Imprints of selection in peripheral and ecologically marginal central-eastern European Scots pine populations.

Knowledge of the molecular mechanisms underlying the stress response in plants is essential to understand evolutionary processes that result in long-term persistence of populations. Populations inhabiting marginal ecological conditions at the distribution range periphery may have preserved imprints of natural selection that have shaped functional genetic variation of the species. Our aim was to evaluate the extent of selection processes in the extremely fragmented, peripheral and isolated populations of Scots pine in central-eastern Europe. Autochthonous populations of the Carpathian Mts. and the Pannonian Basin were sampled and drought stress-related candidate genes were re-sequenced. Neutrality tests and outlier detection approaches were applied to infer the effect and direction of selection. Populations retained high genetic diversity by preserving a high number of alleles and haplotypes, many of them being population specific. Neutrality tests and outlier detection highlighted nucleotide positions that are under divergent selection and may be involved in local adaptation. The detected genetic pattern confirms that natural selection has played an important role in shaping modern-day genetic variation in marginal Scots pine populations, allowing for the long-term persistence of populations. Selection detected at functional regions possibly acts to maintain diversity and counteract the effect of genetic erosion.

A simple molecular identification method of the Thrips tabaci (Thysanoptera: Thripidae) cryptic species complex.

The onion thrips (Thrips tabaci Lindeman, 1889) is a key pest of a wide range of crops because of its ecological attributes such as polyphagy, high reproduction rate, ability to transmit tospoviruses and resistance to insecticides. Recent studies revealed that T. tabaci is a cryptic species complex and it has three lineages (leek-associated arrhenotokous L1-biotype, leek-associated thelytokous L2-biotype and tobacco-associated arrhenotokous T-biotype), however, the adults remain indistinguishable. T. tabaci individuals were collected from different locations of Hungary to create laboratory colonies from each biotypes. Mitochondrial COI (mtCOI) region was sequenced from morphologically identified individuals. After sequence analysis SNPs were identified and used for CAPS marker development, which were suitable for distinguishing the three T. tabaci lineages. Genetic analysis of the T. tabaci species complex based on mtCOI gene confirmed the three well-known biotypes (L1, L2, T) and a new biotype because the new molecular evidence presented in this study suggests T-biotype of T. tabaci forming two distinct (sub)clades (T1 and T2). This genetic finding indicates that the genetic variability of T. tabaci populations is still not fully mapped. We validated our developed marker on thrips individuals from our thrips colonies. The results demonstrated that the new marker effectively identifies the different T. tabaci biotypes. We believe that our reliable genotyping method will be useful in further studies focusing on T. tabaci biotypes and in pest management by scanning the composition of sympatric T. tabaci populations.

Identification of a novel UDP-glycosyltransferase gene from Rhodiola rosea and its expression during biotransformation of upstream precursors in callus culture.

Roseroot (Rhodiola rosea L.) is a medicinal plant with adaptogenic properties and several pharmaceutically important metabolites. In this study, a full length cDNA encoding a UDPG gene of roseroot was identified, cloned and characterized. Its ORF (1425 bp) was transferred into E. coli, where the expression of the recombinant enzyme was confirmed. To monitor the enzyme activity, 3 precursors (tyramine, 4-hydroxyphenylpyruvate & tyrosol) of salidroside biosynthesis pathway were added to roseroot callus cultures and samples were harvested after 1, 6, 12, 24, 48 & 96 h. Along with the controls (without precursor feeding), each sample was subjected to HPLC and qRT-PCR for phytochemical and relative UDP-glycosyltransferase gene expression analysis, respectively. The HPLC analysis showed that the salidroside content significantly increased; reaching 0.5% of the callus dry weight (26-fold higher than the control) after 96 h when 2 mM tyrosol was given to the media. The expression of the UDP-glycosyltransferase increased significantly being the highest at 12 h after the feeding. The effect of tyramine and 4-hydroxyphenylpyruvate was not as pronounced as of tyrosol. Here, we introduce a R. rosea specific UDPG gene and its expression pattern after biotransformation of intermediate precursors in in vitro roseroot callus cultures.

Intercontinental migration pattern and genetic differentiation of arctic-alpine Rhodiola rosea L.: A chloroplast DNA survey.

Our study describes genetic lineages and historical biogeography of Rhodiola rosea a widely distributed arctic-alpine perennial species of the Northern Hemisphere based on sequence analysis of six chloroplast regions. Specimens of 44 localities from the Northern Hemisphere have been sequenced and compared with those available in the GenBank. Our results support the migration of the species into Europe via the Central Asian highland corridor, reaching the European Alpine System (EAS) and also the western European edge, the British Isles. The EAS proved to be an important center of genetic diversity, especially the region of the Eastern Alps and the Dolomites where signs of glacial refugia was observed. Apart from those of the EAS, a common lineage was detected along the Atlantic coast from the British Isles toward Scandinavia as well as Iceland and the eastern parts of North America. Accordingly, the British Isles represent a main link between the northern Atlantic and southern EAS lineages.

Changes in the Content of the Glycosides, Aglycons and their Possible Precursors of Rhodiola rosea during the Vegetation Period.

Phytochemical participants in the biosynthetic pathway of salidroside and cinnamyl alcohol glycosides were studied from seven Rhodiola rosea L. individuals originating from a wild population. Plants were grown in a phytotron and samples were taken at 3 weekly intervals during the vegetation period. Based on HPLC analysis, all the key compounds to which roseroot medicinal property is attributed were detected, with salidrosde being the most dominant, followed by its aglycone, tyrosol. The contents of all compounds were 2-3 times more in the rhizomes than in roots. The highest content of salidroside, tyrosol, rosarin, rosavin and cinnamyl alcohol was recorded in rhizomes and at the beginning of shoot elongation. The seven roseroot individuals showed a very high deviation in their chemical content at each sampling time. Our statistical analysis showed that the trend of salidroside accumulation in the rhizome was the most similar in all studied plants. These results have important implications for choosing a reasonable harvest time to obtain the maximum phytochemical content and a better understanding of active compounds formation in R. rosea L.

Genetic variation of the endangered Gentiana lutea L. var. aurantiaca (Gentianaceae) in populations from the Northwest Iberian Peninsula.

Gentiana lutea L. (G. lutea L.) is an endangered plant, patchily distributed along the mountains of Central and Southern Europe. In this study, inter-simple sequence repeat (ISSR) markers were used to investigate the genetic variation in this species within and among populations of G. lutea L. var. aurantiaca of the Cantabrian Mountains (Northwest Iberian Peninsula). Samples of G. lutea L. collected at different locations of the Pyrenees and samples of G. lutea L. subsp. vardjanii of the Dolomites Alps were also analyzed for comparison. Using nine ISSR primers, 106 bands were generated, and 89.6% of those were polymorphic. The populations from the Northwest Iberian Peninsula were clustered in three different groups, with a significant correlation between genetic and geographic distances. Gentiana lutea L. var. aurantiaca showed 19.8% private loci and demonstrated a remarkable level of genetic variation, both among populations and within populations; those populations with the highest level of isolation show the lowest genetic variation within populations. The low number of individuals, as well as the observed genetic structure of the analyzed populations makes it necessary to protect them to ensure their survival before they are too small to persist naturally.

Non-TIR-NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.).

Genes encoding for proteins with nucleotide-binding site and leucine-rich repeat motifs (NBS-LRR) have been suggested to play a general role in plant defence mechanism. In Prunus species, many TIR (Toll / Interleukin-1 Receptor), and only very few non-TIR sequences were identified, which was explained either by the unequal distribution of TIR/non-TIR sequences in the Prunus genome or by the incapability of primers in the amplification of non-TIR RGAs. The objective of this work was to check whether a new semi-nested PCR strategy can be developed for the targeted isolation of non-TIR-NBS-LRR Resistance Gene Analog (RGA) sequences from apricot. Three primers (CUB-P-loop F, CUB-Kin2 F and CUB-HD R) were designed, from which CUB-Kin2 F and CUB-HD R were constructed to anneal selectively to the non-TIR sequences. A colony Polymerase Chain Reaction (PCR) indicated that out of the 96 clones tested 28 showed amplification using the newly developed primers, while no amplification occurred when using the formerly described primers. Half of the 28 positive clones were sequenced and they turned out to represent 11 different non-TIR RGA sequences. A phylogenetic analysis was carried out based on an alignment containing 293 Rosaceae and 21 non-Rosaceaa sequences. A significantly higher ratio (91%) of non-TIR sequences were arranged in multi-genera clades than that of (57%) the TIR groups confirming that non-TIR sequences might be of more ancient origin than TIR sequences.

Isolation and genotype-dependent, organ-specific expression analysis of a Rhodiola rosea cDNA encoding tyrosine decarboxylase.

Tyrosine decarboxylase (TyrDC) is an important enzyme in the secondary metabolism of several plant species, and was hypothesized to play a key role in the biosynthesis of salidroside, a pharmacologically valuable compound of roseroot. A 1520bp cDNA was cloned and sequenced, and turned out to contain an ORF of 963bp, which encodes a protein of 320 amino acids. The expression of the gene was studied by real-time PCR from leaves and roots of both high and low salidroside producer genotype of roseroot. The gene expression analysis showed the gene to be expressed in leaves as well as in roots; however, the expression was significantly higher in roots, which coincides with the fact that salidroside accumulates preferentially in the underground parts of the plant. The expression was also higher in the line accumulating high levels of salidroside, compared to the line with lower salidroside content. The difference in the expression intensity suggests a decisive role for this enzyme in the salidroside biosynthesis.

Production of cinnamyl glycosides in compact callus aggregate cultures of Rhodiola rosea through biotransformation of cinnamyl alcohol.

Rhodiola rosea is a multipurpose medicinal plant with adaptogenic properties: it increases the body's nonspecific resistance and normalizes body functions. The commercial interest for roseroot-based products has quickly increased worldwide. Nearly all raw-materials originate from natural populations. As a result of the intensive collection the species has become endangered. Production of the pharmaceutical compounds from the medicinal plants in cell cultures is an alternative to field cultivation. The present protocol describes the establishment of compact callus aggregate culture of Rhodiola rosea and the production of cinnamyl alcohol glycosides through biotransformation.

LC/MS/MS identification of glycosides produced by biotransformation of cinnamyl alcohol in Rhodiola rosea compact callus aggregates.

Cinnamyl alcohol was added to the media of compact callus aggregates (CCA) of Rhodiola rosea for stimulating the production of cinnamyl glycosides. The biotransformation reaction produced high amounts of rosin, while only a very low amount of rosavin was produced. As the consumption rate of cinnamyl alcohol was much higher than production of rosin, the aqueous methanol extracts of compact callus aggregates were studied by liquid chromatography-mass spectrometric methods and four new unexpected biotransformation products of cinnamyl alcohol were identified.