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Assembling genomes of non-model plants: A case study with evolutionary insights from Ranunculus (Ranunculaceae).

Whereas genome sequencing and assembly technologies are improving, cost can still be prohibitive for plant species with large, complex genomes. As a consequence, genomics work on some taxa in evolutionarily pivotal positions in the vascular plant tree of life has been hampered. The species-rich genus Ranunculus (Ranunculaceae) is an important angiosperm group for the study of polyploidy, apomixis, and reticulate evolution. However, neither mitochondrial nor high-quality nuclear genome sequences are available. This limits phylogenomic, functional, and taxonomic analyses thus far. Here, we tested Illumina short-read, Oxford Nanopore Technology (ONT) and PacBio (HiFi) long-read, and hybrid-read assembly strategies. We sequenced the diploid progenitor species R. cassubicifolius (R. auricomus species complex) and selected the best assemblies in terms of completeness, contiguity, and quality scores. We first assembled the plastome (156 kbp, 85 genes) and mitogenome (1.18 Mbp, 40 genes) sequences using Illumina and Illumina-PacBio-hybrid strategies, respectively. We also present an updated plastome and the first mitogenome phylogeny of Ranunculaceae, including studies of gene loss (e.g., infA, ycf15, or rps) with evolutionary implications. For the nuclear genome sequence, we favored a PacBio-based assembly polished three times with filtered short reads and subsequently scaffolded into eight pseudochromosomes by chromatin conformation data (Hi-C). We obtained a haploid genome sequence of 2.69 Gbp, with 94.1% complete BUSCO genes found and 35 482 annotated genes, and inferred ancient gene duplications compared to existing Ranunculales genomes. The genomic information presented here will enable advanced evolutionary-functional analyses for the species complex, but also for the genus and beyond Ranunculaceae.

Ranunculus

Molecular phylogenetic study of the Ranunculaceae: utility of the nuclear 26S ribosomal DNA in inferring intrafamilial relationships.

There are only a small number of molecular markers currently proven to be useful for phylogenetic inference within the flowering plants. We demonstrate that the 5' end of the 26S ribosomal DNA (ca. 1100 bp) is of great value for investigating generic to subfamilial relationships. We analyzed DNA sequences from 31 species of the Ranunculaceae and four species of the Berberidaceae to test phylogenetic relationships within the Ranunculaceae. The inferred phylogeny strongly supports the concept that the Thalictrum chromosome group is not monophyletic, but consists of three independent lineages: (1) Hydrastis, (2) Xanthorhiza and Coptis, and (3) Thalictrum, Aquilegia, and Enemion. Based on comparison with conventional taxonomic characters, we propose a hypothesis that the third group also includes the rest of the Thalictrum chromosome taxa that have a base chromosome number of seven. For the Ranunculus chromosome group, our study suggests several relationships that have not been recognized by conventional systematics. The inferred 26S rDNA topology is compared with results from two previously published molecular data sets: DNA sequences from rbcL, atpB, and 18S rDNA genes and restriction fragment length polymorphism data from chloroplast DNA. The three topologies are highly congruent and agree with karyological characters, but not with fruit type, both of which have often been used for the higher classification of the Ra- nunculaceae.

Base Sequence

Counter-irritant and other medicinal uses of plants in Ranunculaceae by native peoples in British Columbia and neighbouring areas.

At least 20 species in Ranunculaceae, the buttercup family, are reported as having been used medicinally by 19 different groups of native peoples in British Columbia and adjacent areas. These species are known to contain the skin-irritating, blister-causing compound, protoanemonin, in their fresh state. Protoanemonin is almost certainly the active principle involved in many of these medicinal applications. A majority involved the use of the plants as external poultices for boils, cuts, abrasions and other skin sores. Other disorders having a high frequency of treatment with ranunculaceous species include: muscular aches, colds and other respiratory ailments, and general, unspecified illness. Native groups in other parts of North America also used many ranunculaceous species as poultices, and for colds, headaches and many other ailments. A number were used for stimulation and "revival" of unconscious persons. It is suggested that the protoanemonin contained in these plants may have, through several different mechanisms, positively influenced the healing process physiologically and not just psychologically. If future research confirms this, these protoanemonin containing plants may have potential in certain treatments in modern medicine.

British Columbia

The genome sequence of Aquilegia vulgaris L., 1753 (Ranunculales: Ranunculaceae).

We present a genome assembly of Aquilegia vulgaris (Columbine; Streptophyta; Magnoliopsida; Ranunculales; Ranunculaceae). The assembly consists of two haplotypes with total lengths of 296.10 megabases and 289.22 megabases. Most of haplotype 1 (98.81%) is scaffolded into 7 chromosomal pseudomolecules. Most of haplotype 2 (94.35%) is scaffolded into 7 chromosomal pseudomolecules. The mitochondrial sequences have lengths of 388.22 and 83.91 kilobases and the plastid genome assembly has a length of 161.5 kilobases. Gene annotation of this assembly on Ensembl identified 23 898 protein-coding genes. This assembly was generated as part of the Darwin Tree of Life project, which produces reference genomes for eukaryotic species found in Britain and Ireland.

Aquilegia vulgaris

[Medicinal plant resources of Ranunculaceae in Zhejiang Province].

This paper reports the general situation on medicinal plant resources of Ranunculaceae in Zhejiang. There are 13 genera and 51 species (including varieties). Suggestions have been made with regard to the preservation, development and utilization of the natural resources.

China

[From Rumanian folk medicine: Non-specific stimulus therapy using transcutaneous implantation of hellebore (Helleborus purpurascens, Fam. Ranunculaceae) in agriculturally useful animals].

In the Rumanian traditional medicine a transcutaneous implantation of the root of Helleborus purpurascens is used to provoke leucocytosis and neutrophils with the aim to activate chronic diseases for better healing. The skin is perforated with a thick needle (in cattle and horses in the area of the thorax, in sheep and pigs in the ear flap), the transplant is introduced and after 24 hours removed. Though the method is more than 100 years old, it is not reported in the literature. The way of action is unknown. The investigation are performed to show the efficacy of this method of Helleborus-implants in comparison with the effect of an placebo. It could be shown that Helleborus-implant increased leucocytes in horses, sheep and pigs and increased neutrophils in all animal species. In the same way the phagocytosis was increased. Only in horses an abscess was observed. As a loss of performance may occur and fever and inappetence is followed by the implantation the authors dissuade from the use of Helleborus-implants in cattle and horses. Pigs and sheep tolerate this procedure without any problems, so that the methods may be used in modern treatment.

Animals

An unusual fatty acid pattern in Eranthis seed oil.

The current discussion on "renewable resources", and the possibility of gene transfer into rapeseed, has led to many investigations into the biosynthetic pathways leading to industrially useful fatty acids. The various tribes and genera of the plant family Ranunculaceae contain a large variety of unusual fatty acids. Seed fatty acid patterns differ considerably from genus to genus and are chemotaxonomically significant indicators. Eranthis seed oil has now been found to contain a fatty acid pattern that deviates significantly from the eleven different fatty acid patterns that had been described in this plant family. The main fatty acid (up to 57%) is 13-cis, 16-cis-docosadienoic acid. Other, minor fatty acids found are delta 5-cismonoenoic, -dienoic, and -trienoic fatty acids that had already been reported to be constituents of the genus-specific seed oil fatty acid patterns of various genera from this plant family. Capillary gas-liquid chromatographic retention data indicate that 22:3 delta 5 cis, 13 cis, 16 cis is probably also present. Seed fatty acid chemotaxonomic evidence thus points to a different position of Eranthis within the tribes of this plant family. These findings again indicate that the plant family Ranunculaceae would be an ideal object to study fatty acid biosynthesis and phylogenetic evolution, because in other genera of this family other types of desaturation and chain elongation mechanisms predominate.

Biological Evolution

Studies on bioactive saponins from Chinese medicinal plants.

In our recent studies on bioactive saponins, two Chinese medicinal plants: Mussaenda pubescens Ait.f (Rubiaceae) and Clematis chinensis Osbeck (Ranunculaceae), were investigated. Of the two medicinal plants, M. pubescens is a Chinese folk medicine which has been used as a diuretic, antiphlogistic, diaphoretic and antipyretic agent, and has also been used to detoxify mushroom poisons and to terminate early pregnancy. Clematis chinensis is a Chinese traditional medicine which has been used as an analgesic, diuretic, antitumor, antiinflammatory and insecticidal agent for ages. As a result of our studies, eighteen saponins were identified from M. pubescens, among which seventeen were new compounds, while eleven saponins were isolated from C. chinensis, three of which were new compounds. In the course of our structural studies, mass fragment analysis in FAB-mass spectra and 1H- and 13C-NMR spectra were used to determine the structures of the sapogenin and oligosaccharide moieties. In those more complicated and minor saponins, various 2D-NMR experiments were carried out on 400-, 500- or 600-MHz NMR instruments, which permitted the identification of new sapogenins in glycoside form. In addition, it was also possible to assign all the proton and carbon signals of the sugar units on the basis of 1H-1H DQF COSY, TOCSY and HMQC spectra, which further permitted the establishment of linkage sites and sequences among the sugar units and aglycones by means of NOESY, ROESY and HMBC spectra. When the proton signals of sugar units are overlapped seriously in 1H-NMR spectra, peracetylation is a very helpful technique which can spread proton signals in wider range, thus simplifying their assignment by means of 2D-NMR spectra. When some of the sugar proton signals of a peracetylate are also overlapped coincidentally, alternation of deuterated solvents for measuring NMR spectra can circumvent the difficulties. Pharmacological tests indicated that mussaendoside O, the most abundant saponin from M. pubescens, can inhibit significantly the secretions of the lachrymal and salivary glands induced by galanthamine, and can also inhibit the contraction of the isolated longitudinal muscle strip from guinea pig ileum evoked by an M-Ach receptor agonist (carbachol, 10(-6) M) at concentrations of 10(-4) and 10(-5) M. From these results, the saponin should be an antagonist of the M-Ach receptor, and was presumed to be responsible for its antitoxicity activity toward some mushroom poisons of medicinal plant origin. In addition, mussaendoside O also showed immunopromotive and hemolytic activities.

Animals

Inhibitory effect of bisbenzylisoquinoline alkaloids on nitric oxide production in activated macrophages.

Bisbenzylisoquinoline (BBI) alkaloids are anti-inflammatory constituents of plants of the families Menispermaceae and Ranunculaceae, which have been used as folk remedies in Japan and China. Five BBI alkaloids (cepharanthine, chondocurine, cycleanine, isotetrandrine and tetrandrine) were tested for suppressive effect on in vitro nitric oxide (NO) production by lipopolysaccharide-stimulated peritoneal macrophages, which were induced with thioglycollate or bacillus Calmette-Guerin in mice. All these BBI alkaloids significantly suppressed NO production at 5 micrograms/mL. Cepharanthine, isotetrandrine and cycleanine were slightly more inhibitory than tetrandrine and chondocurine. The suppression persisted for at least 48 hr. As NO is one of the critical mediators in inflammation, these results may explain some aspects of the anti-inflammatory mechanisms of BBI compounds.

Alkaloids

Phyllosphere microbiomes in grassland plants harbor a vast reservoir of novel antimicrobial peptides and biosynthetic diversity.

INTRODUCTION: The phyllosphere microorganisms colonizing plant surface harbor capacities to synthesize diverse specialized metabolites that mediate communication and interactions with environment and host. However, most known metabolites are derived from a few culturable microorganisms, and the genomic diversity and biosynthetic potential of the vast majority of bacteria associated with plants remain largely unexplored. OBJECTIVES: Here, we aim to explore the genome architecture, biosynthetic ability, and host specific adaptability of grassland ecosystems, uncovering new perspectives on grassland phyllosphere microbial resources. METHODS: We employed ultra-deep metagenomic sequencing, functional analysis, host-associated characterization, and bioactivity assays to explore the phyllosphere microbiome across 221 grassland plant samples representing 45 families. This approach revealed host preference in biosynthetic gene clusters (BGCs) and validated the antimicrobial efficacy of phyllosphere-derived antimicrobial peptides (AMPs). RESULTS: Grassland plant phyllosphere microbiomes encode diverse BGCs. We identified 885,396 potential AMPs from over 68 million non-redundant gene sequences. Then, we reconstructed hundreds of near-complete genomes from phyllosphere metagenomes, and 32.61 % of reconstructed genomes were identified as unclassified genomes, primarily within Pseudomonadota, Actinomycetota, Bacillota and Bacteroidota phyla. Of the near-complete genomes, 91.97 % of the BGCs and 99.76 % of the identified AMPs were previously uncharacterized. Host phylogenetic analysis revealed functional divergence. Poaceae-associated Pseudomonas genomes contain an average of 28 BGCs, significantly higher than those in Asteraceae-associated genomes (mean = 14.76, P = 0.033). Similarly, Poaceae-associated Pantoea genomes carried an average of 9 BGCs, exhibiting significant enrichment compared to genomes from Asteraceae (mean = 7.13, P = 6.1e-05), Lamiaceae (mean = 7, P = 0.015), Ranunculaceae (mean = 8.22, P = 0.0053), and Rosaceae (mean = 7.75, P = 0.00069). ParaFit analyses further confirmed that host phylogeny significantly structures microbial functional repertoires, with intra-family hosts sharing more KEGG pathways than inter-family hosts. These results suggest that host evolutionary relationships are associated with metabolic specialization in phyllosphere microbiomes. All 13 AMPs synthesized via solid-phase peptide synthesis demonstrated antimicrobial activity, inhibiting the growth of at least one tested bacterial strain. CONCLUSION: This study demonstrates the promise of grassland plant phyllosphere microbiome as a rich source for novel antimicrobial agents.

Antimicrobial Peptides