Identification of microsatellite loci from Epimedium koreanum and cross‐species amplification in four species of Epimedium (Berberidaceae)

Nineteen polymorphic microsatellite markers were isolated and characterized from a trinucleotide enriched partial genomic library of Epimedium koreanum. The average allele number of these microsatellites was 5.9 per locus, ranging from two to 11. The observed heterozygosity (H_O) and expected heterozygosity (H_E) at the population level were 0.00–0.90 and 0.12–0.90, respectively. In addition, the results of cross‐species amplification of this set of microsatellite markers in four closely related Epimedium species, E. brevicornum, E. sagittatum, E. pubescens and E. wushanense, revealed that these microsatellite markers were useful for population genetic structure evaluation and genotype analysis of major Epimedium species that have been used as traditional Chinese medicines.     

川产淫羊藿属（Epimedium）药用植物的无性系构件特征和生物量配置比较

在构件水平上,对川产的3种药用淫羊藿属植物（淫羊藿（Epimedium brevicornu）、箭叶淫羊藿（E.sagittatum）、柔毛淫羊藿（E.pubescens））的无性系构件特征、克隆构型及构件生物量配置进行比较,结果表明在野生状态下：该属3个种无性系构件的形态特征差异显著,且存在较大的变异性,变异系数范围在29.29%~48.31%之间;箭叶淫羊藿更趋于“游击型”克隆构型,而淫羊藿和柔毛淫羊藿则更趋于“密集型”克隆构型;该属3个种都将高比例生物量配置到叶片或者根茎,其次为茎,再其次为根。柔毛淫羊藿具有更强的克隆繁殖能力,单位面积上各构件生物量均最大,应作为人工引种栽培的首选种。     

Essential oil in Betula spp. leaves naturally growing in Estonia

The essential oils of the leaves of Betula pendula Roth., Betula pubescens Ehrh., Betula humilis Schrank and Betula nana L. (Betulaceae), and B. pendula buds were obtained using hydrodistillation and their chemical compositions were analyzed using gas chromatography and gas chromatography – mass spectrometry. The yield of essential oil was the highest (0.27%) in leaves of. B. humilis and the lowest in the leaves of B. pendula (0.11%), B. pubescens (0.05%) and B. nana (traces). Overall, 35–60 compounds were identified representing more than 92% of the total volume of the oils investigated. The bicyclical sesquiterpenoids found in the essential oils extracted from the leaves of B. pendula, B. pubescens and B. humilis and from the buds of B. pendula where α-betulenol (20.4–33.1%), α-betulenol acetate (5,6–28.9%), β-betulenal (5.5–6.2%) and β-betulenol (2.0–5.8%). The buds of B. pendula contained more birkenal, (E)-β-caryophyllene, δ-cadinene and caryophyllene oxide, but less α-betulenol acetate, β-betulenal and aliphatic compounds than the oils extracted from the leaves investigated. The oil composition of B. humilis and B. nana leaves was studied for the first time. The content of essential oil of B. nana leaves was much more varied: the oil contained less bicyclical sesquiterpenoids and more aliphatic compounds than the other Betula species investigated. The findings of our study could be used in chemosystematics of birch species, especially for B. nana.     

Variations in Essential Oil Compositions of Lavandula pubescens (Lamiaceae) Aerial Parts Growing Wild in Yemen

Lavandula pubescens Decne . is one of five Lavandula species growing wild in Yemen. The plant is used in Yemeni traditional medicine, and the essential oil tends to be rich in carvacrol. In this work, L. pubescens was collected from eight different locations in Yemen, the essential oils obtained by hydrodistillation, and the oils analyzed by gas chromatography/mass spectrometry (GC/MS). Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to differentiate between the L. pubescens samples. The essential oils were rich in carvacrol (60.9 – 77.5%), with lesser concentrations of carvacrol methyl ether (4.0 – 11.4%), caryophyllene oxide (2.1 – 6.9%), and terpinolene (0.6 – 9.2%). The essential oil compositions in this study showed very high similarity, but it was possible to discern two separate groups based on minor components, in particular the concentrations of terpinolene, carvacrol methyl ether, m‐cymen‐8‐ol, and caryophyllene oxide.     

Pollen and stomata morphometrics and polyploidy in Eriotheca (Malvaceae‐Bombacoideae)

Approximately 70% of the angiosperm species are polyploid, an important phenomenon in the evolution of those plants. But ploidy estimates have often been hindered because of the small size and large number of chromosomes in many tropical groups. Since polyploidy affects cell size, morphometric analyses of pollen grains and stomata have been used to infer ploidy level. Polyploidy is present in many species of the Cerrado, the Neotropical savanna region in Central Brazil, and has been linked to apomixis in some taxa. Eriotheca gracilipes and Eriotheca pubescens are common tree species in this region, and present cytotypes that form reproductive mosaics. Hexaploid individuals (2n = 6x = 276) are polyembryonic and apomictic, while tetraploid and diploid individuals (2n = 2x = 92, 2n = 4x = 184) are sexual and monoembryonic. We tested whether morphometric analysis can be used to estimate ploidy levels in E. gracilipes and E. pubescens individuals. Pollen material from diploid and hexaploid individuals of E. gracilipes, and tetraploid and hexaploid individuals of E. pubescens, were fixed in 50% FAA, and expanded leaves were dried in silica gel. Pollen grains and stomata of at least five individuals from each population were measured. The results demonstrate that all measures were significantly different among cytotypes. Individuals with higher levels of ploidy (hexaploid) all presented measurements that were higher than those with lower levels (diploid and tetraploid). There was no overlap between ploidy levels in each species at 95% confidence interval. Thus, the size of the pollen grains and stomata are effective parameters for analysis of ploidy levels in E. gracilipes and E. pubescens.     

Cloning and characterization of phenylalanine ammonia-lyase in medicinal Epimedium species

Phenylalanine ammonia-lyase (PAL) plays an important role in the phenylpropanoid pathway and in accumulation of major secondary metabolites in medicinal Epimedium species, including icariin, epimedin A, epimedin B, and epimedin C (hereafter designated as active components). In this study, three Epimedium sagittatum PALs (EsPALs) mRNA sequences, designated respectively as EsPAL1, EsPAL2 and EsPAL3 deduced to encode 708, 716, and 739 amino acids, were isolated and characterized. Based on sequence and phylogenetic analyses, EsPAL1 was found to be closer to EsPAL2 than to EsPAL3. Spatio-temporal expression profiles and metabolic accumulation profiles revealed that EsPAL3 was highly expressed in flavonoid-enriched tissues and leaves at certain developmental stages along with high levels of active components, while EsPAL1 was highly expressed in leathery leaves along with high lignin content. Under light stress, the total flavonoid content was enhanced by 100 μM phytohormones tested or 5 % sucrose through upregulating different EsPAL isoform(s). Our findings have laid a solid foundation for improving the content of bioactive components in Epimedium via metabolically engineering EsPAL.     

Application of DNA barcoding in Roscoea (Zingiberaceae) and a primary discussion on taxonomic status of Roscoea cautleoides var. pubescens

DNA barcoding is a useful tool to define operational taxonomic units based on standardized DNA regions. In this study, three chloroplast markers (rbcL, trnH-psbA and matK) and one nrDNA marker (ITS) were tested for species identification in Roscoea. The ITS and trnH-psbA regions showed high success rate of PCR amplification and bidirectional sequencing, as well as perfect discriminatory ability. On the contrary, rbcL possessed no genetic variation and matK was relatively difficult in PCR amplification and DNA sequencing. Combination of multiple markers greatly improved identification ability of DNA barcoding. ITS + trnH-psbA could effectively discriminate 90% species based on method of the Neighbor-Joining (NJ) tree. Awful PCR amplification and DNA sequencing of matK restricted its efficiency, although it showed rich genetic variability in Roscoea. Moreover, it might be more appropriate to treat Roscoea cautleoides var. pubescens as an independent species based on molecular data, namely R. pubescens Z. Y. Zhu.     

Comparative analyses of plastid and AFLP data suggest different colonization history and asymmetric hybridization between Betula pubescens and B. nana

Birches (Betula spp.) hybridize readily, confounding genetic signatures of refugial isolation and postglacial migration. We aimed to distinguish hybridization from range‐shift processes in the two widespread and cold‐adapted species B. nana and B. pubescens, previously shown to share a similarly east–west‐structured variation in plastid DNA (pDNA). We sampled the two species throughout their ranges and included reference samples of five other Betula species and putative hybrids. We analysed 901 individual plants using mainly nuclear high‐resolution markers (amplified fragment length polymorphisms; AFLPs); a subset of 64 plants was also sequenced for two pDNA regions. Whereas the pDNA variation as expected was largely shared between B. nana and B. pubescens, the two species were distinctly differentiated at AFLP loci. In B. nana, both the AFLP and pDNA results corroborated the former pDNA‐based hypothesis that it expanded from at least two major refugia in Eurasia, one south of and one east of the North European ice sheets. In contrast, B. pubescens showed a striking lack of geographic structuring of its AFLP variation. We identified a weak but significant increase in nuclear (AFLP) gene flow from B. nana into B. pubescens with increasing latitude, suggesting hybridization has been most frequent at the postglacial expansion front of B. pubescens and that hybrids mainly backcrossed to B. pubescens. Incongruence between pDNA and AFLP variation in B. pubescens can be explained by efficient expansion from a single large refugium combined with leading‐edge hybridization and plastid capture from B. nana during colonization of new territory already occupied by this more cold‐tolerant species.     

Seed traits,nutrient elements and assessment of genetic diversity for almond (Amygdalus spp.) endangered to China as revealed using SRAP markers

Almond (Amygdalus spp.) is one of the most important nut fruits worldwide and its seed has been recognized as a health nutrient supply for human consumption. In this study, we characterized the major seed traits and fruit nutrient elements among four Chinese wild almonds (Amygdalus mongolica Maxim., Amygdalus pedunculata Pall., Amygdalus tangutica Batal., and Amygdalus triloba (Lindl) Ricker.) in addition to commercial two cultivars (Amygdalus communis L.), as well as their genetic diversity and natural variations using SRAP marker, aiming at exploiting valuable traits for breeding new cultivars. Our results show that the wild almond species are rich in crude fat, Ca, Ma, Fe and Zn compared to the cultivars analyzed. Our study also identified a relatively high level of genetic diversity (h = 0.3363, I = 0.5075) among species analyzed as evidenced by fact that 161 out of a total of 162 bands amplified with fourteen SRAP primer combinations reveal polymorphism among species analyzed. AMOVA analysis reveals a high level of genetic differentiation among populations (Φ_ST = 0.4474) but The UPMGA cluster analysis indicates that A. communis and A. mongolica are more closely related. Potential utilization of the wild almond species and their valuable traits for breeding new cultivars is discussed.     

淫羊藿属植物查耳酮合成酶启动子序列的克隆及比较分析

采用TAIL-PCR技术从箭叶淫羊藿(Epimedium sagittatum)叶片中克隆得到查耳酮合成酶基因(Chalcone synthase)的启动子,命名为ECHSP, 长度为750 bp,其具有TATA BOX、CAAT BOX、MYB结合位点、MYC结合位点等元件。对箭叶淫羊藿、木鱼坪淫羊藿(E. franchetii)、粗毛淫羊藿(E. acuminatum)、直距淫羊藿(E. mikinorii)、黔岭淫羊藿(E. leptorrhizum)等5种植物的CHS启动子序列进行比较分析,结果表明,CHS启动子的核苷酸多态性丰富,表明CHS 的表达可能受到多种因素的灵活调节。ECHSP与其他植物的CHS启动子序列进行比较,其TATA box的位置可能与黄酮类化合物的积累相关。此外,进化分析表明ECHSP的核苷酸序列可应用于淫羊藿属植物的系统进化研究。     

A new coumaronochromone and phenolic constituents from the leaves of Iris bungei Maxim.

A novel compound, irisbungin, which was isolated from the leaves of Iris bungei Maxim., was elucidated as 5,7,5′-trihydroxy-coumaronochromone by spectroscopic methods and literature data. Along with the new coumaronochromone, nine known compounds, ayamenin B, mangiferin, hispidulin, apigenin, 3-phenyllactic acid, syringic acid, vanillic acid, p-coumaric acid and 3′,5′-dimethoxy-4′-O-β-d-glucopyranosyl-cinnamic acid were also isolated. All of the compounds were first isolated from this species. The presence of irisbungin and mangiferin in I. bungei Maxim. has important systematic significance for this species and the Iris genus.     

Genetic similarity among cultivars of Phyllostachys pubescens

Phyllostachys pubescens is the most important economic bamboo species in China, which grows widely in the South of China. There are more than ten cultivars in this species but their genetic relationship still remains unknown. We used both amplified fragment length polymorphism (AFLP) and inter-simple sequence repeat (ISSR) techniques to determine genetic similarity among ten cultivars of P. pubescens and two related species. Eight hundred and twenty seven bands, in which 495 are polymorphic, were detected using 15 pairs of AFLP primers whereas total 231 bands, in which 154 bands are polymorphic, were scored using 16 ISSR primers. Statistic analysis showed that the genetic similarity matrices obtained from these two sets of molecular markers had a significant correlation (R = 0.959, P = 0.013). The dendrogram generated with AFLP and ISSR markers could clearly genetically identify ten cultivars of P. pubescens that had high similarity with genetic distances ranging from 0.023 to 0.108, and could be divided into three groups based on their genetic variation and similarity. Our results suggest that these molecular markers are useful to genetically classify cultivars or varieties of a species, particularly a bamboo species.     

Molecular-networking-guided discovery of species-specific markers for discriminating five medicinal Paris herbs

BackgroundParidis Rhizoma (PR) is a famous traditional herbal medicine. Apart from two officially recorded species, viz. Paris polyphylla Smith var. yunnanensis (Franch.) Hand. - Mazz. (PPY) and P. polyphylla Smith var. chinensis (Franch.) Hara (PPC), there are still many other species used as folk medicine. It is necessary to understand the metabolic differences among Paris species.PurposeTo establish a strategy that can discover species-specific steroidal saponin markers to distinguish closely-related Paris herbs for quality and safety control.MethodsA new strategy of molecular-networking-guided discovery of species-specific markers was proposed. Firstly, the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was applied to obtain the MS and MS/MS data of all samples. Then, molecular networking (MN) was created using MS/MS data to prescreen the steroidal saponins for subsequent analysis. Next, the principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA) models were established to discover potential markers. Finally, the verification, identification and distribution of chemical markers were performed.ResultsA total of 126 steroidal saponins were screened out from five species using MN. Five species were classified successfully by OPLS-DA model, and 18 species-specific markers were discovered combining the variable importance in the projection (VIP) value, P value (one-way ANOVA) and their relative abundance. These markers could predict the species of Paris herbs correctly.ConclusionThese results revealed that this new strategy could be an efficient way for chemical discrimination of medicinal herbs with close genetic relationship.     

Effect of floral morphology on fruit set in Epimedium sagittatum (Berberidaceae)

The relationship between the floral morphology, especially the heterostyly, and the pollination ability in Epimedium sagittatum was investigated. Three distinct kinds of flowers were observed in this species: (1) about 54% of the flowers had pistils that were of equal length or shorter than the stamens (PESS); (2) in 23.5% of the flowers, the pistils were longer than the stamens (PLS), and; (3) in 22.5% of the flowers, the styles were positioned axially away from the anthers (SAA). No significant difference in fruit setting rate was observed between PLS and SAA flowers, but PESS flowers set less fruits than the other two. A breeding experiment indicated that the species was self-incompatible, and revealed that the presence of PLS and SAA could increase outcrossing and fruit set. A field experiment showed that open-pollinated flowers of E. sagittatum produced significantly less fruits than an artificial xenogamy experiment. This may be because most of the flowers in natural populations have pistils and anthers of the same length (PESS). The principal visitors and effective pollinators are Bombus sp1 and Bombus sp2. Attractant experiments indicated that the fruit set of flowers whose spurs were removed was significantly lower than that of flowers whose stamens were removed, suggesting that the spurs of the flowers could strongly increase the pollinator visitation and fruit set. The results therefore showed that floral morphology could significantly affect fruit set in E. sagittatum.     

Chemical and genetic diversity of two Mediterranean subspecies of Teucrium polium L.

Chemical and genetic diversity of Teucrium polium L. subsp. polium from western Algeria and T. polium L. subsp. capitatum from Corsica were investigated. Diversity within and among the two populations of subspecies was assessed according to the chemical composition of their essential oils and the genetic diversity. Chemical analysis was performed using a combination of capillary GC-RI and GC/MS after fractionation using column chromatography. Genetic structures were mapped using three polymorphic genetic markers: two chloroplast markers (RPL32-TRNL and TRNL-F) and ribosomal nuclear markers (ITS region). The statistical analysis showed that both subspecies were clearly distinguished by these chemical and genetic markers. The oil chemical compositions differed qualitatively and quantitatively between the subspecies. Both collective oils were dominated by hydrocarbon compounds however the Algerian sample oils exhibited higher amounts of hydrocarbon sesquiterpenes than those of Corsica (31.2 g/100 g vs. 4.4 g/100 g) while the latter displayed higher amounts of hydrocarbon monoterpenes than the first (59.3 g/100 g vs. 34.3 g/100 g). Neighbor-joining, Maximum likelihood and Bayesian trees constructed from chloroplast markers and nuclear ITS region sequences showed the existence of two groups associated with taxonomic and chemical characteristics. The study indicated that variation in the essential oil composition within subspecies depends on genetic background. The samples of subsp. capitatum from Corsica are a homogeneous group, in contrast to samples of subsp. polium from Algeria which were clustered in two groups. Chemical and genetic diversity of Algerian populations could be explained by geographical isolation of the populations. In addition, the morphological polymorphism observed throughout the colour of flowers could be explained by environmental parameters as well as the soil pH.     

Effect of neighboring seeds associated with habitats on the seed removal of two coexisting myrmecochorous plants in central China

Although neighboring plants can influence animal-seed dispersal interactions, little is known about the effect of neighboring seeds and the influence of habitat on seed dispersal by ants. Here we investigated the influence of neighboring seeds on seed removal in two coexisting myrmecochorous species (Epimedium pubescens and Helleborus thibetanus) from temperate deciduous forests of Qinling Mountains, central China, by examining (1) the potential role of ants and rodents and (2) whether the neighboring seed effect differed between forest edge and interior. We found that, presence of the higher-ESMR (elaiosome: seed mass ratio) E. pubescens did not significantly affect seed removal of the lower-ESMR H. thibetanus. By contrast, the presence of H. thibetanus decreased the seed removal rates of E. pubescens, with only a significant effect in rodent exclusion (ant alone) rather than in both ant exclusion (rodent alone) and full access (rodent + ant). Moreover, we found that those effects were not significantly different between the forest edge and the interior, which may be attributed to a similar pattern of overall seed-dispersing ant abundance in the two habitats. This suggested that neighboring seeds could influence seed removal of the focal plant depending on the absence of rodents; when rodents were present, the interaction of rodents and ants rendered no influence of neighboring seeds on seed removal. Our results show that the neighborhood effect was regulated by both dispersers and predators, and this effect was not context-dependent at a small spatial scale. This study highlights the importance to understand the effect of shared seed-removing animals and habitat context to assess the neighboring seed effect on plant-animal interactions.     

Structural and stereochemical studies of five new pregnane steroids from the stem bark of Toona ciliata var. pubescens

Five new pregnane steroids, toonasterones A (1), B (2), (Z)-aglawone (3), (Z)-toonasterone C (4), and (E)-toonasterone C (5), were isolated from the stem bark of Toona ciliata var. pubescens. Their structures were elucidated by means of detailed spectroscopic (IR, MS, and 2D NMR) analysis, and the stereochemistry of 1 was secured by X-ray diffraction analysis. (Z)-aglawone (3) exhibited moderate inhibitory activity of protein tyrosine phosphatase 1B (PTP1B), a potential drug target for treatment of type-II diabetes and obesity, with an IC₅₀ value of 1.12 μg/mL.