Phylogenomics and biogeographical diversification of Collabieae (Orchidaceae) and its implication in the reconstruction of the dynamic history of Asian evergreen broadleaved forests.

The tribe Collabieae (Epidendroideae, Orchidaceae) comprises approximately 500 species. Generic delimitation within Collabieae are confusing and phylogenetic interrelationships within the Collabieae have not been well resolved. Plastid genomes and nuclear internal transcribed spacer (ITS) sequences were used to estimate the phylogenetic relationships, ancestral ranges, and diversification rates of Collabieae. The results showed that Collabieae was subdivided into nine clades with high support. We proposed to combine Ancistrochilus and Pachystoma into Spathoglottis, merge Collabium and Chrysoglossum into Diglyphosa, and separate Pilophyllum and Hancockia as distinctive genera. The diversification of the nine clades of Collabieae might be associated with the uplift of the Himalayas during the Late Oligocene/Early Miocene. The enhanced East Asian summer monsoon in the Late Miocene may have promoted the rapid diversification of Collabieae at a sustained high diversification rate. The increased size of terrestrial pseudobulbs may be one of the drivers of Collabieae diversification. Our results suggest that the establishment and development of evergreen broadleaved forests facilitated the diversification of Collabieae.

Genome-Wide Identification and Expression Analysis of the GRAS Gene Family and Their Responses to Heat Stress in Cymbidium goeringii.

The GRAS gene family, responsible for encoding transcription factors, serves pivotal functions in plant development, growth, and responses to stress. The exploration of the GRAS gene family within the Orchidaceae has been comparatively limited, despite its identification and functional description in various plant species. This study aimed to conduct a thorough examination of the GRAS gene family in Cymbidum goeringii, focusing on its physicochemical attributes, phylogenetic associations, gene structure, cis-acting elements, and expression profiles under heat stress. The results show that a total of 54 CgGRASs were pinpointed from the genome repository and categorized into ten subfamilies via phylogenetic associations. Assessment of gene sequence and structure disclosed the prevalent existence of the VHIID domain in most CgGRASs, with around 57.41% (31/54) CgGRASs lacking introns. The Ka/Ks ratios of all CgGRASs were below one, indicating purifying selection across all CgGRASs. Examination of cis-acting elements unveiled the presence of numerous elements linked to light response, plant hormone signaling, and stress responsiveness. Furthermore, CgGRAS5 contained the highest quantity of cis-acting elements linked to stress response. Experimental results from RT-qPCR demonstrated notable variations in the expression levels of eight CgGRASs after heat stress conditions, particularly within the LAS, HAM, and SCL4/7 subfamilies. In conclusion, this study revealed the expression pattern of CgGRASs under heat stress, providing reference for further exploration into the roles of CgGRAS transcription factors in stress adaptation.

Genome-Wide Identification and Drought Stress Response Pattern of the NF-Y Gene Family in Cymbidium sinense.

Cymbidium sinense, a type of orchid plant, is more drought-resistant and ornamental than other terrestrial orchids. Research has shown that many members of the NUCLEAR FACTOR Y (NF-Y) transcription factor family are responsive to plant growth, development, and abiotic stress. However, the mechanism of the NF-Y gene family's response to abiotic stress in orchids has not yet been reported. In this study, phylogenetic analysis allowed for 27 CsNF-Y genes to be identified (5 CsNF-YAs, 9 CsNF-YBs, and 13 CsNF-YC subunits), and the CsNF-Ys were homologous to those in Arabidopsis and Oryza. Protein structure analysis revealed that different subfamilies contained different motifs, but all of them contained Motif 2. Secondary and tertiary protein structure analysis indicated that the CsNF-YB and CsNF-YC subfamilies had a high content of alpha helix structures. Cis-element analysis showed that elements related to drought stress were mainly concentrated in the CsNF-YB and CsNF-YC subfamilies, with CsNF-YB3 and CsNF-YC12 having the highest content. The results of a transcriptome analysis showed that there was a trend of downregulation of almost all CsNF-Ys in leaves under drought stress, while in roots, most members of the CsNF-YB subfamily showed a trend of upregulation. Additionally, seven genes were selected for real-time reverse transcription quantitative PCR (qRT-PCR) experiments. The results were generally consistent with those of the transcriptome analysis. The regulatory roles of CsNF-YB 1, 2, and 4 were particularly evident in the roots. The findings of our study may make a great contribution to the understanding of the role of CsNF-Ys in stress-related metabolic processes.

Genome-Wide Identification and Expression Pattern Analysis of TIFY Family Genes Reveal Their Potential Roles in Phalaenopsis aphrodite Flower Opening.

The TIFY gene family (formerly known as the zinc finger proteins expressed in inflorescence meristem (ZIM) family) not only functions in plant defense responses but also are widely involved in regulating plant growth and development. However, the identification and functional analysis of TIFY proteins remain unexplored in Orchidaceae. Here, we identified 19 putative TIFY genes in the Phalaenopsis aphrodite genome. The phylogenetic tree classified them into four subfamilies: 14 members from JAZ, 3 members from ZML, and 1 each from PPD and TIFY. Sequence analysis revealed that all Phalaenopsis TIFY proteins contained a TIFY domain. Exon-intron analysis showed that the intron number and length of Phalaenopsis TIFY genes varied, whereas the same subfamily and subgroup genes had similar exon or intron numbers and distributions. The most abundant cis-elements in the promoter regions of the 19 TIFY genes were associated with light responsiveness, followed by MeJA and ABA, indicating their potential regulation by light and phytohormones. The 13 candidate TIFY genes screened from the transcriptome data exhibited two types of expression trends, suggesting their different roles in cell proliferation and cell expansion of floral organ growth during Phalaenopsis flower opening. Overall, this study serves as a background for investigating the underlying roles of TIFY genes in floral organ growth in Phalaenopsis.

The Complete Chloroplast Genomes of Bulbophyllum (Orchidaceae) Species: Insight into Genome Structure Divergence and Phylogenetic Analysis.

Bulbophyllum is one of the largest genera and presents some of the most intricate taxonomic problems in the family Orchidaceae, including species of ornamental and medical importance. The lack of knowledge regarding the characterization of Bulbophyllum chloroplast (cp) genomes has imposed current limitations on our study. Here, we report the complete cp genomes of seven Bulbophyllum species, including B. ambrosia, B. crassipes, B. farreri, B. hamatum, B. shanicum, B. triste, and B. violaceolabellum, and compared with related taxa to provide a better understanding of their genomic information on taxonomy and phylogeny. A total of 28 Bulbophyllum cp genomes exhibit typical quadripartite structures with lengths ranging from 145,092 bp to 165,812 bp and a GC content of 36.60% to 38.04%. Each genome contained 125-132 genes, encompassing 74-86 protein-coding genes, 38 tRNA genes, and eight rRNA genes. The genome arrangements, gene contents, and length were similar, with differences observed in ndh gene composition. It is worth noting that there were exogenous fragment insertions in the IR regions of B. crassipes. A total of 18-49 long repeats and 38-80 simple sequence repeats (SSRs) were detected and the single nucleotide (A/T) was dominant in Bulbophyllum cp genomes, with an obvious A/T preference. An analysis of relative synonymous codon usage (RSCU) revealed that leucine (Leu) was the most frequently used codon, while cysteine (Cys) was the least used. Six highly variable regions (rpl32-trnLUAG > trnTUGU-trnLUAA > trnFGAA-ndhJ > rps15-ycf1 > rbcL-accD > psbI-trnSGCU) and five coding sequences (ycf1 > rps12 > matK > psbK > rps15) were identified as potential DNA markers based on nucleotide diversity. Additionally, 31,641 molecular diagnostic characters (MDCs) were identified in complete cp genomes. A phylogenetic analysis based on the complete cp genome sequences and 68 protein-coding genes strongly supported that 28 Bulbophyllum species can be divided into four branches, sects. Brachyantha, Cirrhopetalum, and Leopardinae, defined by morphology, were non-monophyly. Our results enriched the genetic resources of Bulbophyllum, providing valuable information to illustrate the complicated taxonomy, phylogeny, and evolution process of the genus.

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.

Molecular mechanism of different flower color formation of Cymbidium ensifolium.

Cymbidium ensifolium is one of the national orchids in China, which has high ornamental value with changeable flower colors. To understand the formation mechanism of different flower colors of C. ensifolium, this research conducted transcriptome and metabolome analyses on four different colored sepals of C. ensifolium. Metabolome analysis detected 204 flavonoid metabolites, including 17 polyphenols, 27 anthocyanins, 75 flavones, 34 flavonols, 25 flavonoids, 18 flavanones, and 8 isoflavones. Among them, purple-red and red sepals contain a lot of anthocyanins, including cyanidin, pelargonin, and paeoniflorin, while yellow-green and white sepals have less anthocyanins detected, and their metabolites are mainly flavonols, flavanones and flavonoids. Transcriptome sequencing analysis showed that the expression levels of the anthocyanin biosynthetic enzyme genes in red and purple-red sepals were significantly higher than those in white and yellow-green sepals of C. ensifolium. The experimental results showed that CeF3'H2, CeDFR, CeANS, CeF3H and CeUFGT1 may be the key genes involved in anthocyanin production in C. ensifolium sepals, and CeMYB104 has been proved to play an important role in the flower color formation of C. ensifolium. The results of transformation showed that the CeMYB104 is involved in the synthesis of anthocyanins and can form a purple-red color in the white perianth of Phalaenopsis. These findings provide a theoretical reference to understand the formation mechanism of flower color in C. ensifolium.

R2R3-MYB genes coordinate conical cell development and cuticular wax biosynthesis in Phalaenopsis aphrodite.

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.

Origin and diversification of a Himalayan orchid genus Pleione.

Pleione is an orchid endemically distributed in high mountain areas across the Hengduan Mountains (HDM), Himalayas, Southeast Asia and South of China. The unique flower shapes, rich colors and immense medicinal importance of Pleione are valuable ornamental and economic resources. However, the phylogenetic relationships and evolutionary history of the genus have not yet been comprehensively resolved. Here, the evolutionary history of Pleione was investigated using single-copy gene single nucleotide polymorphisms and chloroplast genome datasets. The data revealed that Pleione could be divided into five clades. Discordance in topology between the two phylogenetic trees and network and D-statistic analyses indicated the occurrence of reticulate evolution in the genus. The evolution could be attributed to introgression and incomplete lineage sorting. Ancestral area reconstruction suggested that Pleione was originated from the HDM. Uplifting of the HDM drove rapid diversification by creating conditions favoring rapid speciation. This coincided with two periods of consolidation of the Asian monsoon climate, which caused the first rapid diversification of Pleione from 8.87 to 7.83 Mya, and a second rapid diversification started at around 4.05 Mya to Pleistocene. The interaction between Pleione and climate changes, especially the monsoons, led to the current distribution pattern and shaped the dormancy characteristic of the different clades. In addition to revealing the evolutionary relationship of Pleione with orogeny and climate changes, the findings of this study provide insights into the speciation and diversification mechanisms of plants in the East Asian flora.

Transcriptome and metabolome analysis revealed the changes of Geniposide and Crocin content in Gardenia jasminoides fruit.

BACKGROUND: Gardenia jasminoides Ellis is a perennial evergreen shrub of G. jasminoides of Rubiaceae. Geniposide and Crocin are important components in the fruit of G. jasminoides. In addition to being used as medicinal materials, they are also widely used in food, medicine, cosmetics, and other fields. They have high medicinal value, economic value, and ornamental value. However, at present, the utilization rate of G. jasminoides resources is low, mainly focused on germplasm cultivation, primary processing, and clinical pharmacology, and there are few studies on the quality of Gardenia fruit. METHODS AND RESULTS: Based on transcriptome sequencing and metabolic group analysis, the morphological and structural changes of Gardenia fruit with young fruit, middle fruit, and ripe fruit were analyzed, and the formation mechanism and content changes of Geniposide and Crocin in Gardenia fruit were studied. The content of Geniposide decreased with the development of fruit, so did the expression of the main structural gene GES, G10H, and IS in its synthesis pathway, while the content of Crocin increased with the development of fruit, and the expression of the main structural gene CCD, ALDH, and UGT in its synthesis pathway also increased. The relationship between the morphological structure of G. jasminoides and the accumulation of Geniposide and Crocin was summarized. CONCLUSIONS: This study not only provides a theoretical basis for the mining and utilization of Geniposide and Crocin, but also provides a theoretical basis for genetic background for the identification and cloning of bioactive substances in gardenia fruit in future. At the same time, it provides support for increasing the dual-use value of G. jasminoides and breeding excellent germplasm resources.

Genome-Wide Identification Analysis of the R2R3-MYB Transcription Factor Family in Cymbidium sinense for Insights into Drought Stress Responses.

Cymbidium sinense represents a distinctive Orchidaceae plant that is more tolerant than other terrestrial orchids. Studies have shown that many members of the MYB transcription factor (TF) family, especially the R2R3-MYB subfamily, are responsive to drought stress. This study identified 103 CsMYBs; phylogenetic analysis classified these genes into 22 subgroups with Arabidopsis thaliana. Structural analysis showed that most CsMYB genes contained the same motifs, three exons and two introns, and showed a helix-turn-helix 3D structure in each R repeat. However, the members of subgroup 22 contained only one exon and no intron. Collinear analysis revealed that C. sinense had more orthologous R2R3-MYB genes with wheat than A. thaliana and rice. Ka/Ks ratios indicated that most CsMYB genes were under purifying negative selection pressure. Cis-acting elements analysis revealed that drought-related elements were mainly focused on subgroups 4, 8, 18, 20, 21, and 22, and Mol015419 (S20) contained the most. The transcriptome analysis results showed that expression patterns of most CsMYB genes were upregulated in leaves in response to slight drought stress and downregulated in roots. Among them, members in S8 and S20 significantly responded to drought stress in C. sinense. In addition, S14 and S17 also participated in these responses, and nine genes were selected for the real-time reverse transcription quantitative PCR (RT-qPCR) experiment. The results were roughly consistent with the transcriptome. Our results, thus, provide an important contribution to understanding the role of CsMYBs in stress-related metabolic processes.

Genome-Wide Identification and Expression Analysis of the SPL Gene Family in Three Orchids.

SPL transcription factors regulate important processes such as plant growth and development, metabolic regulation, and abiotic stress. They play crucial roles in the development of flower organs. However, little is known about the characteristics and functions of the SPLs in the Orchidaceae. In this study, Cymbidium goeringii Rchb. f., Dendrobium chrysotoxum Lindl., and Gastrodia elata BI. were used as research objects. The SPL gene family of these orchids was analyzed on a genome-wide scale, and their physicochemical properties, phylogenetic relationships, gene structures, and expression patterns were studied. Transcriptome and qRT-PCR methods were combined to investigate the regulatory effect of SPLs on the development of flower organs during the flowering process (bud, initial bloom, and full bloom). This study identifies a total of 43 SPLs from C. goeringii (16), D. chrysotoxum (17), and G. elata (10) and divides them into eight subfamilies according to the phylogenetic tree. Most SPL proteins contained conserved SBP domains and complex gene structures; half of the genes had introns longer than 10 kb. The largest number and variety of cis-acting elements associated with light reactions were enriched, accounting for about 45% of the total (444/985); 13/43 SPLs contain response elements of miRNA156. GO enrichment analysis showed that the functions of most SPLs were mainly enriched in the development of plant flower organs and stems. In addition, expression patterns and qRT-PCR analysis suggested the involvement of SPL genes in the regulation of flower organ development in orchids. There was little change in the expression of the CgoSPL in C. goeringii, but DchSPL9 and GelSPL2 showed significant expression during the flowering process of D. chrysotoxum and G. elata, respectively. In summary, this paper provides a reference for exploring the regulation of the SPL gene family in orchids.

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.

Sex-Related Gene Network Revealed by Transcriptome Differentiation of Bisexual and Unisexual Flowers of Orchid Cymbidium tortisepalum.

Despite extensive research on orchid reproductive strategies, the genetic studies of sex differentiation in the orchid family are still lacking. In this study, we compared three sexual phenotypes of Cymbidium tortisepalum bisexual flowers as well as female and male unisexual mutants. Through comparative transcriptomes, we analyzed the sex-biased differentially expressed genes (DEGs) and gene co-expression networks of sex organs (gynostemium and ovary) among them, identified the candidate genes of sex differentiation, and validated their expression by qRT-PCR. The C. tortisepalum unisexual mutants with degenerated phenotypes were compared to the bisexual plants with respect to both the flower organs and plant morphologies. Totally, 12,145, 10,789, and 14,447 genes were uniquely expressed in the female, male, and hermaphrodite sex organs, respectively. A total of 4291 sex-biased DEGs were detected among them, with 871, 2867, and 1937 DEGs in the comparisons of bisexual vs. female, bisexual vs. male, and male vs. female flowers, respectively. Two co-expressed network modules, with 81 and 419 genes were tightly correlated with female sexual traits, while two others with 265 and 135 genes were highly correlated with male sexual traits. Two female-biased hub genes (CtSDR3b and CtSDR3b-like) nested in the female modules, the homologs of maize sex determinant tasselseed2, may control the feminization of C. tortisepalum. At the same time, two male-biased hub genes (CtYAB2 and CtYAB5) nested in the male modules, the homologs of grape sex determinant VviYABBY3, may control the androphany of C. tortisepalum. This study discovered the molecular regulation networks and proposed a model for orchid sex differentiation, therefore providing for the first time the genetic basis for the sex separation in the orchid family.

Genome-Wide Identification of TCP Gene Family in Dendrobium and Their Expression Patterns in Dendrobium chrysotoxum.

The TCP gene family are plant-specific transcription factors that play important roles in plant growth and development. Dendrobium chrysotoxum, D. nobile, and D. huoshanense are orchids with a high ornamental value, but few studies have investigated the specific functions of TCPs in Dendrobium flower development. In this study, we used these three Dendrobium species to analyze TCPs, examining their physicochemical properties, phylogenetic relationships, gene structures, and expression profiles. A total of 50 TCPs were identified across three Dendrobium species; they were divided into two clades-Class-I (PCF subfamily) and Class-II (CIN and CYC/TB1 subfamilies)-based on their phylogenetic relationships. Our sequence logo analysis showed that almost all Dendrobium TCPs contain a conserved TCP domain, as well as the existence of fewer exons, and the cis-regulatory elements of the TCPs were mostly related to light response. In addition, our transcriptomic data and qRT-PCR results showed that DchTCP2 and DchTCP13 had a significant impact on lateral organs. Moreover, changes in the expression level of DchTCP4 suggested its important role in the phenotypic variation of floral organs. Therefore, this study provides a significant reference for the further exploration of TCP gene functions in the regulation of different floral organs in Dendrobium orchids.

Genome-Wide Identification of PEBP Gene Family in Two Dendrobium Species and Expression Patterns in Dendrobium chrysotoxum.

The PEBP gene family plays a significant role in regulating flower development and formation. To understand its function in Dendrobium chrysotoxum and D. nobile flowering, we identified 22 PEBP genes (11 DchPEBPs and 11 DnoPEBPs) from both species. We conducted analyses on their conserved domains and motifs, phylogenetic relationships, chromosome distribution, collinear correlation, and cis elements. The classification results showed that the 22 PEBPs were mainly divided into three clades, as follows: FT, MFT, and TFL1. A sequence analysis showed that most PEBP proteins contained five conserved domains, while a gene structure analysis revealed that 77% of the total PEBP genes contained four exons and three introns. The promoter regions of the 22 PEBPs contained several cis elements related to hormone induction and light response. This suggests these PEBPs could play a role in regulating flower development by controlling photoperiod and hormone levels. Additionally, a collinearity analysis revealed three pairs of duplicate genes in the genomes of both D. chrysotoxum and D. nobile. Furthermore, RT-qPCR has found to influence the regulatory effect of DchPEBPs on the development of flower organs (sepals, petals, lip, ovary, and gynostemium) during the flowering process (bud, transparent stage, and initial bloom). The results obtained imply that DchPEBP8 and DchPEBP9 play a role in the initial bloom and that DchPEBP7 may inhibit flowering processes. Moreover, DchPEBP9 may potentially be involved in the development of reproductive functionality. PEBPs have regulatory functions that modulate flowering. FT initiates plant flowering by mediating photoperiod and temperature signals, while TFL1 inhibits flowering processes. These findings provide clues for future studies on flower development in Dendrobium.

Genome-Wide Identification and Analysis of bHLH Transcription Factors Related to Anthocyanin Biosynthesis in Cymbidium ensifolium.

The basic helix-loop-helix (bHLH) transcription factors are widely distributed across eukaryotic kingdoms and participate in various physiological processes. To date, the bHLH family has been identified and functionally analyzed in many plants. However, systematic identification of bHLH transcription factors has yet to be reported in orchids. Here, 94 bHLH transcription factors were identified from the Cymbidium ensifolium genome and divided into 18 subfamilies. Most CebHLHs contain numerous cis-acting elements associated with abiotic stress responses and phytohormone responses. A total of 19 pairs of duplicated genes were found in the CebHLHs, of which 13 pairs were segmentally duplicated genes and six pairs were tandemly duplicated genes. Expression pattern analysis based on transcriptome data revealed that 84 CebHLHs were differentially expressed in four different color sepals, especially CebHLH13 and CebHLH75 of the S7 subfamily. The expression profiles of CebHLH13 and CebHLH75 in sepals, which are considered potential genes regulating anthocyanin biosynthesis, were confirmed through the qRT-PCR technique. Furthermore, subcellular localization results showed that CebHLH13 and CebHLH75 were located in the nucleus. This research lays a foundation for further exploration of the mechanism of CebHLHs in flower color formation.

Identification, Molecular Characteristics, and Evolution of YABBY Gene Family in Melastoma dodecandrum.

The YABBY gene family plays an important role in plant growth and development, such as response to abiotic stress and lateral organ development. YABBY TFs are well studied in numerous plant species, but no study has performed a genome-wide investigation of the YABBY gene family in Melastoma dodecandrum. Therefore, a genome-wide comparative analysis of the YABBY gene family was performed to study their sequence structures, cis-acting elements, phylogenetics, expression, chromosome locations, collinearity analysis, protein interaction, and subcellular localization analysis. A total of nine YABBY genes were found, and they were further divided into four subgroups based on the phylogenetic tree. The genes in the same clade of phylogenetic tree had the same structure. The cis-element analysis showed that MdYABBY genes were involved in various biological processes, such as cell cycle regulation, meristem expression, responses to low temperature, and hormone signaling. MdYABBYs were unevenly distributed on chromosomes. The transcriptomic data and real-time reverse transcription quantitative PCR (RT-qPCR) expression pattern analyses showed that MdYABBY genes were involved in organ development and differentiation of M. dodecandrum, and some MdYABBYs in the subfamily may have function differentiation. The RT-qPCR analysis showed high expression of flower bud and medium flower. Moreover, all MdYABBYs were localized in the nucleus. Therefore, this study provides a theoretical basis for the functional analysis of YABBY genes in M. dodecandrum.

Apostasia Mitochondrial Genome Analysis and Monocot Mitochondria Phylogenomics.

Apostasia shenzhenica belongs to the subfamily Apostasioideae and is a primitive group located at the base of the Orchidaceae phylogenetic tree. However, the A. shenzhenica mitochondrial genome (mitogenome) is still unexplored, and the phylogenetic relationships between monocots mitogenomes remain unexplored. In this study, we discussed the genetic diversity of A. shenzhenica and the phylogenetic relationships within its monocotyledon mitogenome. We sequenced and assembled the complete mitogenome of A. shenzhenica, resulting in a circular mitochondrial draft of 672,872 bp, with an average read coverage of 122× and a GC content of 44.4%. A. shenzhenica mitogenome contained 36 protein-coding genes, 16 tRNAs, two rRNAs, and two copies of nad4L. Repeat sequence analysis revealed a large number of medium and small repeats, accounting for 1.28% of the mitogenome sequence. Selection pressure analysis indicated high mitogenome conservation in related species. RNA editing identified 416 sites in the protein-coding region. Furthermore, we found 44 chloroplast genomic DNA fragments that were transferred from the chloroplast to the mitogenome of A. shenzhenica, with five plastid-derived genes remaining intact in the mitogenome. Finally, the phylogenetic analysis of the mitogenomes from A. shenzhenica and 28 other monocots showed that the evolution and classification of most monocots were well determined. These findings enrich the genetic resources of orchids and provide valuable information on the taxonomic classification and molecular evolution of monocots.

Genome-Wide Identification of the YABBY Gene Family in Dendrobium Orchids and Its Expression Patterns in Dendrobium chrysotoxum.

The small plant-specific YABBY gene family plays key roles in diverse developmental processes in plants. Dendrobium chrysotoxum, D. huoshanense, and D. nobile are perennial herbaceous plants belonging to Orchidaceae with a high ornamental value. However, the relationships and specific functions of the YABBY genes in the Dendrobium species remain unknown. In this study, six DchYABBYs, nine DhuYABBYs, and nine DnoYABBYs were identified from the genome databases of the three Dendrobium species, which were unevenly distributed on five, eight, and nine chromosomes, respectively. The 24 YABBY genes were classified into four subfamilies (CRC/DL, INO, YAB2, and FIL/YAB3) based on their phylogenetic analysis. A sequence analysis showed that most of the YABBY proteins contained conserved C2C2 zinc-finger and YABBY domains, while a gene structure analysis revealed that 46% of the total YABBY genes contained seven exons and six introns. All the YABBY genes harbored a large number of Methyl Jasmonate responsive elements, as well as anaerobic induction cis-acting elements in the promoter regions. Through a collinearity analysis, one, two, and two segmental duplicated gene pairs were identified in the D. chrysotoxum, D. huoshanense, and D. nobile genomes, respectively. The Ka/Ks values of these five gene pairs were lower than 0.5, indicating that the Dendrobium YABBY genes underwent negative selection. In addition, an expression analysis revealed that DchYABBY2 plays a role in ovary and early-stage petal development, while DchYABBY5 is essential for lip development and DchYABBY6 is crucial for early sepal formation. DchYABBY1 primarily regulates sepals during blooming. Furthermore, there is the potential involvement of DchYABBY2 and DchYABBY5 in gynostemium development. The results of a comprehensive genome-wide study would provide significant clues for future functional investigations and pattern analyses of YABBY genes in different flower parts during flower development in the Dendrobium species.