Extraction and analysis of high-quality chloroplast DNA with reduced nuclear DNA for medicinal plants.

BACKGROUND: Obtaining high-quality chloroplast genome sequences requires chloroplast DNA (cpDNA) samples that meet the sequencing requirements. The quality of extracted cpDNA directly impacts the efficiency and accuracy of sequencing analysis. Currently, there are no reported methods for extracting cpDNA from Erigeron breviscapus. Therefore, we developed a suitable method for extracting cpDNA from E. breviscapus and further verified its applicability to other medicinal plants. RESULTS: We conducted a comparative analysis of chloroplast isolation and cpDNA extraction using modified high-salt low-pH method, the high-salt method, and the NaOH low-salt method, respectively. Subsequently, the number of cpDNA copies relative to the nuclear DNA (nDNA ) was quantified via qPCR. As anticipated, chloroplasts isolated from E. breviscapus using the modified high-salt low-pH method exhibited intact structures with minimal cell debris. Moreover, the concentration, purity, and quality of E. breviscapus cpDNA extracted through this method surpassed those obtained from the other two methods. Furthermore, qPCR analysis confirmed that the modified high-salt low-pH method effectively minimized nDNA contamination in the extracted cpDNA. We then applied the developed modified high-salt low-pH method to other medicinal plant species, including Mentha haplocalyx, Taraxacum mongolicum, and Portulaca oleracea. The resultant effect on chloroplast isolation and cpDNA extraction further validated the generalizability and efficacy of this method across different plant species. CONCLUSIONS: The modified high-salt low-pH method represents a reliable approach for obtaining high-quality cpDNA from E. breviscapus. Its universal applicability establishes a solid foundation for chloroplast genome sequencing and analysis of this species. Moreover, it serves as a benchmark for developing similar methods to extract chloroplast genomes from other medicinal plants.

Genetic diversities in wild and cultivated populations of the two closely-related medical plants species, Tripterygium Wilfordii and T. Hypoglaucum (Celastraceae).

BACKGROUND: The sustainable supply of medicinal plants is important, and cultivating and domesticating them has been suggested as an optimal strategy. However, this can lead to a loss of genetic diversity. Tripterygium wilfordii Hook. f. is a medicinal plant commonly used in traditional Chinese medicine, but its wild populations are dwindling due to excessive harvesting. To protect the species and meet the increasing demand, it is urgent to cultivate it on a large scale. However, distinguishing between T. wilfordii and T. hypoglaucum, two similar species with different medicinal properties, is challenging. Therefore, it is crucial to understand the genetic diversity and population structure of these species for their sustainable utilization. RESULTS: In this study, we investigated the genetic diversity and population structure of the two traditional medicinal semiwoody vines plant species, Tripterygium wilfordii and T. hypoglaucum, including wild and cultivated populations using chloroplast DNA (cpDNA) sequences and microsatellite loci. Our results indicated that the two species maintain a high level of genetic divergence, indicating possible genetic bases for the different contents of bioactive compounds of the two species. T. wilfordii showed lower genetic diversity and less subdivided population structures of both markers than T. hypoglaucum. The potential factors in shaping these interesting differences might be differentiated pollen-to-seed migration rates, interbreeding, and history of population divergence. Analyses of cpDNA and microsatellite loci supported that the two species are genetically distinct entities. In addition, a significant reduction of genetic diversity was observed for cultivated populations of the two species, which mainly resulted from the small initial population size and propagated vegetative practice during their cultivation. CONCLUSION: Our findings indicate significant genetic divergence between T. wilfordii and T. hypoglaucum. The genetic diversity and population structure analyses provide important insights into the sustainable cultivation and utilization of these medicinal plants. Accurate identification and conservation efforts are necessary for both species to ensure the safety and effectiveness of crude drug use. Our study also highlighted the importance of combined analyses of different DNA markers in addressing population genetics of medicinal plants because of the contrasts of inheritance and rates of gene flow. Large-scale cultivation programs should consider preserving genetic diversity to enhance the long-term sustainability of T. wilfordii and T. hypoglaucum. Our study proposed that some populations showed higher genetic diversity and distinctness, which can be considered with priority for conservation and as the sources for future breeding and genetic improvement.

Population Genetic Analysis of Paris polyphylla var. yunnanensis Based on cpDNA Fragments.

Paris polyphylla var. yunnanensis is a well-known medicinal plant that is mainly distributed in Southwest China; however, its genetic diversity and biodiversity processes are poorly understood. In this study, the sequences of cpDNA trnL-trnF fragments of 15 wild populations and 17 cultivated populations of P. polyphylla var. yunnanensis were amplified, sequenced, and aligned to study the population genetics of this species. Genetic diversity was analyzed based on nucleotide diversity, haplotype diversity, Watterson diversity, population-level diversity, and species-level genetic diversity. Genetic structure and genetic differentiation were explored using haplotype distribution maps and genetic distance matrices. A total of 15 haplotypes were identified in the 32 populations of P. polyphylla var. yunnanensis. Five unique haplotypes were identified from the fourteen haplotypes of the cultivated populations, while only one unique haplotype was identified from the ten haplotypes of the wild populations. The haplotype richness and genetic diversity of the cultivated populations were higher than those of the wild populations (HT = 0.900 vs. 0.861). In addition, there were no statistically significant correlations between geographic distance and genetic distance in the cultivated populations (r = 0.16, p > 0.05), whereas there was a significant correlation between geographical distance and genetic structure in the wild populations (r = 0.32, p > 0.05), indicating that there was a geographical and genetic connection between the wild populations. There was only 2.5% genetic variation between the wild populations and cultivated populations, indicating no obvious genetic differentiation between the wild and cultivated populations. Overall, the genetic background of the cultivated populations was complex, and it was hypothesized that the unique haplotypes and higher diversity of the cultivated populations were caused by the mixed provenance of the cultivated populations.

Population genetic structure of wild Angelica acutiloba, A. acutiloba var. iwatensis, and their hybrids by atpF-atpA intergenic spacer in chloroplast DNA and genome-wide SNP analysis using MIG-seq.

Sampling surveys of Angelica acutiloba and A. acutiloba var. iwatensis, which are medicinal plants endemic to Japan, were conducted in the Chubu region in the central area of the main island of Japan. A. acutiloba grows in riverbeds in mountainous areas, while A. acutiloba. var. iwatensis grows on slopes near mountain ridges at 1000 m above sea level or on constantly collapsing rocky slopes and bare fields on developed land along asphalt roads in valleys of mountainous areas. Specimens of two wild Angelica species collected in this region were examined for maternal lineage by DNA polymorphism analysis of the atpF-atpA region for chloroplast DNA using direct sequencing and genomic component analysis by genome-wide SNP using MIG-seq. In this study area, while all A. acutiloba populations were monophyletic in both maternal and ancestral lineages, A. acutiloba var. iwatensis were genetically heterogeneous due to being composed of three maternal and three ancestral lineages to various degrees. In addition, a natural hybrid population with maternal lineage presumed to be A. acutiloba and paternal lineage A. acutiloba var. iwatensis was also found. In the present study, we report that the combined method of atpF-atpA and MIG-seq analyses is a useful tool for determining the population genetic structure of two wild Angelica species and for identifying hybrids.

Comparisons of Chloroplast Genome Mutations among 13 Samples of Oil-Tea Camellia from South China.

The differences in cpDNA SNPs and InDels of 13 samples from single trees of different species or populations of oil-tea camellia in South China were examined in this study, and phylogenetic trees were reconstructed based on CDSs and non-CDSs of cpDNAs to research the evolutionary relationships among all samples. The SNPs of all samples included all kinds of substitutions, and the frequency of the transition from AT to GC was highest; meanwhile, the frequencies of all kinds of transversions differed among the samples, and the SNPs exhibited polymorphism. The SNPs were distributed in all the different functional regions of cpDNAs, and approximately half of all SNPs in exons led to missense mutations and the gain or loss of termination codons. There were no InDels in the exons of any cpDNA samples, except those retrieved from Camellia gigantocarpa, although this InDel did not lead to a frame shift. The InDels of all cpDNA samples were unevenly distributed in the intergenic region and upstream and downstream of genes. The genes, regions of the same gene, sites and mutation types in the same region related to the distributions of SNPs, and InDels were inconsistent among samples. The 13 samples were divided into 2 clades and 7 or 6 subclades, and the samples of species from the same sections of the Camellia genus did not belong to the same subclades. Meanwhile, the genetic relationship between the samples of Camellia vietnamensis and the undetermined species from Hainan Province or the population of C. gauchowensis in Xuwen was closer than that between C. vietnamensis and the population of C. gauchowensis in Luchuan, and the genetic relationship among C. osmantha, C. vietnamensis and C. gauchowensis was very close. In sum, SNPs and InDels in the different cpDNAs resulted in variable phenotypes among the different species or populations, and they could be developed into molecular markers for studies on species and population identification and phylogenetic relationships. The conclusion from the identification of undetermined species from Hainan Province and the phylogenetic relationships among 13 oil-tea camellia samples based on cpCDS and cpnon-CDS sequences were the same as those from the former report.

Identification of three cultivated varieties of Scutellaria baicalensis using the complete chloroplast genome as a super-barcode.

Scutellaria baicalensis has been one of the most commonly used traditional Chinese medicinal plants in China for more than 2000 years. The three new varieties cultivated could not be distinguished by morphology before flowering. It will hinder the promotion of later varieties. Chloroplast DNA has been widely used in species identification. Moreover, previous studies have shown that complete chloroplast genome sequences have been suggested as super barcodes for identifying plants. Therefore, we sequenced and annotated the complete chloroplast genomes of three cultivated varieties. The chloroplast genomes of SBW, SBR, and SBP were 151,702 bp, 151,799 bp, and 151,876 bp, which contained 85 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. The analysis of the repeat sequences, codon usage, and comparison of chloroplast genomes shared a high degree of conservation. However, the sliding window results show significant differences among the three cultivated varieties in matK-rps16 and petA-psbJ. And we found that the matK-rps16 sequence can be used as a barcode for the identification of three varieties. In addition, the complete chloroplast genome contains more variations and can be used as a super-barcode to identify these three cultivated varieties. Based on the protein-coding genes, the phylogenetic tree demonstrated that SBP was more closely related to SBW, in the three cultivated varieties. Interestingly, we found that S. baicalensis and S. rehderiana are closely related, which provides new ideas for the development of S. baicalensis. The divergence time analysis showed that the three cultivated varieties diverged at about 0.10 Mya. Overall, this study showed that the complete chloroplast genome could be used as a super-barcode to identify three cultivated varieties of S. baicalensis and provide biological information, and it also contributes to bioprospecting.

Comparative Analysis of Chloroplast Genome of Desmodium stryacifolium with Closely Related Legume Genome from the Phaseoloid Clade.

Desmodium styracifolium is a medicinal plant from the Desmodieae tribe, also known as Grona styracifolia. Its role in the treatment of urolithiasis, urinary infections, and cholelithiasis has previously been widely documented. The complete chloroplast genome sequence of D. Styracifolium is 149,155 bp in length with a GC content of 35.2%. It is composed of a large single copy (LSC) of 82,476 bp and a small single copy (SSC) of 18,439 bp, which are separated by a pair of inverted repeats (IR) of 24,120 bp each and has 128 genes. We performed a comparative analysis of the D. styracifolium cpDNA with the genome of previously investigated members of the Sesamoidea tribe and on the outgroup from its Phaseolinae sister tribe. The size of all seven cpDNAs ranged from 148,814 bp to 151,217 bp in length due to the contraction and expansion of the IR/SC boundaries. The gene orientation of the SSC region in D. styracifolium was inverted in comparison with the other six studied species. Furthermore, the sequence divergence of the IR regions was significantly lower than that of the LSC and the SSC, and five highly divergent regions, trnL-UAA-trnT-UGU, psaJ-ycf4, psbE-petL, rpl36-rps8, and rpl32-trnL-UGA, were identified that could be used as valuable molecular markers in future taxonomic studies and phylogenetic constructions.

Differentiation of Lingxiaohua and Yangjinhua by chloroplast genome sequencing and DNA barcoding markers.

Lingxiaohua (Campsis Flos, Campsis grandiflora (Thunb.) K. Schum) is a medicinal herb used for promoting diuresis and treating blood-related disorders by the promotion of blood circulation. It also possesses anti-inflammatory and antioxidative properties. This non-poisonous plant is frequently confused with poisonous Yangjinhua (Daturae Metelis Flos, Datura metel Linnaeus) in the market, resulting in serious anticholinergic poisoning. The confusion of these two herbs is due to the similarity in their appearances. In our study, we compared the complete chloroplast genomes of the two plants and found that they are very different in terms of their gene content and gene arrangement. There were also significant differences in the number and repeating motifs of microsatellites and complex repeats. We used universal primers for the amplification of rbcL, matK, psbA-trnH, and ITS2 regions and successfully differentiated the two plants. Furthermore, we designed two pairs of primers based on the nucleotide differences in chloroplast genomes at the rps14 and rpoC1 regions to provide additional authentication markers. The universal primers and specific primers when used together can accurately discriminate Lingxiaohua and Yangjinhua.

Discrimination of Zicao Samples Based on DNA Barcoding and HPTLC Fingerprints, and Identification of (22E)-Ergosta-4,6,8(14),22-tetraen-3-one As a Marker Compound.

The unambiguous identification of plant material is a prerequisite of rational phytotherapy. Misidentification can even cause serious health problems, as in the case of the Chinese medicinal herb Zicao. Commercial material labelled "Zicao" may be derived from the roots of Arnebia euchroma (ruan zicao), Lithospermum erythrorhizon (ying zicao), or Onosma paniculata (dian zicao). All of these roots contain shikonin derivatives as main bioactive constituents, but ying zicao and dian zicao contain also hepatotoxic pyrrolizidine alkaloids in high amounts. Therefore, the use of A. euchroma with a very low pyrrolizidine alkaloid content is desirable. Confusions of the species occur quite often, indicating an urgent need for an unambiguous identification method. Discrimination of 23 zicao samples has been achieved by analyses of the nuclear internal transcribed spacer ITS2 and trnL-F intergenic spacer of the chloroplast DNA. Data were analyzed using Bioedit, ClustalX, Mega 11 and BLAST. Results indicate that ITS2 barcoding can accurately distinguish Arnebia euchroma from their adulterants. Subsequently, an HPTLC method has been developed allowing a chemical discrimination of the most widely used species. (22E)-Ergosta-4,6,8(14),22-tetraen-3-one has been identified as characteristic marker compound, allowing an unambiguous discrimination of A. euchroma and L. erythrorhizon.

Molecular barcoding of Melissa officinalis L. (badranjboye) in Iran and identification of adulteration in its medicinal services.

Medicinal plants are an important source for treatment of diseases in many countries. Today, controlling the quality of the products of medicinal plants is an important task. Customer health may be in danger due to the fraud and misconduct by the sales associates in the sales centers. Melissa officinalis L. (Lamiaceae) is an important medicinal plant used for treatment of several diseases. In Iran, the species of Dracocephalum, Hymencrater, Nepeta and Stachys are mistakenly sold under the name of Badranjboye that have different pharmaceutical properties. For avoiding this mistake, this investigation was performed with the following aims: 1) Checking for the cheating and identifying the Badranjboye in the Iran's market of medicinal plants, 2) Providing the molecular barcode for the medicinal species of Melissa. For this purpose, Market-sold plant samples (leaves) and original reference plant species were compared by morphology, odor as well as Internal transcribed spacer (ITS) and chloroplast DNA ((psbA-trnH) and (trnL-trnF)) sequences. Various molecular analyses, such as sequencing, determination of genetic distance, and construction of phylogenetic tree were performed. These reports have shown that internal transcribed spacer (ITS) and chloroplast DNA (psbA-trnH) sequences are an efficient molecular marker to produce barcode gap and differentiating Melissa officinalis from other species.

The Chloroplast Genome of Wild Saposhnikovia divaricata: Genomic Features, Comparative Analysis, and Phylogenetic Relationships.

Saposhnikovia divaricata, a well-known Chinese medicinal herb, is the sole species under the genus Saposhnikovia of the Apiaceae subfamily Apioideae Drude. However, information regarding its genetic diversity and evolution is still limited. In this study, the first complete chloroplast genome (cpDNA) of wild S. divaricata was generated using de novo sequencing technology. Similar to the characteristics of Ledebouriella seseloides, the 147,834 bp-long S. divaricata cpDNA contained a large single copy, a small single copy, and two inverted repeat regions. A total of 85 protein-coding, 8 ribosomal RNA, and 36 transfer RNA genes were identified. Compared with five other species, the non-coding regions in the S. divaricata cpDNA exhibited greater variation than the coding regions. Several repeat sequences were also discovered, namely, 33 forward, 14 reverse, 3 complement, and 49 microsatellite repeats. Furthermore, phylogenetic analysis using 47 cpDNA sequences of Apioideae members revealed that L. seseloides and S. divaricata clustered together with a 100% bootstrap value, thereby supporting the validity of renaming L. seseloides to S. divaricata at the genomic level. Notably, S. divaricata was most closely related to Libanotis buchtormensis, which contradicts previous reports. Therefore, these findings provide a valuable foundation for future studies on the genetic diversity and evolution of S. divaricata.

Chloroplast genome sequencing based on genome skimming for identification of Eriobotryae Folium.

BACKGROUND: Whole chloroplast genome (cpDNA) sequence is becoming widely used in the phylogenetic studies of plant and species identification, but in most cases the cpDNA were acquired from silica gel dried fresh leaves. So far few reports have been available to describe cpDNA acquisition from crude drugs derived from plant materials, the DNA of which usually was seriously damaged during their processing. In this study, we retrieved cpDNA from the commonly used crude drug Eriobotryae Folium (Pipaye in Chinese, which is the dried leaves of Eriobotrya japonica, PPY) using genome skimming technique. RESULTS: We successfully recovered cpDNA sequences and rDNA sequences from the crude drug PPY, and bioinformatics analysis showed a high overall consistency between the cpDNA obtained from the crude drugs and fresh samples. In the ML tree, each species formed distinct monophyletic clades based on cpDNA sequence data, while the phylogenetic relationships between Eriobotrya species were poorly resolved based on ITS and ITS2. CONCLUSION: Our results demonstrate that both cpDNA and ITS/ITS2 are effective for identifying PPY and its counterfeits derived from distantly related species (i.e. Dillenia turbinata and Magnolia grandiflora), but cpDNA is more effective for distinguishing the counterfeits derived from the close relatives of Eriobotrya japonica, suggesting the potential of genome skimming for retrieving cpDNA from crude drugs used in Traditional Chinese Medicine for their identification.

Phylogenetic analysis and development of molecular markers for five medicinal Alpinia species based on complete plastome sequences.

BACKGROUND: Alpinia species are widely used as medicinal herbs. To understand the taxonomic classification and plastome evolution of the medicinal Alpinia species and correctly identify medicinal products derived from Alpinia species, we systematically analyzed the plastome sequences from five Alpinia species. Four of the Alpinia species: Alpinia galanga (L.) Willd., Alpinia hainanensis K.Schum., Alpinia officinarum Hance, and Alpinia oxyphylla Miq., are listed in the Chinese pharmacopeia. The other one, Alpinia nigra (Gaertn.) Burtt, is well known for its medicinal values. RESULTS: The four Alpinia species: A. galanga, A. nigra, A. officinarum, and A. oxyphylla, were sequenced using the Next-generation sequencing technology. The plastomes were assembled using Novoplasty and annotated using CPGAVAS2. The sizes of the four plastomes range from 160,590 bp for A. galanga to 164,294 bp for A. nigra, and display a conserved quadripartite structure. Each of the plastomes encodes a total of 111 unique genes, including 79 protein-coding, 28 tRNA, and four rRNA genes. In addition, 293-296 SSRs were detected in the four plastomes, of which the majority are mononucleotides Adenine/Thymine and are found in the noncoding regions. The long repeat analysis shows all types of repeats are contained in the plastomes, of which palindromic repeats occur most frequently. The comparative genomic analyses revealed that the pair of the inverted repeats were less divergent than the single-copy region. Analysis of sequence divergence on protein-coding genes showed that two genes (accD and ycf1) had undergone positive selection. Phylogenetic analysis based on coding sequence of 77 shared plastome genes resolves the molecular phylogeny of 20 species from Zingiberaceae. In particular, molecular phylogeny of four sequenced Alpinia species (A. galanga, A. nigra, A. officinarum, and A. oxyphylla) based on the plastome and nuclear sequences showed congruency. Furthermore, a comparison of the four newly sequenced Alpinia plastomes and one previously reported Alpinia plastomes (accession number: NC_048461) reveals 59 highly divergent intergenic spacer regions. We developed and validated two molecular markers Alpp and Alpr, based on two regions: petN-psbM and psaJ-rpl33, respectively. The discrimination success rate was 100 % in validation experiments. CONCLUSIONS: The results from this study will be invaluable for ensuring the effective and safe uses of Alpinia medicinal products and for the exploration of novel Alpinia species to improve human health.

Plastid Transformation in Sugar Beet: An Important Industrial Crop.

Chloroplast biotechnology has assumed great importance in the past 20 years and, thanks to the numerous advantages as compared to conventional transgenic technologies, has been applied in an increasing number of plant species but still very much limited. Hence, it is of outmost importance to extend the range of species in which plastid transformation can be applied. Sugar beet (Beta vulgaris L.) is an important industrial crop of the temperate zone in which chloroplast DNA is not transmitted trough pollen. Transformation of the sugar beet genome is performed in several research laboratories, conversely sugar beet plastome genetic transformation is far away from being considered a routine technique. We describe here a method to obtain transplastomic sugar beet plants trough biolistic transformation. The availability of sugar beet transplastomic plants should avoid the risk of gene flow between these cultivated genetic modified sugar beet plants and the wild-type plants or relative wild species.

Identification of Valeriana fauriei and other Eurasian medicinal valerian by DNA polymorphisms in psbA-trnH intergenic spacer sequences in chloroplast DNA.

In order to differentiate among Valeriana fauriei Briq. and other Eurasian medicinal valerian (V. dioica L., V. hardwickii Wall., V. jatamansi Jones, and V. officinalis L.), we attempted to establish DNA markers. DNA sequences for the psbA-trnH intergenic spacer region of chloroplast DNA (psbA-trnH) and 18S ribosomal RNA, internal transcribed spacer 1 (ITS1), 5.8S ribosomal RNA, internal transcribed spacer 2 (ITS2), and 28S ribosomal RNA of nuclear DNA in V. fauriei and other Eurasian medicinal valerian were compared. Using partial sequences of psbA-trnH (nucleotide positions 1-75 from the 5' end of the intergenic spacer region), V. fauriei and other Eurasian medicinal valerian could be correctly identified to the species level. In addition, the partial sequences of psbA-trnH in V. fauriei contained five different haplotypes, and it was possible to distinguish the origins of valerian from Japan and Eurasia (China and Korea). On the other hand, individuals had heterogeneous sequences of ITS1 and ITS2, making it impossible to use direct sequencing and DNA markers of ITS1 and ITS2 to distinguish species and origins of V. fauriei and other Eurasian medicinal valerian.

Genetic identification of medicinally used Salacia species by nrDNA ITS sequences and a PCR-RFLP assay for authentication of Salacia-related health foods.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots and stems of several Salacia species have been used as traditional medicines, especially in Ayurvedic medical system for the treatment of diabetes, rheumatism, gonorrhea, amenorrhea, skin diseases, etc. Due to reported evidence supporting Salacia's beneficial effects in early-stage diabetes and other lifestyle-related diseases, Salacia-based dietary supplements and health foods have been gaining popularity in Japan and other countries in recent years. However, due to the morphological similarities between Salacia plants, particularly in the medicinally used parts (roots and stems), the authentication of the botanical identities of Salacia-derived products is challenging. AIM OF THIS STUDY: This study aims to develop a genetic approach to authenticate the medicinally used Salacia species and to determine the botanical sources of the commercially available Salacia-derived products. MATERIALS AND METHODS: The sequences of nuclear DNA internal transcribed spacer (ITS) and chloroplast trnK-rps16 region were determined and compared between 10 plant specimens from three medicinally used Salacia species as well as 48 samples of commercial crude drugs. Moreover, a PCR-restriction fragment length polymorphism (RFLP) assay was developed for rapid identification based on the ITS sequences. RESULTS: The plant specimens from the three medicinally used Salacia species showed three main types of sequences in both ITS (types I, II, III) and trnK-rps16 (i, ii, iii) regions. Combined the sequences of ITS and trnK-rps16 regions, S. reticulata and S. oblonga had type I-i and type III-iii or similar sequences, respectively. S. chinensis had type II-ii or II(536M)-i sequences. Forty-eight samples of commercial crude drugs were identified based on ITS and trnK-rps16 DNA barcode. A convenient PCR-RFLP assay using Cac8I restriction enzyme was established and applied to identify the botanical sources of health food products purchased from online retailers. All the twelve samples were identified as S. chinensis. CONCLUSION: The nrDNA ITS sequences provided useful information to authenticate Salacia species and to elucidate the phylogenetic relationship within the Salacia genus. Genetic identification results revealed that S. chinensis and S. reticulata are the major sources of commercially available Salacia-products. Based on the ITS sequences, a convenient PCR-RFLP assay was established for the identification of the medicinally used Salacia species as well as their derived health food products.

Phylogeographic and demographic modeling analyses of the multiple origins of the rheophytic goldenrod Solidago yokusaiana Makino.

Understanding adaptation mechanisms is important in evolutionary biology. Parallel adaptation provides good opportunities to investigate adaptive evolution. To confirm parallel adaptation, it is effective to examine whether the phenotypic similarity has one or multiple origins and to use demographic modeling to consider the gene flow between ecotypes. Solidago yokusaiana is a rheophyte endemic to the Japanese Archipelago that diverged from Solidago virgaurea. This study examined the parallel origins of S. yokusaiana by distinguishing between multiple and single origins and subsequent gene flow. The haplotypes of noncoding chloroplast DNA and genotypes at 14 nuclear simple sequence repeat (nSSR) loci and single-nucleotide polymorphisms (SNPs) revealed by double-digest restriction-associated DNA sequencing (ddRADseq) were used for phylogeographic analysis; the SNPs were also used to model population demographics. Some chloroplast haplotypes were common to S. yokusaiana and its ancestor S. virgaurea. Also, the population genetic structures revealed by nSSR and SNPs did not correspond to the taxonomic species. The demographic modeling supported the multiple origins of S. yokusaiana in at least four districts and rejected a single origin with ongoing gene flow between the two species, implying that S. yokusaiana independently and repeatedly adapted to frequently flooding riversides.

Complete chloroplast genomes of three important species, Abelmoschus moschatus, A. manihot and A. sagittifolius: Genome structures, mutational hotspots, comparative and phylogenetic analysis in Malvaceae.

Abelmoschus is an economically and phylogenetically valuable genus in the family Malvaceae. Owing to coexistence of wild and cultivated form and interspecific hybridization, this genus is controversial in systematics and taxonomy and requires detailed investigation. Here, we present whole chloroplast genome sequences and annotation of three important species: A. moschatus, A. manihot and A. sagittifolius, and compared with A. esculentus published previously. These chloroplast genome sequences ranged from 163121 bp to 163453 bp in length and contained 132 genes with 87 protein-coding genes, 37 transfer RNA and 8 ribosomal RNA genes. Comparative analyses revealed that amino acid frequency and codon usage had similarity among four species, while the number of repeat sequences in A. esculentus were much lower than other three species. Six categories of simple sequence repeats (SSRs) were detected, but A. moschatus and A. manihot did not contain hexanucleotide SSRs. Single nucleotide polymorphisms (SNPs) of A/T, T/A and C/T were the largest number type, and the ratio of transition to transversion was from 0.37 to 0.55. Abelmoschus species showed relatively independent inverted-repeats (IR) boundary traits with different boundary genes compared with the other related Malvaceae species. The intergenic spacer regions had more polymorphic than protein-coding regions and intronic regions, and thirty mutational hotpots (≥200 bp) were identified in Abelmoschus, such as start-psbA, atpB-rbcL, petD-exon2-rpoA, clpP-intron1 and clpP-exon2.These mutational hotpots could be used as polymorphic markers to resolve taxonomic discrepancies and biogeographical origin in genus Abelmoschus. Moreover, phylogenetic analysis of 33 Malvaceae species indicated that they were well divided into six subfamilies, and genus Abelmoschus was a well-supported clade within genus Hibiscus.

Characteristic and Phylogenetic Analysis of the Complete Chloroplast Genomes of Three Medicinal Plants of Schisandraceae.

Schisandra chinensis, which has a high development value, has long been used as medicine. Its mature fruits (called Wuweizi in Chinese) have long been used in the famous traditional Chinese medicine (TCM) recorded in the "Chinese Pharmacopoeia." Chloroplasts (CP) are the highly conserved primitive organelles in plants, which can serve as the foundation for plant classification and identification. This study introduced the structures of the CP genomes of three Schisandraceae species and analyzed their phylogenetic relationships. Comparative analyses on the three complete chloroplast genomes can provide us with useful knowledge to identify the three plants. In this study, approximately 5 g fresh leaves were harvested for chloroplast DNA isolation according to the improved extraction method. A total of three chloroplast DNAs were extracted. Afterwards, the chloroplast genomes were reconstructed using denovo combined with reference-guided assemblies. General characteristics of the chloroplast genome and genome comparison with three Schisandraceae species was analyzed by corresponding software. The total sizes of complete chloroplast genomes of S. chinensis, S. sphenanthera, and Kadsura coccinea were 146875 bp, 146842 bp, and 145399 bp, respectively. Altogether, 124 genes were annotated, including 82 protein-coding genes, 34 tRNAs, and 8 rRNAs of all 3 species. In SSR analysis, only S. chinensis was annotated to hexanucleotides. Moreover, comparative analysis of chloroplast Schisandraceae genome sequences revealed that the gene order and gene content were slightly different among Schisandraceae species. Finally, phylogenetic trees were reconstructed, based on the genome-wide SNPs of 38 species. The method can be used to identify and differentially analyze Schisandraceae plants and offer useful information for phylogenetics as well as further studies on traditional medicinal plants.

Mitochondrial plastid DNA can cause DNA barcoding paradox in plants.

The transfer of ancestral plastid genomes into mitochondrial genomes to generate mitochondrial plastid DNA (MTPT) is known to occur in plants, but its impacts on mitochondrial genome complexity and the potential for causing a false-positive DNA barcoding paradox have been underestimated. Here, we assembled the organelle genomes of Cynanchum wilfordii and C. auriculatum, which are indigenous medicinal herbs in Korea and China, respectively. In both species, it is estimated that 35% of the ancestral plastid genomes were transferred to mitochondrial genomes over the past 10 million years and remain conserved in these genomes. Some plastid barcoding markers co-amplified the conserved MTPTs and caused a barcoding paradox, resulting in mis-authentication of botanical ingredients and/or taxonomic mis-positioning. We identified dynamic and lineage-specific MTPTs that have contributed to mitochondrial genome complexity and might cause a putative barcoding paradox across 81 plant species. We suggest that a DNA barcoding guidelines should be developed involving the use of multiple markers to help regulate economically motivated adulteration.