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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 ferriceanthocyanin 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. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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]

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. [ABSTRACT FROM AUTHOR]

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 ScF30H1, ScF3050H, 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 ScF305 0H, ScF30H, and ScDFRs) for flower color modification of ornamentals. [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]

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. [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]