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Background: Heterosis breeding is one of the most important breeding methods for chrysanthemum. To date, the genetic mechanisms of heterosis for waterlogging tolerance in chrysanthemum are still unclear. This study aims to analyze the expression profiles and potential heterosis-related genes of two hybrid lines and their parents with extreme differences in waterlogging tolerance under control and waterlogging stress conditions by RNA-seq., Results: A population of 140 F 1 progeny derived from Chrysanthemum indicum (Nanchang) (waterlogging-tolerant) and Chrysanthemum indicum (Nanjing) (waterlogging-sensitive) was used to characterize the extent of genetic variation in terms of seven waterlogging tolerance-related traits across two years. Lines 98 and 95, respectively displaying positive and negative overdominance heterosis for the waterlogging tolerance traits together with their parents under control and waterlogging stress conditions, were used for RNA-seq. In consequence, the maximal number of differentially expressed genes (DEGs) occurred in line 98. Gene ontology (GO) enrichment analysis revealed multiple stress-related biological processes for the common up-regulated genes. Line 98 had a significant increase in non-additive genes under waterlogging stress, with transgressive up-regulation and paternal-expression dominant patterns being the major gene expression profiles. Further, GO analysis identified 55 and 95 transgressive up-regulation genes that overlapped with the up-regulated genes shared by two parents in terms of responses to stress and stimulus, respectively. 6,640 genes in total displaying maternal-expression dominance patterns were observed in line 95. In addition, 16 key candidate genes, including SAP12, DOX1, and ERF017 which might be of significant importance for the formation of waterlogging tolerance heterosis in line 98, were highlighted., Conclusion: The current study provides a comprehensive overview of the root transcriptomes among F 1 hybrids and their parents under waterlogging stress. These findings lay the foundation for further studies on molecular mechanisms underlying chrysanthemum heterosis on waterlogging tolerance., (© 2024. The Author(s).)

Key Message: The dynamic genetic architecture of flowering time in chrysanthemum was elucidated by GWAS. Thirty-six known genes and 14 candidate genes were identified around the stable QTNs and QEIs, among which ERF-1 was highlighted. Flowering time (FT) adaptation is one of the major breeding goals in chrysanthemum, a multipurpose ornamental plant. In order to reveal the dynamic genetic architecture of FT in chrysanthemum, phenotype investigation of ten FT-related traits was conducted on 169 entries in 2 environments. The broad-sense heritability of five non-conditional FT traits, i.e., budding (FBD), visible coloring (VC), early opening (EO), full-bloom (OF) and decay period (DP), ranged from 56.93 to 84.26%, which were higher than that of the five derived conditional FT traits (38.51-75.13%). The phenotypic variation coefficients of OF_EO and DP_OF were relatively large ranging from 30.59 to 36.17%. Based on 375,865 SNPs, the compressed variance component mixed linear model 3VmrMLM was applied for a multi-locus genome-wide association study (GWAS). As a result, 313 quantitative trait nucleotides (QTNs) were identified for the non-conditional FT traits in single-environment analysis, while 119 QTNs and 67 QTN-by-environment interactions (QEIs) were identified in multi-environment analysis. As for the conditional traits, 343 QTNs were detected in single-environment analysis, and 119 QTNs and 83 QEIs were identified in multi- environment analysis. Among the genes around stable QTNs and QEIs, 36 were orthologs of known FT genes in Arabidopsis and other plants; 14 candidates were mined by combining the transcriptomics data and functional annotation, including ERF-1, ACA10, and FOP1. Furthermore, the haplotype analysis of ERF-1 revealed six elite accessions with extreme FBD. Our findings contribute to the understanding of dynamic genetic architecture of FT and provide valuable resources for future chrysanthemum molecular breeding programs., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Main Conclusion: Multi-locus GWAS detected several known and candidate genes responsible for flowering time in chrysanthemum. The associations could greatly increase the predictive ability of genome selection that accelerates the possible application of GS in chrysanthemum breeding. Timely flowering is critical for successful reproduction and determines the economic value for ornamental plants. To investigate the genetic architecture of flowering time in chrysanthemum, a multi-locus genome-wide association study (GWAS) was performed using a collection of 200 accessions and 330,710 single-nucleotide polymorphisms (SNPs) via 3VmrMLM method. Five flowering time traits including budding (FBD), visible colouring (VC), early opening (EO), full-bloom (OF) and senescing (SF) stages, plus five derived conditional traits were recorded in two environments. Extensive phenotypic variations were observed for these flowering time traits with coefficients of variation ranging from 6.42 to 38.27%, and their broad-sense heritability ranged from 71.47 to 96.78%. GWAS revealed 88 stable quantitative trait nucleotides (QTNs) and 93 QTN-by-environment interactions (QEIs) associated with flowering time traits, accounting for 0.50-8.01% and 0.30-10.42% of the phenotypic variation, respectively. Amongst the genes around these stable QTNs and QEIs, 21 and 10 were homologous to known flowering genes in Arabidopsis; 20 and 11 candidate genes were mined by combining the functional annotation and transcriptomics data, respectively, such as MYB55, FRIGIDA-like, WRKY75 and ANT. Furthermore, genomic selection (GS) was assessed using three models and seven unique marker datasets. We found the prediction accuracy (PA) using significant SNPs identified by GWAS under SVM model exhibited the best performance with PA ranging from 0.90 to 0.95. Our findings provide new insights into the dynamic genetic architecture of flowering time and the identified significant SNPs and candidate genes will accelerate the future molecular improvement of chrysanthemum., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Chrysanthemum (Chrysanthemum morifolium Ramat.) is a globally important ornamental plant with great economic, cultural, and symbolic value. However, research on chrysanthemum is challenging due to its complex genetic background. Here, we report a near-complete assembly and annotation for C. morifolium comprising 27 pseudochromosomes (8.15 Gb; scaffold N50 of 303.69 Mb). Comparative and evolutionary analyses reveal a whole-genome triplication (WGT) event shared by Chrysanthemum species approximately 6 million years ago (Mya) and the possible lineage-specific polyploidization of C. morifolium approximately 3 Mya. Multilevel evidence suggests that C. morifolium is likely a segmental allopolyploid. Furthermore, a combination of genomics and transcriptomics approaches demonstrate the C. morifolium genome can be used to identify genes underlying key ornamental traits. Phylogenetic analysis of CmCCD4a traces the flower colour breeding history of cultivated chrysanthemum. Genomic resources generated from this study could help to accelerate chrysanthemum genetic improvement., (© 2023. The Author(s).)

Chrysanthemum morifolium is a well-known edible medicinal plant in Asia and some other regions. Content of selenium in Se-enriched C. morifolium (SeCM) is significantly higher than that in traditional C. morifolium (non-Se-enriched C. morifolium, TCM). In order to understand health effects of SeCM, its chemical composition, lifespan-prolonging activities, and impacts on antioxidant defense-related gene expressions of model organism D. melanogaster were systematically studied. A total of eight phenols, including luteolin-7-O-glucoside, linarin, luteolin, apigenin, diosmetin, acacetin, 3-caffeoylquinic acid and 4,5-dicaffeoylquinic acid, were identified in SeCM extract. Compared with TCM, SeCM exhibited superior antioxidant properties. Intake of SeCM dramatically reduced malondialdehyde level and increased activities of endogenous antioxidant enzymes in fruit flies. SeCM was able to upregulate gene expressions of Cu/Zn-superoxide dismutase, Mn-superoxide dismutase and hydrogen peroxide catalase, and extend lifespans of fruit flies. Comparatively high antioxidant capacities and lifespan-prolonging activities of SeCM might be attributed to its abundant phenols and selenium, which probably ameliorated accumulation of free radicals and susceptibility to oxidative stress. These findings provide clues on further exploitation and utilization of Se-enriched C. morifolium. PRACTICAL APPLICATIONS: Chrysanthemum morifolium has been used for nutraceutical and curative purposes in China for thousands of years. Se-enriched C. morifolium typically contains more selenium than traditional C. morifolium, and is widely consumed in Asia and some other regions. Selenium is an essential micronutrient for humans, and selenium deficiency may result in several diseases such as myocardial infarction. SeCM is one of important selenium supplements. In this study, SeCM was found to upregulate gene expressions of Cu/Zn-superoxide dismutase, Mn-superoxide dismutase, and hydrogen peroxide catalase, and extend lifespans of experimental animals. These results provide supporting information for developing SeCM-based functional foods with distinct health benefits., (© 2022 Wiley Periodicals LLC.)

Fluorescence in situ hybridization (FISH) is a conventional method used to visualize the distribution of DNA elements within a genome. To examine the relationships within the Chrysanthemum genus, ribosomal DNA (rDNA), a popular cytogenetic marker, was utilized as a probe for FISH within this genus. Based on the genome data of Chrysanthemum nankingense, C. seticuspe and its allied genera in the Compositae(Asteraceae), we explored rDNA sequences to design oligonucleotide probes and perform oligonucleotide fluorescence in situ hybridization (Oligo-FISH) in eight Chrysanthemum accessions. The results showed that the majority of 5S rDNA signals were located in subterminal chromosome regions and that the number of 5S rDNA sites might be tightly associated with ploidy. For 45S rDNA sites, the number and intensity of signals differed from those of previously investigated Chrysanthemum resources. These findings may provide an optimally reliable method of examining the chromosome composition and structural variation of Chrysanthemum and its related species and allow researchers to understand the evolutionary history and phylogenetic relationships of Chrysanthemum.

TOPLESS (TPL)/TOPLESS-related (TPR) corepressors are important regulators of plant growth and development, but their functions in chrysanthemum (Chrysanthemum morifolium) are currently unclear. In this study, a chrysanthemum TPL/TPR family gene, designated CmTPL1-1, was characterized. This gene encodes an 1135-amino-acid polypeptide harboring a conserved N-terminal domain and two C-terminal WD40 domains. CmTPL1-1 showed no transcriptional activity in yeast, and a localization experiment indicated that it localized to the nuclei in onion epidermal cells. Transcript profiling established that the gene was most highly expressed in the stem apex. The heterologous expression of CmTPL1-1 in Arabidopsis thaliana produced a pleiotropic phenotype, including smaller leaves, shorter siliques, increased meristem number, asymmetrical petal distribution and reduced stamen number. In transgenic plants, four AtARFs were downregulated, while six AtIAAs and two AtGH3s were upregulated at the transcript level; moreover, the expression of three key class I KNOTTED-like homeobox (KNOX) genes was upregulated. In addition, by yeast two-hybrid screening of a chrysanthemum cDNA library, we found that CmTPL1-1 could interact with CmWOX4, CmLBD38 and CmLBD36, and these interactions were confirmed by bimolecular fluorescence complementation (BiFC) assays. Overall, we speculated that heterologous expression of CmTPL1-1 regulates plant growth and development by interacting with auxin signaling in Arabidopsis., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

For a flowering plant, the transition from vegetative stage to reproductive growth is probably the most critical developmental switch. In the model plant Arabidopsis thaliana, the product of BBX7, group II member of BBX family, acts to delay floral transition. In this study, a presumed chrysanthemum homolog of a second group gene AtBBX8, designated CmBBX8, had been isolated and characterized. The transcription of CmBBX8 followed a diurnal rhythm as the chrysanthemum floral transition regulator. Overexpression of CmBBX8 accelerated flowering, while its (artificial microRNAs) amiR-enabled knockdown delayed flowering in plants grown under both long- and short-day conditions. Global expression analysis revealed that genes associated with photoperiod were down-regulated in amiR-CmBBX8 lines compared with the wild type, which were verified to be up-regulated in overexpressing lines (OX-CmBBX8) by RT-PCR. A number of in vitro assays were used to show that CmBBX8 targets CmFTL1. Furthermore, the function of CmFTL1 as a floral inducer under long-day conditions was confirmed by the behaviour of engineered summer-flowering chrysanthemum plants. The conclusion is that the BBX8-FT regulatory module is an important determinant of reproductive development in summer-flowering chrysanthemum., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Both the presence of, and the important contribution to growth and development made by TCP transcription factors, have been established in various plant species. Here, a TCP4 homolog isolated from Chrysanthemum nankingense was shown to be more strongly transcribed in the diploid than in the autotetraploid form of the species. CnTCP4 was shown to encode a member of the class II TCP family and to be transcribed most strongly in the leaf and ligulate flowers. Its transcription was found to be substantially inhibited by spraying the plant with the synthetic cytokinin 6-benzylaminopurine. The transient expression of CnTCP4 in onion epidermal cells showed that its product localized to the nucleus, and a yeast one hybrid assay suggested that its product had transcriptional activation ability. The constitutive expression of CnTCP4 in fission yeast suppressed cell proliferation, inducing the formation of longer and a higher frequency of multinuclated cells. Its constitutive expression in Arabidopsis thaliana reduced the size of the leaves. The presence of the transgene altered the transcription of a number of cell division-related genes. A yeast one hybrid assay identified a second TCP gene (CnTCP2) able to interact with the CnTCP4 promoter. A transient expression experiment in Nicotiana benthamiana leaves showed that CnTCP2 was able to activate the CnTCP4 promoter. Like CnTCP4, CnTCP2 was shown to encode a member of the class II TCP family, to be transcribed most strongly in the leaf and ligulate flowers, and to be suppressed by exogenous 6-benzylaminopurine treatment. The CnTCP2 protein also localized to the nucleus, but had no transcriptional activation ability. Its constitutive expression in A. thaliana had similar phenotypic consequences to those induced by CnTCP4., Competing Interests: The authors declare no conflict of interest.

Key Message: 81 SNPs were identified for three inflorescence-related traits, in which 15 were highly favorable. Two dCAPS markers were developed for future MAS breeding, and six candidate genes were predicted. Chrysanthemum is a leading ornamental species worldwide and demonstrates a wealth of morphological variation. Knowledge about the genetic basis of its phenotypic variation for key horticultural traits can contribute to its effective management and genetic improvement. In this study, we conducted a genome-wide association study (GWAS) based on two years of phenotype data and a set of 92,617 single nucleotide polymorphisms (SNPs) using a panel of 107 diverse cut chrysanthemums to dissect the genetic control of three inflorescence-related traits. A total of 81 SNPs were significantly associated with the three inflorescence-related traits (capitulum diameter, number of ray florets and flowering time) in at least one environment, with an individual allele explaining 22.72-38.67% of the phenotypic variation. Fifteen highly favorable alleles were identified for the three target traits by computing the phenotypic effect values for the stable associations detected in 2 year-long trials at each locus. Dosage pyramiding effects of the highly favorable SNP alleles and significant linear correlations between highly favorable allele numbers and corresponding phenotypic performance were observed. Two highly favorable SNP alleles correlating to flowering time and capitulum diameter were converted to derived cleaved amplified polymorphic sequence (dCAPS) markers to facilitate future breeding. Finally, six putative candidate genes were identified that contribute to flowering time and capitulum diameter. These results serve as a foundation for analyzing the genetic mechanisms underlying important horticultural traits and provide valuable insights into molecular marker-assisted selection (MAS) in chrysanthemum breeding programs.

To identify the high yield planting date and pinching pattern, we compared the yields of five new varieties (lines) of tea-applied chrysanthemum, the ‘Suju 10’, ‘Suju 12’, ‘Suju 13’, ‘CH1-44’ and ‘CH5-13’. A field experiment with split-split plot design was carried out. The main plot treatments including three (early, middle and late) planting dates, split-plot treatments including 5 new varieties (lines), and split-split-plot treatments including four different kinds of pinching schemes. The results showed that the yield of ‘CH5-13’ and ‘Suju 13’ was relatively higher among the five varieties (lines), followed by ‘CH1-44’ and ‘Suju10’, and ‘Suju 12’ showed the lowest yield. Among the five varieties (lines), planting date on May 27th and two-time pinching treatments showed the highest values of plant height, crown width, flower number, flower diameter, fresh mass, yield per plant and yield per unit area. Compared with the planting date on May 7th and June 13th, the above indexes increased by 16.0% and 19.0%, 18.0% and 22.8%, 36.7% and 42.2%, 11.1% and 2.3%, 13.0% and 4.0%, 47.8% and 36.6%, 48.5% and 36.7%, respectively. With the pinching time postponed, plant height decreased. Compared with the no pinching treatment, plant height of the two-time pinching treatment decreased by 50.2%, and values of crown width, flowers number, the flower fresh mass, the yield per plant and the yield per unit area were highest, with increases of 17.0%, 29.1%, 5.5%, 34.0% and 34.8%, respectively. The impact performance of three main factors on the growth and yield of the tea-applied chrysanthemum was in order of planting date>varieties>pinching time.

Main Conclusion: 37 unconditional QTLs, 51 conditional QTLs and considerable epistatic QTLs were detected for waterlogging tolerance, and six favourable combinations were selected accelerating the possible application of MAS in chrysanthemum breeding. Chrysanthemum is seriously impacted by soil waterlogging. To determine the genetic characteristics of waterlogging tolerance (WAT) in chrysanthemum, a population of 162 F 1 lines was used to construct a genetic map to identify the dynamic and epistatic quantitative trait loci (QTLs) for four WAT traits: wilting index (WI), dead leaf ratio (DLR), chlorosis score (Score) and membership function value of waterlogging (MFVW). The h B for the WAT traits ranged from 0.49 to 0.64, and transgressive segregation was observed in both directions. A total of 37 unconditional consensus QTLs with 5.81-18.21% phenotypic variation explanation (PVE) and 51 conditional consensus QTLs with 5.90-24.56% PVE were detected. Interestingly, three unconditional consensus QTLs were consistently identified across different stages, whereas no conditional consensus QTLs were consistently expressed. In addition, considerable epistatic QTLs, all with PVE values ranging from 0.01 to 8.87%, were detected by a joint analysis of WAT phenotypes. These results illustrated that the QTLs (genes) controlling WAT were environmentally dependent and selectively expressed at different times and indicated that both additive and epistatic effects underlie the inheritance of WAT in chrysanthemum. The findings of the current study provide insights into the complex genetic architecture of WAT, and the identification of favourable alleles represents an important step towards the application of molecular marker-assisted selection (MAS) and QTL pyramiding in chrysanthemum WAT breeding programmes.2 for the WAT traits ranged from 0.49 to 0.64, and transgressive segregation was observed in both directions. A total of 37 unconditional consensus QTLs with 5.81-18.21% phenotypic variation explanation (PVE) and 51 conditional consensus QTLs with 5.90-24.56% PVE were detected. Interestingly, three unconditional consensus QTLs were consistently identified across different stages, whereas no conditional consensus QTLs were consistently expressed. In addition, considerable epistatic QTLs, all with PVE values ranging from 0.01 to 8.87%, were detected by a joint analysis of WAT phenotypes. These results illustrated that the QTLs (genes) controlling WAT were environmentally dependent and selectively expressed at different times and indicated that both additive and epistatic effects underlie the inheritance of WAT in chrysanthemum. The findings of the current study provide insights into the complex genetic architecture of WAT, and the identification of favourable alleles represents an important step towards the application of molecular marker-assisted selection (MAS) and QTL pyramiding in chrysanthemum WAT breeding programmes.

Main Conclusion: Forty-five molecular markers were detected significantly associated with chrysanthemum' waterlogging tolerance, and four favorable parental lines were identified as potential donors for improving waterlogging tolerance in chrysanthemum. The productivity of chrysanthemum is downgraded by waterlogging soils, which has driven a search for germplasm showing an enhanced level of waterlogging tolerance (WT). As yet little is known regarding the mode of inheritance of WT in chrysanthemum. The study set out to characterize the extent of genetic variation for WT represented in a collection of one hundred chrysanthemum accessions by testing them under both greenhouse and field conditions. A membership function value of waterlogging (MFVW), which integrated a wilting index, a chlorosis score and the proportion of dead leaf in waterlogged plants, was used as a measure of WT. The variation for MFVW among plants grown in the greenhouse (two experiments) was generally higher than that generated in field-grown (one experiment) plants. The MFVW broad sense heritability was 0.82, and the phenotypic coefficient of variation (31.8 %) was larger than the genetic one (28.8 %). Association mapping (AM) identified 45 markers related to WT: 25 by applying the general linear model (GLM) + principal component (PC) model, 16 by applying the mixed linear model (MLM), 31 by applying the MLM + Q matrix model and 12 by applying the MLM + PC model. Of the associated markers, eight and two were predictive in two and three experiments within all models, respectively; the proportion of the phenotypic variance explained by the eight associations ranged from 6.3 to 16.4 %. On the basis of their harboring all four of the leading markers E2M16-2, SSR150-6, E19M16-1 and E10M10-12, the varieties 'Nannong Xuefeng', 'Qx097', 'Nannong Xunzhang' and 'Finch' were identified as potential donors for future improvement of WT in chrysanthemum.

Varieties of the economically important ornamental species chrysanthemum have been bred to fit a number of market niches, but the genetic basis and evolutionary relationships among various cultivated types are poorly understood. Here, a DNA marker-based analysis of 199 chrysanthemum entries representing each of the five cultivated types is presented. A set of >90,000 single nucleotide polymorphisms (SNPs) associated with a minor allele frequency of at least 5% was defined, and used to perform a phylogenetic analysis which corresponded well with the phenotypic classification. The analysis revealed that the small-flowered types, spray cut chrysanthemum (SCC) and potted and ground chrysanthemum (PGC), are more closely related to the wild progenitor species (WC) than are the large-flowered ones, disbud cut chrysanthemum (DCC) and traditional chrysanthemum (TC); and the PGC type was closest. Some 550 genetic regions appeared to have experienced selection in the separation of potted and ground-cover types from disbud cut types, and that between potted and ground-cover types from traditional types. A genome-wide association analysis revealed that seven SNPs lying within six genes were predictive of three important traits (ray floret type, cultivated type and flower shape), but no association with flower color was detected. The study has provided a number of novel insights into evolutionary relationships, the population structure and the genetic basis of some key ornamental traits., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Salinity-stressed plants of salinity sensitive ('Qx096') and tolerant ('Qx097') chrysanthemum cultivar were treated with a range of concentrations of spermidine (Spd). Plant performance, as indicated by various parameters associated with growth, was improved by the treatment, as was the tissue content of soluble protein and proline. The extent of both Na(+) accumulation and K(+) loss was reduced. Activity levels of the stress-related enzymes SOD, POD, APX and CAT were significantly increased and the production of malondialdehyde (MDA) decreased. The suggestion was that treatment with 1.5 mM Spd would be an effective means alleviating salinity-stress induced injury through its positive effect on photosynthetic efficiency, reactive oxygen species scavenging ability and the control of ionic balance and osmotic potential. Its protective capacity was more apparent in 'Qx096' than in 'Qx097'., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

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 plant-specific LATERAL ORGAN BOUNDARIES DOMAIN (LBD) genes are important regulators of growth and development. Here, a chrysanthemum class I LBD transcription factor gene, designated CmLBD1, was isolated and its function verified. CmLBD1 was transcribed in both the root and stem, but not in the leaf. The gene responded to auxin and was shown to participate in the process of adventitious root primordium formation. Its heterologous expression in Arabidopsis thaliana increased the number of lateral roots formed. When provided with exogenous auxin, lateral root emergence was promoted. CmLBD1 expression also favored callus formation from A. thaliana root explants in the absence of exogenously supplied phytohormones. In planta, CmLBD1 probably acts as a positive regulator of the response to auxin fluctuations and connects auxin signaling with lateral root formation.

Members of the large WRKY transcription factor family are responsible for the regulation of plant growth, development and the stress response. Here, five WRKY members were isolated from chrysanthemum. They each contained a single WRKY domain and a C2H2 zinc finger motif, so were classified into group II. Transient expression experiments demonstrated that all five were expressed in the nucleus, although CmWRKY42 was also expressed in the cytoplasm. When expressed heterologously in yeast, the products of CmWRKY22 and CmWRKY48 exhibited transactivation activity, while those of CmWRKY21, CmWRKY40 and CmWRKY42 did not. The transcription of the five CmWRKY genes was profiled when the plants were challenged with a variety of abiotic and biotic stress agents, as well as being treated with various phytohormones. CmWRKY21 proved to be markedly induced by salinity stress, and suppressed by high temperature exposure; CmWRKY22 was induced by high temperature exposure; CmWRKY40 was highly induced by salinity stress, and treatment with either abscisic acid (ABA) or methyl jasmonate (MeJA); CmWRKY42 was up-regulated by salinity stress, low temperature, ABA and MeJA treatment and aphid infestation; CmWRKY48 was induced by drought stress, ABA and MeJA treatment and aphid infestation. The function of CmWRKY48 was further investigated by over-expressing it transgenically. The constitutive expression of this transcription factor inhibited the aphids' population growth capacity, suggesting that it may represent an important component of the plant's defense machinery against aphids., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

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, β-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 establishment of a (set of) stably expressed reference gene(s) is required to normalize transcription data. Polyploidy is very common in the plant kingdom, but it is not necessarily the case that a reference gene which works well at the diploid level will also work well at the polyploid level. Here, ten candidate reference genes are compared in the context of gene transcription in the genus Chrysanthemum. The robustness of some, but not all, of these was shown to be high across ploidy levels. MTP (metalloprotease) and ACTIN (actin) were the most stable in diploid and tetraploid C. nankingense, while PSAA (photosynthesis-related plastid gene representing photosystem I) and EF-1α (elongation factor-1α) were the most stable in tetraploid and hexaploid C. zawadskii. EF-1α and PGK (phosphoglycerate kinase) was the best combination for the complete set of four taxa. These results suggest that when making cross-species comparison of transcript abundance involving different ploidy levels, care needs to be taken in the selection of reference gene(s).

Flowering time is an important trait in chrysanthemum, but its genetic basis remains poorly understood. An intra-specific mapping population bred from the cross between the autumn-flowering cultivar 'Yuhualuoying' and the summer-flowering 'Aoyunhanxiao' was used to determine the number and relative effect of QTL segregating for five measures of flowering time. From flowering time data recorded over two consecutive seasons, 35 additive QTL were detected, each explaining between 5.8% and 22.7% of the overall phenotypic variance. Of these, 13 were detected in both years. Nine genomic regions harboring QTL for at least two of the five traits were identified. Ten pairs of loci epistatically determined the flowering time, but their contribution to the overall phenotypic variance was less than for the additive QTL. The results suggest that flowering time in chrysanthemum is principally governed by main effect QTL but that epistasis also contributes to the genetic architecture of the trait, and the major QTL identified herein are useful in our ongoing efforts to streamline the improvement of chrysanthemum via the use of molecular methodology.

The leaves of the chrysanthemum variety 'NAU04-1-31-1' are variegated with distinct green and yellow sectors. The chlorophyll content of the yellow leaf tissue is less than that in the green one. The chloroplasts in the yellow leaf tissue were vacuolated, lacked thylakoid membrane structure and contained clusters of plastoglobuli with few or no starch grains. The yellow leaf tissue was more sensitive to photo-inhibition than the green leaf tissue. Suppression subtractive hybridization (SSHs) libraries were constructed to identify genes which were differentially transcribed in the two tissue types. The sequencing of 339 SSH clones identified 150 unigenes (93 singletons and 57 contigs), of which 85 were differentially transcribed in the green leaf tissue and 65 in the yellow leaf tissue. Unigenes associated with photosynthesis were particularly frequent, and many of these genes were up-regulated in the yellow leaf tissue. Both CmChlH which encodes the large subunit of Mg-chelatase and CmFtsH (ATP-dependent metalloprotease) were up-regulated in the yellow leaf tissue, and their transcription was regulated by light.

Five intergeneric hybrids between the chrysanthemum cultivar 'Zhongshanjingui' (as female) and Ajania przewalskii (as male) were obtained with the help of embryo culture. While 'Zhongshanjingui' bears a standard anemone type flower and A. przewalskii a non-anemone type one, the inflorescence type of the hybrids varied. The diameter of the hybrids' flowers was intermediate between those of the parents. The chromosome number of the hybrids was 2n = 45, of which GISH analysis was able to establish that 27 were inherited from 'Zhongshanjingui' and the other 18 from A. przewalskii. A combination of various assays was used to show that the cold tolerance of the hybrids was equivalent to that of the highly tolerant A. przewalskii parent. Enhanced cold tolerance was correlated with an increase in free proline and a decrease in malondialdehyde content.

An appreciation of the genetic diversity retained in the gene pool of anemone‐type chrysanthemums is needed to drive further genetic improvement of this commercially important ornamental species. Here, a panel of 67 accessions was characterized with respect to both floral morphology, assessed using a set of 12 inflorescence‐associated traits, and to genotype, using sequence‐related amplified polymorphism (SRAP) fingerprinting. Abundant variation and a high level of broad‐sense heritability (>88%) were revealed for all traits except number of tubular floret lobes. Of the possible 66 pairwise inter‐trait combinations, 37 proved to be significantly correlated. A principal component analysis of the trait data showed that the first four principal components explained ~78% of the variance; meanwhile a hierarchical clustering analysis revealed a number of discrete groups reflecting the various cultivated types, but was not predictive of provenance. The SRAP genotyping employed 22 primer combinations and generated 238 informative fragments. A moderately high level of the Jaccard's similarity coefficient (mean 0.67) was calculated for the examined panel. A hierarchical clustering analysis based on the genotypic data revealed four groups, which were clearly reflective of cultivated type; moreover, a comparison of the within‐cluster means for each of the two key anemone‐related traits—tubular floret length and tubular floret length/style length—showed that the genotype‐based clusterings were associated with the anemone levels, albeit imperfectively. A principal coordinate analysis showed that the first two coordinates accounted for about 14% of the total variation. In summary, the present study adds new understanding of the genetic diversity present in the anemone‐type chrysanthemum gene pool, and the findings will lay the foundation for implementing a genotype/phenotype association strategy to reveal the genetic and molecular basis of floral form in this valuable ornamental species. [ABSTRACT FROM AUTHOR]

The phylogenetic relationships among Chrysanthemum and its related genera (Anthemideae, Asteraceae) is poorly understood. In the present study, these relationships were investigated using 45S and 5S ribosomal DNA (rDNA)-targeted fluorescent in situ hybridization. The results showed that there were two 45S rDNA signals present in Crossostephium chinense , four 45S rDNA signals in Cercidiphyllum japonicum , Artemisia sieversiana , Artemisia annua and Artemisia absinthium , six 45S rDNA signals in Chrysanthemum boreale and Pyrethrum parthenium , eight 45S rDNA signals in Chrysanthemum nankingense , Chrysanthemum dichrum , Chrysanthemum lavandulifolium and Tanacetum vulgare , and ten 45S rDNA signals in Ajania przewalskii . For the 5S rDNA locus, two 5S rDNA signals were present in C . nankingense , C . dichrum , C . lavandulifolium , C . boreale , C . japonicum , C. chinense and P. parthenium , four in A. sieversiana , A . annua , A . absinthium and A. przewalskii , and six 5S in T. vulgare . In addition, karyotypes of the 12 species were investigated. From this study, we inferred that Chrysanthemum was closely related to Ajania , and that Chrysanthemum species originating from China and Japan may have evolved differently. These findings add a new level to the understanding of the phylogenetic relationships of Chrysanthemum and related genera. [ABSTRACT FROM AUTHOR]

Aphids have caused great damage to chrysanthemum production, and it's crucial to breed new chrysanthemums with strong aphid resistance. However, little information is available regarding the inheritance of aphid resistance in chrysanthemum. The inheritance pattern of chrysanthemum aphid resistance within an F 1 segregating population was dissected with the major gene plus polygene mixed inheritance model and molecular markers. The result showed that aphid resistance of chrysanthemum is a quantitative trait with a moderate coefficient of variation >50%. The mixed inheritance model based on a single segregating generation suggested the inheritance of aphid resistance was controlled by two pairs of major genes with additive effect 0.68 and 0.39, respectively. The heritability of major gene was calculated at 89.21%. Marker-trait analysis based on one-way variance analysis uncovered 11 markers significantly associated with phenotype, cumulatively explaining ∼74% variation. Bulked segregant analysis (BSA) based on the gene bulks of high resistant and susceptive genotypes in F 1 population identified two SSR markers, SSR145-93 and SSR197-205, linked to high aphid resistance ( r > 0.4). The QTL analysis detected 5 putative QTL for aphid resistance in two successive years, with individual QTL explaining the phenotypic variation of 14.30–28.00%. The inheritance model and molecular markers identified for aphid resistance facilitate the ongoing breeding activities in chrysanthemum. [ABSTRACT FROM AUTHOR]

Abstract: A genetic linkage map of chrysanthemum (Chrysanthemum morifolium) was constructed by genotyping 142 F1 progeny of the bi-parental cross ‘Yuhualuoying’בAoyunhanxiao’ 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 1034cM with an average map distance of 6.2cM 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 1095cM and a mean inter-marker separation of 6.9cM 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. [Copyright &y& Elsevier]

Low temperature is a major stress factor that has adverse effects on chrysanthemum production; however, there is little information regarding the genetic mechanism of cold tolerance. Based on a previously constructed F1 mapping population, genetic variations and QTL effects were investigated for cold tolerance at the vegetative, bud, flower, and rhizome stages. The results showed that the coefficient of variation for cold tolerance at the four growth stages ranged from 22.17 to 46.14%, and heterosis and transgressive segregation were observed in both directions. Mixed inheritance model analysis revealed that cold tolerance at the vegetative and flower stages was controlled by two pairs of major genes that exhibited additivity-dominance-epistasis effects, and the heritabilities of the major genes were 88.56% and 65.86%, whereas no major gene model was identified for cold tolerance at the bud and rhizome stages. A total of 15 cold-tolerance-associated QTLs that explained 6.47–68.89% of the phenotypic variation were detected across the four stages, four of which were synergistic QTLs. Most of the identified QTLs were expressed at 2–4 growth stages, but only one QTL was identified at all four stages. This finding indicated that the QTLs underlying cold tolerance were dependent on the environment and were selectively expressed at different stages. In addition, for cold tolerance at the bud and flower stages, the QTLs qBdsCTM33 and qFfsCTM33 on the M33 linkage group accounted for 65.78% and 68.89% of the phenotypic variation, respectively, but had corresponding negative additive effects of − 5.16 and − 4.57 °C, indicating that these QTLs are major contributors to the improvement of cold tolerance. The findings of the present study reveal the genetic architecture of cold tolerance at different developmental stages, and the major QTLs and linked molecular markers identified represent an important step toward molecular-marker-assisted selection and QTL pyramiding for future breeding of cold-tolerant chrysanthemum. [ABSTRACT FROM AUTHOR]

Understanding the genetic architecture is a prerequisite for crop improvement. The current research aimed to characterize the extent of genetic variation of drought tolerance harbored in a global collection of 159 chrysanthemum cultivars over 2 years. An average subordinate function value (ASFV), integrating the wilting index, the fresh weight retention rate, and the survival rate after re-watering under two drought-stressed trials, was used to quantify the level of drought tolerance. The performance of ASFV was generally correlated between the 2 years; and a high magnitude (0.95) of broad-sense heritability, coupled with the moderate genetic advance, was estimated for the ASFV. By applying MLM model with both population structure and kinship matrix as covariates association mapping identified 16 markers linked to drought tolerance, with the proportion of the phenotypic variation explained by an individual marker ranging from 4.4 to 7.6%. Of the eight markers predictive across the 2 years, four (E11M24-9, E3M2-8, E1M5-5, and EST-SSR34-3) were identified as favorable alleles for drought tolerance. Several cultivars that carry at least three of the four favorable alleles were identified as potential donor parents for future improvement of the drought tolerance. The findings provide an insight into the genetic basis of the drought tolerance in chrysanthemum and will, therefore, aid in developing new cultivars with enhanced tolerance against drought stress. [ABSTRACT FROM AUTHOR]

• Bulked segregant analysis coupled with whole-genome sequencing (BSA-seq) was used to excavate major loci and genes for anemone type flower in chrysanthemum. • Three out of twenty-two key candidate genes were screened out by a combination of gene functional annotation, transcriptome and qRT-PCR analyses. • The developed KASP markers would help in marker-assisted selection of anemone type flower in chrysanthemum molecular breeding. Chrysanthemum is one of the most leading commercial flower crops well-known for its flower shape diversity. Anemone type chrysanthemums receive increasing market attention that are distinguished by their pigmented and elongated disk florets. However, there are few reports on quantitative trait loci (QTL) mapping and the identification genes for anemone in chrysanthemum. In this study, bulked segregant analysis coupled with whole-genome sequencing (BSA-seq) approach was employed to detect candidate genomic regions controlling anemone type flower. Two mixed DNA pools composed of 28 anemone type and 20 non-anemone type F 1 lines derived from a cross between 'Nannong Xuefeng' and 'Monalisa' together with two parents were used for high-throughput sequencing. A total of 42 consistent QTLs covering 30.00 Mb were simultaneously identified by |△(SNP/InDel-index)| and Euclidean distance (ED) algorithms based on 15,783,313 SNPs (single-nucleotide polymorphisms) and 1,546,200 small InDels (insertions and deletions). Twenty-two key candidate genes were screened out by a combination of gene functional annotation and transcriptome analysis, in which evm.model.scaffold_915.436 (CBF5), evm.model.scaffold_24.208 (U2AF65A), and a uncharacterized gene evm.model.scaffold_260.201 were highlighted by qRT-PCR. Furtherly, four associations were successfully converted to KASP (kompetitive allele specific PCR) markers, among them, the KASP markers developed for SNP locus Chr6__33961007 and Chr6__179207697 were proven to efficiently discriminate anemone and non-anemone type chrysanthemums being a couple of diagnostic markers in two populations. The results presented herein provide insights for further research on elucidating the formation mechanism of anemone type flower, and the anemone-specific KASP marker will be highly useful for applying MAS to accelerate breeding improvement of chrysanthemum. [ABSTRACT FROM AUTHOR]

High-density genetic maps are a valuable tool for quantitative trait locus (QTL) mapping and gene discovery for important traits in plants. However, such work in chrysanthemum remains largely unexplored, primarily owing to its large genome and complex genetic background. In this study, a high-density map containing 11,941 SNP bins spanning a total of 2967.76 cM in 27 linkage groups (LGs) with an average intermarker distance of 0.26 cM was developed with a genotyping by sequencing (GBS) approach. A total of 34 QTL clusters for waterlogging tolerance (WAT) were detected via multiple-QTL mapping (MQM) algorithms. A total of 186 candidate genes within these QTL clusters were mined in combination with transcriptome data. One key candidate gene within the QTL cluster cWAT6.1 , which belongs to group VII ethylene response factors (CmWAT6.1), was proven to be a positive regulator of WAT in chrysanthemum. Furthermore, transcriptomic assays of transgenic and wild-type chrysanthemums under normal conditions as well as further experiments provided evidence that CmWAT6.1 regulated WAT by promoting cell wall formation and lignin biosynthesis and coordinating oligosaccharide metabolic processes. This study represents important progress in gene mining through forward genetics, provides valuable genetic resources for further WAT improvement and provides new insight into the molecular mechanism of WAT in chrysanthemum. • A high-density map with an average intermarker distance of 0.26 cM for 27 linkage groups was constructed. • A total of 34 stable QTL clusters for waterlogging tolerance were detected via QTL mapping • 186 candidate genes within QTL clusters were mined in combination with gene functional annotation and transcriptome data. • CmWAT6.1 was proven to be a positive regulator of chrysanthemum waterlogging tolerance by promoting lignin biosynthesis. [ABSTRACT FROM AUTHOR]

• Extensive and continuous variations for leaf angle related traits were observed among 200 cut chrysanthemum accessions across two years. • 100 stable quantitative trait nucleotides (QTNs) and 75 QTN×environments interactions (QEIs) were identified to be associated with leaf angle using multi-locus genome-wide association study via 3VmrMLM. • 26 known and 32 novel genes mined here will contribute to the molecular breeding program of chrysanthemum plant architecture. • Haplotype analysis of ARF16 revealed four accessions with elite leaf angle as potential donors for improving chrysanthemum leaf angle. Chrysanthemum (Chrysanthemum morifolium Ramat.), one of the four most popular cut flowers in the world, occupies a large share of global floriculture trade. Leaf angle (LA) is an important factor affecting plant architecture, photosynthesis and yields of cut chrysanthemum. However, the genetic basis and related genes underlying LA remain largely unknown. In this research, phenotypes of five LA-related traits in 200 cut chrysanthemum accessions were investigated across two consecutive years (Y2020 and Y2021), and their broad-sense genetic heritabilities ranged from 68.91% to 84.70%. Leaf orientation value (LOV) exhibited the maximum phenotypic coefficient of variation of 35.66% in Y2020 and 32.34% in Y2021. A new methodological framework named 3VmrMLM was applied for multi-locus genome-wide association study (GWAS) based on a set of 330,710 single nucleotide polymorphisms (SNPs) to explore the genetic loci and candidate genes of LA. As a result, 303 quantitative trait nucleotides (QTNs) in single-environment, 120 QTNs and 75 QTN × environments interactions (QEIs) in multiple-environments were identified. Among the genes around these QTNs and QEIs, 26 known genes were considered to be associated with LA, including ARF16, IBH1, AN3 , etc. Furthermore, the haplotype analysis of ARF16 revealed four accessions with elite LA. Our findings provide a fundamental understanding to the genetic architecture of LA and contribute to further gene cloning and molecular improvement of chrysanthemum plant architecture. [ABSTRACT FROM AUTHOR]

Abstract: Anther wall development, microsporogenesis and microgametogenesis were compared between a normal male fertile chrysanthemum cultivar ‘NJAU04-29-2’ and the two male sterile selections ‘rm20-11’ (anther indehiscent) and ‘NJAU05-52-2’ (anther aborted). In both of the two male sterile types, the tapetum enlarged abnormally and showed signs of disorganization of walls at the onset of meiosis, the pollen was aborted, the anthers appeared shrunken, and the anther vascular bundle and connective tissue were degenerated by anthesis. In ‘rm20-11’, the two smaller pollen sacs began to degenerate at the microsporogenesis stage, so that only one or two microsporangia developed, while in ‘NJAU05-52-2’, only one or two microsporangia were formed following the archesporial cell stage, and most of the microspore mother cells degenerated during the course of meiosis. [ABSTRACT FROM AUTHOR]

• Inheritance of branching traits in chrysanthemum was dissected by QTL mapping under two contrasting densities. • Twelve and eight additive QTLs were uncovered under the low and high planting density regime conditions, respectively. • Two joint QTLs and four epistatic QTLs were detected. • Only one QTL, TsbnE1 × 4 or TsbnE2 × 4, was stable across two environments. The architecture of chrysanthemum plants raised for cut flowers is strongly influenced by their ability to form branches. The genetic basis of branching was revealed through a quantitative trait locus (QTL) analysis of an F 1 population generated by crossing the varieties 'Nannong Xuefeng' and 'Monalisa' grown under contrasting planting densities. Under the low planting density regime (E1), 12 additive QTLs involving seven branching-associated traits were detected, while under the high planting density regime (E2), the number of QTL detected was only eight. One of the individual QTL accounted for over 10% of the phenotypic variance. Of the 20 QTLs, only one was expressed under both high and low planting densities. A joint QTL analysis across the two environments identified two QTLs which were separately detected in E1. A set of four QTLs exhibiting additive × additive epistasis was identified, few of which had interaction with environments. [ABSTRACT FROM AUTHOR]