Association genetics of phenolic needle compounds in Norway spruce with variable susceptibility to needle bladder rust.

KEY MESSAGE: Accumulation of phenolic needle metabolites in Norway spruce is regulated by many genes with small and additive effects and is correlated with the susceptibility against fungal attack. Norway spruce accumulates high foliar concentrations of secondary phenolic metabolites, with important functions for pathogen defence responses. However, the molecular genetic basis underlying the quantitative variation of phenolic compounds and their role in enhanced resistance of spruce to infection by needle bladder rust are unknown. To address these questions, a set of 1035 genome-wide single nucleotide polymorphisms (SNPs) was associated to the quantitative variation of four simple phenylpropanoids, eight stilbenes, nine flavonoids, six related arithmetic parameters and the susceptibility to infection by Chrysomyxa rhododendri in an unstructured natural population of Norway spruce. Thirty-one significant genetic associations for the flavonoids gallocatechin, kaempferol 3-glucoside and quercetin 3-glucoside and the stilbenes resveratrol, piceatannol, astringin and isorhapontin were discovered, explaining 22-59% of phenotypic variation, and indicating a regulation of phenolic accumulation by many genes with small and additive effects. The phenolics profile differed between trees with high and low susceptibility to the fungus, underlining the importance of phenolic compounds in the defence mechanisms of Norway spruce to C. rhododendri. Results highlight the utility of association studies in non-model tree species and may enable marker-assisted selection of Norway spruce adapted to severe pathogen attack.

Spatial and Ecological Drivers of Genetic Structure in Greek Populations of Alkanna tinctoria (Boraginaceae), a Polyploid Medicinal Herb.

Background and Aims: Quantifying genetic variation is fundamental to understand a species' demographic trajectory and its ability to adapt to future changes. In comparison with diploids, however, genetic variation and factors fostering genetic divergence remain poorly studied in polyploids due to analytical challenges. Here, by employing a ploidy-aware framework, we investigated the genetic structure and its determinants in polyploid Alkanna tinctoria (Boraginaceae), an ancient medicinal herb that is the source of bioactive compounds known as alkannin and shikonin (A/S). From a practical perspective, such investigation can inform biodiversity management strategies. Methods: We collected 14 populations of A. tinctoria within its main distribution range in Greece and genotyped them using restriction site-associated DNA sequencing. In addition, we included two populations of A. sieberi. By using a ploidy-aware genotype calling based on likelihoods, we generated a dataset of 16,107 high-quality SNPs. Classical and model-based analysis was done to characterize the genetic structure within and between the sampled populations, complemented by genome size measurements and chromosomal counts. Finally, to reveal the drivers of genetic structure, we searched for associations between allele frequencies and spatial and climatic variables. Key Results: We found support for a marked regional structure in A. tinctoria along a latitudinal gradient in line with phytogeographic divisions. Several analyses identified interspecific admixture affecting both mainland and island populations. Modeling of spatial and climatic variables further demonstrated a larger contribution of neutral processes and a lesser albeit significant role of selection in shaping the observed genetic structure in A. tinctoria. Conclusion: Current findings provide evidence of strong genetic structure in A. tinctoria mainly driven by neutral processes. The revealed natural genomic variation in Greek Alkanna can be used to further predict variation in A/S production, whereas our bioinformatics approach should prove useful for the study of other non-model polyploid species.

Epigenetic Variability Among Saffron Crocus (Crocus sativus L.) Accessions Characterized by Different Phenotypes.

This work represents the first epigenomic study carried out on saffron crocus. Five accessions of saffron, showing differences in tepal pigmentation, yield of saffron and flowering time, were analyzed at the epigenetic level by applying a methylation-sensitive restriction enzyme-sequencing (MRE-seq) approach. Five accession-specific hypomethylomes plus a reference hypomethylome, generated by combining the sequence data from the single accessions, were obtained. Assembled sequences were annotated against existing online databases. In the absence of the Crocus genome, the rice genome was mainly used as the reference as it is the best annotated genome among monocot plants. Comparison of the hypomethylomes revealed many differentially methylated regions, confirming the high epigenetic variability present among saffron accessions, including sequences encoding for proteins that could be good candidates to explain the accessions' alternative phenotypes. In particular, transcription factors involved in flowering process (MADS-box and TFL) and for the production of pigments (MYB) were detected. Finally, by comparing the generated sequences of the different accessions, a high number of SNPs, likely having arisen as a consequence of the prolonged vegetative propagation, were detected, demonstrating surprisingly high genetic variability. Gene ontology (GO) was performed to map and visualize sequence polymorphisms located within the GOs and to compare their distributions among different accessions. As well as suggesting the possible existence of alternative phenotypes with a genetic basis, a clear difference in polymorphic GO is present among accessions based on their geographic origin, supporting a possible signature of selection in the Indian accession with respect to the Spanish ones.

Development of novel microsatellite markers for Alkanna tinctoria by comparative transcriptomics.

PREMISE: Alkanna tinctoria (Boraginaceae) is an important medicinal herb with its main distribution across the Mediterranean region. To reveal its genetic variation and population structure, microsatellite markers were developed and validated in four Greek populations. METHODS AND RESULTS: RNA-Seq data of the related species Arnebia euchroma and Echium plantagineum were assembled and mined to identify conserved ortholog sets containing simple sequence repeat motifs. Fifty potential loci were identified and then tested on A. tinctoria, of which 17 loci were polymorphic. The number of alleles ranged from one to nine, and the levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and 0.000 to 0.820, respectively. Most of these loci could be successfully amplified in eight other species of Boraginaceae (Alkanna graeca, A. hellenica, A. sfikasiana, Echium vulgare, E. plantagineum, Lithospermum officinale, Borago officinalis, and Anchusa officinalis). CONCLUSIONS: This study provides the first set of microsatellite loci for studying the genetic variation and population structure of A. tinctoria and shows their potential usefulness in other Boraginaceae species.

Drought Sensitivity of Norway Spruce at the Species' Warmest Fringe: Quantitative and Molecular Analysis Reveals High Genetic Variation Among and Within Provenances.

Norway spruce (Picea abies) is by far the most important timber species in Europe, but its outstanding role in future forests is jeopardized by its high sensitivity to drought. We analyzed drought response of Norway spruce at the warmest fringe of its natural range. Based on a 35-year old provenance experiment we tested for genetic variation among and within seed provenances across consecutively occurring strong drought events using dendroclimatic time series. Moreover, we tested for associations between ≈1,700 variable SNPs and traits related to drought response, wood characteristics and climate-growth relationships. We found significant adaptive genetic variation among provenances originating from the species' Alpine, Central and Southeastern European range. Genetic variation between individuals varied significantly among provenances explaining up to 44% of the phenotypic variation in drought response. Varying phenotypic correlations between drought response and wood traits confirmed differences in selection intensity among seed provenances. Significant associations were found between 29 SNPs and traits related to drought, climate-growth relationships and wood properties which explained between 11 and 43% of trait variation, though 12 of them were due to single individuals having extreme phenotypes of the respective trait. The majority of these SNPs are located within exons of genes and the most important ones are preferentially expressed in cambium and xylem expansion layers. Phenotype-genotype associations were stronger if only provenances with significant quantitative genetic variation in drought response were considered. The present study confirms the high adaptive variation of Norway spruce in Central and Southeastern Europe and demonstrates how quantitative genetic, dendroclimatic and genomic data can be linked to understand the genetic basis of adaptation to climate extremes in trees.

Identification and characterization of the Fasciola hepatica sodium- and chloride-dependent taurine transporter.

The parasitic liver fluke Fasciola hepatica infests mainly ruminants, but it can also cause fasciolosis in people, who ingest the metacercariae encysted on plants. The drug of choice to treat fasciolosis is triclabendazole (TBZ), which has been on the market for several decades. This is also true for the other available drugs. Accordingly, drug-resistant flukes have been emerging at an increasing rate making it desirable to identify alternative drug targets. Here, we focused on the fact that adult F. hepatica persists in the hostile environment of the bile ducts of infected organisms. A common way to render bile acids less toxic is to conjugate them to taurine (2-aminoethanesulfonic acid). We cloned a transporter from the solute carrier-6 (SLC6) family, which was most closely related to the GABA-transporter-2 of other organisms. When heterologously expressed, this F. hepatica transporter supported the high-affinity cellular uptake of taurine (KM = 12.0 ± 0.5 µM) but not of GABA. Substrate uptake was dependent on Na+- and Cl- (calculated stoichiometry 2:1). Consistent with the low chloride concentration in mammalian bile, the F. hepatica transporter had a higher apparent affinity for Cl- (EC50 = 14±3 mM) than the human taurine transporter (EC50 = 55±7 mM). We incubated flukes with unconjugated bile acids in the presence and absence of taurine: taurine promoted survival of flukes; the taurine transporter inhibitor guanidinoethansulfonic acid abolished this protective effect of taurine. Based on these observations, we conclude that the taurine transporter is critical for the survival of liver flukes in the bile. Thus, the taurine transporter represents a candidate drug target.

Selecting Hypomethylated Genomic Regions Using MRE-Seq.

Here, we describe a method capable of filtering the hypomethylated part of plant genomes, the so-called hypomethylome. The principle of the method is based on the filtration and sequence analysis of small DNA fragments generated by methylation-sensitive four-cutter restriction endonucleases, possessing ((5me))CpG motifs in their recognition sites. The majority of these fragments represent genes and their flanking regions containing also regulatory elements-the gene space of the genome. Besides the enrichment of the gene space, another advantage of the method is the simultaneous depletion of repetitive elements due to their methylated nature and its easy application on complex and large plant genomes. Additionally to the wet lab procedure, we describe how to analyze the data using bioinformatics methods and how to apply the method to comparative studies.

How to Isolate a Plant's Hypomethylome in One Shot.

Genome assembly remains a challenge for large and/or complex plant genomes due to their abundant repetitive regions resulting in studies focusing on gene space instead of the whole genome. Thus, DNA enrichment strategies facilitate the assembly by increasing the coverage and simultaneously reducing the complexity of the whole genome. In this paper we provide an easy, fast, and cost-effective variant of MRE-seq to obtain a plant's hypomethylome by an optimized methyl filtration protocol followed by next generation sequencing. The method is demonstrated on three plant species with knowingly large and/or complex (polyploid) genomes: Oryza sativa, Picea abies, and Crocus sativus. The identified hypomethylomes show clear enrichment for genes and their flanking regions and clear reduction of transposable elements. Additionally, genomic sequences around genes are captured including regulatory elements in introns and up- and downstream flanks. High similarity of the results obtained by a de novo assembly approach with a reference based mapping in rice supports the applicability for studying and understanding the genomes of nonmodel organisms. Hence we show the high potential of MRE-seq in a wide range of scenarios for the direct analysis of methylation differences, for example, between ecotypes, individuals, within or across species harbouring large, and complex genomes.