As the junction of floral development pathways, the FLOWERING LOCUS T (FT) protein called 'florigen' plays an important role in the process of plant flowering through signal integration. We isolated four transcripts encoding different isoforms of a FT orthologous gene CmFTL1, from Chrysanthemum morifolium cultivar 'Jimba'. Sequence alignments suggested that the four transcripts are related to the intron 1. Expression analysis showed that four alternative splicing (AS) forms of CmFTL1 varied depending on the developmental stage of the flower. The functional complement experiment using an Arabidopsis mutant ft-10 revealed that the archetypal and AS forms of CmFTL1 had the function of complementing late flower phenotype in different levels. In addition, transgenic confirmation at transcript level showed CmFTL1 and CmFTL1ast coexist in the same tissue type at the same developmental stage, indicating a post-transcriptional modification of CmFTL1 in Arabidopsis. Moreover, ectopic expression of different AS forms in chrysanthemum resulted in the development of multiple altered phenotypes, varying degrees of early flowering. We found that an alternative splicing form (CmFTL1-astE134) without the exon 2 lacked the ability causing the earlier flower phenotype. The evidence in this study indicates that complex alternative processing of CmFTL1 transcripts in C. morifolium may be associated with flowering regulation and hold some potential for biotechnical engineering to create early-flowering phenotypes in ornamental cultivars.

Chrysanthemums are sensitive to waterlogging stress, and the development of screening methods for tolerant germplasms or genes and the breeding of tolerant new varieties are of great importance in chrysanthemum breeding. To understand the genetic basis of waterlogging tolerance (WT) in chrysanthemums, we performed a genome-wide association study (GWAS) using 92,811 single nucleotide polymorphisms (SNPs) in a panel of 88 chrysanthemum accessions, including 64 spray cut and 24 disbud chrysanthemums. The results showed that the average MFVW (membership function value of waterlogging) of the disbud type (0.65) was significantly higher than that of the spray type (0.55) at P < 0.05, and the MFVW of the Asian accessions (0.65) was significantly higher than that of the European accessions (0.48) at P < 0.01. The GWAS performed using the general linear model (GLM) and mixed linear model (MLM) identified 137 and 14 SNP loci related to WT, respectively, and 11 associations were commonly predicted. By calculating the phenotypic effect values for 11 common SNP loci, six highly favorable SNP alleles that explained 12.85-21.85% of the phenotypic variations were identified. Furthermore, the dosage-pyramiding effects of the favorable alleles and the significant linear correlations between the numbers of highly favorable alleles and phenotypic values were identified (r 2 = 0.45; P < 0.01). A major SNP locus (Marker6619-75) was converted into a derived cleaved amplified polymorphic sequence (dCAPS) marker that cosegregated with WT with an average efficiency of 78.9%. Finally, four putative candidate genes in the WT were identified via quantitative real-time PCR (qRT-PCR). The results presented in this study provide insights for further research on WT mechanisms and the application of molecular marker-assisted selection (MAS) in chrysanthemum WT breeding programs.

Allopolyploid formation involves two major events: interspecific hybridization and polyploidization. A number of species in the Asteraceae family are polyploids because of frequent hybridization. The effects of hybridization on genomics and transcriptomics in Chrysanthemum nankingense×Tanacetum vulgare hybrids have been reported. In this study, we obtained allopolyploids by applying a colchicine treatment to a synthesized C. nankingense×T. vulgare hybrid. Sequence-related amplified polymorphism (SRAP), methylation-sensitive amplification polymorphism (MSAP), and high-throughput RNA sequencing (RNA-Seq) technologies were used to investigate the genomic, epigenetic, and transcriptomic alterations in both the hybrid and allopolyploids. The genomic alterations in the hybrid and allopolyploids mainly involved the loss of parental fragments and the gain of novel fragments. The DNA methylation level of the hybrid was reduced by hybridization but was restored somewhat after polyploidization. There were more significant differences in gene expression between the hybrid/allopolyploid and the paternal parent than between the hybrid/allopolyploid and the maternal parent. Most differentially expressed genes (DEGs) showed down-regulation in the hybrid/allopolyploid relative to the parents. Among the non-additive genes, transgressive patterns appeared to be dominant, especially repression patterns. Maternal expression dominance was observed specifically for down-regulated genes. Many methylase and methyltransferase genes showed differential expression between the hybrid and parents and between the allopolyploid and parents. Our data indicate that hybridization may be a major factor affecting genomic and transcriptomic changes in newly formed allopolyploids. The formation of allopolyploids may not simply be the sum of hybridization and polyploidization changes but also may be influenced by the interaction between these processes.

MYB transcription factors are widely involved in the development of and physiological processes in plants. Here, we isolated the chrysanthemum R2R3-MYB family transcription factor CmMYB15, a homologous gene of AtMYB15. It was demonstrated that CmMYB15 expression was induced by aphids and that CmMYB15 could bind to AC elements, which usually exist in the promoter of lignin biosynthesis genes. Overexpression of CmMYB15 in chrysanthemum enhanced the resistance of aphids. Additionally, the content of lignin and the expression of several lignin biosynthesis genes increased. In summary, the results indicate that CmMYB15 regulates lignin biosynthesis genes that enhance the resistance of chrysanthemum to aphids.

The Mildew Resistance Locus O (MLO) gene family has been investigated in many species. However, there are few studies on chrysanthemum MLO genes. We report in this study that CmMLO17 in Chrysanthemum morifolium was upregulated after Alternaria alternata infection. Silencing of CmMLO17 by artificial microRNA resulted in reduced susceptibility of chrysanthemum to A. alternata infection. Genes in the abscisic acid (ABA) and Ca2+ signaling pathways were upregulated in the CmMLO17-silenced line R20 compared to the wild-type plants. We speculated that CmMLO17-silenced plants had a faster and stronger defense response that was mediated by the ABA and Ca2+ signaling pathways, resulting in reduced susceptibility of chrysanthemum to A. alternata infection. In addition, a candidate gene, CmKIC, that may interact with CmMLO17 was discovered by the yeast two-hybrid assay. The interaction between CmMLO17 and CmKIC was confirmed using the yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) analysis. CmMLO17 and CmKIC were both located on the plasma membrane, and CmKIC was also located on the nucleus. CmKIC overexpression increased the susceptibility of chrysanthemum to A. alternata, whereas CmKIC silencing resulted in reduced susceptibility. Therefore, CmMLO17 and CmKIC may work together in C. morifolium to support the growth of A. alternata. The results of this study will provide insight into the potential function of MLO and improve the understanding of plant defense responses to necrotrophic pathogens.

The CmBBX8-CmFTL1 regulatory module is a key determinant in the transition from vegetative growth to reproductive development in summer-flowering chrysanthemum. However, the detailed regulatory mechanism of CmBBX8-mediated flowering remains elusive. In this study, we revealed that RADICAL-INDUCED CELL DEATH 1 (CmRCD1) physically associated with CmBBX8 through bimolecular fluorescence complementation (BiFC), pulldown and Coimmunoprecipitation (CoIP) assays. Furthermore, the RCD1-SRO1-TAF4 (RST) domain of CmRCD1 and the B-box of CmBBX8 mediated their interaction. In addition, Luciferase (LUC) assays and electrophoretic mobility shift assay (EMSAs) showed that CmRCD1 repressed the transcriptional activity of CmBBX8 and interfered with its binding to the CmFTL1 promoter, thereby leading to delayed flowering in the summer chrysanthemum 'Yuuka'. These results provide insight into the molecular framework of CmRCD1-CmBBX8-mediated flowering in chrysanthemum.

The enzyme RNAPII CTD phosphatase-like 1 is known as a transcriptional regulator of the plant response to various abiotic stresses. Here, the isolation of CmCPL1, a chrysanthemum (Chrysanthemum morifolium) gene encoding this enzyme is described. Its predicted 955 residue gene product includes the FCPH catalytic domain, two double-stranded RNA binding motifs, and a nuclear localization signal. A sub-cellular localization assay confirmed that CmCPL1 was expressed in the nucleus. CmCPL1 transcription was shown to be significantly inducible by heat stress. The over-expression and knockdown of CmCPL1, respectively, increased and diminished the tolerance of chrysanthemum to heat stress, which maybe dependent on the regulation of CmCPL1 and on the expression of downstream heat stress-responsive genes.

The switch from vegetative growth to reproductive growth is a key event in the development of a plant. Here, the product of the chrysanthemum gene CmMYB2, an R2R3 MYB transcription factor that is localized in the nucleus, was shown to be a component of the switching mechanism. Plants engineered to overexpress CmMYB2 flowered earlier than did wild-type plants, while those in which CmMYB2 was suppressed flowered later. In both the overexpression and RNAi knockdown plants, a number of genes encoding proteins involved in gibberellin synthesis or signaling, as well as in the response to photoperiod, were transcribed at a level that differed from that in the wild type. Both yeast two-hybrid and bimolecular fluorescence complementation assays revealed that CmMYB2 interacts with CmBBX24, a zinc-finger transcription factor known to regulate flowering by its influence on gibberellin synthesis.

Chrysanthemum is frequently attacked by aphids, which greatly hinders the growth and ornamental value of this plant species. WRKY transcription factors play an important role in the response to biotic stresses such as pathogen and insect stresses. Here, chrysanthemum CmWRKY53 was cloned, and its expression was induced by aphid infestation. To verify the role of CmWRKY53 in resistance to aphids, CmWRKY53 transgenic chrysanthemum was generated. CmWRKY53 was found to mediate the susceptibility of chrysanthemum to aphids. The expression levels of secondary metabolite biosynthesis genes, such as peroxidase- and polyphenol oxidase-encoding genes, decreased in CmWRKY53-overexpressing (CmWRKY53-Oe) plants but dramatically increased in chimeric dominant repressor (CmWRKY53-SRDX) plants, suggesting that CmWRKY53 contributes to the susceptibility of chrysanthemum to aphids, possibly due to its role in the regulation of secondary metabolites.

Chrysanthemum (Chrysanthemum morifolium Ramat.) is an economically important plant species growing worldwide. However, its origin, especially as revealed by biogeographic and metabolomics research, remains unclear. To understand the geographic distribution of species diversity and metabolomics in three genera (Chrysanthemum, Ajania, and Phaeostigma), geographic information systems and gas chromatography-mass spectrometry were used in 19, 15, and 4 species respectively. China and Japan were two potential panbiogeographic nodes and diverse hotspots of Chrysanthemum, with species richness ratios of 58.97 and 33.33%. We studied different species from two hotspots which in similar geographical environments had closer chemotaxonomic relationships under the same cultivation conditions based on a cluster of 30 secondary metabolites. The average distribution altitude (ADA) differed significantly among Chrysanthemum, Ajania, and Phaeostigma in which it was 1227.49, 2400.12, and 3760.53 m.a.s.l. respectively, and the presence/absence of ray florets (RF) was significantly correlated with ADA (-0.62). Mountain landform was an important contributor to global Chrysanthemum diversity, playing a key role in the divergence and distribution pattern of Chrysanthemum and its allies. The Hengduan Mountains-Qinling Mountains (HDQ) in China was a potential secondary radiation and evolution center of Chrysanthemum and its related genera in the world. During the Quaternary glacial-interglacial cycles, this region became their refuge, and they radiated and evolved from this center.

The chrysanthemum genome harbors three FT-like genes: CmFTL1 and CmFTL3 are thought to act as regulators of floral induction under long-day (LD) and short-day (SD) conditions, respectively, whereas the function of CmFTL2 is currently unclear. The objective of the present research was to explore the function of CmFTL2 in the determination of flowering time of the photo-insensitive chrysanthemum cultivar 'Floral Yuuka', both in response to variation in the photoperiod and to the exogenous provision of sucrose. Spraying leaves of 'Floral Yuuka' plants with 50 mM sucrose accelerated flowering and increased the level of CmFTL2 transcription in the leaf more strongly than either CmFTL1 or FTL3 under both long and SD conditions. Transcription profiling indicated that all three CmFTL genes were upregulated during floral induction. The relationship of the CmFTL2 sequence with that of other members of the PEBP family suggested that its product contributes to the florigen rather than to the anti-florigen complex. The heterologous expression of CmFTL2 in the Arabidopsis thaliana ft-10 mutant rescued the mutant phenotype, showing that CmFTL2 could compensate for the absence of FT. These results suggest that CmFTL2 acts as a regulator of floral transition and responds to both the photoperiod and sucrose.

Chrysanthemum (Chrysanthemum morifolium) black spot disease (CBS) poses a major threat to Chrysanthemum cultivation owing to suitable climate conditions and current lack of resistant cultivars for greenhouse cultivation. In this study, we identified a number of genes that respond to Alternaria alternata infection in resistant and susceptible Chrysanthemum cultivars. Based on RNA sequencing technology and a weighted gene coexpression network analysis (WGCNA), we constructed a model to elucidate the response of Chrysanthemum leaves to A. alternata infection at different stages and compared the mapped response of the resistant cultivar 'Jinba' to that of the susceptible cultivar 'Zaoyihong'. In the early stage of infection, when lesions had not yet formed, abscisic acid (ABA), salicylic acid (SA) and EDS1-mediated resistance played important roles in the Chrysanthemum defense system. With the formation of necrotic lesions, ethylene (ET) metabolism and the Ca2+ signal transduction pathway strongly responded to A. alternata infection. During the late stage, when necrotic lesions continued to expand, members of the multidrug and toxic compound extrusion (MATE) gene family were highly expressed, and their products may be involved in defense against A. alternata invasion by exporting toxins produced by the pathogen, which plays important roles in the pathogenicity of A. alternata. Furthermore, the function of hub genes was verified by qPCR and transgenic assays. The identification of hub genes at different stages, the comparison of hub genes between the two cultivars and the highly expressed genes in the resistant cultivar 'Jinba' provide a theoretical basis for breeding cultivars resistant to CBS.

Chrysanthemum (Chrysanthemum morifolium) is one of the four major cut-flower plants worldwide and possesses both high ornamental value and cultural connotation. As most chrysanthemum varieties flower in autumn, it is costly to achieve annual production. JAZ genes in the TIFY family are core components of the jasmonic acid (JA) signaling pathway; in addition to playing a pivotal role in plant responses to defense, they are also widely implicated in regulating plant development processes. Here, we characterized the TIFY family gene CmJAZ1-like from the chrysanthemum cultivar 'Jinba'. CmJAZ1-like localizes in the nucleus and has no transcriptional activity in yeast. Tissue expression pattern analysis indicated that CmJAZ1-like was most active in the root and shoot apex. Overexpressing CmJAZ1-like with Jas domain deletion in chrysanthemum resulted in late flowering. RNA-Seq analysis of the overexpression lines revealed some differentially expressed genes (DEGs) involved in flowering, such as the homologs of the flowering integrators FT and SOC1, an FUL homolog involved in flower meristem identity, AP2 domain-containing transcription factors, MADS box genes, and autonomous pathway-related genes. Based on KEGG pathway enrichment analysis, the differentially transcribed genes were enriched in carbohydrate metabolic and fatty acid-related pathways, which are notable for their role in flowering in plants. This study preliminarily verified the function of CmJAZ1-like in chrysanthemum flowering, and the results can be used in molecular breeding programs aimed at flowering time regulation of chrysanthemum.

Distant hybridization is widely used to develop crop cultivars, whereas the hybridization process of embryo abortion often severely reduces the sought-after breeding effect. The LEAFY COTYLEDON1 (LEC1) gene has been extensively investigated as a central regulator of seed development, but it is far less studied in crop hybridization breeding. Here we investigated the function and regulation mechanism of CmLEC1 from Chrysanthemum morifolium during its seed development in chrysanthemum hybridization. CmLEC1 encodes a nucleic protein and is specifically expressed in embryos. CmLEC1's overexpression significantly promoted the seed-setting rate of the cross, while the rate was significantly decreased in the amiR-CmLEC1 transgenic chrysanthemum. The RNA-Seq analysis of the developing hybrid embryos revealed that regulatory genes involved in seed development, namely, CmLEA (late embryogenesis abundant protein), CmOLE (oleosin), CmSSP (seed storage protein), and CmEM (embryonic protein), were upregulated in the OE (overexpressing) lines but downregulated in the amiR lines vs. wild-type lines. Future analysis demonstrated that CmLEC1 directly activated CmLEA expression and interacted with CmC3H, and this CmLEC1-CmC3H interaction could enhance the transactivation ability of CmLEC1 for the expression of CmLEA. Further, CmLEC1 was able to induce several other key genes related to embryo development. Taken together, our results show that CmLEC1 plays a positive role in the hybrid embryo development of chrysanthemum plants, which might involve activating CmLEA's expression and interacting with CmC3H. This may be a new pathway in the LEC1 regulatory network to promote seed development, one perhaps leading to a novel strategy to not only overcome embryo abortion during crop breeding but also increase the seed yield.