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Aim To evaluate the adherence to the Mediterranean diet (MD) and the level of nutritional literacy (NL) among university students from different academic fields of study, within the context of the COVID-19 pandemic.Methods A total of 1114 first-year undergraduate students at the University of Lisbon, Portugal, were included in this study. A self-administered online questionnaire was applied that included questions regarding sociodemographic information, the MD measured by the PREDIMED questionnaire (PREvención con DIeta MEDiterránea) and NL assessed using the Newest Vital Sign questionnaire.Results The average PREDIMED score revealed a low adherence (6.79±2.14 points) to the MD. Notably, students in the Social Sciences and Humanities academic fields showed the highest level of adherence (U=21 071; p

ABSTRACTSalinity is an important problem for agriculture in the Mediterranean area, and thus, it is essential to develop mitigation strategies to reduce its impact. The main objective of this study was to test the effectiveness of halotolerant plant growth-promoting bacteria (H-PGPB) in improving grapevine salt stress tolerance. Grapevines grafted onto a salt-sensitive rootstock were inoculated with a consortium of H-PGPB. The substrate of half of the plants of each treatment was salinized up to 2 dS m−1. Plants grew for six days under these conditions, and afterward, NaCl was removed to assess plant recovery through growth, physiology, and canopy temperature measurements. Inoculation with H-PGPB had a positive effect on plant physiology, but after salt treatment, grapevines stopped their photosynthetic metabolism, leading to severe defoliation. Remarkably, after salt stress removal, inoculated plants re-sprouted faster, demonstrating that H-PGPB inoculation could be a good practice to increase vineyard resilience to salt stress.

Soil characteristics, land management practices, and plant genotypes influence arbuscular mycorrhizal fungi (AMF) communities, leading to the proliferation of AMF taxa with different growth and nutritional outcomes in their hosts. However, the specific patterns driving these relationships are still not well understood. This study aimed to (1) evaluate the influence of soil characteristics, land use, and rootstock on AMF diversity and community structure and (2) assess the effect of those AMF communities on grapevine growth and nutrition. Soil samples were collected from vineyard and non-agricultural areas in Lisbon and Pegões, Portugal, and trap cultures established using Richter 110 and 1103 Paulsen rootstocks. After 3.5 months growth under greenhouse conditions, root-associated AMF communities were assessed by amplicon metagenomic sequencing using AMF-specific primers. Alpha diversity was only influenced by the soil type, while in β-diversity, an interaction was found between the soil type and land use. Both diversity measures were positively correlated with foliar K and negatively with leaf Mn and Mg. Notably, the concentrations of these nutrients were highly correlated with the relative abundance of operational taxonomic units (OTUs) within the genera Glomus, Rhizophagus, and Claroideoglomus. These results are valuable for supporting AMF selection for improved plant nutrition based on varying soil types and land uses.

High temperature (HT) events have negative impact on wheat grains yield and quality. Transcriptome profiles of wheat developing grains of commercial genotypes (Antequera and Bancal) and landraces (Ardito and Magueija) submitted to heatwave-like treatments during grain filling were evaluated. Landraces showed significantly more differentially expressed genes (DEGs) and presented more similar responses than commercial genotypes. DEGs were more associated with transcription and RNA and protein synthesis in Antequera and with metabolism alterations in Bancal and landraces. Landraces upregulated genes encoding proteins already described as HT responsive, like heat shock proteins and cupins. Apart from the genes encoding HSP, two other genes were upregulated in all genotypes, one encoding for Adenylate kinase, essential for the cellular homeostasis, and the other for ferritin, recently related with increased tolerance to several abiotic stress in Arabidopsis. Moreover, a NAC transcription factor involved in plant development, known to be a negative regulator of starch synthesis and grain yield, was found to be upregulated in both commercial varieties and downregulated in Magueija landrace. The detected diversity of molecular processes involved in heat response of commercial and traditional genotypes contribute to understand the importance of genetic diversity and relevant pathways to cope with these extreme events.

MicroRNAs (miRNAs) are non-coding small RNAs that play crucial roles in plant development and stress responses and can regulate plant interactions with beneficial soil microorganisms such as arbuscular mycorrhizal fungi (AMF). To determine if root inoculation with distinct AMF species affected miRNA expression in grapevines subjected to high temperatures, RNA-seq was conducted in leaves of grapevines inoculated with either Rhizoglomus irregulare or Funneliformis mosseae and exposed to a high-temperature treatment (HTT) of 40 °C for 4 h per day for one week. Our results showed that mycorrhizal inoculation resulted in a better plant physiological response to HTT. Amongst the 195 identified miRNAs, 83 were considered isomiRs, suggesting that isomiRs can be biologically functional in plants. The number of differentially expressed miRNAs between temperatures was higher in mycorrhizal (28) than in non-inoculated plants (17). Several miR396 family members, which target homeobox-leucine zipper proteins, were only upregulated by HTT in mycorrhizal plants. Predicted targets of HTT-induced miRNAs in mycorrhizal plants queried to STRING DB formed networks for Cox complex, and growth and stress-related transcription factors such as SQUAMOSA promoter-binding-like-proteins, homeobox-leucine zipper proteins and auxin receptors. A further cluster related to DNA polymerase was found in R. irregulare inoculated plants. The results presented herein provide new insights into miRNA regulation in mycorrhizal grapevines under heat stress and can be the basis for functional studies of plant-AMF-stress interactions.

Students with chronic conditions (CC) tend to experience several barriers in terms of their school participation and performance. Therefore, the present study aims to explore the factors related to the time of diagnosis of CC (recent/non-recent), the barriers to participation and academic success (health condition, people’s attitude towards CC and school physical environment), the physical and mental health (physical/psychological symptoms and concerns) and school-related variables (relationship with teachers and peers), regarding the school participation of first-year students with CC. This work is part of the HOUSE-Colégio F3 Project, University of Lisbon, which includes 1143 first-year university students from 17 Faculties and Institutes of the University of Lisbon. In this specific study, only the subsample of 207 students with CC was considered, 72.4% of which were female, aged between 18 and 54 years (M = 20.00; SD = 4.83). The results showed that students with a recent diagnosis of CC and students with school participation affected by the CC were those who presented more negative indicators regarding barriers to school participation, physical and mental health, and school-related variables. A greater impact of CC in terms of school participation was associated with having a recent diagnosis, with people’s attitude towards CC and with the health condition as barriers, with more psychological symptoms and worse relationships with teachers and peers. This is a relevant message for the organization of health services for students with CC at the beginning of their university studies, especially since they are often displaced from home and managing their health conditions alone (in many cases, for the first time).

Stressful high temperatures on plants can limit whole-plant function and decrease crop productivity. However, little is known regarding heat stress effects on microtubule cytoskeleton and chromatin in roots from intact plants. Here we studied high temperature effects on cell division, microtubule and chromatin organization patterns in rye root tips from intact plants subjected to 40ºC for 4 h and after different recovery periods (0RT, 7RT, 24 RT). We showed that heat stress induced changes in nuclear morphology as detected by the unusual presence of interphase cells with irregularly shaped nuclei, probably associated with changes in chromosome segregation at anaphase, leading to micronuclei formation as well as changes in the mitotic index. These alterations were associated to differential effects in microtubules organization in both heat-stressed interphase and mitotic cells at 0RT and 7RT. Although no changes in the distribution of H3 phosphorylation of Ser 10 residues on chromatin were found in cells from heat-stressed plants, marked alterations in chromatin DNA methylation patterns were detected. These effects included higher agglutination of 5-methylcytosine domains in both interphase and metaphase cells compared to controls. Taken together these results seem to suggest that alterations in microtubule conformation upon heat stress influences nuclear chromatin organization and cell cycle progression. However, when seedlings recovered from stress (24RT), root tip cells presented microtubule configurations and chromatin organization patterns similar to controls. We conclude that in spite of heat stress markedly altered cell cycle progression and distribution of epigenetic marks, these responses are transient to cope with such stress conditions in the roots.

Wheat is a dietary staple consumed worldwide strongly responsible for proteins and carbohydrate population intake. However, wheat production and quality will scarcely fulfill forward demands, which are compounded by high-temperature (HT) events as heatwaves, increasingly common in Portugal. Thus, landraces assume crucial importance as potential reservoirs of useful traits for wheat breeding and may be pre-adapted to extreme environmental conditions. This work evaluates four Portuguese landrace yield and grain composition through attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, particularly protein content, and their responses to HT treatment mimicking a heatwave. Landraces showed distinct yield traits, especially plant height and first spike grain number, and a similar pattern in FTIR spectra, although revealing differences in grain components’ proportions. Comparison between spectra band intensity indicates that Ardito has the highest protein-related peaks, contrary to Magueija, which appears to be the landrace with higher lipid content. In plants submitted to 1 week of HT treatment 10 days after anthesis, the first spike grain size and weight were markedly reduced in all landraces. Additionally, it was observed that a general increase in grain protein content in the four landraces, being the increment observed in Ardito and Grécia, is statistically significant. The comparative assessment of control and HT average FTIR spectra denoted also the occurrence of alterations in grain polysaccharide composition. An integrated assessment of the evaluations performed revealed that Ardito and Magueija landraces presented diverse yield-related characteristics and distinct responses to cope with HT. In fact, the former landrace revealed considerable grain yield diminution along with an increase in grain protein proportion after HT, while the latter showed a significant increase in spikes and grain number, with grain quality detriment. These results reinforce the relevance of scrutinizing old genotype diversity seeking for useful characteristics, particularly considering HT impact on grain production and quality.

Plant inoculation with arbuscular mycorrhizal fungi (AMF) is increasingly employed to enhance productivity and sustainability in agricultural ecosystems. In the present study, the potential benefits of AMF inoculation on young grapevines replanted in pots containing vineyard soil with high Cu concentration were evaluated. For this purpose, one-year-old cv. Touriga Nacional grapevines grafted onto 1103P rootstocks were further inoculated with Rhizoglomus irregulare or Funneliformis mosseae, or left non-inoculated, and maintained in a sterilized substrate under greenhouse conditions for three months. After this time, half of the plants were transplanted to containers filled with an Arenosol from a vineyard which had been artificially contaminated or not with 300 mg kg−1 of Cu. At the end of the growing season, soil nutrient concentration, soil dehydrogenase activity and mycorrhizal colonization rate were analyzed. Grapevine performance was assessed by measuring several vegetative growth and physiological parameters as well as nutrient concentrations in leaves and roots. In the non-contaminated soil, R. irregulare- and F. mosseae-inoculated plants had significantly greater root biomass than the non-inoculated ones. However, the opposite effect was observed in the Cu-contaminated soil, where non-inoculated plants performed better regarding shoot and root development. Concerning nutrient levels, an increase in Cu, Mg and Mn concentrations was observed in the roots of plants growing in the contaminated soil, although only Mn was translocated to leaves. This led to a large increase in leaf Mn concentrations, which was significantly higher in non-inoculated and F. mosseae- inoculated plants than in the R. irregulare- inoculated ones. Copper contamination induced a general decrease in leaf N, P and Fe concentrations as well as chlorosis symptoms. The largest decrease in N and P was observed in F. mosseae- inoculated plants, with 73 and 31.2%, respectively. However, these plants were the ones with the least decrease in Fe concentration (10% vs. almost 30% in the other two inoculation treatments). In conclusion, this study indicates that soil Cu levels can modify the outcome of AMF inoculations in young grapevines, disclosing new AMF-plant associations potentially relevant in vineyards with a tradition of Cu-based fungicide application.

Heat stress negatively affects several physiological and biochemical processes in grapevine plants. In this work, two new methods, calorespirometry, which has been used to determine temperature adaptation in plants, and near-infrared (NIR) spectroscopy, which has been used to determine several grapevine-related traits and to discriminate among varieties, were tested to evaluate grapevine response to high temperatures. ‘Touriga Nacional’ variety grapevines, inoculated or not with Rhizoglomus irregulare or Funneliformis mosseae, were used in this study. Calorespirometric parameters and NIR spectra, as well as other parameters commonly used to assess heat injury in plants, were measured before and after high temperature exposure. Growth rate and substrate carbon conversion efficiency, calculated from calorespirometric measurements, and stomatal conductance, were the most sensitive parameters for discriminating among high temperature responses of control and inoculated grapevines. The results revealed that, although this vine variety can adapt its physiology to temperatures up to 40 °C, inoculation with R. irregulare could additionally help to sustain its growth, especially after heat shocks. Therefore, the combination of calorespirometry together with gas exchange measurements is a promising strategy for screening grapevine heat tolerance under controlled conditions and has high potential to be implemented in initial phases of plant breeding programs.

Wheat is one of the most important cereals for food and feed, and it is, therefore, necessary to determine the effects of short-term high temperature events (heatwaves) during grain filling. These heatwave events are increasingly common, especially in Portugal. In this work, seven commercial varieties recommended for production in Portugal were submitted to one-week high temperature (HT) treatment ten days after anthesis to evaluate heat effects on grain yield and quality. Grain yield parameters, such as grain number and weight, were evaluated as well as grain composition through attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Variation in HT response between varieties was detected. Grain number and weight tended to decrease in most varieties analyzed. However, two varieties proved to be more resilient since grain number and weight remain unaltered in the Bancal variety, which is the one with better yield results, and even increased in the Pata Negra variety. Regarding grain composition, the comparison between ATR-FTIR spectra of milled grains from control and HT plants revealed alterations in peaks assigned to polysaccharides and proteins. Additionally, a model was built based on nitrogen elemental analysis to predict protein content in flour samples through spectral data that corroborated the differences identified by spectra profile comparison. Moreover, both analyses showed that the intervarietal diversity observed in control conditions was significantly reduced in HT treated plants. The results obtained highlight the intervarietal diversity of wheat response to HT, regarding grain yield parameters, grain composition, and particularly, protein content.

Wheat is undoubtedly one of the most important crops worldwide and it is essential to study how the distinct varieties answer to heat waves associated with climatic changes, in order to design adequate wheat breeding strategies. To assess high temperature (HT) impact in wheat grain characteristics, seven commercial varieties, which have been recommended for production in Portugal, were submitted for one-week HT treatment ten days after anthesis. Firstly, predicted grain technological quality was determined by giving high scores for all varieties studied, based on the allelic compositions of genes encoding high molecular weight glutenins, granule-bound starch synthase and puroindolines. The effects of HT on transcription levels of those genes were, for the first time, evaluated in distinct wheat genotypes, in comparison with control plants. Finally, protein fraction content in mature grains were also estimated in untreated and treated plants. Immature grains from plants, maintained in control conditions, showed significant intervarietal differences in transcription levels of genes associated with grain quality traits, a variability that was significantly reduced in grains from HT treated plants. On the other hand, the influence of HT in mature grain protein-fractions and in gliadin/glutenin ratios revealed intervarietal diversity, even with opposite effects in some varieties. The present study, therefore, discloses marked variability in parameters associated with flour quality between the wheat varieties analyzed, which are differentially affected by HT treatments, similar to heat waves frequently observed in climate change scenarios.

The predicted world population increase along with climate changes threatens sustainable agricultural supply in the coming decades. It is therefore vital to understand crops diversity associated to abiotic stress response. Heat stress is considered one of the major constrains on crops productivity thus it is essential to develop new approaches for a precocious and rigorous evaluation of varietal diversity regarding heat tolerance. Plant cell membrane thermostability (CMS) is a widely used method for wheat thermotolerance assessment although its limitations require complementary solutions. In this work we used CMS assay and explored TdHSP101C genes as an additional tool for durum wheat screening. Genomic and transcriptomic analyses of TdHSP101C genes were performed in varieties with contrasting CMS results and further correlated with heat stress tolerance during fertilization and seed development. Although the durum wheat varieties studied presented a very high homology on TdHSP101C genes (>99%) the transcriptomic assessment allowed the discrimination between varieties with good CMS results and its correlation with differential impacts of heat treatment during inflorescence emergence and seed development on grain yield. The evidences here reported indicate that TdHSP101C transcription levels induced by heat stress in fully expanded leaves may be a promising complementary screening tool to discriminate between durum wheat varieties identified as thermotolerant through CMS.

The genus Avena comprises four distinct genomes organized in diploid (AA or CC), tetraploid (AABB or AACC) and hexaploid species (AACCDD), constituting an interesting model for phylogenetic analysis. The aim of this work was to characterize 45S rDNA intergenic spacer (IGS) variability in distinct species representative of Avena genome diversity-A. strigosa (AA), A. ventricosa (CvCv), A. eriantha (CpCp), A. barbata (AABB), A. murphyi (AACC), A. sativa (AACCDD) and A. sterilis (AACCDD) through the assessment of the 5' external transcribed spacer (5'-ETS), a promising IGS region for phylogenetic studies poorly studied in Avena genus. In this work, IGS length polymorphisms were detected mainly due to distinct 5'-ETS sequence types resulting from major differences in the number and organization of repeated motifs. Although species with A genome revealed a 5'-ETS organization (A-organization) similar to the one previously described in A. sativa, a distinct organization was unraveled in C genome diploid species (C-organization). Interestingly, such new organization presents a higher similarity with other Poaceae species than A-genome sequences, supporting the hypothesis of C-genome being the ancestral Avena genome. Additionally, polyploid species with both genomes mainly retain the A-genome 5'-ETS organization, confirming the preferential elimination of C-genome sequences in Avena polyploid species. Moreover, 5'-ETS sequences phylogenetic analysis consistently clustered the species studied according to ploidy and genomic constitution supporting the use of ribosomal genes to highlight Avena species evolutive pathways.

Bisphenol A (BPA) is a widely utilized endocrine disruptor capable of mimicking endogenous hormones, employed in the manufacture of numerous consumer products, thereby interfering with physiological cellular functions. Recent research has shown that BPA alters epigenetic cellular mechanisms in mammals and may be correlated to enhanced cellular senescence. Here, the effects of BPA at 10 ng/mL and 1 µg/mL, concentrations found in human samples, were analyzed on HT29 human colon adenocarcinona cell line and Human Umbilical Vein Endothelial Cells (HUVEC). Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) transcriptional analysis of the Long Interspersed Element-1 (LINE-1) retroelement showed that BPA induces global transcription deregulation in both cell lines, although with more pronounced effects in HUVEC cells. Whereas there was an increase in global transcription in HT29 exclusively after 24 h of exposure, this chemical had prolonged effects on HUVEC. Immunoblotting revealed that this was not accompanied by alterations in the overall content of H3K9me2 and H3K4me3 epigenetic marks. Importantly, cell viability assays and transcriptional analysis indicated that prolonged BPA exposure affects aging processes in senescent HUVEC. To our knowledge this is the first report that BPA interferes with senescence in primary vascular endothelial cells, therefore, suggesting its association to the etiology of age-related human pathologies, such as atherosclerosis.

Rye (Secale cereale L.) is a cereal crop of major importance in many parts of Europe and rye breeders are presently very concerned with the restrict pool of rye genetic resources available. Such narrowing of rye genetic diversity results from the presence of ‘Petkus’ pool in most modern rye varieties as well as ‘Petkus’ x ‘Carsten’ heterotic pool in hybrid rye breeding programs. Previous studies on rye’s genetic diversity revealed moreover a common genetic background on landraces (ex situ) and cultivars, regardless of breeding level or geographical origin. Thus evaluation of in situ populations is of utmost importance to unveil on farm diversity, which is largely undervalued.Here, we perform the first comprehensive assessment of rye’s genetic diversity and population structuring using cultivars, ex situ landraces along a comprehensive sampling of in situ accessions from Portugal, through a molecular-directed analysis using SSRs markers. Rye genetic diversity and population structure analysis does not present any geographical trend but disclosed marked differences between genetic backgrounds of in situ accessions and those of cultivars/ex situ collections. Such genetic distinctiveness of in situ accessions highlights their unexplored potential as new genetic resources, which can be used to boost rye breeding strategies and the production of new varieties. Overall, our study successfully demonstrates the high prospective impact of comparing genetic diversity and structure of cultivars, ex situ and in situ samples in ascertaining the status of plant genetic resources.

Faba bean (Vicia faba L.) is a facultative cross-pollinating legume crop with a great importance for food and feed due to its high protein content as well as the important role in soil fertility and nitrogen fixation. In this work we evaluated genetic diversity and population structure of faba bean accessions from the Western Mediterranean basin and wild related species. For that purpose we screened 53 V. faba, 2 V. johannis and 7 V. narbonensis accessions from Portugal, Spain and Morocco with 28 faba bean Single Sequence Repeats (SSR). SSR genotyping showed that the number of alleles detected per locus for the polymorphic markers ranged between 2 and 10, with Polymorphic Information Content (PIC) values between 0.662 and 0.071, and heterozygosity (HO) between 0-0.467. Heterozygosity and inbreeding coefficient levels indicate a higher level of inbreeding in wild related species than in cultivated Vicia. The analysis of molecular variance (AMOVA) showed a superior genetic diversity within accessions than between accessions even from distant regions. These results are in accordance to population structure analysis showing that individuals from the same accession can be genetically more similar to individuals from far away accessions, than from individuals from the same accession. In all three levels of analysis (whole panel of cultivated and wild accessions, cultivated faba bean accessions and Portuguese accessions) no population structure was observed based on geography or climatic factors. Differences between V. narbonensis and V. johannis are undetectable although these wild taxa are clearly distinct from V. faba accessions. Thus, a limited gene flow occurred between cultivated accessions and wild relatives. Contrastingly, the lack of population structure seems to indicate a high degree of gene flow between V. faba accessions, possibly explained by the partially allogamous habit in association with frequent seed exchange/introduction.

The union of different genomes in the same nucleus frequently results in hybrid genotypes with improved genome plasticity related to both genome remodeling events and changes in gene expression. Most modern cereal crops are polyploid species. Triticale, synthesized by the cross between wheat and rye, constitutes an excellent model to study polyploidization functional implications. We intend to attain a deeper knowledge of dispersed repetitive sequence involvement in parental genome reshuffle in triticale and in wheat-rye addition lines that have the entire wheat genome plus each rye chromosome pair. Through Random Amplified Polymorphic DNA (RAPD) analysis with OPH20 10-mer primer we unraveled clear alterations corresponding to the loss of specific bands from both parental genomes. Moreover, the sequential nature of those events was revealed by the increased absence of rye-origin bands in wheat-rye addition lines in comparison with triticale. Remodeled band sequencing revealed that both repetitive and coding genome domains are affected in wheat-rye hybrid genotypes. Additionally, the amplification and sequencing of pSc20H internal segments showed that the disappearance of parental bands may result from restricted sequence alterations and unraveled the involvement of wheat/rye related repetitive sequences in genome adjustment needed for hybrid plant stabilization.

Faba bean have been grown in Portugal for a long time and locally adapted populations are still maintained on farm. The genetic diversity of four Portuguese faba bean populations that are still cultivated in some regions of the country was evaluated using the Inter Retrotransposons Amplified Polymorphism (IRAP) technique. It was shown that molecular markers based on retrotransposons previously identified in other species can be efficiently used in the genetic variability assessment of Vicia faba. The IRAP experiment targeting Athila yielded the most informative banding patterns. Cluster analysis using the neighbor-joining algorithm generated a dendrogram that clearly shows the distribution pattern of V. faba samples. The four equina accessions are separated from each other and form two distinct clades while the two major faba bean accessions are not unequivocally separated by the IRAP. Fluorescent In Situ Hybridization (FISH) analysis of sequences amplified by IRAP Athila revealed a wide distribution throughout V. faba chromosomes, confirming the whole-genome coverage of this molecular marker. Morphological characteristics were also assessed through cluster analysis of seed characters using the unweighted pair group method arithmetic average (UPGMA) and principal component analysis (PCA), showing a clear discrimination between faba bean major and equina groups. It was also found that the seed character most relevant to distinguish accessions was 100 seed weight. Seed morphological traits and IRAP evaluation give similar results supporting the potential of IRAP analysis for genetic diversity studies.

Genomic diversity of Portuguese accessions of Avena species—diploid A. strigosa and hexaploids A. sativa and A. sterilis—was evaluated through molecular and cytological analysis of 45S rDNA, and other repetitive sequences previously studied in cereal species—rye subtelomeric sequence (pSc200) and cereal centromeric sequence (CCS1). Additionally, retrotransposons and microsatellites targeting methodologies—IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism)—were performed. A very high homology was detected for ribosomal internal transcribed sequences (ITS1 and ITS2) between the species analyzed, although nucleolar organizing regions (NOR) fluorescent in situ hybridization (FISH) analysis revealed distinct number of Nor loci between diploid and hexaploid species. Moreover, morphological diversity, evidenced by FISH signals with different sizes, was observed between distinct accessions within each species. pSc200 sequences were for the first time isolated from Avena species but proven to be highly similar in all genotypes analyzed. The use of primers designed for CCS1 unraveled a sequence homologous to the Ty3/gypsy retrotransposon Cereba, that was mapped to centromeric regions of diploid and hexaploid species, being however restricted to the more related A and D haplomes. Retrotransposon-based methodologies disclosed species- and accessions-specific bands essential for the accurate discrimination of all genotypes studied. Centromeric, IRAP and REMAP profiles therefore allowed accurate assessment of inter and intraspecific variability, demonstrating the potential of these molecular markers on future oat breeding programs.

BackgroundPolyploidization is a major evolutionary process in plants where hybridization and chromosome doubling induce enormous genomic stress and can generate genetic and epigenetic modifications. However, proper evaluation of DNA sequence restructuring events and the precise characterization of sequences involved are still sparse.Methodology/principal findingsInter Retrotransposons Amplified Polymorphism (IRAP), Retrotransposons Microsatellite Amplified Polymorphism (REMAP) and Inter Simple Sequence Repeat (ISSR) largely confirmed the absence of any intraspecific variation in wheat, rye and triticale. The comparative analysis of banding profiles between wheat and rye inbred lines revealed 34% of monomorphic (common to both parental species) bands for the ten different primer combinations used. The analysis of triticale plants uncovered nearly 51% of rearranged bands in the polyploid, being the majority of these modifications, due to the loss of rye bands (83%). Sequence analysis of rye fragments absent in triticale revealed for instance homology with hydroxyproline-rich glycoproteins (HRGP), a protein that belongs to a major family of inducible defence response proteins. Conversely, a wheat-specific band absent in triticale comprises a nested structure of copia-like retrotransposons elements, namely Claudia and Barbara. Sequencing of a polyploid-specific band (absent in both parents) revealed a microsatellite related sequence. Cytological studies using Fluorescent In Situ Hybridization (FISH) with REMAP products revealed a widespread distribution of retrotransposon and/or microsatellite flanking sequences on rye chromosomes, with a preferential accumulation in heterochromatic sub-telomeric domains.Conclusions/significanceHere, we used PCR-based molecular marker techniques involving retrotransposons and microsatellites to uncover polyploidization induced genetic restructuring in triticale. Sequence analysis of rearranged genomic fragments either from rye or wheat origin showed these to be retrotransposon-related as well as coding sequences. Further FISH analysis revealed possible chromosome hotspots for sequence rearrangements. The role of chromatin condensation on the origin of genomic rearrangements mediated by polyploidization in triticale is also discussed.

BACKGROUND: Chromatin organizational and topological plasticity, and its functions in gene expression regulation, have been strongly revealed by the analysis of nucleolar dominance in hybrids and polyploids where one parental set of ribosomal RNA (rDNA) genes that are clustered in nucleolar organizing regions (NORs), is rendered silent by epigenetic pathways and heterochromatization. However, information on the behaviour of dominant NORs is very sparse and needed for an integrative knowledge of differential gene transcription levels and chromatin specific domain interactions. METHODOLOGY/PRINCIPAL FINDINGS: Using molecular and cytological approaches in a wheat-rye addition line (wheat genome plus the rye nucleolar chromosome pair 1R), we investigated transcriptional activity and chromatin topology of the wheat dominant NORs in a nucleolar dominance situation. Herein we report dominant NORs up-regulation in the addition line through quantitative real-time PCR and silver-staining technique. Accompanying this modification in wheat rDNA trascription level, we also disclose that perinucleolar knobs of ribosomal chromatin are almost transcriptionally silent due to the residual detection of BrUTP incorporation in these domains, contrary to the marked labelling of intranucleolar condensed rDNA. Further, by comparative confocal analysis of nuclei probed to wheat and rye NORs, we found that in the wheat-rye addition line there is a significant decrease in the number of wheat-origin perinucleolar rDNA knobs, corresponding to a diminution of the rDNA heterochromatic fraction of the dominant (wheat) NORs. CONCLUSIONS/SIGNIFICANCE: We demonstrate that inter-specific interactions leading to wheat-origin NOR dominance results not only on the silencing of rye origin NOR loci, but dominant NORs are also modified in their transcriptional activity and interphase organization. The results show a cross-talk between wheat and rye NORs, mediated by ribosomal chromatin dynamics, revealing a conceptual shift from differential amphiplasty to 'mutual amphiplasty' in the nucleolar dominance process.

Nucleolar dominance is an epigenetic phenomenon in plant and animal genetic hybrids that describes the expression of 45S ribosomal RNA genes (rRNA genes) inherited from only one progenitor due to the silencing of the other progenitor's rRNA genes. rRNA genes are tandemly arrayed at nucleolus organizer regions (NORs) that span millions of basepairs, thus gene silencing in nucleolar dominance occurs on a scale second only to X-chromosome inactivation in female mammals. In Arabidopsis suecica, the allotetraploid hybrid of A. thaliana and A. arenosa, the A. thaliana -derived rRNA genes are subjected to nucleolar dominance and are silenced via repressive chromatin modifications. However, the developmental stage at which nucleolar dominance is established in A. suecica is currently unknown. We show that nucleolar dominance is not apparent in seedling cotyledons formed during embryogenesis but becomes progressively established during early postembryonic development in tissues derived from both the shoot and root apical meristems. The progressive silencing of A. thaliana rRNA genes correlates with the transition of A. thaliana NORs from a decondensed euchromatic state associated with histone H3 that is trimethylated on lysine 4 (H3K4me3) to a highly condensed heterochromatic state in which the NORs are associated with H3K9me2 and 5-methylcytosine-enriched chromocenters. In RNAi-lines in which the histone deacetylases HDA6 and HDT1 are knocked down, the developmentally regulated condensation and inactivation of A. thaliana NORs is disrupted. Collectively, these data demonstrate that HDA6 and HDT1 function in the postembryonic establishment of nucleolar dominance, a process which recurs in each generation.