Comparative morphoanatomy and transcriptomic analyses reveal key factors controlling floral trichome development in Aristolochia (Aristolochiaceae).

Trichomes are specialized epidermal cells in aerial plant parts. Trichome development proceeds in three stages, determination of cell fate, specification, and morphogenesis. Most genes responsible for these processes have been identified in the unicellular branched leaf trichomes from the model Arabidopsis thaliana. Less is known about the molecular basis of multicellular trichome formation across flowering plants, especially those formed in floral organs of early diverging angiosperms. Here, we aim to identify the genetic regulatory network (GRN) underlying multicellular trichome development in the kettle-shaped trap flowers of Aristolochia (Aristolochiaceae). We selected two taxa for comparison, A. fimbriata, with trichomes inside the perianth, which play critical roles in pollination, and A. macrophylla, lacking specialized trichomes in the perianth. A detailed morphoanatomical characterization of floral epidermis is presented for the two species. We compared transcriptomic profiling at two different developmental stages in the different perianth portions (limb, tube, and utricle) of the two species. Moreover, we present a comprehensive expression map for positive regulators and repressors of trichome development, as well as cell cycle regulators. Our data point to extensive modifications in gene composition, expression, and putative roles in all functional categories when compared with model species. We also record novel differentially expressed genes (DEGs) linked to epidermis patterning and trichome development. We thus propose the first hypothetical genetic regulatory network (GRN) underlying floral multicellular trichome development in Aristolochia, and pinpoint key factors responsible for the presence and specialization of floral trichomes in phylogenetically distant species of the genus.

[Comparison of chloroplast genomes of medicinal plants in Aristolochiaceae].

In this study, the chloroplast genome of Asarum sieboldii f. seoulense was sequenced, analyzed, and compared with chloroplast genomes of other medicinal plants in Aristolochiaceae downloaded from GenBank, aiming to clarify the characteristics of the chloroplast genome of A. sieboldii f. seoulense and the differences in chloroplast genome among medicinal plants of Aristolochiaceae. To be specific, the chloroplast genome of A. sieboldii f. seoulense was sequenced and assembled by high-throughput sequencing, and the general characteristics, repeats, inverted repeat(IR) boundary, and phylogenetic relationship of the chloroplast genomes of 11 medicinal species in Aristolochiaceae were analyzed with REPuter. The result showed that the genome of A. sieboldii f. seoulense was 167 293 bp, with large single-copy(LSC) region of 89 840 bp, small single-copy(SSC) region of 21 415 bp, IR region of 28 019 bp, and GC content of 37.9%. A total of 133 genes were annotated, including 89 protein-coding genes, 36 tRNA genes and 8 rRNA genes. The chloroplast genomes of the 11 medicinal species were 159 308-167 293 bp, with 130-134 genes annotated. Forward(F), reverse(R), complement(C), and palindromic(P) long repeats and simple sequence repeat(SSR) were found in the chloroplast genomes of five species. Among them, A. sieboldii f. seoulense had six types of SSR. In the phylogenetic tree, A. sieboldii f. seoulense and A. heterotropoides were in the same clade. The result is expected to lay a basis for the classification, identification, and phylogeny of medicinal plants in Aristolochiaceae.

The potential of three different PCR-related approaches for the authentication of mixtures of herbal substances and finished herbal medicinal products.

BACKGROUND: Herbal substances and preparations thereof play an important role in healthcare systems worldwide. Due to the variety of these products regarding origin, composition and processing procedures, appropriate methodologies for quality assessment need to be considered. A majority of herbal substances is administered as multicomponent mixtures, especially in the field of Traditional Chinese Medicine and ayurvedic medicine, but also in finished medicinal products. Quality assessment of complex mixtures of herbal substances with conventional methods is challenging. Thus, emphasis of the present work was directed on the development of complementary methods to elucidate the composition of mixtures of herbal substances and finished herbal medicinal products. HYPOTHESIS/PURPOSE: An indispensable prerequisite for the safe and effective use of herbal medicines is the unequivocal authentication of the medicinal plants used therein. In this context, we investigated the potential of three different PCR-related methods in the characterization and authentication of herbal substances. METHODS: A multiplex PCR assay and a quantitative PCR (qPCR) assay were established to analyze defined mixtures of the herbal substances Quercus cortex, Juglandis folium, Aristolochiae herba, Matricariae flos and Salviae miltiorrhizae radix et rhizoma and a finished herbal medicinal product. Furthermore, a standard cloning approach using universal primers targeting the ITS region was established in order to allow the investigation of herbal mixtures with unknown content. RESULTS: The cloning approach had some limitations regarding the detection/recovery of the components in defined mixtures of herbal substances, but the complementary use of two sets of universal primer pairs increased the detection of components out of the mixture. While the multiplex PCR did not retrace all components in the defined mixtures of herbal substances, the established qPCR resulted in simultaneous and specific detection of the five target sequences in all defined mixtures. CONCLUSION: These data indicate that for authentication purposes, complementary PCR-related methods are highly recommendable for the analysis of herbal mixtures in parallel.

Total duplication of the small single copy region in the angiosperm plastome: Rearrangement and inverted repeat instability in Asarum.

PREMISE OF THE STUDY: As more plastomes are assembled, it is evident that rearrangements, losses, intergenic spacer expansion and contraction, and syntenic breaks within otherwise functioning plastids are more common than was thought previously, and such changes have developed independently in disparate lineages. However, to date, the magnoliids remain characterized by their highly conserved plastid genomes (plastomes). METHODS: Illumina HiSeq and MiSeq platforms were used to sequence the plastomes of Saruma henryi and those of representative species from each of the six taxonomic sections of Asarum. Sequenced plastomes were compared in a phylogenetic context provided by maximum likelihood and parsimony inferences made using an additional 18 publicly available plastomes from early-diverging angiosperm lineages. KEY RESULTS: In contrast to previously published magnoliid plastomes and the newly sequenced Saruma henryi plastome published here, Asarum plastomes have undergone extensive disruption and contain extremely lengthy AT-repeat regions. The entirety of the small single copy region (SSC) of A. canadense and A. sieboldii var. sieboldii has been incorporated into the inverted repeat regions (IR), and the SSC of A. delavayi is only 14 bp long. All sampled Asarum plastomes share an inversion of a large portion of the large single copy region (LSC) such that trnE-UUC is adjacent to the LSC-IR boundary. CONCLUSIONS: Plastome divergence in Asarum appears to be consistent with trends seen in highly rearranged plastomes of the monocots and eudicots. We propose that plastome instability in Asarum is due to repetitive motifs that serve as recombinatory substrates and reduce genome stability.

Relative contributions of neutral and non-neutral processes to clinal variation in calyx lobe length in the series Sakawanum (Asarum: Aristolochiaceae).

Background and Aims: Clines, the gradual variation in measurable traits along a geographical axis, play a major role in evolution and can contribute to our understanding of the relative roles of selective and neutral process in trait variation. Using genetic and morphological analyses, the relative contributions of neutral and non-neutral processes were explored to infer the evolutionary history of species of the series Sakawanum (genus Asarum), which shows significant clinal variation in calyx lobe length. Methods: A total of 27 populations covering the natural geographical distribution of the series Sakawanum were sampled. Six nuclear microsatellite markers were used to investigate genetic structure and genetic diversity. The lengths of calyx lobes of multiple populations were measured to quantify their geographical and taxonomic differentiation. To detect the potential impact of selective pressure, morphological differentiation was compared with genetic differentiation (QCT-FST comparison). Key Results: Average calyx lobe length of A. minamitanianum was 124.11 mm, while that of A. costatum was 13.80 mm. Though gradually changing along the geographical axis within series, calyx lobe lengths were significantly differentiated among the taxa. Genetic differentiation between taxa was low (FST = 0.099), but a significant geographical structure along the morphological cline was detected. Except for one taxon pair, pairwise QCT values were significantly higher than the neutral genetic measures of FST and G'ST. Conclusions: Divergent selection may have driven the calyx lobe length variation in series Sakawanum taxa, although the underlying mechanism is still not clear. The low genetic differentiation indicates recent divergence and/or gene flows between geographically close taxa. These neutral processes would also affect the clinal variation in calyx lobe lengths. Overall, this study implies the roles of population history and divergent selection in shaping the current cline of a flower trait in the series Sakawanum.

An integrated system for identifying the hidden assassins in traditional medicines containing aristolochic acids.

Traditional herbal medicines adulterated and contaminated with plant materials from the Aristolochiaceae family, which contain aristolochic acids (AAs), cause aristolochic acid nephropathy. Approximately 256 traditional Chinese patent medicines, containing Aristolochiaceous materials, are still being sold in Chinese markets today. In order to protect consumers from health risks due to AAs, the hidden assassins, efficient methods to differentiate Aristolochiaceous herbs from their putative substitutes need to be established. In this study, 158 Aristolochiaceous samples representing 46 species and four genera as well as 131 non-Aristolochiaceous samples representing 33 species, 20 genera and 12 families were analyzed using DNA barcodes based on the ITS2 and psbA-trnH sequences. Aristolochiaceous materials and their non-Aristolochiaceous substitutes were successfully identified using BLAST1, the nearest distance method and the neighbor-joining (NJ) tree. In addition, based on sequence information of ITS2, we developed a Real-Time PCR assay which successfully identified herbal material from the Aristolochiaceae family. Using Ultra High Performance Liquid Chromatography-Mass Spectrometer (UHPLC-HR-MS), we demonstrated that most representatives from the Aristolochiaceae family contain toxic AAs. Therefore, integrated DNA barcodes, Real-Time PCR assays using TaqMan probes and UHPLC-HR-MS system provides an efficient and reliable authentication system to protect consumers from health risks due to the hidden assassins (AAs).

Genetic structure within and among populations of Saruma henryi, an endangered plant endemic to China.

The endangered perennial plant Saruma henryi Oliv. is endemic to China and has phylogenetic, ecological, and medicinal value. We used 10 microsatellite (SSR) loci to investigate genetic diversity and differentiation in 16 natural populations. Genetic diversity was high at the species level (H(E) = 0.9427, h = 0.9410, I = 3.0213) and low at the population level (H(E) = 0.4441, h = 0.4307, I = 0.6822). Pronounced genetic differentiation was detected among populations (G(ST) = 0.5428, F(ST) = 0.5524), in line with the limited among-population gene flow (Nm = 0.21). The significant isolation-by-distance pattern revealed by a Mantel test (r = 0.311, P = 0.001) suggested a clear geographic tendency in the distribution of genetic variability. Bayesian assignment and principal coordinates analyses supported the clustering of 16 populations into three groups. The present SSR results were also compared with previously published ISSR results. These results have significant implications for conservation of the species.

Strong phylogeographic pattern of cpDNA variation reveals multiple glacial refugia for Saruma henryi Oliv. (Aristolochiaceae), an endangered herb endemic to China.

Saruma henryi Oliv. (Aristolochiaceae) is an endangered herb endemic to China. In this study, chloroplast microsatellites (cpSSRs) and sequences of the atpB-rbcL intergenic spacers were employed to reveal its genetic diversity and phylogeographic patterns. We detected high within-species genetic diversity (H(T)=0.939 for cpSSR; H(T)=0.862 for atpB-rbcL) and pronounced among-population genetic differentiation (H(S)=0.182, G(ST)=0.811, R(ST)=0.9, F(ST)=0.93 for cpSSR; H(S)=0.238, G(ST)=0.724, N(ST)=0.758, F(ST)=0.79 for atpB-rbcL) with a strong phylogeographic pattern (R(ST)>G(ST), P<0.01 for cpSSR; N(ST)>G(ST), U=0.25 for atpB-rbcL). Eleven haplotypes were distinguished by cpSSRs and atpB-rbcL intergenic spacers, respectively. The molecular phylogenetic data, together with the geographic distribution of the haplotypes, suggested the existence of multiple localized glacial refugia in Mts. Qinling, eastern Mts. Bashan and the southeastern edge of Yunnan-Guizhou Plateau. Nested clade analysis (NCA) and population genetic analyses supported the limited gene flow (caused by low dispersal capacity and complex topography of its habitats) as the major factor responsible for the strong population differentiation and phylogeographic pattern. Past fragmentation and allopatric fragmentation were inferred as important processes responsible for the modern phylogeograhpic pattern. In addition, contiguous range expansions occurred in western Mts. Qinling and eastern Mts. Bashan.