Orchids constitute approximately 10% of flowering plant species. However, only about 10 orchid genomes have been published. Metabolites are the main way through which orchids respond to their environment. Dendrobium nobile, belonging to Dendrobium, the second largest genus in Orchidaceae, has high ornamental, medicinal, and ecological value. D. nobile is the source of many popular horticultural varieties. Among the Dendrobium species, D. nobile has the highest amount of dendrobine, which is regarded as one of the criteria for evaluating medicinal quality. Due to lack of data and analysis at the genomic level, the biosynthesis pathways of dendrobine and other related medicinal ingredients in D. nobile are unknown. In this paper, we report a chromosome-scale reference genome of D. nobile to facilitate the investigation of its genomic characteristics for comparison with other Dendrobium species. The assembled genome size of D. nobile was 1.19 Gb. Of the sequences, 99.45% were anchored to 19 chromosomes. Furthermore, we identified differences in gene number and gene expression patterns compared with two other Dendrobium species by integrating whole-genome sequencing and transcriptomic analysis [e.g., genes in the polysaccharide biosynthesis pathway and upstream of the alkaloid (dendrobine) biosynthesis pathway]. Differences in the TPS and CYP450 gene families were also found among orchid species. All the above differences might contribute to the species-specific medicinal ingredient biosynthesis pathways. The metabolic pathway-related analysis will provide further insight into orchid responses to the environment. Additionally, the reference genome will provide important insights for further molecular elucidation of the medicinal active ingredients of Dendrobium and enhance the understanding of orchid evolution.

P. wenshanica S.C.Chen & Z.H.Tsi and P. subcalceata Gagnep. have long been recognized as synonyms of P. leveilleana Schltr. In the present study, detailed morphological comparisons suggest that specimens referred to as P. wenshanica and P. subcalceata differ significantly in both vegetative and floral characters from those of P. leveilleana. Here we resurrect P. wenshanica and P. subcalceata as independent species. Key diagnostic characters essential for delineating identities of these species are presented.

Cymbidium nanulum Y.S.Wu & S.C.Chen is an IUCN Red listed Endangered species and distributes in South-Central China and Hainan. Here, we report the complete chloroplast (cp) genome sequence and the cp genome features of C. nanulum. The complete chloroplast (cp) genome sequence of C. nanulum was 149,776 bp in length. It presented a typical quadripartite structure including one large single-copy region (LSC, 85,392 bp), one small single-copy region (SSC, 14,210 bp), and two inverted repeat regions (IRs, 25,087 bp). The cp genome encoded 133 genes, of which 104 were unique genes (77 protein-coding genes, 23 tRNAs, and 4 rRNAs). The maximum-likelihood phylogenetic analysis showed that C. nanulum was closely related to other species of genus Cymbidium.

Phalaenopsis orchids are popular ornamental plants worldwide. The application of the efficient multiplex genome editing tools in Phalaenopsis , will greatly accelerate the development of orchid gene function and breeding research. In this study, we establish a fast and convenient Phalaenopsis protoplast platform for the identification of functional genome editing tools. Two multiplex genome editing tools, PTG-Cas9 (PTG, polycistronic tRNA gRNA) system and PTGm-Cas9 (PTG-Cas9 system with modified sgRNA structure) system are designed to edit PDS gene of commercial Phalaenopsis ST166 at four target sites. We find that both PTG-Cas9 and PTGm-Cas9 system are functional in Phalaenopsis , and the PTGm-Cas9 system with modified sgRNA has a higher editing efficiency than PTG-Cas9 system. Further, we design another multiplex genome editing tool, termed as DPII-Cpf1 system (dual Pol II promoter to drive the expression of Cpf1 endonuclease and crRNA), to edit PDS gene of Phalaenopsis at four target sites likewise. All the four targets are efficiently edited by DPII-Cpf1 system, and the total mutation rate is about 3 times higher than that of PTGm-Cas9 system. Taken together, using the Phalaenopsis protoplast platform, we successfully establish two efficient multiplex genome editing tools for Phalaenopsis research, PTGm-Cas9 and DPII-Cpf1. The multiplex genome editing tools established in this study have great application potentials in efficiently constructing large-scale knockout mutant libraries of orchid and speeding up orchid precise breeding.