play critical mutagenic and regulatory roles during oncogenesis in humans and model species. However, it is not known whether recent nLTR activity in the genome is related to the lifetime cancer risk of a species beyond humans and conventional model organisms. We examined whether recent nLTR activity predicts cancer prevalence across mammals using comparative analyses of de novo whole-genome repeat annotations from 55 species, each with over 20 published zoo pathology records. We quantified nLTR activity as the number of potentially active elements, their proximity to protein-coding genes and cancer gene orthologs (CGOs), and insertions within these genes. Across all three metrics, neoplasia prevalence was associated with both L1 and combined L1-SINE activity, while malignancy was linked exclusively to the L1-SINE predictors. This pattern suggests a complementary and escalating trajectory, where L1s contribute to early tumorigenic events, while SINE activity, driven by L1s, amplifies their impact and fuels the transition to malignancy. Moreover, genomes harboring more CGOs tended to exhibit higher neoplasia prevalence, and the number of fusion cancer genes was strongly correlated with the number of potentially active L1s across species. Our results further revealed a pattern wherein species with minimal cancer prevalence exhibit restricted activity of at least one major nLTR superfamily, suggesting that preserving genome stability through limited retrotransposition may serve as a protective mechanism against cancer.

Cancer Genes

Chromosome-level genome assembly of Manglietia pachyphylla.

Manglietia pachyphylla, an endangered evergreen tree within the Magnoliaceae family, is renowned for its exceptional ornamental value in landscape horticulture. Despite its classification as a Category II nationally protected plant species in China, the genetic basis of its adaptive traits and conservation priorities remains poorly understood. To address this, we present the first chromosome-scale genome assembly of M. pachyphylla utilizing an integrated approach combining PacBio HiFi long-read and Hi-C chromosome conformation capture sequencing technologies. The assembled genome spans 2.15&#x2009;Gb (contig N50&#x2009;=&#x2009;43.57&#x2009;Mb), exhibiting a heterozygosity rate of 0.78% and repeat content of 78.64%, predominantly comprising long terminal repeat (LTR) retrotransposons (52.86%). Hi-C scaffolding anchored 99.57% of the assembly to 19 pseudochromosomes, achieving a BUSCO completeness score of 96.4%. Annotation revealed 42,505 putative protein-coding genes, with 84.46% of predicted genes were functionally annotated. Phylogenomic analysis positioned M. pachyphylla and Oyama sieboldii clustered together in a well-supported group. This high-contiguity genome assembly enables future investigations into adaptive evolution, functional genomics, and evidence-based conservation strategies for this endangered species.

Chromosomes, Plant

Genome analysis of the glycosphingolipid-producing green alga tetraselmis sp. NKG400013.

Microalgae are gaining attention as sustainable resources for the production of valuable compounds, including biofuels, pigments, and bioactive metabolites. To support metabolic engineering and genome editing approaches aimed at enhancing these traits, high-quality genome assemblies are essential; however, genomic information remains limited for many microalgal lineages. Tetraselmis sp. NKG400013 is a green alga known for high glycosphingolipid accumulation with distinctive structural features. Here, we report a draft genome assembly of this strain generated using PacBio HiFi sequencing and transcriptome-supported annotation. The assembled genome spans 423.7&#x2005;Mbp, with 74.5% repetitive sequences and 15,322 predicted protein-coding genes. Comparative analyses across 11 green algal species revealed a positive correlation between genome sizes and repeat contents, indicating that transposable element expansion, particularly long terminal repeat retrotransposons, has substantially contributed to genome enlargement in Tetraselmis. Genome-wide functional annotation and ortholog inference identified core enzymes required for glycosylceramide biosynthesis. Both sphingolipid &#x394;4 and &#x394;8 desaturases were identified in Tetraselmis and their coexistence suggests an expanded capacity for long-chain base modification that may underlie its distinctive glycosphingolipid profile. These results establish a genomic framework for understanding the high glycosphingolipid-producing capacity of NKG400013 and provide insights into the evolutionary diversification of sphingolipid metabolism in green algae.

Chlorophyta

Hide and seek: de novo identification in sugar beet reveals impact of non-autonomous LTR retrotransposons.

Plant genomes are filled with retrotransposons and their derivatives, constantly undergoing sequence diversification and structural rearrangement. Among them, short, non-autonomous retrotransposons lack full coding capacity and often form subfamilies. As a result, non-autonomous retrotransposons are incompletely identified in most to all genome assemblies.Here, we capitalize on our comprehensive understanding of the transposable element (TE) landscape in sugar beet (Beta vulgaris) to assess the extent of the blind spot for non-autonomous long terminal repeat (LTR) retrotransposons. This use case serves to answer if all of these sequences are derivatives of easier-to-identify full-length elements or if there is more variability that is currently overlooked.For this we applied a semi-automated structural discovery workflow followed by in-depth manual verification to characterize non-autonomous LTR retrotransposons in sugar beet. We retrieve more than 100 non-autonomous LTR retrotransposon families that lack complete autonomous coding capacity, including canonical terminal-repeat retrotransposons in miniature (TRIMs), elongated non-coding derivatives and families retaining fragmented coding remnants. The identified families span a broad range, including elements exceeding 15,000&#xa0;bp in length and display evidence for reshuffling and modular evolution. Only a subset of families could be confidently linked to autonomous retrotransposons, showing sequence diversification within the non-autonomous LTR retrotransposon fraction beyond the autonomous genomic templates.We highlight that a large fraction of non-autonomous LTR retrotransposons is incompletely recovered with the current TE identification workflows, even if the output is well-curated and condensed into TE libraries and suggest procedures to remedy this gap. This study gives a genome-wide view into the non-autonomous LTR retrotransposon landscape of a single plant genome and highlights the importance of structure-based approaches for their identification and classification.

LTR retrotransposons

A chromosome-level genome assembly of Coffea arabica L. var. 'Kona Typica'.

Coffea arabica L. var. 'Kona Typica' is renowned for its premium cup quality, but its vulnerability to pests and diseases limits production. To accelerate cultivar improvement, we generated a chromosome-level genome assembly of 'Kona Typica' using PacBio HiFi sequencing and Hi-C scaffolding technology. The final assembly spans 1.13&#x2009;Gb, with a scaffold N50 of 50.50&#x2009;Mb, organized into 22 chromosomes. BUSCO assessment indicated a high completeness at 99.1%. We annotated 65,458 protein-coding genes and identified 1,073,545 interspersed repeats, accounting for 65.16% of the genome. Analysis of transposon insertion ages revealed that most long terminal repeat retrotransposons proliferated after the polyploidization event. This high-quality genome assembly of 'Kona Typica' provides a valuable resource for exploring coffee genomic evolution and genetic mechanisms of complex traits, facilitating genomics studies and the development of improved coffee cultivars with enhanced disease resistance and quality traits.

Coffea

A novel allele of Sh1 facilitates the development of waxy-sweet corn from waxy corn.

Waxy corn and sweet corn represent 2 major classes of fresh-eating corn, each with distinct sensory attributes and nutritional compositions. Developing a new variety that combines both waxy and sweet traits would address rising consumer demand and expand new market potential. From a fast neutron-mutagenized population of the waxy corn inbred line HB522, we isolated a novel mutant, designated as wx-sweet, whose kernels simultaneously exhibit waxy and sweet characteristics at the milk-filling stage. Through bulked segregant analysis combined with fine mapping, we mapped the causal locus to SHRUNKEN1 (Sh1) on chromosome 9, which was confirmed by an allelism test with a characterized Mu-insertion allele of Sh1. A 7,227-bp Copia-type long terminal repeat retrotransposon insertion was identified in exon 2 of Sh1 in the wx-sweet mutant by long-read sequencing. Consistently, the novel sh1 allele significantly reduced sucrose synthase activity. Genetic and physiological analyses demonstrate that sh1 and wx1 act synergistically to fine-tune carbohydrate metabolism in the endosperm. Integrated transcriptomic and metabolomic profiling uncover extensive transcriptional reprogramming and redirected metabolic flux, leading to substantial accumulation of sucrose and a range of oligosaccharides. These metabolic shifts underlie the unique simultaneous dual waxy-sweet texture in fresh-eating wx-sweet kernels. In summary, our work not only provides valuable genetic resources for breeding next-generation fresh-eating corn but also, for the first time, elucidates the molecular mechanism by which the sh1 and wx1 mutations cooperatively shape the waxy-sweet endosperm phenotype.

Zea mays

Hijacking pre-tRNA enables LTR-retrotransposon-initiated constitutive heterochromatin formation.

Pericentric heterochromatin serves as a fundamental component of eukaryotic chromosomes, endowing specialized genomic architecture with broad functional consequences. Although it is universally marked by H3K9me3 modification, the underlying pericentric DNA sequences diverge substantially across species. Here, by leveraging a transposition reporter system combined with a genome-wide RNA interference (RNAi) screen, we identified a specialized mechanism for recruiting SUV39H methyltransferase to initiate pericentric heterochromatin formation. This pathway depends on a highly ordered complex comprising the Puf68, pre-transfer RNAs (tRNAs), and the primer binding site (PBS). Puf68 binds with high affinity to poly-U tracts in pre-tRNA 3' trailer, forming a Puf68/pre-tRNA complex that subsequently base-pairs with the PBS of nascent long terminal repeat (LTR)-retrotransposons. Through direct interaction, Puf68 recruits Su(var)3-9 to these regions, catalyzing H3K9 trimethylation. Notably, Puf68 is sufficient to initiate de novo heterochromatin assembly both at pericentric and ectopically integrated LTR-retrotransposon regions. Our findings not only uncover a previously unrecognized mechanism of heterochromatin initiation but also resolve a long-standing question of how hosts harness nascent LTR-retrotransposon transcripts.

Heterochromatin

Genome assembly of Astatotilapia latifasciata uncovers B chromosome-linked chromatin reorganization.

B chromosomes (Bs) are supernumerary genomic elements found in many eukaryotes, yet their full sequence composition, functional potential, and regulatory impact on the host genome remain unclear. Here, we present a chromosome-level genome assembly of the cichlid fish Astatotilapia latifasciata, integrating PacBio long reads, Illumina short reads, and Hi-C chromatin contact maps to resolve both A and B chromosomes. The 0.93&#x2009;Gb assembly (N50&#x2009;=&#x2009;36.2&#x2009;Mb) includes a 34&#x2009;Mb B chromosome containing 789 predicted protein-coding genes and a markedly higher density of transposable elements (TEs), especially long terminal repeats (LTR) retrotransposons. Transcriptome profiling revealed that B-linked genes are predominantly transcriptionally repressed relative to their A chromosome paralogs. Hi-C-based chromatin modeling uncovered distinct 3D structural configurations associated with the B chromosome, including fewer topologically associating domains (TADs), reduced loop formation, and altered compartmentalization. These changes are linked to long-range chromatin interactions and genomic rearrangements, suggesting that the B chromosome reshapes the nuclear architecture of the host genome. Our study proposes a potential regulatory role of Bs in genome and provides a genomic resource for investigating chromosome evolution in cichlids.

Animals

Diving Deeper Into Mechanisms of Acrylamide-Induced Toxicity: RNA Sequencing Reveals Transcriptomic Alteration and Retrotransposon Expression in Drosophila melanogaster.

Given the inevitability of human and animal exposure to acrylamide, there is increasing concern regarding its potential health risks. While a number of molecular mechanisms have been proposed, the complexity of acrylamide toxicological pathways and interactions remains incompletely characterized. In this study, we employed a transcriptomic approach to investigate the transcriptional responses of Drosophila melanogaster following exposure to acrylamide (100&#x2009;mg/kg). Our analysis identified 634 differentially expressed genes (DEGs), with 362 upregulated and 272 downregulated. Functional analysis revealed these DEGs are enriched in pathways related to reproduction, detoxification, cellular and metabolic processes, signaling, synaptic formation and organization. Notably, acrylamide exposure upregulated the expression of tau and beta-amyloid protein precursor-like genes, both implicated in Alzheimer's disease pathology. An aversive memory test further demonstrated that acrylamide impaired the short-term memory of treated flies. Additionally, acrylamide-induced toxicity altered the expression of nine long terminal repeat retrotransposons, belonging to the gypsy and pao superfamilies. By exploring the potential role of transposable element activity in acrylamide-mediated toxicity, this study provides novel insights into the molecular mechanisms underlying its effects. Collectively, these findings offer a more comprehensive understanding of the mechanisms and pathways associated with the toxic action and detoxification of acrylamide in D. melanogaster.

Animals

Chromosome-level genome assembly of the ornamental plant Alcea rosea.

Alcea rosea, a member of the Malvaceae family, is celebrated for its rich floral palette and global horticultural significance. Here, we present a high-quality reference genome for A. rosea, achieving a genome assembly size of 1.01 Gbp, with a Contig N50 length of 36.61 Mbp. The genome sequence was successfully mapped to 21 chromosomes, and the scaffold N50 length reached 52.57 Mbp, with a scaffold genome completeness of 99.6%. A total of 565.84 Mbp (comprising 56% of the genome) of repetitive sequences were identified, with transposable elements being predominant, particularly long terminal repeat (LTR) elements, which accounted for 48.44% of the genome. 51,436 genes were annotated. Among these predicted genes, the average gene length and coding sequence (CDS) length were 2739.92&#x2009;bp and 1242.54&#x2009;bp, respectively.

Genome, Plant

A chromosome-level genome assembly and annotation of Cercis chuniana (Fabaceae).

The genus Cercis L., at the base of the subfamily Cercidoideae of Fabaceae, is known for its ecological adaptability and significant medicinal, ornamental, and economic value. However, the lack of a high-quality genome hinders the understanding of the evolution of Cercis and Fabaceae. In this study, we present a chromosome-level genome of Cercis chuniana by combining Illumina short reads, PacBio HiFi long reads, and Hi-C data. The final genome size is 355.53&#x2009;Mb, consisting of 12 contigs with a N50 of 42.34&#x2009;Mb. Notably, 344.24&#x2009;Mb, corresponding to 96.82% of the genome, was anchored to seven chromosomes. The assembly comprises 24.83% repetitive sequences, including 19.32% long terminal repeats. Additionally, a total of 33,837 protein-coding genes were predicted in the genome, with 32,709 (96.67%) genes successfully annotated. The high-quality genome assembly of C. chuniana not only bridges the existing gap in genomic data and offers important resources for molecular studies of this species, but also provides essential insights for future studies on speciation, functional and comparative genomics within the Fabaceae family.

Genome, Plant

Simplifying multiplex genome engineering in Saccharomyces cerevisiae with intron-mediated Random Assembly and INtegration (RAIN).

Engineering of multistep enzymatic pathways often involves extensive optimization of heterologous gene expression levels and requires cloning of promoter and open reading frames (ORFs) to generate expression cassettes. We present work on a nascent method for multiplex genome engineering in&#xa0;Saccharomyces cerevisiae that negates the requirement for cloning of expression cassettes. Our system, Random Assembly and INtegration (RAIN), uses intron-mediated homologous recombination (HR) for random&#xa0;in vivo assembly of exogenous promoter and ORF libraries, which are combined and cotransformed in a one-pot method. The libraries include consensus homology arms which target long terminal repeat regions of the Ty1 retrotransposon, providing over a hundred possible integration loci. In this way, our developmental system aims to negate the need for&#xa0;in vitro combinatorial cloning of promoters and ORFs to generate expression cassettes, simplifying in vitro DNA preparation before multiplex genome engineering. This paper presents findings from a series of experiments to demonstrate a proof of concept for the RAIN system. These include: the first reported use of intron-mediated assembly of promoters and ORFs for expression of a functional gene product; up to three markerless genomic integrations; and up to five integrations with antibiotic selection. We also present a number of innovations to improve integration efficiency during multiplex engineering in S. cerevisiae including: SGS1 gene knockout; disruption of heteroduplex rejection; modified Cas9 expression architecture; and overexpression of HR genes RAD52, MRE11, and RAD59. To demonstrate how our system can be used for single transformation phenotype engineering of multiple strains, we also transformed a library of methylotrophy associated genes to generate four new strains that were able to grow on a solid minimal medium with methanol as the sole additional carbon source. Our findings contribute to the ongoing efforts to improve multiplex genome engineering tools in S. cerevisiae, and provide the foundations for further development of a novel toolbox for generating useful genetic diversity for metabolic pathway engineering.

Saccharomyces cerevisiae

Revisiting the genome assembly of Lupinus species reveals differential diploidization after a shared whole-genome duplication.

Accurate genome assemblies are essential for comparative genomics, yet Hi-C-guided scaffolding can introduce structural errors that misrepresent chromosome architecture and bias evolutionary inferences. Here, we identified pervasive scaffolding errors-including artificial fusions, internal inversions, and incomplete contig mounting-in 2 previously published Lupinus genomes (L. cosentinii and L. digitatus) using a segmentation method based on long terminal repeat (LTR) retrotransposon density. We reassembled both genomes, producing chromosome-level references of 472.7 Mb (16 chromosomes) and 427.2 Mb (21 chromosomes), with BUSCO completeness >98.5%. Synteny validation and reapplication of LTR profiling confirmed that all prior errors were resolved. Using these corrected genomes together with 4 additional Lupinus species and 2 outgroup legumes, we investigated postpolyploid evolution. Synonymous substitution rate (Ks) analysis revealed a genus-specific whole-genome duplication (WGD) event (Ks = 0.17) shared by all 6 Lupinus species. The proportion of WGD-derived genes varied markedly, from 60% in L. digitatus to only 36% in L. mutabilis, indicating differential diploidization. While all species retained a core set of WGD duplicates enriched in cytoskeleton organization, ion transport, and defense responses, each exhibited lineage-specific functional trajectories: cell wall modification in L. cosentinii and L. digitatus, nitrogen metabolism in L. albus and L. angustifolius, flower development in L. luteus, and stress/lipid metabolism in L. mutabilis. Our corrected assemblies provide optimal references for Lupinus comparative genomics, and our findings demonstrate that a shared WGD event can lead to both conserved and highly divergent postpolyploid fates, likely underpinning adaptive diversification within the genus.

Lupinus

A Chromosome-Level Genome Assembly of the Potato Leafhopper Empoasca fabae (Hemiptera: Cicadellidae).

The potato leafhopper, Empoasca fabae (Harris, 1841), is a highly polyphagous, migratory insect pest of eastern North America that feeds on more than 200 herbaceous and woody plant species, causing substantial losses to forage and field crops. Despite its agricultural and ecological importance, no genome has been available for this species. Here, we present the first chromosome-level genome assembly of E. fabae, generated from Oxford Nanopore long reads, Illumina short reads, and Omni-C proximity-ligation data. The final assembly spans 908&#x2005;Mb across 132 scaffolds, with 99.8% of the assembly captured in ten chromosome-length scaffolds (nine autosomes and an X chromosome) with a scaffold N50 of 96.2&#x2005;Mb. The assembly is highly complete, recovering 92.9% of conserved hemipteran single-copy orthologs from protein annotations, and is composed of 47.6% repetitive sequence, dominated by long terminal repeat retrotransposons and unclassified elements. Read-depth comparison between male and female individuals supports assignment of a single sex-linked chromosome, consistent with an XO sex determination system. BRAKER3 gene annotation predicted 31,406 protein-coding genes after retaining the longest isoform per locus. Comparative genome analysis of the two closest related Typhlocybinae species with genomes available, Matsumurasca onukii and Hebata decipiens, revealed extensive chromosome-scale collinearity while defining a shared core gene repertoire. This reference genome provides a foundation for comparative and population genomic studies and for investigating genetic traits in this economically important crop pest species.

Animals

Telomere-to-telomere genome of Phoebe chekiangensis reveals that age-dependent CHG hypomethylation promotes floral transition via MADS-box gene activation.

Phoebe species are renowned for their highly valuable 'golden thread' timber; however, their protracted juvenile phase presents a significant obstacle to mechanistic investigations of floral induction. Phoebe chekiangensis, a rare early-flowering representative within this genus, provides a unique model system for dissecting the vegetative-to-reproductive phase transition. Nevertheless, the absence of a high-quality reference genome has severely hindered molecular insights into its developmental regulation. Here, we present the first telomere-to-telomere (T2T) genome assembly for P. chekiangensis, comprising two completely gap-free haplotypes with contig N50 values exceeding 65&#xa0;Mb, base-level quality scores >36, and Long Terminal Repeat Assembly Index scores surpassing the gold standard threshold of 20. Approximately 29&#x2009;000 genes were annotated per haplotype, supported by a BUSCO completeness score of&#x2009;>97%. Age-resolved transcriptomic landscapes identified two MADS-box transcription factors, PcMADS5 (AP1-like) and PcMADS19.1 (SOC1-like), as core activators of the floral transition. Both genes triggered precocious flowering when ectopically expressed in Arabidopsis thaliana. Whole-genome bisulfite sequencing revealed a progressive, age-dependent decline in CHG (where H is A, C, or T) DNA methylation, which was particularly pronounced at the PcMADS19.1 locus. Notably, DML1/2, which mediate active DNA demethylation, were coordinately upregulated during the onset of reproductive growth. Chemical demethylation using 5-azacytidine further diminished CHG methylation and selectively enhanced PcMADS19.1 expression, confirming a causal relationship between CHG hypomethylation and transcriptional activation. This work delivers the first chromosome-scale T2T genome within the genus Phoebe and uncovers CHG demethylation as a previously unrecognized epigenetic switch governing reproductive competence in woody perennials.

Journal Article

The piRNA pathway mediates transcriptional silencing of LTR retrotransposons in ovaries and somatic tissues of&#xa0;Aedes mosquitoes.

The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by suppressing transposable elements in animal germlines. Despite its well-established function in the animal germline, piRNAs and PIWI proteins are expressed in somatic tissues across arthropod species, and their functions outside the gonads remain poorly understood.&#xa0;Aedes albopictus mosquitoes express four PIWI genes, Piwi4, Piwi5, Piwi6, and&#xa0;Ago3, in both gonadal and somatic tissues. Here, we generated Piwi6 knockout (KO) Ae. albopictus cell lines and observed a substantial upregulation of long terminal repeat retrotransposons, including a full-length endogenous retrovirus that we named Aedes albopictus Endogenous Retrovirus-1 (AalERV1). Nascent RNA sequencing and Cleavage Under Targets and Tagmentation (CUT&Tag) analyses revealed that Piwi6 silences&#xa0;AalERV1 transcriptionally by guiding the deposition of the repressive H3K9me3 histone mark. Consistently, Piwi6 localized to both the cytoplasm and nucleus, with sequences in the intrinsically disordered region guiding nuclear translocation. Reintroduction of full-length GFP-Piwi6, but not a mutant GFP-Piwi6 defective in nuclear localization, rescued AalERV1 repression in Piwi6 KO cells. Importantly, Piwi6-mediated control of AalERV1 was recapitulated in vivo as Piwi6 knockdown increased AalERV1 expression in both ovaries and somatic tissues of Ae. albopictus mosquitoes. These results establish Aedes mosquitoes as a model to study nuclear PIWI functions and suggest that somatic piRNA-mediated transposon silencing is evolutionarily conserved across arthropod species.

Animals