Phylogenomics of mulberries (Morus, Moraceae) inferred from plastomes and single copy nuclear genes.

Mulberries (genus Morus), belonging to the order Rosales, family Moraceae, are important woody plants due to their economic values in sericulture, as well as for nutritional benefits and medicinal values. However, the taxonomy and phylogeny of Morus, especially for the Asian species, remains challenging due to its wide geographical distribution, morphological plasticity, and interspecific hybridization. To better understand the evolutionary history of Morus, we combined plastomes and a large-scale nuclear gene analyses to investigate their phylogenetic relationships. We assembled the plastomes and screened 211 single-copy nuclear genes from 13 Morus species and related taxa. The plastomes of Morus species were relatively conserved in terms of genome size, gene content, synteny, IR boundary and codon usage. Using nuclear data, our results elucidated identical topologies based on coalescent and concatenation methods. The genus Morus was supported as monophyletic, with M. notabilis as the first diverging lineage and the two North American Morus species, M. microphylla and M. rubra, as sister to the other Asian species. In the Asian Morus species, interspecific relationships were completely resolved. However, cyto-nuclear discordances and gene tree-species tree conflicts were detected in the phylogenies of Morus, with multiple evidences supporting hybridization/introgression as the main cause of discordances between nuclear and plastid phylogenies, while gene tree-species tree conflicts were mainly caused by ILS.

Impacts of oxbow lake evolution on sediment microbial community structure in the Yellow River source region.

Oxbow lake formation and evolution have significant impacts on the fragile Yellow River Basin ecosystem. However, the effects of different oxbow lake evolutionary stages on sediment microbial community structure are not yet understood comprehensively. Therefore, microbial community structure in three stages of oxbow lake succession, namely, lotic lake (early stage), semi-lotic lake (middle stage), and lentic lake (late stage), was investigated in the present study in the Yellow River Basin on the Qinghai-Tibet Plateau. Amplicon sequencing was employed to reveal differences in microbial community diversity and composition. The bacterial and fungal communities in sediment were significantly different among the three succession stages and were driven by different environmental factors. In particular, bacterial community structure was influenced primarily by nitrate-nitrogen (N), microbial biomass phosphorus, and total carbon (C) and organic C in the early, middle, and late stages, respectively. Conversely, fungal community structure was influenced primarily by ammonium-N in the early stage and by moisture content in the middle and late stages. However, the predicted functions of the microbial communities did not exhibit significant differences across the three succession stages. Both bacteria and fungi were influenced significantly by stochastic factors. Homogeneous selection had a high relative contribution to bacteria community assembly in the middle stage, whereas the relative contributions of heterogeneous selection processes to fungal community assembly increased through the three stages. As succession time increased, the total number of keystone species increased gradually, and the late succession stage had high network complexity and the highest network stability. The findings could facilitate further elucidation of the evolution mechanisms of oxbow lake source area, high-altitude river evolution dynamics, in addition to aiding a deeper understanding of the long-term ecological evolution patterns of source river ecosystems.

Multi-omics analyses provide insights into the genomic basis of differentiation among four sweet osmanthus groups.

Sweet osmanthus (Osmanthus fragrans) is famous in China for its flowers and contains four groups: Albus, Luteus, Aurantiacus, and Asiaticus. Understanding the relationships among these groups and the genetic mechanisms of flower color and aroma biosynthesis are of tremendous interest. In this study, we sequenced representative varieties from two of the four sweet osmanthus groups. Multi-omic and phylogenetic analyses of varieties from each of the four groups showed that Asiaticus split first within the species, followed by Aurantiacus and the sister groups Albus and Luteus. We show that the difference in flower color between Aurantiacus and the other three groups was caused by a 4-bp deletion in the promoter region of carotenoid cleavage dioxygenase 4 (OfCCD4) that leads to expression decrease. In addition, we identified 44 gene pairs exhibiting significant structural differences between the multi-seasonal flowering variety 'Rixianggui' in the Asiaticus group and other autumn flowering varieties. Through correlation analysis between intermediate products of aromatic components and gene expression, we identified eight genes associated with the linalool, α- and ß-ionone biosynthesis pathways. Overall, our study offers valuable genetic resources for sweet osmanthus, while also providing genetic clues for improving the flower color and multi-season flowering of osmanthus and other flowers.

Full-Length Transcriptome Sequencing and Comparative Transcriptomic Analyses Provide Comprehensive Insight into Molecular Mechanisms of Flavonoid Metabolites Biosynthesis in Styphnolobium japonicum.

Styphnolobium japonicum L. is a commonly consumed plant in China, known for its medicinal and nutritional benefits. This study focuses on the medicinal properties influenced by flavonoid metabolites, which vary during flower development. Utilizing full-length transcriptome sequencing on S. japonicum flowers, we observed changes in gene expression levels as the flowers progressed through growth stages. During stages S1 and S2, key genes related to flavonoid synthesis (PAL, 4CL, CHS, F3H, etc.) exhibited heightened expression. A weighted gene co-expression network analysis (WGCNA) identified regulatory genes (MYB, bHLH, WRKY) potentially involved in the regulatory network with flavonoid biosynthesis-related genes. Our findings propose a regulatory mechanism for flavonoid synthesis in S. japonicum flowers, elucidating the genetic underpinnings of this process. The identified candidate genes present opportunities for genetic enhancements in S. japonicum, offering insights into potential applications for improving its medicinal attributes.

High-quality chromosome-level genome assembly and multi-omics analysis of rosemary (Salvia rosmarinus) reveals new insights into the environmental and genome adaptation.

High-quality genome of rosemary (Salvia rosmarinus) represents a valuable resource and tool for understanding genome evolution and environmental adaptation as well as its genetic improvement. However, the existing rosemary genome did not provide insights into the relationship between antioxidant components and environmental adaptability. In this study, by employing Nanopore sequencing and Hi-C technologies, a total of 1.17 Gb (97.96%) genome sequences were mapped to 12 chromosomes with 46 121 protein-coding genes and 1265 non-coding RNA genes. Comparative genome analysis reveals that rosemary had a closely genetic relationship with Salvia splendens and Salvia miltiorrhiza, and it diverged from them approximately 33.7 million years ago (MYA), and one whole-genome duplication occurred around 28.3 MYA in rosemary genome. Among all identified rosemary genes, 1918 gene families were expanded, 35 of which are involved in the biosynthesis of antioxidant components. These expanded gene families enhance the ability of rosemary adaptation to adverse environments. Multi-omics (integrated transcriptome and metabolome) analysis showed the tissue-specific distribution of antioxidant components related to environmental adaptation. During the drought, heat and salt stress treatments, 36 genes in the biosynthesis pathways of carnosic acid, rosmarinic acid and flavonoids were up-regulated, illustrating the important role of these antioxidant components in responding to abiotic stresses by adjusting ROS homeostasis. Moreover, cooperating with the photosynthesis, substance and energy metabolism, protein and ion balance, the collaborative system maintained cell stability and improved the ability of rosemary against harsh environment. This study provides a genomic data platform for gene discovery and precision breeding in rosemary. Our results also provide new insights into the adaptive evolution of rosemary and the contribution of antioxidant components in resistance to harsh environments.

A new mechanism of flowering regulation by the competition of isoforms in Osmanthus fragrans.

The regulation of flowering time is typically governed by transcription factors or epigenetic modifications. Transcript isoforms can play important roles in flowering regulation. Recently, transcript isoforms were discovered in the key genes, OfAP1 and OfTFL1, of the flowering regulatory network in Osmanthus fragrans. OfAP1-b generates a full-length isoform of OfAP1-b1 as well as an isoform of OfAP1-b2 that lacks the C-terminal domain. Although OfAP1-b2 does not possess an activation domain, it has a complete K domain that allows it to form heterodimers. OfAP1-b2 competes with OfAP1-b1 by binding with OfAGL24 to create non-functional and functional heterodimers. As a result, OfAP1-b1 promotes flowering while OfAP1-b2 delays flowering. OfTFL1 produces two isoforms located in different areas: OfTFL1-1 in the cytoplasm and OfTFL1-2 in the nucleus. When combined with OfFD, OfTFL1-1 does not enter the nucleus to repress AP1 expression, leading to early flowering. Conversely, when combined with OfFD, OfTFL1-2 enters the nucleus to repress AP1 expression, resulting in later flowering. Tissue-specific expression and functional conservation testing of OfAP1 and OfTFL1 support the new model's effectiveness in regulating flowering. Overall, this study provides new insights into regulating flowering time by the competition of isoforms.

Mechanisms for leaf color changes in Osmanthus fragrans 'Ziyan Gongzhu' using physiology, transcriptomics and metabolomics.

BACKGROUND: Color-leaved O. fragrans is a variety of Osmanthus fragrans, which has both the fragrance of Osmanthus and the color of color-leaved plants. However, the molecular mechanism of color change of color-leaved O. fragrans is not clear. In this study, we analyzed the regulatory mechanism of four different color leaves of 'Ziyan Gongzhu' through physiological, transcriptome and metabolome levels. RESULTS: Firstly, we measured the leaf pigments content and leaf chromatic parameters for correlation analysis, indicating a significant correlation between them. Overall, the content of chlorophyll a + b is low and the content of anthocyanin is high in T1 and T2 leaves, along with low expression of chlorophyll synthesis genes (HEMA, CHLG, and CAO, etc.) and high expression of anthocyanin synthesis genes (F3H, F3'H, DFR and ANS, etc.), resulting purple red and light purple in T1 and T2 leaves, respectively. It was also found that the pigment closely related to the color leaves of 'Ziyan Gongzhu' was cyanidin. The content anthocyanins, may be regulated by two putative MYB activators (OfMYB3 and OfMYB4) and two putative MYB repressors (OfMYB1 and OfMYB2). In contrast, the content of chlorophyll a + b is high and the content of anthocyanin is low in T3 and T4 leaves, along with high expression of chlorophyll synthesis genes and low expression of anthocyanin synthesis genes, resulting yellow green and dark green in T3 and T4 leaves, respectively. And abnormal chloroplast development affects chlorophyll content in T1, T2, and T3 leaves. Although the content of carotenoids first dropped in T2 leaves, it then rapidly accumulated in T4 leaves, in sync with the increase in the expression of genes related to carotenoid biosynthesis (ZDS, LHYB, and ZEP, for example). Analysis of photosynthetic, carbohydrate and hormone-related differentially abundant metabolites (DAMs) and DEGs found that they may participate in the regulation of leaf color change of 'Ziyan Gongzhu' by affecting pigment synthesis. CONCLUSION: Our results pave the way for a comprehensive knowledge of the regulatory processes governing leaf color in 'Ziyan Gongzhu' and identify possible genes for application regarding molecular colored-leaf cultivar breeding.

Phylogenomics insights into gene evolution, rapid species diversification, and morphological innovation of the apple tribe (Maleae, Rosaceae).

Maleae is one of the most widespread tribes of Rosaceae and includes several important fruit crops and ornamental plants. We used nuclear genes from 62 transcriptomes/genomes, including 26 newly generated transcriptomes, to reconstruct a well-supported phylogeny and study the evolution of fruit and leaf morphology and the possible effect of whole genome duplication (WGD). Our phylogeny recovered 11 well-supported clades and supported the monophyly of most genera (except Malus, Sorbus, and Pourthiaea) with at least two sampled species. A WGD was located to the most recent common ancestor (MRCA) of Maleae and dated to c. 54 million years ago (Ma) near the Early Eocene Climatic Optimum, supporting Gillenieae (x = 9) being a parental lineage of Maleae (x = 17) and including duplicate regulatory genes related to the origin of the fleshy pome fruit. Whole genome duplication-derived paralogs that are retained in specific lineages but lost in others are predicted to function in development, metabolism, and other processes. An upshift of diversification and innovations of fruit and leaf morphologies occurred at the MRCA of the Malinae subtribe, coinciding with the Eocene-Oligocene transition (c. 34 Ma), following a lag from the time of the WGD event. Our results provide new insights into the Maleae phylogeny, its rapid diversification, and morphological and molecular evolution.

Phylogenomic and syntenic data demonstrate complex evolutionary processes in early radiation of the rosids.

Some of the most vexing problems of deep level relationship that remain in angiosperms involve the superrosids. The superrosid clade contains a quarter of all angiosperm species, with 18 orders in three subclades (Vitales, Saxifragales and core rosids) exhibiting remarkable morphological and ecological diversity. To help resolve deep-level relationships, we constructed a high-quality chromosome-level genome assembly for Tiarella polyphylla (Saxifragaceae) thus providing broader genomic representation of Saxifragales. Whole genome microsynteny analysis of superrosids showed that Saxifragales shared more synteny clusters with core rosids than Vitales, further supporting Saxifragales as more closely related with core rosids. To resolve the ordinal phylogeny of superrosids, we screened 122 single copy nuclear genes from genomes of 36 species, representing all 18 superrosid orders. Vitales were recovered as sister to all other superrosids (Saxifragales + core rosids). Our data suggest dramatic differences in relationships compared to earlier studies within core rosids. Fabids should be restricted to the nitrogen-fixing clade, while Picramniales, the Celastrales-Malpighiales (CM) clade, Huerteales, Oxalidales, Sapindales, Malvales and Brassicales formed an "expanded" malvid clade. The Celastrales-Oxalidales-Malpighiales (COM) clade (sensu APG IV) was not monophyletic. Crossosomatales, Geraniales, Myrtales and Zygophyllales did not belong to either of our well-supported malvids or fabids. There is strong discordance between nuclear and plastid phylogenetic hypotheses for superrosid relationships; we show that this is best explained by a combination of incomplete lineage sorting and ancient reticulation.

The complete chloroplast genome of Ulmus mianzhuensis with insights into structural variations, adaptive evolution, and phylogenetic relationships of Ulmus (Ulmaceae).

BACKGROUND: Ulmus mianzhuensis is an endemic tree species in China with high ornamental and economic value. Currently, little is known regarding its genomic architecture, phylogenetic position, or adaptive evolution. Here, we sequenced the complete chloroplast genome (cp genome) of U. mianzhuensis and further compared the variations in gene organization and structure within Ulmus species to define their genomic evolution, then reconstructed the phylogenomic relationship of 31 related Ulmus species to explore the systematic position of U. mianzhuensis and the utility of cp genome for resolving phylogenetics among Ulmus species. RESULTS: Our results revealed that all the Ulmus species exhibited a typical quadripartite structure, with a large single copy (LSC) region of 87,170 - 88,408 bp, a small single copy (SSC) region of 18,650 - 19,038 bp and an inverted repeat (IR) region of 26,288 - 26,546 bp. Within Ulmus species, gene structure and content of cp genomes were highly conserved, although slight variations were found in the boundary of SC/IR regions. Moreover, genome-wide sliding window analysis uncovered the variability of ndhC-trnV-UAC, ndhF-rpl32, and psbI-trnS-GCU were higher among 31 Ulmus that may be useful for the population genetics and potential DNA barcodes. Two genes (rps15 and atpF) were further detected under a positive selection of Ulmus species. Comparative phylogenetic analysis based on the cp genome and protein-coding genes revealed consistent topology that U. mianzhuensis is a sister group to U. parvifolia (sect. Microptelea) with a relatively low-level nucleotide variation of the cp genome. Additionally, our analyses also found that the traditional taxonomic system of five sections in Ulmus is not supported by the current phylogenomic topology with a nested evolutionary relationship between sections. CONCLUSIONS: Features of the cp genome length, GC content, organization, and gene order were highly conserved within Ulmus. Furthermore, molecular evidence from the low variation of the cp genome suggested that U. mianzhuensis should be merged into U. parvifolia and regarded as a subspecies of U. parvifolia. Overall, we demonstrated that the cp genome provides valuable information for understanding the genetic variation and phylogenetic relationship in Ulmus.

Intraspecific Chloroplast Genome Variation and Domestication Origins of Major Cultivars of Styphnolobium japonicum.

Styphnolobium japonicum is a significant resource of ornamental and medicinal plants. In this study, we employed high-throughput sequencing to assemble nine chloroplast genomes of S. japonicum. We compared and reconstructed the phylogenetic relationships of these genomes, along with three publicly available chloroplast genomes. Our results showed that the length of the 12 S. japonicum chloroplast genomes ranged from 158,613 bp to 158,837 bp, all containing 129 unique functional genes. The genetic diversity within S. japonicum chloroplast genomes was relatively low, with π = 0.00029, Theta-W = 0.00028, and an indel frequency of 0.62 indels/1 kb. Among the four regions, the SSC region exhibited the highest genetic diversity and indel frequency, while the IR region had the lowest. Non-coding regions displayed greater genetic variation compared to coding regions, with a few highly variable regions identified. The phylogenetic tree constructed revealed that the major cultivars of S. japonicum originated from two genetic 'sources. S. japonicum 'JinhuaiJ2' had an independent origin and showed close relatedness to S. japonicum var. violacea, S. japonicum var. japonicum, and S. japonicum f. oligophylla. On the other hand, other major cultivars shared a common genetic origin and were closely related to S. japonicum f. pendula. This study highlights the variability of chloroplast genomes within S. japonicum and provides insights into the genetic origins of major cultivars and their relationships with different varieties and forma.

Comparative methylomics and chromatin accessibility analysis in Osmanthus fragrans uncovers regulation of genic transcription and mechanisms of key floral scent production.

Linalool and ionone are two important aromatic components in sweet osmanthus petals, and the regulatory mechanisms that produce these two components remain unclear. In this study, we employed whole-genome methylation sequencing and ATAC-seq technology to analyze the genomic DNA methylation status and chromatin accessibility of the sweet osmanthus cultivars 'Zaohuang' and 'Chenghong Dangui'. Results showed that the promoter region of TPS2, a key gene in the linalool synthesis pathway, was less methylated in 'Chenghong Dangui' than in 'Zaohuang'. The chromatin was more accessible in 'Chenghong Dangui' than in 'Zaohuang', which resulted in a much stronger expression of this gene in 'Chenghong Dangui' than in 'Zaohuang'. This eventually led to a high quantity of linalool and its oxides in the petals of 'Chenghong Dangui', but there were lower levels present in the petals of 'Zaohuang'. These results suggest that DNA methylation and chromatin accessibility play major roles in linalool synthesis in sweet osmanthus. The methylation level of the promoter region of CCD4, a key gene for ionone synthesis, was higher in 'Zaohuang' than in 'Chenghong Dangui'. The chromatin accessibility was lower in 'Zaohuang' than in 'Chenghong Dangui', although the expression of this gene was significantly higher in 'Zaohuang' than in 'Chenghong Dangui'. ChIP-seq analysis and a series of experiments showed that the differential expression of CCD4 and CCD1 in the two cultivars may predominantly be the result of regulation by ERF2 and other transcription factors. However, a 183-bp deletion involving the CCD4 promoter region in 'Chenghong Dangui' may be the main reason for the low expression of this gene in its petals. This study provides an important theoretical basis for improving selective breeding of key floral fragrance components in sweet osmanthus.

Spatiotemporal Variation of Osmanthus fragrans Phenology in China in Response to Climate Change From 1973 to 1996.

Climate change greatly affects spring and autumn plant phenology around the world consequently, and significantly impacts ecosystem function and the social economy. However, autumn plant phenology, especially autumn flowering phenology, has not been studied so far. In this study, we examined the spatiotemporal pattern of Osmanthus fragrans phenology, including both leaf phenology (the date of bud-bust, BBD; first leaf unfolding, FLD; and 50% of leaf unfolding, 50 LD) and flowering phenology (the date of first flowering, FFD; peak of flowering, PFD; and end of flowering, EFD). Stepwise multiple linear regressions were employed to analyze the relationships between phenophases and climatic factors in the long term phenological data collected by the Chinese Phenological Observation Network from 1973 to 1996. The results showed that spring leaf phenophases and autumn flowering phenophases were strongly affected by latitude. BBD, FLD, and 50LD of O. fragrans were delayed by 3.98, 3.93, and 4.40 days as per degree of latitude increased, while FFD, PFD and EFD in O. fragrans advanced 3.11, 3.26, and 2.99 days, respectively. During the entire study period, BBD was significantly delayed across the region, whereas no significant trends were observed either in FLD or 50LD. Notably, all flowering phenophases of O. fragrans were delayed. Both leaf and flowering phenophases negatively correlated with growing degree-days (GDD) and cold degree-days (CDD), respectively. BBD and FLD were negatively correlated with total annual precipitation. In addition to the effects of climate on autumn flowering phenology, we found that earlier spring leaf phenophases led to delayed autumn flowering phenophases. Our results suggest that future climate change and global warming might delay the phenological sequence of O. fragrans. Our findings also advanced the flowering mechanism study of autumn flowering plants, and facilitated the accurate prediction of future phenology and climate change.

The complete chloroplast genome sequence of Malus halliana (Rosaceae), an important ornamental plant.

Malus halliana is an important ornamental plant resource. Herein, we determined the complete chloroplast (cp) genome sequence of M. halliana using Illumina sequencing data. The whole cp genome is 160,089 bp in size, consisting of a pair of inverted repeats (IR 52,706 bp), a large single-copy region (LSC 88,189 bp), and a small single-copy region (SSC 19,194 bp). The plastid genome contains 129 genes, 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes. In addition, a maximum-likelihood phylogenetic analysis demonstrated that M. halliana was most closely related to Malus hupehensis. The complete plastome sequence will provide useful genetic information for phylogenetic studies, the resolution of taxonomic discrepancies, and molecular breeding.

Plastome Evolution in Saxifragaceae and Multiple Plastid Capture Events Involving Heuchera and Tiarella.

Saxifragaceae, a family of over 600 species and approximately 30 genera of herbaceous perennials, is well-known for intergeneric hybridization. Of the main lineages in this family, the Heuchera group represents a valuable model for the analysis of plastid capture and its impact on phylogeny reconstruction. In this study, we investigated plastome evolution across the family, reconstructed the phylogeny of the Heuchera group and examined putative plastid capture between Heuchera and Tiarella. Seven species (11 individuals) representing Tiarella, as well as Mitella and Heuchera, were selected for genome skimming. We assembled the plastomes, and then compared these to six others published for Saxifragaceae; the plastomes were found to be highly similar in overall size, structure, gene order and content. Moreover, ycf15 was lost due to pseudogenization and rpl2 lost its only intron for all the analyzed plastomes. Comparative plastome analysis revealed that size variations of the plastomes are purely ascribed to the length differences of LSC, SSC, and IRs regions. Using nuclear ITS + ETS and the complete plastome, we fully resolved the species relationships of Tiarella, finding that the genus is monophyletic and the Asian species is most closely related to the western North American species. However, the position of the Heuchera species was highly incongruent between nuclear and plastid data. Comparisons of nuclear and plastid phylogenies revealed that multiple plastid capture events have occurred between Heuchera and Tiarella, through putative ancient hybridization. Moreover, we developed numerous molecular markers for Tiarella (e.g., plastid hotspot and polymorphic nuclear SSRs), which will be useful for future studies on the population genetics and phylogeography of this disjunct genus.

Characterization of the complete chloroplast genome sequence of Malus toringoides (Rosaceae).

Malus toringoides belongs to the Malus genus (Rosaceae) and is a precious resource among wild plants. In this study, we report the first complete chloroplast (cp) genome sequence of M. toringoides. The whole cp genome contains 126 genes, 83 protein-coding genes, 35 tRNA genes, and eight rRNA genes. A maximum-likelihood phylogenetic tree analysis based on 12 complete chloroplast genomes indicated that M. toringoides clustered closely with Malus hupehensis. Thus, the chloroplast genome can provide valuable genetic information for the protection and exploitation of M. toringoides.

Mechanism of floral scent production in Osmanthus fragrans and the production and regulation of its key floral constituents, ß-ionone and linalool.

Sweet osmanthus (Osmanthus fragrans Lour.) is among the top ten most well-known flowers in China and is recognized as both an aromatic plant and ornamental flower. Here, manual sectioning, scanning electron microscopy, and transmission electron microscopy of sweet osmanthus petals revealed that large amounts of lipids are present inside the petal cells and on the cell surfaces. However, no secretory structures were observed. Instead, the petal cells protrude slightly outward, and the surfaces of the cells are adorned with highly regular brush-shaped hairs. The surfaces of the 'Yingui' petals possessed mostly curled and more numerous hairs, whereas the 'Dangui' petals possessed fewer brush-shaped and more sparsely arranged hairs. In addition, many granular substances were attached to the brush-shaped hairs, and the granules were denser on the hairs of the 'Yingui' petals compared to the hairs on the 'Dangui' petals. Furthermore, 35 aromatic components in the 'Yingui' petals and 30 aromatic components in the 'Dangui' petals were detected via GC-MS. The main aromatic component of the 'Yingui' petals was ß-ionone, whereas that of the 'Dangui' petals was linalool and its oxides. Transcriptome sequencing and qRT-PCR indicated that the high ß-ionone content in the 'Yingui' petals was due to the overexpression of CCD1 and CCD4 and that the high linalool content in the 'Dangui' petals was due to the overexpression of MECS, HDR, IDI1, and LIS1, which function upstream of the linalool synthetic pathway. In particular, the expression levels of CCD4 and LIS1 were upregulated by 5.5- and 5.1-fold in the 'Yingui' and 'Dangui' petals, respectively. One transcription factor (ERF61) was cloned and named, and the expression pattern of ERF61 in sweet osmanthus petals was found to be generally consistent with that of CCD4. Tobacco transformation experiments, yeast one-hybrid experiments, and electrophoretic mobility shift assays indicated that ERF61 binds to the CCD4 promoter and stimulates CCD4 expression, thereby regulating the synthesis of ß-ionone in sweet osmanthus petals.

Rapid recovery of high content phytosterols from corn silk.

BACKGROUND: Phytosterols have important physiological and officinal function. METHODS: An efficient ultrasonic assisted extraction, purification and crystallization procedure of phytosterols was established from corn silk for the first time. RESULTS: The orthogonal test was applied to optimize the process parameters and a maximum phytosterols recovery as high as 10.5886 mg/g was achieved by ultrasonic treatment for 55 min with liquid-solid ratio of 12:1 at 35 °C, 220 w. The ultrasonic extraction temperature (T, °C) has the most significant effect on extraction yield of phytosterols. An orthogonal crystallization test was performed and the optimal conditions [crystallization temperature of 8 °C, time of 12 h and solid-liquid ratio of 1:1 (g/ml)] afforded maximum phytosterols purity of 92.76 ± 0.43%. CONCLUSIONS: An efficient extraction and crystallization procedure was established.

The First Genetic Map in Sweet Osmanthus (Osmanthus fragrans Lour.) Using Specific Locus Amplified Fragment Sequencing.

Osmanthus fragrans is an ornamental plant of substantial commercial value, and no genetic linkage maps of this species have previously been reported. Specific-locus amplified fragment sequencing (SLAF-seq) is a recently developed technology that allows massive single nucleotide polymorphisms (SNPs) to be identified and high-resolution genotyping. In our current research, we generated the first genetic map of O. fragrans using SLAF-seq, which is composed with 206.92 M paired-end reads and 173,537 SLAF markers. Among total 90,715 polymorphic SLAF markers, 15,317 polymorphic SLAFs could be used for genetic map construction. The integrated map contained 14,189 high quality SLAFs that were grouped in 23 genetic linkage groups, with a total length of 2962.46 cM and an average distance of 0.21 cM between two adjacent markers. In addition, 23,664 SNPs were identified from the mapped markers. As far as we know, this is the first of the genetic map of O. fragrans. Our results are further demonstrate that SLAF-seq is a very effective method for developing markers and constructing high-density linkage maps. The SNP markers and the genetic map reported in this study should be valuable resource in future research.

Characterization of OfWRKY3, a transcription factor that positively regulates the carotenoid cleavage dioxygenase gene OfCCD4 in Osmanthus fragrans.

The sweet osmanthus carotenoid cleavage dioxygenase 4 (OfCCD4) cleaves carotenoids such as ß-carotene and zeaxanthin to yield ß-ionone. OfCCD4 is a member of the CCD gene family, and its promoter contains a W-box palindrome with two reversely oriented TGAC repeats, which are the proposed binding sites of WRKY transcription factors. We isolated three WRKY cDNAs from the petal of Osmanthus fragrans. One of them, OfWRKY3, encodes a protein containing two WRKY domains and two zinc finger motifs. OfWRKY3 and OfCCD4 had nearly identical expression profile in petals of 'Dangui' and 'Yingui' at different flowering stages and showed similar expression patterns in petals treated by salicylic acid, jasmonic acid and abscisic acid. Activation of OfCCD4pro:GUS by OfWRKY3 was detected in coinfiltrated tobacco leaves and very weak GUS activity was detected in control tissues, indicating that OfWRKY3 can interact with the OfCCD4 promoter. Yeast one-hybrid and electrophoretic mobility shift assay showed that OfWRKY3 was able to bind to the W-box palindrome motif present in the OfCCD4 promoter. These results suggest that OfWRKY3 is a positive regulator of the OfCCD4 gene, and might partly account for the biosynthesis of ß-ionone in sweet osmanthus.