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Petals of the monocot Phalaenopsis aphrodite (Orchidaceae) possess conical epidermal cells on their adaxial surfaces, and a large amount of cuticular wax is deposited on them to serve as a primary barrier against biotic and abiotic stresses. It has been widely reported that subgroup 9A members of the R2R3-MYB gene family, MIXTA and MIXTA-like in eudicots, act to regulate the differentiation of conical epidermal cells. However, the molecular pathways underlying conical epidermal cell development and cuticular wax biosynthesis in monocot petals remain unclear. Here, we characterized two subgroup 9A R2R3-MYB genes, PaMYB9A1 and PaMYB9A2 (PaMYB9A1/2), from P. aphrodite through the transient overexpression of their coding sequences and corresponding chimeric repressors in developing petals. We showed that PaMYB9A1/2 function to coordinate conical epidermal cell development and cuticular wax biosynthesis. In addition, we identified putative targets of PaMYB9A1/2 through comparative transcriptome analyses, revealing that PaMYB9A1/2 acts to regulate the expression of cell wall-associated and wax biosynthetic genes. Furthermore, a chemical composition analysis of cuticular wax showed that even-chain n-alkanes and odd-chain primary alcohols are the main chemical constituents of cuticular wax deposited on petals, which is inconsistent with the well-known biosynthetic pathways of cuticular wax, implying a distinct biosynthetic pathway occurring in P. aphrodite flowers. These results reveal that the function of subgroup 9A R2R3-MYB family genes in regulating the differentiation of epidermal cells is largely conserved in monocots and dicots. Furthermore, both PaMYB9A1/2 have evolved additional functions controlling the biosynthesis of cuticular wax., Two subgroup 9A R2R3-MYB genes act to regulate the development of conical epidermal cells and the biosynthesis of cuticular wax in Phalaenopsis petals.

Orchid gynostemium, the fused organ of the androecium and gynoecium, and ovule development are unique developmental processes. Two DROOPING LEAF/CRABS CLAW (DL/CRC) genes, PeDL1 and PeDL2, were identified from the Phalaenopsis orchid genome and functionally characterized. Phylogenetic analysis indicated that the most recent common ancestor of orchids contained the duplicated DL/CRC-like genes. Temporal and spatial expression analysis indicated that PeDL genes are specifically expressed in the gynostemium and at the early stages of ovule development. Both PeDLs could partially complement an Arabidopsis crc-1 mutant. Virus-induced gene silencing (VIGS) of PeDL1 and PeDL2 affected the number of protuberant ovule initials differentiated from the placenta. Transient overexpression of PeDL1 in Phalaenopsis orchids caused abnormal development of ovule and stigmatic cavity of gynostemium. PeDL1, but not PeDL2, could form a heterodimer with Phalaenopsis equestris CINCINNATA 8 (PeCIN8). Paralogous retention and subsequent divergence of the gene sequences of PeDL1 and PeDL2 in P. equestris might result in the differentiation of function and protein behaviors. These results reveal that the ancestral duplicated DL/CRC-like genes play important roles in orchid reproductive organ innovation.

Background Transposable elements (TEs) are fragments of DNA that can insert into new chromosomal locations. They represent a great proportion of eukaryotic genomes. The identification and characterization of TEs facilitates understanding the transpositional activity of TEs with their effects on the orchid genome structure. Results We combined the draft whole-genome sequences of Phalaenopsis equestris with BAC end sequences, Roche 454, and Illumina/Solexa, and identified long terminal repeat (LTR) retrotransposons in these genome sequences by using LTRfinder and classified by using Gepard software. Among the 10 families Gypsy-like retrotransposons, three families Gypsy1, Gypsy2, and Gypsy3, contained the most copies among these predicted elements. In addition, six high-copy retrotransposons were identified according to their reads in the sequenced raw data. The 12-kb Orchid-rt1 contains 18,000 copies representing 220 Mbp of the P. equestris genome. Southern blot and slot blot assays showed that these four retrotransposons Gypsy1, Gypsy2, Gypsy3, and Orchid-rt1 contained high copies in the large-genome-size/large-chromosome species P. violacea and P. bellina. Both Orchid-rt1 and Gypsy1 displayed various ratios of copy number for the LTR sequences versus coding sequences among four Phalaenopsis species, including P. violacea and P. bellina and small-genome-size/small-chromosome P. equestris and P. ahprodite subsp. formosana, which suggests that Orchid-rt1 and Gypsy1 have been through various mutations and homologous recombination events. FISH results showed amplification of Orchid-rt1 in the euchromatin regions among the four Phalaenopsis species. The expression levels of Peq018599 encoding copper transporter 1 is highly upregulated with the insertion of Orchid-rt1, while it is down regulated for Peq009948 and Peq014239 encoding for a 26S proteasome non-ATP regulatory subunit 4 homolog and auxin-responsive factor AUX/IAA-related. In addition, insertion of Orchid-rt1 in these three genes are all in their intron regions. Conclusion Orchid-rt1 and Gypsy1–3 have amplified within Phalaenopsis orchids concomitant with the expanded genome sizes, and Orchid-rt1 and Gypsy1 may have gone through various mutations and homologous recombination events. Insertion of Orchid-rt1 is in the introns and affects gene expression levels.

Analyses of the genome sequences and expression data for two closely related mycoheterotrophic orchid species provide insights into the genomic basis underlying the evolution of mycoheterotrophy. To improve our understanding of the origin and evolution of mycoheterotrophic plants, we here present the chromosome-scale genome assemblies of two sibling orchid species: partially mycoheterotrophic Platanthera zijinensis and holomycoheterotrophic Platanthera guangdongensis. Comparative analysis shows that mycoheterotrophy is associated with increased substitution rates and gene loss, and the deletion of most photoreceptor genes and auxin transporter genes might be linked to the unique phenotypes of fully mycoheterotrophic orchids. Conversely, trehalase genes that catalyse the conversion of trehalose into glucose have expanded in most sequenced orchids, in line with the fact that the germination of orchid non-endosperm seeds needs carbohydrates from fungi during the protocorm stage. We further show that the mature plant of P. guangdongensis, different from photosynthetic orchids, keeps expressing trehalase genes to hijack trehalose from fungi. Therefore, we propose that mycoheterotrophy in mature orchids is a continuation of the protocorm stage by sustaining the expression of trehalase genes. Our results shed light on the molecular mechanism underlying initial, partial and full mycoheterotrophy.

Background Orchids produce a colorless protocorm by symbiosis with fungi upon seed germination. For mass production of orchids, the prevailing approaches are both generation of protocorm-like bodies (PLBs) from callus and multiplication of adventitious buds on inflorescence. However, somaclonal variations occur during micropropagation. Results We isolated the two most expressed transposable elements belonging to P Instability Factor (PIF)-like transposons. Among them, a potential autonomous element was identified by similarity analysis against the whole-genome sequence of Phalaenopsis equestris and named PePIF1. It contains a 19-bp terminal inverted repeat flanked by a 3-bp target site duplication and two coding regions encoding ORF1- and transposase-like proteins. Phylogenetic analysis revealed that PePIF1 belongs to a new P-lineage of PIF. Furthermore, two distinct families, PePIF1a and PePIF1b, with 29 and 37 putative autonomous elements, respectively, were isolated, along with more than 3000 non-autonomous and miniature inverted-repeat transposable element (MITE)-like elements. Among them, 828 PePIF1-related elements were inserted in 771 predicted genes. Intriguingly, PePIF1 was transposed in the somaclonal variants of Phalaenopsis cultivars, as revealed by transposon display, and the newly inserted genes were identified and sequenced. Conclusion A PIF-like element, PePIF1, was identified in the Phalaenopsis genome and actively transposed during micropropagation. With the identification of PePIF1, we have more understanding of the Phalaenopsis genome structure and somaclonal variations during micropropagation for use in orchid breeding and production. Electronic supplementary material The online version of this article (10.1186/s12864-018-5420-4) contains supplementary material, which is available to authorized users.

The ovules and egg cells are well developed to be fertilized at anthesis in many flowering plants. However, ovule development is triggered by pollination in most orchids. In this study, we characterized the function of a Bsister gene, named PeMADS28, isolated from Phalaenopsis equestris, the genome-sequenced orchid. Spatial and temporal expression analysis showed PeMADS28 predominantly expressed in ovules between 32 and 48 days after pollination, which synchronizes with integument development. Subcellular localization and protein–protein interaction analyses revealed that PeMADS28 could form a homodimer as well as heterodimers with D-class and E-class MADS-box proteins. In addition, ectopic expression of PeMADS28 in Arabidopsis thaliana induced small curled rosette leaves, short silique length and few seeds, similar to that with overexpression of other species’ Bsister genes in Arabidopsis. Furthermore, complementation test revealed that PeMADS28 could rescue the phenotype of the ABS/TT16 mutant. Together, these results indicate the conserved function of BsisterPeMADS28 associated with ovule integument development in orchid.

The plant YABBY transcription factors are key regulators in the lamina development of lateral organs. Orchid is one of the largest families in angiosperm and known for their unique floral morphology, reproductive biology, and diversified lifestyles. However, nothing is known about the role of YABBY genes in orchids, although biologists have never lost their fascination with orchids. In this study, a total of 54 YABBY genes, including 15 genes in CRC/DL, eight in INO, 17 in YAB2, and 14 in FIL clade, were identified from the eight orchid species. A sequence analysis showed that all protein sequences encoded by these YABBY genes share the highly conserved C2C2 zinc-finger domain and YABBY domain (a helix-loop-helix motif). A gene structure analysis showed that the number of exons is highly conserved in the same clades. The genes in YAB2 clade have six exons, and genes in CRC/DL, INO, and FIL have six or seven exons. A phylogenetic analysis showed all 54 orchid YABBY genes could be classified into four major clades, including CRC/DL, INO, FIL, and YAB2. Many of orchid species maintain more than one member in CRC/DL, FIL, and YAB2 clades, implying functional differentiation among these genes, which is supported by sequence diversification and differential expression. An expression analysis of PhalaenopsisYABBY genes revealed that members in the CRC/DL clade have concentrated expressions in the early floral development stage and gynostemium, the fused male and female reproductive organs. The expression of PeINO is consistent with the biological role it played in ovule integument morphogenesis. Transcripts of members in the FIL clade could be obviously detected at the early developmental stage of the flowers. The expression of three genes, PeYAB2,PeYAB3, and PeYAB4, in the YAB2 clade could be revealed both in vegetative and reproductive tissues, and PeYAB4 was transcribed at a relatively higher level than that of PeYAB2 and PeYAB3. Together, this comprehensive analysis provides the basic information for understanding the function of the YABBY gene in Orchidaceae.

Orchid (Orchidaceae) is one of the largest families in angiosperms and presents exceptional diversity in lifestyle. Their unique reproductive characteristics of orchid are attracted by scientist for centuries. One of the synapomorphies of orchid plants is that their seeds do not contain endosperm. Lipids are used as major energy storage in orchid seeds. However, regulation and mobilization of lipid usage during early seedling (protocorm) stage of orchid is not understood. In this study, we compared transcriptomes from developing Phalaenopsis aphrodite protocorms grown on 1/2-strength MS medium with sucrose. The expression of P. aphrodite MALATE SYNTHASE (PaMLS), involved in the glyoxylate cycle, was significantly decreased from 4 days after incubation (DAI) to 7 DAI. On real-time RT-PCR, both P. aphrodite ISOCITRATE LYASE (PaICL) and PaMLS were down-regulated during protocorm development and suppressed by sucrose treatment. In addition, several genes encoding transcription factors regulating PaMLS expression were identified. A gene encoding homeobox transcription factor (named PaHB5) was involved in positive regulation of PaMLS. This study showed that sucrose regulates the glyoxylate cycle during orchid protocorm development in asymbiotic germination and provides new insights into the transcription factors involved in the regulation of malate synthase expression.

Oxalidaceae is one of the most important plant families in horticulture, and its key commercially relevant genus, Averrhoa, has diverse growth habits and fruit types. Here, we describe the assembly of a high-quality chromosome-scale genome sequence for Averrhoa carambola (star fruit). Ks distribution analysis showed that A. carambola underwent a whole-genome triplication event, i.e., the gamma event shared by most eudicots. Comparisons between A. carambola and other angiosperms also permitted the generation of Oxalidaceae gene annotations. We identified unique gene families and analyzed gene family expansion and contraction. This analysis revealed significant changes in MADS-box gene family content, which might be related to the cauliflory of A. carambola. In addition, we identified and analyzed a total of 204 nucleotide-binding site, leucine-rich repeat receptor (NLR) genes and 58 WRKY genes in the genome, which may be related to the defense response. Our results provide insights into the origin, evolution and diversification of star fruit.

The orchid flower is renowned for complexity of flower organ morphogenesis and has attracted great interest from scientists. The YABBY genes encode plant-specific transcription factors with important roles in vegetative and reproductive development in seed plants.DROOPING LEAF/CRABS CLAW(DL/CRC) orthologs are involved in reproductive organ development (especially carpels) of angiosperms. Orchid gynostemium (the fused organ of the androecium and gynoecium) and ovule development are unique developmental processes. Understanding theDL/CRC-likegenes controlling the developmental program of the gynostemium and ovule could provide accessible information for reproductive organ molecular regulation in orchids. TwoDL/CRC-likegenes, namedPeDL1andPeDL2, were cloned fromPhalaenopsis equestris. The orchid DL/CRC forms a monophyletic clade with two subclades including AshDL, PeDL1 and DcaDL1 in subclade I, and PeDL2 and DcaDL2 in subclade II. The temporal and spatial expression analysis indicatedPeDLgenes are specifically expressed in the gynostemium and at the early stages of ovule development. BothPeDLscould partially complement anArabidopsis crc-1mutant. Transient overexpression ofPeDL1inPhalaenopsisorchids caused abnormal development of ovule and stigmatic cavity of gynostemium. PeDL1, instead of PeDL2, could form a heterodimer with PeCIN8. Paralogue retention and subsequent divergence of the gene sequence ofPeDL1andPeDL2inP. equestrismight result in the differentiation of function and protein behaviors. These results reveal the important roles ofPeDLsinvolved in orchid gynostemium and ovule development and provide new insights for further understanding the molecular mechanisms underlying orchid reproductive organ development.

The laurel family within the Magnoliids has attracted attentions owing to its scents, variable inflorescences, and controversial phylogenetic position. Here, we present a chromosome-level assembly of the Litsea cubeba genome, together with low-coverage genomic and transcriptomic data for many other Lauraceae. Phylogenomic analyses show phylogenetic discordance at the position of Magnoliids, suggesting incomplete lineage sorting during the divergence of monocots, eudicots, and Magnoliids. An ancient whole-genome duplication (WGD) event occurred just before the divergence of Laurales and Magnoliales; subsequently, independent WGDs occurred almost simultaneously in the three Lauralean lineages. The phylogenetic relationships within Lauraceae correspond to the divergence of inflorescences, as evidenced by the phylogeny of FUWA, a conserved gene involved in determining panicle architecture in Lauraceae. Monoterpene synthases responsible for production of specific volatile compounds in Lauraceae are functionally verified. Our work sheds light on the evolution of the Lauraceae, the genetic basis for floral evolution and specific scents., Litsea cubeba belongs to the Lauraceae family within the Magnoliids clade. Here, the authors assemble its genome and reveal divergence of inflorescence and sexual differentiation, the phylogenetic relationships across the Lauraceae and related species, and biosynthetic genes related to essential oil synthesis.

WebComparing the whole genome sequence of Apostasia shenzhenica with transcriptome and genome data from five orchid subfamilies permits the reconstruction of an ancestral gene toolkit, providing insight into orchid origins, evolution and diversification. Supplementary information The online version of this article (doi:10.1038/nature23897) contains supplementary material, which is available to authorized users., Orchid origins Around 10 per cent of flowering plant species are orchids, with a broad diversity in both morphology and lifestyle. Apostasia is one of the earliest-diverging genera of Orchidaceae. To study the evolution and diversity of Orchidaceae, Zhong-Jian Liu, Yves Van de Peer and colleagues sequenced the genome of Apostasia shenzhenica, a self-pollinating species found in southeast China. The authors also report improved genomes for two species of Epidendroideae, Phalaenopsis equestris and Dendrobium catenatum, as well as transcriptome analysis of representatives of subfamilies of Orchidaceae. Their analyses provide insights into orchid origins, genome evolution, adaptation and diversification. Supplementary information The online version of this article (doi:10.1038/nature23897) contains supplementary material, which is available to authorized users., Constituting approximately 10% of flowering plant species, orchids (Orchidaceae) display unique flower morphologies, possess an extraordinary diversity in lifestyle, and have successfully colonized almost every habitat on Earth1,2,3. Here we report the draft genome sequence of Apostasia shenzhenica4, a representative of one of two genera that form a sister lineage to the rest of the Orchidaceae, providing a reference for inferring the genome content and structure of the most recent common ancestor of all extant orchids and improving our understanding of their origins and evolution. In addition, we present transcriptome data for representatives of Vanilloideae, Cypripedioideae and Orchidoideae, and novel third-generation genome data for two species of Epidendroideae, covering all five orchid subfamilies. A. shenzhenica shows clear evidence of a whole-genome duplication, which is shared by all orchids and occurred shortly before their divergence. Comparisons between A. shenzhenica and other orchids and angiosperms also permitted the reconstruction of an ancestral orchid gene toolkit. We identify new gene families, gene family expansions and contractions, and changes within MADS-box gene classes, which control a diverse suite of developmental processes, during orchid evolution. This study sheds new light on the genetic mechanisms underpinning key orchid innovations, including the development of the labellum and gynostemium, pollinia, and seeds without endosperm, as well as the evolution of epiphytism; reveals relationships between the Orchidaceae subfamilies; and helps clarify the evolutionary history of orchids within the angiosperms. Supplementary information The online version of this article (doi:10.1038/nature23897) contains supplementary material, which is available to authorized users.

The oomycete genus Phytophthora includes devastating plant pathogens that are found in almost all ecosystems. We sequenced the genomes of two quarantined Phytophthora species–P. fragariae and P. rubi. Comparing these Phytophthora species and related genera allowed reconstruction of the phylogenetic relationships within the genus Phytophthora and revealed Phytophthora genomic features associated with infection and pathogenicity. We found that several hundred Phytophthora genes are putatively inherited from red algae, but Phytophthora does not have vestigial plastids originating from phototrophs. The horizontally-transferred Phytophthora genes are abundant transposons that “transmit” exogenous gene to Phytophthora species thus bring about the gene recombination possibility. Several expansion events of Phytophthora gene families associated with cell wall biogenesis can be used as mutational targets to elucidate gene function in pathogenic interactions with host plants. This work enhanced the understanding of Phytophthora evolution and will also be helpful for the design of phytopathological control strategies., Phytophthora; Genome; Phylogenetic; Pathogenicity; Horizontally gene transfer.

Highlight Of twenty-three TCP genes identified from the genome of Phalaenopsis orchid, PePCF10 and PeCIN8 play important roles in orchid ovule development by modulating cell division., TEOSINTE-BRANCHED/CYCLOIDEA/PCF (TCP) proteins are plant-specific transcription factors known to have a role in multiple aspects of plant growth and development at the cellular, organ and tissue levels. However, there has been no related study of TCPs in orchids. Here we identified 23 TCP genes from the genome sequence of Phalaenopsis equestris. Phylogenetic analysis distinguished two homology classes of PeTCP transcription factor families: classes I and II. Class II was further divided into two subclasses, CIN and CYC/TB1. Spatial and temporal expression analysis showed that PePCF10 was predominantly expressed in ovules at early developmental stages and PeCIN8 had high expression at late developmental stages in ovules, with overlapping expression at day 16 after pollination. Subcellular localization and protein–protein interaction analyses revealed that PePCF10 and PeCIN8 could form homodimers and localize in the nucleus. However, PePCF10 and PeCIN8 could not form heterodimers. In transgenic Arabidopsis thaliana plants (overexpression and SRDX, a super repression motif derived from the EAR-motif of the repression domain of tobacco ETHYLENE-RESPONSIVE ELEMENT-BINDING FACTOR 3 and SUPERMAN, dominantly repressed), the two genes helped regulate cell proliferation. Together, these results suggest that PePCF10 and PeCIN8 play important roles in orchid ovule development by modulating cell division.

Constituting approximately 10% of flowering plant species, orchids (Orchidaceae) display unique flower morphologies, possess an extraordinary diversity in lifestyle, and have successfully colonized almost every habitat on Earth. Here we report the draft genome sequence of Apostasia shenzhenica, a representative of one of two genera that form a sister lineage to the rest of the Orchidaceae, providing a reference for inferring the genome content and structure of the most recent common ancestor of all extant orchids and improving our understanding of their origins and evolution. In addition, we present transcriptome data for representatives of Vanilloideae, Cypripedioideae and Orchidoideae, and novel third-generation genome data for two species of Epidendroideae, covering all five orchid subfamilies. A. shenzhenica shows clear evidence of a whole-genome duplication, which is shared by all orchids and occurred shortly before their divergence. Comparisons between A. shenzhenica and other orchids and angiosperms also permitted the reconstruction of an ancestral orchid gene toolkit. We identify new gene families, gene family expansions and contractions, and changes within MADS-box gene classes, which control a diverse suite of developmental processes, during orchid evolution. This study sheds new light on the genetic mechanisms underpinning key orchid innovations, including the development of the labellum and gynostemium, pollinia, and seeds without endosperm, as well as the evolution of epiphytism; reveals relationships between the Orchidaceae subfamilies; and helps clarify the evolutionary history of orchids within the angiosperms.

Summary A thin-profile buckling-restrained brace (thin-BRB) consists of a rectangular steel casing and a flat steel core that is parallel to a gusset plate. A thin configuration reduces the width of the restraining member and thus saves usable space in buildings. However, deformable debonding layers, which cover the steel core plate in order to mitigate the difference between the peak tensile and compressive axial forces, provide a space for the steel core to form high mode buckling waves when the thin-BRB is under compression. The wave crests squeeze the debonding layers and produce outward forces on the inner surface of the restraining member. If the restraining member is too weak in sustaining the outward forces, local bulging failure occurs and the thin-BRB loses its compression capacity immediately. In order to investigate local bulging behavior, a total of 22 thin-BRB specimens with a ratio of steel core plate to restraining steel tube depth ranging from 0.3 to 0.7 and axial yield force capacities ranging from 421 kN to 3036 kN were tested by applying either cyclically increasing, decreasing, or constant axial strains. The restraining steel tube widths of all the specimens were smaller than 200 mm and were infilled with mortar with a compressive >strength of 97 MPa or 55 MPa. Thirteen of the 22 thin-BRB specimens' restraining members bulged out when the compressive core strains exceeded 0.03. A seismic design method of the thin-BRB in preventing local bulging failure is proposed in this study. Test and finite element model (FEM) analysis results suggest that the outward forces can be estimated according to the BRB compressive strength, steel core high mode buckling wavelength, and the debonding layer thickness. In addition, the capacity of the restraining member in resisting the outward forces can be estimated by using the upper bound theory in plastic analysis. Both the FEM analysis and test results indicate that the proposed method is effective in predicting the possibility of local bulging failure. Test results indicate that the proposed design method is conservative for thin-BRB specimens with a large steel core plate to restraining steel tube depth ratio. This paper concludes with design recommendations for thin-BRBs for severe seismic services. Copyright © 2015 John Wiley & Sons, Ltd.

We have identified an often observed yet unresolved intermediate structure in a popular processing with dimethylformamide solutions of lead chloride and methylammonium iodide for perovskite solar cells. With subsecond time-resolved grazing-incidence X-ray scattering and X-ray photoemission spectroscopy, supplemental with ab initio calculation, the resolved intermediate structure (CH3NH3)2PbI2Cl2·CH3NH3I features two-dimensional (2D) perovskite bilayers of zigzagged lead-halide octahedra and sandwiched CH3NH3I layers. Such intermediate structure reveals a hidden correlation between the intermediate phase and the composition of the processing solution. Most importantly, the 2D perovskite lattice of the intermediate phase is largely crystallographically aligned with the [110] planes of the three-dimensional perovskite cubic phase; consequently, with sublimation of Cl ions from the organo-lead octahedral terminal corners in prolonged annealing, the zigzagged octahedral layers of the intermediate phase can merge with the intercalated methylammonium iodide layers for templated growth of perovskite crystals. Regulated by annealing temperature and the activation energies of the intermediate and perovskite, deduced from analysis of temperature-dependent structural kinetics, the intermediate phase is found to selectively mature first and then melt along the layering direction for epitaxial conversion into perovskite crystals. The unveiled epitaxial conversion under growth kinetics controls might be general for solution-processed and intermediate-templated perovskite formation.

Among 300,000 species in angiosperms, Orchidaceae containing 30,000 species is one of the largest families. Almost every habitats on earth have orchid plants successfully colonized, and it indicates that orchids are among the plants with significant ecological and evolutionary importance. So far, four orchid genomes have been sequenced, including Phalaenopsis equestris, Dendrobium catenatum, Dendrobium officinale, and Apostaceae shengen. Here, we review the current progress and the direction of orchid research in the post genomics era. These include the orchid genome evolution, genome mapping (genome-wide association analysis, genetic map, physical map), comparative genomics (especially receptor-like kinase and terpene synthase), secondary metabolomics, and genome editing.

Orchidaceae, renowned for its spectacular flowers and other reproductive and ecological adaptations, is one of the most diverse plant families. Here we present the genome sequence of the tropical epiphytic orchid Phalaenopsis equestris, a frequently used parent species for orchid breeding. P. equestris is the first plant with crassulacean acid metabolism (CAM) for which the genome has been sequenced. Our assembled genome contains 29,431 predicted protein-coding genes. We find that contigs likely to be underassembled, owing to heterozygosity, are enriched for genes that might be involved in self-incompatibility pathways. We find evidence for an orchid-specific paleopolyploidy event that preceded the radiation of most orchid clades, and our results suggest that gene duplication might have contributed to the evolution of CAM photosynthesis in P. equestris. Finally, we find expanded and diversified families of MADS-box C/D-class, B-class AP3 and AGL6-class genes, which might contribute to the highly specialized morphology of orchid flowers.

Orchids are known for their beauty and complexity of flower and ecological strategies. The evolution in orchid floral morphology, structure, and physiological properties has held the fascination of botanists for centuries, from Darwin through to the present. In floral studies, MADS-box genes contributing to the now famous ABCDE model of floral organ identity control have dominated conceptual thinking. The sophisticated orchid floral organization offers an opportunity to discover new variant genes and different levels of complexity to the ABCDE model. Recently, several remarkable research reports on orchid MADS-box genes, especially B-class MADS-box genes, have revealed the evolutionary track and important functions on orchid floral development. Diversification and fixation of both paleoAP3 gene sequences and expression profiles might be explained by subfunctionalization and even neofunctionalization. Knowledge about MADS-box genes encoding ABCDE functions in orchids will give insights into the highly evolved floral morphogenetic networks of orchids.

Orchidaceae constitute one of the largest families of angiosperms. They are one of the most ecological and evolutionary significant plants and have successfully colonized almost every habitat on earth. Because of the significance of plant biology, market needs and the current level of breeding technologies, basic research into orchid biology and the application of biotechnology in the orchid industry are continually endearing scientists to orchids in Taiwan. In this introductory review, we give an overview of the research activities in orchid biology and biotechnology, including the status of genomics, transformation technology, flowering regulation, molecular regulatory mechanisms of floral development, scent production and color presentation. This information will provide a broad scope for study of orchid biology and serve as a starting point for uncovering the mysteries of orchid evolution.

The orchid floral organs represent novel and effective structures for attracting pollination vectors. In addition, to avoid inbreeding, the androecium and gynoecium are united in a single structure termed the gynostemium. Identification of C-class MADS-box genes regulating reproductive organ development could help determine the level of homology with the current ABC model of floral organ identity in orchids. In this study, we isolated and characterized two C-class AGAMOUS-like genes, denoted CeMADS1 and CeMADS2, from Cymbidium ensifolium. These two genes showed distinct spatial and temporal expression profiles, which suggests their functional diversification during gynostemium development. Furthermore, the expression of CeMADS1 but not CeMADS2 was eliminated in the multitepal mutant whose gynostemium is replaced by a newly emerged flower, and this ecotopic flower continues to produce sepals and petals centripetally. Protein interaction relationships among CeMADS1, CeMADS2 and E-class PeMADS8 proteins were assessed by yeast two-hybrid analysis. Both CeMADS1 and CeMADS2 formed homodimers and heterodimers with each other and the E-class PeMADS protein. Furthermore, transgenic Arabidopsis plants overexpressing CeMADS1 or CeMADS2 showed limited growth of primary inflorescence. Thus, CeMADS1 may have a pivotal C function in reproductive organ development in C. ensifolium.

Geranyl diphosphate is the precursor for monoterpenes, the major floral scent in Phalaenopsis bellina. The cDNA of P. bellina GDP synthase (PbGDPS) was cloned, and its sequence contained a glutamate-rich motif (Glu-rich) corresponding to the second aspartate-rich motif (SARM) but not any aspartate-rich motifs (Asp-rich). The recombinant PbGDPS enzyme revealed dual prenyltransferase activities by the production of both GDP and farnesyl diphosphate (FDP). Spatial and temporal expression analyses illustrated that the expression of PbGDPS was flower specific and concomitant with maximal emission of monoterpenes on day 5 post-anthesis. In addition, the expression was strongly related to the intensity of the emitted scent among different plant varieties. This is the first report of a functional GDPS without an Asp-rich motif functioning as a homodimer in a plant (P. bellina, Orchidaceae, monocot).

Orchids are known for both their floral diversity and ecological strategies. The versatility and specialization in orchid floral morphology, structure and physiological properties have fascinated botanists for centuries. In floral studies, MADS-box genes contributing to the now famous ABC model of floral organ identity control have dominated conceptual thinking. The sophisticated orchid floral organization, such as labellum and gynostemium, offers an opportunity to discover new variant genes and different levels of complexity to the ABC model. In our studies, we suggest that the duplication and differential expression of B and C class genes may contribute to the specificity of orchid floral morphology. We also proposed a molecular model to explain the development of sterile and fertile floral organs in orchids. Knowledge of MADS-box genes encoding ABC functions in orchids will give insights into the highly evolved floral morphogenetic networks and the diversification of orchids.

Somaclonal variation occurs during plant tissue culture and introduces changes that can result in the development of desirable traits. Using cDNA-amplified restriction fragment length polymorphism (cDNA-AFLP) analysis, we compared gene expression patterns between flower buds from wild type (donor) plants of Phalaenopsis Hsiang Fei cv. H. F., whose flowers are bronze in color, and from its somaclonal variants, whose flowers have a mosaic yellowish color, and sometimes an aberrant shape. Using 128 fluorescently labeled primer sets, a total of 2269 transcript-derived fragments (TDFs) were analyzed. Among them, 25 TDFs were differentially expressed between the wild type plant and its variant. After cloning and sequencing these differentially expressed TDFs, we found that they contained 27 distinct sequences. Further confirmation of the differential expression of these sequences was carried out by using semi-quantitative RT-PCR. We found that five sequences showed higher expression levels in the wild type plant compared to those in the variant plant. These corresponded to sequences that encoded casein kinase, isocitrate dehydrogenase, cytochrome P450, EMF2, and a no hits found protein. In contrast, two other sequences, whose roles were unknown, were expressed to a higher level in the variant plant compared to those in the wild type plant. The differential expression of these genes may lead to the mosaic color patterns as well as the aberrant flower shapes in the somaclonal variants of Phalaenopsis Hsiang Fei cv. H. F.

*† Contributed equally to this work. Summary Geranyl diphosphate (GDP) is the precursor of monoterpenes, which are the major floral scent compounds in Phalaenopsis bellina. The cDNA of P. bellina GDP synthase (PbGDPS) was cloned, and its sequence corresponds to the second Asp-rich motif (SARM), but not to any aspartate-rich (Asp-rich) motif. The recombinant PbGDPS enzyme exhibits dual prenyltransferase activity, producing both GDP and farnesyl diphosphate (FDP), and a yeast two-hybrid assay and gel filtration revealed that PbGDPS was able to form a homodimer. Spatial and temporal expression analyses showed that the expression of PbGDPS was flower specific, and that maximal PbGDPS expression was concomitant with maximal emission of monoterpenes on day 5 post-anthesis. Homology modelling of PbGDPS indicated that the Glu-rich motif might provide a binding site for Mg 2+ and catalyze the formation of prenyl products in a similar way to SARM. Replacement of the key Glu residues with alanine totally abolished enzyme activity, whereas their mutation to Asp resulted in a mutant with two-thirds of the activity of the wild-type protein. Phylogenetic analysis indicated that plant GDPS proteins formed four clades: members of both GDPS-a and GDPS-b clades contain Asp-rich motifs, and function as homodimers. In contrast, proteins in the GDPS-c and GDPS-d clades do not contain Asp-rich motifs, but although members of the GDPS-c clade function as heterodimers, PbGDPS, which is more closely related to the GDPS-c clade proteins than to GDPS-a and GDPS-b proteins, and is currently the sole member of the GDPS-d clade, functions as a homodimer.

Orchids are known for their unique reproductive biology and ecological strategies. One of the remarkable reproductive characters of most orchid species is that ovule development is precisely initiated following pollination. Thus, orchids are attractive systems for investigating both the initiation and subsequent development of the ovule. Ethylene plays a critical role in ovary maturation and ovule differentiation in orchids. Genes corresponding to ethylene biosynthesis in orchid reproductive organs have been identified and characterized. The phosphorylation states of proteins involved in ethylene biosynthesis or ethylene signaling may be correlated with ovule development. Recently, an orchid GLOBOSA-like MADS-box gene was isolated from Phalaenopsis and shown to have a role in ovule development via auxin/ethylene signaling. Adopting functional genomics strategies will help us to get insights into the regulation network between ethylene signaling pathway and ovule developmental program in orchids.

Orchids make up about 10% of all seed plant species, have great economical value and are of specific scientific interest because of their renowned flowers and ecological adaptations. Here, we report the first draft genome sequence of a lithophytic orchid, Dendrobium catenatum. We predict 28,910 protein-coding genes and find evidence of a whole genome duplication shared with Phalaenopsis. We observed the expansion of many resistance-related genes, suggesting a powerful immune system responsible for adaptation to a wide range of ecological niches. We also discovered extensive duplication of genes involved in glucomannan synthase activities, likely related to the synthesis of medicinal polysaccharides. Expansion of MADS-box gene clades ANR1, StMADS11, and MIKC*, involved in the regulation of development and growth, suggests that these expansions are associated with the astonishing diversity of plant architecture in the genus Dendrobium. On the contrary, members of the type I MADS box gene family are missing, which might explain the loss of the endospermous seed. The findings reported here will be important for future studies into polysaccharide synthesis, adaptations to diverse environments and flower architecture of Orchidaceae.

Orchids have profound diversity of specialized pollination and ecological strategies and provide a rich setting for studying evolutionary relationships and molecular biology. The sophisticated orchid flower morphology includes two whorls of perianth segments, three sepals and three petals, one of which is modified as labellum (or lip). In addition, the male and female reproductive organs are fused to form a gynostemium. In order to study gene expression in orchid reproductive organs, a cDNA library of mature flower buds of Phalaenopsis equestris, a native diploid species of Phalaenopsis in Taiwan, was constructed. A total of 5593 expressed sequence tags (ESTs) from randomly selected clones were identified and characterized. Cluster analysis allowed the identification of a unigene set of 3688 sequences. This abundance of transcripts with predicted cellular roles was functionally characterized by the BLASTX matches to known proteins. Comparison of the relative EST frequencies based on functional categories among floral tissues of five species including P. equestris, Acorus Americanus, Asparagus officinalis, Oryza sativa and Arabidopsis thaliana was performed. The most highly transcribed genes in Phalaenopsis floral buds are those coding for RNA-dependent RNA polymerase of Cymbidium mosaic virus, followed by heat shock protein genes. A total 217 putative transcription factor related ESTs were identified. C3H and trihelix families occupied 25% of transcribed transcription factor genes, indicating that the profile of the transcription factors in orchid flower buds is polarized. The extensive analysis of the genes in floral organs adds to the growing repertoire of known plant genes and may also reveal unique features of the reproductive organs of orchids.