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The understanding of capitulum morphogenesis is critical for modification of chrysanthemum flower type. Previous studies showed candidate genes which involved in the inflorescence architecture, such as PIN-FORMED 1 (PIN1), AUXIN RESPONSE FACTOR 5 (ARF5) and LEAFY (LFY), acted dominant role during early flower development. However, it is not known whether those candidate genes play parts in the capitulum morphogenesis. In the present study, floral morphogenesis and the corresponding external morphological changes in Chrysanthemum lavandulifolium were observed using scanning electron microscope and light microscope technology. Capitulum morphogenesis showed that ray florets primordia initiated later than the outermost disc florets primordia at the middle stage of florets primordia differentiation (stage 6), breaking the acropetal initiation of organ primordia on capitulum. Organogenesis of disc and ray florets showed that the early generation of florets corollas primordia was the key differentiation stages, in which disc and ray florets fused with actinomorphic and zygomorphic corollas, respectively. Furthermore, the expression patterns of inflorescence architecture related genes were detected at key stages of capitulum morphogenesis as well as the different organs on capitulum using quantitative real time PCR (qRT-PCR). The transcripts of ClARF5 specially increased at stage 5 and stage 6, as well as in ray florets corollas. The results indicated that ClARF5 might be responsible for the orientation and initiation of disc and ray florets during early capitulum morphogenesis. Studies on the capitulum morphogenesis of C. lavandulifolium not only bring molecular insights into capitulum morphogenesis, but also lay foundation for the flower type-oriented modification in cultivated chrysanthemums.

Flower type is an important quality trait of ornamental plants. Mastering genetic variation in flower types is of practical significance for breeding improved flower types. Chrysanthemum (non-anemone type) flowers consist of several elements; both ray floret shape and the relative number of ray florets (RNRF) on the capitulum are very important factors. However, until recently, few reports have standardized chrysanthemum flower type evaluations, hindering the development of genetic studies of chrysanthemum flower types. In this study, as an important index of ray floret shape, the corolla tube merged degree (CTMD) was defined as the corolla tube length/ray floret length (CTL/RFL). In addition, the number of ray florets/number of florets (NRF/NF) was used to describe the RNRF on the capitulum. Major gene and polygene mixed inheritance analyses were conducted based on 2 F1 populations from 2 pairs of parents that strongly differed in the CTMD and RNRF. ANOVA revealed that all traits strongly significantly differed among the different hybrids, and correlation analysis revealed that the CTMD and RNRF might have evolved independently. Additionally, according to the Q-mode cluster analysis results, we divided the RNRF into 4 levels: single, semi-double I, semi-double II and double. Genetic analysis revealed that both the CTMD and RNRF could be described by a B-2 genetic model via two additive-dominance major genes. In addition, the heritability of the major genes for these traits was greater than 50%, indicating that the CTMD and RNRF were controlled mainly by genetic factors. These results will provide a new theoretical basis for further improvement and breeding of chrysanthemum flower types.

Flower color, which varies widely in potted multiflora chrysanthemum, is an extremely important character for classification. A quantitative definition of the flower color of potted multiflora chrysanthemums and the establishment of a standard color classification system may help us to evaluate the germplasm resources effectively. In this study, 273 potted multiflora chrysanthemum advanced lines were selected as materials. First, color classification results were compared between two methods: one is a combination of CIELAB and Munsell color systems, and the other one applying Q clustering analysis based on the CIELAB color system. We then divided the 273 lines into six color groups. The composition and content of anthocyanins and carotenoids in the different color advanced lines were identified by HPLC-MS. Two derivatives of cyanidin, cyanidin-3-O-(6″-O-malonyl-glucoside) and cyanidin-3-O-(3″,6″-O-dimalonyl-β-glucopyanoside), were identified in the pink, purple, orange-brown, and red lines. The carotenoids, primarily consisting of violanthin, z-violanthin, 9′z-lutein 5,6-epoxide, lutein, α-carotene, β-carotene, and 9z-β-carotene, were identified in all chrysanthemum advanced lines. Additionally, a*, represents the green-red component with green in the negative direction and red in the positive direction, was strongly positively correlated with cyanidin-3-O-(6″-O-malonyl-glucoside) (r = 0.884, P

Chrysanthemum (Chrysanthemum × morifolium) is an important horticultural crop with great ornamental and economic value. Most chrysanthemum cultivars are self-incompatible, which limits the creation of pure chrysanthemum lines which are the material basis for breeding and theoretical research. Here, artificial self-pollination and natural cross-pollination were used to analyze the self-compatibility (SC) and cross-compatibility (CC) of 85 chrysanthemums cultivated by our research group. By conducting a phenotypic study, observing of pollen germination in vitro and in vivo and gene expression analyses, we explored the reasons for the self-compatibility of chrysanthemum and identified self-compatible mutants. It was found that there were wide variations in self-compatibility among different chrysanthemum cultivars and the SC index ranged from 0 to 5.64, which could classify the chrysanthemum cultivars into three types: self-compatibility (SC index ≥ 1), self-incompatibility (SI, 1 > SC index > 0) and high self-incompatibility (SC index = 0). Seven chrysanthemum cultivars with self-pollinated progenies (‘Hongguan’, K183−8, A22, B12, D87, D76 and S8) were selected, and the pollen germination rates in vitro and in vivo were not significantly related to SC. The pollen tube of ‘Hongguan’ could pass through its own stigma and grow toward the embryo sac along the style, and there was no obvious inbreeding depression in self-pollinated progenies of ‘Hongguan’. In addition, we found that one S-locus receptor kinase (SRK), two S-locus cysteine-rich protein (SCR), two exocyst complex component EXo70A1 (Exo70A1) and one thioredoxin h-like 1 (THL1) were important for regulating self-compatibility of C. × morifolium. Our results not only laid a material foundation for creating pure lines through continuous self-pollination and analyzing the regulation mechanism underlying SC variation but also provided the possibility for theoretical research on the important ornamental traits of chrysanthemums in the future.

AP1/FUL-like genes encode class-specific MADS box transcription factors in angiosperms and play important roles in the plant floral transition and floral organ identity. Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is one of the original species of Chrysanthemum. Understanding the function of AP1/FUL-like genes in C. lavandulifolium provide molecular-level insight into the flowering mechanism of Chrysanthemum. Here, an AP1/FUL-like gene was isolated from C. lavandulifolium. Sequence and phylogenetic analyses indicated that this gene, ClM8, belongs to the euFUL clade of the AP1/FUL-like genes. We observed different expression levels of ClM8 in C. lavandulifolium roots, stems, fruits and leaves and high expression in the flowers. A subcellular localisation assay showed that ClM8 is localised to the nucleus in vivo. The over-expression of ClM8 in Arabidopsis resulted in altered flowering time and inflorescence structure. These aberrant, transgenic Arabidopsis plants showed early flowering following the production of four to six small rosette leaves, lateral flowers and a composite terminal flower. The terminal flower consisted of two or four pistils surrounded by an abnormal number of sepals, petals and stamens. In addition, the morphology of the style of 35S::ClM8 plants silique differed from wild type, appearing thinner and longer. Further investigation suggested that the expression levels of FLOWERING LOCUS T (FT), LEAFY (LFY) and APETALA1 (AP1) were apparently increased in ClM8-transgenic Arabidopsis. Thus, we speculate that ClM8 promoted flowering in Arabidopsis due to the upregulation of FT. Taken together, our results suggest that ClM8 is involved in flowering induction and floral development that might also play a role in the development of fruits and leaves.

Cineraria (Senecio cruentus Masson ex L’Herit) is a groundsel perennial herbaceous plant that has a variety of colors and is an ideal model used to research the mechanism of flower color variation. Anthocyanin is the major component that determines the flower color, and studying the regulation of endogenous anthocyanin biosynthesis-related genes is important for understanding the molecular mechanism of flower color variation. Selecting suitable reference genes is a prerequisite for identifying the accurate expression pattern of these genes. We analyzed the stability of eight reference genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein phosphatase 2A (PP2A), β-tubulin, Actin, TIP41-like family protein (TIP41), elongation factor 1α (EF-1α), SAND family protein (SAND) and S-adenosylmethionine synthetase (SAMS) in different flower developmental stages of four different color lines in cineraria. The results showed that SAND and Actin were the most stable genes in the blue and pink lines, while the TIP41 and Actin genes performed best in the white line; however, the PP2A and Actin genes were the least variable in the carmine line. SAND and Actin genes were the most stable gene in all samples. SAMS was the least stable gene in all samples, so it is not suitable as a reference gene. In addition, the relative expression level of Senecio cruentus Flavanone 3-hydroxylase gene (ScF3H), the upstream gene in the anthocyanin biosynthesis pathway, in the different developmental stages of the flower in the carmine line was analyzed to verify the reference genes selected in this study. We recommended the combination of SAND, Actin and PP2A were suitable for normalization the target genes in different color lines of cineraria during their flower developmental stages. This study presented the first systematic study of reference genes stability and provided a guide to research of the molecular mechanism of the anthocyanin biosynthesis pathway and flower development in cineraria.