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Polyploidy is an important driving force in speciation and evolution; however, the genomic basis for parallel selection of a particular trait between polyploids and ancestral diploids remains unexplored. Here we construct graph-based pan-genomes for diploid (A2) and allotetraploid (AD1) cotton species, enabled by an assembly of 50 genomes of genetically diverse accessions. We delineate a mosaic genome map of tetraploid cultivars that illustrates genomic contributions from semi-wild forms into modern cultivars. Pan-genome comparisons identify syntenic and hyper-divergent regions of continued variation between diploid and tetraploid cottons, and suggest an ongoing process of sequence evolution potentially linked to the contrasting genome size change in two subgenomes. We highlight 43% of genetic regulatory relationships for gene expression in diploid encompassing sequence divergence after polyploidy, and specifically characterize six underexplored convergent genetic loci contributing to parallel selection of fiber quality. This study offers a framework for pan-genomic dissection of genetic regulatory components underlying parallel selection of desirable traits in organisms.

Plant-herbivore interactions dominate food web links in terrestrial settings, thereby shaping ecosystem structure and functioning. Within a given plant, both primary and secondary metabolites mediate the interplay between above- and below-ground herbivores. Here, we investigate how root feeding by larvae of the turnip moth Agrotis segetum (Lepidoptera: Noctuidae) affects development and feeding behavior of an above-ground sap-feeder i.e., the large cotton aphid Acyrthosiphon gossypii (Hemiptera: Aphididae) on cotton. We further draw upon transcriptomics, metabolomics and in-vitro bioassays to elucidate how plant metabolites mediate these interactions. Root feeding reduces A. gossypii adult survival and fecundity by 35.1%. Aphids on A. segetum-infested cotton spend 7% longer probing cells than on un-infested plants. Root and stem herbivory by A. segetum larvae alters gene transcripts and metabolites in leaves, inducing biosynthesis of three flavonoids i.e., dihydromyricetin, phloridzin and dihydroquercetin. In-vitro bioassays show that the latter two compounds markedly decrease A. gossypii adult survival and fecundity. Below-ground herbivory thus elicits biosynthesis and systemic translocation of phloridzin and dihydroquercetin, with antagonistic effects on above-ground sap-feeders. Our study sheds light on the underlying mechanisms of herbivore-triggered plant defenses in cotton and reveals the interplay between herbivore guilds across ecological realms. We discuss the implications of these findings for pest management.

Cotton is an irreplaceable economic crop currently domesticated in the human world for its extremely elongated fiber cells specialized in seed epidermis, which makes it of high research and application value. To date, numerous research on cotton has navigated various aspects, from multi-genome assembly, genome editing, mechanism of fiber development, metabolite biosynthesis, and analysis to genetic breeding. Genomic and 3D genomic studies reveal the origin of cotton species and the spatiotemporal asymmetric chromatin structure in fibers. Mature multiple genome editing systems, such as CRISPR/Cas9, Cas12 (Cpf1) and cytidine base editing (CBE), have been widely used in the study of candidate genes affecting fiber development. Based on this, the cotton fiber cell development network has been preliminarily drawn. Among them, the MYB-bHLH-WDR (MBW) transcription factor complex and IAA and BR signaling pathway regulate the initiation; various plant hormones, including ethylene, mediated regulatory network and membrane protein overlap fine-regulate elongation. Multistage transcription factors targeting CesA 4, 7, and 8 specifically dominate the whole process of secondary cell wall thickening. And fluorescently labeled cytoskeletal proteins can observe real-time dynamic changes in fiber development. Furthermore, research on the synthesis of cotton secondary metabolite gossypol, resistance to diseases and insect pests, plant architecture regulation, and seed oil utilization are all conducive to finding more high-quality breeding-related genes and subsequently facilitating the cultivation of better cotton varieties. This review summarizes the paramount research achievements in cotton molecular biology over the last few decades from the above aspects, thereby enabling us to conduct a status review on the current studies of cotton and provide strong theoretical support for the future direction.

Background: CRISPR/Cas-derived base editor enables precise editing of target sites and has been widely used for basic research and crop genetic improvement. However, the editing efficiency of base editors at different targets varies greatly. Results: Here, we develop a set of highly efficient base editors in cotton plants. GhABE8e, which is fused to conventional nCas9, exhibits 99.9% editing efficiency, compared to GhABE7.10 with 64.9%, and no off-target editing is detected. We further replace nCas9 with dCpf1, which recognizes TTTV PAM sequences, to broaden the range of the target site. To explore the functional divergence of TERMINAL FLOWER 1 (TFL1), we edit the non-coding and coding regions of GhTFL1with 26 targets to generate a comprehensive allelic population including 300 independent lines in cotton. This allows hidden pleiotropic roles for GhTFL1to be revealed and allows us to rapidly achieve directed domestication of cotton and create ideotype germplasm with moderate height, shortened fruiting branches, compact plant, and early-flowering. Further, by exploring the molecular mechanism of the GhTFL1L86Pand GhTFL1K53G+S78Gmutations, we find that the GhTFL1L86Pmutation weakens the binding strength of the GhTFL1 to other proteins but does not lead to a complete loss of GhTFL1 function. Conclusions: This strategy provides an important technical platform and genetic information for the study and creation of ideal plant architecture.

Calcium-dependent protein kinases (CDPKs) act as key signal transduction enzymes in plants, especially in response to diverse stresses, including herbivory. In this study, a comprehensive analysis of the CDPKgene family in upland cotton revealed that GhCPKsare widely expressed in multiple cotton tissues and respond positively to various biotic and abiotic stresses. We developed a strategy for screening insect-resistance genes from a CRISPR–Cas9 mutant library of GhCPKs. The library was created using 246 single-guide RNAs targeting the GhCPKgene family to generate 518 independent T0 plants. The average target-gene coverage was 86.18%, the genome editing rate was 89.49%, and the editing heritability was 82%. An insect bioassay in the field led to identification of 14 GhCPKmutants that are resistant or susceptible to insects. The mutant that showed the clearest insect resistance, cpk33/74(in which the homologous genes GhCPK33and GhCPK74were knocked out), was selected for further study. Oral secretions from Spodoptera liturainduced a rapid influx of Ca2+in cpk33/74leaves, resulting in a significant increase in jasmonic acid content. S-adenosylmethionine synthase is an important protein involved in plant stress response, and protein interaction experiments provided evidence for interactions of GhCPK33 and GhCPK74 with GhSAMS1 and GhSAM2. In addition, virus-induced gene silencing of GhSAMS1and GhSAM2in cotton impaired defense against S. litura. This study demonstrates an effective strategy for constructing a mutant library of a gene family in a polyploid plant species and offers valuable insights into the role of CDPKs in the interaction between plants and herbivorous insects.

Retrons are a class of multigene antiphage defense systems typically consisting of a retron reverse transcriptase, a non-coding RNA, and a cognate effector. Although triggers for several retron systems have been discovered recently, the complete mechanism by which these systems detect invading phages and mediate defense remains unclear. Here, we focus on the retron Ec86 defense system, elucidating its modes of activation and mechanisms of action. We identified a phage-encoded DNA cytosine methyltransferase (Dcm) as a trigger of the Ec86 system and demonstrated that Ec86 is activated upon multicopy single-stranded DNA (msDNA) methylation. We further elucidated the structure of a tripartite retron Ec86-effector filament assembly that is primed for activation by Dcm and capable of hydrolyzing nicotinamide adenine dinucleotide (NAD+). These findings provide insights into the retron Ec86 defense mechanism and underscore an emerging theme of antiphage defense through supramolecular complex assemblies.

Phenotypic diversity and evolutionary innovation ultimately trace to variation in genomic sequence and rewiring of regulatory networks. Here, we constructed a pan-genome of the Gossypiumgenus using ten representative diploid genomes. We document the genomic evolutionary history and the impact of lineage-specific transposon amplification on differential genome composition. The pan-3D genome reveals evolutionary connections between transposon-driven genome size variation and both higher-order chromatin structure reorganization and the rewiring of chromatin interactome. We linked changes in chromatin structures to phenotypic differences in cotton fiber and identified regulatory variations that decode the genetic basis of fiber length, the latter enabled by sequencing 1,005 transcriptomes during fiber development. We showcase how pan-genomic, pan-3D genomic and genetic regulatory data serve as a resource for delineating the evolutionary basis of spinnable cotton fiber. Our work provides insights into the evolution of genome organization and regulation and will inform cotton improvement by enabling regulome-based approaches.

Cotton (Gossypium hirsutum) is an important fiber crop worldwide. Insect attack causes cotton yield and quality losses. However, little is known about the mechanism of cotton response to insect attack. We simulated insect feeding by applying insect oral secretions (OS) to wounds, and combined transcriptome and metabolome analysis to investigate how OS from two major pest species (Helicoverpa armigeraand Spodoptera litura) affect cotton defense responses. We found that respectively 12,668 and 13,379 genes were differentially expressed in comparison with wounding alone. On addition of OS, the jasmonic acid signaling pathway was rapidly and strongly induced, whereas genes involved in salicylic acid biosynthesis were downregulated. On constructing a coexpression gene network, we identified a hub gene encoding a leucine-rich repeat receptor kinase that may play an important role in early signal recognition and transduction. OS from the two insect species altered the abundance of flavonoid-related compounds in different patterns. Gossypol remained in lower concentration after OS application than after wounding alone, suggesting a suppressive effect of OS on cotton defense response. This study illustrated transcriptional and metabolic changes of cotton in responding to OS from two chewing insect species, identified potential key response genes, and revealed evidence for OS inhibition of wounding-induced cotton defense response.

Increasing soil salinization has led to severe reductions in plant yield and quality, and investigating the molecular mechanism of salt stress response is therefore an urgent priority. In this study, we systematically analyzed the response of cotton roots to salt stress using single-cell transcriptomics technology; 56 281 high-quality cells were obtained from 5-day-old lateral root tips of Gossypium arboreumunder natural growth conditions and different salt treatments. Ten cell types with an array of novel marker genes were identified and confirmed by in situRNA hybridization, and pseudotime analysis of some specific cell types revealed their potential differentiation trajectories. Prominent changes in cell numbers under salt stress were observed for outer epidermal and inner endodermal cells, which were significantly enriched in response to stress, amide biosynthetic process, glutathione metabolism, and glycolysis/gluconeogenesis. Analysis of differentially expressed genes identified in multiple comparisons revealed other functional aggregations concentrated on plant-type primary cell wall biogenesis, defense response, phenylpropanoid biosynthesis, and metabolic pathways. Some candidate differentially expressed genes encoding transcription factors or associated with plant hormones also responsive to salt stress were identified, and the function of Ga03G2153, annotated as auxin-responsive GH3.6, was confirmed by virus-induced gene silencing. The GaGH3.6-silenced plants showed a severe stress-susceptible phenotype, and physiological and biochemical measurements indicated that they suffered more significant oxidative damage. These results suggest that GaGH3.6might participate in cotton salt tolerance by regulating redox processes. We thus construct a transcriptional atlas of salt-stressed cotton roots at single-cell resolution, enabling us to explore cellular heterogeneity and differentiation trajectories and providing valuable insights into the molecular mechanisms that underlie plant stress tolerance.

Jute (Corchorus capsularisL.) is the second most important natural plant fiber source after cotton. However, developing an efficient gene editing system for jute remains a challenge. In this study, the transgenic hairy root system mediated by Agrobacterium rhizogenesstrain K599 was developed for Meifeng 4, an elite jute variety widely cultivated in China. The transgenic hairy root system for jute was verified by subcellular localization and bimolecular fluorescence complementation (BiFC) assays. The CHLOROPLASTOS ALTERADOS 1(CcCLA1) gene, which is involved in the development of chloroplasts, was targeted for editing at two sites in Meifeng 4. Based on this hairy root transformation, the gRNA scaffold was placed under the control of cotton ubiquitin-GhU6.7 and −GhU6.9 promoters, respectively, to assess the efficiency of gene editing. Results indicated the 50.0% (GhU6.7) and 38.5 % (GhU6.9) editing events in the target 2 alleles (gRNA2), but no mutation was detected in the target 1 allele (gRNA1) in transgenic-positive hairy roots. CcCLA1gene editing at gRNA2 under the control of GhU6.7 in Meifeng 4 was also carried out by Agrobacterium tumefaciens-mediated transformation. Two CcCLA1mutants were albinic, with a gene editing efficiency of 5.3%. These findings confirm that the CRISPR/Cas9 system, incorporating promoter GhU6.7, can be used as a gene editing tool for jute.

Allotetraploid cotton species (Gossypium hirsutumand Gossypium barbadense) have long been cultivated worldwide for natural renewable textile fibers. The draft genome sequences of both species are available but they are highly fragmented and incomplete1–4. Here we report reference-grade genome assemblies and annotations for G. hirsutumaccession Texas Marker-1 (TM-1) and G. barbadenseaccession 3–79 by integrating single-molecule real-time sequencing, BioNano optical mapping and high-throughput chromosome conformation capture techniques. Compared with previous assembled draft genomes1,3, these genome sequences show considerable improvements in contiguity and completeness for regions with high content of repeats such as centromeres. Comparative genomics analyses identify extensive structural variations that probably occurred after polyploidization, highlighted by large paracentric/pericentric inversions in 14 chromosomes. We constructed an introgression line population to introduce favorable chromosome segments from G. barbadenseto G. hirsutum, allowing us to identify 13 quantitative trait loci associated with superior fiber quality. These resources will accelerate evolutionary and functional genomic studies in cotton and inform future breeding programs for fiber improvement.

Background: Somatic embryogenesis is a major process for plant regeneration. However, cell communication and the gene regulatory network responsible for cell reprogramming during somatic embryogenesis are still largely unclear. Recent advances in single-cell technologies enable us to explore the mechanism of plant regeneration at single-cell resolution. Results: We generate a high-resolution single-cell transcriptomic landscape of hypocotyl tissue from the highly regenerable cotton genotype Jin668 and the recalcitrant TM-1. We identify nine putative cell clusters and 23 cluster-specific marker genes for both cultivars. We find that the primary vascular cell is the major cell type that undergoes cell fate transition in response to external stimulation. Further developmental trajectory and gene regulatory network analysis of these cell clusters reveals that a total of 41 hormone response-related genes, including LAX2, LAX1, and LOX3, exhibit different expression patterns in the primary xylem and cambium region of Jin668 and TM-1. We also identify novel genes, including CSEF, PIS1, AFB2, ATHB2, PLC2, and PLT3, that are involved in regeneration. We demonstrate that LAX2, LAX1and LOX3play important roles in callus proliferation and plant regeneration by CRISPR/Cas9 editing and overexpression assay. Conclusions: This study provides novel insights on the role of the regulatory network in cell fate transition and reprogramming during plant regeneration driven by somatic embryogenesis.

Genes encoding reporter proteins are used as visual marker-assisted tools in genetic transformation as well as plant breeding. In this study, the red fluorescent protein identified in Discosomasp. coral (DsRed2) was successfully used as a visual marker for cotton genetic engineering. DsRed2 was successfully expressed in two cotton cultivars, JIN668 and YZ1, driven by the CaMV-35S promoter via the Agrobacterium-mediated transformation. Our results suggest that DsRed2expression provides an early-stage selection tool for the transgenic calli via visual observation. Red fluorescence can be detected not only in callus and somatic embryos but also in most tissues and organs of mature plants. The transgenic line Yz-2-DsRed2was crossed with four different cotton cultivars to assess the transgene heritability and stability in different genetic backgrounds. The heritability of the red color was highly stable when Yz-2-DsRed2was used as a male parent. The DsRed2gene expressed 100% in the F1hybrids. To investigate the relationship between DsRed2transcription and DNA methylation, a methylation-specific PCR approach was applied to the CaMV-35S promoter region. The results showed a negative association between DNA methylation level in the promoter region and the transgene transcription. Taken together, these findings suggest DsRed2a visual reporter gene for cotton genetic transformation and molecular breeding programs.

Comparative population genomics offers an excellent opportunity for unraveling the genetic history of crop domestication. Upland cotton (Gossypium hirsutum) has long been an important economic crop, but a genome-wide and evolutionary understanding of the effects of human selection is lacking. Here, we describe a variation map for 352 wild and domesticated cotton accessions. We scanned 93 domestication sweeps occupying 74 Mb of the A subgenome and 104 Mb of the D subgenome, and identified 19 candidate loci for fiber-quality-related traits through a genome-wide association study. We provide evidence showing asymmetric subgenome domestication for directional selection of long fibers. Global analyses of DNase I–hypersensitive sites and 3D genome architecture, linking functional variants to gene transcription, demonstrate the effects of domestication on cis-regulatory divergence. This study provides new insights into the evolution of gene organization, regulation and adaptation in a major crop, and should serve as a rich resource for genome-based cotton improvement.

A promoter of the PNZIP(Pharbitis nilleucine zipper) gene (1.459 kb) was cloned from Pharbitis niland fused to the GUS(β-glucuronidase) and Bacillus thuringiensisendotoxin (Cry9C) genes. Several transgenic PNZIP::GUS and PNZIP::Cry9C cotton lines were developed by Agrobacterium-mediated transformation. Strong GUS staining was detected in the green tissues of the transgenic PNZIP::GUS cotton plants. In contrast, GUS staining in the reproductive structures such as petals, anther, and immature seeds of PNZIP::GUS cotton was very faint. Two transgenic PNZIP::Cry9C lines and one transgenic cauliflower mosaic virus (CaMV) 35S::Cry9C line were selected for enzyme-linked immunosorbent assay (ELISA) and insect bioassays. Expression of the Cry9C protein in the 35S::Cry9C line maintained a high level in most tissues ranging from 24.6 to 45.5 μg g-1fresh weight. In green tissues such as the leaves, boll rinds, and bracts of the PNZIP::Cry9C line, the Cry9C protein accumulated up to 50.2, 39.7, and 48.3 μg g-1fresh weight respectively. In contrast, seeds of the PNZIP::Cry9C line (PZ1.3) accumulated only 0.26 μg g-1fresh weight of the Cry9C protein, which was 100 times lower than that recorded for the seeds of the CaMV 35S::Cry9C line. The insect bioassay showed that the transgenic PNZIP::Cry9C cotton plant exhibited strong resistance to both the cotton bollworm and the pink bollworm. The PNZIP promoter could effectively drive Bt toxin expression in green tissues of cotton and lower accumulated levels of the Bt protein in seeds. These features should allay public concerns about the safety of transgenic foods. We propose the future utility of PNZIP as an economical, environmentally friendly promoter in cotton biotechnology.

A successful transformation program relies on the number of survival plants in soil that can be obtained. Low recovery of transgenic plants is still a key restrictive factor for transgenic cotton production. In order to utilize genetic transformation in cotton breeding program effectively, an efficient grafting system for recovering plants derived from somatic embryogenesis following Agrobacteriuminfection and kanamycin selection was developed. Various aspects of in vitrografting were examined in efforts to improve the efficiency of transformant recovery. Using strong seedling rootstocks was the first important step to obtain high rate of successful grafts. Scion size >0.6 cm and seedling rootstock at age of 6–12 days were appropriate for grafting. The successful grafting ratio was higher when using hypocotyls without radicle. Shoot-tip and shoot stem with axillary bud were also suitable for in vitrografting, which meant we could significantly improve the survival ratio of transgenic plantlets, because one plantlet has a shoot-tip but several axillary buds. Based on our data, the period from in vitroseedling rootstock germination to transplant of grafts to field usually takes one month. Over 90% successful grafting ratio could be obtained under optimal conditions, which represented a significant improvement over currently available methods for recovery of cotton plantlet from somatic embryogenesis after transformation. Ex vitrografting could also be used for plant recovery, which gave an average of successful grafting ratio of 71.9%. However, this method was strongly affected by environmental factors.A successful transformation program relies on the number of survival plants in soil that can be obtained. Low recovery of transgenic plants is still a key restrictive factor for transgenic cotton production. In order to utilize genetic transformation in cotton breeding program effectively, an efficient grafting system for recovering plants derived from somatic embryogenesis following Agrobacteriuminfection and kanamycin selection was developed. Various aspects of in vitrografting were examined in efforts to improve the efficiency of transformant recovery. Using strong seedling rootstocks was the first important step to obtain high rate of successful grafts. Scion size >0.6 cm and seedling rootstock at age of 6–12 days were appropriate for grafting. The successful grafting ratio was higher when using hypocotyls without radicle. Shoot-tip and shoot stem with axillary bud were also suitable for in vitrografting, which meant we could significantly improve the survival ratio of transgenic plantlets, because one plantlet has a shoot-tip but several axillary buds. Based on our data, the period from in vitroseedling rootstock germination to transplant of grafts to field usually takes one month. Over 90% successful grafting ratio could be obtained under optimal conditions, which represented a significant improvement over currently available methods for recovery of cotton plantlet from somatic embryogenesis after transformation. Ex vitrografting could also be used for plant recovery, which gave an average of successful grafting ratio of 71.9%. However, this method was strongly affected by environmental factors.

Abstract A reliable and high-efficiency system of transforming embryogenic callus (EC) mediated by Agrobacterium tumefaciens was developed in cotton. Various aspects of transformation were examined in efforts to improve the efficiency of producing transformants. LBA4404 and C58C3, harboring the p?gusBin19 plasmid containing neomycin phosphortransferase II ( npt -II) gene as a selection marker, were used for transformation. The effects of Agrobacterium strains, acetosyringone (AS), co-cultivation temperature, co-cultivation duration, Agrobacterium concentration and physiological status of EC on transformation efficiency were evaluated. Strain LBA4404 proved significantly better than C58C3. Agrobacterium at a concentration of 0.5 × 10 8 cells ml -1 (OD 600 =0.5) improved the efficiency of transformation. Relatively low co-cultivation temperature (19 °C) and short co-cultivation duration (48 h) were optimal for developing a highly efficient method of transforming EC. Concentration of AS at 50 mg l -1 during co-cultivation significantly increased transformation efficiency. EC growing 15 days after subculture was the best physiological status for transformation. An overall scheme for producing transgenic cotton is presented, through which an average transformation rate of 15% was obtained.

Summary: A new protocol has been developed for the highly efficient somatic embryogenesis and plant regeneration of 10 recalcitrant Chinese cotton cultivars. Calluses and embryogenic calluses were induced on MSB1 medium containing the optimal combination of indolebutyric acid (IBA; 2.46 μM) and kinetin (KT; 2.32 μM). Up to 86.7% of embryogenic calluses differentiated into globular somatic embryos 2 mo. after culture on MSB2 medium containing double KNO3 and free of growth regultors. Up to 38.3% of the somatic embryos were converted into complete plants in 8 wk on MSB3 medium with l-asparagine (Asn)/l-glutamine (Gln) (7.6/13.6 mM). The plants were successfully transferred to soil and grew to maturity. With the protocol described here, we have obtained hundreds of regenerating plantlets from 10 recalcitrant cultivars, which is important for the application of tissue culture to cotton breeding and biotechnology.

A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotyl-derived cultures and suspension cultures of Gossypium klotzschianumAnderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9 μM 2,4-D and 2.32 μM kinetin. MSB medium containing 0.045 μM 2,4-D, 0.93 μM kinetin, 2.46 μM IBA promoted embryogenic culture proliferation and embryo development. Suspension cultures with 0.23 μM 2,4-D and 0.93 μM kinetin also produced many embryos. Somatic embryos cultured on MSB medium with PGRs produced secondary embryos, and embryos developed into normal plantlets on PGR-free MSB medium. Regenerated plantlets were transferred onto the quarter-strength MSB medium with 0.5% active charcoal to avoid recallusing. Hypocotyls were better than cotyledons for culture induction and plant regeneration. 2,4-D and kinetin were essential for culture induction and maintenance.

Background: Millennia of directional human selection has reshaped the genomic architecture of cultivated cotton relative to wild counterparts, but we have limited understanding of the selective retention and fractionation of genomic components. Results: We construct a comprehensive genomic variome based on 1961 cottons and identify 456 Mb and 357 Mb of sequence with domestication and improvement selection signals and 162 loci, 84 of which are novel, including 47 loci associated with 16 agronomic traits. Using pan-genome analyses, we identify 32,569 and 8851 non-reference genes lost from Gossypium hirsutumand Gossypium barbadensereference genomes respectively, of which 38.2% (39,278) and 14.2% (11,359) of genes exhibit presence/absence variation (PAV). We document the landscape of PAV selection accompanied by asymmetric gene gain and loss and identify 124 PAVs linked to favorable fiber quality and yield loci. Conclusions: This variation repertoire points to genomic divergence during cotton domestication and improvement, which informs the characterization of favorable gene alleles for improved breeding practice using a pan-genome-based approach.

Background: Wintersweet (Chimonanthus praecox), an important ornamental plant, has evolved unique fragrant aroma and winter-flowering properties, which are critical for its successful sexual reproduction. However, the molecular mechanisms underlying these traits are largely unknown in this species. In addition, wintersweet is also a typical representative species of the magnoliids, where the phylogenetic position of which relative to eudicots and monocots has not been conclusively resolved. Results: Here, we present a chromosome-level wintersweet genome assembly with a total size of 695.36 Mb and a draft genome assembly of Calycanthus chinensis. Phylogenetic analyses of 17 representative angiosperm genomes suggest that Magnoliids and eudicots are sister to monocots. Whole-genome duplication signatures reveal two major duplication events in the evolutionary history of the wintersweet genome, with an ancient one shared by Laurales, and a more recent one shared by the Calycantaceae. Whole-genome duplication and tandem duplication events have significant impacts on copy numbers of genes related to terpene and benzenoid/phenylpropanoid (the main floral scent volatiles) biosynthesis, which may contribute to the characteristic aroma formation. An integrative analysis combining cytology with genomic and transcriptomic data reveals biological characteristics of wintersweet, such as floral transition in spring, floral organ specification, low temperature-mediated floral bud break, early blooming in winter, and strong cold tolerance. Conclusions: These findings provide insights into the evolutionary history of wintersweet and the relationships among the Magnoliids, monocots, and eudicots; the molecular basis underlying floral scent biosynthesis; and winter flowering, and highlight the utility of multi-omics data in deciphering important ornamental traits in wintersweet.