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Cardi T.,Centro Of Ricerca Per Lorticoltura | D'Agostino N.,Centro Of Ricerca Per Lorticoltura | Tripodi P.,Centro Of Ricerca Per Lorticoltura
Frontiers in Plant Science | Year: 2017

In the frame of modern agriculture facing the predicted increase of population and general environmental changes, the securement of high quality food remains a major challenge to deal with. Vegetable crops include a large number of species, characterized by multiple geographical origins, large genetic variability and diverse reproductive features. Due to their nutritional value, they have an important place in human diet. In recent years, many crop genomes have been sequenced permitting the identification of genes and superior alleles associated with desirable traits. Furthermore, innovative biotechnological approaches allow to take a step forward towards the development of new improved cultivars harboring precise genome modifications. Sequence-based knowledge coupled with advanced biotechnologies is supporting the widespread application of new plant breeding techniques to enhance the success in modification and transfer of useful alleles into target varieties. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system, zinc-finger nucleases, and transcription activator-like effector nucleases represent the main methods available for plant genome engineering through targeted modifications. Such technologies, however, require efficient transformation protocols as well as extensive genomic resources and accurate knowledge before they can be efficiently exploited in practical breeding programs. In this review, we revise the state of the art in relation to availability of such scientific and technological resources in various groups of vegetables, describe genome editing results obtained so far and discuss the implications for future applications. © 2017 Cardi, D’Agostino and Tripodi.


Molesini B.,University of Verona | Zanzoni S.,University of Verona | Mennella G.,Centro Of Ricerca Per Lorticoltura | Francese G.,Centro Of Ricerca Per Lorticoltura | And 3 more authors.
Plant and Cell Physiology | Year: 2017

Arabidopsis thaliana At4g17830 codes for a protein showing sequence similarity with the Escherichia coli N-acetylornithine deacetylase (EcArgE), an enzyme implicated in the linear ornithine (Orn) biosynthetic pathway. In plants, N-acetylornithine deacetylase (NAOD) activity has yet to be demonstrated; however, At4g17830-silenced and mutant (atnaod) plants display an impaired reproductive phenotype and altered foliar levels of Orn and polyamines (PAs). Here, we showed the direct connection between At4g17830 function and Orn biosynthesis, demonstrating biochemically that At4g17830 codes for a NAOD. These results are the first experimental proof that Orn can be produced in Arabidopsis via a linear pathway. In this study, to identify the role of AtNAOD in reproductive organs, we carried out a transcriptomic analysis on atnaod mutant and wild-type flowers. In the atnaod mutant, the most relevant effects were the reduced expression of cysteine-rich peptidecoding genes, known to regulate male-female cross-talk during reproduction, and variation in the expression of genes involved in nitrogen:carbon (N:C) status. The atnaod mutant also exhibited increased levels of sucrose and altered sensitivity to glucose. We hypothesize that AtNAOD participates in Orn and PA homeostasis, contributing to maintain an optimal N:C balance during reproductive development. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.


Nendel C.,Leibniz Center for Agricultural Landscape Research | Venezia A.,Centro Of Ricerca Per Lorticoltura | Piro F.,Centro Of Ricerca Per Lorticoltura | Ren T.,China Agricultural University | And 2 more authors.
Journal of Agricultural Science | Year: 2013

The EU-Rotate-N model was developed as a tool to estimate the growth and nitrogen (N) uptake of vegetable crop rotations across a wide range of European climatic conditions and to assess the economic and environmental consequences of alternative management strategies. The model has been evaluated under field conditions in Germany and Norway and under greenhouse conditions in China. The present work evaluated the model using Italian data to evaluate its performance in a warm and dry environment. Data were collected from four 2-year field rotations, which included lettuce (Lactuca sativa L.), fennel (Foeniculum vulgare Mill.), spinach (Spinacia oleracea L.), broccoli (Brassica oleracea L. var. italica Plenck) and white cabbage (B. oleracea convar. capitata var. alba L.); each rotation used three different rates of N fertilizer (average recommended N1, assumed farmer's practice N2=N1+0·3×N1 and a zero control N0). Although the model was not calibrated prior to running the simulations, results for above-ground dry matter biomass, crop residue biomass, crop N concentration and crop N uptake were promising. However, soil mineral N predictions to 0·6 m depth were poor. The main problem with the prediction of the test variables was the poor ability to capture N mineralization in some autumn periods and an inappropriate parameterization of fennel. In conclusion, the model performed well, giving results comparable with other bio-physical process simulation models, but for more complex crop rotations. The model has the potential for application in Mediterranean environments for field vegetable production. Copyright © Cambridge University Press 2012.


Cardi T.,Centro Of Ricerca Per Lorticoltura | Neal Stewart C.,University of Tennessee at Knoxville
Plant Cell Reports | Year: 2016

Transgene integration in plants is based on illegitimate recombination between non-homologous sequences. The low control of integration site and number of (trans/cis)gene copies might have negative consequences on the expression of transferred genes and their insertion within endogenous coding sequences. The first experiments conducted to use precise homologous recombination for gene integration commenced soon after the first demonstration that transgenic plants could be produced. Modern transgene targeting categories used in plant biology are: (a) homologous recombination-dependent gene targeting; (b) recombinase-mediated site-specific gene integration; (c) oligonucleotide-directed mutagenesis; (d) nuclease-mediated site-specific genome modifications. New tools enable precise gene replacement or stacking with exogenous sequences and targeted mutagenesis of endogeneous sequences. The possibility to engineer chimeric designer nucleases, which are able to target virtually any genomic site, and use them for inducing double-strand breaks in host DNA create new opportunities for both applied plant breeding and functional genomics. CRISPR is the most recent technology available for precise genome editing. Its rapid adoption in biological research is based on its inherent simplicity and efficacy. Its utilization, however, depends on available sequence information, especially for genome-wide analysis. We will review the approaches used for genome modification, specifically those for affecting gene integration and modification in higher plants. For each approach, the advantages and limitations will be noted. We also will speculate on how their actual commercial development and implementation in plant breeding will be affected by governmental regulations. © 2016 Springer-Verlag Berlin Heidelberg


PubMed | University of Tennessee at Knoxville and Centro Of Ricerca Per Lorticoltura
Type: Journal Article | Journal: Plant cell reports | Year: 2016

Transgene integration in plants is based on illegitimate recombination between non-homologous sequences. The low control of integration site and number of (trans/cis)gene copies might have negative consequences on the expression of transferred genes and their insertion within endogenous coding sequences. The first experiments conducted to use precise homologous recombination for gene integration commenced soon after the first demonstration that transgenic plants could be produced. Modern transgene targeting categories used in plant biology are: (a) homologous recombination-dependent gene targeting; (b) recombinase-mediated site-specific gene integration; (c) oligonucleotide-directed mutagenesis; (d) nuclease-mediated site-specific genome modifications. New tools enable precise gene replacement or stacking with exogenous sequences and targeted mutagenesis of endogeneous sequences. The possibility to engineer chimeric designer nucleases, which are able to target virtually any genomic site, and use them for inducing double-strand breaks in host DNA create new opportunities for both applied plant breeding and functional genomics. CRISPR is the most recent technology available for precise genome editing. Its rapid adoption in biological research is based on its inherent simplicity and efficacy. Its utilization, however, depends on available sequence information, especially for genome-wide analysis. We will review the approaches used for genome modification, specifically those for affecting gene integration and modification in higher plants. For each approach, the advantages and limitations will be noted. We also will speculate on how their actual commercial development and implementation in plant breeding will be affected by governmental regulations.


PubMed | University of Tuscia, University Utrecht, CNR Institute of Neuroscience, Centro Of Ricerca Per Lorticoltura and University of Naples Federico II
Type: | Journal: Journal of plant physiology | Year: 2016

Trichoderma species include widespread rhizosphere-colonising fungi that may establish an opportunistic interaction with the plant, resulting in growth promotion and/or increased tolerance to biotic and abiotic stresses. For this reason, Trichoderma-based formulations are largely used in agriculture to improve yield while reducing the application of agro-chemicals. By using the Suppression Subtractive Hybridization method, we identified molecular mechanisms activated during the in vitro interaction between tomato (Solanum lycopersicum L.) and the selected strain MK1 of Trichoderma longibrachiatum, and which may participate in the stimulation of plant growth and systemic resistance. Screening and sequence analysis of the subtractive library resulted in forty unique transcripts. Their annotation in functional categories revealed enrichment in cell defence/stress and primary metabolism categories, while secondary metabolism and transport were less represented. Increased transcription of genes involved in defence, cell wall reinforcement and signalling of reactive oxygen species suggests that improved plant pathogen resistance induced by T. longibrachiatum MK1 in tomato may occur through stimulation of the above mechanisms. The array of activated defence-related genes indicates that different signalling pathways, beside the jasmonate/ethylene-dependent one, collaborate to fine-tune the plant response. Our results also suggest that the growth stimulation effect of MK1 on tomato may involve a set of genes controlling protein synthesis and turnover as well as energy metabolism and photosynthesis. Transcriptional profiling of several defence-related genes at different time points of the tomato-Trichoderma interaction, and after subsequent inoculation with the pathogen Botrytis cinerea, provided novel information on genes that may specifically modulate the tomato response to T. longibrachiatum, B. cinerea or both.


PubMed | University of Tuscia, Centro Of Ricerca Per Lorticoltura and University of Naples Federico II
Type: Journal Article | Journal: PloS one | Year: 2015

During its evolution and domestication Solanum lycopersicum has undergone various genetic bottlenecks and extreme inbreeding of limited genotypes. In Europe the tomato found a secondary centre for diversification, which resulted in a wide array of fruit shape variation given rise to a range of landraces that have been cultivated for centuries. Landraces represent a reservoir of genetic diversity especially for traits such as abiotic stress resistance and high fruit quality. Information about the variation present among tomato landrace populations is still limited. A collection of 123 genotypes from different geographical areas was established with the aim of capturing a wide diversity. Eighteen morphological traits were evaluated, mainly related to the fruit. About 45% of morphological variation was attributed to fruit shape, as estimated by the principal component analysis, and the dendrogram of relatedness divided the population in subgroups mainly on the basis of fruit weight and locule number. Genotyping was carried out using the tomato array platform SolCAP able to interrogate 7,720 SNPs. In the whole collection 87.1% markers were polymorphic but they decreased to 44-54% when considering groups of genotypes with different origin. The neighbour-joining tree analysis clustered the 123 genotypes into two main branches. The STRUCTURE analysis with K = 3 also divided the population on the basis of fruit size. A genomic-wide association strategy revealed 36 novel markers associated to the variation of 15 traits. The markers were mapped on the tomato chromosomes together with 98 candidate genes for the traits analyzed. Six regions were evidenced in which candidate genes co-localized with 19 associated SNPs. In addition, 17 associated SNPs were localized in genomic regions lacking candidate genes. The identification of these markers demonstrated that novel variability was captured in our germoplasm collection. They might also provide a viable indirect selection tool in future practical breeding programs.


PubMed | University of Verona, Consiglio per la ricerca in agricoltura e lanalisi delleconomia agraria and Centro Of Ricerca Per Lorticoltura
Type: | Journal: Plant & cell physiology | Year: 2017

Arabidopsis thaliana At4g17830 codes for a protein showing sequence similarity with the Escherichia coli N-acetylornithine deacetylase (EcArgE), an enzyme implicated in the linear ornithine (Orn) biosynthetic pathway. In plants, N-acetylornithine deacetylase (NAOD) activity has yet to be demonstrated; however, At4g17830-silenced and mutant (atnaod) plants display an impaired reproductive phenotype and altered foliar levels of Orn and polyamines (PAs). Here, we showed the direct connection between At4g17830 function and Orn biosynthesis, demonstrating biochemically that At4g17830 codes for a NAOD. These results are the first experimental proof that Orn can be produced in Arabidopsis via a linear pathway. In this study, to identify the role of AtNAOD in reproductive organs, we carried out a transcriptomic analysis on atnaod mutant and wild-type flowers. In the atnaod mutant, the most relevant effects were the reduced expression of cysteine-rich peptide-coding genes, known to regulate male-female cross-talk during reproduction, and variation in the expression of genes involved in nitrogen:carbon (N:C) status. The atnaod mutant also exhibited increased levels of sucrose and altered sensitivity to glucose. We hypothesize that AtNAOD participates in Orn and PA homeostasis, contributing to maintain an optimal N:C balance during reproductive development.


Scotti R.,Centro Of Ricerca Per Lorticoltura | Iovieno P.,Centro Of Ricerca Per Lorticoltura | Zaccardelli M.,Centro Of Ricerca Per Lorticoltura
Biology and Fertility of Soils | Year: 2015

A forest soil located in the “Parco Nazionale del Cilento e del Vallo di Diano”, Salerno, Italy, has been partly converted to a crop field by deforestation and a deep tillage (mouldboard plowing), characterized by an inversion of soil profile at a depth of 1 m, followed by plowing. In this study, we compare soil quality in a crop field and in the adjacent unchanged forest. Soil samples were collected from the two sites few months after the use change and characterized for main chemical (pH, electrical conductivity, organic C, total N, available P, cation exchange capacity) and microbiological (enzyme activities related to cycles of major nutrients, catabolic response profiles, microbial and fungal biomass) properties. We found a depletion of 50 % in soil organic C content in the crop field in comparison with forest, which indicates that the quality of the top layer of the soil decreased, with consequent deep alteration of the C cycle. The drop of organic matter content determined serious effects on soil microbial activity. Microbial and fungal biomass, as well as all enzyme activities, were decreased. The soil catabolic response profiles showed a significant reduction in functional diversity in the cultivated compared to the forest soil. Among the investigated properties, dehydrogenase and β-galactosidase activities, ergosterol content and catabolic evenness showed the highest sensitivity to the soil use change, suggesting that they could be assumed as indicators of the stress induced by this use change. © 2015, Springer-Verlag Berlin Heidelberg.


Cantarella C.,Centro Of Ricerca Per Lorticoltura | D'Agostino N.,Centro Of Ricerca Per Lorticoltura
BMC Research Notes | Year: 2015

Background: With the advent of high-throughput sequencing technologies large-scale identification of microsatellites became affordable and was especially directed to non-model species. By contrast, few efforts have been published toward the automatic identification of polymorphic microsatellites by exploiting sequence redundancy. Few tools for genotyping microsatellite repeats have been implemented so far that are able to manage huge amount of sequence data and handle the SAM/BAM file format. Most of them have been developed for and tested on human or model organisms with high quality reference genomes. Results: In this note we describe polymorphic SSR retrieval (PSR), a read counter and simple sequence repeat (SSR) length polymorphism detection tool. It is written in Perl and was developed to identify length polymorphisms in perfect microsatellites exploiting next generation sequencing (NGS) data. PSR has been developed bearing in mind plant non-model species for which de novo transcriptome assembly is generally the first sequence resource available to be used for SSR-mining. PSR is divided into two modules: the read-counting module (PSR-read-retrieval) identifies all the reads that cover the full-length of perfect microsatellites; the comparative module (PSR-poly-finder) detects both heterozygous and homozygous alleles at each microsatellite locus across all genotypes under investigation. Two threshold values to call a length polymorphism and reduce the number of false positives can be defined by the user: the minimum number of reads overlapping the repetitive stretch and the minimum read depth. The first parameter determines if the microsatellite-containing sequence must be processed or not, while the second one is decisive for the identification of minor alleles. PSR was tested on two different case studies. The first study aims at the identification of polymorphic SSRs in a set of de novo assembled transcripts defined by RNA-sequencing of two different plant genotypes. The second research activity aims to investigate sequence variations within a collection of newly sequenced chloroplast genomes. In both the cases PSR results are in agreement with those obtained by capillary gel separation. Conclusion: PSR has been specifically developed from the need to automate the gene-based and genome-wide identification of polymorphic microsatellites from NGS data. It overcomes the limits related to the existing and time-consuming efforts based on tools developed in the pre-NGS era. © 2015 Cantarella and D'Agostino.

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