Genome assembly and microsatellite marker development using Illumina and PacBio sequencing in Persicaria maackiana (Polygonaceae) from Korea.

BACKGROUND: Persicaria maackiana (Regel) is a potential medicinal plant that exerts anti-diabetic effects. However, the lack of genomic information on P. maackiana hinders research at the molecular level. OBJECTIVE: Herein, we aimed to construct a draft genome assembly and obtain comprehensive genomic information on P. maackiana using high-throughput sequencing tools PacBio Sequel II and Illumina. METHODS: Persicaria maackiana samples from three natural populations in Gaecheon, Gichi, and Uiryeong reservoirs in South Korea were used to generate genomic DNA libraries, perform genome de novo assembly, gene ontology analysis, phylogenetic tree analysis, genotyping, and identify microsatellite markers. RESULTS: The assembled P. maackiana genome yielded 32,179 contigs. Assessment of assembly integrity revealed 1503 (93.12%) complete Benchmarking Universal Single-Copy Orthologs. A total of 64,712 protein-coding genes were predicted and annotated successfully in the protein database. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs, 13,778 genes were annotated into 18 categories. Genes that activated AMPK were identified in the KEGG pathway. A total of 316,992 microsatellite loci were identified, and primers targeting the flanking regions were developed for 292,059 microsatellite loci. Of these, 150 primer sets were randomly selected for amplification, and 30 of these primer sets were identified as polymorphic. These primers amplified 3-9 alleles. The mean observed and expected heterozygosity were 0.189 and 0.593, respectively. Polymorphism information content values of the markers were 0.361-0.754. CONCLUSION: Collectively, our study provides a valuable resource for future comparative genomics, phylogeny, and population studies of P. maackiana.

Genome Assembly and Microsatellite Marker Development Using Illumina and PacBio Sequencing in the Carex pumila (Cyperaceae) from Korea.

This study is the first to report the characterization of Carex pumila genomic information. Assembly of the genome generated a draft of C. pumila based on PacBio Sequel II and Illumina paired-end sequencing, which was assembled from 2941 contigs with an estimated genome size of 0.346 Gb. The estimate of repeats in the genome was 31.0%, and heterozygosity ranged from 0.426 to 0.441%. The integrity evaluation of the assembly revealed 1481 complete benchmarked universal single-copy orthologs (BUSCO) (91.76%), indicating the high quality of the draft assembly. A total of 23,402 protein-coding genes were successfully predicted and annotated in the protein database. UpsetR plots showed that 7481 orthogroups were shared by all species. The phylogenetic tree showed that C. pumila is a close but distant relative of Ananas comosus. C. pumila had greater contraction (3154) than expansion (392). Among the extended gene families, aquaporins have been found to be enriched. Primers for microsatellite markers determined 30 polymorphic markers out of 100. The average number of alleles amplified by these 30 polymorphic markers was 4 to 12, with an average polymorphism information content (PIC) value of 0.660. In conclusion, our study provides a useful resource for comparative genomics, phylogeny, and future population studies of C. pumila.

Phytochemical Investigation of Equisetum arvense and Evaluation of Their Anti-Inflammatory Potential in TNFα/INFγ-Stimulated Keratinocytes.

Equisetum arvense L. (Equisetaceae), widely known as 'horsetail', is a perennial plant found extensively across Asia. Extracts of E. arvense have been used in traditional medicine, particularly for the treatment of inflammatory disorders. This study aimed to determine the phytochemical compounds in E. arvense ethanolic extract and their anti-inflammatory properties. Subsequently, we isolated and identified nine secondary metabolites, including kaempferol 3,7-di-O-ß-D-glucopyranoside (1), icariside B2 (2), (Z)-3-hexenyl ß-D-glucopyranoside (3), luteolin 5-O-ß-D-glucopyranoside (4), 4-O-ß-D-glucopyranosyl caffeic acid (5), clemastanin B (6), 4-O-caffeoylshikimic acid (7), (7S,8S)-threo-7,9,9'-trihydroxy-3,3'-dimethoxy-8-O-4'-neolignan-4-O-ß-D-glucopyranoside (8), and 3-O-caffeoylshikimic acid (9). The chemical structures of the isolated compounds (1-9) were elucidated using HR-ESI-MS data, NMR spectra, and ECD data. Next, the anti-inflammatory effects of the isolates were evaluated in tumor necrosis factor (TNF)α/interferon (IFN)γ-induced HaCaT, a human keratinocyte cell line. Among the isolates, compound 3 showed the highest inhibitory effect on the expression of pro-inflammatory chemokines, followed by compounds 6 and 8. Correspondingly, the preceding isolates inhibited TNFα/IFNγ-induced activation of pro-inflammatory transcription factors, signal transducer and activator of transcription 1, and nuclear factor-κB. Collectively, E. arvense could be employed for the development of prophylactic or therapeutic agents for improving dermatitis.

Anti-Adipogenic Effects of Salicortin from the Twigs of Weeping Willow (Salix pseudolasiogyne) in 3T3-L1 Cells.

Salix pseudolasiogyne (Salicaceae), the "weeping willow," has been used in traditional Korean medicine to treat pain and fever due to its high concentrations of salicylic acid and salicin. The present study investigated bioactive compounds from S. pseudolasiogyne twigs to discover bioactive natural products. Phytochemical investigation of the ethanol (EtOH) extract of S. pseudolasiogyne twigs followed by liquid chromatography-mass spectrometry (LC/MS)-based analysis led to the isolation of two salicin derivatives, salicortinol and salicortin, the structures of which were determined by interpretation of their NMR spectra and data from the LC/MS analysis. To the best of our knowledge, this is the first report of salicortinol isolated from S. pseudolasiogyne. The isolated compounds were evaluated for their anti-adipogenic effects in 3T3-L1 cells. Both salicortinol and salicortin were found to significantly inhibit adipocyte differentiation in 3T3-L1 cells. In particular, salicortin exhibited a strong inhibitory effect on lipid accumulation. Furthermore, salicortin inhibited the expression of lipogenic and adipogenic transcription factors, including FASN, FABP4, C/EBPα, C/EBPß, and PPARγ, without inducing cytotoxicity. These results suggest that salicortin could be a potential therapeutic compound for the prevention or treatment of metabolic disorders such as obesity.

Bioactive Phytochemicals from Salix pseudolasiogyne Twigs: Anti-Adipogenic Effect of 2'-O-Acetylsalicortin in 3T3-L1 Cells.

Salix pseudolasiogyne (Salicaceae) is a willow tree and has been used as a medicinal herb in Korea to treat pain and fever. As a part of an ongoing study to identify bioactive natural products, potential anti-adipogenic compounds were investigated using the ethanol (EtOH) extract of S. pseudolasiogyne twigs. Phytochemical investigation of the EtOH extracts using liquid chromatography-mass spectrometry (LC/MS) led to the separation of two compounds, oregonin (1) and 2'-O-acetylsalicortin (2). The structures of the isolates were identified using nuclear magnetic resonance spectroscopy and LC/MS analysis. To the best of our knowledge, it is the first report identifying oregonin (1) in twigs of S. pseudolasiogyne. Here, we found that the isolated compounds, oregonin (1) and 2'-O-acetylsalicortin (2), showed anti-adipogenic effects during 3T3-L1 cell differentiation. Notably, 2'-O-acetylsalicortin (2), at a concentration of 50 µM, significantly suppressed lipid accumulation. Moreover, the mRNA and protein levels of lipogenic and adipogenic transcription factors were reduced in 2'-O-acetylsalicortin (2)-treated 3T3-L1 cells. Taken together, these results indicate that 2'-O-acetylsalicortin (2), isolated from S. pseudolasiogyne twigs, has the potential to be applied as a therapeutic agent to effectively control adipocyte differentiation, a critical stage in the progression of obesity.

Genetic diversity and structure of an endangered medicinal herb: implications for conservation.

Human-driven habitat fragmentation leads to spatial isolation of endangered plant species increasing extinction risk. Understanding genetic variability and population structure of rare and isolated plant species is of great importance for assessing extinction risk and setting up conservation plans. Aconitum austrokoreense, an endangered and endemic species in Korea, is a perennial herb commonly used for medicinal purposes. We used five nuclear microsatellites and one chloroplast marker to investigate genetic diversity and population structure for 479 individuals of A. austrokoreense from seven populations throughout South Korea. A multivariate approach, discriminant analysis of principal components analysis, revealed broad-scale spatial patterns of A. austrokoreense populations across three major mountains that were composed of seven genetically distinct subgroups. High pairwise FST values (mean FST = 0.35; highest FST = 0.55) suggested significant differentiation among populations. Overall within population genetic variation was low. Based on Mantel test, there was significant correlation between geographical and genetic distances indicating pattern of isolation by distance. Our results suggest that A. austrokoreense populations may have undergone recent population bottlenecks. Given the limited dispersal ability of the species and ongoing habitat fragmentation, population isolation may further be exacerbated leading to increased extinction risk.