Dynamic N6 -methyladenosine RNA modification regulates peanut resistance to bacterial wilt.

N6 -methyladenosine (m6 A) is the most abundant mRNA modification in eukaryotes and is an important regulator of gene expression as well as many other critical biological processes. However, the characteristics and functions of m6 A in peanut (Arachis hypogea L.) resistance to bacterial wilt (BW) remain unknown. Here, we analyzed the dynamic of m6 A during infection of resistant (H108) and susceptible (H107) peanut accessions with Ralstonia solanacearum (R. solanacearum), the causative agent of BW. Throughout the transcriptome, we identified 'URUAY' as a highly conserved motif for m6 A in peanut. The majority of differential m6 A located within the 3' untranslated region (UTR) of the transcript, with fewer in the exons. Integrative analysis of RNA-Seq and m6 A methylomes suggests the correlation between m6 A and gene expression in peanut R. solanacearum infection, and functional analysis reveals that m6 A-associated genes were related to plant-pathogen interaction. Our experimental analysis suggests that AhALKBH15 is an m6 A demethylase in peanut, leading to decreased m6 A levels and upregulation of the resistance gene AhCQ2G6Y. The upregulation of AhCQ2G6Y expression appears to promote BW resistance in the H108 accession.

PSW1, an LRR receptor kinase, regulates pod size in peanut.

Pod size is a key agronomic trait that greatly determines peanut yield, the regulatory genes and molecular mechanisms that controlling peanut pod size are still unclear. Here, we used quantitative trait locus analysis to identify a peanut pod size regulator, POD SIZE/WEIGHT1 (PSW1), and characterized the associated gene and protein. PSW1 encoded leucine-rich repeat receptor-like kinase (LRR-RLK) and positively regulated pod stemness. Mechanistically, this allele harbouring a 12-bp insertion in the promoter and a point mutation in the coding region of PSW1 causing a serine-to-isoleucine (S618I) substitution substantially increased mRNA abundance and the binding affinity of PSW1 for BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). Notably, PSW1HapII (super-large pod allele of PSW1) expression led to up-regulation of a positive regulator of pod stemness PLETHORA 1 (PLT1), thereby resulting in larger pod size. Moreover, overexpression of PSW1HapII increased seed/fruit size in multiple plant species. Our work thus discovers a conserved function of PSW1 that controls pod size and provides a valuable genetic resource for breeding high-yield crops.

DWARF AND ROBUST PLANT regulates plant height via modulating gibberellin biosynthesis in chrysanthemum.

YABBY (YAB) genes are specifically expressed in abaxial cells of lateral organs and determine abaxial cell fate. However, most studies have focused on few model plants, and the molecular mechanisms of YAB genes are not well understood. Here, we identified a YAB transcription factor in chrysanthemum (Chrysanthemum morifolium), Dwarf and Robust Plant (CmDRP), that belongs to a distinct FILAMENTOUS FLOWER (FlL)/YAB3 sub-clade lost in Brassicaceae. CmDRP was expressed in various tissues but did not show any polar distribution in chrysanthemum. Overexpression of CmDRP resulted in a semi-dwarf phenotype with a significantly decreased active GA3 content, while reduced expression generated the opposite phenotype. Furthermore, plant height of transgenic plants was partially rescued through the exogenous application of GA3 and Paclobutrazol, and expression of the GA biosynthesis gene CmGA3ox1 was significantly altered in transgenic plants. Yeast one-hybrid, luciferase, and chromatin immunoprecipitation-qPCR analyses showed that CmDRP could directly bind to the CmGA3ox1 promoter and suppress its expression. Our research reveals a nonpolar expression pattern of a YAB family gene in dicots and demonstrates it regulates plant height through the GA pathway, which will deepen the understanding of the genetic and molecular mechanisms of YAB genes.

Regulation of lignin biosynthesis by an atypical bHLH protein CmHLB in Chrysanthemum.

Stem mechanical strength is one of the most important agronomic traits that affects the resistance of plants against insects and lodging, and plays an essential role in the quality and yield of plants. Several transcription factors regulate mechanical strength in crops. However, mechanisms of stem strength formation and regulation remain largely unexplored, especially in ornamental plants. In this study, we identified an atypical bHLH transcription factor CmHLB (HLH PROTEIN INVOLVED IN LIGNIN BIOSYNTHESIS) in chrysanthemum, belonging to a small bHLH sub-family - the PACLOBUTRAZOL RESISTANCE (PRE) family. Overexpression of CmHLB in chrysanthemum significantly increased mechanical strength of the stem, cell wall thickness, and lignin content, compared with the wild type. In contrast, CmHLB RNA interference lines exhibited the opposite phenotypes. RNA-seq analysis indicated that CmHLB promoted the expression of genes involved in lignin biosynthesis. Furthermore, we demonstrated that CmHLB interacted with Chrysanthemum KNOTTED ARABIDOPSIS THALIANA7 (CmKNAT7) through the KNOX2 domain, which has a conserved function, i.e. it negatively regulates secondary cell wall formation of fibres and lignin biosynthesis. Collectively, our results reveal a novel role for CmHLB in regulating lignin biosynthesis by interacting with CmKNAT7 and affecting stem mechanical strength in Chrysanthemum.

The core regulatory networks and hub genes regulating flower development in Chrysanthemum morifolium.

KEY MESSAGE: The study has facilitated important insights into the regulatory networks involved in flower development in chrysanthemum (Asteraceae), and is informative with respect to the mechanism of flower shape determination. Chrysanthemum morifolium, valued as an ornamental species given the diversity of its inflorescence form, is viewed as a model for understanding flower development in the Asteraceae. Yet, the underlying regulatory networks remain largely unexplored. Here, a transcriptomic survey of the Chrysanthemum morifolium variety 'Jinba' was undertaken to uncover the global gene expression profiles and identify the modules of co-transcribed genes associated with flower development. The weighted gene coexpression network analysis revealed important networks and hub genes including ray floret petals-specific coexpression network, disc floret petals-specific network, B and E class genes involved network and CYC2 genes network. Three ray floret petal-specific hub genes were also strongly transcribed in the ray florets of a selection of six diverse varieties and especially so in those which form ligulate ray floret petals. CmCYC2c was strongly transcribed in the distal and lateral regions of the ray floret petals, and also, along with CmCYC2d, in the tubular ray florets. Furthermore, CmOFP, belonging to the family of ovate proteins, was identified in the CYC2 genes network. CmOFP can interact with CmCYC2d that physically interact with CmCYC2c. This work provides important insights into the regulatory networks involved in flower development in chrysanthemum, and is informative with respect to the mechanistic basis of the regulation of flower shape.

Genome-wide identification and analysis of long noncoding RNAs (lncRNAs) during seed development in peanut (Arachis hypogaea L.).

BACKGROUND: Long noncoding RNAs (lncRNAs) have several known functions involving various biological regulatory processes in plant. However, the possible roles of lncRNAs during peanut seed development have not been fully explored. RESULTS: In this study, two peanut recombinant inbred lines (RIL8) that differ in seed size were used to investigate comprehensive lncRNA profiles derived from the seed development at 15 and 35 days after flowering (DAF). We identified a total of 9388 known and 4037 novel lncRNAs, from which 1437 were differentially expressed lncRNAs. Interestingly, the expression patterns of a number of lncRNAs can be very different between two closely related inbred lines and these lncRNAs were expressed predominantly in only one RIL at 35 DAF. Some differentially expressed lncRNAs were found related to putative cis-acting target genes and predicted to be involved in transcription, transport, cell division, and plant hormone biosynthesis. The expression patterns of several representative lncRNAs and 12 protein-coding genes were validated by qPCR. Same expression pattern was observed between most lncRNAs and their target genes. 11 lncRNAs, XR_001593099.1, MSTRG.18462.1, MSTRG.34915.1, MSTRG.41848.1, MSTRG.22884.1, MSTRG.12404.1, MSTRG.26719.1, MSTRG.35761.1, MSTRG.20033.1, MSTRG.13500.1, and MSTRG.9304.1 and their cis-acting target genes may play key roles in peanut seed development. CONCLUSIONS: These results provided new information on lncRNA-mediated regulatory roles in peanut seed development, contributing to the comprehensive understanding of the molecular mechanisms involved in peanut seed development.

Comparative Analyses of Five Complete Chloroplast Genomes from the Genus Pterocarpus (Fabacaeae).

Pterocarpus is a genus of trees mainly distributed in tropical Asia, Africa, and South America. Some species of Pterocarpus are rosewood tree species, having important economic value for timber, and for some species, medicinal value as well. Up to now, information about this genus with regard to the genomic characteristics of the chloroplasts has been limited. Based on a combination of next-generation sequencing (Illumina Hiseq) and long-read sequencing (PacBio), the whole chloroplast genomes (cp genomes) of five species (rosewoods) in Pterocarpus (Pterocarpus macrocarpus, P. santalinus, P. indicus, P. pedatus, P. marsupium) have been assembled. The cp genomes of five species in Pterocarpus have similar structural characteristics, gene content, and sequence to other flowering plants. The cp genomes have a typical four-part structure, containing 110 unique genes (77 protein coding genes, 4 rRNAs, 29 tRNAs). Through comparative genomic analysis, abundant simple sequence repeat (SSR)loci (333-349) were detected in Pterocarpus, among which A /T single nucleotide repeats accounted for the highest proportion (72.8-76.4%). In the five cp genomes of Pterocarpus, eight hypervariable regions, including trnH-GUG_psbA, trnS-UGA_psbC, accD-psaI, ndhI-exon2_ndhI-exon1, ndhG_ndhi-exon2, rpoC2-exon2, ccsA, and trnfM-CAU, are proposed for use as DNA barcode regions. In the comparison of gene selection pressures (P. santalinus as the reference genome), purifying selection was inferred as the primary mode of selection in maintaining important biological functions. Phylogenetic analysis shows that Pterocarpus is a monophyletic group. The species P. tinctorius is resolved as early diverging in the genus. Pterocarpus was resolved as sister to the genus Tipuana.

Genome-wide identification of circular RNAs in peanut (Arachis hypogaea L.).

BACKGROUND: Circular RNAs (circRNAs), a class of widely expressed endogenous regulatory RNAs, are involved in diverse physiological and developmental processes in eukaryotic cells. However, there have been no related studies on the number of circRNAs and their overall characteristics including circRNA abundance and expression profiles in peanut, which is one of the most important edible oil seed crops in the world. RESULTS: We performed a genome-wide identification of circular RNAs using ribosomal-depleted RNA-sequencing from the seeds of two peanut eighth-generation recombinant inbred lines (RIL8): 'RIL 8106' (a medium-pod variety) and 'RIL 8107' (a super-pod variety), at 15 and 35 days after flowering (DAF), respectively. A total of 347 circRNA candidates were detected by two computational pipelines: CIRCexplorer and CIRI, with at least two supporting junction reads. All these circRNAs were generated from exons of annotated genes, and widespread on the 20 peanut chromosomes. The expression profiles revealed that circRNAs were differentially expressed between two stages and between two lines. GO enrichment analysis of the host genes produced differentially-expressed circRNAs suggested that circRNAs are involved in seed development and regulation of seed size. Fifteen circRNAs were experimentally analyzed by qRT-PCR with divergent primers, and six circRNAs were resistant to digestion with RNase R exonuclease, and the back-splicing sites were further validated by Sanger DNA sequencing. CONCLUSIONS: We present the first systematical investigation of the genomic characteristics and expression profiles of circRNAs in peanut. The results revealed that circRNAs are abundant and widespread in peanut, and the differentially-expressed circRNAs between two lines suggested that they might play regulatory roles in peanut seeds development.

Comprehensive characterization of a floral mutant reveals the mechanism of hooked petal morphogenesis in Chrysanthemum morifolium.

The diversity of form of the chrysanthemum flower makes this species an ideal model for studying petal morphogenesis, but as yet, the molecular mechanisms underlying petal shape development remain largely unexplored. Here, a floral mutant, which arose as a bud sport in a plant of the variety 'Anastasia Dark Green', and formed straight, rather than hooked petals, was subjected to both comparative morphological analysis and transcriptome profiling. The hooked petals only became discernible during a late stage of flower development. At the late stage of 'Anastasia Dark Green', genes related to chloroplast, hormone metabolism, cell wall and microtubules were active, as were cell division-promoting factors. Auxin concentration was significantly reduced, and a positive regulator of cell expansion was down-regulated. Two types of critical candidates, boundary genes and adaxial-abaxial regulators, were identified from 7937 differentially expressed genes in pairwise comparisons, which were up-regulated at the late stage in 'Anastasia Dark Green' and another two hooked varieties. Ectopic expression of a candidate abaxial gene, CmYAB1, in chrysanthemum led to changes in petal curvature and inflorescence morphology. Our findings provide new insights into the regulatory networks underlying chrysanthemum petal morphogenesis.

Genome-Wide Analysis of the Growth-Regulating Factor Family in Peanut (Arachis hypogaea L.).

Growth-regulating factors (GRFs) are plant-specific transcription factors that perform important functions in plant growth and development. Herein, we identified and characterised 24 AhGRF genes in peanut (Arachis hypogaea). AhGRF family genes were divided into six classes with OLQ and WRC domains. Transcriptome expression profile showed that more AhGRF genes, such as AhGRF5a gene, were at higher expression during pod development in Arachis monticola than cultivated species, especially at the pod rapid-expansion stage. AhGRF5a and AhGRF5b genes expressed at higher levels in pods than roots, leaves and stems tissues, existing in the difference between Arachis monticola and H8107. Exogenous GA3 application can activate AhGRF5a and AhGRF5b genes and H8107 line showed more positive response than Arachis monticola species. These results imply that these two AhGRF genes may be active during the peanut pod development.

Transcriptome-Wide Identification and Expression Profiling Analysis of Chrysanthemum Trihelix Transcription Factors.

Trihelix transcription factors are thought to feature a typical DNA-binding trihelix (helix-loop-helix-loop-helix) domain that binds specifically to the GT motif, a light-responsive DNA element. Members of the trihelix family are known to function in a number of processes in plants. Here, we characterize 20 trihelix family genes in the important ornamental plant chrysanthemum (Chrysanthemum morifolium). Based on transcriptomic data, 20 distinct sequences distributed across four of five groups revealed by a phylogenetic tree were isolated and amplified. The phylogenetic analysis also identified four pairs of orthologous proteins shared by Arabidopsis and chrysanthemum and five pairs of paralogous proteins in chrysanthemum. Conserved motifs in the trihelix proteins shared by Arabidopsis and chrysanthemum were analyzed using MEME, and further bioinformatic analysis revealed that 16 CmTHs can be targeted by 20 miRNA families and that miR414 can target 9 CmTHs. qPCR results displayed that most chrysanthemum trihelix genes were highly expressed in inflorescences, while 20 CmTH genes were in response to phytohormone treatments and abiotic stresses. This work improves our understanding of the various functions of trihelix gene family members in response to hormonal stimuli and stress.

Comprehending the cuproptosis and cancer-immunity cycle network: delving into the immune landscape and its predictive role in breast cancer immunotherapy responses and clinical endpoints.

Background: The role of cuproptosis, a phenomenon associated with tumor metabolism and immunological identification, remains underexplored, particularly in relation to the cancer-immunity cycle (CIC) network. This study aims to rigorously examine the impact of the cuproptosis-CIC nexus on immune reactions and prognostic outcomes in patients with breast cancer (BC), striving to establish a comprehensive prognostic model. Methods: In the study, we segregated data obtained from TCGA, GEO, and ICGC using CICs retrieved from the TIP database. We constructed a genetic prognostic framework using the LASSO-Cox model, followed by its validation through Cox proportional hazards regression. This framework's validity was further confirmed with data from ICGC and GEO. Explorations of the tumor microenvironment were carried out through the application of ESTIMATE and CIBERSORT algorithms, as well as machine learning techniques, to identify potential treatment strategies. Single-cell sequencing methods were utilized to delineate the spatial distribution of key genes within the various cell types in the tumor milieu. To explore the critical role of the identified CICs, experiments were conducted focusing on cell survival and migration abilities. Results: In our research, we identified a set of 4 crucial cuproptosis-CICs that have a profound impact on patient longevity and their response to immunotherapy. By leveraging these identified CICs, we constructed a predictive model that efficiently estimates patient prognoses. Detailed analyses at the single-cell level showed that the significance of CICs. Experimental approaches, including CCK-8, Transwell, and wound healing assays, revealed that the protein HSPA9 restricts the growth and movement of breast cancer cells. Furthermore, our studies using immunofluorescence techniques demonstrated that suppressing HSPA9 leads to a notable increase in ceramide levels. Conclusion: This research outlines a network of cuproptosis-CICs and constructs a predictive nomogram. Our model holds great promise for healthcare professionals to personalize treatment approaches for individuals with breast cancer. The work provides insights into the complex relationship between the cuproptosis-CIC network and the cancer immune microenvironment, setting the stage for novel approaches to cancer immunotherapy. By focusing on the essential gene HSPA9 within the cancer-immunity cycle, this strategy has the potential to significantly improve the efficacy of treatments against breast cancer.

Transferable non-invasive modal fusion-transformer (NIMFT) for end-to-end hand gesture recognition.

Objective.Recent studies have shown that integrating inertial measurement unit (IMU) signals with surface electromyographic (sEMG) can greatly improve hand gesture recognition (HGR) performance in applications such as prosthetic control and rehabilitation training. However, current deep learning models for multimodal HGR encounter difficulties in invasive modal fusion, complex feature extraction from heterogeneous signals, and limited inter-subject model generalization. To address these challenges, this study aims to develop an end-to-end and inter-subject transferable model that utilizes non-invasively fused sEMG and acceleration (ACC) data.Approach.The proposed non-invasive modal fusion-transformer (NIMFT) model utilizes 1D-convolutional neural networks-based patch embedding for local information extraction and employs a multi-head cross-attention (MCA) mechanism to non-invasively integrate sEMG and ACC signals, stabilizing the variability induced by sEMG. The proposed architecture undergoes detailed ablation studies after hyperparameter tuning. Transfer learning is employed by fine-tuning a pre-trained model on new subject and a comparative analysis is performed between the fine-tuning and subject-specific model. Additionally, the performance of NIMFT is compared to state-of-the-art fusion models.Main results.The NIMFT model achieved recognition accuracies of 93.91%, 91.02%, and 95.56% on the three action sets in the Ninapro DB2 dataset. The proposed embedding method and MCA outperformed the traditional invasive modal fusion transformer by 2.01% (embedding) and 1.23% (fusion), respectively. In comparison to subject-specific models, the fine-tuning model exhibited the highest average accuracy improvement of 2.26%, achieving a final accuracy of 96.13%. Moreover, the NIMFT model demonstrated superiority in terms of accuracy, recall, precision, and F1-score compared to the latest modal fusion models with similar model scale.Significance.The NIMFT is a novel end-to-end HGR model, utilizes a non-invasive MCA mechanism to integrate long-range intermodal information effectively. Compared to recent modal fusion models, it demonstrates superior performance in inter-subject experiments and offers higher training efficiency and accuracy levels through transfer learning than subject-specific approaches.

Potential planting regions of Pterocarpus santalinus (Fabaceae) under current and future climate in China based on MaxEnt modeling.

This study modeled the habitat distribution of Pterocarpus santalinus, a valuable rosewood species, across China under current and future climate scenarios (SSPs126, SSPs245, and SSPs585) using MaxEnt. Our findings reveal that the current suitable habitat, spanning approximately 409,600 km2, is primarily located in the central and southern parts of Guangdong, Guangxi, Fujian, and Yunnan, as well as in the Hainan provinces, along with the coastal regions of Taiwan, and the Sichuan-Chongqing border. The habitat's distribution is significantly influenced by climatic factors such as temperature seasonality (bio4), mean temperature of the wettest quarter (bio8), annual mean temperature (bio1), and annual precipitation (bio12), while terrain and soil factors play a lesser role. Under future climate scenarios, the suitable habitat for P. santalinus is projected to expand, with a northeastward shift in its distribution center. This research not only sheds light on the geoecological characteristics and geographical distribution of P. santalinus in China but also offers a scientific basis for planning its cultivation areas and enhancing cultivation efficiency under changing climate conditions.

Natural resistance-associated macrophage proteins are involved in tolerance to heavy metal Cd2+ toxicity and resistance to bacterial wilt of peanut (Arachis hypogaea L.).

Peanut (Arachis hypogaea L.) is one of the most important oil and industrial crops. However, heavy-metal pollution and frequent soil diseases, poses a significant threat to the production of green and healthy peanuts. Herein, we investigated the effects of heavy metal Cd2+ toxicity to the peanuts, and screened out two peanut cultivars H108 and YZ 9102 with higher Cd2+-tolerance. RNA-seq revealed that Natural resistance-associated macrophage proteins (NRAMP)-like genes were involved in the Cd2+ stress tolerance in H108. Genome-wide identification revealed that 28, 13 and 9 Nramp-like genes existing in the A. hypogaea, A. duranensis and A. ipaensis, respectively. The 50 peanut NRAMP genes share conserved architectural characters, and they were classified into two groups. Expressions of AhNramps, particularly AhNramp4, AhNramp12, AhNramp19, and AhNramp25 could be greatly induced by not only cadmium toxicity, but also copper and zinc stresses. The expression profiles of AhNramp14, AhNramp16 and AhNramp25 showed significant differences in the H108 (resistance) and H107 (susceptible) under the infection of bacterial wilt. In addition, we found that the expression profiles of AhNramp14, AhNramp16, and AhNramp25 were greatly up- or down-regulated by the application of exogenous salicylic acid, methyl jasmonate, and abscisic acid. The AhNramp25, of which expression was affected by both heavy metal toxicity and bacterial wilt infection, were selected as strong candidate genes for peanut stress breeding. Our findings will provide an additional information required for further analysis of AhNramps involved in tolerance to heavy metal toxicity and resistance to bacterial wilt of peanut.

Coordinated Lipid Mobilization during Seed Development and Germination in Peanut (Arachis hypogaea L.).

Peanut (Arachis hypogaea L.) is one of the most important oil crops in the world due to its lipid-rich seeds. Lipid accumulation and degradation play crucial roles in peanut seed maturation and seedling establishment, respectively. Here, we utilized lipidomics and transcriptomics to comprehensively identify lipids and the associated functional genes that are important in the development and germination processes of a large-seed peanut variety. A total of 332 lipids were identified; triacylglycerols (TAGs) and diacylglycerols were the most abundant during seed maturation, constituting 70.43 and 16.11%, respectively, of the total lipids. Significant alterations in lipid profiles were observed throughout seed maturation and germination. Notably, TAG (18:1/18:1/18:2) and (18:1/18:2/18:2) peaked at 23386.63 and 23392.43 nmol/g, respectively, at the final stage of seed development. Levels of hydroxylated TAGs (HO-TAGs) increased significantly during the initial stage of germination. Accumulation patterns revealed an inverse relationship between free fatty acids and TAGs. Lipid degradation was determined to be regulated by diacylglycerol acyltransferase, triacylglycerol lipase, and associated transcription factors, predominantly yielding oleic acid, linoleic acid, and linolenic acid. Collectively, the results of this study provide valuable insights into lipid dynamics during the development and germination of large-seed peanuts, gene resources, and guiding future research into lipid accumulation in an economically important crop.

Novel variants of clade 2.3.4 highly pathogenic avian influenza A(H5N1) viruses, China.

We characterized 7 highly pathogenic avian influenza A(H5N1) viruses isolated from poultry in China during 2009-2012 and found that they belong to clade 2.3.4 but do not fit within the 3 defined subclades. Antigenic drift in subtype H5N1 variants may reduce the efficacy of vaccines designed to control these viruses in poultry.

Prevalence of low pathogenic avian influenza virus in one live bird market in Eastern China from 2002-2011.

The role of live bird markets (LBMs) in the perpetuation of avian influenza virus (AIV) has been well studied worldwide; however, there is a paucity of information on the prevalence of different AIV subtypes in LBMs in Eastern China. In this study, long-term surveillance was conducted to investigate the prevalence of AIV in chickens, ducks, and geese in an LBM in Yangzhou city, Jiangsu province, Eastern China, between July 2002 and May 2011. The study used primary virus isolation and subtype-specific reverse-transcription-PCR. In total, 23 different HA-NA subtype combinations were detected, mainly in domestic ducks. This result suggests that domestic ducks play a more important role in LBMs in Eastern China.

Morphological Characteristics and Expression Patterns of CmCYC2c of Different Flower Shapes in Chrysanthemum morifolium.

The chrysanthemum is widely used as a cut flower, potted flower, and garden flower worldwide and has high ornamental, edible, and medicinal value. The flower heads, composed of ray florets and disc florets, are the most diverse in terms of morphology among ornamental plants. Here, we compared and analyzed the developmental processes of different capitulum types as well as ray florets and disc florets. Morphological differentiation of the two florets occurred on the dorsal domain of the petals at stage Ⅳ of flower development, and differences in stamen development occurred at stage Ⅴ. The dorsal domain of the ray florets and the early stage of flower development were also an essential site and period, respectively, for the differences among capitulum types. In situ hybridization revealed that CmCYC2c, whose homologs are involved in the specification of floret identity in Asteraceae, was expressed in both the dorsal and ventral domains of the ray petals in the tubular-type chrysanthemum, whereas, it was differentially transcribed in the ray petals of flat- and spoon-type chrysanthemum cultivars and had lower or no expression in the dorsal domain and higher expression in the ventral domain at stage Ⅳ. Our study indicates that the expression pattern of CmCYC2c on the dorsal domain of the ray floret at stage Ⅳ contributes to the formation of diverse flower head types in chrysanthemums.