Chloroplast Microsatellite-Based High-Resolution Melting Analysis for Authentication and Discrimination of <i>Ilex</i> Species

Ilex species are important sources of high-quality raw plant materials for the production of drugs and functional foods. The precise identification of different species within the Ilex genus would greatly facilitate authentication and certification as well as forest resource monitoring in plantations. Combining DNA barcoding with high-resolution melting (HRM) analysis represents a robust strategy for species discrimination, as demonstrated in recent DNA barcoding studies. Here, using concatenated and aligned complete chloroplast genomes of different Ilex species, we conducted a sliding window analysis to identify regions of high nucleotide diversity (Pi). We optimized and validated the utility of PCR-based HRM coupled with microsatellite markers to discriminate among the four Ilex species, Ilex integra Thunb., Ilex rotunda Thunb., Ilex cornuta Lindl. and Paxton, and Ilex x wandoensis C.F. Mill and M. Kim, from wild populations in southwestern Korea. The marker trnSUGA-psbZ produced clear melting patterns and distinct melting curve profiles for the four Ilex species using HRM analysis. We applied this protocol to commercially available Ilex accessions and consistently identified the correct species for all 15 accessions tested. Therefore, combining DNA barcoding with HRM analysis is a powerful method for identifying different species within the same genus, which could be used for quality control of raw materials in the functional food/medicinal plant industry

Chloroplast Microsatellite-Based High-Resolution Melting Analysis for Authentication and Discrimination of Ilex Species

Ilex species are important sources of high-quality raw plant materials for the production of drugs and functional foods. The precise identification of different species within the Ilex genus would greatly facilitate authentication and certification as well as forest resource monitoring in plantations. Combining DNA barcoding with high-resolution melting (HRM) analysis represents a robust strategy for species discrimination, as demonstrated in recent DNA barcoding studies. Here, using concatenated and aligned complete chloroplast genomes of different Ilex species, we conducted a sliding window analysis to identify regions of high nucleotide diversity (Pi). We optimized and validated the utility of PCR-based HRM coupled with microsatellite markers to discriminate among the four Ilex species, Ilex integra Thunb., Ilex rotunda Thunb., Ilex cornuta Lindl. and Paxton, and Ilex x wandoensis C.F. Mill and M. Kim, from wild populations in southwestern Korea. The marker trnSUGA-psbZ produced clear melting patterns and distinct melting curve profiles for the four Ilex species using HRM analysis. We applied this protocol to commercially available Ilex accessions and consistently identified the correct species for all 15 accessions tested. Therefore, combining DNA barcoding with HRM analysis is a powerful method for identifying different species within the same genus, which could be used for quality control of raw materials in the functional food/medicinal plant industry

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) &gt; 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

The complete mitochondrial genome of Neoporphyra dentata (Bangiales, Rhodophyta)

Neoporphyra dentata (Kjellman) L.-E. Yang & J. Brodie, 2020 is an economically valuable species in seaweed aquaculture in the southwest coastal regions of Korea. Here, we report the complete mitogenome information of N. dentata using Illumina Miseq platform permitted assembly of a circular mitochondrial genome of 26,807 bp from N. dentata consisting of 29.9% GC contents, 9 protein coding genes (PCGs), 2 ribosomal RNA genes (12S rRNA and 16S rRNA), 23 transfer RNA (tRNA) genes, and a non-coding region. The overall nucleotide composition was A: 38%, T: 32%, C: 14.7%, and G: 15.2%. The mitochondrial genome of N. dentata contributes to revealing the phylogenetic relationships among species of the Bangiaceae family

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 &lt;2.0%. The average recoveries of orientin ranged 98.30–101.57%, whereas isoorientin ranged 97.81–102.14% with RSD values &lt;2.0%. Quantitative analysis found that VB extract contained 2.90 mg/g of orientin and 3.45 mg/g of isoorientin

Complete chloroplast genome sequences of Vaccinium bracteatum Thunb., V. vitis-idaea L., and V. uliginosum L. (Ericaceae)

Members of the Vaccinium genus (Ericaceae family) have excellent medicinal and nutritional properties. In this study, we sequenced the complete chloroplast (cp) genomes of three species, Vaccinium bracteatum Thunb., V. vitis-idaea L., and V. uliginosum L., to investigate their phylogenetic relationships within the Ericaceae. The chloroplast genomes of these species are 174,404 bp, 173,967 bp, and 173,356 bp, respectively. These genomes have a typical quadripartite structure, with an LSC region (106,565 bp, 106,013 bp, and 105,856 bp) and an SSC region (2979 bp, 3518 bp, and 3146 bp) separated by a pair of IRs (32,430 bp, 32,218 bp, and 32,177 bp, respectively). These cp genomes are composed of 139 genes, including 93 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Maximum likelihood phylogenetic analysis indicated that V. bracteatum is most closely related to V. oldhamii. Our findings should facilitate further evolutionary and genetic studies

The complete chloroplast genome of Artemisia Montana (Nakai) Pamp. (Asteraceae), a traditional medicinal herb in Korea

Artemisia Montana (Nakai) Pamp. is a widely used heath food and a well-known traditional Korean herbal medicine. The complete chloroplast genome sequence of A. Montana was determined using high-throughput sequencing technology. Chloroplast genome was 151,133 bp in length, with a large single-copy (LSC) region of 98,497 bp, a small single-copy (SSC) region of 18,352 bp, separated by two inverted repeat (IR) regions of 17,142 bp each. It contained a total of 113 genes, with an overall GC content of 37.5%. The phylogenetic analysis showed that A. montana most closely related to A. feddei. This result will enrich the genetic resources of medicinal plant and useful for future investigation of genetics, evolution and identification of Artemisia species