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Ocimum basilicum (sweet basil) is an economically important aromatic herb; in Italy, approximately 1000 ha of “Genovese-type” basil are grown annually in greenhouses and open fields and are subjected to Downy Mildew (DM) disease, caused by Peronospora belbahrii, leading to huge crop losses. Mutation of the Susceptibility (S) gene DMR6 (Downy Mildew Resistant 6) has been proven to confer a broad-spectrum resistance to DM. In this work, an effective Genome Editing (GE) approach mediated by CRISPR/Cas9 in O. basilicum ‘Italiko’, the élite cultivar used to produce “Pesto Genovese D.O.P”, was developed. A highly efficient genetic transformation method mediated by A. tumefaciens has been optimized from cotyledonary nodes, obtaining 82.2% of regenerated shoots, 84.6% of which resulted in Cas9+ plants. Eleven T0 lines presented different type of mutations in ObDMR6; 60% were indel frameshift mutations with knock-out of ObDMR6 of ‘FT Italiko’. Analysis of six T1 transgene-free seedlings revealed that the mutations of T0 plants were inherited and segregated. Based on infection trials conducted on T0 plants, clone 22B showed a very low percentage of disease incidence after 14 days post infection. The aromatic profile of all in vitro edited plants was also reported; all of them showed oxygenated monoterpenes as the major fraction.

Key message We review the main genes underlying commercial traits in cut flower species and critically discuss the possibility to apply genome editing approaches to produce novel variation and phenotypes. Abstract Promoting flowering and flower longevity as well as creating novelty in flower structure, colour range and fragrances are major objectives of ornamental plant breeding. The novel genome editing techniques add new possibilities to study gene function and breed new varieties. The implementation of such techniques, however, relies on detailed information about structure and function of genomes and genes. Moreover, improved protocols for efficient delivery of editing reagents are required. Recent results of the application of genome editing techniques to elite ornamental crops are discussed in this review. Enabling technologies and genomic resources are reviewed in relation to the implementation of such approaches. Availability of the main gene sequences, underlying commercial traits and in vitro transformation protocols are provided for the world’s best-selling cut flowers, namely rose, lily, chrysanthemum, lisianthus, tulip, gerbera, freesia, alstroemeria, carnation and hydrangea. Results obtained so far are described and their implications for the improvement of flowering, flower architecture, colour, scent and shelf-life are discussed. Electronic supplementary material The online version of this article (10.1007/s00299-020-02632-x) contains supplementary material, which is available to authorized users.

We have established a DNA-free genome editing method via ribonucleoprotein-based CRISPR/Cas9 in cultivated tomato and obtained mutant plants regenerated from transfected protoplasts with a high mutation rate. The application of genome editing as a research and breeding method has provided many possibilities to improve traits in many crops in recent years. In cultivated tomato (Solanum lycopersicum), so far only stable Agrobacterium-mediated transformation carrying CRISPR/Cas9 reagents has been established. Shoot regeneration from transfected protoplasts is the major bottleneck in the application of DNA-free genome editing via ribonucleoprotein-based CRISPR/Cas9 method in cultivated tomato. In this study, we report the implementation of a transgene-free breeding method for cultivated tomato by CRISPR/Cas9 technology, including the optimization of protoplast isolation and overcoming the obstacle in shoot regeneration from transfected protoplasts. We have identified that the shoot regeneration medium containing 0.1 mg/L IAA and 0.75 mg/L zeatin was the best hormone combination with a regeneration rate of up to 21.3%. We have successfully obtained regenerated plants with a high mutation rate four months after protoplast isolation and transfection. Out of 110 regenerated M0 plants obtained, 35 (31.8%) were mutated targeting both SP and SP5G genes simultaneously and the editing efficiency was up to 60% in at least one allele in either SP or SP5G genes.

The possibility to produce plants edited in multiple genes by means of DNA-free approaches opens new perspectives for breeding purposes and acceptance of resultant genotypes. In this work, we have explored the polyethylene glycol (PEG)-mediated delivery of ribonucleoproteins (RNPs) in tomato protoplasts using a multiplexing approach (i.e. two genes targeted simultaneously using two sgRNAs per gene) for the first time. We have analysed the editing outcome in fully developed green calli and demonstrated that tomato protoplasts are a valid cell target for RNP-mediated multiplexed genome editing with high efficiency. RNP could be applied with high efficiency in a multiplexing genome editing approach in tomato protoplasts.

Background: Wild rocket (Diplotaxis tenuifolia) is prone to soil-borne stresses under intensive cultivation systems devoted to ready-to-eat salad chain, increasing needs for external inputs. Early detection of the abiotic and biotic stresses by using digital reflectance-based probes may allow optimization and enhance performances of the mitigation strategies.Methods: Hyperspectral image analysis was applied to D. tenuifolia potted plants subjected, in a greenhouse experiment, to five treatments for one week: a control treatment watered to 100% water holding capacity, two biotic stresses: Fusarium wilting and Rhizoctonia rotting, and two abiotic stresses: water deficit and salinity. Leaf hyperspectral fingerprints were submitted to an artificial intelligence pipeline for training and validating image-based classification models able to work in the stress range. Spectral investigation was corroborated by pertaining physiological parameters. Results: Water status was mainly affected by water deficit treatment, followed by fungal diseases, while salinity did not change water relations of wild rocket plants compared to control treatment. Biotic stresses triggered discoloration in plants just in a week after application of the treatments, as evidenced by the colour space coordinates and pigment contents values. Some vegetation indices, calculated on the bases of the reflectance data, targeted on plant vitality and chlorophyll content, healthiness, and carotenoid content, agreed with the patterns of variations observed for the physiological parameters. Artificial neural network helped selection of VIS (492-504, 540-568 and 712-720 nm) and NIR (855, 900-908 and 970 nm) bands, whose read reflectance contributed to discriminate stresses by imaging. Conclusions: This study provided significative spectral information linked to the assessed stresses, allowing the identification of narrowed spectral regions and single wavelengths due to changes in photosynthetically active pigments and in water status revealing the etiological cause.

Wild rocket (Diplotaxis tenuifolia, Brassicaceae) is a baby-leaf vegetable crop of high economic interest, used in ready-to-eat minimally processed salads, with an appreciated taste and nutraceutical features. Disease management is key to achieving the sustainability of the entire production chain in intensive systems, where synthetic fungicides are limited or not permitted. In this context, soil-borne pathologies, much feared by growers, are becoming a real emergency. Digital screening of green beds can be implemented in order to optimize the use of sustainable means. The current study used a high-resolution hyperspectral array (spectroscopy at 350–2500 nm) to attempt to follow the progression of symptoms of Rhizoctonia, Sclerotinia, and Sclerotium disease across four different severity levels. A Random Forest machine learning model reduced dimensions of the training big dataset allowing to compute de novo vegetation indices specifically informative about canopy decay caused by all basal pathogenic attacks. Their transferability was also tested on the canopy dataset, which was useful for assessing the health status of wild rocket plants. Indeed, the progression of symptoms associated with soil-borne pathogens is closely related to the reduction of leaf absorbance of the canopy in certain ranges of visible and shortwave infrared spectral regions sensitive to reduction of chlorophyll and other pigments as well as to modifications of water content and turgor.

BackgroundCapsicum is one of the major vegetable crops grown world-wide. Current subdivision in clades and species is based on morphological traits and coarse sets of genetic markers. Fruits broad variability has been driven by breeding programs and has been mainly studied by linkage analysis.ResultsWe discovered 746k variable sites by sequencing 1.8% of the genome in a collection of 373 accessions belonging to 11 Capsicum species from 51 countries. We describe genomic variation at population-level, confirm major subdivision in clades and species, and show that the known subdivision of C. annuum in two groups separates large and bulky fruits form small ones. In C. annuum, we identify four novel loci associated with phenotypes determining the fruit shape, including a non-synonymous mutation in the gene Longifolia 1-like (CA03g16080).ConclusionsOur collection covers all the economically important species of Capsicum widely used in breeding programs, and represent the widest and largest study so far in terms of the number of species and genetic variants analyzed. We identified a large set of markers that can be used for population genetic studies and genetic association analyses. Our results foster fine genetic association studies and foresee genomic variability at population-level.

The accessibility and use of natural genetic variability in crops is essential for the management of genetic resources and the establishment of innovative breeding programs. Nuclear whole-genome sequences of several cultivated and wild peppers have been recently generated in order to provide crucial information on the evolution, domestication and divergence of pepper species (Capsicum spp.). Despite next generation sequencing (NGS) technologies providing a significant advance in generation of high throughput data, the analysis of a large-scale data and of individuals is still a challenging task. An alternative strategy to Whole Genome Sequencing is to generate a reduced representation of the genome using target enrichment strategies. Among them, genotyping-by-sequencing (GBS) provides a rapid, highly informative, cost-effective tool for exploring genetic diversity on a genome-wide scale. We applied GBS for SNP marker discovery in nuclear genomes and population structure analysis in a collection of cultivated and domesticated peppers. Information generated in this study represents a first step towards future genome-wide association mapping studies and marker-assisted selection programs. In addition, results from this analysis can be used to gain a better understanding of the genetic relationships within the Capsicum genus. In order to provide a further contribution to that issue, we also determined the complete chloroplast DNA (cpDNA) sequence of 11 Capsicum accessions for a total of 8 species generally included in the C. annuum (7), C. baccatum (3) or C. pubescens/eximium complex (1). We performed comparative analysis and identified potential molecular markers able to discriminate among Capsicum species.

For a long time, plastid transformation has been a routine technology only in tobacco due to lack of effective selection and regeneration protocols, and, for some species, due to inefficient recombination using heterologous flanking regions in transformation vectors. Nevertheless, the availability of this technology to economically important crops offers new possibilities in plant breeding to manage pathogen resistance or improve nutritional value. Herein we describe an efficient plastid transformation protocol for potato (Solanum tuberosum subsp. tuberosum), achieved by the optimization of the tissue culture procedures and using transformation vectors carrying homologous potato flanking sequences. This protocol allowed to obtain up to one shoot per shot, an efficiency comparable to that usually accomplished in tobacco. Further, the method described in this chapter has been successfully used to regenerate potato transplastomic plants expressing recombinant GFP protein in chloroplasts and amyloplasts or long double-stranded RNAs for insect pest control.

Research has been increasingly focusing on the selection of novel and effective biological control agents (BCAs) against soil-borne plant pathogens. The large-scale application of BCAs requires fast and robust screening methods for the evaluation of the efficacy of high numbers of candidates. In this context, the digital technologies can be applied not only for early disease detection but also for rapid performance analyses of BCAs. The present study investigates the ability of different Trichoderma spp. to contain the development of main baby-leaf vegetable pathogens and applies functional plant imaging to select the best performing antagonists against multiple pathosystems. Specifically, sixteen different Trichoderma spp. strains were characterized both in vivo and in vitro for their ability to contain R. solani, S. sclerotiorum and S. rolfsii development. All Trichoderma spp. showed, in vitro significant radial growth inhibition of the target phytopathogens. Furthermore, biocontrol trials were performed on wild rocket, green and red baby lettuces infected, respectively, with R. solani, S. sclerotiorum and S. rolfsii. The plant status was monitored by using hyperspectral imaging. Two strains, Tl35 and Ta56, belonging to T. longibrachiatum and T. atroviride species, significantly reduced disease incidence and severity (DI and DSI) in the three pathosystems. Vegetation indices, calculated on the hyperspectral data extracted from the images of plant-Trichoderma-pathogen interaction, proved to be suitable to refer about the plant health status. Four of them (OSAVI, SAVI, TSAVI and TVI) were found informative for all the pathosystems analyzed, resulting closely correlated to DSI according to significant changes in the spectral signatures among health, infected and bio-protected plants. Findings clearly indicate the possibility to promote sustainable disease management of crops by applying digital plant imaging as large-scale screening method of BCAs' effectiveness and precision biological control support.

Wild rocket is a widely cultivated salad crop. Typical signs and symptoms of powdery mildew were observed on leaves of Diplotaxis tenuifolia, likely favored by climatic conditions occurring in a greenhouse. Based on morphological features and molecular analysis, the disease agent was identified as the fungal pathogen Erysiphe cruciferarum. To the best of our knowledge, this is the first report of E. cruciferarum on D. tenuifolia. Moreover, the present study provides a non-destructive high performing digital approach to efficiently detect the disease. Hyperspectral image analysis allowed to characterize the spectral response of wild rocket affected by powdery mildew and the adopted machine-learning approach (a trained Random Forest model with the four most contributory wavelengths falling in the range 403–446 nm) proved to be able to accurately discriminate between healthy and diseased wild rocket leaves. Shifts in the irradiance absorption by chlorophyll a of diseased leaves in the spectrum blue range seems to be at the base of the hyperspectral imaging detection of wild rocket powdery mildew.

For a long time, plastid transformation has been a routine technology only in tobacco due to lack of effective selection and regeneration protocols, and, for some species, due to inefficient recombination using heterologous flanking regions in transformation vectors. Nevertheless, the availability of this technology to economically important crops offers new possibilities in plant breeding to manage pathogen resistance or improve nutritional value. Herein we describe an efficient plastid transformation protocol for potato (Solanum tuberosum subsp. tuberosum), achieved by the optimization of the tissue culture procedures and using transformation vectors carrying homologous potato flanking sequences. This protocol allowed to obtain up to one shoot per shot, an efficiency comparable to that usually accomplished in tobacco. Further, the method described in this chapter has been successfully used to regenerate potato transplastomic plants expressing recombinant GFP protein in chloroplasts and amyloplasts or long double-stranded RNAs for insect pest control.

[EN] Double digest restriction-site associated sequencing (ddRAD-seq) is a flexible and cost-effective strategy for providing in-depth insights into the genetic architecture of germplasm collections. Using this methodology, we investigated the genomic diversity of a panel of 288 diverse tomato (Solanum lycopersicum L.) accessions enriched in 'da serbo' (called 'de penjar' in Spain) long shelf life (LSL) materials (152 accessions) mostly originating from Italy and Spain. The rest of the materials originate from different countries and include landraces for fresh consumption, elite cultivars, heirlooms, and breeding lines. Apart from their LSL trait, 'da serbo' landraces are of remarkable interest for their resilience. We identified 32,799 high-quality SNPs, which were used for model ancestry population structure and non-parametric hierarchical clustering. Six genetic subgroups were revealed, clearly separating most 'da serbo' landraces, but also the Spanish germplasm, suggesting a subdivision of the population based on type and geographical provenance. Linkage disequilibrium (LD) in the collection decayed very rapidly within, The authors thank the European Union Horizon 2020 Research and Innovation program for funding this research under grant agreement No 774244 (Breeding for Resilient, Efficient and Sustainable Organic Vegetable Production; BRESOV).

[EN] Cultivated rocket (Eruca vesicaria) is a leafy vegetable highly appreciated for its healthpromoting virtues and consumed both raw and cooked as ready-to-use vegetable. Despite Eruca being cultivated worldwide, only a few cultivars are available and limited breeding activities have been carried out so far. Therefore, the genetic resources available represent an unexploited potential source of variation for breeding. In the present study, 155 E. vesicaria accessions from 30 countries across Europe, Asia, Africa, and America have been characterized for 54 qualitative and quantitative morphological and quality traits. Conventional descriptors and automated tools for the determination of the quality, morphology, and colour of leaves have been used. Genetic diversity was assessed using 15 inter simple sequence repeat and simple sequence repeat markers. A high level of diversity was evidenced in the collection. Significant differences were found in most of the traits with the exception of five pseudo-qualitative descriptors. The first and second dimensions of the principal components analysis with phenotypic traits accounted for 25.69% of total variation showing a stratification of the genotypes according to the European and Asian origins. In total, 75% of the variation was contained in the first 15 components having eigenvalues higher than 1.0. Also, the population structure divided the collection into two main clusters separating European genotypes from the rest. Furthermore, hierarchical cluster analysis confirmed a geographical separation, grouping the accessions into three major clusters, which were differentiated by plant architecture, leaf and flower colour, leaf water status, leaf blade shape and hairiness of the leaves and stem. Our approach has broadened the knowledge of the diversity within the Eruca gene pool, thus contributing to identify sources of variation and to select the best candidates for cultivated rocket breeding programs, as well as to determine the genetic basis of plant and leaf traits in future genome-wide association studies., The work was supported by 'RGV-FAO' project funded by the Italian Ministry of Agriculture, Food and Forestry. C. Guijarro-Real thanks the Ministerio de Educacion, Cultura y Deporte of Spain (MECD) for its financial support by means of a predoctoral FPU Grant (FPU14/06798), and for the specific grant for mobility (EST17/00354) from the same Organization.

Hot pepper (Capsicum spp.) is an economically considerable crop, particularly appreciated for its nutritional properties and antioxidants content. Levels of the latter depend on several factors including the cultivar and the environment. In the present study, diversity in morpho-agronomic traits and phytochemical compounds was investigated in 14 hot pepper accessions cultivated in two different pedoclimatic locations. The main source of variation was due to the genotype which showed significant differences for all traits at both locations. With regard to the bioactive compounds, the environment accounted for over 30% of variation for carotenoids, ascorbic acid and tocopherols and less than 0.5% for capsaicinoids. A strong genotype × environment interaction (p

Baby leaf vegetables constitute a significant segment of the convenience fresh food market. Due to cultivation conditions under plastic tunnels favourable to pathogen and to restrictions about synthetic fungicide applications, these crops are prone to soil-borne diseases and need effective biological management. Real-time tracking by digital means of the performances of antagonistic agents against plant pathogens may be a great opportunity to optimize field practices and increase disease biocontrol efficacy. In this study, a non-linear machine learning approach, based on Artificial Neural Networks technique, was used to assess the biocontrol efficacy of Trichoderma spp. from VIS-NIR spectral reflectance, estimating disease severity in baby leaf young plants during specific plant-pathogen-antagonist interactions. The most successful accurate model architecture achieved a predictive success rate of 74%. The variable impact analysis on the 207 variables considered showed that the 20 most important frequencies lie in the intervals Δω1 = [426,460] nm Δω2 = [495,530] nm Δω3 = [570,667] nm Δω4 = [770,880] nm Δω5 = [940,1000] nm. The model with improved classification accuracy is highly suitable for the automated detection of healthy status considering a wide spectrum of crop/pathogen targets under Trichoderma spp. beneficial dealings. Findings indicate that hyperspectral image-derived features could be used as proxy for disease level tracking under biological control of telluric pathogens in baby leaf vegetable cultivations.

The consumers' perception of the importance of healthy food has promoted the interest towards fruit, vegetables and spices rich in antioxidants. Moreover, changing in eating habits has encouraged the diffusion of ready-to-eat commodities with specific merceological standards. In the present study, 12 hot pepper varieties devoted to powder production, canned processing, blister packaging have been evaluated for morpho-agronomic traits and bioactive compounds in replicated trials in three locations and two growing seasons.For all traits, the genotypic component (G) and the genotype × location interaction (G×L) were significant (P 0.05). Morphological fruit traits evidenced a strong effect of the genotype (95%) while yield and biochemical traits showed a variable response to environmental effects. Capsaicin and the yellow fraction of carotenoids were the only nutraceutical compounds showing a relatively high G effect, contrariwise, ascorbic acid, carotenoids and tocopherols were more influenced by location (L) and year of cultivation (Y). Results showed how breeding for quality traits in chilli is still challenging. Multivariate analysis allowed to identify pairs of traits distinguishing the different market typologies: powder types exhibited a lower productivity and higher pungency while those for blister packaging had a remarkable antioxidant capacity.A comprehensive approach was used to study the plasticity of agronomic and nutritional traits in chilli pepper. The set of cultivars analysed produced fruits with considerable levels of several health-related metabolites, including capsaicin, carotenoids and antioxidant phytochemicals. This information is useful for precision breeding of novel varieties.

Mitochondrial genomes (mitogenomes) in higher plants can induce cytoplasmic male sterility and be somehow involved in nuclear-cytoplasmic interactions affecting plant growth and agronomic performance. They are larger and more complex than in other eukaryotes, due to their recombinogenic nature. For most plants, the mitochondrial DNA (mtDNA) can be represented as a single circular chromosome, the so-called master molecule, which includes repeated sequences that recombine frequently, generating sub-genomic molecules in various proportions. Based on the relevance of the potato crop worldwide, herewith we report the complete mtDNA sequence of two S. tuberosum cultivars, namely Cicero and D&eacute, sir&eacute, e, and a comprehensive study of its expression, based on high-coverage RNA sequencing data. We found that the potato mitogenome has a multi-partite architecture, divided in at least three independent molecules that according to our data should behave as autonomous chromosomes. Inter-cultivar variability was null, while comparative analyses with other species of the Solanaceae family allowed the investigation of the evolutionary history of their mitogenomes. The RNA-seq data revealed peculiarities in transcriptional and post-transcriptional processing of mRNAs. These included co-transcription of genes with open reading frames that are probably expressed, methylation of an rRNA at a position that should impact translation efficiency and extensive RNA editing, with a high proportion of partial editing implying frequent mis-targeting by the editing machinery.

Plant genetic resources (PGR) represent valuable sources of genetic variability for crop breeding. The development of novel biotechnologies is necessary for increasing the efficiency of their use in pre-breeding and breeding work. The genome sequencing of hundreds of genotypes and the mining of allele diversity in major crops and populations of landraces and wild relatives allow the isolation of genes underlying characters of interest and their precise modification or transfer into targeted varieties. The technological developments and applications of new plant breeding techniques (NPBT) that maximize the similarity with gene transfer by crossing (cisgenesis/intragenesis) or the accuracy of biotechnological approaches (genome editing) are reviewed. Their potentialities and current limitations as well as the possible advantages of using them separately or combined for the exploitation of PGR in crop breeding are also discussed. Above-mentioned NPBT tackle some objections to the application of biotechnologies in agriculture and are under review worldwide to assess the possible exclusion from the current regulation systems for genetically modified plants.

Members of the genus Capsicum are of great economic importance, including both wild forms and cultivars of peppers and chilies. The high number of potentially informative characteristics that can be identified through next-generation sequencing technologies gave a huge boost to evolutionary and comparative genomic research in higher plants. Here, we determined the complete nucleotide sequences of the plastomes of eight Capsicum species (eleven genotypes), representing the three main taxonomic groups in the genus and estimated molecular diversity. Comparative analyses highlighted a wide spectrum of variation, ranging from point mutations to small/medium size insertions/deletions (InDels), with accD, ndhB, rpl20, ycf1, and ycf2 being the most variable genes. The global pattern of sequence variation is consistent with the phylogenetic signal. Maximum-likelihood tree estimation revealed that Capsicum chacoense is sister to the baccatum complex. Divergence and positive selection analyses unveiled that protein-coding genes were generally well conserved, but we identified 25 positive signatures distributed in six genes involved in different essential plastid functions, suggesting positive selection during evolution of Capsicum plastomes. Finally, the identified sequence variation allowed us to develop simple PCR-based markers useful in future work to discriminate species belonging to different Capsicum complexes.

Crops are the major source of food supply and raw materials for the processing industry. A balance between crop production and food consumption is continually threatened by plant diseases and adverse environmental conditions. This leads to serious losses every year and results in food shortages, particularly in developing countries. Presently, cutting-edge technologies for genome sequencing and phenotyping of crops combined with progress in computational sciences are leading a revolution in plant breeding, boosting the identification of the genetic basis of traits at a precision never reached before. In this frame, machine learning (ML) plays a pivotal role in data-mining and analysis, providing relevant information for decision-making towards achieving breeding targets. To this end, we summarize the recent progress in next-generation sequencing and the role of phenotyping technologies in genomics-assisted breeding toward the exploitation of the natural variation and the identification of target genes. We also explore the application of ML in managing big data and predictive models, reporting a case study using microRNAs (miRNAs) to identify genes related to stress conditions.

Increasing the ability to investigate plant functions and structure through non-invasive methods with high accuracy has become a major target in plant breeding and precision agriculture. Emerging approaches in plant phenotyping play a key role in unraveling quantitative traits responsible for growth, production, quality, and resistance to various stresses. Beyond fully automatic phenotyping systems, several promising technologies can help accurately characterize a wide range of plant traits at affordable costs and with high-throughput. In this review, we revisit the principles of proximal and remote sensing, describing the application of non-invasive devices for precision phenotyping applied to the protected horticulture. Potentiality and constraints of big data management and integration with “omics” disciplines will also be discussed.

Landraces are an important resource for crop breeding, due to their resilience and content of quality traits. However, genetic and phenotypic variability needs to be carefully characterized for proper direct and indirect use. In the present study, a multidisciplinary approach was carried out to assess the Italian sweet pepper landrace ‘Friariello’. A total of 18 traditional accessions were compared with five hybrids and two ecotypes with similar fruit typology. Genetic and morpho-agronomic characterization allowed us to distinguish five different group types of ‘Friariello’. Accessions showing two/three lobes at the blossom end of the fruit were found to be the most productive, whereas the genotypes showing one/two lobes at the blossom end were the most homogeneous. A total of 167 volatile organic compounds (VOCs) were identified in the collection analysed. Moreover, of the 37 targeted VOCs, 29 showed significant differences in content among the pepper genotypes studied. Of such VOCs related to main flavours described for pepper in the literature, ten were found to be the major determinants of variability among the derived ‘Friariello’ groups. A slightly negative, albeit not significant, correlation was observed between ascorbic acid (AsA) content and agronomic traits, suggesting a better quality for less productive accessions, but also the possibility to improve yield without significantly reducing the AsA levels. The approach used allowed us to define how the different typologies can be used for different breeding purposes, integrating the peculiar properties in order to establish a desirable landrace ideotype. Furthermore, valuable sources for improving quality traits in pepper breeding were identified.

The use of germplasm as source of resistances to biotic stresses represents an important strategy in pepper in order to improve susceptible cultivars and release new resistant varieties. The study of the relationships among genotypes within germplasm collections is useful for genetic improvement as well as for the management of genetic resources. In the present study, 59 accessions belonging to nine different species have been genotyped with a set of ten simple sequence repeats (SSR) and eleven allele specific molecular markers linked to major genes and QTLs for pathogen resistance and pungency. Results allowed to select promising genotypes in terms of content of resistance alleles and to identify highly polymorphic SSR pairs which can be used for species discrimination and hybrid identification. The diversity pattern of the SSR loci revealed a number of distinct alleles per locus, ranging from 3 to 11 with an average of 6.3. Hierarchical analysis allowed to distinguish several clusters each including the different species under study with genetic similarities in the range of 7–100 %. The most distant genotypes from the C. annuum accessions belong to C. baccatum and C. pubescens. The differentiation of the accessions on the basis of the presence of resistance and pungency associated markers, allowed to identify 17 clusters in which the resistance genes were differently distributed. Tsw, L 4 and Pvr4 resulted to be most represented within the genotypes of the collection. Accessions belonging to C. chinense and C. frutescens resulted to be the most interesting ones for pyramiding multiple resistance genes, confirming the need of interspecific crosses in breeding programs in order to transfer useful alleles from domesticated Capsicum spp. to varieties and landraces of cultivated pepper.

Tomato (Solanum lycopersicum L.) plants are characterized by having a variety of fruit colours that reflect the composition and accumulation of diverse carotenoids in the berries. Carotenoids are extensively studied for their health-promoting effects and this explains the great attention these pigments received by breeders and researchers worldwide. In this work we applied Agilent’s SureSelect liquid-phase sequence capture and Illumina targeted re-sequencing of 34 tomato genes belonging to the methylerythritol phosphate (MEP) carotenoid pathway on a panel of 48 genotypes which differ for carotenoid content calculated as the sum of β-carotene, cis- and trans-lycopene. We targeted 230 kb of genomic regions including all exons and regulatory regions and observed ~40% of on-target capture. We found ample genetic variation among all the genotypes under study and generated an extensive catalog of SNPs/InDels located in both genic and regulatory regions. SNPs/InDels were also classified based on genomic location and putative biological effect. With our work we contributed to the identification of allelic variations possibly underpinning a key agronomic trait in tomato. Results from this study can be exploited for the promotion of novel studies on tomato bio-fortification as well as of breeding programs related to carotenoid accumulation in fruits.

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 allowing the identification of genes and superior alleles associated with desirable traits. Furthermore, innovative biotechnological approaches provide a step forward in 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 (NPBT) 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 (Cas9) system, zinc-finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs) 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

Gluten is a protein composite found in wheat and related grains including barley, rye, oat, and all their species and hybrids. Gluten matrix is a biomolecular network of gliadins and glutenins that contribute to the texture of pastries, breads, and pasta. Gliadins are mainly responsible for celiac disease, one of the most widespread food-related pathologies in Western world. In view of the importance of gliadin proteins, by combining the quartz crystal microbalance technology, a cheap and robust piezoelectric transducer, with the so-called photonic immobilization technique, an effective surface functionalization method that provides spatially oriented antibodies on gold substrates, we realized a sensitive and reliable biosensor for quantifying these analytes extracted from real samples in a very short time. The resulting immunosensor has a limit of detection of about 4 ppm and, more remarkably, shows excellent sensitivity in the range 7.5-15 ppm. This feature makes our device reliable and effective for practical applications since it is able to keep low the influence of false positives.

Vegetables include high-value crops with health-promoting effects and reduced environmental impact. The availability of genomic and biotechnological tools in certain species, coupled with the recent development of new breeding techniques based on precise editing of DNA, provides unique opportunities to finally take advantage of the past decades of detailed genetic analyses, thus making improvement of traits related to quality and stress tolerance achievable in a reasonable time frame. Recent reports of such approaches in vegetables illustrate the feasibility of obtaining multiple homozygous mutations in a single generation, heritable by the progeny, using stable or transient transformation approaches, which may not rely on the integration of unwanted foreign DNA. Application of these approaches to currently non-sequenced/tissue culture recalcitrant crops will contribute to meet the challenges posed by the increase in population and climate change.

Chloroplast genetic engineering has long been recognised as a powerful technology to produce recombinant proteins. To date, however, little attention has been given to the causes of pleiotropic effects reported, in some cases, as consequence of the expression of foreign proteins in transgenic plastids. In this study, we investigated the phenotypic alterations observed in transplastomic tobacco plants accumulating the Pr55(gag) polyprotein of human immunodeficiency virus (HIV-1). The expression of Pr55(gag) at high levels in the tobacco plastome leads to a lethal phenotype of seedlings grown in soil, severe impairment of plastid development and photosynthetic activity, with chloroplasts largely resembling undeveloped proplastids. These alterations are associated to the binding of Pr55(gag) to thylakoids. During particle assembly in HIV-1 infected human cells, the binding of Pr55(gag) to a specific lipid [phosphatidylinositol-(4-5) bisphosphate] in the plasma membrane is mediated by myristoylation at the amino-terminus and the so-called highly basic region (HBR). Surprisingly, the non-myristoylated Pr55(gag) expressed in tobacco plastids was likely able, through the HBR motif, to bind to nonphosphorous glycerogalactolipids or other classes of lipids present in plastidial membranes. Although secondary consequences of disturbed chloroplast biogenesis on expression of nuclear-encoded plastid proteins cannot be ruled out, results of proteomic analyses suggest that their altered accumulation could be due to retrograde control in which chloroplasts relay their status to the nucleus for fine-tuning of gene expression.

Background Knowledge on population structure and genetic diversity in vegetable crops is essential for association mapping studies and genomic selection. Genotyping by sequencing (GBS) represents an innovative method for large scale SNP detection and genotyping of genetic resources. Herein we used the GBS approach for the genome-wide identification of SNPs in a collection of Capsicum spp. accessions and for the assessment of the level of genetic diversity in a subset of 222 cultivated pepper (Capsicum annum) genotypes. Results GBS analysis generated a total of 7,568,894 master tags, of which 43.4% uniquely aligned to the reference genome CM334. A total of 108,591 SNP markers were identified, of which 105,184 were in C. annuum accessions. In order to explore the genetic diversity of C. annuum and to select a minimal core set representing most of the total genetic variation with minimum redundancy, a subset of 222 C. annuum accessions were analysed using 32,950 high quality SNPs. Based on Bayesian and Hierarchical clustering it was possible to divide the collection into three clusters. Cluster I had the majority of varieties and landraces mainly from Southern and Northern Italy, and from Eastern Europe, whereas clusters II and III comprised accessions of different geographical origins. Considering the genome-wide genetic variation among the accessions included in cluster I, a second round of Bayesian (K = 3) and Hierarchical (K = 2) clustering was performed. These analysis showed that genotypes were grouped not only based on geographical origin, but also on fruit-related features. Conclusions GBS data has proven useful to assess the genetic diversity in a collection of C. annuum accessions. The high number of SNP markers, uniformly distributed on the 12 chromosomes, allowed the accessions to be distinguished according to geographical origin and fruit-related features. SNP markers and information on population structure developed in this study will undoubtedly support genome-wide association mapping studies and marker-assisted selection programs. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3297-7) contains supplementary material, which is available to authorized users.

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.

In the past decades, the progress made in plant biotechnology has made possible the use of plants as a novel production platform for a wide range of molecules. In this context, the transformation of the plastid genome has given a huge boost to prove that plants are a promising system to produce recombinant proteins. In this review, we provide a background on plastid genetics and on the principles of this technology in higher plants. Further, we discuss the most recent biotechnological applications of plastid transformation for the production of enzymes, therapeutic proteins, antibiotics, and proteins with immunological properties. We also discuss the potential of plastid biotechnology and the novel tools developed to overcome some limitations of chloroplast transformation. © 2011 Elsevier Inc.

Mitochondrial genomes in plants are larger and more complex than in other eukaryotes due to their recombinogenic nature as widely demonstrated. The mitochondrial DNA (mtDNA) is usually represented as a single circular map, the so-called master molecule. This molecule includes repeated sequences, some of which are able to recombine, generating sub-genomic molecules in various amounts, depending on the balance between their recombination and replication rates. Recent advances in DNA sequencing technology gave a huge boost to plant mitochondrial genome projects. Conventional approaches to mitochondrial genome sequencing involve extraction and enrichment of mitochondrial DNA, cloning, and sequencing. Large repeats and the dynamic mitochondrial genome organization complicate de novo sequence assembly from short reads. The PacBio RS long-read sequencing platform offers the promise of increased read length and unbiased genome coverage and thus the potential to produce genome sequence data of a finished quality (fewer gaps and longer contigs). However, recently published articles revealed that PacBio sequencing is still not sufficient to address mtDNA assembly-related issues. Here we present a preliminary hybrid assembly of a potato mtDNA based on both PacBio and Illumina reads and debate the strategies and obstacles in assembling genomes containing repeated sequences that are recombinationally active and serve as a constant source of rearrangements.