A high-density genetic map and QTL mapping of leaf traits and glucosinolates in Barbarea vulgaris.

BACKGROUND: Barbarea vulgaris is a wild cruciferous plant and include two distinct types: the G- and P-types named after their glabrous and pubescent leaves, respectively. The types differ significantly in resistance to a range of insects and diseases as well as glucosinolates and other chemical defenses. A high-density linkage map was needed for further progress to be made in the molecular research of this plant. RESULTS: We performed restriction site-associated DNA sequencing (RAD-Seq) on an F2 population generated from G- and P-type B. vulgaris. A total of 1545 SNP markers were mapped and ordered in eight linkage groups, which represents the highest density linkage map to date for the crucifer tribe Cardamineae. A total of 722 previously published genome contigs (50.2 Mb, 30% of the total length) can be anchored to this high density genetic map, an improvement compared to a previously published map (431 anchored contigs, 38.7 Mb, 23% of the assembly genome). Most of these (572 contigs, 31.2 Mb) were newly anchored to the map, representing a significant improvement. On the basis of the present high-density genetic map, 37 QTL were detected for eleven traits, each QTL explaining 2.9-71.3% of the phenotype variation. QTL of glucosinolates, leaf size and color traits were in most cases overlapping, possibly implying a functional connection. CONCLUSIONS: This high-density linkage map and the QTL obtained in this study will be useful for further understanding of the genetic of the B. vulgaris and molecular basis of these traits, many of which are shared in the related crop watercress.

Consequences of combined herbivore feeding and pathogen infection for fitness of Barbarea vulgaris plants.

Plants are often attacked by pathogens and insects. Their combined impact on plant performance and fitness depends on complicated three-way interactions and the plant's ability to compensate for resource losses. Here, we evaluate the response of Barbarea vulgaris, a wild crucifer, to combined attack by an oomycete Albugo sp., a plant pathogen causing white rust, and a flea beetle, Phyllotreta nemorum. Plants from two B. vulgaris types that differ in resistance to P. nemorum were exposed to Albugo and P. nemorum alone and in combination and then monitored for pathogen infection, herbivore damage, defence compounds, nutritional quality, biomass and seed production. Albugo developed infections in the insect-resistant plants, whereas insect-susceptible plants were scarcely infected. Concentrations of Albugo DNA were higher in plants also exposed to herbivory; similarly, flea beetle larvae caused more damage on Albugo-infected plants. Concentrations of saponins and glucosinolates strongly increased when the plants were exposed to P. nemorum and when the insect-susceptible plants were exposed to Albugo, and some of these compounds increased even more in the combined treatment. The biomass of young insect-susceptible plants was lower following exposure to flea beetles, and the number of leaves of both plant types was negatively affected by combined exposure. After flowering, however, adult plants produced similar numbers of viable seeds, irrespective of treatment. Our findings support the concept that pathogens and herbivores can affect each other's performance on a host plant and that the plant reacts by inducing specific and general defences. However, plants may be able to compensate for biomass loss from single and combined attacks over time.

Different geographical distributions of two chemotypes of Barbarea vulgaris that differ in resistance to insects and a pathogen.

The interactions of plants with herbivores and pathogens have been suggested to drive the evolution of resistances in plants and in some cases new lineages and taxa. However, such divergence may require reproductive isolation, e.g., in allopatry. In the crucifer Barbarea vulgaris, some plants are resistant to the flea beetle Phyllotreta nemorum, due to production of specific saponins, whereas others are susceptible. Resistant and susceptible plants additionally differ in resistance to the pathogen Albugo candida, content of glucosinolates, and leaf pubescence, and they are genetically strongly divergent and partially reproductively incompatible. This suggests that at some point they were separated for a considerable length of time. Previously, the insect susceptible P-type had been described only from Denmark, Sweden, and Estonia, whereas the resistant G-type is widely distributed in Western Europe. Here, we tested whether the two plant types have divergent geographical distributions and maintain their distinct trait associations throughout their range. The insect-susceptible type was found in Russia, the Baltics, and parts of Fennoscandia, but not in Central Europe. In contrast, the insect resistant type was found from Finland and westwards. Their different trait associations were consistent within the two ranges. We therefore suggest that the two plant types diverged in allopatry at some time in the past, and evolved different resistances in response to local antagonists. The two plant types probably maintain their distinctness due to a hybridization barrier. Thus, the present distributions of the two types may be shaped by both historical processes and current differential biotic selection.

Plant metabolomics: resolution and quantification of elusive peaks in liquid chromatography-mass spectrometry profiles of complex plant extracts using multi-way decomposition methods.

Previous studies on LC-MS metabolomic profiling of 127 F2 Barbarea vulgaris plants derived from a cross of parental glabrous (G) and pubescent (P) type, revealed four triterpenoid saponins (hederagenin cellobioside, oleanolic acid cellobioside, epihederagenin cellobioside, and gypsogenin cellobioside) that correlated with resistance of plants against the insect herbivore, Phyllotreta nemorum. In this study, for the first time, we demonstrate the efficiency of the multi-way decomposition method PARAllel FACtor analysis 2 (PARAFAC2) for exploring complex LC-MS data. PARAFAC2 enabled automated resolution and quantification of several elusive chromatographic peaks (e.g. overlapped, elution time shifted and low s/n ratio), which could not be detected and quantified by conventional chromatographic data analysis. Raw LC-MS data of 127 F2 B. vulgaris plants were arranged in a three-way array (elution time point×mass spectra×samples), divided into 17 different chromatographic intervals and each interval were individually modeled by PARAFAC2. Three main outputs of the PARAFAC2 models described: (1) elution time profile, (2) relative abundance, and (3) pure mass spectra of the resolved peaks modeled from each interval of the chromatographic data. PARAFAC2 scores corresponding to relative abundances of the resolved peaks were extracted and further used for correlation and partial least squares (PLS) analysis. A total of 71 PARAFAC2 components (which correspond to actual peaks, baselines and tails of neighboring peaks) were modeled from 17 different chromatographic retention time intervals of the LC-MS data. In addition to four previously known saponins, correlation- and PLS-analysis resolved five unknown saponin-like compounds that were significantly correlated with insect resistance. The method also enabled a good separation between resistant and susceptible F2 plants. PARAFAC2 spectral loadings corresponding to the pure mass spectra of chromatographic peaks matched well with experimentally recorded mass spectra (correlation based similarity >95%). This enabled to extract pure mass spectra of highly overlapped and low s/n ratio peaks.

UDP-glycosyltransferases from the UGT73C subfamily in Barbarea vulgaris catalyze sapogenin 3-O-glucosylation in saponin-mediated insect resistance.

Triterpenoid saponins are bioactive metabolites that have evolved recurrently in plants, presumably for defense. Their biosynthesis is poorly understood, as is the relationship between bioactivity and structure. Barbarea vulgaris is the only crucifer known to produce saponins. Hederagenin and oleanolic acid cellobioside make some B. vulgaris plants resistant to important insect pests, while other, susceptible plants produce different saponins. Resistance could be caused by glucosylation of the sapogenins. We identified four family 1 glycosyltransferases (UGTs) that catalyze 3-O-glucosylation of the sapogenins oleanolic acid and hederagenin. Among these, UGT73C10 and UGT73C11 show highest activity, substrate specificity and regiospecificity, and are under positive selection, while UGT73C12 and UGT73C13 show lower substrate specificity and regiospecificity and are under purifying selection. The expression of UGT73C10 and UGT73C11 in different B. vulgaris organs correlates with saponin abundance. Monoglucosylated hederagenin and oleanolic acid were produced in vitro and tested for effects on P. nemorum. 3-O-ß-d-Glc hederagenin strongly deterred feeding, while 3-O-ß-d-Glc oleanolic acid only had a minor effect, showing that hydroxylation of C23 is important for resistance to this herbivore. The closest homolog in Arabidopsis thaliana, UGT73C5, only showed weak activity toward sapogenins. This indicates that UGT73C10 and UGT73C11 have neofunctionalized to specifically glucosylate sapogenins at the C3 position and demonstrates that C3 monoglucosylation activates resistance. As the UGTs from both the resistant and susceptible types of B. vulgaris glucosylate sapogenins and are not located in the known quantitative trait loci for resistance, the difference between the susceptible and resistant plant types is determined at an earlier stage in saponin biosynthesis.