Physiological and transcriptome analyses of Chinese cabbage in response to drought stress

Chinese cabbage is an important leafy vegetable crop with high water demand and susceptibility to drought stress.To explore the molecular mechanisms underlying the response to drought,we performed a transcriptome analysis of drought-tolerant and-sensitive Chinese cabbage genotypes under drought stress,and uncovered core drought-responsive genes and key signaling pathways.A co-expression network was constructed by a weighted gene coexpression network analysis(WGCNA)and candidate hub genes involved in drought tolerance were identified.Furthermore,abscisic acid(ABA)biosynthesis and signaling pathways and their drought responses in Chinese cabbage leaves were systemically explored.We also found that drought treatment increased the antioxidant enzyme activities and glucosinolate contents significantly.These results substantially enhance our understanding of the molecular mechanisms underlying drought responses in Chinese cabbage.

Spatio-temporal dynamics of phytohormones in the tomato graft healing process

Graft healing involves a series of cytological and molecular events including wound responses, callus formation and vascular bundle remodelling. Hormones are important signalling molecules regulating plant development and responses to environmental stimuli. However,the detailed dynamics of phytohormones in graft healing remain elusive. In this research, internodes above and below the graft site were harvested from 0 to 168 h after grafting(HAG), and liquid chromatography tandem mass spectrometry(LC-MS/MS) was used to determinate jasmonic acid, auxin, cytokinin, ethylene, salicylic acid, abscisic acid and gibberellin levels during the graft healing process. Uniform manifold approximation and projection(UMAP) and k-means analyses were performed to explore hormone spatio-temporal dynamics. We found the stage-specific and asymmetric accumulation of phytohormones in the tomato graft healing process. At the early healing stage(before vascular bundle reconnection), IAA, cZ, ABA, JA and SA mainly accumulated above the graft site, while tZ and ACC mainly accumulated below the graft site. MEIAA, ICAld and IP mainly accumulated at the later stage. Comminated with the healing process, we suggested that JA is mainly involved in wound responses, IAA is beneficial to the formation of callus and vascular cell development, tZ promotes cell division, and IP is linked to vascular bundle remodelling. In addition, expression of JA-related genes SlMYC2 and SlJAZ2, IAA-related gene SlIAA1, tZ-related genes SlHP2 and SlRR8, and IP-related gene SlRR9 correlated with hormone accumulation. The findings provide important information about the hormones and genes involved in the tomato graft healing process.

A mutation in the promoter of the yellow stripe-like transporter gene in cucumber results in a yellow cotyledon phenotype

Leaf color mutants in higher plants are considered to be ideal materials for studying the chlorophyll biosynthesis,photosynthesis mechanism and chloroplast development.Herein,we identified a spontaneous mutant,yc412,in cultivated cucumber that exhibited yellow cotyledons.The yellow-lethal mutant was diagnosed with an abnormal chloroplast ultrastructure,and reduced photosynthetic capacity and pigment content.Through bulked segregant analysis-based whole-genome sequencing and fine genetic mapping,we narrowed the yellow cotyledons (yc) locus to a 96.8 kb interval on chromosome 3.By resequencing and molecular cloning,we showed that Csyc is a potential candidate gene,which encodes a yellow stripe-like (YSL) transporter.The T to C mutation in the promoter region of Csyc caused the yellow cotyledon phenotype in yc412.Compared to YZU027A (WT),the expression of Csyc was significantly downregulated in the cotyledons of yc412.Silencing of Csyc in cucumber via virus-induced gene silencing resulted in chlorotic leaves,mainly by suppressing the chlorophyll content.Furthermore,a comparative transcriptome analysis revealed that chloroplast-related genes and chlorophyll biosynthesis genes were significantly downregulated in yc412 cotyledons.Our results provide new insights into the molecular function of the YSL transporter in plant chloroplast development and chlorophyll synthesis.

Improved genome annotation of Brassica oleracea highlights the importance of alternative splicing

Brassica oleracea has been developed into many important crops,including cabbage,kale,cauliflower,broccoli and so on.The genome and gene annotation of cabbage(cultivar JZS),a representative morphotype of B.oleracea,has been widely used as a common reference in biological research.Although its genome assembly has been updated twice,the current gene annotation still lacks information on untranslated regions(UTRs)and alternative splicing(AS).Here,we constructed a high-quality gene annotation(JZSv3)using a full-length transcriptome acquired by nanopore sequencing,yielding a total of 59452 genes and 75684 transcripts.Additionally,we re-analyzed the previously reported transcriptome data related to the development of different tissues and cold response using JZSv3 as a reference,and found that 3843 out of 11908 differentially expressed genes(DEGs)underwent AS during the development of different tissues and 309 out of 903 cold-related genes underwent AS in response to cold stress.Meanwhile,we also identified many AS genes,including BolLHCB5 and BolHSP70,that displayed distinct expression patterns within variant transcripts of the same gene,highlighting the importance of JZSv3 as a pivotal reference for AS analysis.Overall,JZSv3 provides a valuable resource for exploring gene function,especially for obtaining a deeper understanding of AS regulation mechanisms.

Monitoring Xylem Transport in the Stem of Lilium lancifolium Using Fluorescent Dye 5(6)-Carboxyfluorescein Diacetate

The xylem undergoes physiological changes in response to various environmental conditions during the process of plant growth.To understand these physiological changes,it is extremely important to observe the transport of xylem.In this study,the distribution and structure of vascular bundle in Lilium lancifolium were observed using the method of semithin section.Methods for introducing a fluorescent tracer into the xylem of the stems were evaluated.Then,the transport rule of 5(6)-Carboxyfluorescein diacetate(CFDA)in the xylem of the stem of L.lancifolium was studied by fluorescence dye in live cells tracer technology.The results showed that the vascular bundles of L.lancifolium were scattered in the basic tissue,the peripheral vascular bundles were smaller and densely distributed,and the closer to the center,the larger the volume of vascular bundles and the more sparsely distributed.The vascular bundles of L.lancifolium are limited external tenacity vascular bundles,which are composed of phloem and xylem.The most suitable method for CFDA labeling the xylem of isolated stem segments of L.lancifolium was solution soaking for 24 h.The running speed of CF in the isolated stem was 0.3 cm/h,which was consistent with the running speed of the material in the field.CF could be transported between the xylem and parenchyma cells,indicating that the material transport in the xylem could be through the symplastic pathway.The above results laid a foundation for the study of the xylem transport mechanism and the xylem pathogen disease of lily.

Editing of eIF(iso)4E.c confers resistance against Turnip mosaic virus in Brassica rapa

Turnip mosaic virus(TuMV)constitutes one of the primary diseases affecting Brassica rapa,severely impacting its production and resulting in crop failures in various regions worldwide.Recent research has demonstrated the significance of plant translation initiation factors,specifically the eIF4E and eIF4G family genes,as essential recessive disease resistance genes.In our study,we conducted evolutionary and gene expression studies,leading us to identify e IF(iso)4E.c as a potential TuMV-resistant gene.Leveraging CRISPR/Cas9 technology,we obtained mutant B.rapa plants with edited eIF(iso)4E.c gene.We confirmed eIF(iso)4E.c confers resistance against TuMV through phenotypic observations and virus content evaluations.Furthermore,we employed ribosome profiling assays on eif(iso)4e.c mutant seedlings to unravel the translation landscape in response to TuMV.Interestingly,we observed a moderate correlation between the fold changes in gene expression at the transcriptional and translational levels(R^(2)=0.729).Comparative analysis of ribosome profiling and RNA-seq data revealed that plant-pathogen interaction,and MAPK signaling pathway-plant pathways were involved in eIF(iso)4E.c-mediated TuMV resistance.Further analysis revealed that sequence features,coding sequence length,and normalized minimal free energy,influenced the translation efficiency of genes.Our study highlights that the loss of e IF(iso)4E.c can result in a highly intricate translation mechanism,acting synergistically with transcription to confer resistance against TuMV.

Molecular cloning and functional analysis of the pepper resistance gene Me3 to root-knot nematode

Root-knot nematodes(RKNs)cause severe diseases in peppers annually around the world.In pepper,the Me3 gene provides a heat-stable and broad-spectrum resistance to RKNs.In this study,several simple sequence repeat(SSR)markers and insertion/deletion(In Del)markers were developed to fine map the Me3 gene.Analysis of 2272 individuals(F2progenies)revealed that Me3 was located in a 45-kb DNA region between markers SSR784 and SSR339,in which there were three candidate genes.Among them,as a novel nucleotide binding site and leucine rich repeat(NBS-LRR)family gene,the DNA sequence of Capana09g000163 of pepper line‘HDA149’was 6348 bp in length,with a 2802-bp open reading frame encoding 933 amino acids,including NB-ARC and LRR domains.Tobacco transient expression assays demonstrated that expression of Capana09g000163 triggered a hypersensitive response(HR)in Nicotiana benthamiana leaves.Subcellular localization results showed that the Capana09g000163 protein was localized in the cell nucleus.Ectopic expression of Capana09g000163 in Arabidopsis significantly increased resistance against Meloidogyne incognita compared with the wild-type(WT)Arabidopsis.Furthermore,M.incognita was almost unable to develop in transgenic Arabidopsis expressing Capana09g000163.Taken together,we cloned the Me3 gene and verified that it induced resistance against M.incognita with the methods of map-based cloning and transgenic technology,which may be of great significance to pepper breeding for resistance against RKNs.

Dissection of two QTL clusters underlying yield-related heterosis in the cabbage founder parent 01–20

Cabbage has significant heterosis and most commercial cultivars are hybrids.To explore genetic basis of cabbage heterosis and promote cabbage heterosis utilization,we constructed two populations by crossing 100 DH lines derived from a cabbage hybrid 01–20×96–100 with two female parents.Hybrids exhibited different extents of heterosis,the mean value of economic yield was 2.6 times bigger than parents.We identified 66 and 73 QTLs associated with mid-parent heterosis and transgressive heterosis of twelve yield-related traits,respectively.Some QTLs could be detected under the two-year experiment existed in two populations with different testers,showing relatively high phenotypic contribution rate(15.8%–20.0%).Heterosis QTLs exhibited clustered distribution in several cabbage chromosome regions.Two dominant genetic regions,mk300–316 and mk258–268,originated from the elite parent 01–20,exhibited significant genetic effects for yield-related heterosis,which were first identified.Three elite DH lines(D22,D46,D83)harboring these two dominant regions were selected as having strong heterosis in cabbage production.Candidate gene analysis revealed that some genes participating in biosynthetic processes of carbohydrates and some responses to auxin might affect cabbage yield heterosis.QTL identification and genetic dissection of yield-related traits provide new insights into the genetic effects of cabbage heterosis.

Large-scale population structure and genetic architecture of agronomic traits of garlic

Garlic,an asexually propagated crop,is the second important bulb crop after the onion and is used as a vegetable and medicinal plant.Abundant and diverse garlic resources have been formed over thousands of years of cultivation.However,genome variation,population structure and genetic architecture of garlic agronomic traits were still not well elucidated.Here,1100258 single nucleotide polymorphisms(SNPs)were identified using genotyping-by-sequencing in 606 garlic accessions collected from43 countries.Population structure,principal component and phylogenetic analysis showed that these accessions were divided into five subpopulations.Twenty agronomic traits,including above-ground growth traits,bulb-related and bolt-related traits in two consecutive years were implemented in a genome-wide association study.In total,542 SNPs were associated with these agronomic traits,among which 188 SNPs were repeatedly associated with more than two traits.One SNP(chr6:1896135972)was repeatedly associated with ten traits.These associated SNPs were located within or near 858 genes,56 of which were transcription factors.Interestingly,one non-synonymous SNP(Chr4:166524085)in ribosomal protein S5 was repeatedly associated with above-ground growth and bulb-related traits.Additionally,gene ontology enrichment analysis of candidate genes for genomic selection regions between complete-bolting and non-bolting accessions showed that these genes were significantly enriched in‘vegetative to reproductive phase transition of meristem’,‘shoot system development’,‘reproductive process’,etc.These results provide valuable information for the reliable and efficient selection of candidate genes to achieve garlic genetic improvement and superior varieties.

Mapping and validation of the epistatic D and P genes controlling anthocyanin biosynthesis in the peel of eggplant (Solanum melongena L.) fruit

Fruit color is an important trait inf luencing the commercial value of eggplant fruits.Three dominant genes(D,P and Y)cooperatively control the anthocyanin coloration in eggplant fruits,but none has been mapped.In this study,two white-fruit accessions(19141 and 19147)and their F2 progeny,with 9:7 segregation ratio of anthocyanin pigmented versus non-pigmented fruits,were used for mapping the D and P genes.A high-density genetic map was constructed with 5270 SNPs spanning 1997.98 cM.Three QTLs were identified,including two genes on chromosome 8 and one on chromosome 10.Gene expression analyses suggested that the SmANS on chromosome 8 and SmMYB1 on chromosome 10 were the putative candidate genes for P and D,respectively.We further identified(1)a SNP leading to a premature stop codon within the conserved PLN03176 domain of SmANS in 19141,(2)a G base InDel in the promoter region leading to an additional cis-regulatory element and(3)a 6-bp InDel within the R2-MYB DNA binding domain of SmMYB1,in 19147.Subsequently,these three variations were validated by PARMS technology as related to phenotypes in the F2 population.Moreover,silencing of SmANS or SmMYB1 in the purple red fruits of F1(E3316)led to inhibition of anthocyanin biosynthesis in the peels.Conversely,overexpression of SmANS or SmMYB1 restored anthocyanin biosynthesis in the calli of 19141 and 19147 respectively.Our findings demonstrated the epistatic interactions underlying the white color of eggplant fruits,which can be potentially applied to breeding of eggplant fruit peel color.

Characterization of the chemosensory protein EforCSP3 and its potential involvement in host location by Encarsia formosa

Chemosensory proteins(CSPs) perform several functions in insects.This study performed the gene expression,ligand-binding,and molecular docking assays on the EforCSP3 identified in the parasitoid wasp Encarsia formosa,to determine whether EforCSP3 functions in olfaction,especially in host location and host preference.The results showed that EforCSP3 was highly expressed in the female head,and its relative expression was much higher in adults than in other developmental stages.The fluorescence binding assays suggested that the EforCSP3 exhibited high binding affinities to a wide range of host-related volatiles,among which dibutyl phthalate,1-octene,β-elemene,and tridecane had the strongest binding affinity with EforCSP3,besides α-humulene and β-myrcene,and should be assessed as potential attractants.Protein structure modeling and molecular docking predicted the amino acid residues of EforCSP3possibly involved in volatile binding.α-Humulene and β-myrcene attracted E.formosa in a previous study and exhibited strong binding affinities with EforCSP3 in the current study.In conclusion,EforCSP3 may be involved in semiochemical reception by E.formosa.

Overexpression of galactinol synthase 1 from Solanum commersonii(ScGolS1) confers freezing tolerance in transgenic potato

Potato(Solanum tuberosum L.) is the fourth largest food crop in the world. Low temperatures cause serious damage to potato plants every year, and freezing tolerance has become a hot spot in potato research. Galactinol synthase(GolS) is a key enzyme in the synthesis of raffinose family oligosaccharides(RFOs), and plays an important role in the response of plants to abiotic stress. In this study, the ScGolS1 gene from Solanum commersonii was cloned and introduced into the S. tuberosum cultivars 'Atlantic' and 'Desiree' via Agrobacterium-mediated transformation. Phenotyping assays showed that overexpression of ScGolS1 could significantly improve freezing tolerance in transgenic potato plants.Further physiological and biochemical experiments showed that the transgenic lines had lower relative conductivity, malondialdehyde content,and 3,3-diaminobenzidine staining and a higher plant survival rate compared with wild type(WT) under cold stress. Moreover, the C-repeat binding factors(CBF1, CBF2 and CBF3), the downstream cold-responsive genes COR413 and COR47, and the ethylene-responsive factor(ERF)transcription factor genes ERF3, ERF4 and ERF6, which function in the ethylene signaling pathway, were all induced by freezing treatment and expressed at higher levels in the ScGolS1 overexpression lines compared with WT. Besides, the expression of some genes such as MIPS, STS and RS from the RFO metabolic pathway was up-regulated under cold stress, resulting in changes in the content of some soluble sugars. This indicated that ScGolS1 overexpression altered the sugar composition and enhanced freezing tolerance in transgenic potato by inducing the ethylene and CBF signaling pathways. These results provided theoretical support and genetic resources for freezing tolerance breeding in potato.

CRISPR/Cas9-based functional characterization of Px ABCB1 reveals its roles in the resistance of Plutella xylostella(L.)to Cry1Ac,abamectin and emamectin benzoate

The identification of functional midgut receptors for pesticidal proteins produced by Bacillus thuringiensis(Bt)is critical for deciphering the molecular mechanism of Bt resistance in insects.Reduced expression of the PxABCB1 gene was previously found to be associated with Cry1Ac resistance in the diamondback moth,Plutella xylostella(L.).To directly validate the potential receptor role of PxABCB1 and its contribution to Bt Cry1Ac toxicity in P.xylostella,we used CRISPR/Cas9 to generate a homozygous knockout ABCB1KO strain with a 5-bp deletion in exon 3 of its gene.The ABCB1KO strain exhibited a 63-fold resistance to Cry1Ac toxin compared to the parental DBM1Ac-S strain.Intriguingly,the ABCB1KO strain also exhibited significant increases in susceptibility to abamectin and emamectin benzoate.No changes in susceptibility to various other Bt Cry proteins or synthetic insecticides were observed.The knockout strain exhibited no significant fitness costs.Overall,our study indicates that PxABCB1 can protect the insect against avermectin insecticides on one hand,while on the other hand it facilitates the toxic effect of the Bt Cry1Ac toxin.The results of this study will help to inform integrated pest management approaches against this destructive pest.

Manipulation of plant height in garden asparagus (Asparagus officinalis L.) through CRISPR/Cas9-mediated aspSPL14 allele editing

Dear Editor,Reduction in plant height has been associated with yield increases and yield stability in a number of important crop species,such as wheat and rice[1].In these plants,dwarfing is mainly attributed to the inability to synthesize or respond to certain phytohormones,predominantly gibberellin(GA)[2].Ideal Plant Architecture 1(IPA1),an miR156 target gene,encodes SPL14 and it is able to bind directly to the promoters of multiple GA biosynthetic,signal,and deactivating genes in rice[3].Moreover,IPA1 loss-of-function mutants exhibit dwarf phenotypes[4].

An optimized protocol using Steedman’s wax for high-sensitivity RNA in situ hybridization in shoot apical meristems and flower buds of cucumber

In situ mRNA hybridization(ISH)is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber.The most common ISH protocol uses paraffin wax;however,embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals.Here,we developed an optimized protocol to simplify the process and improve RNA sensitivity.We combined embedding tissue in low melting-point Steedman’s wax with processing tissue sections in solution,as in the whole-mount ISH method in the optimized protocol.Using the optimized protocol,we examined the expression patterns of the CLAVATA3(CLV3)and WUSCHEL(WUS)genes in shoot apical meristems and floral meristems of Cucumis sativus(cucumber)and Arabidopsis thaliana(Arabidopsis).The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin wax.Moreover,the optimized protocol achieved high signal sensitivity.The optimized protocol was successful for both cucumber and Arabidopsis,which indicates it might have general applicability to most plants.

A multi-omics approach identifies bHLH71-like as a positive regulator of yellowing leaf pepper mutants exposed to high-intensity light

Light quality and intensity can have a significant impact on plant health and crop productivity.Chlorophylls and carotenoids are classes of plant pigments that are responsible for harvesting light energy and protecting plants from the damaging effects of intense light.Our understanding of the role played by plant pigments in light sensitivity has been aided by light-sensitive mutants that change colors upon exposure to light of variable intensity.In this study,we conducted transcriptomic,metabolomic,and hormone analyses on a novel yellowing mutant of pepper(yl1)to shed light on the molecular mechanism that regulates the transition from green to yellow leaves in this mutant upon exposure to high-intensity light.Our results revealed greater accumulation of the carotenoid precursor phytoene and the carotenoids phytofluene,antheraxanthin,and zeaxanthin in yl1 compared with wild-type plants under high light intensity.A transcriptomic analysis confirmed that enzymes involved in zeaxanthin and antheraxanthin biosynthesis were upregulated in yl1 upon exposure to high-intensity light.We also identified a single basic helix–loop–helix(bHLH)transcription factor,bHLH71-like,that was differentially expressed and positively correlated with light intensity in yl1.Silencing of bHLH71-like in pepper plants suppressed the yellowing phenotype and led to reduced accumulation of zeaxanthin and antheraxanthin.We propose that the yellow phenotype of yl1 induced by high light intensity could be caused by an increase in yellow carotenoid pigments,concurrent with a decrease in chlorophyll accumulation.Our results also suggest that bHLH71-like functions as a positive regulator of carotenoid biosynthesis in pepper.

Application and Popularization of New Green Prevention and Control Technology for Greenhouse Vegetables in Shouguang City,Shandong Province

In recent years,through financial subsidies,Shouguang City has promoted the application of electrostatic sprayer,dual-purpose fog and mist sprinkler machine,Bacillus cereus,flame disinfection service based on fine rotary tillage and multi-functional plant protection machine and other new green prevention and control products and technologies for the greenhouse vegetable in the city. As a result,the utilization rate of pesticides was increased by more than 5%,and the application rate was reduced by more than 10%.

Pseudomonas Cyclic Lipopeptide Medpeptin:Biosynthesis and Modulation of Plant Immunity

The multifunctional secondary metabolites known as cyclic lipopeptides(CLPs),which are produced by a large variety of bacteria,have become a key category of plant immunity elicitors.Pseudomonas-CLPs(PsCLPs)are extremely diverse in structure and biological activity.However,an understanding of CLP-plant structure–function interactions currently remains elusive.Here,we identify medpeptin,a novel CLP from Pseudomonas mediterranea that consists of 22 amino acids.Medpeptin is synthesized by a non-ribosomal peptide synthase(NRPS)gene cluster and regulated by a quorum-sensing system.Further research indicates that medpeptin does not exhibit antimicrobial activity;instead,it induces plant cell death immunity and confers resistance to bacterial infection.Comparative transcriptome analysis and virus-induced gene silencing(VIGS)reveal a set of immune signaling candidates involved in medpeptin perception.Silencing of a cell-wall leucine-rich repeat extensin protein(NbLRX3)or a receptor-like protein kinase(NbRLK25)—but not BAK1 or SGT1—compromises medpeptin-triggered cell death and resistance to pathogen infection in Nicotiana benthamiana.Our findings point to a noncanonical mechanism of CLP sensing and suggest perspectives for the development of plant disease resistance.

Lack of fitness cost and inheritance of resistance to abamectin based on the establishment of a near-isogenic strain of Tetranychus urticae

Many populations of the two-spotted spider mite, Tetranychus urticae Koch, have developed high levels of resistance to the pesticide abamectin in China and other countries. This study developed a near-isogenic line to understand better the inheritance, cross-resistance, and fitness costs associated with abamectin resistance in the field population of T. urticae in China. We introduced the trait that confers extremely high abamectin resistance in a field-collected population of T. urticae into a susceptible laboratory strain(IPP-SS) to generate an abamectin-resistant near-isogenic line(NIL-Aba).This process was carried out through multiple backcrossing to IPP-SS and via parthenogenesis and abamectin screening. Compared with IPP-SS, the NIL-Aba strain had a 25 147-fold resistance to abamectin and a high level of cross-resistance to bifenthrin(288.17-fold), an intermediate level to emamectin benzoate(42.57-fold), and low levels to bifenazate, chlorfenapyr, cyflumetofen, cyenopyrafen, and cyetpyrafen with resistance ranging from 3.18-to 9.31-fold.But it had no cross-resistance to profenofos. The resistance to abamectin in NIL-Aba was autosomal, incompletely dominant, and polygenic. Based on two sex life table parameters, no fitness cost was found in NIL-Aba. Establishing the NIL-Aba strain provides a reliable basis for an in-depth study of abamectin resistance in T. urticae. New information on toxicological characteristics and fitness cost should facilitate the management of abamectin resistance in field populations of T. urticae.

High-quality assembly and methylome of a Tibetan wild tree peony genome(Paeonia ludlowii)reveal the evolution of giant genome architecture

Tree peony belongs to one of the Saxifragales families,Paeoniaceae.It is one of the most famous ornamental plants,and is also a promising woody oil plant.Although two Paeoniaceae genomes have been released,their assembly qualities are still to be improved.Additionally,more genomes from wild peonies are needed to accelerate genomic-assisted breeding.Here we assemble a high-quality and chromosome-scale 10.3-Gb genome of a wild Tibetan tree peony,Paeonia ludlowii,which features substantial sequence divergence,including around 75%specific sequences and gene-level differentials compared with other peony genomes.Our phylogenetic analyses suggest that Saxifragales and Vitales are sister taxa and,together with rosids,they are the sister taxon to asterids.The P.ludlowii genome is characterized by frequent chromosome reductions,centromere rearrangements,broadly distributed heterochromatin,and recent continuous bursts of transposable element(TE)movement in peony,although it lacks recent whole-genome duplication.These recent TE bursts appeared during the uplift and glacial period of the Qinghai-Tibet Plateau,perhaps contributing to adaptation to rapid climate changes.Further integrated analyses with methylome data revealed that genome expansion in peony might be dynamically affected by complex interactions among TE proliferation,TE removal,and DNA methylation silencing.Such interactions also impact numerous recently duplicated genes,particularly those related to oil biosynthesis and flower traits.This genome resource will not only provide the genomic basis for tree peony breeding but also shed light on the study of the evolution of huge genome structures as well as their protein-coding genes.