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Traditional methods for the analysis of repeat expansions, which underlie genetic disorders, such as fragile X syndrome (FXS), lack single-nucleotide resolution in repeat analysis and the ability to characterize causative variants outside the repeat array. These drawbacks can be overcome by long-read and short-read sequencing, respectively. However, the routine application of next-generation sequencing in the clinic requires target enrichment, and none of the available methods allows parallel analysis of long-DNA fragments using both sequencing technologies. In this study, we investigated the use of indirect sequence capture (Xdrop technology) coupled to Nanopore and Illumina sequencing to characterize FMR1, the gene responsible of FXS. We achieved the efficient enrichment (> 200×) of large target DNA fragments (~60–80 kbp) encompassing the entire FMR1 gene. The analysis of Xdrop-enriched samples by Nanopore long-read sequencing allowed the complete characterization of repeat lengths in samples with normal, pre-mutation, and full mutation status (> 1 kbp), and correctly identified repeat interruptions relevant for disease prognosis and transmission. Single-nucleotide variants (SNVs) and small insertions/deletions (indels) could be detected in the same samples by Illumina short-read sequencing, completing the mutational testing through the identification of pathogenic variants within the FMR1 gene, when no typical CGG repeat expansion is detected. The study successfully demonstrated the parallel analysis of repeat expansions and SNVs/indels in the FMR1 gene at single-nucleotide resolution by combining Xdrop enrichment with two next-generation sequencing approaches. With the appropriate optimization necessary for the clinical settings, the system could facilitate both the study of genotype–phenotype correlation in FXS and enable a more efficient diagnosis and genetic counseling for patients and their relatives.

Helianthus tuberosus L., known as the Jerusalem artichoke, is a hexaploid plant species, adapted to low-nutrient soils, that accumulates high levels of inulin in its tubers. Inulin is a fructose-based polysaccharide used either as dietary fiber or for the production of bioethanol. Key enzymes involved in inulin biosynthesis are well known. However, the gene networks underpinning tuber development and inulin accumulation in H. tuberous remain elusive. To fill this gap, we selected 6,365 expressed sequence tags (ESTs) from an H. tuberosus library to set up a microarray platform and record their expression across three tuber developmental stages, when rhizomes start enlarging (T0), at maximum tuber elongation rate (T3), and at tuber physiological maturity (Tm), in “VR” and “K8-HS142”clones. The former was selected as an early tuberizing and the latter as a late-tuberizing clone. We quantified inulin and starch levels, and qRT-PCR confirmed the expression of critical genes accounting for inulin biosynthesis. The microarray analysis revealed that the differences in morphological and physiological traits between tubers of the two clones are genetically determined since T0 and that is relatively low the number of differentially expressed ESTs across the stages shared between the clones (93). The expression of ESTs for sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT), the two critical genes for fructans polymerization, resulted to be temporarily synchronized and mirror the progress of inulin accumulation and stretching. The expression of ESTs for starch biosynthesis was insignificant throughout the developmental stages of the clones in line with the negligible level of starch into their mature tubers, where inulin was the dominant polysaccharide. Overall, our study disclosed candidate genes underpinning the development and storage of carbohydrates in the tubers of two H. tuberosus clones. A model according to which the steady-state levels of 1-SST and 1-FFT transcripts are developmentally controlled and might represent a limiting factor for inulin accumulation has been provided. Our finding may have significant repercussions for breeding clones with improved levels of inulin for food and chemical industry.

Several research studies were focused to understand how grapevine cultivars respond to environment; nevertheless, the biological mechanisms tuning this phenomenon need to be further deepened. Particularly, the molecular processes underlying the interplay between clones of the same cultivar and environment were poorly investigated. To address this issue, we analyzed the transcriptome of berries from three "Nebbiolo" clones grown in different vineyards, during two ripening seasons. RNA-sequencing data were implemented with analyses of candidate genes, secondary metabolites, and agronomical parameters. This multidisciplinary approach helped to dissect the complexity of clone × environment interactions, by identifying the molecular responses controlled by genotype, vineyard, phenological phase, or a combination of these factors. Transcripts associated to sugar signalling, anthocyanin biosynthesis, and transport were differently modulated among clones, according to changes in berry agronomical features. Conversely, genes involved in defense response, such as stilbene synthase genes, were significantly affected by vineyard, consistently with stilbenoid accumulation. Thus, besides at the cultivar level, clone-specific molecular responses also contribute to shape the agronomic features of grapes in different environments. This reveals a further level of complexity in the regulation of genotype × environment interactions that has to be considered for orienting viticultural practices aimed at enhancing the quality of grape productions.

Plants develop in a microbe-rich environment and must interact with a plethora of microorganisms, both pathogenic and beneficial. Indeed, such is the case of Pseudomonas, and its model organisms P. fluorescens and P. syringae, a bacterial genus that has received particular attention because of its beneficial effect on plants and its pathogenic strains. The present study aims to compare plant-beneficial and pathogenic strains belonging to the P. syringae species to get new insights into the distinction between the two types of plant–microbe interactions. In assays carried out under greenhouse conditions, P. syringae pv. syringae strain 260-02 was shown to promote plant-growth and to exert biocontrol of P. syringae pv. tomato strain DC3000, against the Botrytis cinerea fungus and the Cymbidium Ringspot Virus. This P. syringae strain also had a distinct volatile emission profile, as well as a different plant-colonization pattern, visualized by confocal microscopy and gfp labeled strains, compared to strain DC3000. Despite the different behavior, the P. syringae strain 260-02 showed great similarity to pathogenic strains at a genomic level. However, genome analyses highlighted a few differences that form the basis for the following hypotheses regarding strain 260-02. P. syringae strain 260-02: (i) possesses non-functional virulence genes, like the mangotoxin-producing operon Mbo; (ii) has different regulation pathways, suggested by the difference in the autoinducer system and the lack of a virulence activator gene; (iii) has genes encoding DNA methylases different from those found in other P. syringae strains, suggested by the presence of horizontal-gene-transfer-obtained methylases that could affect gene expression.

CLN1 disease (OMIM #256730) is an early childhood ceroid-lipofuscinosis associated with mutated CLN1, whose product Palmitoyl-Protein Thioesterase 1 (PPT1) is a lysosomal enzyme involved in the removal of palmitate residues from S-acylated proteins. In neurons, PPT1 expression is also linked to synaptic compartments. The aim of this study was to unravel molecular signatures connected to CLN1. We utilized SH-SY5Y neuroblastoma cells overexpressing wild type CLN1 (SH-p.wtCLN1) and five selected CLN1 patients’ mutations. The cellular distribution of wtPPT1 was consistent with regular processing of endogenous protein, partially detected inside Lysosomal Associated Membrane Protein 2 (LAMP2) positive vesicles, while the mutants displayed more diffuse cytoplasmic pattern. Transcriptomic profiling revealed 802 differentially expressed genes (DEGs) in SH-p.wtCLN1 (as compared to empty-vector transfected cells), whereas the number of DEGs detected in the two mutants (p.L222P and p.M57Nfs*45) was significantly lower. Bioinformatic scrutiny linked DEGs with neurite formation and neuronal transmission. Specifically, neuritogenesis and proliferation of neuronal processes were predicted to be hampered in the wtCLN1 overexpressing cell line, and these findings were corroborated by morphological investigations. Palmitoylation survey identified 113 palmitoylated protein-encoding genes in SH-p.wtCLN1, including 25 ones simultaneously assigned to axonal growth and synaptic compartments. A remarkable decrease in the expression of palmitoylated proteins, functionally related to axonal elongation (GAP43, CRMP1 and NEFM) and of the synaptic marker SNAP25, specifically in SH-p.wtCLN1 cells was confirmed by immunoblotting. Subsequent, bioinformatic network survey of DEGs assigned to the synaptic annotations linked 81 DEGs, including 23 ones encoding for palmitoylated proteins. Results obtained in this experimental setting outlined two affected functional modules (connected to the axonal and synaptic compartments), which can be associated with an altered gene dosage of wtCLN1. Moreover, these modules were interrelated with the pathological effects associated with loss of PPT1 function, similarly as observed in the Ppt1 knockout mice and patients with CLN1 disease.

Rootstocks are among the main factors that influence grape development as well as fruit and wine composition. In this work, rootstock/scion interactions were studied using transcriptomic and metabolic approaches on leaves of the “Gaglioppo” variety, grafted onto 13 different rootstocks growing in the same vineyard. The whole leaf transcriptome of “Gaglioppo” grafted onto five selected rootstocks showed high variability in gene expression. In particular, significant modulation of transcripts linked to primary and secondary metabolism was observed. Interestingly, genes and metabolites involved in defense responses (e.g., stilbenes and defense genes) were strongly activated particularly in the GAG-41B combination, characterized in addition by the down-regulation of abscisic acid (ABA) metabolism. On the contrary, the leaves of “Gaglioppo” grafted onto 1103 Paulsen showed an opposite regulations of those transcripts and metabolites, together with the greater sensitivity to downy mildew in a preliminary in vitro assay. This study carried out an extensive transcriptomic analysis of rootstock effects on scion leaves, helping to unravel this complex interaction, and suggesting an interesting correlation among constitutive stilbenes, ABA compound, and disease susceptibility to a fungal pathogen.

Studies regarding macroalgal responses to ocean acidification (OA) are mostly limited to short-term experiments in controlled conditions, which hamper the possibility to scale up the observations to long-term effects in the natural environment. To gain a broader perspective, we utilized volcanic CO2 vents as a “natural laboratory” to study OA effects on Sargassum vulgare at different time scales. We measured photosynthetic rates, oxidative stress levels, antioxidant contents, antioxidant enzyme activities, and activities of oxidative metabolic enzymes in S. vulgare growing at a natural acidified site (pH 6.7) compared to samples from a site with current pH (pH 8.2), used as a control one. These variables were also tested in plants transplanted from the control to the acidified site and vice-versa. After short-term exposure, photosynthetic rates and energy metabolism were increased in S. vulgare together with oxidative damage. However, in natural populations under long-term conditions photosynthetic rates were similar, the activity of oxidative metabolic enzymes was maintained, and no sign of oxidative damages was observed. The differences in the response of the macroalga indicate that the natural population at the acidified site is adapted to live at the lowered pH. The results suggest that this macroalga can adopt biochemical and physiological strategies to grow in future acidified oceans.