Development of SSR and SNP markers for identifying opium poppy.

Papaver somniferum L. (opium poppy) is the original plant of heroin, which is a major narcotic drug, and this plant has brought great harm to human health. However, the ban on opium poppy cultivation and trafficking is facing great challenges because of abnormal profits. Therefore, rapid and accurate identification is important to address the abovementioned problems. In this study, eleven simple sequence repeats (SSR) markers and two single nucleotide polymorphism (SNP) markers were mined to distinguish opium poppy from other six Papaver species. These molecular markers were further verified through a large number of plant materials of these seven Papaver species. An excellent multiplex polymerase chain reaction (PCR) system that simultaneously amplifies the three of eleven SSR markers was developed, which effectively improves the efficiency and speed of identification. The present research is of great implication for identifying and investigating the illegal cultivation and trafficking of opium poppy.

Molecular Network-Guided Alkaloid Profiling of Aerial Parts of Papaver nudicaule L. Using LC-HRMS.

Papaver nudicaule L. (Iceland poppy) is widely used for ornamental purposes. A previous study demonstrated the alleviation of lipopolysaccharide-induced inflammation mediated by P. nudicaule extract through nuclear factor-kappa B and signal transducer and activator of transcription 3 inactivation. As isoquinoline alkaloids are chemical markers and bioactive constituents of Papaver species, the present study investigated the alkaloid profile of aerial parts of five P. nudicaule cultivars with different flower colors and a P. rhoeas cropped for two years. A combination of liquid chromatography high-resolution mass spectrometry and molecular networking was used to cluster isoquinoline alkaloids in the species and highlight the possible metabolites. Aside from the 12 compounds, including rotundine, muramine, and allocryptopine, identified from Global Natural Products Social library and reported information, 46 structurally related metabolites were quantitatively investigated. Forty-two and 16 compounds were proposed for chemical profiles of P. nudicaule and P. rhoeas, respectively. Some species-specific metabolites showed similar fragmentation patterns. The alkaloid abundance of P. nudicaule differed depending on the flower color, and the possible chemical markers were proposed. These results show that molecular networking-guided dereplication allows investigation of unidentified metabolites. The derived chemical profile may facilitate evaluation of P. nudicaule quality for pharmacological applications.

[Screening potential DNA barcode regions of genus Papaver].

DNA barcoding is an effective technique in species identification. To determine the candidate sequences which can be used as DNA barcode to identify in Papaver genus, five potential sequences (ITS, matK, psbA-trnH, rbcL, trnL-trnF) were screened. 69 sequences were downloaded from Genbank, including 21 ITS sequences, 10 matK sequences, 8 psbA-trnH sequences, 14 rbcL sequences and 16 trnL-trnF sequences. Mega 6.0 was used to analysis the comparison of sequences. By the methods of calculating the distances in intraspecific and interspecific divergences, evaluating DNA barcoding gap and constructing NJ and UPMGA phylogenetic trees. The sequence trnL-trnF performed best. In conclusion, trnL-trnF can be considered as a novel DNA barcode in Papaver genus, other four sequences can be as combination barcode for identification.

Rapid identification of a narcotic plant Papaver bracteatum using flow cytometry.

In May 2011, numerous poppy plants closely resembling Papaver bracteatum Lindl., a type of narcotic plant that is illegal in Japan, were distributed directly from several large flower shops or through online shopping throughout Japan, including the Tokyo Metropolitan area. In order to better identify the narcotic plants, the relative nuclear DNA content at the vegetative stage was measured by flow cytometric (FCM) analysis in 3 closely-related species of the genus Papaver section Oxytona, namely P. orientale, P. pseudo-orientale, and P. bracteatum, based on the difference between the chromosome numbers of these species. The results showed that the nuclear DNA content differed between these 3 species, and that most of the commercially distributed plants examined in this study could be identified as P. bracteatum. The remaining plants were P. pseudo-orientale, a non-narcotic plant. In addition, the FCM results for the identification of P. bracteatum completely agreed with the results obtained by the morphological analysis, the inter-genic spacer sequence of rpl16-rpl14 (PS-ID sequence) of chloroplast DNA, and the presence of thebaine. These results clearly indicate the usefulness of FCM analysis for the identification of P. bracteatum plants, including when they are in their vegetative stage.

Himalayan-Tibetan plateau uplift drives divergence of polyploid poppies: Meconopsis Viguier (Papaveraceae).

Meconopsis Viguier (Papaveraceae) is an iconic genus of alpine forbs that includes medicinal and ornamental species. This study extends previous phylogenetic analyses of Meconopsis, using ITS sequences representing all the major Meconopsis clades. Phenotypic traits are also analysed for all described species. Our results show that Meconopsis evolved as a ≥ octaploid clade, with considerable interior structure reflecting further changes in ploidy levels as well as phenotypic differentiation. We support the exclusion of a few species as Cathcartia or Papaver, making Meconopsis a Tibetan region clade. Based on average rates of nucleotide substitution in angiosperm herbs we estimate that the Meconopsis clade diverged from the Meconella clade of Papaver approximately 16.6 Ma. This is soon after the 'hard' collision of the Indian continent with Asia caused uplift of the Himalaya and Hengduan ranges, greatly extended the Tibetan plateau, and initiated monsoonal climates. Eight major clades within Meconopsis are well supported and these correspond closely to previously recognised subgenus groups. However, the relationship among the clades is poorly resolved, probably because they diverged rapidly ∼15-11 Ma. Two of these clades are ∼dodecaploid but appear to have originated independently. The eight clades have distinct distributions, variously associated with the Himalaya, the eastern Plateau and Hengduan ranges. Some Meconopsis species were not monophyletic, suggesting that a combination of multilocus molecular and phenotypic traits is required when defining and revising species.

A comparison of ice cold water pretreatment and α-bromonaphthalene cytogenetic method for identification of Papaver species.

The plants belonging to many species in genus Papaver are very similar and have very small chromosomes that make identification very difficult. The study aimed to compare the effects of α-bromonaphtalene and ice cold water pretreatment to identify chromosomes of Papaver species collected from different areas of Iranian West Azerbaijan and Turkish Van, Agri and, Hakkari provinces. The seeds were germinated in Jacobson trays at 24°C under continuous light. Thereafter, roots from 1.5 cm long plantlets were pretreated with α bromonaphtalene for 15, 30, and 45 min or at 0°C in ice cold water for 24 h before fixing, hydrolyzation, and feulgen staining. The ice cold water pretreatment was more appropriate and easy to determine chromosomes. Seeds from seven samples did not germinate. Sixty samples out of the rest of 62 samples were identified as P. pseudo orientale, one sample was identified as P. bracteatum, and another as P. orientale. This is the first study that used ice cold water to determine the chromosomes in papaver species. It is hoped that it will also facilitate to determine chromosome number and identify other papver species.

Analysis of fluid extracts obtained from Papaver rhoeas petals contaminated with Papaver bracteatum petals.

In this paper, we report a case of misidentification of medicinal plants involving dried petals of Papaver rhoeas (red poppy) contaminated with Papaver bracteatum (scarlet poppy) petals. Preliminary TLC analysis indicated the presence of thebaine either in the fluid extracts or in the petals. It was therefore necessary to carry out an accurate botanic examination of the plant material, which revealed contamination of the red poppy petals with scarlet poppy petals. Moreover, to confirm the adulteration, we developed and validated an efficient, reversed-phase ion pair HPLC method for determination of the alkaloids specific for the Papaver species. Six petal batches and five commercial fluid extracts were analyzed. Only one petal batch from Iran contained thebaine and its analogue oripavine while the alkaloids typical for the Papaver bracteatum species were identified in all fluid extracts, meaning that they were all prepared with contaminated petals.

Evolution of gynoecium morphology in Old World Papaveroideae: a combined phylogenetic/ontogenetic approach.

PREMISE OF THE STUDY: The correct assessment of homology is an important prerequisite for reconstructing phylogenetic relationships and character evolution. Old World Papaveroideae (Papaver, Meconopsis, Roemeria, Stylomecon) show substantial diversity in gynoecium and capsule morphology. In particular, capsules can have distinct styles (Meconopsis p.p., Stylomecon) or a sessile stigmatic disc (Papaver). Molecular phylogenetic analyses of Old World Papaveroideae had shown that neither taxa with styles nor those with stigmatic discs represent monophyletic lineages. We here investigate whether either styles or stigmatic discs have arisen repeatedly during the diversification of Old World Papaveroideae. METHODS: We investigated gynoecium ontogeny in Papaver rhoeas, P. californicum, Meconopsis cambrica, and Stylomecon heterophylla by scanning electron microscopy for the first time. Our observations were interpreted on the background of a well-resolved molecular phylogeny of the taxa investigated. KEY RESULTS: Papaver rhoeas and P. californicum share the presence of a developmentally complex garland-like stage in gynoecium ontogeny. The styles of M. cambrica and S. heterophylla result from growth in a ring-like zone beneath the carpel tips. This zone is also present in Papaver. In M. cambrica, traces of a garland-like stage can be seen. Style formation and stigma formation begin more or less simultaneously in M. cambrica, but style formation clearly precedes stigma formation in S. heterophylla. CONCLUSIONS: The styles present in M. cambrica and S. heterophylla are considered to have arisen in parallel from ancestors with a stigmatic disc. We speculate that style formation may have been a means to reduce selfing.

[Identification of Papaver somni ferum L. and Papaver rhoeas using DSWT-FTIR-RBFNN].

Infrared spectra of Papaver somniferum L. and Papaver rhoeas were obtained directly, quickly and accurately by Fourier transform infrared spectroscopy (FTIR)with OMNI sampler. Discrete stationary wavelet transform was used to extrude local region of infrared spectra of Papaver somniferum L. and Papaver rhoeas. The difference of infrared spectra between Papaver somniferum L. and Papaver rhoeas was extruded. Accurate identification rate is improved greatly. One dimensional discrete stationary wavelet transform was implemented to the infrared spectra of Papaver somniferum L. and Papaver rhoeas. The difference between Papaver somniferum L. and Papaver rhoeas was observed at all scales in wavelet domain. Two scales, at which the difference between Papaver somniferum L and Papaver rhoeas is the most obvious, were selected to extract the features of Papaver somniferum L. and Papaver rhoeas. A feature vector including eight feature parameters was constructed. The feature vector was input to RBFNN for training in order to accurately identify Papaver somniferum L. and Papaver rhoeas. In experiment, the authors used one hundred and twenty-eight couples of data of Papaver somniferum L. and Papaver rhoeas (including seventy-eight couples of training samples and fifty couples of testing samples). The experimental results show that it is effective to apply discrete stationary wavelet transform on the basis of FTIR to identify the Papaver somniferum L. and Papaver rhoeas. The accurate identification rate of Papaver somniferum L and Papaver rhoeas is 99.8% and 99.9% respectively.

Differentiation of opium and poppy straw using capillary electrophoresis and pattern recognition techniques.

Opium samples from four different locations and poppy straw from different plant varieties have been assayed using micellar capillary electrophoresis incorporating a sweeping technique. Individual alkaloids (morphine, codeine, papaverine, noscapine, thebaine, oripavine, reticuline and narceine) were quantitatively determined in the different samples by a validated capillary electrophoresis method. Unsupervised pattern recognition of the opium samples and the poppy straw samples using hierarchical cluster analysis (HCA) and principal component analysis (PCA), showed distinct clusters. Supervised pattern recognition using soft independent modelling of class analogy (SIMCA) was performed to show individual groupings and allow unknown samples to be classified according to the models built using the CZE assay results.

Functional analyses of genetic pathways controlling petal specification in poppy.

MADS-box genes are crucial regulators of floral development, yet how their functions have evolved to control different aspects of floral patterning is unclear. To understand the extent to which MADS-box gene functions are conserved or have diversified in different angiosperm lineages, we have exploited the capability for functional analyses in a new model system, Papaver somniferum (opium poppy). P. somniferum is a member of the order Ranunculales, and so represents a clade that is evolutionarily distant from those containing traditional model systems such as Arabidopsis, Petunia, maize or rice. We have identified and characterized the roles of several candidate MADS-box genes in petal specification in poppy. In Arabidopsis, the APETALA3 (AP3) MADS-box gene is required for both petal and stamen identity specification. By contrast, we show that the AP3 lineage has undergone gene duplication and subfunctionalization in poppy, with one gene copy required for petal development and the other responsible for stamen development. These differences in gene function are due to differences both in expression patterns and co-factor interactions. Furthermore, the genetic hierarchy controlling petal development in poppy has diverged as compared with that of Arabidopsis. As these are the first functional analyses of AP3 genes in this evolutionarily divergent clade, our results provide new information on the similarities and differences in petal developmental programs across angiosperms. Based on these observations, we discuss a model for how the petal developmental program has evolved.

Narcotic alkaloids of four papaver species from Iran.

Four native Papaver species of Iran, i. e. P. glaucum, P. tenuifolium, P. dubium and P. fugax, were collected from their natural habitat and subjected to HPLC analysis for determination of their morphine, codeine and thebaine content. P. dubium and R. glaucum contained all of the three mentioned narcotic alkaloids, while morphine was not found in P. fugax, and P. tenuifolium was free from codeine.

Phylogenetics of Papaver and related genera based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnL intron and trnL-F intergenic spacers.

BACKGROUND AND AIMS: Representatives from Papaver, Roemeria, Stylomecon and Meconopsis were studied to elucidate phylogenetic relationships between Papaver and these closely allied genera. METHODS: Two molecular data sets were used individually and combined and included sequences from the internally transcribed spacer region (ITS) of 18S-26S nuclear ribosomal DNA and the trnL intron and the trnL-trnF intergenic spacer region of plastid DNA. KEY RESULTS: Parsimony analysis demonstrated that the genus is not monophyletic unless the closely related Roemeria, Stylomecon and Meconopsis cambrica are included in a revised circumscription of Papaver. Three distinct clades are resolved in a combined ITS and trnL-F analysis. Clade 1 consists of Papaver sect. Meconella and Asian Meconopsis. Clade 2 contains a group here identified as Papaver s.s., comprising sections Carinatae, Meconidium, Oxytona, Papaver, Pilosa, Pseudopilosa and Rhoeadium. Clade 3 consists of Papaver sect. Argemonidium and Roemeria refracta. A number of diagnostic indels support these groupings. Within clade 2, sects. Papaver and Rhoeadium are either not monophyletic or lack evidence supporting their monophyly. CONCLUSIONS: The results of this molecular analysis indicate that a number of morphological characters such as valvate capsule dehiscence, dark or light filaments and sessile stigmatic discs have arisen in parallel. The phylogenetic trees are incongruent with the existing taxonomy of Papaver, and a revised classification is suggested.

Flavonols and an indole alkaloid skeleton bearing identical acylated glycosidic groups from yellow petals of Papaver nudicaule.

From yellow petals of Iceland poppy, besides the known flavonoid gossypitrin, seven kaempferol derivatives were isolated. In addition to kaempferol 3-O-beta-sophoroside and kaempferol 3-O-beta-sophoroside-7-O-beta-glucoside, known from other plants, the mono- and dimalonyl conjugates of the latter were identified by MS and NMR spectroscopy. Structure analyses of a set of co-occurring pigments, the nudicaulins, revealed that they have the identical acylated glycoside moieties attached to a pentacyclic indole alkaloid skeleton for which the structure of 19-(4-hydroxyphenyl)-10H-1,10-ethenochromeno[2,3-b]indole-6,8,18-triol was deduced from MS and NMR as well as chemical and chiroptical methods.

[Minor alkaloids from the capsule of Papaver nudicaule L].

Two minor alkaloids were isolated from the capsule of Papaver nudicaule. On the basis of physicochemical methods and spectroscopic analysis, they were identified as chelidonine, 5-hydroxy-2-hydroxymethylpyridine. Chelidonine was isolated from this plant and 5-hydroxy-2-hydroxymethylpyridine was isolated from genus for the first time.

[Differentiation of Corydalis yanhusuo W. T. Wang ex Z. Y. Su et C. Y. Wu and its application].

There are differences between the big and small leaf types of Corydalis yanhusuo. The big leaf type is better and yields about 10% more than the small one. By comparing the big leaf type in terms of leaf charactistics, inflorescence and flower number, we name the small leaf one as C. yanhusuo f. floribunda.

Rapid semi-quantitative spot test for determination of thebaine and differentiation of Papaver bracteatum from P. orientale and P. pseudo-orientale.

The alkaloids, orientalidine, oripavine, thebaine, narceine, narceine, codeine, papaverine, isothebaine, alpenigenine and morphine react with a solution of sulfuric acid containing molybdate to give specific colors. Using the acid solution permitted the differentiation of Papaver bracteatum latex from P. orientale latex and P. pseudo-orientale latex.