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Although some species that accumulate only cyanidin (Cy) in nature can produce blue flowers through iron ions, there has been no evidence of blue chrysanthemums being generated in this manner. This study revealed that flavonoid extracts from the ray florets of the chrysanthemum cultivar ‘Wandai Fengguang’ turned blue when exposed to Fe3+. Samples that could turn blue were labeled as CB (Cy-determined blue flowers), while samples that did not turn blue were labeled as CN (Cy-determined non-blue flowers). After a series of experiments, a stable screening system was established using flavonoid extracts containing NaAc buffer at pH 5.5 and a total anthocyanin concentration (TAC) of 30 μmol·L−1, and the addition of Fe3+ from 0 to 0.25 μmol·L−1 allowed for the selection of five CB samples from 39 chrysanthemum cultivars. All five CB samples exhibited flower color phenotypes that belonged to Cluster-I with redness (a∗) values ranging from 29.03 to 45.99, yellowness (b∗) values from −11.31 to 3.77, and brightness (L∗) values from 29.07 to 45.99. Additionally, the ratio of TAC to total luteolin concentration (TLC) was found to be a critical factor for distinguishing between CB and CN samples. To realize the desired blue hue in the flavonoid extracts with the participation of Fe3+, a TAC to TLC ratio of 2.25 and above is required. Moreover, the protoplasts and ray florets of CB samples that turned blue with the involvement of Fe2+ showed great potential for cultivating blue chrysanthemums through ferric–anthocyanin chelate. Overall, this study reveals that blue flowers can be cultivated through the increase in the iron ion concentration, combined with the accumulation of Cy.

MYBs constitute the second largest transcription factor (TF) superfamily in flowering plants with substantial structural and functional diversity, which have been brought into focus because they affect flower colors by regulating anthocyanin biosynthesis. Up to now, the genomic data of several Chrysanthemum species have been released, which provides us with abundant genomic resources for revealing the evolution of the MYB gene family in Chrysanthemum species. In the present study, comparative analyses of the MYB gene family in six representative species, including C. lavandulifolium, C. seticuspe, C. ×morifolium, Helianthus annuus, Lactuca sativa, and Arabidopsis thaliana, were performed. A total of 1104 MYBs, which were classified into four subfamilies and 35 lineages, were identified in the three Chrysanthemum species (C. lavandulifolium, C. seticuspe, and C. ×morifolium). We found that whole-genome duplication and tandem duplication are the main duplication mechanisms that drove the occurrence of duplicates in CmMYBs (particularly in the R2R3-MYB subfamily) during the evolution of the cultivated chrysanthemums. Sequence structure and selective pressure analyses of the MYB gene family revealed that some of R2R3-MYBs were subjected to positive selection, which are mostly located on the distal telomere segments of the chromosomes and contain motifs 7 and 8. In addition, the gene expression analysis of CmMYBs in different organs and at various capitulum developmental stages of C. ×morifolium indicated that CmMYBS2, CmMYB96, and CmMYB109 might be the negative regulators for anthocyanin biosynthesis. Our results provide the phylogenetic context for research on the genetic and functional evolution of the MYB gene family in Chrysanthemum species and deepen our understanding of the regulatory mechanism of MYB TFs on the flower color of C. ×morifolium.

Abstract Background RWP-RKs are plant specific transcription factors, which are widely distributed in plants in the form of polygenic families and play key role in nitrogen absorption and utilization, and are crucial to plant growth and development. However, the genome-wide identification and function of RWP-RK in Compositae plants are widely unknown. Results In this study, 101 RWP-RKs in Chrysanthemum lavandulifolium were identified and tandem repeat was an important way for the expansion of RWP-RKs in Compositae species. 101 RWP-RKs contain 38 NIN-like proteins (NLPs) and 31 RWP- RK domain proteins (RKDs), as well as 32 specific expansion members. qRT-PCR results showed that 7 ClNLPs in leaves were up-regulated at the floral transition stage, 10 ClNLPs were negatively regulated by low nitrate conditions, and 3 of them were up-regulated by optimal nitrate conditions. In addition, the flowering time of Chrysanthemum lavandulifolium was advanced under optimal nitrate conditions, the expression level of Cryptochromes (ClCRYs), phytochrome C (ClPHYC) and the floral integration genes GIGANTEA (ClGI), CONSTANS-LIKE (ClCOL1, ClCOL4, ClCOL5), FLOWERING LOCUS T (ClFT), FLOWERING LOCUS C (ClFLC), SUPPRESSOR OF OVER-EXPRESSION OF CONSTANS 1 (ClSOC1) also were up-regulated. The expression level of ClCRY1a, ClCRY1c, ClCRY2a and ClCRY2c in the vegetative growth stage induced by optimal nitrate reached the expression level induced by short-day in the reproductive growth stage, which supplemented the induction effect of short-day on the transcription level of floral-related genes in advance. Conclusions It was speculated that ClNLPs may act on the photoperiodic pathway under optimal nitrate environment, and ultimately regulate the flowering time by up-regulating the transcription level of ClCRYs. These results provide new perspective for exploring the mechanism of nitrate/nitrogen affecting flowering in higher plants.

Flower color is an important trait for ornamental plants and is determined by pigments. Variations in the flower color of chrysanthemum are abundant, which is an important feature related to its ornamental and commercial value. However, few reports have elucidated the inheritance of the flower color and pigmentation of chrysanthemum. In this study, an F1 hybrid population, including 319 hybrids of small-flowered chrysanthemum cultivars with different flower color, was constructed. The genetic variation rules for some color and pigment-related traits were discussed based on major gene and polygene mixed inheritance analyses. Both the total anthocyanin and carotenoid contents were controlled by two pairs of additive dominant major genes and the heritability of the major genes was 70.44% and 86.03%, respectively. Based on the high-density genetic map constructed for the hybrid population, QTL analysis for the above traits was carried out using the interval mapping method. A total of 25 related QTLs and 517 closely linked markers were detected, including three QTLs controlling the total anthocyanin content and two QTLs controlling the total carotenoid content, all of which were major QTLs. Furthermore, 17 unigenes related to pigments were identified via BLAST searches with Chrysanthemum nankingense genome. For such genes, the collinearity was observed in four chromosome level genomes, including three genomes of the genus Chrysanthemum and the first genome of cultivated chrysanthemum. The results lay a foundation for in-depth exploration of flower color and pigmentation in chrysanthemum and provide a reference for future research in other ornamental plants.

TCP proteins, part of the transcription factors specific to plants, are recognized for their involvement in various aspects of plant growth and development. Nevertheless, a thorough investigation of TCPs in Chrysanthemum lavandulifolium, a prominent ancestral species of cultivated chrysanthemum and an excellent model material for investigating ray floret (RF) and disc floret (DF) development in Chrysanthemum, remains unexplored yet. Herein, a comprehensive study was performed to analyze the genome-wide distribution of TCPs in C. lavandulifolium. In total, 39 TCPs in C. lavandulifolium were identified, showing uneven distribution on 8 chromosomes. Phylogenetic and gene structural analyses revealed that ClTCPs were grouped into classes I and II. The class II genes were subdivided into two subclades, the CIN and CYC/TB1 subclades, with members of each clade having similar conserved motifs and gene structures. Four CIN subclade genes (ClTCP24, ClTCP25, ClTCP26, and ClTCP27) contained the potential miR319 target sites. Promoter analysis revealed that ClTCPs had numerous cis-regulatory elements associated with phytohormone responses, stress responses, and plant growth/development. The expression patterns of ClTCPs during capitulum development and in two different florets were determined using RNA-seq and qRT-PCR. The expression levels of TCPs varied in six development stages of capitula; 25 out of the 36 TCPs genes were specifically expressed in flowers. Additionally, we identified six key ClCYC2 genes, which belong to the class II TCP subclade, with markedly upregulated expression in RFs compared with DFs, and these genes exhibited similar expression patterns in the two florets of Chrysanthemum species. It is speculated that they may be responsible for RFs and DFs development. Subcellular localization and transactivation activity analyses of six candidate genes demonstrated that all of them were localized in the nucleus, while three exhibited self-activation activities. This research provided a better understanding of TCPs in C. lavandulifolium and laid a foundation for unraveling the mechanism by which important TCPs involved in the capitulum development.

Abstract Background The ray and disc florets on the chrysanthemum capitulum are morphologically diverse and have remarkably abundant variant types, resulting in a rich variety of flower types. An anemone shape with pigmented and elongated disk florets is an important trait in flower shape breeding of chrysanthemums. The regulatory mechanism of their anemone-type disc floret formation was not clear, thus limiting the directional breeding of chrysanthemum flower types. In this study, we used morphological observation, transcriptomic analysis, and gene expression to investigate the morphogenetic processes and regulatory mechanisms of anemone-type chrysanthemum. Result Scanning electron microscopy (SEM) observation showed that morphological differences between non-anemone-type disc florets and anemone-type disc florets occurred mainly during the petal elongation period. The anemone-type disc florets elongated rapidly in the later stages of development. Longitudinal paraffin section analysis revealed that the anemone-type disc florets were formed by a great number of cells in the middle layer of the petals with vigorous division. We investigated the differentially expressed genes (DEGs) using ray and disc florets of two chrysanthemum cultivars, 082 and 068, for RNA-Seq and their expression patterns of non-anemone-type and anemone-type disc florets. The result suggested that the CYCLOIDEA2 (CYC2s), MADS-box genes, and phytohormone signal-related genes appeared significantly different in both types of disc florets and might have important effects on the formation of anemone-type disc florets. In addition, it is noteworthy that the auxin and jasmonate signaling pathways might play a vital role in developing anemone-type disc florets. Conclusions Based on our findings, we propose a regulatory network for forming non-anemone-type and anemone-type disc florets. The results of this study lead the way to further clarify the mechanism of the anemone-type chrysanthemum formation and lay the foundation for the directive breeding of chrysanthemum petal types.

Chrysanthemum ×morifolium is well known throughout the world for its diverse and exquisite flower types. However, due to the complicated genetic background of C. ×morifolium, it is difficult to understand the molecular mechanism of its flower development. And it limits the molecular breeding of improving chrysanthemum flower types. C. ×morifolium has the typical radial capitulum, and many researches showed that the members of the MIKCc-type MADS box gene family play a key role in the formation and development of the capitulum. However, it has been difficult to isolate the important MIKCc and investigate their roles in this process due to the lack of genomic information in chrysanthemum. Here, we identified MIKCc-type MADS box genes at whole genome-wide level in C. lavandulifolium, a diploid species closely related to C. ×morifolium, and investigated their roles in capitulum development by gene expression pattern analysis and protein interaction analysis. A total of 40 ClMIKCc were identified and were phylogenetically grouped into 12 clades. Members of all clades showed different enriched expression patterns during capitulum formation. We speculate that the E-class genes in C. lavandulifolium underwent subfunctionalization because they have a significantly expanded, more diverse expression patterns, and specifically tissue expression than AtSEPs. Meanwhile, we detected the C-class expressed in disc floret corolla, which could be the clue to explore the morphological differences between disc and ray floret corolla. In addition, the potential roles of some MIKCcs in complex inflorescence formation were explored by comparing the number and phylogenetic relationship of MIKCc subfamily members in Asteraceae with different capitulum types. Members of the FLC branch in Asteraceae were found to be possibly related to the differentiation and development of the ray floret.

Day-neutral multiflora chrysanthemums can flower throughout the year without being influenced by daylength and have great application value in gardens. Studying heterosis and the genetic basis of important traits in day-neutral chrysanthemums can accelerate the breeding of new cultivars. In this research, a genetic population was constructed by crossing 135 F1 hybrid progeny from the day-neutral chrysanthemum ‘82-81-19’ (female parent) and the late-flowering chrysanthemum ‘388Q-76’ (male parent). Six traits, including abnormal (crown) bud, plant height, plant crown width, budding date, full flowering date, and number of petal layers, were selected for inheritance and heterosis analyses, and a single-generation major gene plus polygene mixed inheritance model was used to perform mixed inheritance analysis on these traits. The results indicated that the six traits were widely segregated in the F1 population, with the coefficient of variation (CV) ranging from 30% to 84%. The phenomena of heterosis and extra-parent segregation existed generally in F1 progeny, and the ratio of heterosis value of mid-parents (RHm) for the six traits was 45.5%, 2%, 2%, 6%, 6%, and −0.3%, respectively. The mixed genetic analysis showed that the abnormal (crown) bud and budding date were fitted to the B-3 model and controlled by two pairs of additive major genes. The plant height and plant crown width were fitted to the A-0 model, and no major gene was detected. The full flowering date was fitted to the A-1 model and was controlled by one pair of major genes. The number of petal layers was fitted to the B-1 model and controlled by two pairs of additive–dominant major genes. The heritabilities of major genes for abnormal bud, budding date, full flowering date, and the number of petal layers were 1.0, 0.9871, 0.7240, and 0.5612, respectively, indicating that these traits were less affected by environmental factors. Using a percentile scoring method, eight day-neutral chrysanthemum genotypes were selected from the hybrid progeny.

Abstract 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 L 2 -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.

The traditional Chinese large-flowered chrysanthemum is one of the cultivar groups of chrysanthemum (Chrysanthemum × morifolium Ramat.) with great morphological variation based on many cultivars. Some experts have established several large-flowered chrysanthemum classification systems by using the method of comparative morphology. However, for many cultivars, accurate recognition and classification are still a problem. Combined with the comparative morphological traits of selected samples, we proposed a multi-information model based on deep learning to recognize and classify large-flowered chrysanthemum. In this study, we collected the images of 213 large-flowered chrysanthemum cultivars in two consecutive years, 2018 and 2019. Based on the 2018 dataset, we constructed a multi-information classification model using non-pre-trained ResNet18 as the backbone network. The model achieves 70.62% top-5 test accuracy for the 2019 dataset. We explored the ability of image features to represent the characteristics of large-flowered chrysanthemum. The affinity propagation (AP) clustering shows that the features are sufficient to discriminate flower colors. The principal component analysis (PCA) shows the petal type has a better interpretation than the flower type. The training sample processing, model training scheme, and learning rate adjustment method affected the convergence and generalization of the model. The non-pre-trained model overcomes the problem of focusing on texture by ignoring colors with the ImageNet pre-trained model. These results lay a foundation for the automated recognition and classification of large-flowered chrysanthemum cultivars based on image classification.

Virus-induced gene silencing (VIGS) is a genetic tool used to assess gene function. Tobacco rattle virus (TRV) is a VIGS vector commonly used to induce endogenous gene silencing in plants. However, there is no VIGS system established for Centaurea spp. We evaluated the effectiveness of a TRV-based VIGS system using phytoene desaturase (PDS) as a reporter gene in Centaurea cyanus. Three methods including pressure-, vacuum- and apical meristem-infiltration were tested to infect C. cyanus seedlings. Photobleached leaves were only obtained using apical meristem-infiltration after a 14 d treatment. The CcPDS transcripts in photobleached leaves were significantly reduced compared with that in green leaves treated with empty TRV. Four C. cyanus cultivars were tested to detect their VIGS responses, and ‘Dwarf Tom Pouce Blue’ was the most sensitive. The agro-infiltration condition was optimized by screening for the optimal seedling stage as well as the optimum Agrobacterium density for efficient silencing. Seedlings with four true leaves and infiltration with an Agrobacterium density of OD600 0.5 were optimal conditions to obtain more photobleached leaves and more intense photobleached phenotype. The results demonstrated the feasibility of TRV-based VIGS for functional analysis of genes in C. cyanus.

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.

Abstract 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.

Abstract 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.

Abstract Background Roses are important plants for human beings with pivotal economical and biological traits like continuous flowering, flower architecture, color and scent. Due to frequent hybridization and high genome heterozygosity, classification of roses and their relatives remains a big challenge. Results Here, to identify potential markers for phylogenetic reconstruction and to reveal the patterns of natural selection in roses, we generated sets of high quality and comprehensive reference transcriptomes for Rosa chinensis ‘Old Blush’ (OB) and R. wichuriana ‘Basye’s Thornless’ (BT), two species exhibiting contrasted traits of high economical importance. The assembled reference transcriptomes showed transcripts N50 above 2000 bp. Two roses shared about 10,073 transcripts (N50 = 2282 bp), in which a set of 5959 transcripts was conserved within genera of Rosa. Further comparison with species in Rosaceae identified 4447 transcripts being common (Rosaceae-common) in Rosa, Malus, Prunus, Rubus, and Fragaria, while a pool of 164 transcripts being specific for roses (Rosa-specific). Among the Rosaceae-common transcripts, 409 transcripts showed a signature of positive selection and a clustered expression in different tissues. Interestingly, nine of these rapidly evolving genes were related to DNA damage repair and responses to environmental stimulus, a potential associated with genome confliction post hybridization. Coincident with this fast evolution pattern in rose genes, 24 F-box and four TMV resistant proteins were significantly enriched in the Rosa-specific genes. Conclusions We expect that these Rosaceae-common and Rosa-specific transcripts should facilitate the phylogenetic analysis of Rosaceae plants as well as investigations of Rosa-specific biology. The data reported here could provide fundamental genomic tools and knowledge critical for understanding the biology and domestication of roses and for roses breeding.

Polyacylated anthocyanins with multiple glycosyl and aromatic acyl groups tend to make flowers display bright and stable blue colours. However, there are few studies on the isolation and functional characterization of genes involved in the polyacylated anthocyanin biosynthesis mechanism, which limits the molecular breeding of truly blue flowers. Senecio cruentus is an important potted ornamental plant, and its blue flowers contain 3′,7-polyacylated delphinidin-type anthocyanins that are not reported in any other plants, suggesting that it harbours abundant gene resources for the molecular breeding of blue flowers. In this study, using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis of blue, carmine and white colours of cineraria cultivars “Venezia” (named VeB, VeC, and VeW, respectively), we found that 3′,7-polyacylated anthocyanin, cinerarin, was the main pigment component that determined the blue colour of ray florets of cineraria. Based on the transcriptome sequencing and differential gene expression (DEG) analysis combined with RT- and qRT-PCR, we found two genes encoding uridine diphosphate glycosyltransferase, named ScUGT1 and ScUGT4; two genes encoding acyl-glucoside-dependent glucosyltransferases which belong to glycoside hydrolase family 1 (GH1), named ScAGGT11 and ScAGGT12; one gene encoding serine carboxypeptidase-like acyltransferase ScSCPL2; and two MYB transcriptional factor genes ScMYB2 and ScMYB4, that were specifically highly expressed in the ray florets of VeB, which indicated that these genes may be involved in cinerarin biosynthesis. The function of ScSCPL2 was analysed by virus-induced gene silencing (VIGS) in cineraria leaves combined with HPLC-MS/MS. ScSCPL2 mainly participated in the 3′ and 7-position acylation of cinerarin. These results will provide new insight into the molecular basis of the polyacylated anthocyanin biosynthesis mechanism in higher plants and are of great significance for blue flower molecular breeding of ornamental plants.

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.

Leaf shape is an important quality trait of agronomic crops, and to control the law of genetic variation of leaf shape is of practical significance for improving the early identification and selection of agronomic crops. Variations in the leaf morphology of chrysanthemum cultivars are abundant, and previous studies have quantitatively defined and classified the leaf morphology of chrysanthemum; however, the genetic architecture of chrysanthemum leaves has not been elucidated to date. In this study, two pairs of F1 hybrid populations were constructed by using small-flower chrysanthemum varieties with differences in leaf traits, and the genetic variation rules of these important quantitative traits were further discussed based on the major gene and polygene mixed inheritance analyses. The results showed that the leaves in blade shape (LBS), leaf length/width is controlled by two pairs of additive-dominant major genes (B-1), the widest part length/leaf length is controlled by two completely dominant genes (B-5); in leaf lobe shape (LLS), the lobe length/vein length is controlled by one pair of additive dominant major genes (A-1); and the lobe length/lobe width is controlled by two pairs of additive dominant major genes (B-2). The heritability of major genes was greater than 30%. For the leaf petiole shape (LPS), the petiole length is controlled by a pair of additive-dominant major genes (A-1). The results showed that the leaf traits were mainly controlled by genetic factors. In addition, based on the high-density genetic map of one F1 hybrid population, it was found that 51 quantitative trait loci (QTL) were used to control the leaf traits, including two QTLs that controlled the LBS. There were 18 QTLs that controlled LLS. Moreover, the primary QTLs that controlled leaf width and lobe length were obtained. The results of this study may establish a theoretical foundation for the in-depth exploration of leaf-shape-related genes in chrysanthemum and may provide a reference for future research investigating leaf-shape genetics in other agronomic crops.

The generation of chrysanthemum (Chrysanthemum × 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 < 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 < 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.

The cyanidin (Cy), pelargonidin (Pg) and delphinidin (Dp) pathways are the three major branching anthocyanin biosynthesis pathways that regulate flavonoid metabolic flux and are responsible for red, orange and blue flower colors, respectively. Different species have evolved to develop multiple regulation mechanisms that form the branched pathways. In the current study, five Senecio cruentus cultivars with different colors were investigated. We found that the white and yellow cultivars do not accumulate anthocyanin and that the blue, pink and carmine cultivars mainly accumulate Dp, Pg and Cy in differing densities. Subsequent transcriptome analysis determined that there were 43 unigenes encoding anthocyanin biosynthesis genes in the blue cultivar. We also combined chemical and transcriptomic analyses to investigate the major metabolic pathways that are related to the observed differences in flower pigmentation in the series of S. cruentus. The results showed that mutations of the ScbHLH17 and ScCHI1/2 coding regions abolish anthocyanin formation in the white and the yellow cultivars; the competition of the ScF3’H1, ScF3’5’H and ScDFR1/2 genes for naringenin determines the differences in branching metabolic flux of the Cy, Dp and Pg pathways. Our findings provide new insights into the regulation of anthocyanin branching and also supplement gene resources (including ScF3’5’H, ScF3’H and ScDFRs) for flower color modification of ornamentals.

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.

Genetic transformation is one of the key steps in the molecular breeding of chrysanthemum, which relies on an optimal regeneration and transformation system. However, the regeneration system of different chrysanthemum cultivars varies, and the regeneration time of most cultivars is long. To screen cultivars with highly efficient regeneration, leaves and shoot tip thin cell layers (tTCL) from eight chrysanthemum cultivars with different flower colors and flower types were cultured on Murashige and Skoog media (MS) supplemented with 1.0-5.0 mg L-1 6-benzylaminopurine (6-BA) and 0.1-1.0 mg L-1 α-naphthaleneacetic (NAA). The results showed that the most efficient regeneration media were MS + 6-BA 1.0 mg L-1 + NAA 0.5 mg L-1 for leaf explants and MS + 6-BA 5.0 mg L-1 + NAA 0.1 mg L-1 for tTCL explants. Subsequently, another 13 chrysanthemum cultivars were screened by using the media, and finally, three cultivars with high regeneration efficiency were obtained from 21 cultivars. Among these, C1 had the highest regeneration efficiency: the regeneration rate of leaf explants reached 80.0% after 42 days of culture, and the regeneration rate of tTCL explants reached 100% after 31 days of culture. Furthermore, we also established the transformation system for C1 as follows: preculturing for one day, infecting with Agrobacterium suspension (OD600 = 0.6) for 10 min, and cultivating in the regeneration medium with 350 mg L-1 carbenicillin and 10 mg L-1 kanamycin, thus ultimately achieving a transformation rate of 4.0%. In this study, a new chrysanthemum cultivar with an efficient regeneration and transformation system was screened, which is beneficial to enrich the flower color of chrysanthemum transgenic plant recipients and to the functional research of flower color or type-related genes. [ABSTRACT FROM AUTHOR]

In this study, five cultivars of cut chrysanthemum Chrysanthemum×morifolium Ramat., 'Jinba', 'Yuuka', 'Fenguiren', 'Xueshen', and 'Huangjin' were used to explore the functions of 5-azacytidine (5-azaC) on chrysanthemum growth and flower development. The results showed that 5-azaC had different effects on the growth of the five cultivars during in vitro culture. The final statistics showed that low concentrations promoted plant growth, whereas high concentrations inhibited growth; however, each cultivar had different growth curves, demonstrating that 5-azaC had no consistent inhibitory actions on growth. On the basis of the squaring time and flowering time statistics, we found that 5-azaC had a certain effect on the flowering time of all cut chrysanthemums, and all of these cultivars showed extremely early strains. Summer chrysanthemum ('Yuuka', 'Fenguiren', 'Xueshen', and 'Huangjin') treatments led to both early and delayed flowering. When the statistics were analyzed for different individuals, we found that the treatments shortened the squaring time in early-flowering plants. In 'Jinba', an autumn chrysanthemum, the treatment helped broken juvenile limitations and allowed plants to undergo photoperiod induction in the early stage. Additionally, we also determined the flower diameter differences in these treatments; ray florets from 'Yuuka' and 'Huangjin' trended to show tubular florets, and the location of tubular and ray florets were changed in 'Xueshen' capitulum. In conclusion, on the basis of flowering time in five early varieties of cut chrysanthemum, we propose that 5-azaC may regulate the methylation level of genes that control flower induction and flower development. These results provide phenotypic data and material for exploring the function of DNA methylation in regulating flowering. [ABSTRACT FROM AUTHOR]

Quantitative real-time PCR (qPCR) is a popular and powerful tool used to understand the molecular mechanisms of flower development. However, the accuracy of this approach depends on the stability of reference genes. The capitulum of chrysanthemums is very special, which is consisting of ray florets and disc florets. There are obvious differences between the two types of florets in symmetry, gender, histological structure, and function. Furthermore, the ray florets have various shapes. The objective of present study was to identify the stable reference genes in Chrysanthemum morifolium and Chrysanthemum lavandulifolium during the process of flower development. In this study, nine candidate reference genes were selected and evaluated for their expression stability acrosssamples during the process of flower development, and their stability was validated by four different algorithms (Bestkeeper, NormFinder, GeNorm, and Ref-finder). SAND (SAND family protein) was found to be the most stably expressed gene in all samples or different tissues during the process of C. lavandulifolium development. Both SAND and PGK (phosphoglycerate kinase) performed most stable in Chinese large-flowered chrysanthemum cultivars, and PGK was the best in potted chrysanthemums. There were differences in best reference genes among varieties as the genetic background of them were complex. These studies provide guidance for selecting reference genes for analyzing the expression pattern of floral development genes in chrysanthemums. [ABSTRACT FROM AUTHOR]

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 x 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. [ABSTRACT FROM AUTHOR]

Abstract  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. [ABSTRACT FROM AUTHOR]

Chrysanthemum (Chrysanthemum × morifolium) is one of the most important ornamental plants in the world. They are typically used as cut flowers or potted plants. Chrysanthemum can exhibit red, purple, pink, yellow and white flowers, but lack bright red and blue flowers. In this study, we identified two chrysanthemum cultivars, C × morifolium ‘LPi’ and C × morifolium ‘LPu’, that only accumulate flavonoids in their ligulate flowers. Next, we isolated seven anthocyanin biosynthesis genes, namely CmCHS, CmF3H, CmF3’H, CmDFR, CmANS, CmCHI and Cm3GT in these cultivars. RT-PCR and qRT-PCR analyses showed that CmF3′H was the most important enzyme required for cyanidin biosynthsis. To rebuild the delphinidin pathway, we downregulated CmF3’H using RNAi and overexpressed the Senecio cruentus F3′5′H (PCFH) gene in chrysanthemum. The resultant chrysanthemum demonstrated a significantly increased content of cyanidin and brighter red flower petals but did not accumulate delphinidin. These results indicated that CmF3′H in chrysanthemum is important for anthocyanin accumulation, and Senecio cruentus F3′5′H only exhibited F3′H activity in chrysanthemum but did not rebuild the delphinidin pathway to form blue flower chrysanthemum. [ABSTRACT FROM AUTHOR]

The large-flowered Chinese chrysanthemum is one of the most morphologically complex ornamental plants, and its identification and classification requires a welldefined and reproducible system. The diversity of the capitulum is determined mainly by multiple shapes of ray florets. However, the existing classification systems for ray floret types are incomplete and unsystematic. In this study, 299 ray florets from 151 large- flowered chrysanthemum varieties in China, as well as 12 related traits of ray florets, were selected for quantitative classification. First, as one of the most important indices of ray floret shape, the corolla tube merged degree (CTMD) was defined as the corolla tube length/ray floret length (CTL/RFL). Combined with a probability grading method and linear regression analysis, the CTMD was divided into three groups, flat, spoon, and tubular, of which the CTL/RFL ranged from 0 to 0.20, 0.20 to 0.60, and 0.60 to 1.00, respectively. Second, Q-mode cluster analysis indicated that each group could be further categorized into three types (straight, curved, and atypical), based on other important variables in the ray floret. Finally, the ray floret was classified into nine types, including flat-straight, flat-curve, flat-atypical, spoon-straight, spoon-curve, spoon-atypical, tubular-straight, tubular-curve, and tubular-atypical. This ray floret classification system will be valuable in the classification of capitulum shape and has significance for the identification, breeding, and international standardization of chrysanthemum cultivars. [ABSTRACT FROM AUTHOR]

Conventional crossbreeding remains an effective technique for chrysanthemum (Chrysanthemum ×morifolium Ramat.) breeding. However, there are always many problems when breeding chrysanthemum because of its complex genetic background, such as difficulty matching parents, selecting superior hybrid progenies, quantitatively describing certain target traits, and evaluating breeding results. A recent mathematical analysis method is an effective method for evaluating plant breeding progress. In this study, we used 505 multiflora chrysanthemum germplasm resources as test materials; we divided the flowering time into five groups using a grading analysis method, including extremely early group (genotypes that flowered when daylength was longer than 13.5 hours), early group (genotypes that flowered when daylength was 13.5-12.0 hours), medium group (genotypes that flowered when daylength was 12.0-11.0 hours), late group (genotypes that flowered when daylength was 11.0-10.0 hours), and extremely late group (genotypes that flowered when daylength was shorter than 10.0 hours). Moreover, the breeding objective was to breed early-flowering genotypes. Using 15 phenotypic characters as evaluation factors, 37 excellent genotypes, including four early-flowering genotypes, were screened out from the aforementioned resources according to an analytic hierarchy process (AHP) and weighting of the gray relational grade. We selected one early-flowering genotype and eight medium-flowering genotypes from these 37 genotypes and matched six hybridized combinations based on the genetic distance between genotypes calculated by theQcluster analysis method.Weused a comprehensive evaluation method combining AHP and the gray relational analysis (GRA) method for the evaluation of 367 progenies. Moreover, we screened out 52 superior hybrids, including 36 early-flowering hybrids. The results of this study demonstrate that the mathematical analysis method is an immensely effective method to breed new cultivars of early-flowering multiflora chrysanthemum. This study also provides an effective method to define and improve the flowering time of other cultivated plants. [ABSTRACT FROM AUTHOR]

Abstract: Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is a halophyte species that belongs to the Asteraceae family, and the genus Chrysanthemum. It is one of the ancestors of C. × morifolium Ramatella. Understanding the tolerance mechanism associated with salt stress in C. lavandulifolium could provide important information for explaining the salt tolerance of higher plants and could also help enhancing breeding programs of cultivated Chrysanthemum. In this study, cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to detect differential gene expression in leaves of C. lavandulifolium in response to NaCl treatment. The determination of membrane permeablility, peroxidase activity (POD), malon-dialdehyde (MDA), as well as proline and leaf chlorophyll contents under different NaCl concentrations showed that a 200mM NaCl treatment was an optimal condition for the cDNA-AFLP experiment. Using this concentration during different times (0, 3h, 12h, 24h and 48h), we obtained 1930 cDNA fragments using 64 primers. After sequencing 234 randomly chosen cDNA clones and BLASTx analyzing, we got 129 expressed sequence tags (ESTs) which had no significant homology with other sequences, 85 ESTs were homologous to genes with known functions, whereas the rest of ESTs showed homology to unclassified or putative proteins. 25 ESTs that were similar to known functional genes involved in several abiotic and biotic stresses were confirmed by semi-quantitative RT-PCR and qRT-PCR. The expression patterns of these salt-responsive genes not only responded to salt stress but also to plant hormones, such as abscisic acid (ABA), and to other abiotic stresses such as drought and cold. These results indicate an extensive cross-talk among several stresses. Our results provide interesting information for further understanding the molecular mechanisms of salt tolerance in C. lavandulifolium. [Copyright &y& Elsevier]