Flower color is an important trait for ornamental plants and is determined by pigments. Variations in the flower color of chrysanthemum are abundant, which is an important feature related to its ornamental and commercial value. However, few reports have elucidated the inheritance of the flower color and pigmentation of chrysanthemum. In this study, an F1 hybrid population, including 319 hybrids of small-flowered chrysanthemum cultivars with different flower color, was constructed. The genetic variation rules for some color and pigment-related traits were discussed based on major gene and polygene mixed inheritance analyses. Both the total anthocyanin and carotenoid contents were controlled by two pairs of additive dominant major genes and the heritability of the major genes was 70.44% and 86.03%, respectively. Based on the high-density genetic map constructed for the hybrid population, QTL analysis for the above traits was carried out using the interval mapping method. A total of 25 related QTLs and 517 closely linked markers were detected, including three QTLs controlling the total anthocyanin content and two QTLs controlling the total carotenoid content, all of which were major QTLs. Furthermore, 17 unigenes related to pigments were identified via BLAST searches with Chrysanthemum nankingense genome. For such genes, the collinearity was observed in four chromosome level genomes, including three genomes of the genus Chrysanthemum and the first genome of cultivated chrysanthemum. The results lay a foundation for in-depth exploration of flower color and pigmentation in chrysanthemum and provide a reference for future research in other ornamental plants.

The formation mechanism of different ray floret shapes of chrysanthemum (Chrysanthemum × morifolium) remains elusive due to its complex genetic background. C. vestitum, with the basic ray floret shapes of the flat, spoon, and tubular types, is considered a model material for studying ray floret morphogenesis. In this study, the flat and tubular type lines of C. vestitum at specific stages were used to investigate the key genes that regulate morphological differences in ray florets. We found that the expression levels of genes related to auxin synthesis, transport, and response were generally higher in the tubular type than in the flat type. CvARF3 was highly expressed in the flat type, while CvARF5 and CvARF6 were highly expressed in the tubular type. Additionally, the transcription levels of Class B and E genes closely related to petal development, including CvPI, CvAP3, Cvdefh21, CvSEP3, and CvCDM77, were expressed at higher levels in the tubular type than the flat type. Based on the results, it is proposed that auxin plays a key role in the development of ray florets, and auxin-related genes, especially CvARFs, may be key genes to control the morphological difference of ray florets. Simultaneously, MADS-box genes are involved in the co-regulation of ray floret morphogenesis. The results provide novel insights into the molecular mechanism of different petal type formation and lay a theoretical foundation for the directional breeding of petal type in chrysanthemums.

Day-neutral multiflora chrysanthemums can flower throughout the year without being influenced by daylength and have great application value in gardens. Studying heterosis and the genetic basis of important traits in day-neutral chrysanthemums can accelerate the breeding of new cultivars. In this research, a genetic population was constructed by crossing 135 F₁ hybrid progeny from the day-neutral chrysanthemum ‘82-81-19’ (female parent) and the late-flowering chrysanthemum ‘388Q-76’ (male parent). Six traits, including abnormal (crown) bud, plant height, plant crown width, budding date, full flowering date, and number of petal layers, were selected for inheritance and heterosis analyses, and a single-generation major gene plus polygene mixed inheritance model was used to perform mixed inheritance analysis on these traits. The results indicated that the six traits were widely segregated in the F₁ population, with the coefficient of variation (CV) ranging from 30% to 84%. The phenomena of heterosis and extra-parent segregation existed generally in F₁ progeny, and the ratio of heterosis value of mid-parents (RHm) for the six traits was 45.5%, 2%, 2%, 6%, 6%, and −0.3%, respectively. The mixed genetic analysis showed that the abnormal (crown) bud and budding date were fitted to the B-3 model and controlled by two pairs of additive major genes. The plant height and plant crown width were fitted to the A-0 model, and no major gene was detected. The full flowering date was fitted to the A-1 model and was controlled by one pair of major genes. The number of petal layers was fitted to the B-1 model and controlled by two pairs of additive–dominant major genes. The heritabilities of major genes for abnormal bud, budding date, full flowering date, and the number of petal layers were 1.0, 0.9871, 0.7240, and 0.5612, respectively, indicating that these traits were less affected by environmental factors. Using a percentile scoring method, eight day-neutral chrysanthemum genotypes were selected from the hybrid progeny.

Chrysanthemum × morifolium has great ornamental and economic value on account of its exquisite capitulum. However, previous studies have mainly focused on the corolla morphology of the capitulum. Such an approach cannot explain the variable inflorescence architecture of the chrysanthemum. Previous research from our group has shown that NO APICAL MERISTEM (ClNAM) is likely to function as a hub gene in capitulum architecture in the early development stage. In the present study, ClNAM was used to investigate the function of these boundary genes in the capitulum architecture of Chrysanthemum lavandulifolium, a closely related species of C. × morifolium in the genus. Modification of ClNAM in C. lavandulifolium resulted in an advanced initiation of the floral primordium at the capitulum. As a result, the receptacle morphology was altered and the number of florets decreased. The ray floret corolla was shortened, but the disc floret was elongated. The number of capitula increased significantly, arranged in more densely compounded corymbose synflorescences. The yeast and luciferase reporter system revealed that ClAP1, ClRCD2, and ClLBD18 target and activate ClNAM. Subsequently, ClNAM targets and activates ClCUC2a/c, which regulates the initiation of floral and inflorescence in C. lavandulifolium. ClNAM was also targeted and cleaved by cla-miR164 in this process. In conclusion, this study established a boundary gene regulatory network with cla-miR164-ClNAM as the hub. This network not only influences the architecture of capitulum, but also affects compound corymbose synflorescences of the C. lavandulifolium. These results provide new insights into the mechanisms regulating inflorescence architecture in chrysanthemum.

Chrysanthemum (Chrysanthemum × morifolium Ramat.) is a globally popular ornamental plant, but most cultivars lack efficient petal-based transient transformation systems, limiting floral trait molecular mechanism exploration. Protoplasts are versatile tools for gene localization, interaction, and functional characterization. Here, we established a petal protoplast isolation and transient transformation system for C. morifolium ‘Wandai Fengguang’ via L₉(3⁴) orthogonal design: optimal isolation (0.6 M mannitol, 8 h enzymatic digestion time, 0.4% macerozyme R-10, 4% cellulase R-10) and transformation (40% PEG4000, 12 μg plasmid, 10 min transfection, a protoplast density of 1 × 10⁶ protoplasts mL⁻¹). Under these conditions, protoplast yield was 5.14 × 10⁶ protoplasts g⁻¹·FW, viability 87.41%, and transformation efficiency 51.50%, with good applicability for six additional germplasms. We further analyzed CmVIT1 protein localization. Compared with the previous system, this system significantly improved protoplast yield and transformation efficiency, facilitating the transient transformation of genes related to floral traits in chrysanthemum and providing a methodological framework for other horticultural crops.