SNP double peak methods through chloroplast genome profiling for identifying Gentiana crassicaulis and two closely related species / 药学学报

italic>Gentiana crassicaulis Duthie ex Burk. is one of the plant sources of Gentianae Macrophyllae Radix (QinJiao). Gentiana tibetica King ex Hook. f. and Gentiana robusta King ex Hook. f. are relative species of G. crassicaulis. Due to the large intraspecific morphological variation, G. crassicaulis showed high morphological similarity with G. tibetica and G. robusta. And the distribution area of the three species overlaps to some extent, which makes it difficult to identify them. On the basis of morphological identification, the method of molecular identification of the three species was constructed in this study based on chloroplast genomes. The chloroplast genome of Gentiana tibetica is 148 765bp long, with LSC, SSC and IR 81 163 bp, 17 070 bp and 25 266 bp, respectively. The structure of the three is consistent. The chloroplast genome sequences of G. tibetica and G. crassicaulis are highly similar, and the number of variable sites is 9 (149 267 bp in total). Diagnostic SNP that could effectively identify the three species was screened and verified, and a dual-peak SNP detection method was established for the effective identification of each species and mixed samples. Our study provides basic data for the molecular identification of G. crassicaulis and its related species, and the arrangement of related Tibetan medicine.

Molecular markers based upon whole chloroplast genomes and identifying alpine Gentiana waltonii and G. lhassica (Gentianaceae) / 药学学报

As two original plants of Tibetan herb Jieji, Gentiana waltonii Burk. and Gentiana lhassica Burk. belong to Section Cruciata of Gentiana, Gentianaceae. Here, we report on whole chloroplast genome sequences in the alpine species, respectively, and the features of plastomes were investigated. The plastome of G. waltonii is 148 705 bp long (148 652 bp in G. lhassica) and encodes 112 genes, including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. Two pseudogenes, namely ψrps16 and ψinfA, were found in plastomes. In addition, two novel loci were detected, and a species-specific polymerase chain reaction assay was developed for differentiating G. waltonii and G. lhassica from 10 alpine species in Section Cruciata. Gentiana. Our study provides basic data for identifying Tibetan herbs, alpine species conservation and molecular phylogenetic studies of Gentiana and Gentianaceae.

DNA fingerprinting and genetic diversity in a threatened alpine medicinal plant, Gentiana crassicaulis (Gentianaceae) using AFLP / 药学学报

Jieji Nabao is a common Tibetan herb. According to our ethnobotanical studies, one of its original plants is identified as Gentiana crassicaulis Duthie ex Burk. (Gentianaceae). Endemic to the Qinghai-Tibet Plateau, this medicinal alpine plant is a threatened species. In this study, 163 individuals from 20 populations of G. crassicaulis were collected throughout its geographical range and amplified fragment length polymorphism (AFLP) was used to investigate genetic variation in this species. A cluster analysis was performed on the AFLP data with Halenia elliptica and Gentiana straminea as the outgroups. From 64 pairs of AFLP primer combinations, 12 pairs were selected for amplification and a total of 315 bands were amplified, of which 254 bands were polymorphic, accounting for 80.63%. High genetic differentiation was detected between populations (87%), and low within populations (13%). The UPGMA (unweighted pair-group method with arithmetic means) tree was topologically consistent with the traditional taxonomic treatments at the species level, and the populations of G. crassicaulis were divided into two branches: one from Yunnan and Guizhou, the other from Tibet, Qinghai, Sichuan and Gansu. PCA analysis and the Mantel test showed that there was a positive correlation between genetic distance and geographical distance. In addition, combined with SSR and SNP markers within cpDNA, the genetic differentiation within the Sichuan population S1 was validated.

Analysis of transcriptomes to explore genes contributing to iridoid biosynthesis in Gentiana waltonii and Gentiana robusta (Gentianaceae) / 药学学报

The roots and flowers of Gentiana waltonii and Gentiana robusta are used as Tibetan herb Jie-Ji in traditional Tibetan medicine, with iridoids as the main active ingredient and index components. To study the pathway of iridoid biosynthesis, roots, stems, leaves and flowers of G. waltonii and G. robusta were subjected to a high-throughput transcriptomic sequencing analysis by Illumina HiseqXTen. After removing insignificant reads and de novo splicing, 79 455 and 78 466 unigenes were obtained from G. waltonii and G. robusta respectively, with average length as 834 bp and 862 bp. The unigene GO functions could be divided into three categories of 65 branches. The unigenes were aligned in KOG database and were classified into 25 classes according to function. In KEGG database, 315 and 340 unigenes of G. waltonii and G. robusta were implicated in 20 standard secondary metabolic pathways, respectively. Furthermore, 80 and 57 unigenes of the two species were analyzed to encode 24 key enzymes in the pathway related to iridoid biosynthesis. There were differences in gene expression among different organs. Based on sequence data, significant amounts of SSRs, SNPs and InDels were detected in each dataset. This study provides a platform for further development of molecular markers, excavation of functional genes, and research into metabolic pathways and their regulatory mechanism within G. waltonii and G. robusta.

The identification of Sect. Cruciata (Gentiana) species using mtDNA nad1/b-c and nad5/d-e fragments / 药学学报

italic>Gentiana section Cruciata (Gentianaceae) is a medicinally important section of herbs, including Chinese traditional medicine Gentianae Macrophyllae Radix and Tibetan herb Jieji. Here, we assess the taxonomic significance using mtDNA nad1/b-c and nad5/d-e sequence data. A total of 144 nad1/b-c and nad5/d-e sequences from 11 species within Gentianaceae were obtained, including 138 sequences from 10 species within Gentiana section Cruciata and 6 sequences from Halenia elliptica (outgroup). The results showed that mtDNA nad1/b-c has species- level resolution within the section of Cruciata, i.e. the variable in the position 45 “C” could be used as a stable marker locus to distinguish G. robusta from other taxa; the variable in the position 352 and 353 “GA” could distinguish G. crassicaulis and G. tibetica from other taxa within the section. Intraspecies genotype variability was detected in nad1/b-c sequences of G. officinalis and G. siphonantha, respectively. These genotypes could be used as potential DNA barcode. In addition, intraspecies genotype variability was detected in nad5/d-e sequences of G. macrophylla, G. officinalis and G. siphonantha, respectively. Based on the stable marker locus, a species-specific PCR protocol was developed using the primer PF to identifying G. robusta in the section. This study could expand the understanding of the diversity of mtDNA nad1/b-c and nad5/d-e in the genus Gentiana, and provide the essence for the species identification within Gentiana section Cruciata.

Taxonomic identification of Tibetan herb Bawo Sebo and its chloroplast genome structure / 药学学报

As a common Tibetan herb, Bawo Sebo was mainly used in the treatment of rheumatoid arthritis and urarthritis in Traditional Tibetan medicine. Based on our ethnobotanical survey, the origin of the herb was determined as Swertia verticillifolia T. N. Ho et S. W. Liu (Gentianaceae), endemic to the region of the Qinghai-Tibet Plateau. The diagnostic characters:perennial; stem leaves in whorls; corolla campanulate, yellow-green, 4-lobed; nectary 1 per corolla lobe, naked. Also, its complete chloroplast (cp) genome was sequenced. It is 151 682 bp in length, including a large single copy (LSC) region of 82 623 bp, a small single copy (SSC) region of 18 335 bp and a pair of inverted repeats (IRs) of 25 362 bp. It contains 129 unique genes, including 84 protein-coding genes, 37 tRNAs and 8 rRNAs. This study provides information for understanding the diversity of Swertia cp genomes, and the alpine species identification, conservation and molecular phylogenetic researches of Swertia and Gentianaceae.

Chloroplast genome structures in Gentiana (Gentianaceae), based on three medicinal alpine plants used in Tibetan herbal medicine.

The genus Gentiana is the largest in the Gentianaceae family with ca. 400 species. However, with most species growing on the Qinghai-Tibet plateau, the processes of adaptive evolution and speciation within the genus is not clear. Also, the genomic analyses could provide important information. So far, the complete chloroplast (cp) genome data of the genus are still deficient. As the second and third sequenced members within Gentianaceae, we report the construction of complete cp sequences of Gentiana robusta King ex Hook. f. and Gentiana crassicaulis Duthie ex Burk., and describe a comparative study of three Gentiana cp genomes, including the cp genome of Gentiana straminea Maxim. published previously. These cp genomes are highly conserved in gene size, gene content, and gene order and the rps16 pseudogene with exon2 missing was found common. Three repeat types and five SSR types were investigated, and the number and distribution are similar among the three genomes. Sixteen genome divergent hotspot regions were identified across these cp genomes that could provide potential molecular markers for further phylogenetic studies in Gentiana. The IR/SC boundary organizations in Gentianales cp genomes were compared and three different types of boundaries were observed. Six data partitions of cp genomes in Gentianales were used for phylogenetic analyses and different data partitions were largely congruent with each other. The ML phylogenetic tree was constructed based on the fragments in cp genomes commonly available in 33 species from Lamiids, including 12 species in Gentianales, 1 in Boraginaceae, 10 in Solanales, and 10 in Lamiales. The result strongly supports the position of Boraginaceae (Ehretia acuminata) as the sister of Solanales, with the bootstrap values of 97 %. This study provides a platform for further research into the molecular phylogenetics of species in the order Gentianales (family Gentianaceae) notably in respect of speciation and species identification.

The Complete Chloroplast Genome of Ye-Xing-Ba (Scrophularia dentata; Scrophulariaceae), an Alpine Tibetan Herb.

Scrophularia dentata is an important Tibetan medicinal plant and traditionally used for the treatment of exanthema and fever in Traditional Tibetan Medicine (TTM). However, there is little sequence and genomic information available for S. dentata. In this paper, we report the complete chloroplast genome sequence of S. dentata and it is the first sequenced member of the Sect. Tomiophyllum within Scrophularia (Scrophulariaceae). The gene order and organization of the chloroplast genome of S. dentata are similar to other Lamiales chloroplast genomes. The plastome is 152,553 bp in length and includes a pair of inverted repeats (IRs) of 25,523 bp that separate a large single copy (LSC) region of 84,058 bp and a small single copy (SSC) region of 17,449 bp. It has 38.0% GC content and includes 114 unique genes, of which 80 are protein-coding, 30 are transfer RNA, and 4 are ribosomal RNA. Also, it contains 21 forward repeats, 19 palindrome repeats and 41 simple sequence repeats (SSRs). The repeats and SSRs within S. dentata were compared with those of S. takesimensis and present certain discrepancies. The chloroplast genome of S. dentata was compared with other five publicly available Lamiales genomes from different families. All the coding regions and non-coding regions (introns and intergenic spacers) within the six chloroplast genomes have been extracted and analysed. Furthermore, the genome divergent hotspot regions were identified. Our studies could provide basic data for the alpine medicinal species conservation and molecular phylogenetic researches of Scrophulariaceae and Lamiales.

The complete chloroplast genome of Gentiana straminea (Gentianaceae), an endemic species to the Sino-Himalayan subregion.

Endemic to the Sino-Himalayan subregion, the medicinal alpine plant Gentiana straminea is a threatened species. The genetic and molecular data about it is deficient. Here we report the complete chloroplast (cp) genome sequence of G. straminea, as the first sequenced member of the family Gentianaceae. The cp genome is 148,991bp in length, including a large single copy (LSC) region of 81,240bp, a small single copy (SSC) region of 17,085bp and a pair of inverted repeats (IRs) of 25,333bp. It contains 112 unique genes, including 78 protein-coding genes, 30 tRNAs and 4 rRNAs. The rps16 gene lacks exon2 between trnK-UUU and trnQ-UUG, which is the first rps16 pseudogene found in the nonparasitic plants of Asterids clade. Sequence analysis revealed the presence of 13 forward repeats, 13 palindrome repeats and 39 simple sequence repeats (SSRs). An entire cp genome comparison study of G. straminea and four other species in Gentianales was carried out. Phylogenetic analyses using maximum likelihood (ML) and maximum parsimony (MP) were performed based on 69 protein-coding genes from 36 species of Asterids. The results strongly supported the position of Gentianaceae as one member of the order Gentianales. The complete chloroplast genome sequence will provide intragenic information for its conservation and contribute to research on the genetic and phylogenetic analyses of Gentianales and Asterids.

A strategy for identifying six species of Sect. Cruciata (Gentiana) in Gansu using DNA barcode sequences / 药学学报

Located in the transition zone between the Qinghai-Tibet Plateau and the Loess Plateau, Gansu province is one of the distribution centers of Sect. Cruciata, Gentiana (Gentianaceae) in China. Six species in the section, G. crassicaulis, G. straminea, G. siphonantha, G. officinalis, G. dahurica and G. macrophylla, are native to Gansu. In this paper, samples of 6 species and Halenia elliptica (outgroup) were collected. Nuclear DNA ITS, chloroplast DNA matK, rbcL, rpoC1, trnL (UAA) intron, psbA-trnH, atpB-rbcL, trnS (GCU)-trnG (UCC), rpl20-rps12 and trnL (UAA)-trnF (GAA) were sequenced from these samples. Based on the sequence analyses, high intragenomic polymorphisms were detected in ITS regions of G. crassicaulis, G. straminea, G. siphonantha, G. officinalis and G. dahurica, and they showed incomplete concerted evolution. A methodological study to identifying such close-related species as G. macrophylla, G. officinalis and G. dahurica was carried out based on the special genotypes. The results showed that 7 cp DNA sequence fragments could be used to identify G. crassicaulis, G. straminea and G. siphonantha. With nr ITS genotype II, III and IV of G. dahurica, the species can be distinguished from the close-related G. officinalis using 12 cloned sequences in a sample (with statistical significance). The cp DNA sequences of G. macrophylla were classified into two genotypes, and with genotype II, the species can be distinguished from the close-related G. officinalis and G. dahurica using 6 test samples each (with statistical significance). Furthermore, DNA barcode sequences were determined for all 6 species in Gansu. Also, the studies provide some basic data for analyses of genetic diversity and identification of Gentiana species.

19(4→3)-abeo-abietane diterpenoids from Scrophularia dentata Royle ex Benth.

Five 19(4→3)-abeo-abietane diterpenoids, scrodentoids A-E (1-5), were isolated from the whole plant of Scrophularia dentata. Planar structures of scrodentoids A-E were elucidated mainly by using 1D, 2D NMR and MS data. The absolute configurations of compounds 1 and 2 were established using X-ray crystallographic analysis. The absolute configurations of other compounds were confirmed using HPLC-UV/CD detection. The immunosuppressive effects of compounds 1-5 were studied using a ConA-induced splenocyte proliferation model. These compounds significantly inhibited ConA-induced splenocyte proliferation, with IC50 values in the range of 3.49-133.86 µM. Compounds 1-5 (IC50>10 µM) showed no discernible cytotoxic activity against B16 or MCF-7 cells.

DNA-based identification of Gentiana robusta and related species / 中国中药杂志

The alpine plant Gentiana robusta is an endemic species to the Sino-Himalayan subregion. Also, it is one of the original plants used as traditional Tibetan medicine Jie-Ji. We sequence the nuclear ribosomal internal transcribed spacer (ITS) regions, matK, rbcL, rpoC1, trnL (UAA), psbA-trnH, atpB-rbcL, trnS( GCU)-trnG(UCC), rpl20-rps12, trnL(UAA)-trnF( GAA) fragments of cp DNA in both G. robusta and such relative species as G. straminea, G. crassicaulis and G. waltonii. With Halenia elliptica as the outgroup, molecular systematic analysis reveals that G. robusta is a natural hybrid. G. straminea is the mother of hybrids, but the father is not very clear. In addition, the molecular markers for distinguishing G. robusta from the parental species or closely related species are identified, respectively. Our studies may provide valuable reference for the species identifications of medicinal plants with complex genetic backgrounds.

Studies on genetic diversity of three Tibetan herbs / 中国中药杂志

The genetic diversity of three Tibetan herbs, i. e., Sang-Di, E-Dewa and Ye-Xingba (Tibetan names), was studied based on the field collection, specimen identification and DNA sequence analysis. Swertia hispidicalyx, Gentiana lhassica and Scrophularia dentata, as the original plants of the three Tibetan herbs, were collected and identified. The regions of ITS, matK, rbcL, rpoC1, trnL(UAA), psbA-trnH, atpB-rbcL, trnS (GCU)-trnG(UCC), rpl20-rps12, trnL(UAA)-trnF(GAA) and nadl 2nd intron were amplified and sequenced. The ITS regions of S. hispidicalyx and S. dentata were cloned and sequenced, and the sequences were classified into different genotypes. All the sequences were analyzed and compared with those of closely related species. Our studies may provide reference for the genetic diversity analysis and molecular identification of the three Tibetan herbs.

Iridoid glycosides isolated from Scrophularia dentata Royle ex Benth. and their anti-inflammatory activity.

Scrodentosides A-E (1-5), five new acylated iridoid glycosides, together with 19 known ones, were isolated from the whole plant of Scrophularia dentata Royle ex Benth. The structures of these isolated glycosides were elucidated by spectroscopic methods. Bioassay showed that compounds 7 and 11 had significant inhibitory effect against NF-κB activation with IC50 value of 43.7 µM and 1.02 µM respectively.

Molecular Pharmacognosy on Tibetan Traditional Medicine Dong-Na Duan-Chi / 中国中医药信息杂志

Objective To identify the common Tibetan herb Dong-Na Duan-Chi by molecular pharmacognosy. Methods Ethnopharmacology investigation was carried out and local herbalists were visited to observe which plants were being used as Dong-Na Duan-Chi. Then, nuclear ribosomal DNA ITS sequences were amplified and sequenced for molecular identification. Results The botanic origins of Dong-Na Duan-Chi in Tibet Autonomous Region were authenticated as two species, i.e. Veronica eriogyne H.Winkl. and Veronica ciliata Fisch.. The sequences of ITS for Veronica eriogyne and Veronica ciliata are 551 bp and 552 bp in size. The results showed that nrDNA ITS sequences could be used to discriminate V.eriogyne and V.ciliata from closely related species. Conclusion nrDNA ITS sequences can be used as the molecular markers to identify the Tibetan herb Dong-Na Duan-Chi from other species of the same genus Veronica L.

Identification of medicinal plants used as Tibetan traditional medicine Chuan-Bu based on nrDNA markers / 中国中药杂志

<p><b>OBJECTIVE</b>To identify the common Tibetan herb Chuan-Bu.</p><p><b>METHOD</b>Local herbalists were visited to observe which plants were being used as Chuan-Bu. Samples of the indigenous plants were collected at the same time. Leaf materials were collected from field surveys. Total genomic DNA was extracted from silica gel-dried leaf samples. The PCR products were purified and directly sequenced.</p><p><b>RESULT</b>As the origin of Chuan-Bu in Tibet autonomous region was authenticated, two species were determined, i. e. Euphorbia stracheyiand E. wallichii. Also, based on our earlier research, the origin of Chuan-Bu in Gansu province, is from E. kansuensis. The sequences of ITS1 for E. stracheyi and E. wallichii were 261 bp in size, and 221 bp in ITS2, respectively. The size of the 5.8S coding region was 164 bp for all species examined in the genus. Especially, there was a heterozygous locus in ITS1 (C/G; position 72) for E. stracheyi. The nucleotide divergence between sequences of the 6 species in pairwise comparisons was calculated and the result showed that the variable site could be detected in each pairwise comparison of sequences. Also, there were 8 point mutations in the 5.8S coding region.</p><p><b>CONCLUSION</b>nrDNA ITS sequences can be used as the molecular markers to identify the Tibetan herb Chuan-Bu and such Traditional Chinese Medicines from the same genus Euphorbia as E. lathyris, E. humifusa and E. pekinensis.</p>

Identification of medicinal plants used as Tibetan Traditional Medicine jie-ji.

AIM OF THE STUDY: Based on the authors' collection of specimens used as jie-ji in local Tibetan areas, China, and taxonomic determination, this paper aims to give a list of medicinal plants as jie-ji, formally identify the ones recognized as jie-ji ga-bao or jie-ji na-bao and to offer basic data for further studies on these Tibetan herbs. MATERIALS AND METHODS: Local herbalists were visited in Tibetan areas, China to observe which plants were being used as jie-ji. Samples of the indigenous plants were collected at the same time. Also, the medicinal plants as jie-ji were taxonomically identified. RESULTS: A list of medicinal plants including 10 species of jie-ji in local Tibetan areas is given, including their morphological pictures used for identification. CONCLUSIONS: The origin of jie-ji is from 10 species of the Section Cruciata, Genus Gentiana (Gentianaceae). five species with dark blue flowers are used as jie-ji na-bao, the other five with white flowers are used as jie-ji ga-bao. Also, Gentiana macrophylla Pall. with dark blue flowers in the Section Cruciata, Genus Gentiana is not the original plant of jie-ji na-bao. The species endemic to the province are used as the original plants of jie-ji only in local Tibetan area of the province. Finally, the drug use of jie-ji in Traditional Tibetan Medicine is reasonable and it is efficacious.