Sweet osmanthus (Osmanthus fragrans Lour.) is among the top ten most well-known flowers in China and is recognized as both an aromatic plant and ornamental flower. Here, manual sectioning, scanning electron microscopy, and transmission electron microscopy of sweet osmanthus petals revealed that large amounts of lipids are present inside the petal cells and on the cell surfaces. However, no secretory structures were observed. Instead, the petal cells protrude slightly outward, and the surfaces of the cells are adorned with highly regular brush-shaped hairs. The surfaces of the ‘Yingui’ petals possessed mostly curled and more numerous hairs, whereas the ‘Dangui’ petals possessed fewer brush-shaped and more sparsely arranged hairs. In addition, many granular substances were attached to the brush-shaped hairs, and the granules were denser on the hairs of the ‘Yingui’ petals compared to the hairs on the ‘Dangui’ petals. Furthermore, 35 aromatic components in the ‘Yingui’ petals and 30 aromatic components in the ‘Dangui’ petals were detected via GC-MS. The main aromatic component of the ‘Yingui’ petals was β-ionone, whereas that of the ‘Dangui’ petals was linalool and its oxides. Transcriptome sequencing and qRT-PCR indicated that the high β-ionone content in the ‘Yingui’ petals was due to the overexpression of CCD1 and CCD4 and that the high linalool content in the ‘Dangui’ petals was due to the overexpression of MECS, HDR, IDI1, and LIS1, which function upstream of the linalool synthetic pathway. In particular, the expression levels of CCD4 and LIS1 were upregulated by 5.5- and 5.1-fold in the ‘Yingui’ and ‘Dangui’ petals, respectively. One transcription factor (ERF61) was cloned and named, and the expression pattern of ERF61 in sweet osmanthus petals was found to be generally consistent with that of CCD4. Tobacco transformation experiments, yeast one-hybrid experiments, and electrophoretic mobility shift assays indicated that ERF61 binds to the CCD4 promoter and stimulates CCD4 expression, thereby regulating the synthesis of β-ionone in sweet osmanthus petals.

Saxifragaceae, a family of over 600 species and approximately 30 genera of herbaceous perennials, is well-known for intergeneric hybridization. Of the main lineages in this family, the Heuchera group represents a valuable model for the analysis of plastid capture and its impact on phylogeny reconstruction. In this study, we investigated plastome evolution across the family, reconstructed the phylogeny of the Heuchera group and examined putative plastid capture between Heuchera and Tiarella. Seven species (11 individuals) representing Tiarella, as well as Mitella and Heuchera, were selected for genome skimming. We assembled the plastomes, and then compared these to six others published for Saxifragaceae; the plastomes were found to be highly similar in overall size, structure, gene order and content. Moreover, ycf15 was lost due to pseudogenization and rpl2 lost its only intron for all the analyzed plastomes. Comparative plastome analysis revealed that size variations of the plastomes are purely ascribed to the length differences of LSC, SSC, and IRs regions. Using nuclear ITS+ETS and the complete plastome, we fully resolved the species relationships of Tiarella, finding that the genus is monophyletic and the Asian species is most closely related to the western North American species. However, the position of Heuchera species was highly incongruent between nuclear and plastid data. Comparisons of nuclear and plastid phylogenies revealed that multiple plastid capture events have occurred between Heuchera and Tiarella, through putative ancient hybridization. Moreover, we developed numerous molecular markers for Tiarella (e.g., plastid hotspot and polymorphic nuclear SSRs), which will be useful for future studies on the population genetics and phylogeography of this disjunct genus.

Linalool and ionone are two important aromatic components in sweet osmanthus petals, and the regulatory mechanisms that produce these two components remain unclear. In this study, we employed whole-genome methylation sequencing and ATAC-seq technology to analyze the genomic DNA methylation status and chromatin accessibility of the sweet osmanthus cultivars ‘Zaohuang’ and ‘Chenghong Dangui’. Results showed that the promoter region of TPS2, a key gene in the linalool synthesis pathway, was less methylated in ‘Chenghong Dangui’ than in ‘Zaohuang’. The chromatin was more accessible in ‘Chenghong Dangui’ than in ‘Zaohuang’, which resulted in a much stronger expression of this gene in ‘Chenghong Dangui’ than in ‘Zaohuang’. This eventually led to a high quantity of linalool and its oxides in the petals of ‘Chenghong Dangui’, but there were lower levels present in the petals of ‘Zaohuang’. These results suggest that DNA methylation and chromatin accessibility play major roles in linalool synthesis in sweet osmanthus. The methylation level of the promoter region of CCD4, a key gene for ionone synthesis, was higher in ‘Zaohuang’ than in ‘Chenghong Dangui’. The chromatin accessibility was lower in ‘Zaohuang’ than in ‘Chenghong Dangui’, although the expression of this gene was significantly higher in ‘Zaohuang’ than in ‘Chenghong Dangui’. ChIP-seq analysis and a series of experiments showed that the differential expression of CCD4 and CCD1 in the two cultivars may predominantly be the result of regulation by ERF2 and other transcription factors. However, a 183-bp deletion involving the CCD4 promoter region in ‘Chenghong Dangui’ may be the main reason for the low expression of this gene in its petals. This study provides an important theoretical basis for improving selective breeding of key floral fragrance components in sweet osmanthus.

Osmanthus fragrans is an ornamental plant of substantial commercial value, and no genetic linkage maps of this species have previously been reported. Specific-locus amplified fragment sequencing (SLAF-seq) is a recently developed technology that allows massive single nucleotide polymorphisms (SNPs) to be identified and high-resolution genotyping. In our current research, we generated the first genetic map of O. fragrans using SLAF-seq, which is composed with 206.92 M paired-end reads and 173,537 SLAF markers. Among total 90,715 polymorphic SLAF markers, 15,317 polymorphic SLAFs could be used for genetic map construction. The integrated map contained 14,189 high quality SLAFs that were grouped in 23 genetic linkage groups, with a total length of 2962.46 cM and an average distance of 0.21 cM between two adjacent markers. In addition, 23,664 SNPs were identified from the mapped markers. As far as we know, this is the first of the genetic map of O. fragrans. Our results are further demonstrate that SLAF-seq is a very effective method for developing markers and constructing high-density linkage maps. The SNP markers and the genetic map reported in this study should be valuable resource in future research.