Exploiting genetic and genomic resources to increase the ascorbic acid content in the tomato fruit

The wild tomato species such as Solanum pennellii are an important source of genes that were lost during tomato selection and cultivation. The S. pennellii Introgression lines (ILs), which carry defined homozygous segments of the wild genome in the cultivated genetic background of cv.M82, are an important genetic resource to map quantitative traits loci (QTLs), such as those controlling plant yield and fruit quality, and to exploit the genetic diversity present in the wild species. Two introgression lines (IL12-4 and IL7-3) harbouring QTLs for ascorbic acid content were previously identified in the laboratory of Structural and Functional Genomics at the Department of Agricultural Sciences of University of Naples Federico II. These two ILs showed increased content of antioxidant compounds in the fruit compared to the cultivated S. lycopersicum cv. M82. Afterwards, sub-lines with reduced sizes of the introgressed region were obtained from IL7-3 and IL12-4. The first aim of the present thesis was to perform the phenotypic selection and characterization of the S. pennellii sub-lines in different environmental conditions. In the fruit of the different sub-lines, the level of soluble solids content in terms of °Brix, firmness and ascorbic acid was highly variable in three different environmental conditions. The sub-lines R182 and B27, deriving from IL7-3 and IL12-4, respectively, were selected for their better performances in terms of fruit quality since they exhibited a significantly higher firmness, °Brix and ascorbic acid content compared to M82. Moreover, the two sub-lines also showed a production comparable to that of the control line M82 in all the environment conditions tested. The second aim of the present thesis was to identify candidate genes involved in determing the high level of ascorbic acid in the fruit and mapping in the introgressed regions of the sub-lines R182 and B27. Since today the only reference genome used to investigate gene positions and functions in tomato still remains that of the first completely sequenced genome, i.e. that of S. lycopersicum cv. Heinz, the first step to reach this second aim was to reassembly the S. pennellii IL7-3 and IL12-4 genomes. In order to reassemble the genome of M82 and of the ILs, several resources were used. These consisted of Next Generation Sequence resources available today, such as the sequence data of the S. pennellii and different RNA-seq data related to the IL populations. By using an integrated bioinformatic approach a new reference genome and annotation for IL12-4 and the IL7-3 was built. Moreover, to confirm the reliability of the IL7-3 assembly and define the size of S. pennellii introgression region in the sub-line R182, a group of species-specific molecular markers were designed based on polymorphisms found comparing the genomes of the cultivated and the wild species. Finally, to identify candidate genes mapping in the wild regions better defined in the present thesis, a transcriptomic approach with RNA-Seq was carried out. Transcriptome analyses allowed identifying three candidate genes in the two sub-lines: the Solyc12g098480 encoding for the UDP-glucoronic-acid-4-epimerase in the wild introgressed region of B27 and the Solyc07go49310 and Solyc07g049290 corresponding to two Major facilitator superfamily (MFS) proteins in in the wild introgressed region of R182. The third aim of the present thesis was to carry out the functional validation of candidate genes potentially involved in the control of ascorbic acid content in the red ripe fruit. The first gene selected was the UDP-glucoronic-acid-4-epimerase gene mapping in the introgressed region of the sub-line B27. Its role in increasing ascorbic acid in this sub-line fruit was investigated through its over-expression in tomato fruit by stable and transient techniques. In addition, the genotyping and phenotyping evaluation of CRISP/Cas9 knock-out mutants for a non-canonical uORF carried by two GDP-L-galactosephosphorylases (GGP1, Solyc06g073320 and GGP2, Solyc02g091510) allowed demonstrating that these two genes are involved in the regulation of the ascorbate biosynthesis in tomato. In conclusion, the results obtained in the present thesis, allowed selecting two elite sub-lines that in the near future could be used as breeding material to improve tomato fruit for nutritional traits. In addition, the results achieved allowed increasing knowledge about genes involved in the control of ascorbic acid content in tomato fruit. Indeed, three candidate genes were identified exploiting the genomics resources available for tomato, and other two genes have been functionally validated. The transfer of these genes by conventional and innovative strategies will aid in the future the creation of new improved varieties

Phenotypic and molecular selection of a superior Solanum pennellii introgression sub-line suitable for improving quality traits of cultivated tomatoes

The Solanum pennellii Introgression Line (IL) population can be exploited to identify favorable alleles that can improve yield and fruit quality traits in commercial tomato varieties. Over the past few years, we have selected ILs that exhibit increased content of antioxidant compounds in the fruit compared to the cultivar M82, which represents the genetic background in which the different wild regions of the S. pennellii ILs were included. Recently, we have identified seven sub-lines of the IL7-3 accumulating different amounts of antioxidants in the ripe fruit. Since the wild region carried on chromosome 7 induces a low fruit production in IL7-3, the first aim of the present work was to evaluate yield performances of the selected sub-lines in three experimental fields located in the South of Italy. Another aim was to confirm in the same lines the high levels of antioxidants and evaluate other fruit quality traits. On red ripe fruit, the levels of soluble solids content, firmness, and ascorbic acid (AsA) were highly variable among the sub-lines grown in three environmental conditions, evidencing a significant genotype by environment interaction for soluble solids and AsA content. Only one sub-line (coded R182) exhibited a significantly higher firmness, even though no differences were observed for this trait between the parental lines M82 and IL7-3. The same sub-line showed significantly higher AsA content compared to M82, thus resembling IL7-3. Even though IL7-3 always exhibited a significantly lower yield, all the sub-lines showed yield variability over the three trials. Interestingly, the sub-line R182, selected for its better performances in terms of fruit quality, in all the trials showed a production comparable to that of the control line M82. A group of species-specific molecular markers was tested on R182 and on the parental genotypes in order to better define the wild genomic regions carried by the elite line R182. In these regions three candidate genes that could increase the level of AsA in the fruit were identified. In the future, the line R182 could be used as pre-breeding material in order to obtain new varieties improved for nutritional traits.info:eu-repo/semantics/publishedVersio

Rare earth elements in human and animal health: State of art and research priorities

"Background: A number of applications have been developed using rare earth elements (REE), implying several human exposures and raising unsolved questions as to REE-associated health effects. Methods: A MedLine survey was retrieved from early reports (1980s) up to June 2015, focused on human and animal exposures to REE. Literature from animal models was selected focusing on REE-associated health effects. Results: Some REE occupational exposures, in jobs such as glass polishers, photoengravers and movie projectionists showed a few case reports on health effects affecting the respiratory system. No case-control or cohort studies of occupational REE exposures were retrieved. Environmental exposures have been biomonitored in populations residing in REE mining areas, showing REE accumulation. The case for a iatrogenic REE exposure was raised by the use of gadolinium-based contrast agents for nuclear magnetic resonance. Animal toxicity studies have shown REE toxicity, affecting a number of endpoints in liver, lungs and blood. On the other hand, the use of REE as feed additives in livestock is referred as a safe and promising device in zootechnical activities, possibly suggesting a hormetic effect both known for REE and for other xenobiotics. Thus, investigations on long-term exposures and observations are warranted. Conclusion: The state of art provides a limited definition of the health effects in occupationally or environmentally REE-exposed human populations. Research priorities should be addressed to case-control or cohort studies of REE-exposed humans and to life-long animal experiments. (C) 2015 Elsevier Inc. All rights reserved.

Fertilisation with compost mitigates salt stress in tomato by affecting plant metabolomics and nutritional profiles

Abstract Background Salinity is one of the major threats for crop growth and yield and its rate of expansion is expected to increase. We conducted a pot experiment to evaluate and compare the effect of a green compost addition and mineral fertilisation, on growth, nutrition and metabolites of tomato plants, exposed to increasing doses of NaCl. Results Although the development of stressed plants was lower than the corresponding controls, compost-treated plants performed better than mineral-amended plants watered with the same amount of salt. The different plant growth was related to an increased nutritional status. Namely, compost-treated plants showed a larger content of macro- and micronutrients, and a greater accumulation of osmoprotectants, such as soluble sugars and amino acids. Moreover, compost-treated plants showed a larger content of metabolites involved in modulating the response to salt stress, such as molecules related to energy transfer in plants and precursors of Reactive Oxygen Species scavenging compounds. Overall, the better performance of compost-added plants may be attributed to a greater availability of the organic forms of nutrients and to the positive bioactivity of compost-derived humic substances. Conclusions Compost application efficiently mitigated salt stress in tomato plants in respect to mineral fertilisation. This alleviating role was associated to the induction of a more efficient metabolic response that increased accumulation of metabolites involved in modulating the salinity stress. Therefore, fertilising with composted agricultural residue may represent a convenient alternative to mineral fertilisers to grow tomato plants in the presence of salt stress. Graphical Abstrac

What is in your cup of tea? DNA Verity Test to characterize black and green commercial teas

In this study, we used several molecular techniques to develop a fast and reliable protocol (DNA Verity Test, DVT) for the characterization and confirmation of the species or taxa present in herbal infusions. As a model plant for this protocol, Camellia sinensis, a traditional tea plant, was selected due to the following reasons: its historical popularity as a (healthy) beverage, its high selling value, the importation of barely recognizable raw product (i.e., crushed), and the scarcity of studies concerning adulterants or contamination. The DNA Verity Test includes both the sequencing of DNA barcoding markers and genotyping of labeled-PCR DNA barcoding fragments for each sample analyzed. This protocol (DVT) was successively applied to verify the authenticity of 32 commercial teas (simple or admixture), and the main results can be summarized as follows: (1) the DVT protocol is suitable to detect adulteration in tea matrices (contaminations or absence of certified ingredients), and the method can be exported for the study of other similar systems; (2) based on the BLAST analysis of the sequences of rbcL+matK±rps7-trnV(GAC) chloroplast markers, C. sinensis can be taxonomically characterized; (3) rps7-trnV(GAC) can be employed to discriminate C. sinensis from C. pubicosta; (4) ITS2 is not an ideal DNA barcode for tea samples, reflecting potential incomplete lineage sorting and hybridization/introgression phenomena in C. sinensis taxa; (5) the genotyping approach is an easy, inexpensive and rapid pre-screening method to detect anomalies in the tea templates using the trnH(GUG)-psbA barcoding marker; (6) two herbal companies provided no authentic products with a contaminant or without some of the listed ingredients; and (7) the leaf matrices present in some teabags could be constituted using an admixture of different C. sinensis haplotypes and/or allied species (C. pubicosta)

Ecosostenibilità come scelta di vita

Alcuni nuovi insediamenti possono essere definiti, a buon diritto, comunità. La pratica dell’ecosostenibilità, infatti, si sostanzia di comportamenti collettivi e condivisi, di attività comuni in cooperazione e, più in generale, di una fitta rete di relazioni solidali. Ai benefici di costruzioni a basso impatto, preferibilmente concepite secondo i dettami della bioarchitettura, questi quartieri affiancano i vantaggi di servizi avanzati spesso gestiti in cooperazione fra gli abitanti. La condivisione di spazi e obiettivi, e il reciproco controllo sociale, determinano un’incidenza di comportamenti virtuosi molto maggiore che negli altri insediamenti, nei quali si lamenta spesso l’effetto paradosso di Jevons (il paradosso di Jevons ammonisce che quando gli “effetti collaterali” di un’attività si riducono molto - che siano il costo o l’impatto ambientale - essa appare così a buon mercato da accrescersi in misura anche maggiore del risparmio unitario). La complessità funzionale è un’altra peculiarità di questi quartieri che replicano l’intreccio di funzioni tipico delle comunità di villaggio: i campi comuni, ad esempio, sono uno scenario frequente nei paesi dell’Europa centro-settentrionale, così come laboratori artigianali e altre attività produttive specifiche

Genomic Dissection of a Wild Region in a Superior Solanum pennellii Introgression Sub-Line with High Ascorbic Acid Accumulation in Tomato Fruit

The Solanum pennellii introgression lines (ILs) have been exploited to map quantitative trait loci (QTLs) and identify favorable alleles that could improve fruit quality traits in tomato varieties. Over the past few years, ILs exhibiting increased content of ascorbic acid in the fruit have been selected, among which the sub-line R182. The aims of this work were to identify the genes of the wild donor S. pennellii harbored by the sub-line and to detect genes controlling ascorbic acid accumulation by using genomics tools. A Genotyping-By-Sequencing (GBS) approach confirmed that no wild introgressions were present in the sub-line besides one region on chromosome 7. By using a dense single nucleotide polymorphism (SNP) map obtained by RNA sequencing (RNA-Seq), the wild region of the sub-line was finely identified; thus, defining 39 wild genes that replaced 33 genes of the ILs genetic background (cv. M82). The differentially expressed genes mapping in the region and the variants detected among the cultivated and the wild alleles evidenced the potential role of the novel genes present in the wild region. Interestingly, one upregulated gene, annotated as a major facilitator superfamily protein, showed a novel structure in R182, with respect to the parental lines. These genes will be further investigated using gene editing strategies

Correction: What is in your cup of tea? DNA Verity Test to characterize black and green commercial teas(PLoS ONE (2017) 12:5 (e0178262) DOI: 10.1371/journal.pone.0178262)

In this study, we used several molecular techniques to develop a fast and reliable protocol (DNA Verity Test, DVT) for the characterization and confirmation of the species or taxa present in herbal infusions. As a model plant for this protocol, Camellia sinensis, a traditional tea plant, was selected due to the following reasons: its historical popularity as a (healthy) beverage, its high selling value, the importation of barely recognizable raw product (i.e., crushed), and the scarcity of studies concerning adulterants or contamination. The DNA Verity Test includes both the sequencing of DNA barcoding markers and genotyping of labeled-PCR DNA barcoding fragments for each sample analyzed. This protocol (DVT) was successively applied to verify the authenticity of 32 commercial teas (simple or admixture), and the main results can be summarized as follows: (1) the DVT protocol is suitable to detect adulteration in tea matrices (contaminations or absence of certified ingredients), and the method can be exported for the study of other similar systems; (2) based on the BLAST analysis of the sequences of rbcL+matK±rps7-trnV(GAC) chloroplast markers, C. sinensis can be taxonomically characterized; (3) rps7-trnV(GAC) can be employed to discriminate C. sinensis from C. pubicosta; (4) ITS2 is not an ideal DNA barcode for tea samples, reflecting potential incomplete lineage sorting and hybridization/introgression phenomena in C. sinensis taxa; (5) the genotyping approach is an easy, inexpensive and rapid pre-screening method to detect anomalies in the tea templates using the trnH(GUG)-psbA barcoding marker; (6) two herbal companies provided no authentic products with a contaminant or without some of the listed ingredients; and (7) the leaf matrices present in some teabags could be constituted using an admixture of different C. sinensis haplotypes and/or allied species (C. pubicosta)