Chrysanthemum Breeding Research Articles

Cross‐resistance of chrysanthemum to western flower thrips, celery leafminer, and two‐spotted spider mite

AbstractChrysanthemum [Chrysanthemum × morifolium Ramat. (Asteraceae)] is one of the economically most important greenhouse ornamentals worldwide. A major constraint in chrysanthemum production is adequate pest management, requiring the use of different tactics, such as improving host plant resistance, in the framework of an integrated pest management (IPM) approach. In this study, we investigated cross‐resistance of chrysanthemum to its three major pests: western flower thrips [Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae)], celery leafminer [Liriomyza trifolii (Burgess) (Diptera: Agromyzidae)], and two‐spotted spider mite [Tetranychus urticae Koch (Acari: Tetranychidae)]. We quantified resistance to each pest by performing greenhouse bioassays with a broad range of chrysanthemum types from commercial germplasm provided by Dutch breeding companies. Considerable variation was detected among the chrysanthemum cultivars in thrips silver damage and growth damage, leafminer damage, measured as number of mines and pupae, and spider mite numbers and damage. We observed significant positive correlations between thrips damage (both silver and growth damage) vs. leafminer numbers (both mines and pupae), and between leafminer numbers (both mines and pupae) vs. spider mite numbers. Our results indicate an overlap in resistance to all three herbivores. The important implications of this result for chrysanthemum breeding are discussed.

Assessing the Genetic Diversity of Chrysanthemum Cultivars with Microsatellites

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

Transcriptome sequencing and whole genome expression profiling of chrysanthemum under dehydration stress

BackgroundChrysanthemum is one of the most important ornamental crops in the world and drought stress seriously limits its production and distribution. In order to generate a functional genomics resource and obtain a deeper understanding of the molecular mechanisms regarding chrysanthemum responses to dehydration stress, we performed large-scale transcriptome sequencing of chrysanthemum plants under dehydration stress using the Illumina sequencing technology.ResultsTwo cDNA libraries constructed from mRNAs of control and dehydration-treated seedlings were sequenced by Illumina technology. A total of more than 100 million reads were generated and de novo assembled into 98,180 unique transcripts which were further extensively annotated by comparing their sequencing to different protein databases. Biochemical pathways were predicted from these transcript sequences. Furthermore, we performed gene expression profiling analysis upon dehydration treatment in chrysanthemum and identified 8,558 dehydration-responsive unique transcripts, including 307 transcription factors and 229 protein kinases and many well-known stress responsive genes. Gene ontology (GO) term enrichment and biochemical pathway analyses showed that dehydration stress caused changes in hormone response, secondary and amino acid metabolism, and light and photoperiod response. These findings suggest that drought tolerance of chrysanthemum plants may be related to the regulation of hormone biosynthesis and signaling, reduction of oxidative damage, stabilization of cell proteins and structures, and maintenance of energy and carbon supply.ConclusionsOur transcriptome sequences can provide a valuable resource for chrysanthemum breeding and research and novel insights into chrysanthemum responses to dehydration stress and offer candidate genes or markers that can be used to guide future studies attempting to breed drought tolerant chrysanthemum cultivars.

Genetic Engineering of Novel Bluer-Colored Chrysanthemums Produced by Accumulation of Delphinidin-Based Anthocyanins

Chrysanthemums (Chrysanthemum morifolium Ramat.) have no purple-, violet- or blue-flowered cultivars because they lack delphinidin-based anthocyanins. This deficiency is due to the absence of the flavonoid 3',5'-hydroxylase gene (F3'5'H), which encodes the key enzyme for delphinidin biosynthesis. In F3'5'H-transformed chrysanthemums, unpredictable and unstable expression levels have hampered successful production of delphinidin and reduced desired changes in flower color. With the aim of achieving delphinidin production in chrysanthemum petals, we found that anthocyanin biosynthetic gene promoters combined with a translational enhancer increased expression of some F3'5'H genes and accompanying delphinidin-based anthocyanin accumulation in transgenic chrysanthemums. Dramatic accumulation of delphinidin (up to 95%) was achieved by simple overexpression of Campanula F3'5'H controlled by a petal-specific flavanone 3-hydroxylase promoter from chrysanthemum combined with the 5'-untranslated region of the alcohol dehydrogenase gene as a translational enhancer. The flower colors of transgenic lines producing delphinidin-based anthocyanins changed from a red-purple to a purple-violet hue in the Royal Horticultural Society Colour Charts. This result represents a promising step toward molecular breeding of blue chrysanthemums.

Isolation and functional analysis of a homolog of flavonoid 3′,5′‐hydroxylase gene from Pericallis × hybrida

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

Altered expression of CmNRRa changes flowering time of Chrysanthemum morifolium

Flowering time is an important ornamental trait for chrysanthemum (Chrysanthemum morifolium, Dendranthema x grandiflorum) floricultural production. In this study, CmNRRa, an orthologous gene of OsNRRa that regulates root growth in response to nutrient stress in rice, was identified from Chrysanthemum and its role in flowering time was studied. The entire CmNRRa cDNA sequence was determined using a combinatorial PCR approach along with 5' and 3' RACE methods. CmNRRa expression levels in various tissues were monitored by real-time RT-PCR. CmNRRa was strongly expressed in flower buds and peduncles, suggesting that CmNRRa plays a regulatory role in floral development. To investigate the biological function of CmNRRa in chrysanthemums, overexpression and knockdown of CmNRRa were carried out using transgenic Chrysanthemum plants generated through Agrobacterium-mediated transformation. CmNRRa expression levels in the transgenic plants were assayed by real-time RT-PCR and Northern blot analysis. The transgenic plants showed altered flowering times compared with nontransgenic plants. CmNRRa-RNAi transgenic plants flowered 40-64days earlier, while CmNRRa-overexpressing plants exhibited a delayed flowering phenotype. These results revealed a negative effect of CmNRRa on flowering time modulation. Alteration of CmNRRa expression levels might be an effective means of controlling flowering time in Chrysanthemum. These results possess potential application in molecular breeding of chrysanthemums that production year-round, and may improve commercial chrysanthemum production in the flower industry.

菊属与蒿属植物苗期抗蚜虫性鉴定

Chrysanthemum(Dendranthema×grandiflorum Tzvel.) is one of the most economically important ornamental plants all over the world.Aphid is one of major agricultural pests and specialized to feed on phloem,they penetrate plant tissues via a primarily intercellular route,and ingest phloem sap from Chrysanthemum plants through stylets.Aphids not only result in a loss in quality,but also are an important vector of plant viruses besides damaging Chrysanthemum plants directly.Chrysanthemum is particularly susceptible to aphid infestation from the time of its seedling stage to its flowering as a crop.The phenotypes caused by aphids include the leaf rolling,chloroplast degradation,and folding of leaves or inflorescence of Chrysanthemum.Given the evolution of aphid resistance to pesticides,there is a growing need to screen aphid-resistant germplasm from Dendranthema and its closely related genera Artemisia L.for the breeding of chrysanthemum.The aphid resistance of eleven species from Dendranthema and Artemisia L.were evaluated at seedling stage under the field in Chrysanthemum Germplasm Resource Preserving Centre,Nanjing Agricultural University,China and lab conditions in no-insect nets room of Nanjing Agricultural University.The results showed that the aphid-resistance reduced with average damage index of aphid(I*) value increased under the field condition.A.vulgaris 'Variegate',A.scoparia and A.annua that all had 0.00 I* value were all aphid immunity species;A.japonica with 0.13 of the I* value was belonging to high resistant level;The I* value of aphid resistance of the D.crassum belonging to resistant level was 0.25;D.dichrum with 0.37 I* value was belonging to moderately resistant grade;D.yoshinaganthum and D.japonicum whose I* value were 0.51—0.75 were belonging to aphid sensitive grade;D.boreale,D.zawadskii and D.nankingense with the 0.75 I* value were highly sensitive to aphids.Under lab condition,aphid resistance varied among species.Eleven species can be divided into five different grades in terms of aphid resistance according to the multiplication rate(MR).For example,the A.vulgaris 'Variegate',A.japonica,A.scoparia and A.annua with 0.00 MR all were the highest resistance species;D.crassum that had 5.86 MR belonged to moderate resistant type;D.dichrum,D.yoshinaganthum and D.japonicum with 7—10 MR were belonging to low resistant grade;D.boreale,D.zawadskii and D.nankingense whose MR were all 10,were non-resistant species.The aphid resistance under field condition was overall consistent with that under lab condition.The aphid resistance of eight species such as A.vulgaris 'Variegate' and A.scoparia were confirmed under field and lab conditions.However,A.japonica was high resistant specie under field condition while belonging to the highest resistant grade under lab condition;D.crassum was resistant to aphid under field condition but was moderate resistant one under lab condition;and D.dichrum had moderate resistance under field condition but low resistance to the pests under lab condition.Therefore,A.vulgaris 'Variegate',A.scoparia,A.japonica,A.annua and D.crassum were promising germplasms for aphid-resistance breeding of chrysanthemum based on our results.

Isolation of a gibberellin 20‐oxidase cDNA from and characterization of its expression in chrysanthemum

With 5 figures and 1 table AbstractA PCR strategy was used to isolate a full‐length GA20‐oxidase cDNA from chrysanthemum. The gene putatively encodes a 377 residue polypeptide, which shares 87% homology with lettuce GA20ox. A Southern blot analysis indicated that the hexaploid chrysanthemum genome probably contains several GA20ox copies. The DgGA20ox gene is expressed in the leaf, stem, flower and root, but most strongly in the young leaf. The application of the GA biosynthesis inhibitor paclobutrazol to the fourth internode increased the expression of DgGA20ox, while exogenously applied GA3 decreased it. This supports the commonly observed phenomenon that the expression level of GA20ox genes is regulated by the local level of physiologically active GA. The relevance of the DgGA20ox in relation to chrysanthemum breeding was discussed as well.

A preliminary genetic linkage map of chrysanthemum ( Chrysanthemum morifolium) cultivars using RAPD, ISSR and AFLP markers

A genetic linkage map of chrysanthemum ( Chrysanthemum morifolium) was constructed by genotyping 142 F 1 progeny of the bi-parental cross ‘Yuhualuoying’ × ‘Aoyunhanxiao’ with a combination of RAPD, ISSR and AFLP markers in a double pseudo-testcross mapping strategy. A total of 567 polymorphic markers, including 153 RAPDs, 61 ISSRs and 353 AFLPs, were used in linkage mapping. 336 of 567 (60%) markers were grouped on the two parental maps, leaving 231 (40%) markers unlinked. In the ‘Yuhualuoying’ linkage map, 210 markers including 116 testcross and 94 intercross markers were placed in 12 major and 32 minor (8 triplets and 24 doublets) linkage groups, covering 1034 cM with an average map distance of 6.2 cM between adjacent markers. In ‘Aoyunhanxiao’ linkage map, 190 markers consisting of 113 testcross and 77 intercross markers were resolved into 9 major and 24 minor linkage groups, with genome coverage of 1095 cM and a mean inter-marker separation of 6.9 cM between adjacent markers. Six pairs of homologous linkage groups were established on the basis of 64 intercross markers shared by the two parental maps. The maps lay a foundation for further quantitative traits loci (QTL) mapping and marker-assisted breeding of chrysanthemum.

Interspecific hybrids between Chrysanthemum grandiflorum (Ramat.) Kitamura and C. indicum (L.) Des Moul. and their drought tolerance evaluation

Chrysanthemum grandiflorum ‘Yuhuaxingchen’ is an important commercial chrysanthemum cultivar with excellent ornamental quality but low drought tolerance, whereas C. indicum has exceptional drought tolerance. In our earlier study, many hybrid seeds between them were obtained through interspecific hybridization. In the present study, we selected six putative hybrid lines with most drought tolerance from all the hybrid lines by withholding water, indentified their facticity by chromosome counting, and then evaluated their drought tolerance through determining foliar electrolyte leakage (EL), contents of malondialdehyde (MDA) and proline, and plant survival rate after 20% polyethylene glycol 6000 treatment. It was found that 155 out of 282 seeds germinated and only 132 seedlings survived. In addition, chromosome and morphological analysis showed that the six putative hybrids were real hybrids and their morphological features were intermediate between their parents. Furthermore, the density of leaf epidermal hair, proline content, and plant survival rate were the highest in C. indicum and the lowest in C. grandiflorum among the six hybrids and their parents. In contrast, EL value and MDA content were the highest in C. grandiflorum and the lowest in C. indicum. These results suggest that some true hybrids with improved drought tolerance can be obtained through interspecific hybridization in chrysanthemum breeding. Therefore, interspecific hybridization between chrysanthemum cultivars and their wild species may become a promising way to improve their biotic and abiotic resistance in the future breeding.

Improved translation efficiency in chrysanthemum and torenia with a translational enhancer derived from the tobacco alcohol dehydrogenase gene

We investigated the efficiency of the 5� -untranslated region (UTR) of the tobacco alcohol dehydrogenase gene (NtADH-5� UTR) as a translational enhancer in chrysanthemum (Chrysanthemum morifolium) and torenia (Torenia fournieri). Three constructs were introduced: control, ADH� , cauliflower mosaic virus 35S RNA (CaMV 35S) promoter::b-glucuronidase (GUS); NtADH-5� UTR with a spacer, ADHS, CaMV 35S promoter::NtADH-5� UTR::25-bp spacer::GUS; and NtADH-5� UTR with no spacer, ADH� , CaMV 35S promoter::NtADH-5� UTR::GUS. The highest GUS activity in ADHS and ADHfor chrysanthemum was about 45 and 190 times, respectively, and for torenia was 12 and 22 times, respectively, than that for the ADHplants. NtADH-5� UTR enhanced translational efficiency in both species. With the lowest translational efficiency set to 1, the relative translational efficiencies were 1 to 15 (ADH� ), 47 to 568 (ADHS), and 267 to 1360 (ADH� ) for chrysanthemum, and 1 to 3 (ADH� ), 47 to 114 (ADHS), and 85 to 226 (ADH� ) for torenia. NtADH-5� UTR would facilitate practical breeding and fundamental genetic research in chrysanthemum and torenia.

In vitro propagation using adventitious buds technique as a source of new variability in chrysanthemum

Eleven cultivars of Chrysanthemum × grandiflorum (Ramat.) Kitam.: ‘Richmond’ and its 10 radiomutants, representing the Lady group, were propagated in vitro with shoot tips and leaves as explants. The aim of this study was to investigate if the explant type used for micropropagation affects the genotype and phenotype of chrysanthemums. Plants grown from shoot tips and adventitious buds formed on leaves were rooted in vitro, acclimatized and cultivated in glasshouse up to full-flowering. The colour and shape of inflorescences of plants obtained from two different explant types were compared within the cultivars. All plants derived from shoot-tip explants showed the inflorescence colour and shape typical for the cultivars. Inflorescence colour of plants derived from adventitious buds were true-to-type in four cultivars: ‘Richmond’, ‘Lady Amber’, ‘Lady White’ and ‘Lady Yellow’. All plants of ‘Lady Apricot’ (originally: golden beet) and ‘Lady Salmon’ (salmon) propagated from adventitious buds technique showed altered inflorescence colour (respectively: purple gold; pink and white). ‘Lady Bronze’ (originally: reddish brown), ‘Lady Orange’ (orange brown) and ‘Lady Rosy’ (purple gold) propagated with adventitious buds had both typical and changed inflorescence colours (respectively: yellow; yellow and red; reddish pink). ‘Lady Vitroflora’ showed altered number of ligulate florets grown into tubes in inflorescence when propagated with shoot tips and leaves as explants. Those changes might be an effect of either chimeral structure or somaclonal variation of the plants investigated. The variation appears only if non-meristematical explants were used. The adventitious buds technique might be useful in chrysanthemum breeding as a source of a new variability.

Phenotypic markers for selection of winter hardy garden chrysanthemum ( Dendranthema× grandiflora Tzvelv.) genotypes

Consumer interest in winter hardy herbaceous perennials has precipitated a re-prioritization of breeding program objectives for garden chrysanthemums. The objective of this study was to determine which phenotypic marker(s) in garden chrysanthemum crowns are correlated with winter hardiness for use as non-destructive, inexpensive selection tools in breeding programs. An experiment was conducted over 2 years (fall 1999, 2000) using plants grown in field trials at 45°N latitude (−34.5 °C, minimum temperature). Germplasm for fall 1999, included n=5 numbered genotypes, n=8 cultivars from the University of Minnesota breeding program, and n=2 cultivars from Yoder and GroLink. In fall 2000 studies, germplasm consisted of n=5 cultivars (University of Minnesota) and n=10 cultivars (Yoder, GroLink). Four genotypes were included in both years. Ramets ( n=24) of each genotype were planted in May of each year. First-year, field-grown crowns ( n=3 replicatios/genotype) were dug each fall. Winter hardiness classifications were assigned to genotypes based on winter survival data for clones collected over the 2 years of the study. Data was collected on the number of emergent rhizomes, number of non-emergent rhizomes, proportion of total rhizomes that were emergent, fresh/dry weights, and measurements (length, diameter, % branching) of the five most basipetal rhizomes. Discriminant analysis was conducted on the pooled data for the 2 years. The data were divided into training and evaluation sets for analysis. The best discriminant function model included the variables as the number of emergent and non-emergent rhizomes, and average rhizome length. The success rate of the discriminant analysis was 88.9% for both the training and validation sets. Emergent rhizome number would be the easiest and only non-destructive trait for field selection. A second discriminant analysis was conducted using only number of emergent rhizomes. The one-variable model resulted in a success rate of 93.3% for the training set and 86.7% for the evaluation set. A garden chrysanthemum breeding program could collect data on emergent rhizome number in the fall of the first year’s growth to determine winter hardiness with 86.7% accuracy, confirm this with % winter survival (in test winters), and/or laboratory freezing tests.

468 Inheritance of Day Neutrality and Heat-delay Insensitivity in Dendranthema grandiflora Tzvelv.

Greenhouse and garden chrysanthemums are quantitative short-day (SD) plants for flower bud initiation (FBI) and qualitative (obligate) SD plants for flower bud development (FBD). Continuous or intermittent application of red light in the middle of the dark period (night), inhibits FBI. The chrysanthemum breeding program has been selecting for day-neutral (DN) types, i.e. that will undergo FBI and FBD under any photoperiod. The inheritance of DN was studied using six cultivars (n = 2 SD types, n = 4 DN types) that were crossed in a complete diallel over two crossing periods. Pollinations were replicated and ovules were counted. Histograms of self and cross seed set showed a distribution from 0% to 100%, with the majority of pollinations below 30%. Mean self seed set (2.6%) was less than the mean cross seed set (32.8%), indicating the presence of a self incompatibility system. Parents and F1 progeny were grown under LD conditions (red light, night interruption, 2200-0200 HR) and high temperatures (30 °C day/25 °C night, to screen for heat delay insensitivity). F2 progeny could not be generated due to self incompatibility. The fraction of flowering: non-flowering progeny and the number of days to first flower was recorded on the flowering individuals for comparison with the parents. Due to small progeny numbers, reciprocal crosses were bulked prior to Chi-square tests (1:1, 3:1, 1:3). The number of days to first flower ranged from 27 to 93+ in all progeny with significantly earlier and later outliers present. Most Chi-square tests were not significant, indicating that the inheritance of DN and heat delay insensitivity are not controlled by a single gene. Additive and epistatic effects may also be present.

Application of in vitro techniques in mutation breeding of chrysanthemum

Rooted cuttings of Chrysanthemum morifolium cv. Maghi, a small flowered, late blooming cultivar, were treated with different doses of gamma rays. Somatic mutations in flower colour (light mauve, white, light yellow and dark yellow) and chlorophyll variegation in leaves were detected as chimeras in treated populations. Attempts were made to standardize a microtechnique for plant regeneration from mutated tissues of stem node, stem internode, shoot tip and ray floret. All these explants were cultured on Murashige and Skoog's medium with 3% sucrose, 0.8% agar and different concentrations and combinations of growth regulators. Plant regeneration was successful from all of the mutated tissues. Plants with chlorophyll variegation in leaves and two new flower colours (light mauve and white) were isolated in pure form with 64% and 100% efficiency of mutant recovery, respectively. Attempts are being made to use this technique to establish new varieties from chimeric tissues to meet the increasing demand of the floriculture trade.

Chrysanthemum Breeding for Resistance to White Rust

A series of experiments were conducted to promote chrysanthemum breeding for resistance to white rust (Puccinia horiana P. Hennings), For testing the resistance to white rust, three plants of each species and cultivar were incubated in a box kept at 17'C under 100% relative humidity and the inoculation was effected by hanging leaves with telia actively producing sporidia from the ceiling of the box. The inoculum concentration was restricted to ca. 70 sporidia per 1mm2 leaf area. Reliable criteria for grading and rating inLection were established based principally on the extent of telium formation on the fully unfolded top leaves. White rust resistance of chrysanthernum cultivars could be evaluated by this testing method at an early stage of the plants, for their reactions to white rust at the young plant stage and the flowering stage were highly correlated. Variation in resistance depending on the fungal isolates was found in the tests of 40 cultivars inoculated with 6 typical isolates in Japan, suggesting physiological specialization in the pathogen. Among 6 isolates, some showed a wide range of pathogenicity and others medium and narrow ranges. Besids the existence of unstable cultivars showing various reactions to the fungal isolates, cultivars with stable resistance to all the isolates were also found.