Comparative and phylogenetic analysis of Chiloschista (Orchidaceae) species and DNA barcoding investigation based on plastid genomes.

BACKGROUND: Chiloschista (Orchidaceae, Aeridinae) is an epiphytic leafless orchid that is mainly distributed in tropical or subtropical forest canopies. This rare and threatened orchid lacks molecular resources for phylogenetic and barcoding analysis. Therefore, we sequenced and assembled seven complete plastomes of Chiloschista to analyse the plastome characteristics and phylogenetic relationships and conduct a barcoding investigation. RESULTS: We are the first to publish seven Chiloschista plastomes, which possessed the typical quadripartite structure and ranged from 143,233 bp to 145,463 bp in size. The plastomes all contained 120 genes, consisting of 74 protein-coding genes, 38 tRNA genes and eight rRNA genes. The ndh genes were pseudogenes or lost in the genus, and the genes petG and psbF were under positive selection. The seven Chiloschista plastomes displayed stable plastome structures with no large inversions or rearrangements. A total of 14 small inversions (SIs) were identified in the seven Chiloschista plastomes but were all similar within the genus. Six noncoding mutational hotspots (trnNGUU-rpl32 > rpoB-trnCGCA > psbK-psbI > psaC-rps15 > trnEUUC-trnTGGU > accD-psaI) and five coding sequences (ycf1 > rps15 > matK > psbK > ccsA) were selected as potential barcodes based on nucleotide diversity and species discrimination analysis, which suggested that the potential barcode ycf1 was most suitable for species discrimination. A total of 47-56 SSRs and 11-14 long repeats (> 20 bp) were identified in Chiloschista plastomes, and they were mostly located in the large single copy intergenic region. Phylogenetic analysis indicated that Chiloschista was monophyletic. It was clustered with Phalaenopsis and formed the basic clade of the subtribe Aeridinae with a moderate support value. The results also showed that seven Chiloschista species were divided into three major clades with full support. CONCLUSION: This study was the first to analyse the plastome characteristics of the genus Chiloschista in Orchidaceae, and the results showed that Chiloschista plastomes have conserved plastome structures. Based on the plastome hotspots of nucleotide diversity, several genes and noncoding regions are suitable for phylogenetic and population studies. Chiloschista may provide an ideal system to investigate the dynamics of plastome evolution and DNA barcoding investigation for orchid studies.

Research Progress on Anthocyanin-Mediated Regulation of 'Black' Phenotypes of Plant Organs.

The color pattern is one of the most important characteristics of plants. Black stands out among the vibrant colors due to its rare and distinctive nature. While some plant organs appear black, they are, in fact, dark purple. Anthocyanins are the key compounds responsible for the diverse hues in plant organs. Cyanidin plays an important role in the deposition of black pigments in various plant organs, such as flower, leaf, and fruit. A number of structural genes and transcription factors are involved in the metabolism of anthocyanins in black organs. It has been shown that the high expression of R2R3-MYB transcription factors, such as PeMYB7, PeMYB11, and CsMYB90, regulates black pigmentation in plants. This review provides a comprehensive overview of the anthocyanin pathways that are involved in the regulation of black pigments in plant organs, including flower, leaf, and fruit. It is a great starting point for further investigation into the molecular regulation mechanism of plant color and the development of novel cultivars with black plant organs.

Complete Chloroplast Genomes and Comparative Analyses of Three Paraphalaenopsis (Aeridinae, Orchidaceae) Species.

Paraphalaenopsis, a genus of perennial herbs from the family Orchidaceae, contains a number of ornamental species. However, there is no information on the chloroplast genomes of Paraphalaenopsis, which limits our studies of this genus. In this study, we reported the chloroplast genomes of three species of Paraphalaenopsis (P. labukensis, P. denevel, and P. laycockii 'Semi-alba') and performed comprehensive comparative analysis. These three chloroplast genomes showed a typical quadripartile structure. Their lengths ranged from 147,311 bp to 149,240 bp. Each genome contained 120 unique genes, including 74 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Comparative analysis revealed major differences in sequence divergence in the three chloroplast genomes. In addition, six hypervariable regions were identified (psbM-trnDGUC, psbB, ccsA, trnKUUU, trnSGCU-trnGUCC, rps16-trnQUUG) that can be used as DNA molecular markers. Phylogenetic relationships were determined using the chloroplast genomes of 28 species from 12 genera of Aeridinae. Results suggested that Paraphalaenopsis was a clade of Aeridinae that was sister to the Holcoglossum-Vanda clade, with 100% bootstrap support within Aeridinae. The findings of this study provided the foundation for future studies on the phylogenetic analysis of Aeridinae.

Comparative Phylogenetic Analysis for Aerides (Aeridinae, Orchidaceae) Based on Six Complete Plastid Genomes.

Aerides Lour. (Orchidaceae, Aeridinae) is a group of epiphytic orchids with high ornamental value, mainly distributed in tropical and subtropical forests, that comprises approximately 20 species. The species are of great value in floriculture and garden designing because of their beautiful flower shapes and colors. Although the morphological boundaries of Aerides are clearly defined, the relationship between Aerides and other closely related genera is still ambiguous in terms of phylogeny. To better understand their phylogenetic relationships, this study used next-generation sequencing technology to investigate the phylogeny and DNA barcoding of this taxonomic unit using genetic information from six Aerides plastid genomes. The quadripartite-structure plastomes ranged from 147,244 bp to 148,391 bp and included 120 genes. Among them, 74 were protein coding genes, 38 were tRNA genes and 8 were rRNA genes, while the ndh genes were pseudogenized or lost. Four non-coding mutational hotspots (rpl20-rpl33, psbM, petB, rpoB-trnCGCA, Pi > 0.06) were identified. A total of 71-77 SSRs and 19-46 long repeats (>30 bp) were recognized in Aerides plastomes, which were mostly located in the large single-copy region. Phylogenetic analysis indicated that Aerides was monophylic and sister to Renanthera. Moreover, our results confirmed that six Aerides species can be divided into three major clades. These findings provide assistance for species identification and DNA barcoding investigation in Aerides, as well as contributes to future research on the phylogenomics of Orchidaceae.

Genome-Wide Identification and Characterization of the GRF Gene Family in Melastoma dodecandrum.

Growth-regulating factor (GRF) is a kind of transcription factor unique to plants, playing an important role in the flowering regulation, growth, and development of plants. Melastoma dodecandrum is an important member of Melastomataceae, with ornamental, medicinal, and edible benefits. The identification of the GRF gene family in M. dodecandrum can help to improve their character of flavor and continuous flowering. The members of the GRF gene family were identified from the M. dodecandrum genome, and their bioinformatics, selective pressure, and expression patterns were analyzed. The results showed that there were 20 GRF genes in M. dodecandrum. Phylogenetic analysis showed that the 71 GRF genes from M. dodecandrum, Arabidopsis thaliana, Camellia sinensis, and Oryza sativa can be divided into three clades and six subclades. The 20 GRF genes of M. dodecandrum were distributed in twelve chromosomes and one contig. Furthermore, the gene structure and motif analysis showed that the intron and motif within each clade were very similar, but there were great differences among different clades. The promoter contained cis-acting elements related to hormone induction, stress, and growth and development. Different transcriptomic expression of MdGRFs indicated that MdGRFs may be involved in regulating the growth and development of M. dodecandrum. The results laid a foundation for further study on the function and molecular mechanism of the M. dodecandrum GRF gene family.

Genome-Wide Identification and Analysis of WRKY Gene Family in Melastoma dodecandrum.

WRKY is one of the largest transcription factor families in plants and plays an important role in plant growth and development as well as in abiotic and biological stresses. However, there is little information about the WRKY family in Melastoma dodecandrum. In this study, 126 WRKY members were identified in M. dodecandrum. According to phylogenetic analysis, they were divided into three major groups, and group II was further divided into five subgroups. MedWRKY genes were unevenly distributed on 12 chromosomes. Additionally, the gene structure and sequence composition were similar within the same group and differed between groups, suggesting their functional diversity. The promoter sequence analysis identified a number of cis-acting elements related to plant growth and development, stress response, and secondary metabolite synthesis in the WRKY gene family. The collinearity analysis showed that gene replication events were the main driving force of MedWRKY gene evolution. The transcriptome data and RT-qPCR analysis suggested that MedWRKY genes had higher expression in the roots and ripe fruit of M. dodecandrum. In short, this paper lays a foundation for further study of the functions and molecular mechanism of M. dodecandrum WRKY gene family.

Comprehensive Analysis for GRF Transcription Factors in Sacred Lotus (Nelumbo nucifera).

Sacred lotus (Nelumbo nucifera) is an aquatic perennial plant with essential food, ornamental, and pharmacological value. Growth-regulating factor (GRF) is a transcription factor (TF) family that plays an important role in regulating the growth and development of plants. In this study, a comprehensive analysis of the GRF family in N. nucifera was performed, and its role in N. nucifera development was studied. A total of eight GRF genes were identified in the N. nucifera genome. Phylogenetic analysis divided the 38 GRF genes into six clades, while the NuGRFs only contained five clades. The analyses of gene structures, motifs, and cis-acting regulatory elements of the GRF gene family were performed. In addition, the chromosome location and collinearity were analyzed. The expression pattern based on transcriptomic data and real-time reverse transcription-quantitative PCR (qRT-PCR) revealed that the GRF genes were expressed in multiple organs and were abundant in actively growing tissues, and the expression levels decreased as the age of N. nucifera increased. Then, 3D structures of the NuGRF proteins were predicted by homology modeling. Finally, the subcellular localization of GRF1 was ascertained in the tobacco leaf through a vector. Therefore, this study provides a comprehensive overview of the GRF TF family in N. nucifera.

Plastid phylogenomics improves resolution of phylogenetic relationship in the Cheirostylis and Goodyera clades of Goodyerinae (Orchidoideae, Orchidaceae).

Goodyerinae are one of phylogenetically unresolved groups of Orchidaceae. The lack of resolution achieved through the analyses of previous molecular sequences from one or a few markers has long confounded phylogenetic estimation and generic delimitation. Here, we present large-scale phylogenomic data to compare the plastome structure of the two main clades (Goodyera and Cheirostylis) in this subtribe and further adopt two strategies, combining plastid coding sequences and the whole plastome, to investigate phylogenetic relationships. A total of 46 species in 16 genera were sampled, including 39 species in 15 genera sequenced in this study. The plastomes of heterotrophic species are not drastically reduced in overall size, but display a pattern congruent with a loss of photosynthetic function. The plastomes of autotrophic species ranged from 147 to 165 kb and encoded from 132 to 137 genes. Three unusual structural features were detected: a 1.0-kb inversion in the large single-copy region of Goodyera schlechtendaliana; the loss and/or pseudogenization of ndh genes only in two species, Cheirostylis chinensis and C. montana; and the expansion of inverted repeat regions and contraction of small single-copy region in Hetaeria oblongifolia. Phylogenomic analyses provided improved resolution for phylogenetic relationships. All genera were recovered as monophyletic, except for Goodyera and Hetaeria, which were each recovered as non-monophyletic. Nomenclatural changes are needed until the broader sampling and biparental inherited markers. This study provides a phylogenetic framework of Goodyerinae and insight into plastome evolution of Orchidaceae.

Comparative Analysis of Luisia (Aeridinae, Orchidaceae) Plastomes Shed Light on Plastomes Evolution and Barcodes Investigation.

Luisia, a genus of the subtribe Aeridinae of Orchidaceae, comprises ca. 40 species. Members of Luisia exhibit unique morphological characteristics and represent a valuable ornamental orchid genus. However, due to the scarcity of distinct morphological characters, species identification within this genus is ambiguous and controversial. In the present study, next-generation sequencing (NGS) methods were used to assemble the plastomes of five Luisia species and compare them with one publicly available Luisia plastid genome data. The plastomes of Luisia possessed a quadripartite structure, with sizes ranging from 146,243 bp to 147,430 bp. The plastomes of six Luisia species contained a total of 120 genes, comprising 74 protein-coding genes, 38 tRNA genes and eight rRNA genes. Notably, all ndh genes were pseudogenized or lost. An analysis of codon usage bias showed that leucine (Leu) exhibited the highest frequency, while cysteine (Cys) exhibited the lowest frequency. A total of 57 to 64 SSRs and 42 to 49 long repeats were identified. Five regions and five coding sequences were identified for DNA barcodes, based on the nucleotide diversity (Pi) analysis. The species of Luisia constituted a monophyletic group and were sister to Paraphalaenopsis with strong support. Our study deepens the understanding of species identification, plastome evolution and the phylogenetic positions of Luisia.

Why Black Flowers? An Extreme Environment and Molecular Perspective of Black Color Accumulation in the Ornamental and Food Crops.

Pollinators are attracted to vibrant flower colors. That is why flower color is the key agent to allow successful fruit set in food or ornamental crops. However, black flower color is the least attractive to pollinators, although a number of plant species produce black flowers. Cyanidin-based anthocyanins are thought to be the key agents to induce black color in the ornamental and fruit crops. R2R3-MYB transcription factors (TFs) play key roles for the tissue-specific accumulation of anthocyanin. MYB1 and MYB11 are the key TFs regulating the expression of anthocyanin biosynthesis genes for black color accumulation. Post-transcriptional silencing of flavone synthase II (FNS) gene is the technological method to stimulate the accumulation of cyanidin-based anthocyanins in black cultivars. Type 1 promoter of DvIVS takes the advantage of FNS silencing to produce large amounts of black anthocyanins. Exogenous ethylene application triggers anthocyanin accumulation in the fruit skin at ripening. Environment cues have been the pivotal regulators to allow differential accumulation of anthocyanins to regulate black color. Heat stress is one of the most important environmental stimulus that regulates concentration gradient of anthocyanins in various plant parts, thereby affecting the color pattern of flowers. Stability of black anthocyanins in the extreme environments can save the damage, especially in fruits, caused by abiotic stress. White flowers without anthocyanin face more damages from abiotic stress than dark color flowers. The intensity and pattern of flower color accumulation determine the overall fruit set, thereby controlling crop yield and human food needs. This review paper presents comprehensive knowledge of black flower regulation as affected by high temperature stress, and the molecular regulators of anthocyanin for black color in ornamental and food crops. It also discusses the black color-pollination interaction pattern affected by heat stress for food and ornamental crops.

Transcriptional Proposition for Uniquely Developed Protocorm Flowering in Three Orchid Species: Resources for Innovative Breeding.

During orchid seed culture, seeds germinate as protocorms, and protocorms normally develop into plant with leaves and roots. Orchids require many years of vegetative development for flowering. However, under a certain combination of growth cultures, we observed that protocorms can directly flower without leaves and roots. Therefore, we performed comparative transcriptome analysis to identify the different transcriptional regulators of two types of protocorms of Cymbidium ensifolium, Cymbidium sinense, and Cymbidium goeringii. Zinc finger, MYB, AP2, and bHLH were the most abundant transcription factor (TF) families in the transcriptome. Weighted gene coexpression network analysis (WGCNA) was performed to identify hub genes related to leaf and flower development. The key hubs included SPL6, SVP, SEP2, KNOX1, AP2, OFP1, COL12, MYB13, MYB36, MYB59, bHLH086, and ARF7. The hub genes were further validated through statistical tools to propose the roles of key TFs. Therefore, this study initiates to answer that why there is no leaf initiation and root development and how can protocorm bypass the vegetative phase to flower? The outcomes can direct future research on short-span flowering in orchids through protocorms.

Transcriptome mining of hormonal and floral integrators in the leafless flowers of three cymbidium orchids.

Flowering is the most studied ornamental trait in orchids where long vegetative phase may span up to three years. Cymbidium orchids produce beautiful flowers with astonishing shapes and pleasant scent. However, an unusually long vegetative phase is a major drawback to their ornamental value. We observed that under certain culture conditions, three cymbidium species (Cymbidium ensifolium, C. goeringii and C. sinense) skipped vegetative growth phase and directly flowered within six months, that could be a breakthrough for future orchids with limited vegetative growth. Hormonal and floral regulators could be the key factors arresting vegetative phase. Therefore, transcriptomic analyses were performed for leafless flowers and normal vegetative leaves to ascertain differentially expressed genes (DEGs) related to hormones (auxin, cytokinin, gibberellin, abscisic acid and ethylene), floral integrators and MADS-box genes. A significant difference of cytokinin and floral regulators was observed among three species as compared to other hormones. The MADS-box genes were significantly expressed in the leafless flowers of C. sinense as compared to other species. Among the key floral regulators, CONSTANS and AGAMOUS-like genes showed the most differential expression in the leafless flowers as compared to leaves where the expression was negligible. However, CONSTANS also showed downregulation. Auxin efflux carriers were mainly downregulated in the leafless flowers of C. ensifolium and C. sinense, while they were upregulated in C. goeringii. Moreover, gibberellin and cytokinin genes were also downregulated in C. ensifolium and C. sinense flowers, while they were upregulated in C. goeringii, suggesting that species may vary in their responses. The data mining thus, outsources the valuable information to direct future research on orchids at industrial levels.

The Melastoma dodecandrum genome and the evolution of Myrtales.

Melastomataceae has abundant morphological diversity with high economic and ornamental merit in Myrtales. The phylogenetic position of Myrtales is still contested. Here, we report the chromosome-level genome assembly of Melastoma dodecandrum in Melastomataceae. The assembled genome size is 299.81 Mb with a contig N50 value of 3.00 Mb. Genome evolution analysis indicated that M. dodecandrum, Eucalyptus grandis, and Punica granatum were clustered into a clade of Myrtales and formed a sister group with the ancestor of fabids and malvids. We found that M. dodecandrum experienced four whole-genome polyploidization events: the ancient event was shared with most eudicots, one event was shared with Myrtales, and the other two events were unique to M. dodecandrum. Moreover, we identified MADS-box genes and found that the AP1-like genes expanded, and AP3-like genes might have undergone subfunctionalization. The SUAR63-like genes and AG-like genes showed different expression patterns in stamens, which may be associated with heteranthery. In addition, we found that LAZY1-like genes were involved in the negative regulation of stem branching development, which may be related to its creeping features. Our study sheds new light on the evolution of Melastomataceae and Myrtales, which provides a comprehensive genetic resource for future research.

Genomes of leafy and leafless Platanthera orchids illuminate 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.

The complete plastid genome of Holcoglossum singchianum (Orchidaceae, Vandeae).

The complete plastid genome of Holcoglossum singchianum was determined and analyzed in this work. The plastome was 147,715 bp in length with 84,094 bp of the large single-copy (LSC) region, 12,073 bp of the small single-copy (SSC) region and 25,774 bp of the inverted repeat (IRs) regions. The genome contained 120 genes, 74 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Phylogenetic analysis of 20 Aeridinae plastomes suggested three groups of Holcoglossum were divided, and H. singchianum was sister to H. lingulatum.

Complete plastid genome of Vanda concolor (Orchidaceae, Aeridinae).

Vanda is one of the five most horticulturally important genera in Orchidaceae. In this study, we assembled the complete plastid genome of an important cultivated species, V. concolor. The plastome was 149,474 bp in length, containing a large single-copy region (LSC) of 85,678 bp, a small single-copy region (SSC) of 12,002 bp, and two inverted repeat regions (IR) of 25,897 bp. A total of 127 genes were predicted, including 38 tRNA, 8 rRNA, and 74 protein-coding genes. Phylogenetic analysis of 21 representative plastome of the subtribe Aeridinae suggested V. concolor to be sister to V. brunnea and that this pair is sister to Neofinetia.

The complete chloroplast genome of Pleione pleionoides (Orchidaceae).

Pleione pleionoides is a vulnerable orchid with significant ornamental values. Here, we report the first complete chloroplast genome of P. pleionoides. The circular genome was 159,468 bp in length and consisted of a pair of inverted repeats (IR 26,651 bp), which were separated by a large single copy region (LSC 87,461 bp) and a small single copy region (SSC 18,705 bp). It contained 115 unique genes, including 87 protein-coding genes, 38 tRNAs, and 8 rRNAs. The maximum likelihood phylogenetic analysis indicated that P. pleionoides and P. bulbocodioides cluster together and closely related to P. formosana.

The complete chloroplast genome of a widespread ornamental shrub in China, Magnolia figo (Magnoliaceae).

Plants in Michelia, presented by Magnolia figo DC, are wonderful resources in Magnoliaceae, covering a series of aromatic plants. Despite extensive studies in this family, the M. figo complete chloroplast genome and the taxonomical status based on the whole chloroplast sequences remain unclear. Herein, we report the complete chloroplast genome of M. figo. The chloroplast genome was 160,113 bp in length, with a large single-copy (LSC) region of 88,113 bp and a small single-copy (SSC) region of 18,797 bp, separated by two inverted repeat (protein-coding) regions of 26,602 bp. A total of 135 CDSs were found, including 129 genes, 85 protein-coding mRNAs, 36 tRNA genes, and eight rRNA genes. The overall GC content was 39.3%, and GC percentages range from 34.3% to 43.2% throughout LSC, IRs, and SSC regions. Phylogenetic analysis showed that M. figo is most closely to Michelia odora and displayed a relationship that three Michelia were nested inside Magnolia. This announcement of the complete M. figo cp genome sequence could provide valuable information for further breeding, cp genetic modification, and phylogenetic study in Magnoliaceae.