Molecular Insights into the Temperature-dependent Super-double-flower-like Malformation in Carnation (Dianthus caryophyllus L.)

Malformed flowers similar to super-double-flowers were observed in potted ‘Cherie’ carnation (Dianthus caryophyllus L.) plants. In the malformed flower (mlf) lines, most flowers were malformed at 15°C, but not at 20°C. Thus, we hypothesized that the malformation was due to a mutation associated with morphological responses to temperature. In this study, RNA-sequencing analysis of young flower buds and whole-genome re-sequencing of leaves were performed using wild-type (WT) and mlf plants to identify malformation-related candidate genes. The RNA-sequencing analysis revealed 691 significant differentially expressed genes (DEGs) between WT flower buds at 15 or 20°C and mlf flower buds at 15°C. The Gene Ontology (GO) analysis indicated that metal ion binding, transmembrane transport, and anaphase-promoting complex enriched GO terms in mlf, whereas translation and ribosome enriched GO in terms of WT. The Kyoto Encyclopedia of Genes and Genomes analysis revealed an increase in the expression of 9-cis-epoxycarotenoid dioxygenase (NCED), Pyrabactin Resistance 1-Like (PYL), and Calmodulin (CAM), but a decrease in the expression of Histone H4, in mlf. The fragments per kilobase per million reads (FPKM) values were used to select candidate malformation-related DEGs. Transcription factor genes, including WUSCHEL (WUS) and STERILE APETALA, were upregulated in mlf, whereas PISTILLATA-like protein, MADS-box protein CMB2, and F-box UNUSUAL FLORAL ORGANS were downregulated. Heat Shock Cognate 70 kDa (HSC70) and Temperature-induced lipocalin-1 were upregulated in mlf, but genes encoding histones and ribosomal proteins were downregulated. Moreover, NCED1, PYL8 and 9, and cytokinin-related genes were upregulated in mlf. Using whole-genome re-sequencing data, sequence variants were detected in the upstream regions and exons of WUS, HSC70-1 and 2, CAM7, and ribosomal protein-encoding genes. Furthermore, examination of the F₁ progeny derived from WT and mlf crosses with cultivars producing fertile pollen revealed a significant difference in the proportion of malformed flower phenotypes between WT and mlf regardless of temperature, suggesting that the malformed flower phenotypes of mlf can be inherited. Candidate genes associated with the temperature-dependent super-double-flower phenotypes were examined.