Characterization and Comparative Analysis of Chloroplast Genomes of Medicinal Herb Scrophularia ningpoensis and Its Common Adulterants (Scrophulariaceae).

Scrophularia ningpoensis, a perennial medicinal plant from the Scrophulariaceae family, is the original species of Scrophulariae Radix (SR) in the Chinese Pharmacopoeia. This medicine is usually deliberately substituted or accidentally contaminated with other closely related species including S. kakudensis, S. buergeriana, and S. yoshimurae. Given the ambiguous identification of germplasm and complex evolutionary relationships within the genus, the complete chloroplast genomes of the four mentioned Scrophularia species were sequenced and characterized. Comparative genomic studies revealed a high degree of conservation in genomic structure, gene arrangement, and content within the species, with the entire chloroplast genome spanning 153,016-153,631 bp in full length, encoding 132 genes, including 80 protein-coding genes, 4 rRNA genes, 30 tRNA genes, and 18 duplicated genes. We identified 8 highly variable plastid regions and 39-44 SSRs as potential molecular markers for further species identification in the genus. The consistent and robust phylogenetic relationships of S. ningpoensis and its common adulterants were firstly established using a total of 28 plastid genomes from the Scrophulariaceae family. In the monophyletic group, S. kakudensis was determined to be the earliest diverging species, succeeded by S. ningpoensis. Meanwhile, S. yoshimurae and S. buergeriana were clustered together as sister clades. Our research manifestly illustrates the efficacy of plastid genomes in identifying S. ningpoensis and its counterfeits and will also contribute to a deeper understanding of the evolutionary processes within Scrophularia.

Comparative and Phylogenetic Analyses of Complete Chloroplast Genomes of Scrophularia incisa Complex (Scrophulariaceae).

The Scrophularia incisa complex is a group of closely related desert and steppe subshrubs that includes S. incisa, S. kiriloviana and S. dentata, which are the only S. sect. Caninae components found in Northwest China. Based on earlier molecular evidence, the species boundaries and phylogenetic relationships within this complex remain poorly resolved. Here, we characterized seven complete chloroplast genomes encompassing the representatives of the three taxa in the complex and one closely related species, S. integrifolia, as well as three other species of Scrophularia. Comparative genomic analyses indicated that the genomic structure, gene order and content were highly conserved among these eleven plastomes. Highly variable plastid regions and simple sequence repeats (SSRs) were identified. The robust and consistent phylogenetic relationships of the S. incisa complex were firstly constructed based on a total of 26 plastid genomes from Scrophulariaceae. Within the monophyletic complex, a S. kiriloviana individual from Pamirs Plateau was identified as the earliest diverging clade, followed by S. dentata from Tibet, while the remaining individuals of S. kiriloviana from the Tianshan Mountains and S. incisa from Qinghai-Gansu were clustered into sister clades. Our results evidently demonstrate the capability of plastid genomes to improve phylogenetic resolution and species delimitation, particularly among closely related species, and will promote the understanding of plastome evolution in Scrophularia.

[Transcriptome characterization for Scrophularia ningpoensis based on high-throughput sequencing technology and related genes for synthesis of terpenoid compounds].

To investigate the profile of gene function and search for SSR, a new technology of high-throughput Solexa/Illumina sequencing was used to generate the root transcriptome of Scrophularia ningpoensis, and 65 602 036 raw reads were obtained. Based on the bioinformatics analysis and Trinity, 73 983 unigenes were obtained with an average length of 823 bp. The comparison of sequence homology in database showed that 56 389 unigenes had different degrees of homology. A total of 520 metabolic pathways related genes and 191 relDODO transcription factors were identified by the Swiss-Prot, GO, KEGG and COG.The 11 659 SSRs were found by MISA and the highest frequency was AG/CT. In this study, we obtained numerous SSRs to provide references for the study of functional gene cloning and genetic diversity of S. ningpoensis. The key genes involved in the secondary metabolism are the basis for the study of biosynthesis and regulatory mechanism of the secondary metabolites.

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.

Induction of aromatic amino acids and phenylpropanoid compounds in Scrophularia striata Boiss. cell culture in response to chitosan-induced oxidative stress.

Manipulation of cell culture media by elicitors is one of most important strategies to inducing secondary metabolism for the production of valuable metabolites. In this investigation, inducing effect of chitosan on physiological, biochemical, and molecular parameters were investigated in cell suspension cultures of Scrophularia striata Boiss. The results showed that chitosan concentration and time of elicitation are determinants of the effectiveness of the elicitor. Accumulation of aromatic amino acids (phenylalanine [Phe] and tyrosine [Tyr]), phenylpropanoid compounds (phenolic acids [PAs] and echinacoside [ECH]), hydrogen peroxide (H2O2) production, phenylalanine ammonia-lyase (PAL) activity and gene expression, and antioxidant enzymes (superoxide dismutase [SOD], peroxidase [POX], catalase [CAT]) activities were altered by changing the exposure time of elicitation. Results showed that, upon elicitation with chitosan, oxidative events were induced, antioxidant responses of S. striata cells were boosted through enhanced activity of an effective series of scavenging enzymes (SOD, CAT, and POX), and biosynthesis of non-enzymatic antioxidants (ECH and PAs [cinnamic, p-coumaric and, caffeic acids]). The increase in amino acid content and PAL activity at early days of exposure to chitosan was related with rises in phenolic compounds. These results provide evidence that chitosan by up-regulation of PAL gene differentially improves the production of phenylpropanoid compounds, which are of medical commercial value with good biotechnological prospects.

The complete chloroplast genome sequence of the Korean endemic plant Scrophularia takesimensis.

The chloroplast genome sequence of Scrophularia takesimensis (Scrophulariaceae) was determined in this study. The cpDNA was 152,425 bp in length, containing a pair of 25,478 bp inverted repeat regions (IR), which were separated by small and large single copy region (SSC and LSC) of 17,938 bp and 83,531 bp, respectively. There are total of 113 genes in the genome, including 79 protein coding genes, 29 tRNA genes, 4 rRNA genes. 18 genes encoded one or two introns. The overall AT contents of the cpDNA were 61.9% and in the LSC, SSC and IR regions were 63.8%, 67.8% and 56.8%, respectively.

[Studies on genetic diversity of three Tibetan herbs].

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.

The population genetics of cultivation: domestication of a traditional Chinese medicine, Scrophularia ningpoensis Hemsl. (Scrophulariaceae).

BACKGROUND: Domestic cultivation of medicinal plants is an important strategy for protecting these species from over harvesting. Some species of medicinal plants have been brought into cultivation for more than hundreds years. Concerns about severe loss of genetic diversity and sustainable cultivation can potentially limit future use of these valuable plants. Genetic studies with comprehensive sampling of multiple medicinal species by molecular markers will allow for assessment and management of these species. Here we examine the population genetic consequences of cultivation and domestication in Scrophularia ningpoensis Hemsl. We used chloroplast DNA and genomic AFLP markers to clarify not only the effects of domestication on genetic diversity, but also determine the geographic origins of cultivars and their genetic divergence from native populations. These results will allow both better management of cultivated populations, but also provide insights for crop improvement. RESULTS: Twenty-one cpDNA haplotypes of S. ningpoensis were identified. Wild populations contain all haplotypes, whereas only three haplotypes were found in cultivated populations with wild populations having twice the haplotype diversity of cultivated populations. Genetic differentiation between cultivated populations and wild populations was significant. Genomic AFLP markers revealed similar genetic diversity patterns. Furthermore, Structure analysis grouped all wild populations into two gene pools; two of which shared the same gene pool with cultivated S. ningpoensis. The result of Neighbor-Joining analysis was consistent with the structure analysis. In principal coordinate analysis, three cultivated populations from Zhejiang Province grouped together and were separated from other cultivated populations. CONCLUSIONS: These results suggest that cultivated S. ningpoensis has experienced dramatic loss of genetic diversity under anthropogenic influence. We postulate that strong artificial selection for medicinal quality has resulted in genetic differentiation between cultivated and wild populations. Furthermore, it appears that wild populations in Jiangxi-Hunan area were involved in the origin of cultivated S. ningpoensis.

Diversification of Scrophularia (Scrophulariaceae) in the Western Mediterranean and Macaronesia--phylogenetic relationships, reticulate evolution and biogeographic patterns.

The flora of the Mediterranean region and Macaronesia is characterized by high levels of species diversity and endemism. We examined phylogenetic relationships of Scrophularia within one of its secondary centers of diversity located in the Iberian Peninsula and adjacent Macaronesia. In total, 65 ingroup accessions from 45 species, representing an almost complete sampling of the region, were analyzed using sequences from the internal transcribed spacer region (ITS) and the plastid trnQ-rps16 intergenic spacer. Phylogenetic relationships were inferred using Bayesian inference, maximum likelihood and statistical parsimony networking. Incongruence between datasets was assessed with statistical tests and displayed by split networks. Biogeographic inferences incorporating information from both markers (despite low resolution in some parts of the trees) and all incongruent taxa were accomplished with a novel combination of methods, using trees generated with the taxon duplication approach as input for Bayesian binary MCMC (BBM) analysis as implemented in RASP. Nuclear and chloroplast markers support a clade which comprises the majority of Iberian and Macaronesian species and consists of three subclades. Analyses of the substantial incongruence observed among markers indicate reticulate evolution and suggest that Scrophularia species diversity in this region is largely attributable to hybridization; a combination of both polyploidy and dysploidy in the karyotypic evolution of Western Mediterranean Scrophularia taxa is proposed. Our results provide support for an ancient hybridization event between two widespread lineages, which resulted in an allopolyploid ancestor of the Iberian - Macaronesian group with 2n=58 chromosomes. The ancestor then diverged into the three main lineages present in the Iberian Peninsula, Northern Africa and Macaronesia today. Subsequent interspecific hybridizations at different ploidy levels additionally generated new species. Presumably, hybridization and diversification within the genus in the Western Mediterranean have not been restricted to one particular event, but occurred repeatedly. It can be assumed that the topographical complexity found in the Iberian Peninsula has promoted diversification and hybrid speciation processes in Scrophularia, and that isolation in glacial refugia has preserved recent and ancient lineages. For the Macaronesian taxa, biogeographic analyses support several origins, by colonizations from at least four distinct lineages.

The role of birds and insects in pollination shifts of Scrophularia (Scrophulariaceae).

The mixed vertebrate-insect pollination system is rare in Holarctic plants. Phylogenetic relationships of 116 Scrophularia taxa were investigated based on two plastid (ndhF and trnL-trnF) and one nuclear (ITS) DNA regions. A wider time-calibrated analysis of ndhF sequences of the Lamiales revealed that Scrophularia diverged as early as in the Miocene (<22 Ma). Results of maximum-likelihood optimizations supported wasp pollination as the ancestral pollination system from which other systems derived (hoverfly, mixed vertebrate-insect and bird systems). Four origins for a mixed vertebrate-insect (MVI) pollination system were inferred, in which two western Mediterranean species (S. sambucifolia and S. grandiflora) and two island species (the Tirrenian S. trifoliata and the Canarian S. calliantha) were involved. S. calliantha is the only species in which a more complex MVI system, including pollination by the lizard Gallotia stehlini, has evolved. In addition, bird (hummingbird) floral traits found in the New Mexican S. macrantha appear to have been independently acquired. In contrast, we failed to find evidence for an ancient role of hummingbirds in the evolution of European Scrophularia. Indeed, paleontological data revealed that extinction of European hummingbirds (30-32 Ma) occurred earlier than the divergence of European MVI lineages of Scrophularia. In conclusion, our results showed that a role of birds in pollination of Scrophularia may not have been effective in the Miocene-Pliocene, but bird pollination that shows its origin in the Pliocene-Pleistocene is still operating independently in different islands and continents.

[Genetic diversity of Scrophularia ningpoensis based on SCoT analysis].

OBJECTIVE: To explore the genetic diversity and relationship of different Scrophularia ningpoensis cultivars. METHOD: Forty-eight germplasmic resources of S. ningpoensis cultivars were analyzed by Start Codon Targeted Polymorphism(SCOT) molecular markers. Genetic distance was calculated by TREECONW software and the systematic diagram of genetic relationship was clustered by UPGMA method. RESULT: A total of 279 bands were detected using 48 primers, among which 214 were polymorphic bands. The average percentage of polymorphic bands was 76.7%. Genetic distance was changed from 0.1507 to 0.4933. Clustering results showed that the genetic relationship of S. ningpoensis cultivars was more complex. There was significant correlation between some germplasm and its geographic origin while geographical distribution of some germplasm was not very obvious, but it was also showed that some of the S. ningpoensis from the same region were in the same group which presented the law of geographical distribution in the tested materials. CONCLUSION: Significant polymorphism and genetic diversity can be observed among S. ningpoensis germplasm resources which provided a wealth of genetic basis for cultivating fine varieties.

Molecular authentication of geo-authentic Scrophularia ningpoensis.

Scrophularia ningpoensis has long been used in the Chinese Materia Medica for inflammation. Like other herbal medicines, S. ningpoensis collected from different localities may considerably differ in their therapeutic efficacy, and the one grown in Zhejiang Province is recognized as geo-authentic. However, it is difficult to confirm the geographical authenticity by similar morphological characteristics. In the present study, inter-simple sequence repeat (ISSR) markers were conducted to detect S. ningpoensis from different origins. A 1 259-bp fragment amplified by primer UBC874 was found only in geo-authentic ones. By cloning and sequencing that specific band, sequence characterized amplified region (SCAR) markers were designed to distinguish geo-authentic S. ningpoensis from others. This is a rapid and easy method that can be used to identify the geographical authenticity of S. ningpoensis.

Association between chemical and genetic variation of wild and cultivated populations of Scrophularia ningpoensis Hemsl.

Scrophularia ningpoensis Hemsl. is an important Chinese medicinal herb with a domestication history of more than one thousand years. Although a number of studies have focused on either chemical or genetic variation, none have dealt with their association to discuss the formation of chemical diversity. We applied HPLC fingerprinting with identification of four predominant bioactive compounds using LC-ESI-MS to assess chemical variation among 6 cultivated and 5 wild populations of S. ningpoensis. Significant chemical differences were revealed between wild and cultivated populations in terms of chromatographic profiles, principal component analysis (PCA) plots, and bioactive compounds contents. Compared to cultivated populations, the chemical profiles varied considerably among wild populations, of which some were remarkably similar to cultivated populations. Inter simple sequence repeats (ISSR) fingerprinting indicated a genetic differentiation pattern parallel to chemical variation. Evidence strongly supported the association between chemical and genetic variation of S. ningpoensis. Based on both sets of data, suggestions are proposed for the conservation of genetic diversity, crop improvement, and good agricultural practice. The present results will also facilitate our theoretical understanding of the selective and adaptive evolutionary processes of medicinal plant species impacted by domestication and a changing environment.

[Analysis of genetic difference among Scrophularia ningpoensis cultivars by SRAP].

OBJECTIVE: The genetic difference among Scrophularia ningpoensis cultivars were analyzed in molecular level. METHOD: Ninety-two individuls of three S. ningpoensis cultivars were employed to be analyzed by the approach of Sequence-related Amplified Polymorphism (SRAP).The parameters were calculated by POPGENE1.31 and the relationship was constructed based on UPGMA method. RESULT: 1) A total of 227 bands were scored and 199 bands of them were polymorphic. 2) The result is showed that there is a medium level of genetic diversity among three cultivars. At species level: percentage of polymorphic loci PPB=52.42%, effective number of alleles N(e)=1.2812, Nei's gene diversity H=0.1671 and Shannon's information index H(sp)= 0.2526; At cultivar level: PPB=21.44%, N(e)=1.1216, Nei's gene diversity H=0.0725 and Shannon's information index H(pop)= 0.1083. 3) The Nei's coefficient of genetic differentiation was 0.5625, which was consistent with the Shannon's coefficient of genetic differentiation (0.5713). Most of the genetic variation existed among cultivars. 4) The gene flow (N(m)=0.3889) was less among cultivars, indicating that the degree of genetic differentiation was higher. 5) Genetic similarity coefficient were changed from 0.8082 to 0.9133. By clustering analysis, the classified result of SRAP marker between traditional modal character was almost same. CONCLUSION: The genetic diversity of samples of S. ningpoensis is medium. The genetic difference among cultivar is higher than that within cultivar.

Intraspecific morphological and genetic differentiation in Scrophularia grayana (Scrophulariaceae).

Scrophularia grayana, which is distributed throughout northern Japan and Sakhalin, and its locally endemic variety var. grayanoides, have been examined morphometrically and genetically. Principal-component analysis using a total of 26 morphological characteristics revealed that these taxa are morphologically differentiated, but that the difference is not distinct. These two taxa have the same number of chromosomes in the somatic cells, 2n = 94, suggesting that ploidal level difference is not relevant to their divergence. The distributions of the taxa are adjoining in the north of Japanese mainland Honshu. Nevertheless, principal-coordinate analysis using putative 112 ISSR loci indicated they are genetically very distinct. Many taxon-specific alleles were found, and many of the alleles were fixed in each taxon. This genetic information suggests that a relatively long time has passed since the taxa became differentiated and that gene flow has rarely occurred between them, although morphological similarity has been maintained, probably because of natural selective forces.

Genetic transformation of the figwort, Scrophularia buergeriana Miq., an Oriental medicinal plant.

Scrophularia buergeriana Miq. (figwort) contains a diverse group of bioactive natural products and is used to treat a variety of ailments, including fever, constipation, neuritis, and laryngitis. A transformation protocol was established for S. buergeriana using Agrobacterium tumefaciens. Kanamycin-resistant plants were regenerated from leaf explants co-cultivated with A. tumefaciens strain GV3101. The shoot regeneration medium was supplemented with 2 mg l(-1) 6-benzylaminopurine and 70 mg l(-1) putrescine to improve the efficiency of organogenesis. Detection of the neomycin phosphotransferase gene, the presence of high levels of beta-glucuronidase (GUS) transcripts and enzyme activity, and the histochemical localization of GUS confirmed the genetic transformation of S. buergeriana. This work demonstrates the potential of using A. tumefaciens to efficiently transfer foreign genes into a commercially and culturally important Oriental medicinal plant.