Orchids possess significant ornamental value and are highly esteemed, especially among horticulturists. Numerous Dendrobium species are cultivated extensively due to their substantial commercial importance and the vast diversity in flower color and morphology (Teixeira da Silva et al., 2014). Dendrobium thyrsiflorum, characterized by its white flowers, yellow lips and globular inflorescences (Figure 1a), serves as a representative species. We sequenced its genome, providing insights into its characteristics and evolutionary background. The transcriptomic analysis for various flower segments (Figure 1b) of D. thyrsiflorum revealed expression patterns associated with flower color, shape and senescence. Additionally, we identified and characterized genes linked to crucial horticultural traits, including flower development and environmental adaptability. This genomic investigation offers valuable perspectives on significant innovations in the economic horticultural value of D. thyrsiflorum and provides important genomic data for the cultivation of Dendrobium. The genome size of D. thyrsiflorum was estimated to be approximately 1.438 Gb, with a heterozygosity rate of 2.15%, as determined by 17-mer analysis (Figure S1 and Table S1). Utilizing PacBio sequencing and high-throughput chromosome conformation capture (Hi-C) technologies, we achieved a final assembly of 1.427 Gb (representing 99.51% of the genome) organized into 20 pseudochromosomes (2N = 2X = 40) (Figure S2 and Tables S2–S4), with an N50 value of 72.88 Mb. Gene density was observed to increase towards the chromosome ends, in contrast to non-coding elements, which showed a decreasing trend (Figure 1c and Table S5). Assessment using benchmarking universal single-copy orthologues (BUSCO) indicated a 97.5% completeness for the assembled genome (Table S6). Repetitive sequences constituted approximately 70.64% of the D. thyrsiflorum genome (Table S7). We annotated 28 702 protein-coding genes (Tables S8–S10), in addition to non-coding RNA genes (Table S11), identifying 319 microRNAs (miRNAs) and 534 ribosomal RNA (rRNA) genes. The expansion of terpenoid biosynthetic genes enhances the diversity of terpenoid compounds (Table S12 and Figure 1d). We identified 247 genes under significant selection in comparison to other sequenced Dendrobium species (Tables S13 and S14). Our analysis reveals that the genes encoding tropinone reductase (dth.13G01329.1) are associated with alkaloid biosynthesis, suggesting their potential as candidate genes for investigating alkaloid composition variations among Dendrobium orchids. Notably, no whole-genome duplication events were detected within the Dendrobium genus (Figure S3). In D. thyrsiflorum, chromosome 20 originates near the centromere of Chromosome 9, a region shared by the ancestors of three Dendrobium orchids (Figure 1e). Chromosome 20 demonstrates high conservation without significant structural variation compared to C. sinense and D. thysiflorum (Figure S4). This indicates that the presence of Chromosome 20 reflects the ancestral state of D. thysiflorum; its fusion with Chromosome 9 occurred post-divergence from the ancestors of D. nobile and D. chrysotoxum. The flowers of D. thyrsiflorum feature white petals with a yellow to orange lip, a characteristic highly regarded for its aesthetic value (Figure 1a,b). The development of the yellow hue is closely associated with carotenoids (Watkins and Pogson, 2020). The enzyme CrtP primarily facilitates the conversion of phytoene into lycopene (Figure 1f). In D. thyrsiflorum, the CrtP-like gene dth.16G00352.1 was the only one significantly up-regulated in the lip during critical stages of flower colour development (Stages 2 and 3), suggesting that it plays a crucial role in determining the yellow coloration of the lip. The absence of the ZDS gene results in a loss of pigmentation (Avendaño-Vázquez et al., 2014). Similarly, crtH dysfunction leads to prolycopene accumulation in tomatoes, altering their coloration (Isaacson et al., 2002). Among the homologues of ZDS and crtH, one ZDS-like gene, dth.11G01227.1, and two crtH-like genes, dth.12G00312.1 and dth.17G01686.1, exhibited significantly elevated expression in the lip during Stages 2, 3, and 4 (Figure 1f), indicating their pivotal roles in carotenoid biosynthesis associated with yellow lip coloration. In Brassica juncea, two homologous genes, BjA02.PC1 and BjB04.PC2, promote the development of carotenoid-enriched plastoglobules (PGs), leading to the production of yellow floral pigments (Li et al., 2023). Mutations in these genes result in the whitening of flowers. A similar expression pattern was observed in D. thyrsiflorum, where two BjPC-like genes, dth.06G00155.1 and dth.19G01519.1, demonstrated significant differential expression in the lip petals during the critical coloration phase (Figure 1f), underscoring their importance in carotenoid metabolism. Notable yellow secondary metabolites include 4,2′,4′,6′-tetrahydroxychalcone, isosalipurposide and aurones (Teixeira da Silva et al., 2014). Gene expression analysis revealed several genes associated with synthesis showing high expression levels (Figure 1f), including the AS1-like gene dth.16G00457.1, and genes within the CHI family, such as dth.09G02022.1 and dth.18G01034.1. The expression of these genes is the reason behind the yellow lip in the D. thyrsiflorum orchid (Figure 1g). The flowering period of D. thyrsiflorum is notably brief, limiting its economic viability. Ethylene, a crucial hormone, plays a significant role in flower senescence. In our study, we identified ethylene biosynthesis and signal transduction pathway genes, observing organ-specific expression among different gene copies. This suggests divergent functional roles for these homologous genes, with specialization in various floral parts (Figure 1h). Similar expression patterns were noted in D. densiflorum flowers. The MADS-box family, known for its pivotal role in flower development (Mondragón-Palomino and Theißen, 2009), was further explored. We constructed a phylogenetic tree and assessed the expression of MADS-box genes in D. thyrsiflorum (Figure S5), revealing higher copy numbers in the A, D, and SVP classes compared to those in Phalaenopsis or Apostasia. Conversely, D. thyrsiflorum exhibited fewer copies in the Bs, SOC1 and AGL6 classes, indicating a complex role of the MADS-box in flower development that is not strictly tied to gene copy number. In the context of environmental stress responses, volatile terpenes are essential for the survival of Dendrobium orchids under various stress conditions such as chilling, high temperatures and water scarcity (Yu et al., 2020). Our findings suggest a significant role for subclade A TPS-b within a monocot-specific clade in D. thyrsiflorum, contributing to the diversification of the TPS family and influencing the production of secondary metabolites (Figure S5). This project was supported by the National Natural Science Foundation of China (32300189), the Talent Project of Guangzhou Medical University High-level University Construction (06-410-2106132), the Forestry Peak Discipline Construction Project of Fujian Agriculture and Forestry University (72202200205) and the National Key Research and Development Program of China (No. 2203YFD1600504). The authors declare no conflict of interest. Q.X., Z.J.L. and G.Q.Z. conceived this project. B.J.C., Q.X., J.Y.W. and P.J.Z. analysed the data and wrote the manuscript. Other authors carried out analyses and experiments. Genome sequences and whole-genome assemblies have been submitted to the National Center for Biotechnology Information (NCBI) database under BioProject accession number PRJNA837390. The annotations of D. thyrsiflorum and other orchids have been uploaded to Figshare under DOI: 10.6084/m9.figshare.25378522. Figure S1–S5 Supplementary Figures. Table S1–S14 Supplementary Tables. Data S1 Experimental procedures. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
