Migration without interbreeding: Evolutionary history of a highly selfing Mediterranean grass inferred from whole genomes.

Wild plant populations show extensive genetic subdivision and are far from the ideal of panmixia which permeates population genetic theory. Understanding the spatial and temporal scale of population structure is therefore fundamental for empirical population genetics - and of interest in itself, as it yields insights into the history and biology of a species. In this study we extend the genomic resources for the wild Mediterranean grass Brachypodium distachyon to investigate the scale of population structure and its underlying history at whole-genome resolution. A total of 86 accessions were sampled at local and regional scales in Italy and France, which closes a conspicuous gap in the collection for this model organism. The analysis of 196 accessions, spanning the Mediterranean from Spain to Iraq, suggests that the interplay of high selfing and seed dispersal rates has shaped genetic structure in B. distachyon. At the continental scale, the evolution in B. distachyon is characterized by the independent expansion of three lineages during the Upper Pleistocene. Today, these lineages may occur on the same meadow yet do not interbreed. At the regional scale, dispersal and selfing interact and maintain high genotypic diversity, thus challenging the textbook notion that selfing in finite populations implies reduced diversity. Our study extends the population genomic resources for B. distachyon and suggests that an important use of this wild plant model is to investigate how selfing and dispersal, two processes typically studied separately, interact in colonizing plant species.

Metabolomic Variation Aligns with Two Geographically Distinct Subpopulations of Brachypodium Distachyon before and after Drought Stress.

Brachypodium distachyon (Brachypodium) is a non-domesticated model grass that has been used to assess population level genomic variation. We have previously established a collection of 55 Brachypodium accessions that were sampled to reflect five different climatic regions of Turkey; designated 1a, 1c, 2, 3 and 4. Genomic and methylomic variation differentiated the collection into two subpopulations designated as coastal and central (respectively from regions 1a, 1c and the other from 2, 3 and 4) which were linked to environmental variables such as relative precipitation. Here, we assessed how far genomic variation would be reflected in the metabolomes and if this could be linked to an adaptive trait. Metabolites were extracted from eight-week-old seedlings from each accession and assessed using flow infusion high-resolution mass spectrometry (FIE-HRMS). Principal Component Analysis (PCA) of the derived metabolomes differentiated between samples from coastal and central subpopulations. The major sources of variation between seedling from the coastal and central subpopulations were identified. The central subpopulation was typified by significant increases in alanine, aspartate and glutamate metabolism and the tricarboxylic acid (TCA) cycle. Coastal subpopulation exhibited elevated levels of the auxin, indolacetic acid and rhamnose. The metabolomes of the seedling were also determined following the imposition of drought stress for seven days. The central subpopulation exhibited a metabolomic shift in response to drought, but no significant changes were seen in the coastal one. The drought responses in the central subpopulation were typified by changes in amino acids, increasing the glutamine that could be functioning as a stress signal. There were also changes in sugars that were likely to be an osmotic counter to drought, and changes in bioenergetic metabolism. These data indicate that genomic variation in our Turkish Brachypodium collection is largely reflected as distinctive metabolomes ("metabolotypes") through which drought tolerance might be mediated.

Allotetraploidization in Brachypodium May Have Led to the Dominance of One Parent's Metabolome in Germinating Seeds.

Seed germination is a complex process during which a mature seed resumes metabolic activity to prepare for seedling growth. In this study, we performed a comparative metabolomic analysis of the embryo and endosperm using the community standard lines of three annual Brachypodium species, i.e., B. distachyon (Bd) and B. stacei (Bs) and their natural allotetraploid B. hybridum (BdBs) that has wider ecological range than the other two species. We explored how far the metabolomic impact of allotetraploidization would be observable as over-lapping changes at 4, 12, and 24 h after imbibition (HAI) with water when germination was initiated. Metabolic changes during germination were more prominent in Brachypodium embryos than in the endosperm. The embryo and endosperm metabolomes of Bs and BdBs were similar, and those of Bd were distinctive. The Bs and BdBs embryos showed increased levels of sugars and the tricarboxylic acid cycle compared to Bd, which could have been indicative of better nutrient mobilization from the endosperm. Bs and BdBs also showed higher oxalate levels that could aid nutrient transfer through altered cellular events. In Brachypodium endosperm, the thick cell wall, in addition to starch, has been suggested to be a source of nutrients to the embryo. Metabolites indicative of sugar metabolism in the endosperm of all three species were not prominent, suggesting that mobilization mostly occurred prior to 4 HAI. Hydroxycinnamic and monolignol changes in Bs and BdBs were consistent with cell wall remodeling that arose following the release of nutrients to the respective embryos. Amino acid changes in both the embryo and endosperm were broadly consistent across the species. Taking our data together, the formation of BdBs may have maintained much of the Bs metabolome in both the embryo and endosperm during the early stages of germination. In the embryo, this conserved Bs metabolome appeared to include an elevated sugar metabolism that played a vital role in germination. If these observations are confirmed in the future with more Brachypodium accessions, it would substantiate the dominance of the Bs metabolome in BdBs allotetraploidization and the use of metabolomics to suggest important adaptive changes.

Defining the Cell Wall, Cell Cycle and Chromatin Landmarks in the Responses of Brachypodium distachyon to Salinity.

Excess salinity is a major stress that limits crop yields. Here, we used the model grass Brachypodium distachyon (Brachypodium) reference line Bd21 in order to define the key molecular events in the responses to salt during germination. Salt was applied either throughout the germination period ("salt stress") or only after root emergence ("salt shock"). Germination was affected at ≥100 mM and root elongation at ≥75 mM NaCl. The expression of arabinogalactan proteins (AGPs), FLA1, FLA10, FLA11, AGP20 and AGP26, which regulate cell wall expansion (especially FLA11), were mostly induced by the "salt stress" but to a lesser extent by "salt shock". Cytological assessment using two AGP epitopes, JIM8 and JIM13 indicated that "salt stress" increases the fluorescence signals in rhizodermal and exodermal cell wall. Cell division was suppressed at >75 mM NaCl. The cell cycle genes (CDKB1, CDKB2, CYCA3, CYCB1, WEE1) were induced by "salt stress" in a concentration-dependent manner but not CDKA, CYCA and CYCLIN-D4-1-RELATED. Under "salt shock", the cell cycle genes were optimally expressed at 100 mM NaCl. These changes were consistent with the cell cycle arrest, possibly at the G1 phase. The salt-induced genomic damage was linked with the oxidative events via an increased glutathione accumulation. Histone acetylation and methylation and DNA methylation were visualized by immunofluorescence. Histone H4 acetylation at lysine 5 increased strongly whereas DNA methylation decreased with the application of salt. Taken together, we suggest that salt-induced oxidative stress causes genomic damage but that it also has epigenetic effects, which might modulate the cell cycle and AGP expression gene. Based on these landmarks, we aim to encourage functional genomics studies on the responses of Brachypodium to salt.

MS Analysis of DNA Modifications in Urinary/Body Fluids.

Analytical techniques based on mass spectrometry allow to analyze DNA modifications in body fluids. Here we describe two chromatographic methods that can be used for the simultaneous determination of the modified DNA bases and nucleosides in the same urine sample: isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) and high-performance liquid chromatography coupled with gas chromatography and mass spectrometry (HPLC/GC/MS).

Preparation of Internal Standards for 2D-UPLC-MS/MS Quantification of Noncanonical DNA Bases.

Reliable quantitative analysis of DNA modification using liquid chromatography coupled with tandem mass spectrometry requires stable isotope-labeled internal standards. Only some of them are commercially available. Here we present a method allowing for the synthesis of [13C10,15N2]-5-methyl-2'-deoxycytidine from [13C10,15N2]-2'-deoxythymidine. We also describe an approach for the oxidation of [13C10,15N2]-5-methyl-2'-deoxycytidine and [13C10,15N2]-2'-deoxythymidine with Na2S2O8, leading to the generation of [13C10,15N2]-5-formyl-2'-deoxycytidine, [13C10,15N2]-5-carboxy-2'-deoxycytidine or [13C10,15N2]-5-(hydroxymethyl)-2'-deoxyuridine, correspondingly. Moreover, we provide optimized protocols for the oxidation of [13C5,15N2]-thymine to [13C10,15N2]-5-hydroxymethyluracil, [13C10,15N2]-5-formyluracil, and [13C10,15N2]-5-carboxyuracil using Na2S2O8.

Genetic and Methylome Variation in Turkish Brachypodium Distachyon Accessions Differentiate Two Geographically Distinct Subpopulations.

Brachypodium distachyon (Brachypodium) is a non-domesticated model grass species that can be used to test if variation in genetic sequence or methylation are linked to environmental differences. To assess this, we collected seeds from 12 sites within five climatically distinct regions of Turkey. Seeds from each region were grown under standardized growth conditions in the UK to preserve methylated sequence variation. At six weeks following germination, leaves were sampled and assessed for genomic and DNA methylation variation. In a follow-up experiment, phenomic approaches were used to describe plant growth and drought responses. Genome sequencing and population structure analysis suggested three ancestral clusters across the Mediterranean, two of which were geographically separated in Turkey into coastal and central subpopulations. Phenotypic analyses showed that the coastal subpopulation tended to exhibit relatively delayed flowering and the central, increased drought tolerance as indicated by reduced yellowing. Genome-wide methylation analyses in GpC, CHG and CHH contexts also showed variation which aligned with the separation into coastal and central subpopulations. The climate niche modelling of both subpopulations showed a significant influence from the "Precipitation in the Driest Quarter" on the central subpopulation and "Temperature of the Coldest Month" on the coastal subpopulation. Our work demonstrates genetic diversity and variation in DNA methylation in Turkish accessions of Brachypodium that may be associated with climate variables and the molecular basis of which will feature in ongoing analyses.

Relationships between soil parameters and physiological status of Miscanthus x giganteus cultivated on soil contaminated with trace elements under NPK fertilisation vs. microbial inoculation.

Crop growth and development can be influenced by a range of parameters, soil health, cultivation and nutrient status all play a major role. Nutrient status of plants can be enhanced both through chemical fertiliser additions (e.g. N, P, K supplementation) or microbial fixation and mobilisation of naturally occurring nutrients. With current EU priorities discouraging the production of biomass on high quality soils there is a need to investigate the potential of more marginal soils to produce these feedstocks and the impacts of soil amendments on crop yields within them. This study investigated the potential for Miscanthus x giganteus to be grown in trace element (TE)-contaminated soils, ideally offering a mechanism to (phyto)manage these contaminated lands. Comprehensive surveys are needed to understand plant-soil interactions under these conditions. Here we studied the impacts of two fertiliser treatments on soil physico-chemical properties under Miscanthus x giganteus cultivated on Pb, Cd and Zn contaminated arable land. Results covered a range of parameters, including soil rhizosphere activity, arbuscular mycorrhization (AM), as well as plant physiological parameters associated with photosynthesis, TE leaf concentrations and growth performance. Fertilization increased growth and gas exchange capacity, enhanced rhizosphere microbial activity and increased Zn, Mg and N leaf concentration. Fertilization reduced root colonisation by AMF and caused higher chlorophyll concentration in plant leaves. Microbial inoculation seems to be a promising alternative for chemical fertilizers, especially due to an insignificant influence on the mobility of toxic trace elements (particularly Cd and Zn).