Comparative Analysis of Morphologic Traits of 50 Large-flowered Chrysanthemum Varieties

With 50 large-flowered Chrysanthemum varieties from germplasm nursery of Wunaoshan Forest Farm in Macheng City as research objects, 64 morphological traits were investigated by field experiments adopting randomized block design. The morphological differences were observed by uniformity analysis, variability analysis, principal component analysis and cluster analysis. The result showed that the vari- able coefficients of 59 traits were greater than 15%; the contribution rate of first seven principal components reached 81.45%; and it was found by clustering analy- sis that the 50 germplasm resources could be divided into four clusters with obvious morphological differences, and plant type could be used as an index for classifica- tion.

CmbHLH110,a novel bHLH transcription factor,accelerates flowering in chrysanthemum

Basic helix-loop-helix(bHLH)transcription factor gene family in plants controls various growth and development aspects;however,the actual roles of these genes in flowering plants are not well known.In this study,a novel bHLH protein CmbHLH110 was found to interact with CmERF110 by in vitro and in vivo experiments,a chrysanthemum ERF110 homolog that acts as a positive flowering regulator.In addition,CmbHLH110 was also found to regulate the flowering of chrysanthemums,overexpression of CmbHLH110 causes chrysanthemums to flower earlier,and suppressed CmbHLH110 leads to delayed flowering.Furthermore,the loss-of-function Arabidopsis mutant of its homologue PERICYCLE FACTOR TYPE-A 5(PFA5)had a noticeable late flowering phenotype,and CmbHLH110 completely complemented the late flowering phenotype of the pfa5 mutant,whereas heterologous overexpression of CmbHLH110 in Arabidopsis Col-0 caused early flowering.Transcriptome sequencing revealed significant differential expression of flowering-related and circadian clock-related genes in transgenic chrysanthemum.Therefore,we concluded that CmbHLH110,as a novel flowering regulator,could interact with CmERF110 to regulate flowering in chrysanthemum.

Transcriptomic Analysis Reveals the Formation Mechanism of Anthocyanins Light-Independent Synthesis in Chrysanthemum

Chrysanthemum×morifolium is a horticultural crop which plays a vital role in theflower industry with signifi-cant economic value and has a cultivation history of over three thousand years in China.The accumulation of anthocyanins is always affected by light.Here,we revealed that anthocyanin accumulation is highly dependent on light in‘2021135’genotype chrysanthemum,while it is light-independent in‘2001402’genotype chrysanthe-mum.However,no literature has been reported regarding the non-photosensitive chrysanthemum in anthocya-nins light-independent synthesis pathways.Through the phenotype analysis of 44 F1 generations,we found that light-independence is a dominant trait which can be stable inherited by progeny.The transcriptome of the rayflorets of‘2021135’and‘2001402’under light and bagging treatment were sequenced and analyzed.Based on weighted gene co-expression network analysis(WGCNA),K-means analysis,and Real-Time Quantitative Poly-merase Chain Reaction(RT-qPCR)analysis,16 genes were highly correlated with the anthocyanin content.The anthocyanin content of rayflorets treated with different light-quality conditions indicated that blue light signifi-cantly affected anthocyanin accumulations.Through Yeast one-hybrid analysis,CmBIC1.1 and CmBIC1.2 can directly regulate the anthocyanin structural gene CmCHS2.In our study,we revealed the important characteristics of light-independent anthocyanin synthesis in chrysanthemums and screened regulatory factors in light-depen-dent and light-independent anthocyanin synthesis pathways.The results laid the groundwork for subsequent ana-lysis of the molecular mechanism involved in the light-independent synthesis of anthocyanins in chrysanthemums.

Flavonoid extracts from chrysanthemum with appropriate anthocyanins turn blue when exposed to iron ions

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 Fe^(3+).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 Fe^(3+)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 Fe^(3+),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 Fe^(2+)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.

FLOWERING LOCUS T(FT)in Photosensitive Type Chrysanthemum Accelerates Flowering in Arabidopsis

FLOWERING LOCUS T(FT)is an important factor for integrating flowering signals through the photoperiod pathway,which significantly promotes flowering.Most chrysanthemum varieties are short-day plants,sensitive to the photoperiod,which limits their ornamental and annual production supply.In this study,we aim to analyze the nutrients and flowering-related genes of chrysanthemums with different photoperiod types and to clone and verify the function of the flowering-related gene CmFT.We found that the formation of floral buds requires the accumulation of starch while consuming soluble sugars and the expression patterns of flowering-related genes GIGANTEA(GI),CONSTANS(CO),and FT in C.morifolium‘Zilian’and C.morifolium‘Zihongtuogui’had a certain synchronization during floral buds differentiation according to our quantitative validation,and the expression levels of CmGI,CmCO and CmFT in C.morifolium‘Zihongtuogui’were higher than those in C.morifolium.‘Zilian’in the later stage of differentiation.CmFT was cloned from photosensitive chrysanthemums-C.morifolium‘Zihongtuogui’and polypeptide alignment and phylogenetic analysis showed that CmFT was clustered in FT-like subfamily.In further functional verification,we obtained two Arabidopsis transgenic lines.Our results showed that CmFT transgenic ft mutant lines can significantly accelerate flowering in Arabidopsis.Thus,we can initially confirm that CmFT plays an important role in promoting flowering,which may be the key reason for the photosensitivity of C.morifolium‘Zihongtuogui’.Overall,the results of this study are of great importance in revealing the flowering mechanism of different photoperiod types of chrysanthemums.

Pharmacological effects of volatile oil from chrysanthemum and its associated mechanisms:a review

Volatile oil(VO)is the main chemical component of common plants in Chrysanthemum genus,and it possesses several beneficial pharmacological properties,including bacteriostatic,antioxidant,anti-tumor,anti-inflammatory,antipyretic,analgesic,antiosteoporotic,antihypertensive,sedative,and hypnotic effects.To date,research on the effective components of Chrysanthemum extract has mainly focused on flavonoids,whereas limited data are available on the chemical constituents and underlying mechanisms of action of the VO components.In this review,the pharmacological activities and mechanisms of VO are comprehensively reviewed with the aim of providing a foundation for further development for medicinal,aromatherapy,and diet therapy applications.

CmMYB3-like negatively regulates anthocyanin biosynthesis and flower color formation during the post-flowering stage in Chrysanthemum morifolium

Color fading caused by a decrease in anthocyanin accumulation during the post-flowering stage significantly affects postharvest quality of chrysanthemum.However,the underlying mechanism by which anthocyanin accumulation decreases during the post-flowering stage still unclear,which greatly restricts design of molecular breeding in chrysanthemum.Here,a chrysanthemum SG7 R2R3 MYB transcription factor(TF),CmMYB3-like,was identified to have a function in regulating anthocyanin biosynthesis during the post-flowering stage.Quantitative real time PCR(qRT-PCR)assays showed that the expression of CmMYB3-like was gradually downregulated when anthocyanin content increased during the flowering stage and was significantly upregulated during the post-flowering stage.Genetic transformation of chrysanthemum and dual-luciferase assays in N.benthamiana leaves showed that CmMYB3-like suppressed anthocyanin accumulation by inhibiting the transcription of CmCHS and CmANS directly and that of CmF3H indirectly.However,overexpression or suppression of CmMYB3-like did not affect the biosynthesis of flavones or flavonols.Genetic transformation of chrysanthemum revealed that the overexpression of CmMYB3-like inhibited anthocyanin accumulation,but its suppression prevented the decrease in anthocyanin accumulation during the post-flowering stage.Our results revealed a crucial role of CmMYB3-like in regulating the color of petals during the post-flowering stage and provided a target gene for molecular design breeding to improve the postharvest quality of chrysanthemum.

Effect of Different Arbuscular Mycorrhizal Fungi Strains on Mineral Nutrition and Antioxidant Enzymes of Chrysanthemum morifolium

[Objective] The purpose was to study the effects of different arbuscular mycorrhizal fungi strains（AMF）on the mineral nutrition and antioxidant enzymes of Chrysanthemum morifolium.[Method] The pot experiment was conducted in the greenhouse,C.morifolium ＇Jinba＇ was inoculated with five kinds of AMF,N,P,K,malondial dehyde（MDA）content,as well as the superoxide dismutase（SOD）,peroxidase（POD） and catalase（CAT） activity in roots,leaves and petals of C.morifolium were measured at seedling and flowering stages.[Result] The G.i,G.e and G.m treatments could promote mineral nutrient absorption,increase N,P,K content in roots,leaves and petals of C.morifolium compared with the control without inoculation.The G.d、G.e and G.m treatments could significantly reduce MDA content in roots and petals,thus alleviating membrane permeability and lipid peroxidation.The G.i treatments could also improve the SOD,POD and CAT activities of C.morifolium,thereby increasing the capability of scavenging oxygen free radicals.[Conclusion] According to the comprehensive analysis,G.i was screened out as the best strain to improve mineral nutrition and antioxidant enzyme activities of C.morifolium.

Antioxidase Activities of Tetraploid Lines and Diploid Control Line of Chrysanthemum cinerariifolium

Objective The SOD, POD and APX enzyme activities of tetraploid lines and diploid control line of Chrysanthemum cinerariifolium in test-tube and field were determined in order to provide a reference for the selective breeding of high resistance good tetraploid varieties of C. cinerariifolium. Method The SOD, POD and APX activities of each sample were determined. Every material should have 3 replicates which been determined and calculated mean value to analyze. Result The various kinds of antioxidase activities in protective enzyme system of tetraploid lines were increased generally in comparison with that of diploid control line. And there is certain relativity between the samples in field and in test-tube of these lines. Conclusion The antioxidase activities of each line in test-tube could be considered as reference index to screen high resistance good tetraploid varieties of C. cinerariifolium.

Effect of Sound Stimulation on the Lipid Physical States and Metabolism of Plasma Membrane from Chrysanthemum Callus

Plasma membrane vesicles of chrysanthemum ( Dendranthema morifolium (Ramat.) Tzvel.) callus was purified by sucrose gradient centrifugation to investigate the influence of sound stimulation on the lipid physical states and metabolism of plasma membrane. The results showed that sound stimulation decreased the content of phosphodiesters and the fluorescent intensity of DPH, but increased the light scattering value of the membrane, the fluorescent intensity of MC540 and the content of phosphomonoesters, indicating that the vesicles got looser, the charge density and hydrophobicity of membrane surface decreased under sound stimulation of some strength and frequency. However, the membrane fluidity increased under the condition. Meanwhile, the anabolism of membrane lipid increased and the catabolism decreased. It can be seen that the physical state and metabolism of membrane lipid is sensitive to sound stimulation.

Research on Pollen Grains of Tetraploid Lines and Diploid Control Line of Chrysanthemum cinerariifolium (Trev.) Vis.

[Objective] The aim was to study characters of pollen grains of tetraploid lines and diploid control line of Chrysanthemum cinerariifolium (Trev.) Vis.,morphological characters,fertility of pollen grain and germination percentage of seeds. [Method] Pollen grains were prepared by sulphuric acid-acetyl oxide decomposition method. The lengths of polar axis and equatorial axis of pollen grains were determined with general optical microscope. The morphology of pollen grains was observed with SEM (scanning electron microscope) and the typical visual fields of 2 500× (or 2 000×),7 000× were taken pictures. [Result] Comparing with the diploid control line,the pollen grains of five tetraploid lines which were tested were different from the diploid line in morphology,sculpture,etc.. 4 of the 5 tested samples were significant larger than the diploid line in size and one was similar to the diploid line. [Conclusion] This research provided references for breeding tetraploid improved varieties of Chrysanthemum cinerariifolium (Trev.) Vis. with good fertility and high germination percentage.

Cloning and Analysis of the cDNA Sequence of XTH Gene from Chrysanthemum morifolium Petal

[Objective] This study aimed to investigate the structural character of xy- Ioglucan endotransglycosylase/hydrolase （XTH） gene in ethylene-insensitive feverfew. [Method] The total RNA was extracted from Chrysanthemum rnorifolium petal using Trizol reagent, and the cDNA fragment of XTH gene was cloned by RT-PCR and T/A cloning. [Result] The sequencing result showed that the cloned cDNA sequence was 911 bp. It was predicted to encode a polypeptide of 293 amino acids and had seven active sites of XTH family, and then named as CmXTH （gene accession number HM752243）. In addition, The BLAST analysis showed that the deduced amino acid sequence of CmXTH showed high homology with other 19 chosen plant XTHs. Among of these, CmXTH shared closer genetic relationship with Gerbera jamesonii, Solanum lycopersicum, whereas had relatively distant relationship with Populus euparatica, Fragaria ananassa, Actinidia deliciosa, etc. [Conclusion] The cloned fragment was certainly the cDNA of XTH gene, which was associated with the petal growth and senescence in Chrysanthemum morifolium.

Optimization on Identification Standards and Artificial Inoculation Methods In Vitro on Resistance to Chrysanthemum White Rust

[Objective] This research aimed at exploring an effective way for inoculation and identification of chrysanthemum white rust under controlled conditions. [Method] By combining the observation methods with the naked eye and under optical microscope, we had established the identification standards for chrysanthemum white rust with six classifications and optimized artificial inoculation methods in vitro. [Result] The results showed that bottled cuttings identification method and petri dished leaves identification method both can be used for identification in vitro of chrysanthemum white rust, bottled cuttings identification method had shown better effects than petri dished leaves identification method, and was supposed to be best artificial inoculation and identification method in vitro. [Conclusion] This research had provided a scientific method for safe and effective researches on chrysanthemum white rust, in order to control the occurrence and diffusion of this quarantine disease.

The Narrative Technique of Odour of Chrysanthemums

Odour of Chrysanthemums is one of the short stories by D. H. Lawrence. The fiction shows how humanity was ruined by bourgeois industrial civilization through the death of a miner. In embedding themes, the narrative technique of Odour of Chrysanthemums is worth our attention. This paper is intended to make an analysis of point of view, focalization and symbol in the light of narrative theory.

Chrysanthemum extract alleviates acetaminophen-induced liver injury by inhibiting oxidative stress via AMPK pathway in rats

OBJECTIVE Acetaminophen(APAP),also known as paracetamol,is a commonly used antipyretic,anal⁃gesic and anti-inflammatory drug.However,during the use of APAP for more than half a century,people have not only used APAP to fight diseases but have also suffered the adverse effects brought about by APAP for more than half a cen⁃tury.The most serious adverse reaction to APAP is hepatotoxicity caused by overdose or long-term use.In Chinese tra⁃ditional medicine,chrysanthemums have the functions of dispelling wind,dissipating heat,clearing the liver and improv⁃ing eyesight.Although the chrysanthemum variety named Bianliang ziyu from Kaifeng is not a medicinal variety,it has good value for medicine and food.The aim of this study was to investigate the protective effect of Bianliang Ziyu extract(BZE)on APAP-damaged rats and the potential molecular mechanism.METHODS Male Sprague-Dawley rats(200-220 g)were intragastrically administered BZE(110,220 and 440 mg·kg^-1)for 8 d.On the ninth day,APAP(800 mg·kg^-1)was administered intragastrically to the rats 0.5 h after BZE administration to induced drug-induced liver injury.The serum and liver samples were collected after 24 h.The levels of alanine aminotransferase(ALT),aspartic aminotransferase(AST),reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione(GSH)in serum and liver tissue of rats were detected by kit method.HE staining was used to observe the histopathological changes in the liver of rat.The effects of BZE on the expression of the oxidative stress related proteins and the mitochondrial biosyn⁃thesis related proteins were detected by Western blot.RESULTS The results showed that BZE significantly reduced the levels of ALT,AST,MDA and ROS and increased the levels of GSH and SOD caused by APAP.Moreover,BZE increased phosphorylation of AMP-activated protein kinase(AMPK)and glycogen synthase kinase 3β(GSK3β),promoted the nuclear translocation of nuclear factor-erythroid 2-related factor 2(Nrf2).BZE also upregulated the expression of mitochondrial biosynthesis related proteins such as peroxisome proliferator-activated receptorγ(PPAR-γ),peroxisome proliferator-activated receptorγcoactivator-1α(PGC-1α),mitochondrial transcription factor(TFAM)and nuclear respira⁃tory factor 1(NRF1).CONCLUSION BZE alleviates APAP-induced liver injury in rats by inhibiting oxidative stress via GSK3β-Nrf2 signaling and the mitochondrial biosynthesis pathway mediated by AMPK.

The Dynamic Changes in Cold Tolerance of Ground-cover Chrysanthemum Growing in the Open Field during the Overwintering

The ground-cover chrysanthemum （Dendranthemaxgrandiflorum Kitamura） ＇Zixunzhang＇ was used as the experimental material in this study. The physiological and biochemical indexes of leaves, emergent rhizomes and roots from ＇Zixunzhang＇ growing in the open field during the autumn and winter in Beijing area were deter- mined. These indexes showed that root activities decreased slowly with the drop in temperature. The relative water contents of leaves and emergent rhizomes, and the water content of roots decreased. The activities of SOD, CAT and POD from leaves and emergent rhizomes were affected significantly by low temperature, but their change trends were different, which suggested that these 3 enzymes might play an interacted role in the formation of the cold tolerance of ＇Zixunzhang＇ mum, while the changes of the three enzyme activities in roots were little when the tem- perature dropped. With the significant increasing of the content of soluble sugar from leaves, the contents of soluble protein and proline declined overall; and the contents of osmotic regulation substances of emergent rhizomes and roots increased at first and then decreased. The correlation analysis indicated that the cold toler- ance of ground-cover mum ＇Zixunzhang＇ was related closely to relative water con- tents, SOD enzyme activity, soluble protein, soluble sugar and root activity. The morphological observation of leaves, emergent rhizomes and roots also showed that the survival of the ground-cover mum mainly depended on the cold tolerance of roots during the overwintering.

Chrysanthemum weyrichii基因组测定及rDNA定位

以Chrysanthemum weyrichii为试材,采用流式细胞术估算其基因组大小,同时利用染色体计数法统计了C.weyrichii的染色体条数并对其rDNA染色体定位进行了研究。结果表明:C.weyrichii基因组大小为10.02Gb,染色条数为2n=8x=72,5S rDNA杂交信号有4对位点,位于染色体的亚中部;45S rDNA杂交信号有8对位点,位于染色体的末端。该研究结果为该物种基因组分析以及菊属植物染色体进化研究提供了依据。

Current achievements and future prospects in the genetic breeding of chrysanthemum:a review

Chrysanthemum(Chrysanthemum morifolium Ramat.)is a leading flower with applied value worldwide.Developing new chrysanthemum cultivars with novel characteristics such as new flower colors and shapes,plant architectures,flowering times,postharvest quality,and biotic and abiotic stress tolerance in a time-and cost-efficient manner is the ultimate goal for breeders.Various breeding strategies have been employed to improve the aforementioned traits,ranging from conventional techniques,including crossbreeding and mutation breeding,to a series of molecular breeding methods,including transgenic technology,genome editing,and marker-assisted selection(MAS).In addition,the recent extensive advances in high-throughput technologies,especially genomics,transcriptomics,proteomics,metabolomics,and microbiomics,which are collectively referred to as omics platforms,have led to the collection of substantial amounts of data.Integration of these omics data with phenotypic information will enable the identification of genes/pathways responsible for important traits.Several attempts have been made to use emerging molecular and omics methods with the aim of accelerating the breeding of chrysanthemum.However,applying the findings of such studies to practical chrysanthemum breeding remains a considerable challenge,primarily due to the high heterozygosity and polyploidy of the species.This review summarizes the recent achievements in conventional and modern molecular breeding methods and emerging omics technologies and discusses their future applications for improving the agronomic and horticultural characteristics of chrysanthemum.

Effects of Acetylsalicylic Acid and Calcium Chloride on Photosynthetic Apparatus and Reactive Oxygen-Scavenging Enzymes in Chrysanthemum Under Low Temperature Stress with Low Light

The effects of acetylsalicylic acid （ASA）, CaCl2, and ASA ＋ CaCl2 on the photosynthetic apparatus and antioxidant enzyme activities were investigated in chrysanthemum Jinba （a cut flower cultivar） under low temperature stress with low light （TL stress） （16/12℃, day/night, PFD 100 μmol m^-2 s-1）. The results showed that under TL stress, the net photosynthesis rate （Pn）, carboxylation efficiency （CE）, apparent quantum yield （AQY）, maximal photochemical efficiency （Fv/Fm） of PSII, quantum yield of PSII electron transport （ФPSII）, and photochemical quenching （qP） of the chrysanthemum leaves in all treatments were significantly decreased, but the decreases were alleviated by ASA, CaCl2, and ASA ＋ CaCl2 treatments compared with the controls. The alleviating effect of ASA ＋ CaCI2 was better than either ASA or CaCl2 single treatment. Moreover, the ASA ＋ CaCl2 treatment highly improved the chlorophyll content, relatively improved the number and size of chloroplast and starch grain in the leaves of chrysanthemum plants compared with ASA and CaCl2 treatments. It was indicated that ASA and/or CaCI2 could regulate the photosynthetic functions in the leaves of chrysanthemum plants to enhance the resistance against TL stress. On the other hand, reduction in relative conductance rate implied that ASA and/ or CaCl2 could protect from membrane injury in leaves of chrysanthemum plants. The activities of SOD, POD, and CAT in the treated leaves of chrysanthemum were increased as compared with the controls. It was suggested that ASA and/or CaCl2 had positive regulation effects on the defence enzyme activities in chrysanthemum leaves which could protect the photosynthetic apparatus to a certain degree under the TL stress. In brief, the treatment of ASA together with CaCl2 was better for chrysanthemum plants to adapt TL stress than single ASA or CaCl2 treatments.

Genome-wide association study identifies favorable SNP alleles and candidate genes for waterlogging tolerance in chrysanthemums

Chrysanthemums are sensitive to waterlogging stress,and the development of screening methods for tolerant germplasms or genes and the breeding of tolerant new varieties are of great importance in chrysanthemum breeding.To understand the genetic basis of waterlogging tolerance(WT)in chrysanthemums,we performed a genome-wide association study(GWAS)using 92,811 single nucleotide polymorphisms(SNPs)in a panel of 88 chrysanthemum accessions,including 64 spray cut and 24 disbud chrysanthemums.The results showed that the average MFVW(membership function value of waterlogging)of the disbud type(0.65)was significantly higher than that of the spray type(0.55)at P<0.05,and the MFVW of the Asian accessions(0.65)was significantly higher than that of the European accessions(0.48)at P<0.01.The GWAS performed using the general linear model(GLM)and mixed linear model(MLM)identified 137 and 14 SNP loci related to WT,respectively,and 11 associations were commonly predicted.By calculating the phenotypic effect values for 11 common SNP loci,six highly favorable SNP alleles that explained 12.85—21.85%of the phenotypic variations were identified.Furthermore,the dosage-pyramiding effects of the favorable alleles and the significant linear correlations between the numbers of highly favorable alleles and phenotypic values were identified(r2=0.45;P<0.01).A major SNP locus(Marker6619-75)was converted into a derived cleaved amplified polymorphic sequence(dCAPS)marker that cosegregated with WT with an average efficiency of 78.9%.Finally,four putative candidate genes in the WT were identified via quantitative real-time PCR(qRT-PCR).The results presented in this study provide insights for further research on WT mechanisms and the application of molecular marker-assisted selection(MAS)in chrysanthemum WT breeding programs.