Cloning and Functional Characterization of Dihydroflavonol 4-Reductase Gene Involved in Anthocyanin Biosynthesis of Chrysanthemum.

Dihydroflavonol 4-reductase (DFR) catalyzes a committed step in anthocyanin and proanthocyanidin biosynthesis by reducing dihydroflavonols to leucoanthocyanidins. However, the role of this enzyme in determining flower color in the economically important crop chrysanthemum (Chrysanthemum morifolium Ramat.) is unknown. Here, we isolated cDNAs encoding DFR from two chrysanthemum cultivars, the white-flowered chrysanthemum "OhBlang" (CmDFR-OB) and the red-flowered chrysanthemum "RedMarble" (CmDFR-RM) and identified variations in the C-terminus between the two sequences. An enzyme assay using recombinant proteins revealed that both enzymes catalyzed the reduction of dihydroflavonol substrates, but CmDFR-OB showed significantly reduced DFR activity for dihydrokaempferol (DHK) substrate as compared with CmDFR-RM. Transcript levels of anthocyanin biosynthetic genes were consistent with the anthocyanin contents at different flower developmental stages of both cultivars. The inplanta complementation assay, using Arabidopsis thaliana dfr mutant (tt3-1), revealed that CmDFR-RM, but not CmDFR-OB, transgenes restored defective anthocyanin biosynthesis of this mutant at the seedling stage, as well as proanthocyanidin biosynthesis in the seed. The difference in the flower color of two chrysanthemums can be explained by the C-terminal variation of CmDFR combined with the loss of CmF3H expression during flower development.

Molecular and morphological discrimination of Chrysanthemum indicum using allele-specific PCR and T-shaped trichome.

Chrysanthemum indicum L. is a traditional oriental medicinal herb prepared as a tea from flowers that have been used in China and South Korea since ancient times. It has a long history in the treatment of hypertension, inflammation, and respiratory diseases. Among Chrysanthemum species, C. indicum has more active chemical components as well as better therapeutic effects, and C. indicum is mostly used for medicinal purposes in South Korea. However, the usage of C. indicum has become problematic over the years due to the abundance of adulterated Chrysanthemum and confusion with morphologically related species such as C. morifolium, C. boreale, and Aster spathulifolius. Thus, here we developed a method for molecular authentication using chloroplast universal region rpoC2 and morphological authentication based on T-shaped trichomes of the adaxial leaf surface. By using a species-specific primer derived from the rpoC2 region, we established a multiplex allele-specific PCR for the discrimination of C. indicum. Amplicons of 675 bp for C. indicum and 1026 bp for other Chrysanthemum species were produced using both rpoC2-specific and common primers. These primers can be used to analyze dried samples of Chrysanthemum. Morphological discrimination was performed using T-shaped trichomes present only on the adaxial leaf surface of C. indicum species, and then molecular markers were utilized to authenticate C. indicum products from adulterant samples available in the market. Our results indicate that these molecular markers in combination with morphological differentiation can serve as an effective tool for identifying C. indicum.

Discrimination of Chrysanthemum Varieties Using Hyperspectral Imaging Combined with a Deep Convolutional Neural Network.

Rapid and accurate discrimination of Chrysanthemum varieties is very important for producers, consumers and market regulators. The feasibility of using hyperspectral imaging combined with deep convolutional neural network (DCNN) algorithm to identify Chrysanthemum varieties was studied in this paper. Hyperspectral images in the spectral range of 874⁻1734 nm were collected for 11,038 samples of seven varieties. Principal component analysis (PCA) was introduced for qualitative analysis. Score images of the first five PCs were used to explore the differences between different varieties. Second derivative (2nd derivative) method was employed to select optimal wavelengths. Support vector machine (SVM), logistic regression (LR), and DCNN were used to construct discriminant models using full wavelengths and optimal wavelengths. The results showed that all models based on full wavelengths achieved better performance than those based on optimal wavelengths. DCNN based on full wavelengths obtained the best results with an accuracy close to 100% on both training set and testing set. This optimal model was utilized to visualize the classification results. The overall results indicated that hyperspectral imaging combined with DCNN was a very powerful tool for rapid and accurate discrimination of Chrysanthemum varieties. The proposed method exhibited important potential for developing an online Chrysanthemum evaluation system.

Sequencing and Analysis of Chrysanthemum carinatum Schousb and Kalimeris indica. The Complete Chloroplast Genomes Reveal Two Inversions and rbcL as Barcoding of the Vegetable.

Chrysanthemum carinatum Schousb and Kalimeris indica are widely distributed edible vegetables and the sources of the Chinese medicine Asteraceae. The complete chloroplast (cp) genome of Asteraceae usually occurs in the inversions of two regions. Hence, the cp genome sequences and structures of Asteraceae species are crucial for the cp genome genetic diversity and evolutionary studies. Hence, in this paper, we have sequenced and analyzed for the first time the cp genome size of C. carinatum Schousb and K. indica, which are 149,752 bp and 152,885 bp, with a pair of inverted repeats (IRs) (24,523 bp and 25,003) separated by a large single copy (LSC) region (82,290 bp and 84,610) and a small single copy (SSC) region (18,416 bp and 18,269), respectively. In total, 79 protein-coding genes, 30 distinct transfer RNA (tRNA) genes, four distinct rRNA genes and two pseudogenes were found not only in C. carinatum Schousb but also in the K. indica cp genome. Fifty-two (52) and fifty-nine (59) repeats, and seventy (70) and ninety (90) simple sequence repeats (SSRs) were found in the C. carinatum Schousb and K. indica cp genomes, respectively. Codon usage analysis showed that leucine, isoleucine, and serine are the most frequent amino acids and that the UAA stop codon was the significantly favorite stop codon in both cp genomes. The two inversions, the LSC region ranging from trnC-GCA to trnG-UCC and the whole SSC region were found in both of them. The complete cp genome comparison with other Asteraceae species showed that the coding area is more conservative than the non-coding area. The phylogenetic analysis revealed that the rbcL gene is a good barcoding marker for identifying different vegetables. These results give an insight into the identification, the barcoding, and the understanding of the evolutionary model of the Asteraceae cp genome.

GC-MS Analysis of the Composition of the Essential Oil from Dendranthema indicum Var. Aromaticum Using Three Extraction Methods and Two Columns.

Dendranthema indicum var. aromaticum, which is an aromatic plant with a strong and special fragrance throughout the whole plant, is used for the treatment of colds and headaches, and as a mosquito repellant in Shennongjia, Hubei province, China. To analyze the composition of the essential oil from this medicinal herb, we developed a gas chromatography-mass Spectrometry (GC-MS) method including microwave-assisted extraction, hydrodistillation and direct headspace analysis in two different stationary phase columns. In total, 115 volatile compounds were identified, of which 90 compounds were identified using Rxi-5MS and 78 using HP-INNOWAX. Our results revealed that the oil was mainly composed of five categories of compound: oxygenated monoterpenes (28.76-78.10%), oxygenated sesquiterpenes (4.27-38.06%), sesquiterpenes (3.22-11.57%), fatty hydrocarbons (1.65-9.81%) and monoterpenes (0-3.32%). The major constituents are α-thujone, ß-thujone, cis-sabinol, sabinyl acetate and (-)-neointermedeol.However, the essential oil composition in the published literature differs significantly. Therefore, a cluster analysis was carried out using the top ten compositions in the reported literature as well as this study, using Minitab software. To provide detailed information on plant origin, the ITS1-5.8s-ITS2 region was amplified and sequenced (Accession No. MF668250). Besides, in order to provide a macroscopic view of the chemical composition, the biosynthetic pathway of the main components was summarized according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the published literatures.

Analysis of Floral Volatile Components and Antioxidant Activity of Different Varieties of Chrysanthemum morifolium.

This study investigated the volatile flavor compounds and antioxidant properties of the essential oil of chrysanthemums that was extracted from the fresh flowers of 10 taxa of Chrysanthemum morifolium from three species; namely Dendranthema morifolium (Ramat.) Yellow, Dendranthema morifolium (Ramat.) Red, Dendranthema morifolium (Ramat.) Pink, Dendranthema morifolium (Ramat.) White, Pericallis hybrid Blue, Pericallis hybrid Pink, Pericallis hybrid Purple, Bellis perennis Pink, Bellis perennis Yellow, and Bellis perennis White. The antioxidant capacity of the essential oil was assayed by spectrophotometric analysis. The volatile flavor compounds from the fresh flowers were collected using dynamic headspace collection, analyzed using auto thermal desorber-gas chromatography/mass spectrometry, and identified with quantification using the external standard method. The antioxidant activities of Chrysanthemum morifolium were evaluated by DPPH and FRAP assays, and the results showed that the antioxidant activity of each sample was not the same. The different varieties of fresh Chrysanthemum morifolium flowers were distinguished and classified by fingerprint similarity evaluation, principle component analysis (PCA), and cluster analysis. The results showed that the floral volatile component profiles were significantly different among the different Chrysanthemum morifolium varieties. A total of 36 volatile flavor compounds were identified with eight functional groups: hydrocarbons, terpenoids, aromatic compounds, alcohols, ketones, ethers, aldehydes, and esters. Moreover, the variability among Chrysanthemum morifolium in basis to the data, and the first three principal components (PC1, PC2, and PC3) accounted for 96.509% of the total variance (55.802%, 30.599%, and 10.108%, respectively). PCA indicated that there were marked differences among Chrysanthemum morifolium varieties. The cluster analysis confirmed the results of the PCA analysis. In conclusion, the results of this study provide a basis for breeding Chrysanthemum cultivars with desirable floral scents, and they further support the view that some plants are promising sources of natural antioxidants.

Genetic diversity, population structure and association analysis in cut chrysanthemum (Chrysanthemum morifolium Ramat.).

Characterizing the genetic diversity present in a working set of plant germplasm can contribute to its effective management and genetic improvement. The cut flower chrysanthemum (Chrysanthemum morifolium Ramat.) is an economically important ornamental species. With the repeated germplasm exchange and intensive breeding activities, it remains a major task in genetic research. The purpose of the present study was to characterize the genetic diversity and the population structure of a worldwide collection of 159 varieties, and to apply an association mapping approach to identify DNA-based markers linked to five plant architecture traits and six inflorescence traits. The genotyping demonstrated that there was no lack of genetic diversity in the collection and that pair-wise kinship values were relatively low. The clustering based on a Bayesian model of population structure did not reflect known variation in either provenance or inflorescence type. A principal coordinate analysis was, however, able to discriminate most of the varieties according to both of these criteria. About 1 in 100 marker pairs exhibited a degree of linkage disequilibrium. The association analysis identified a number of markers putatively linked to one or more of the traits. Some of these associations were robust over two seasons. The findings provide an in-depth understanding of genetic diversity and population structure present in cut flower chrysanthemum varieties, and an insight into the genetic control of plant architecture and inflorescence-related traits.

The complete chloroplast genome sequence of Chrysanthemum indicum.

Chrysanthemum indicum, an important medicinal plant of Asteraceae, had a long history in use for medicine in China. In this study, the complete chloroplast genome of C. indicum was sequenced by a 454 sequencing platform, and the structure of the obtained chloroplast genome was also analyzed. The complete chloroplast genome of C. indicum was 150 972 bp in length and had a pair of inverted repeats (IR, 24 956 bp) separated by a large (LSC, 82 741 bp) and small single copy (SSC, 18 319 bp) regions. Its total GC content was 37.48%. There were 126 chloroplast genes including 83 protein-coding genes, 35 tRNAs and eight rRNAs were successfully annotated. Sixteen genes contained one or two introns. Phylogenetic analyses declared that the chloroplast genome could distinguish C. indicum from its closely related species and might become a potential super barcode for the identification of these species.

CmWRKY15 Facilitates Alternaria tenuissima Infection of Chrysanthemum.

Abscisic acid (ABA) has an important role in the responses of plants to pathogens due to its ability to induce stomatal closure and interact with salicylic acid (SA) and jasmonic acid (JA). WRKY transcription factors serve as antagonistic or synergistic regulators in the response of plants to a variety of pathogens. Here, we demonstrated that CmWRKY15, a group IIa WRKY family member, was not transcriptionally activated in yeast cells. Subcellular localization experiments in which onion epidermal cells were transiently transfected with CmWRKY15 indicated that CmWRKY15 localized to the nucleus in vivo. The expression of CmWRKY15 could be markedly induced by the presence of Alternaria tenuissima inoculum in chrysanthemum. Furthermore, the disease severity index (DSI) data of CmWRKY15-overexpressing plants indicated that CmWRKY15 overexpression enhanced the susceptibility of chrysanthemum to A. tenuissima infection compared to controls. To illustrate the mechanisms by which CmWRKY15 regulates the response to A. tenuissima inoculation, the expression levels of ABA-responsive and ABA signaling genes, such as ABF4, ABI4, ABI5, MYB2, RAB18, DREB1A, DREB2A, PYL2, PP2C, RCAR1, SnRK2.2, SnRK2.3, NCED3A, NCED3B, GTG1, AKT1, AKT2, KAT1, KAT2, and KC1were compared between transgenic plants and controls. In summary, our data suggest that CmWRKY15 might facilitate A. tenuissima infection by antagonistically regulating the expression of ABA-responsive genes and genes involved in ABA signaling, either directly or indirectly.

Transcriptomic analyses reveal species-specific light-induced anthocyanin biosynthesis in chrysanthemum.

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

Identification of floral scent in chrysanthemum cultivars and wild relatives by gas chromatography-mass spectrometry.

The objective of this study was to identify the major volatile compounds and their relative concentrations in flowers of different chrysanthemum cultivars and their wild relatives. The volatile organic components of fresh flowers were analyzed using a headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. In total, 193 volatile organic components were detected; the major scent components were monoterpenoids and oxygenated monoterpenoids, which accounted for 68.59%-99.93% of the total volatiles in all tested materials except for Chrysanthemum indicum collected from Huangshan, in which they accounted for only 37.45% of total volatiles. The major volatile compounds were camphor, α-pinene, chrysanthenone, safranal, myrcene, eucalyptol, 2,4,5,6,7,7ab-hexahydro-1H-indene, verbenone, ß-phellandrene and camphene. In a hierarchical cluster analysis, 39 accessions of Chrysanthemum and its relatives formed six clusters based on their floral volatile compounds. In a principal component analysis, only spider type flowers were located closely on the score plot. The results of this study provide a basis for breeding chrysanthemum cultivars which desirable floral scents.

[The analyses and identification of Flos rhododendri mollis and Flos chrysanthemi indici via infrared spectroscopy].

In this study, major chemical components of Flos rhododendri mollis and Flos chrysanthemi indici were characterized using Fourier transform infrared spectroscopy (FTIR). For Flos rhododendri mollis, the bands at 1,648 and 1,543 cm(-1) were attributed to amide I and amide II , respectively, indicating that it contained proteins probably resulting in immunization. In case of Flos chrysanthemi indici, stretching vibration of C==O function group was responsible for the bands at 1,734 and 1,515 cm(-1), as a result of essential oils, lipids, etc. Since FTIR spectra of Flos rhododendri mollis and Flos chrysanthemi indici are almost identical and it is difficult to discriminate them, two-step identification was investigated via secondary derivative of the FTIR spectra. The bands at 1,656 and 1,515 cm(-1) corresponds to flavonoides in Flos rhododendri mollis and Flos chrysanthemi indici. In the secondary derivative of the FTIR spectrum of Flos chrysanthemi indici, characteristic bands of inulin were present at 1,163, 1,077, 1,026, 986 and 869 cm(-1), and therefore Flos chrysanthemi indici contained inulin as well. Tri-step identification was carried out for Flos rhododendri mollis and Flos chrysanthemi indici by means of comparing their 2D-IR correlation spectra in different wave number range. In the characteristic range of flavonoides (1,700-1,400 cm(-1)), Flos rhododendri mollis exhibited 3 obvious autopeaks, while 10 autopeaks were visualized in the 2D-IR correlation spectrum of Flos chrysanthemi indici Moreover, in the characteristic range of glucoside (1,250-900 cm(-1)), 10 and 9 autopeaks were present in the 2D-IR correlation spectra of Flos rhododendri mollis and Flos chrysanthemi indici, respectively. Therefore, the tri-step identification of FTIR is a time-saving; accurate, cost-saving and convenient method to effectively distinguish traditional Chinese medicines.

Comparative analysis of genetic diversity among species of Chrysanthemum and its related genera using inter-simple sequence repeat and sequence-related amplified polymorphism markers.

In this study, inter-simple sequence repeats (ISSRs) and sequence-related amplified polymorphism (SRAP) were applied to assess the genetic diversity in 38 species of Chrysanthemum and related genera. A total of 204 and 567 bands were amplified by 24 ISSR and 25 SRAP primers, of which 196 (97%) and 557 (99%) were polymorphic, respectively. The ISSR-based genetic similarity ranged from 0.016 to 0.886 and averaged 0.201, while the SRAP-based genetic similarity varied from 0.010 to 0.811 and averaged 0.122. Both the ISSR and SRAP techniques revealed similar clustering patterns and grouped species of Chrysanthemum and Ajania together. The results of principal coordinate analysis corroborated the unweighted pair group method with arithmetic average clustering. Additionally, results from ISSR and SRAP data were significantly correlated (r = 0.89, P < 0.001). Knowledge about genetic diversity among species can aid the transfer of traits of interest from the wild into cultivated chrysanthemum in future distant interspecific breeding.

Phylogenetic and transcription analysis of chrysanthemum WRKY transcription factors.

WRKY transcription factors are known to function in a number of plant processes. Here we have characterized 15 WRKY family genes of the important ornamental species chrysanthemum (Chrysanthemum morifolium). A total of 15 distinct sequences were isolated; initially internal fragments were amplified based on transcriptomic sequence, and then the full length cDNAs were obtained using RACE (rapid amplification of cDNA ends) PCR. The transcription of these 15 genes in response to a variety of phytohormone treatments and both biotic and abiotic stresses was characterized. Some of the genes behaved as would be predicted based on their homology with Arabidopsis thaliana WRKY genes, but others showed divergent behavior.

Application of genomic SSR locus polymorphisms on the identification and classification of chrysanthemum cultivars in China.

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.

Phylogenetic relationships of chrysanthemums in Korea based on novel SSR markers.

Chrysanthemums are well known for their esthetic and medicinal values. Characterization of chrysanthemums is vital for their conservation and management as well as for understanding their genetic relationships. We found 12 simple sequence repeat markers (SSRs) of 100 designed primers to be polymorphic. These novel SSR markers were used to evaluate 95 accessions of chrysanthemums (3 indigenous and 92 cultivated accessions). Two hundred alleles were identified, with an average of 16.7 alleles per locus. KNUCRY-77 gave the highest polymorphic information content value (0.879), while KNUCRY-10 gave the lowest (0.218). Similar patterns of grouping were observed with a distance-based dendrogram developed using PowerMarker and model-based clustering with Structure. Three clusters with some admixtures were identified by model-based clustering. These newly developed SSR markers will be useful for further studies of chrysanthemums, such as taxonomy and marker-assisted selection breeding.

[Study on inflorescences microscopic characteristics of five wild medicinal plants from Chrysanthemum genus in Anhui].

OBJECTIVE: To probe into the disparities in the microscopic characteristics of each part of inflorescences of five wild medicinal plants from Chrysanthemum genus in Anhui Province, and explore the correlation between the distribution of plants and ecological environment. METHODS: The transverse sections of peduncles of the five Chrysanthemum species were observed; The surface piles of the involucre, ray flower, and tubular flower were compared; The ultra-morphology of pollen was observed. RESULTS: There were evident differences, which were associated with ecological environment, among the structures of peduncles of the five species. The morphology of non-glandular hairs on the external involucre presented significant differences, but the morphology of non-glandular hairs tended to be similar gradually from the external to the internal layer of the involucre, and the morphology of surface piles of each part of the inflorescences also tended to converge from the external to the internal. The disparities of glandular hairs among different species were their sizes; As for each species, the size of glandular hairs grew bigger from the involucre to ray flower, and to tubular flower, the density of glandular hairs decreased from the external to the internal layer of the involucre, while it increased on ray flower and tubular flower. The ultra-morphology of pollen of C. zawadskii was distinctly different from that of other species, and disparities of pollen morphology also existed between C. nankingense and C. indicum. CONCLUSION: The structure of peduncles and the disparities in morphology of non-glandular hairs on the surface of external involucre can serve as significant basis for differentiating the inflorescences of Chrysanthemum plants. The categorizing of designating C. nankingense as an separate species is supported.

Expression profile analysis of genes involved in horizontal gravitropism bending growth in the creeping shoots of ground-cover chrysanthemum by suppression subtractive hybridization.

The molecular mechanisms underlying gravitropic bending of shoots are poorly understood and how genes related with this growing progress is still unclear. To identify genes related to asymmetric growth in the creeping shoots of chrysanthemum, suppression subtractive hybridization was used to visualize differential gene expression in the upper and lower halves of creeping shoots of ground-cover chrysanthemum under gravistimulation. Sequencing of 43 selected clones produced 41 unigenes (40 singletons and 1 unigenes), which were classifiable into 9 functional categories. A notable frequency of genes involve in cell wall biosynthesis up-regulated during gravistimulation in the upper side or lower side were found, such as beta tubulin (TUB), subtilisin-like protease (SBT), Glutathione S-transferase (GST), and expensing-like protein (EXP), lipid transfer proteins (LTPs), glycine-rich protein (GRP) and membrane proteins. Our findings also highlighted the function of some metal transporter during asymmetric growth, including the boron transporter (BT) and ZIP transporter (ZT), which were thought primarily for maintaining the integrity of cell walls and played important roles in cellulose biosynthesis. CmTUB (beta tubulin) was cloned, and the expression profile and phylogeny was examined, because the cytoskeleton of plant cells involved in the plant gravitropic bending growth is well known.

[Rapid identification system for seedlings of medicinal Chrysanthemum morifolium].

OBJECTIVE: To achieve the rapid identification for seedlings of medicinal Chrysanthemum morifolium, the discriminant equation was established and the software for rapid identification was designed. METHOD: Leaf structure of medicinal Chrysanthemum of 12 cultivars was analyzed to establish the discriminant equation based on variance analysis and discriminant analysis. On this basis, the identification program and software (based on the python language) were designed. RESULT: Through the analysis of variance and multiple comparisons for the 11 leaf parameter index data of 12 different cultivars, it was found that that the leaf parameters were significant different from each other and reached significant levels. The discriminant equation and the rapid identification software were set up based on the analysis of various indicators. CONCLUSION: The rapid identification system of seedlings of medicinal Chrysanthemum could be achieved through the establishment of discriminant equation combined with computer technology.

Functional characterization of a Chrysanthemum dichrum stress-related promoter.

A 1,474-bp stress-inducible CdDREBa promoter was identified from Chrysanthemum dichrum, revealing several candidate stress-related cis-acting elements (MYC-box, MYB site, GT-1, and W-box) within it. In Arabidopsis leaf tissues transformed with a CdDREBa promoter-ß-glucuronidase (GUS) gene fusion, serially 5'-deleted CdDREBa promoters were differentially activated by cold and salinity. Histochemical and quantitative assays of GUS expression allowed us to localize a critical part of the promoter located between upstream 430 and 351 nt. This 80-bp fragment enhanced GUS expression under salinity stress when fused to -90/+8 CaMV 35S minimal promoter. Further promoter internal-deletion assays indicated that a low temperature-responsive element was located between positions -430 and -390, and a salinity inducible one between -385 and -351. Our results showed that there was a novel stress-related critical region except for the known cis-acting element (MYC-box, GT-1) in CdDREBa promoter.