Chloroplast Genome-Based Hypervariable Markers for Rapid Authentication of Six Korean Pyropia Species

We previously established that polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using partial plastid rbcL and mitochondrial trnC–trnP gene sequences can be used to distinguish the six representative Pyropia species produced via mariculture in Korea. In this study, we develop progressive InDel markers by comparing seven complete Pyropia chloroplast genomes obtained from The National Center of Biotechnology Informnation (NCBI) GenBank. Comparative analyses of nucleotide diversity among the genomes revealed seven hypervariable sites (cemA, rps13, trnM-argB, petD-petB, trnR-trnQ, ccs1-orf24, and ycf12-ftrB) among 637 sliding windows with nucleotide diversity > 0.025 (Pi). These sites included two genes and five gene-intergenic regions, three of which (cemA, trnM-argB, trnR-trnQ) showed complete amplification for all six test species. Finally, trnM-argB, an InDel-variable locus with high discriminatory power, was selected as a DNA barcode candidate. These results suggest that the obtained trnM-argB region can be used for the effective exploration of the variation present in six Korean Pyropia and for further evolutionary, phylogenetic, barcoding and genetic engineering studies of Pyropia species

An Efficient PCR-RFLP Method for the Rapid Identification of Korean Pyropia Species

The present study utilizes polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using partial plastid rbcL and mitochondrial trnC–trnP gene sequences to distinguish the six representative Pyropia species produced via mariculture in Korea. The rbcL, trnC, and trnP sequences of 15 Pyropia species from the NCBI database were aligned to determine specific restriction enzyme sites of the six Pyropia species. To confirm the presence of restriction sites of eight enzymes, PCR amplicons were digested as follows: a 556 bp fragment within the rbcL region of chloroplast DNA was confirmed in P. yezoensis using BglI, whereas Tth111I, AvaII, BsrI, and BsaAI enzymes produced fragments of 664, 271, 600, and 510 bp, respectively, from the rps11–trnG region of mitochondrial DNA in P. seriata, P. dentata, P. suborbiculata, and P. haitanensis. In the case of P. pseudolinearis, HindIII, SacII, and SphI enzymes each had two cleavage sites, at positions 174 and 825, 788 and 211, and 397 and 602 bp, respectively. All six species were successfully distinguished using these eight restriction enzymes. Therefore, we propose that PCR-RFLP analysis is an efficient tool for the potential use of distinguishing between the six Pyropia species cultivated via mariculture in Korea

Complete chloroplast genome sequences of Pyropia dentata (Bangiales, Rhodophyta)

Pyropia dentata is an economically valuable species in seaweed aquaculture in the southwest coastal regions of Korea. Herein, we determined the complete chloroplast (cp) genome sequence of P. dentata using PacBio resequencing data. The whole cp genome is 192,266 bp in size, encodes 148 genes (including 92 protein-coding, 44 tRNA-coding, and 12 rRNA-coding genes), and lacks inverted repeat (IR) regions. A maximum likelihood phylogenetic analysis demonstrated that P. dentata was most closely related to P. haitanensis. The cp genome will provide a useful reference for further studies of P. dentata

Complete chloroplast genome sequence of Stauntonia hexaphylla (Ranunculales: Lardizabalaceae), a species endemic to Korea

The complete chloroplast (cp) genome of Stauntonia hexaphylla, a monotypic genus native to Korea, was determined. The whole cp genome is 158,390 bp in size, containing a large single-copy (LSC) region of 87,115 bp and a small single-copy region (SSC) of 18,928 bp, separated by a pair of inverted repeats (IRs) of 26,174 bp. The cp genome encodes 117 genes, including 79 protein-coding, 38 tRNA-coding, and 8 rRNA-coding genes. The overall GC content is 37.8%. A phylogenetic analysis demonstrated a close relationship between S. hexaphylla and S. obovatifoliola subsp. urophylla. The cp genome will provide new insight into the evolution of Lardizabalaceae

Comparative Analysis of Complete Chloroplast Genome Sequences and Insertion-Deletion (Indel) Polymorphisms to Distinguish Five Vaccinium Species

We report the identification of interspecific barcoding InDel regions in Vaccinium species. We compared five complete Vaccinium chloroplast (cp) genomes (V. bracteatum, V. vitis-idaea, V. uliginosum, V. macrocarpon, and V. oldhamii) to identify regions that can be used to distinguish them. Comparative analysis of nucleotide diversity from five cp genomes revealed 25 hotspot coding and noncoding regions, occurring in 65 of a total of 505 sliding windows, that exhibited nucleotide diversity (Pi) > 0.02. PCR validation of 12 hypervariable InDel regions identified seven candidate barcodes with high discriminatory powers: accD-trnT-GGU, rpoB-rpoA, ycf2-trnL-GAA, rps12-ycf15, trnV-GAC, and ndhE-ndhF. Among them, the rpoB-rpoA(2) and ycf2-trnL-CAA sequences clearly showed the intraspecific and interspecific distance among five Vaccinium species by using a K2P technique. In phylogenetic analysis, included five Vaccinium species (n = 19) in the Bayesian and Neighbor-Joining (NJ) analysis revered all species in two major clades and resolved taxonomic position within species groups. These two locus provide comprehensive information that aids the phylogenetics of this genus and increased discriminatory capacity during species authentication

Cynanchum wilfordii Ameliorates Testosterone-Induced Benign Prostatic Hyperplasia by Regulating 5α-Reductase and Androgen Receptor Activities in a Rat Model

Benign prostatic hyperplasia (BPH) is characterized by uncontrolled proliferation of the prostate gland. Cynanchum wilfordii has been reported to improve sexual behavior in male rats. In this study, we investigated the protective effect of an aqueous extract of C. wilfordii (CWW) against BPH development in a testosterone-induced BPH rat model. The rats were divided into the following six groups: sham/vehicle; BPH/vehicle; BPH/finasteride; and three CWW doses (50, 100, and 200 mg/kg). After a 4-week treatment with CWW, the rats were euthanized at scheduled times, and their prostates were weighed, followed by a histopathological examination. Prostate growth inhibition rates in rats administered CWW 50, 100, and 200 mg/kg were 54.5%, 51.8%, and 50.1%, respectively. The BPH/CWW group showed decreased serum testosterone and dihydrotestosterone (DHT) levels compared to the BPH/vehicle group. Furthermore, the BPH/CWW group showed reduced prostate testosterone and DHT levels compared to the BPH/vehicle group. Mechanistically, the reverse transcription-polymerase chain reaction revealed downregulated mRNA expression levels of the androgen receptor, 5α-reductase, and B-cell lymphoma-2 (Bcl-2) in the BPH/CWW200 group compared with those in the testosterone-induced groups. In conclusion, these findings show the effectiveness of CWW in slowing the progression of testosterone-induced BPH in rats

Complete chloroplast genomes of E. umbellata Thunb., E. multiflora Thunb., E. macrophylla Thunb., and E. glabra Thunb. (Elaeagnaceae)

Elaeagnus is a genus which consists about 70 species of flowering plants in the family Elaeagnaceae, and its edible fruit is a natural product used as food and in traditional medicine. In this study, we sequenced the complete chloroplast (cp) genome of four species, namely Elaeagnus umbellate Thunb., E. multiflora Thunb., E. macrophylla Thunb., and E. glabra Thunb., to study their phylogenetic relationships within the Elaeagnaceae. Total lengths of the chloroplast genome were 152,261 bp, 152,267 bp, 152,224 bp, and 152,227 bp, respectively. The four genomes had representative quadripartite structures, with an LSC region (82,207 bp, 82,191 bp, 82,136 bp, and 82,139 bp) and an SSC region (18,262 bp, 18,282 bp,and 18,278 bp for both species) separated by a pair of IRs (25,896 bp, 25,897 bp, and 25,905 bp for the latter two species), respectively. Moreover, they were composed of 136–137 genes, including 88 protein-coding genes, 40–41 tRNA genes, and 8 rRNA genes. A maximum likelihood phylogenetic analysis indicated that E. umbellata was most closely related to E. multiflora, whereas E. macrophylla was close to E. glabra

Isolation and Analytical Method Validation for Phytocomponents of Aqueous Leaf Extracts from Vaccinium bracteatum Thunb. in Korea

In this study, major phytochemical compounds of Vaccinium bracteatum Thunb. (VB) aqueous leaf extract were isolated and analyzed using a HPLC-based method, followed by method validation in accordance with the International Conference on Harmonisation (ICH) guidelines for drug development. Five major compounds were isolated in VB extract. Apart from vaccinoside, which had been the only compound isolated in VB extract to date, vanillic acid and protocatechuic acid were isolated for the first time. Isolation of orientin and isoorientin in the VB extract helped validate the reverse-phase analytical method. A new simple and rapid high-performance liquid chromatography (HPLC)-based method was developed for the validation of orientin and isoorientin in VB extract and was determinated according to the ICH guidelines. The analytical method was validated through a Waters Alliance HPLC System containing an e2695 separation module and a 2998 photodiode array (PDA) detector. The VB extract and solutions of orientin and isoorientin were analyzed using a reverse-phase Eclipse XDB-C18 column (4.6 × 250 mm ID, 5 µm, Waters), which was maintained at 30 °C. A mobile phase of methanol and 0.01% formic acid in water was used at a flow rate of 1.0 mL/min to achieve gradient elution. The linearity of the orientin and isoorientin was excellent results (R2 ≥ 0.9999) in the concentration range of 1.0–50.0 μg/mL. Precision values ranged 98.55–101.70% and 98.70–101.18%, respectively. The intra-day and inter-day relative standard deviation (RSD) values of the orientin and isoorientin were all <2.0%. The average recoveries of orientin ranged 98.30–101.57%, whereas isoorientin ranged 97.81–102.14% with RSD values <2.0%. Quantitative analysis found that VB extract contained 2.90 mg/g of orientin and 3.45 mg/g of isoorientin