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.

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.

Genome-Wide Identification of the MYB Gene Family in Cymbidiumensifolium and Its Expression Analysis in Different Flower Colors.

MYB transcription factors of plants play important roles in flavonoid synthesis, aroma regulation, floral organ morphogenesis, and responses to biotic and abiotic stresses. Cymbidium ensifolium is a perennial herbaceous plant belonging to Orchidaceae, with special flower colors and high ornamental value. In this study, a total of 136 CeMYB transcription factors were identified from the genome of C. ensifolium, including 27 1R-MYBs, 102 R2R3-MYBs, 2 3R-MYBs, 2 4R-MYBs, and 3 atypical MYBs. Through phylogenetic analysis in combination with MYB in Arabidopsis thaliana, 20 clusters were obtained, indicating that these CeMYBs may have a variety of biological functions. The 136 CeMYBs were distributed on 18 chromosomes, and the conserved domain analysis showed that they harbored typical amino acid sequence repeats. The motif prediction revealed that multiple conserved elements were mostly located in the N-terminal of CeMYBs, suggesting their functions to be relatively conserved. CeMYBs harbored introns ranging from 0 to 13 and contained a large number of stress- and hormone-responsive cis-acting elements in the promoter regions. The subcellular localization prediction demonstrated that most of CeMYBs were positioned in the nucleus. The analysis of the CeMYBs expression based on transcriptome data showed that CeMYB52, and CeMYB104 of the S6 subfamily may be the key genes leading to flower color variation. The results lay a foundation for the study of MYB transcription factors of C. ensifolium and provide valuable information for further investigations of the potential function of MYB genes in the process of anthocyanin biosynthesis.

The Cymbidium genome reveals the evolution of unique morphological traits.

The marvelously diverse Orchidaceae constitutes the largest family of angiosperms. The genus Cymbidium in Orchidaceae is well known for its unique vegetation, floral morphology, and flower scent traits. Here, a chromosome-scale assembly of the genome of Cymbidium ensifolium (Jianlan) is presented. Comparative genomic analysis showed that C. ensifolium has experienced two whole-genome duplication (WGD) events, the most recent of which was shared by all orchids, while the older event was the τ event shared by most monocots. The results of MADS-box genes analysis provided support for establishing a unique gene model of orchid flower development regulation, and flower shape mutations in C. ensifolium were shown to be associated with the abnormal expression of MADS-box genes. The most abundant floral scent components identified included methyl jasmonate, acacia alcohol and linalool, and the genes involved in the floral scent component network of C. ensifolium were determined. Furthermore, the decreased expression of photosynthesis-antennae and photosynthesis metabolic pathway genes in leaves was shown to result in colorful striped leaves, while the increased expression of MADS-box genes in leaves led to perianth-like leaves. Our results provide fundamental insights into orchid evolution and diversification.

Chloroplast characterization and phylogenetic relationship of Cymbidium aloifolium (Orchidaceae).

Cymbidium aloifolium is an epiphytic orchid with high medicinal and ornamental value. In order to get a deeper understanding of C. aloifolium, we determined the complete chloroplast genome of C. aloifolium by Illumina sequencing data. The length of this genome is 157,328 bp, including a couple of inverted repeat (IR) regions of 26,829 bp, a large single-copy (LSC) region of 85,793 bp, and a small single-copy (SSC) region of 17,877 bp. The chloroplast genome comprised of 139 genes, including 78 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. In addition, the phylogenetic analysis based on 17 chloroplast genomes of Orchidaceae indicated that C. mannii was closely related to C. aloifolium. This study will provide more valuable information for the classification and phylogenetic research of Cymbidium genus.

The complete chloroplast genome sequence of Goodyera foliosa（Orchidaceae）.

Goodyera foliosa is a terrestrial orchid in Asia and has been listed as an endangered species in the Red List. In this study, we assembled the complete chloroplast genome of G. foliosa using Illumina sequencing data. Its full-length of 154,008 bp including a pair of invert repeats (IR) regions of 25,045 bp, large single-copy (LSC) region of 83,248 bp, and small single-copy (SSC) region of 20,670 bp. The chloroplast genome contains 127 genes, including 80 protein-coding genes, 39 tRNA genes, and 8 rRNA genes. In addition, the phylogenetic analysis base on 12 chloroplast genomes of Orchidaceae indicates that G. schlechtendaliana is closely related to G. foliosa. Our study would be helpful for the formulation of conservation strategies and further research of G. foliosa.

The complete chloroplast genome sequence of Habenaria ciliolaris (Orchidaceae).

Habenaria ciliolaris is a kind of orchid with ornamental value. In this study, we reported the complete chloroplast genome of H. ciliolaris. The complete chloroplast genome is 154,544 bp in length, consists of a pair of inverted repeat (IR, 25,455 bp) regions, a large single-copy region (LSC, 84,032 bp) and a small single-copy region (SSC, 19,602 bp). It contains 179 genes, including 133 protein-coding genes, 38 tRNAs, and 8 rRNAs. A maximum-likelihood phylogenetic analysis demonstrated a close relationship between H. ciliolaris and Habenaria radiata. This work will be valuable for genetic and phylogenetic studies on H. ciliolaris.

The complete chloroplast genome of Eria corneri (Orchidaceae).

Eria corneri is a perennial epiphytic orchid distributed in southeastern China with high value of ornamental and medicinal. In this study, the complete chloroplast genome sequence of E. corneri was determined from Illumina pair-end sequencing data. The complete chloroplast genome sequence of E. corneri is 150,538 base pairs (bp) in length, including one large single-copy region (LSC, 85,941 bp), one small single-copy region (SSC, 13,099 bp), and a pair of inverted repeat regions (IRs) of 25,749 bp. Besides, the complete chloroplast genome contains 132 genes, including 77 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Phylogenetic analysis showed that E. corneri was most closely related to Calanthe triplicata and Calanthe davidii. Our study provides a foundation for the identification and genotyping of Eria species.

The complete chloroplast genome sequence of Tainia cordifolia (Orchidaceae).

Tainia cordifolia is a subtropical plant with significant ornamental value. Herein, we determined the complete chloroplast (cp) genome sequence of T. cordifolia using Illumina sequencing data. The whole cp genome is 158,089 bp in size, consisting of a pair of inverted repeats (IR 25,260 bp), a large single-copy region (LSC 86,876 bp), and a small single-copy region (SSC 20,693 bp). Plastid genome contains 136 genes, 88 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. What is more, a maximum-likelihood phylogenetic analysis demonstrated that T. cordifolia was most closely related to Oberonia japonica and Dendrobium salaccense. The cp genome will provide reference for the further investigation and research of T. cordifolia.

The complete chloroplast genome of Tainia dunnii (Orchidaceae): genome structure and evolution.

Tainia dunnii is a terrestrial orchid with high ornamental value. Herein, we assembled the complete chloroplast genome of Tainia dunnii by next-generation sequencing technologies. The complete chloroplast genome sequence of Tainia dunnii is 158,305 base pairs (bp) in length, including a pair of inverted repeat regions (IRs, 25,244 bp), one large single-copy region (LSC, 86,819 bp), one small single-copy region (SSC, 20,998 bp). Besides, the complete chloroplast genome contains 136 genes in total, including 88 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Phylogenetic analysis showed that Tainia dunnii has the closest relationship with Calanthe davidii and Calanthe triplicata. Our study lay a foundation for further research of Tainia dunnii.