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The formation mechanism of different ray floret shapes of chrysanthemum (Chrysanthemum × morifolium) remains elusive due to its complex genetic background. C. vestitum, with the basic ray floret shapes of the flat, spoon, and tubular types, is considered a model material for studying ray floret morphogenesis. In this study, the flat and tubular type lines of C. vestitum at specific stages were used to investigate the key genes that regulate morphological differences in ray florets. We found that the expression levels of genes related to auxin synthesis, transport, and response were generally higher in the tubular type than in the flat type. CvARF3 was highly expressed in the flat type, while CvARF5 and CvARF6 were highly expressed in the tubular type. Additionally, the transcription levels of Class B and E genes closely related to petal development, including CvPI, CvAP3, Cvdefh21, CvSEP3, and CvCDM77, were expressed at higher levels in the tubular type than the flat type. Based on the results, it is proposed that auxin plays a key role in the development of ray florets, and auxin-related genes, especially CvARFs, may be key genes to control the morphological difference of ray florets. Simultaneously, MADS-box genes are involved in the co-regulation of ray floret morphogenesis. The results provide novel insights into the molecular mechanism of different petal type formation and lay a theoretical foundation for the directional breeding of petal type in chrysanthemums.

Background The study of plant phenotype by deep learning has received increased interest in recent years, which impressive progress has been made in the fields of plant breeding. Deep learning extremely relies on a large amount of training data to extract and recognize target features in the field of plant phenotype classification and recognition tasks. However, for some flower cultivars identification tasks with a huge number of cultivars, it is difficult for traditional deep learning methods to achieve better recognition results with limited sample data. Thus, a method based on metric learning for flower cultivars identification is proposed to solve this problem. Results We added center loss to the classification network to make inter-class samples disperse and intra-class samples compact, the script of ResNet18, ResNet50, and DenseNet121 were used for feature extraction. To evaluate the effectiveness of the proposed method, a public dataset Oxford 102 Flowers dataset and two novel datasets constructed by us are chosen. For the method of joint supervision of center loss and L2-softmax loss, the test accuracy rate is 91.88%, 97.34%, and 99.82% across three datasets, respectively. Feature distribution observed by T-distributed stochastic neighbor embedding (T-SNE) verifies the effectiveness of the method presented above. Conclusions An efficient metric learning method has been described for flower cultivars identification task, which not only provides high recognition rates but also makes the feature extracted from the recognition network interpretable. This study demonstrated that the proposed method provides new ideas for the application of a small amount of data in the field of identification, and has important reference significance for the flower cultivars identification research.

DNA methylation is an important epigenetic modification, that is involved in the regulation of gene expression and cell differentiation, and plays an important regulatory role in flower development in higher plants. There are two types of florets on the capitulum in the genus Chrysanthemum, the flower symmetry factor CYCLOIDEA (CYC) 2-like genes may be important candidate genes for determining the identity of the two types of florets. In this study, the diploid plant Chrysanthemum lavandulifolium was used as the research material, and qRT-PCR and bisulfite sequencing polymerase chain reaction (BSP) were used to identify the expression and DNA methylation pattern of CYC2-like genes in the two types of florets. Gene expression analysis showed that the six ClCYC2-like genes were significantly different in the two types of florets, and the expression levels of ClCYC2c, ClCYC2d, ClCYC2e and ClCYC2f in the ray florets were significantly higher than those in the disc florets. For the DNA methylation analysis of the three genes ClCYC2c, ClCYC2d, and ClCYC2e, it was found that the DNA methylation levels of these three genes were negative correlated with their expression levels, and the ways in which the three genes were regulated by the DNA methylation were different. It is speculated that the different DNA methylation of ClCYC2-like genes in the two types of florets may affect the differentiation and development of the two types of florets. This study provides new clues about epigenetics for the analysis of capitulum formation in Asteraceae.

Chrysanthemum lavandulifolium (Fischer ex Trautv.) Makino is a diploid plant belonging to the Asteraceae family, with typical capitula composed of female ray florets and bisexual disc florets. The differentiation and development of these two types of florets have long been important research focuses; however, the potential epigenetic mechanisms governing these processes have not been elucidated. In the present study, methylation-sensitive amplification polymorphism method was used to trace the dynamic changes of DNA methylation during capitulum development in C. lavandulifolium. DNA methylation patterns and levels were detected in the whole capitula during seven developmental stages, and the obtained results revealed that DNA demethylation was dominant during this process. In addition, DNA methylation patterns and levels showed significant differences between ray and disc florets. Moreover, the expression patterns of candidate genes potentially involved in the development processes of two types of florets were analyzed by real-time quantitative reverse transcription polymerase chain reaction, and correlation analysis indicated that the expression levels of ClPI, ClAG2, ClSEP1, ClCYC2c, ClCYC2d, and ClCYC2e were highly correlated with DNA methylation levels. These results indicate that DNA methylation may be involved in the differentiation and development of ray and disc florets. This study provides epigenetic insights into the capitulum development in C. lavandulifolium.

Anthocyanins in cornflower (Centaurea cyanus) is catalysed by a set of biosynthesis genes, however, the potential mechanism of transcriptional regulation remains unclear. In the present study, we traced the dynamic changes of petal colour development from white to violet and finally to blue on the same petal in cornflower. Pigment analysis showed that anthocyanin accumulation dramatically increased with petal colour development. Subsequently, nine libraries from above three colour regions were constructed for RNA-seq and 105,506 unigenes were obtained by de novo assembling. The differentially expressed genes among three colour regions were significantly enriched in the phenylpropanoid biosynthesis and flavonoid biosynthesis pathways, leading to the excavation and analysis of 46 biosynthesis genes involved in this process. Furthermore, four R2R3-CcMYBs clustered into subgroup 4 or subgroup 6 and one CcbHLH1 clustered into IIIf subgroup were screened out by phylogenetic analysis with Arabidopsis homologues. The promoters of flavanone 3-hydroxylase (CcF3H) and dihydroflavonol 4-reductase (CcDFR) were further isolated to investigate upstream regulation mechanism. CcMYB6-1 significantly upregulated the activity of above two promoters and stimulated anthocyanin accumulation by dual luciferase assay and transient expression in tobacco leaves, and its activity was obviously enhanced when co-infiltrated with CcbHLH1. Moreover, both yeast two-hybrid and bimolecular fluorescence complementation assays indicated the protein-protein interaction between these two activators. Based on these obtained results, it reveals that CcMYB6-1 and CcbHLH1 are two novel transcription factors synergistically involved in regulating anthocyanin biosynthesis. This study provides insights into the regulatory mechanism of anthocyanin accumulation in cornflower.

Virus-induced gene silencing (VIGS) is a technology for rapid gene functional analysis that depends on the degradation of viral RNA and is part of the natural defense mechanism in plants. Senecio cruentus is an important Compositae ornamental species that is plentiful and available in a variety of colors and has a typical blue variety that is rare in Compositae. These advantages make it a good material for studying the anthocyanin biosynthesis and blue flower formation mechanism. With the development of gene sequencing technology, the functions of many candidate genes that may be involved in anthocyanin biosynthesis in S. cruentus need to be identified. However, a stable and rapid genetic transformation system of S. cruentus is still lacking. Here, we screened two cultivars, ‘Venezia’ and ‘Jseter’, selected ScPDS and ScANS as test genes, and investigated the effect of developmental periods, bacterial cell concentrations and infection methods on gene silencing efficiency. The results showed that the silencing efficiency of S. cruentus leaves was low (13%), and it was less affected by the parameters. However, the transcription factor gene ScbHLH17 was still silenced by VIGS, which resulted in the loss of anthocyanin accumulation in leaves, and the expression levels of anthocyanin biosynthesis pathway (ABP) structural genes, including ScCHI, ScDFR3 and ScANS, were decreased significantly. The result proved that ScbHLH17 was an important transcription factor that regulated flower color formation in S. cruentus. In addition, ScANS-silencing phenotypes were observed in S. cruentus capitulum by vacuum-infiltrating S1 stage buds for 10 min after scape injection. In general, the present study provided an important technical support for the study of anthocyanin metabolism pathways in S. cruentus.

Carotenoids are one of the most important pigments for the coloring of many plants, fruits, and flowers. Recently, significant progress has been made in carotenoid metabolism. However, our specific understanding of the transcriptional regulation that controls the expression of carotenoid metabolic genes remains extremely limited. Anemone-type chrysanthemums, a special group of chrysanthemum cultivars, contain elongated disc florets in the capitulum that usually differ in color from the ray florets because of their different carotenoid contents. In this study, the carotenoid composition and content of ray and disc florets from the anemone-type chrysanthemum cultivar “Dong Li Fen Gui” were analyzed by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS), and the key structural gene CmCCD4a-2, whose differential expression resulted in different carotenoid contents in these two types of florets, was identified. The promoter sequence of CmCCD4a-2 was then used as bait to screen a chrysanthemum flower cDNA library, and the transcription factors (TFs) CmAP3 and CmUIF1 were identified. Y2H, BiFC, and Y3H experiments demonstrated that these two TFs were connected by CmPI to form a CmAP3-CmPI-CmUIF1 TF complex. This TF complex regulated carotenoid metabolism by directly activating the expression of CmCCD4a-2. A large number of target genes regulated directly by the CmAP3-CmPI-CmUIF1 TF complex, including carotenoid biosynthetic genes, flavonoid biosynthetic genes, and flower development-related genes, were identified by DNA-affinity purification sequencing (DAP-seq). This result indicated that the CmAP3-CmPI-CmUIF1 TF complex may participate in multiple processes. These findings expand our knowledge of the transcriptional regulation of carotenoid metabolism in plants and will be helpful for manipulating carotenoid accumulation in chrysanthemum.

Cineraria (Senecio cruentus) is an ornamental plant with pure colour and bicolour cultivars, widely used for landscaping. Anthocyanin biosynthesis influences coloration patterns in cineraria. However, how anthocyanins accumulate and distribute in cineraria is poorly understood. This study investigated the molecular mechanisms underlying anthocyanin biosynthesis and bicolour formation in cineraria using pure colour and bicolour cultivars. Transcriptome and gene expression analysis showed that five genes, ScCHS2, ScF3H1, ScDFR3, ScANS, and ScbHLH17, were inhibited in the white cultivar and colourless regions of bicolour cultivars. In contrast, two MADS-box genes, ScAG and ScAGL11, showed significantly higher expression in the colourless regions of bicolour cultivars. ScAG and ScAGL11 were localized in the nucleus and co-expressed with the bicolour trait. Further functional analysis verified that ScAG inhibits anthocyanin accumulation in tobacco (Nicotiana tabacum). However, virus-induced gene silencing (VIGS) experiments showed that silencing of ScAG and ScAGL11 increases anthocyanin content in cineraria leaves. Similar results were observed when ScAG and ScAGL11 were silenced in the cineraria capitulum, accompanied by the smaller size of the colourless region, specifically in the ScAG/ScAGL11-silenced plants. The expression of ScCHS2, ScDFR3, and ScF3H1 increased in silenced cineraria leaves and capitulum. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that ScAG interacts with ScAGL11. Moreover, ScAG directly inhibited the transcription of ScF3H1 while ScAGL11 inhibited ScDFR3 expression by binding to their promoters separately. The findings reported herein indicate that ScAG and ScAGL11 negatively regulate anthocyanin biosynthesis in cineraria ray florets, and their differential expression in ray florets influences the bicolour pattern appearance.

Cultivated chrysanthemum (Chrysanthemum × morifolium Ramat.) is a beloved ornamental crop due to the diverse capitula types among varieties, but the molecular mechanism of capitulum development remains unclear. Here, we report a 2.60 Gb chromosome-scale reference genome of C. lavandulifolium, a wild Chrysanthemum species found in China, Korea and Japan. The evolutionary analysis of the genome revealed that only recent tandem duplications occurred in the C. lavandulifolium genome after the shared whole genome triplication (WGT) in Asteraceae. Based on the transcriptomic profiling of six important developmental stages of the radiate capitulum in C. lavandulifolium, we found genes in the MADS-box, TCP, NAC and LOB gene families that were involved in disc and ray floret primordia differentiation. Notably, NAM and LOB30 homologs were specifically expressed in the radiate capitulum, suggesting their pivotal roles in the genetic network of disc and ray floret primordia differentiation in chrysanthemum. The present study not only provides a high-quality reference genome of chrysanthemum but also provides insight into the molecular mechanism underlying the diverse capitulum types in chrysanthemum.

The variation of flower color of chrysanthemum (Chrysanthemum×morifolium) is extremely rich, and carotenoids, which are mainly stored in the plastid, are important pigments that determine the color of chrysanthemum. However, the genetic regulation of the carotenoid metabolism pathway in this species still remains unclear. In this study, a pink chrysanthemum cultivar, 'Jianliuxiang Pink', and its three bud sport mutants (including white, yellow and red color mutants, 'Jianliuxiang White', 'Jianliuxiang Yellow' and 'Jianliuxiang Red', respectively) were used as experimental materials to analyze the dynamic changes of carotenoid components and plastid ultrastructure at different developmental stages of ray florets. We found that the carotenoid components and plastid ultrastructure of the four color cultivars in the early developmental stage of the chrysanthemum capitulum (S1) were almost identical, and the carotenoids mainly included violaxanthin, lutein and β-carotene, which exist in proplastids and immature chloroplasts. With the development of capitulum, the chloroplasts in 'Jianliuxiang White' and 'Jianliuxiang Pink' were degraded, and the protoplasts did not transform but rather formed vesicles that accumulated trace amounts of carotenoids. The proplastids and chloroplasts in 'Jianliuxiang Yellow' and 'Jianliuxiang Red' were all transformed into chromoplasts and consist of lutein as well as lutein's isomer and derivatives. Using comparative transcriptomics combined with gene expression analysis, we found that CmPg-1, CmPAP10, and CmPAP13, which were involved in chromoplast transformation, CmLCYE, which was involved in carotenoid biosynthesis, and CmCCD4a-2, which was involved in carotenoid degradation, were differentially expressed between four cultivars, and these key genes therefore should affect the accumulation of carotenoids in chrysanthemum. In addition, six transcription factors, CmMYB305, CmMYB29, CmRAD3, CmbZIP61, CmAGL24, CmNAC1, were screened using weighted gene co-expression correlation network analysis (WGCNA) combined with correlative analysis to determine whether they play an important role in carotenoid accumulation by regulating structural genes related to the carotenoid metabolism pathway and plastid development. This study analyzed dynamic changes of carotenoid components and plastid ultrastructure of the four bud mutation cultivars of chrysanthemum and identified structural genes and transcription factors that may be involved in carotenoid accumulation. The above results laid a solid foundation for further analysis of the regulatory mechanism of the carotenoid biosynthesis pathway in chrysanthemum.

Plant growth and development are significantly affected by salt stress. Chrysanthemum lavandulifolium is a halophyte species and one of the ancestors of chrysanthemum (C. ×morifolium). Understanding how this species tolerates salt stress could provide vital insight for clarifying the salt response systems of higher plants, and chrysanthemum-breeding programs could be improved. In this study, salt tolerance was compared among C. lavandulifolium and three chrysanthemum cultivars by physiological experiments, among which C. lavandulifolium and Jinba displayed better tolerance to salt stress than the other two cultivars, whereas Xueshan was a salt-sensitive cultivar. Using the transcriptome database of C. lavandulifolium as a reference, we used digital gene expression technology to analyze the global gene expression changes in C. lavandulifolium seedlings treated with 200 mm NaCl for 12 hours compared with seedlings cultured in normal conditions. In total, 2254 differentially expressed genes (DEGs), including 1418 up-regulated and 836 down-regulated genes, were identified. These DEGs were significantly enriched in 35 gene ontology terms and 29 Kyoto Encyclopedia of Genes and Genomes pathways. Genes related to signal transduction, ion transport, proline biosynthesis, reactive oxygen species scavenging systems, and flavonoid biosynthesis pathways were relevant to the salt tolerance of C. lavandulifolium. Furthermore, comparative gene expression analysis was conducted using reverse transcription polymerase chain reaction to compare the transcriptional levels of significantly up-regulated DEGs in C. lavandulifolium and the salt-sensitive cultivar Xueshan, and species-specific differences were observed. The analysis of one of the DEGs, ClAKT, an important K+ transport gene, was found to enable transgenic Arabidopsis thaliana to absorb K+ and efflux Na+ under salt stress and to absorb K+ under drought stress. The present study investigated potential genes and pathways involved in salt tolerance in C. lavandulifolium and provided a hereditary resource for the confinement of genes and pathways responsible for salt tolerance in this species. This study provided a valuable source of reference genes for chrysanthemum cultivar transgenesis breeding.

The NAC transcription factor is a peculiar kind of transcription factor in plants. Transcription factors are involved in the expression of plant genes under different conditions, and they play a crucial role in plant response to various biotic and abiotic stress. We transferred the ClNAC9 gene into Chrysanthemum grandiflora ‘niu9717’ by Agrobacterium tumefaciens–mediated transformation. The results of kanamycin-resistant screening, polymerase chain reaction (PCR) detection, and Northern blot analysis proved that the target gene had been integrated into the genome of the target plants. Wild-type (WT) plants and transgenic plants were treated with different concentrations of NaCl, NaHCO3, and drought stress, and physiological indexes, such as antioxidant system activity (superoxide dismutase, peroxidase, catalase), malondialdehyde accumulation, and leaf relative water content, were measured. We also observed changes in plant morphology. The physiological indexes’ changing range and extreme values suggested that transgenic plants’ resistance to salinity, alkali, and drought stress was significantly higher than WT plants. Transgenic plant growth was less inhibited compared with WT plants, indicating that the ClNAC9 gene increased the resistance of transgenic plants under the stress of salinization, alkalization, and drought.

The head-like inflorescence in C. × morifolium was considered as a pseudianthia that bears numerous types of floral organs. ABCE-class and CYC2-like genes have been proven to play a key role in the identity of floral organs. To further understand their roles in capitulum development, expression pattern and protein interactions of ABCE-class and CYC2-like genes were detected using quantitative PCR and yeast-two hybrid. A-class genes, DenFUL and ClAP2, particularly accumulated in ray floret corollas and formed heterodimers with CYC2-like proteins. CYC2-like genes are particularly expressed in ray floret corollas, and these proteins form homodimers and heterodimers. The present study indicated that A-class genes in chrysanthemums interacted with CYC2-like genes and might be involved in the identity of reproductive organs and ray florets, especially the differentiation of disc and ray floret corollas. Moreover, the floral organ identity model in Chrysanthemums was proposed based on insights from the gene expression and protein interactions of ABCE-class and CYC2-like genes.

Background The ray floret shapes referred to as petal types on the chrysanthemum (Chrysanthemum × morifolium Ramat.) capitulum is extremely abundant, which is one of the most important ornamental traits of chrysanthemum. However, the regulatory mechanisms of different ray floret shapes are still unknown. C. vestitum is a major origin species of cultivated chrysanthemum and has flat, spoon, and tubular type of ray florets which are the three basic petal types of chrysanthemum. Therefore, it is an ideal model material for studying ray floret morphogenesis in chrysanthemum. Here, using morphological, gene expression and transcriptomic analyses of different ray floret types of C. vestitum, we explored the developmental processes and underlying regulatory networks of ray florets. Results The formation of the flat type was due to stagnation of its dorsal petal primordium, while the petal primordium of the tubular type had an intact ring shape. Morphological differences between the two ray floret types occurred during the initial stage with vigorous cell division. Analysis of genes related to flower development showed that CYCLOIDEA genes, including CYC2b, CYC2d, CYC2e, and CYC2f, were differentially expressed in different ray floret types, while the transcriptional levels of others, such as MADS-box genes, were not significantly different. Hormone-related genes, including SMALL AUXIN UPREGULATED RNA (SAUR), GRETCHEN HAGEN3 (GH3), GIBBERELLIN 2-BETA-DIOXYGENASE 1 (GA2OX1) and APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF), were identified from 1532 differentially expressed genes (DEGs) in pairwise comparisons among the flat, spoon, and tubular types, with significantly higher expression in the tubular type than that in the flat type and potential involvement in the morphogenesis of different ray floret types. Conclusions Our findings, together with the gene interactional relationships reported for Arabidopsis thaliana, suggest that hormone-related genes are highly expressed in the tubular type, promoting petal cell division and leading to the formation of a complete ring of the petal primordium. These results provide novel insights into the morphological variation of ray floret of chrysanthemum.

Flower type is an important and extremely complicated trait of chrysanthemum. The corolla tube merged degree (CTMD) and the relative number of ray florets (RNRF) are the two key factors affecting chrysanthemum flower type. However, few reports have clarified the inheritance of these two complex traits, which limits directed breeding for flower-type improvement. In this study, 305 F1hybrids were obtained from two parents with obvious differences in CTMD and RNRF performance. Using specific-locus amplified fragment sequencing (SLAF-seq) technology, we constructed a high-density genetic linkage map with an average map distance of 0.76 cM. Three major QTLs controlling CTMD and four major QTLs underlying RNRF were repeatedly detected in the 2 years. Moreover, the synteny between the genetic map and other Compositae species was investigated, and weak collinearity was observed. In QTL regions with a high degree of genomic collinearity, eight annotated genes were probed in theHelianthus annuusL. andLactuca sativaL. var. ramosa Hort. genomes. Furthermore, 20 and 11 unigenes were identified via BLAST searches between the SNP markers of the QTL regions and theC. vestitumandC. lavandulifoliumtranscriptomes, respectively. These results lay a foundation for molecular marker-assisted breeding and candidate gene exploration in chrysanthemum without a reference assembly.

Plants sense photoperiod signals to confirm the optimal flowering time. Previous studies have shown that Cryptochrome2 (CRY2) functions to promote floral transition in the long-day plant (LDP) Arabidopsis; however, the function and molecular mechanism by which CRY2 regulates floral transition in short-day plants (SDPs) is still unclear. In this study, we identified a CRY2 homologous gene, ClCRY2, from Chrysanthemum lavandulifolium, a typical SDP. The morphological changes in the C. lavandulifolium shoot apex and ClFTs expression analysis under SD conditions showed that adult C. lavandulifolium completed the developmental transition from vegetative growth to reproductive growth after eight SDs. Meanwhile, ClCRY2 mRNA exhibited an increasing trend from 0 to 8 d of SD treatment. ClCRY2 overexpression in wild-type (WT) Arabidopsis and C. lavandulifolium resulted in early flowering. The transcript levels of the CONSTANS-like (COL) genes ClCOL1, ClCOL4, and ClCOL5, and FLOWERING LOCUS T (FT) homologous gene ClFT1 were upregulated in ClCRY2 overexpression (ClCRY2-OE) C. lavandulifolium under SD conditions. The transcript levels of some circadian clock-related genes, including PSEUDO-REPONSE REGULATOR 5 (PRR5), ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1), and GIGANTEA (GI-1 and GI-2), were upregulated in ClCRY2-OE C. lavandulifolium, while the expression levels of other circadian clock-related genes, such as EARLY FLOWERING 3 (ELF3), ELF4, LATE ELONGATED HYPOCOTYL (LHY), PRR73, and REVEILLE8 (RVE8), were downregulated in ClCRY2-OE C. lavandulifolium under SD conditions. Taken together, the results suggest that ClCRY2 promotes floral transition by fine-tuning the expression of circadian clock-related gene, ClCOLs and ClFT1 in C. lavandulifolium under SD conditions., Genetics: Harnessing flower power in chrysanthemums Researchers have identified the genes that control the timing of flowering in chrysanthemums. For most plants, day-length indicates that it is time to flower. Greenhouse growers can produce out-of-season flowers by manipulating day length, but the process is expensive. Breeding varieties that respond more quickly could reduce costs, but requires a better understanding of the underlying genetics, especially in the subset of plants triggered by short days. Si-lan Dai and co-workers at Beijing Forestry University in China investigated which genes trigger flowering in Chrysanthemum lavandulifolium, a typical short-day plant. They found that flowering of chrysanthemum is controlled by a gene previously identified in long-day plants, and that amplifying the gene produced plants that flowered much sooner than ordinary plants. These findings may help in breeding varieties that are easily induced to bloom.

The generation of chrysanthemum (Chrysanthemum &times, morifolium) flower color is mainly attributed to the accumulation of anthocyanins. Light is one of the key environmental factors that affect the anthocyanin biosynthesis, but the deep molecular mechanism remains elusive. In our previous study, a series of light-induced structural and regulatory genes involved in the anthocyanin biosynthetic pathway in the chrysanthemum were identified using RNA sequencing. In the present study, differentially expressed proteins that are in response to light with the capitulum development of the chrysanthemum &lsquo, Purple Reagan&rsquo, were further identified using isobaric tags for relative and absolute quantification (iTRAQ) technique, and correlation between the proteomic and the transcriptomic libraries was analyzed. In general, 5106 raw proteins were assembled based on six proteomic libraries (three capitulum developmental stages &times, two light treatments). As many as 160 proteins were differentially expressed between the light and the dark libraries with 45 upregulated and 115 downregulated proteins in response to shading. Comparative analysis between the pathway enrichment and the gene expression patterns indicated that most of the proteins involved in the anthocyanin biosynthetic pathway were downregulated after shading, which was consistent with the expression patterns of corresponding encoding genes, while five light-harvesting chlorophyll a/b-binding proteins were initially downregulated after shading, and their expressions were enhanced with the capitulum development thereafter. As revealed by correlation analysis between the proteomic and the transcriptomic libraries, GDSL esterase APG might also play an important role in light signal transduction. Finally, a putative mechanism of light-induced anthocyanin biosynthesis in the chrysanthemum was proposed. This study will help us to clearly identify light-induced proteins associated with flower color in the chrysanthemum and to enrich the complex mechanism of anthocyanin biosynthesis for use in cultivar breeding.

Background Cultivar recognition is a basic work in flower production, research, and commercial application. Chinese large-flowered chrysanthemum (Chrysanthemum × morifolium Ramat.) is miraculous because of its high ornamental value and rich cultural deposits. However, the complicated capitulum structure, various floret types and numerous cultivars hinder chrysanthemum cultivar recognition. Here, we explore how deep learning method can be applied to chrysanthemum cultivar recognition. Results We propose deep learning models with two networks VGG16 and ResNet50 to recognize large-flowered chrysanthemum. Dataset A comprising 14,000 images for 103 cultivars, and dataset B comprising 197 images from different years were collected. Dataset A was used to train the networks and determine the calibration accuracy (Top-5 rate of above 98%), and dataset B was used to evaluate the model generalization performance (Top-5 rate of above 78%). Moreover, gradient-weighted class activation mapping (Grad-CAM) visualization and feature clustering analysis were used to explore how the deep learning model recognizes chrysanthemum cultivars. Conclusion Deep learning method applied to cultivar recognition is a breakthrough in horticultural science with the advantages of strong recognition performance and high recognition speed. Inflorescence edge areas, disc floret areas, inflorescence colour and inflorescence shape may well be the key factors in model decision-making process, which are also critical in human decision-making.

Anthocyanins are important flavonoid pigments involved in the colouring of flowers and fruits. They are synthesized on the cytoplasmic surface of the endoplasmic reticulum and transported into the vacuole for storage. Previous reports have suggested that glutathione S-transferase (GST) is involved in anthocyanin transport. However, due to the limitation of plant materials, most GSTs only participate in the cyanidin or delphinidin transport pathway. Here, an anthocyanin-related GST, ScGST3, was identified from the transcriptome of cineraria. The expression pattern of ScGST3 was highly consistent with anthocyanin accumulation in ray florets. Molecular complementation of Arabidopsis tt19 indicated that the overexpression of ScGST3 restores the anthocyanin-deficient phenotype of the mutant. Virus-induced gene silencing (VIGS) of ScGST3 in carmine and blue cineraria leaves could inhibit anthocyanin accumulation, further confirming the function of ScGST3 in anthocyanin accumulation. In vitro assays showed that ScGST3 increases the water solubility of cyanidin-3-O-glucoside (C3G) and delphinidin-3-O-glucosid (D3G). In addition, we also identified two anthocyanin-related MYB transcription factors, ScMYB3 and ScMYB6. The expression pattern of these two genes was also highly consistent with anthocyanin accumulation. Faded abaxial leaf phenotypes were observed after the silencing of ScMYB3 and ScMYB6, and the expression levels of partial structural genes were repressed. Based on the results from dual-luciferase assays and yeast one-hybrid assays, ScMYB3 can activate the promoter of ScGST3. Collectively, the transcription of ScGST3 is regulated by ScMYB3, which plays an important role in the transport of C3G and D3G in cineraria.

Cryptochrome (CRY), a vital photoreceptor which mediates light signals, controls photomorphogenesis in higher plants. However, the function of CRY in mediating light to regulate growth and development of ornamental plants is still unclear. In this study, we identified two CRY1 homologous genes, ClCRY1a and ClCRY1b, from Chrysanthemum lavandulifolium, a diploid wild chrysanthemum species. Expression analysis demonstrated that these two ClCRY1 genes showed the highest expression levels in seedlings leaves that were transferred to short day (SD) conditions for eight days. ClCRY1a was expressed at a higher level in the dark phase of SD, while ClCRY1b was expressed more highly in SD than that in long day (LD) conditions. Overexpression of either ClCRY1a or ClCRY1b in wild-type (WT) Arabidopsis resulted in early flowering under both LD and SD. The expression levels of GIGANTEA (GI) and FLOWERING LOCUS T (FT) were significantly up-regulated in ClCRY1a overexpressors under both LD and SD. Moreover, the transcript levels of GI, CONSTANS (CO) and FT were markedly increased in ClCRY1b overexpressors under LD; nevertheless, only the expression levels of CO and FT were up-regulated under SD. Taken together, the above results indicated that these two ClCRY1 genes could regulate flowering time via different pathways in C. lavandulifolium under LD and SD conditions. Our results provided evidence for the role of ClCRY1s in controlling photomorphogenesis and laid a foundation for further study on the molecular mechanism of ClCRYs mediating light signals to control floral transition.

Cymbidium longibracteatum is a common cultivated species in the genus Cymbidium due to its elegant appearance, rich flower colors and strong fragrance, but its classification is quite controversial. In this study, the complete chloroplast genome of C. longibracteatum was obtained by Illumina sequencing. The chloroplast genome of C. longibracteatum is 150,070 bp in length with an overall GC content of 37.12%, which contains a large single-copy (LSC;84,949 bp) region, a small single-copy (SSC;13,745bp) region, and a pair of inverted repeats (IRs; 25,688 bp) regions. The genome contains 130 genes, namely 84 protein-coding genes, 38 tRNA genes and 8 rRNA genes. The maximum-likelihood phylogenetic tree has proved that C. longibracteatum should exist as an independent species in the genus Cymbidium, and it is most closely related to C. tortisepalum. This study provides valuable sequence resources for further study of C. longibracteatum.

The generation of chrysanthemum (Chrysanthemum &times, morifolium) flower color is mainly attributed to the accumulation of anthocyanins. In the anthocyanin biosynthetic pathway in chrysanthemum, although all of the structural genes have been cloned, the regulatory function of R2R3-MYB transcription factor (TF) genes, which play a crucial role in determining anthocyanin accumulation in many ornamental crops, still remains unclear. In our previous study, four light-induced R2R3-MYB TF genes in chrysanthemum were identified using transcriptomic sequencing. In the present study, we further investigated the regulatory functions of these genes via phylogenetic and alignment analyses of amino acid sequences, which were subsequently verified by phenotypic, pigmental, and structural gene expression analyses in transgenic tobacco lines. As revealed by phylogenetic and alignment analyses, CmMYB4 and CmMYB5 were phenylpropanoid and flavonoid repressor R2R3-MYB genes, respectively, while CmMYB6 was an activator of anthocyanin biosynthesis, and CmMYB7 was involved in regulating flavonol biosynthesis. Compared with wild-type plants, the relative anthocyanin contents in the 35S:CmMYB4 and 35S:CmMYB5 tobacco lines significantly decreased (p &lt, 0.05), while for 35S:CmMYB6 and 35S:CmMYB7, the opposite result was obtained. Both in the 35S:CmMYB4 and 35S:CmMYB5 lines, the relative expression of several anthocyanin biosynthetic genes in tobacco was significantly downregulated (p &lt, 0.05), on the contrary, several genes were upregulated in the 35S:CmMYB6 and 35S:CmMYB7 lines. These results indicate that CmMYB4 and CmMYB5 negatively regulate anthocyanin biosynthesis in chrysanthemum, while CmMYB6 and CmMYB7 play a positive role, which will aid in understanding the complex mechanism regulating floral pigmentation in chrysanthemum and the functional divergence of the R2R3-MYB gene family in higher plants.

Centaurea cyanus is a popular garden plant native to Europe. Although their petals show abundant colour variations, the flavonoid profiling and the potential molecular mechanisms remain unclear. In the present study, we collected six cornflower cultivars with white, pink, red, blue, mauve and black petals. Ultra-performance liquid chromatography coupled with photodiode array and tandem mass spectrometry (UPLC-MS/MS) was used to investigate the comparative profiling of flavonoids both qualitatively and quantitatively. Ten anthocyanins, six flavones and two flavonols were separated and putatively identified. Except for white petals without any anthocyanins, both pink and red flowers contained pelargonidin derivatives, whereas blue, mauve and black petals accumulated cyanidins. The expression patterns of genes involved in the flavonoid biosynthesis were performed by real-time quantitative reverse transcription-PCR. The anthocyanin biosynthetic pathway in white petals was inhibited starting from flavanone 3-hydroxylase, resulting in the absence of anthocyanin accumulation. The open reading frame of flavonoid 3'-hydroxylase in pink and red petals was truncated; this led to loss of a haem binding site, a conserved motif in the cytochrome P450 family, and loss of conversion from dihydrokaempferol to dihydroquercetin. The significantly higher expression of structural genes corresponding to the hyper-accumulation of flavonoids in black petals may play an important role in black coloration. Remarkably, the mauve and blue petals accumulated the same cyanidin derivative but contained apigenin with different modifications on the 4' position, which may cause the coloration differences. The results obtained in this study will provide insights into the mechanisms of vivid colour diversities in cornflower.

Light is one of the key environmental factors that affect anthocyanin biosynthesis. However, the underlying molecular mechanism remains unclear, and many problems regarding phenotypic change and corresponding gene regulation have not been solved. In the present study, comparative analyses of light-induced anthocyanin accumulation and gene expression between the ray florets and leaves were performed in Chrysanthemum × morifolium 'Purple Reagan'. After contrasting the variations in the flower color phenotype and relative pigment content, as well as expression patterns of structural and regulator genes responsible for anthocyanin biosynthesis and photoreceptor between different plant organs under light and dark conditions, we concluded that (1) both the capitulum and foliage are key organs responding to light for chrysanthemum coloration; (2) compared with flavones, shading makes a greater decrease on the anthocyanins accumulation; (3) most of the structural and regulatory genes in the light-induced anthocyanin pathway specifically express in the ray florets; and (4) CmCHS, CmF3H, CmF3'H, CmANS, CmDFR, Cm3GT, CmMYB5-1, CmMYB6, CmMYB7-1, CmbHLH24, CmCOP1 and CmHY5 are key genes for light-induced anthocyanin biosynthesis in chrysanthemum ray florets, while on the transcriptional level, the expressions of CmPHYA, CmPHYB, CmCRY1a, CmCRY1b and CmCRY2 are insignificantly changed. Moreover, the inferred comprehensive effect of multiple signals on the accumulation of anthocyanins and transmission channel of light signal that exist between the leaves and ray florets were further discussed. These results further our understanding of the relationship between the gene expression and light-induced anthocyanin biosynthesis, and lay foundations for the promotion of the molecular breeding of novel flower colors in chrysanthemums.

The light-dependent coloration of the vital organs of horticultural crops affects multiple parts of production and sales. The simplicity of the metabolic pathways of anthocyanins and the characteristics of light-dependent coloration make chrysanthemum (Chrysanthemum ×morifolium) an ideal subject for studying the mechanism of light-regulated anthocyanin biosynthesis. In this study, real-time quantitative reverse transcription–polymerase chain reaction (PCR) was used in the analysis of the expression levels of anthocyanin biosynthesis genes in C. ×morifolium ‘Reagan’. The reference genes selected were those assumed to remain at constant levels in three flower color lines at five floral developmental stages and at two light conditions. Using digital gene expression technology, we selected nine reference genes with moderate expression in the chrysanthemum ray florets at various floral developmental stages under illuminated and dark conditions as the candidate reference genes for further study. After comprehensively analyzing the stability of gene expression with three distinct statistical algorithms, geNorm, NormFinder, and qBase plus, we found that F-box and PP2A were the most stable genes in all of the samples. In addition, we analyzed the relative expression level of the CmF3H gene in different samples to verify the reference genes that we selected. This study provides a consensus list of validated reference genes that will benefit future studies of the expression of chrysanthemum genes involved in anthocyanin biosynthesis and floral development under various light conditions. Moreover, this information will also promote the molecular breeding of horticultural crops for their color modification.

This paper reports the first study on plant CBF transcription factors (TF) in salt and drought stress responses in Chrysanthemum lavandulifolium. A CBF homolog gene, named as ClCBF1, from C. lavandulifolium was isolated using rapid amplification of cDNA ends (RACE). The deduced peptide is comprised of 210 amino acids (AA) containing an AP2 structural domain characteristic of the AP2 gene family. Quantitative real-time PCR revealed that ClCBF1 gene exhibit differential expression patterns across root, leaf and stem tissues, and it was strongly induced under salt and drought treatments of C. lavandulifolium. Overexpression of ClCBF1 in C. morifolium ‘White Snow’ resulted in stronger tolerance to salt and drought stresses. The ClCBF1 expression level, enzymatic activities of superoxide dismutase and peroxidase, and contents of proline and soluble proteins were enhanced in these transgenic lines, they were repressed in the antisense transgenic lines under the same stress conditions. Results indicate that ClCBF1 represents a promising candidate gene in improving abiotic stress tolerance among ornamental plants.

In Arabidopsis, circadian clock genes play important roles in photoperiod pathway by regulating the daytime expression of CONSTANS (CO), but related reports for chrysanthemum are notably limited. In this study, we isolated eleven circadian clock genes, which lie in the three interconnected negative and positive feedback loops in a wild diploid chrysanthemum, Chrysanthemum lavandulifolium. With the exception of ClELF3, ClPRR1 and ClPRR73, most of the circadian clock genes are expressed more highly in leaves than in other tested tissues. The diurnal rhythms of these circadian clock genes are similar to those of their homologs in Arabidopsis. ClELF3 and ClZTL are constitutively expressed at all time points in both assessed photoperiods. The expression succession from morning to night of the PSEUDO RESPONSE REGULATOR (PRR) gene family occurs in the order ClPRR73/ClPRR37, ClPRR5, and then ClPRR1. ClLHY is expressed during the dawn period, and ClGIs is expressed during the dusk period. The peak expression levels of ClFKF1 and ClGIs are synchronous in the inductive photoperiod. However, in the non-inductive night break (NB) condition or non-24 h photoperiod, the peak expression level of ClFKF1 is significantly changed, indicating that ClFKF1 itself or the synchronous expression of ClFKF1 and ClGIs might be essential to initiate the flowering of C. lavandulifolium. This study provides the first extensive evaluation of circadian clock genes, and it presents a useful foundation for dissecting the functions of circadian clock genes in C. lavandulifolium.

Proline plays a significant role in plant resistance to abiotic stresses, and its level is determined by a combination of synthesis, catabolism and transport. The primary proteins involved are Δ(1)-pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (PDH) and proline transporter (ProT). To utilise proline metabolism to improve the stress resistance of Chrysanthemum×morifolium, we isolated two P5CS-homologous genes (ClP5CS1 and ClP5CS2), one PDH gene (ClPDH) and four ProT-homologous genes (ClProT1-4) (GenBANK accession numbers: KF743136-KF743142) from Chrysanthemum lavandulifolium, which is closely related to chrysanthemums and exhibits strong resistance to stresses. Expression analysis of these genes in different organs and under various stresses indicated that ClP5CSs showed substantial constitutive expression, while ClPDH was only strongly expressed in the capitulum and was inhibited under most stresses. The expression patterns of four ClProT genes presented characteristics of organ specificity and disparity under stresses. Above all, the expression of ClProT2 was restricted to above-ground organs, especially strong in the capitulum and could be obviously induced by various stress conditions. Promoters of ClPDH and ClProTs contained many cis-acting regulatory elements involved in stress responses and plant growth and development. High levels of free proline were found in flower buds, the capitulum under the non-stress condition and later periods of stress conditions except cold treatment. Interestingly, organ specificity and disparity also exist in the level of free proline under different stress conditions. Our study indicates that ClProTs play significant roles in proline accumulation and stress responses, and that ClProT2 could be used to genetically modify the stress resistance of chrysanthemums. In addition, proline metabolism might be closely related to plant flowering and floral development.

Chrysanthemum is one of the most important commercially used floral crops. The flowering feature of chrysanthemum has considerable ecological and cultural value; however, it’s flowering mechanisms has not been well characterized to date because of a lack of genomic information. In this study, we firstly present extensive sequence and transcript abundance data for the transcriptome of a key chrysanthemum-related species, Chrysanthemum lavandulifolium (Fisch. ex Trautv), which was obtained using Illumina HiSeq™ 2000 sequencing platform. There were 108,737 high quality unigenes that were assembled based on 4 GB sequencing data, of which 58,093 were annotated by a similarity search with known proteins (E-value

The morphological characteristics of chrysanthemum (Chrysanthemum ×morifolium) are rich in variation. However, as a result of the aneuploid polyploidy of the chrysanthemum genome and the lack of proper tools, the genomic information of this crop is limited. Development of microsatellite markers has been an increasing trend in crop genetic studies because of the applicability of these markers in breeding programs. In this study, we reported the development of a simple sequence repeat in chrysanthemums using a magnetic beads enrichment method. An enriched genomic library with AC and GT microsatellite motifs was constructed, and 53 positive clones were detected by a colony polymerase chain reaction (PCR) technique. Of these clones, 35 showed high-quality sequences, and 35 primer pairs were designed accordingly. Twenty-six (74.29%) of the 35 primer pairs revealed polymorphisms on a set of 40 chrysanthemum cultivars. There were 172 alleles amplified over 26 loci with an average of 6.615 alleles per locus. The mean values of gene diversity corrected for the sample size and the inbreeding coefficient were 0.609 and 0.119 over 26 loci, respectively, which indicated that the majority of the microsatellite loci is highly informative. Cluster analysis based on 26 polymorphic loci demonstrated that the selected cultivars were clustered according to geographical origin. This study shows the isolation efficiency of the magnetic beads technique; the abundance of microsatellites in chrysanthemum; and the potential application for the cultivar classification, the studies on genetic diversity, and molecular breeding of chrysanthemums, which is beneficial to promoting the conservation and sustainable use of this crop.

Quantitative real-time RT-PCR (RT-qPCR) is a technology that can be used to analyze the abundance of gene expression. Reference genes, which are assumed to remain at constant levels in different tissues at various developmental stages and photoperiodic treatments, were selected to analyze the expression levels of flowering time genes and floral development genes. Using digital gene expression technology, nine reference genes with moderate expression in the leaves of Chrysanthemum lavandulifolium at the juvenile phase (CK1) and the squaring stage (W1) were selected as the candidate reference genes for further study. A total of 115 biological samples of C. lavandulifolium were analyzed, including different tissues under various developmental stages and leaves with varied photoperiodic treatments. The stability of the nine reference genes was slightly variable across the samples, but MTP, SKIP16 and PGK were the most stable genes overall. In addition, the relative expression level of ClFT in different tissues of plants with the competence to flower was analyzed to verify the reference genes selected in this study. These studies provide a guide for selecting reference genes for analyzing the expression pattern of flowering time genes and floral development genes in C. lavandulifolium.

The plant-specific NAC (NAM, ATAF, and CUC) transcription factor family plays a vital role in various plant growth and developmental processes as well as in stress resistance. Using RNA sequencing, we found that the ClNAC genes (ClNAC1-44) were the most strongly up-regulated transcription factor family in Chrysanthemum lavandulifolium leaves under salt treatment. We carried out reverse transcriptase polymerase chain reaction to monitor ClNAC genes response against multiple stresses and hormonal treatments including salt, drought, cold, heat, abscisic acid and salicylic acid treatments. The results showed that 35 ClNAC genes were differentially expressed in different organ, and 32 ClNAC genes could respond to at least 2 kinds of treatments. Quantitative real time polymerase chain reaction showed that 10 ClNAC genes belonging to 7 different subfamilies could respond to at least 5 kinds of treatments. Over 50-fold variation in transcriptional levels of ClNAC17 and ClNAC21 genes was observed under 6 different types of treatments. In the present study, high-level expression of ClNAC genes under abiotic stresses and hormonal treatments suggests that the NAC transcription factors play important roles in abiotic stress tolerance and adaptation.

Nudix hydrolases are associated with detoxification processes in plants under abiotic stress and regulate disease resistance signal transduction pathways. Investigating expression patterns and phyletic evolution of eight Nudix hydrolase gene family members in Chrysanthemum lavandulifolium will provide insights into detoxification pathways. In this study, full-length open reading frames of eight Nudix hydrolase genes (ClNUDX1-8) were obtained using a C. lavandulifolium expressed sequence tag (EST) database, EST splicing, and rapid-amplification of cDNA ends. Sequence similarity analysis of the eight ClNUDXs using MEGA4.0 software revealed that these genes belonged to seven subfamilies of the plant Nudix hydrolase gene family. Reverse transcription-polymerase chain reaction analysis demonstrated that five genes exhibited organ-specific expression; these included ClNUDX1, ClNUDX2, ClNUDX3, CINUDX7, and ClNUDX8. Among those, ClNUDX1, ClNUDX3, ClNUDX7, and ClNUDX8 were induced by salt, drought, heat, and cold stresses. ClNUDX2 was only influenced by salt and drought. Transcription levels of both ClNUDX1 and ClNUDX2 were also regulated by abscisic acid and salicylic acid. The promoter of ClNUDX1, CN1P, was cloned. Transient expression analysis indicated that CN1P was capable of driving expression of the GUS reporter gene. Thus, it was concluded that different members of Nudix hydrolase genes in C. lavandulifolium demonstrated varied expression patterns in response to different abiotic stresses. These findings further elucidated knowledge on the response mechanisms to abiotic stresses in C. lavandulifolium.

A subtractive cDNA library was developed to study genes associated with the release of dormant buds in tree peony. To identify genes that are highly expressed in buds released from dormancy, 588 clones were examined by differential screening and then 185 clones were selected to be sequenced. A total of 31 unique genes were obtained, of which only 25 sequences had matches in the NCBI database or Arabidopsis thaliana protein database while 6 sequences with no matches. Many of the different genes were identified as having unknown or hypothetical functions while others were speculated to have different molecular functions. The expression profiles of the selected “candidate” genes which may be associated with dormancy release according to their putative function and previous reports were assessed by northern blot and semi-quantitative RT-PCR. The results indicated that the transcriptional expressions of the isolated genes are related to growth regulation and stress response. Our results provide interesting information for further understanding the molecular mechanism of bud dormancy release in tree peony.

A subtractive cDNA library was developed to study genes associated with bud dormancy release in tree peonies. In order to identify genes that are highly expressed in buds released from dormancy, 588 clones were examined by differential screening. Of these, 185 clones were selected to be sequenced. A total of 37 unique sequences were obtained of which only 31 sequences have matches in the NCBI database or the Arabidopsis thaliana protein database. Semi-quantitative RT-PCR was used to confirm further the expression profiles for 12 transcripts identified within the subtractive cDNA library. Gene ontology analyses indicated that many of the different genes identified have unknown or hypothetical functions while it is speculated that other genes play different molecular roles. In our study, genes involved in bud dormancy release were growth-related or stress-responsive, while low-temperature-induced ribosomal proteins may also play a role in bud dormancy release. Our results provide interesting information for further understanding of the molecular mechanism of bud dormancy release in tree peonies.

Background The flower colour of agricultural products is very important for their commercial value, which is mainly attributed to the accumulation of anthocyanins. Light is one of the key environmental factors that affect the anthocyanin biosynthesis. However, the deep molecular mechanism remains elusive, and many problems regarding the phenotypic change and the corresponding gene regulation are still unclear. In the present study, Chrysanthemum × morifolium ‘Purple Reagan’, a light-responding pigmentation cultivar, was selected to investigate the mechanism of light-induced anthocyanin biosynthesis using transcriptomic analyses. Results Only cyanidin derivatives were identified based on the analyses of the pigmentation in ray florets. Shading experiments revealed that the capitulum was the key organ and that its bud stage was the key phase responding to light. These results were used to design five libraries for transcriptomic analyses, including three capitulum developmental stages and two light conditions. RNA sequences were de novo assembled into 103,517 unigenes, of which 60,712 were annotated against four public protein databases. As many as 2,135 unigenes were differentially expressed between the light and dark libraries with 923 up-regulated and 1,212 down-regulated unigenes in response to shading. Next, interactive pathway analysis showed that the anthocyanin biosynthetic pathway was the only complete metabolic pathway both modulated in response to light and related to capitulum development. Following the shading treatment, nearly all structural genes involved in the anthocyanin biosynthetic pathway were down-regulated. Moreover, three CmMYB genes and one CmbHLH gene were identified as key transcription factors that might participate in the regulation of anthocyanin biosynthesis under light conditions based on clustering analysis and validation by RT-qPCR. Finally, a light-induced anthocyanin biosynthesis pathway in chrysanthemums was inferred. Conclusion The pigmentation of the ray florets of chrysanthemum cultivar ‘Purple Reagan’ is dependent on light. During the light-induced pigmentation process, the expression of seven structural genes in the anthocyanin biosynthetic pathway (regulated by at least four transcription factors in response to light) are the main contributors to the pigmentation of chrysanthemums. This information will further our understanding of the molecular mechanisms governing light-induced anthocyanin biosynthesis in ornamental plants. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1428-1) contains supplementary material, which is available to authorized users.

The CONSTANS (CO) and CONSTANS-like (COL) genes play key roles in the photoperiodic flowering pathways, and studying their functions can elucidate the molecular mechanisms underlying flowering control in photoperiod-regulated plants. We identified eleven COL genes (ClCOL1–ClCOL11) in Chrysanthemum lavandulifolium and divided them into three groups that are conserved among the flowering plants based on phylogenetic analysis. Most of the ClCOL genes are primarily expressed in the leaf and shoot apices, except for ClCOL6–ClCOL9, which belong to Group II. The expression levels of ClCOL4–ClCOL5 and ClCOL7–ClCOL8 are up-regulated under inductive short-day (SD) conditions, whereas ClCOL6 is down-regulated under inductive SD conditions. The ClCOL genes exhibit four different diurnal rhythm expressions (Type I–Type IV). The Type I genes (ClCOL4–ClCOL5) are highly transcribed under light. The Type II genes (ClCOL1–ClCOL2, ClCOL10) display increased expression in darkness and are rapidly suppressed under light. Transcripts of ClCOL6–ClCOL9 and ClCOL11, belonging to Type III, are abundant in the late light period or at the beginning of the dark period. ClCOL3 belongs to Type IV, with high expression in the early light period and dark period. The peak expression levels of ClCOL4–ClCOL6 are decreased and postponed in the non-inductive night break (NB) and under long-day (LD) conditions, indicating that those genes may play an essential role in the flowering regulation of C. lavandulifolium. The overexpression of ClCOL5 promotes the flowering of Arabidopsis grown under LD conditions, suggesting that ClCOL5 may function as a flowering enhancer in C. lavandulifolium. This study will be useful not only for the study of the C. lavandulifolium photoperiod-dependent flowering process but also for the genetic manipulation of flowering time-related genes to change the flowering time in the chrysanthemum.

The Chinese traditional chrysanthemum is a notable group of chrysanthemums (Chrysanthemum×morifolium Ramat.) in which the phenotypic characteristics richly vary. At present, there is a serious controversy regarding homonyms and synonyms within this group. Moreover, the current international chrysanthemum classification systems are not comprehensive enough to be used on Chinese traditional chrysanthemums. Thus, we first identified a broad collection of 480 Chinese traditional chrysanthemum cultivars using the unique DNA fingerprints and molecular identities that were established by 20 simple sequence repeat markers. Five loci, which distinguished all of the selected cultivars, were identified as the core loci to establish unique fingerprints and molecular identities with 19 denary digits for each cultivar. A cluster analysis based on Nei's genetic distance indicated that the selected cultivars were clustered according to their horticultural classification. Population structure analysis was subsequently performed with K values ranging from 2 to 14, and the most likely estimate for the population structure was ten subpopulations, which was nearly consistent with the clustering result. Principal component analysis was further performed to verify the classification results. On the basis of the Q-matrices of K = 10, a total of 19 traits were found to be associated with 42 markers. Taken together, these results can serve as starting points for the identification and classification of chrysanthemums based on the polymorphism of microsatellite markers, which is beneficial to promote the marker-assisted breeding and international communication of this marvelous crop.

The FLOWERING LOCUS T (FT) gene plays crucial roles in regulating the transition from the vegetative phase to the reproductive phase. In this study, we isolated an FT homologous gene (denoted as ClFT) from Chrysanthemum lavandulifolium. The sequencing analysis indicated that the promoter of the ClFT gene contains many elements, such as light response, abscisic acid, drought-inducibility response and CIRCADIAN clock elements. The expression patterns of ClFT in different tissues/organs at different developmental stages and its responses to different photoperiods were observed. ClFT is expressed in all tested organs/tissues, with the highest expression level being observed in the leaves of plants with visible floral buds under the short day (SD) condition. Next, we studied the rhythmic expression of ClFT during different photoperiod treatments and found that the level of ClFT increases with additional hours of continuous dark. ClFT accumulates when the continuous dark period is 12 h, regardless of the duration of light period. The ectopic expression of the ClFT gene in wild type Arabidopsis (Col-0) results in early flowering, with high expression levels of endogenous LFY and SOC1 being observed in transgenic Arabidopsis. All results indicated that the ClFT gene plays an evolutionarily conserved role in promoting flowering in inductive short days in C. lavandulifolium and that this gene could serve as a vital target for the genetic manipulation of flowering time in chrysanthemums.

As the key enzyme in the biosynthesis of blue flower color pigments, flavonoid 3',5'-hydroxylase (F3'5'H) can catalyze the conversion of its major substrates, 2-S naringenin and dihydrokaempferol, into 3',4',5'-hydroxylated pentahydroxyflavanone and dihydromyricetin, respectively. Unlike other F3'5'Hs belonging to the CYP75A subfamily, Asteraceae-specific F3'5'Hs belong to the CYP75B subfamily. Furthermore, cineraria F3'5'H expressed in yeast exhibited not only F3'H (flavonoid 3'-hydroxylase) activity but also F3'5'H activity in vitro. In this study, Southern blotting showed that there was only one copy of a homolog of the F3'5'H gene PCFH in the Pericallis × hybrida genome. This gene could be detected by Northern blot in the primary developmental stages of ligulate florets of the purple- and blue-flowered cultivars, and its transcripts also accumulated in the leaves. Heterologous expression of PCFH could produce new delphinidin derivatives in the corollas of transgenic tobacco plants, increased the content of cyanidin derivatives and lead to the blue- and red-shifting of flower color in T₀ generation plants. These results indicate that cineraria F3'5'H exhibited both F3'5'H- and F3'H-activity in vivo. The types and contents of anthocyanins and flower color phenotypes of the T₁ generation were similar to those of T₀ generation plants. PCFH exhibited stable inheritance and normal functions between generations. This study supplies new evidence to understand Asteraceae-specific F3'5'Hs and provides important references for the further study of molecular breeding of blue-flowered chrysanthemums using the PCFH gene.

To investigate the diversity of root endophytes in Rhododendron fortunei, fungal strains were isolated from the hair roots of plants from four habitats in subtropical forests of China. In total, 220 slow-growing fungal isolates were isolated from the hair roots of R. fortunei. The isolates were initially grouped into 17 types based on the results of internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with known sequences of ericoid mycorrhizal endophytes and selected ascomycetes or basidiomycetes. Based on phylogenetic analysis of the ITS sequences in GenBank, 15 RFLP types were confirmed as ascomycetes, and two as basidiomycetes; nine of these were shown to be ericoid mycorrhizal endophytes in experimental cultures. The only common endophytes of R. fortunei were identified as Oidiodendron maius at four sites, although the isolation frequency (3-65%) differed sharply according to habitat. Phialocephala fortinii strains were isolated most abundantly from two habitats which related to the more acidic soil and pine mixed forests. A number of less common mycorrhizal RFLP types were isolated from R. fortunei at three, two, or one of the sites. Most of these appeared to have strong affinities for some unidentified root endophytes from Ericaceae hosts in Australian forests. We concluded that the endophyte population isolated from R. fortunei is composed mainly of ascomycete, as well as a few basidiomycete strains. In addition, one basidiomycete strain was confirmed as a putative ericoid mycorrhizal fungus.

A cDNA clone was isolated from tree peony (Paeonia suffruticosa) subtractive cDNA library of burst buds and characterized with regard to its sequence, expression in response to chilling treatment during the release of bud dormancy, and its function in transgenic Arabidopsis thaliana. The clone, designated as PsMPT, contains 1,615 nucleotides with an open reading frame of 1,119 nucleotides, and the deduced amino acid sequence shows high homology with mitochondrial phosphate transporters (MPTs) from various organisms. The mRNA accumulation of PsMPT in tree peony was strongly induced by chilling treatment during the release of bud dormancy. When the treated plants were transferred to normal growth conditions, the level of PsMPT transcripts induced by sufficient chilling could be maintained high, whereas that induced by insufficient chilling decreased sharply. The transgenic Arabidopsis plants that overexpress PsMPT showed rapid growth and earlier flowering than wild-type plants. ATP contents in the transgenic plants were much higher than that in wild-type plants through various developmental stages. Together, these results suggest that the product of PsMPT is a MPT and might play an important role during the release of bud dormancy in tree peony.