Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum)

Key message 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 M-0 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

Functional Hyperspectral Imaging by High-Related Vegetation Indices to Track the Wide-Spectrum Trichoderma Biocontrol Activity Against Soil-Borne Diseases of Baby-Leaf Vegetables

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

Genomic diversity and novel genome-wide association with fruit morphology in <i>Capsicum</i>, from 746k polymorphic sites

Capsicum is one of the major vegetable crops grown worldwide. Current subdivision in clades and species is based on morphological traits and coarse sets of genetic markers. Broad variability of fruits has been driven by breeding programs and has been mainly studied by linkage analysis. We 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 major subdivision of C. annuum separates large and bulky fruits from 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). Our 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 number of genetic variants analyzed. We identified a large set of markers that can be used for population genetic studies and genetic association analyses. Our results provide a comprehensive and precise perspective on genomic variability in Capsicum at population-level and suggest that future fine genetic association studies will yield useful results for breeding

Cultivated Tomato (Solanum lycopersicum L.) Suffered a Severe Cytoplasmic Bottleneck during Domestication: Implications from Chloroplast Genomes

In various crops, genetic bottlenecks occurring through domestication can limit crop resilience to biotic and abiotic stresses. In the present study, we investigated nucleotide diversity in tomato chloroplast genome through sequencing seven plastomes of cultivated accessions from the Campania region (Southern Italy) and two wild species among the closest (Solanum pimpinellifolium) and most distantly related (S. neorickii) species to cultivated tomatoes. Comparative analyses among the chloroplast genomes sequenced in this work and those available in GenBank allowed evaluating the variability of plastomes and defining phylogenetic relationships. A dramatic reduction in genetic diversity was detected in cultivated tomatoes, nonetheless, a few de novo mutations, which still differentiated the cultivated tomatoes from the closest wild relative S. pimpinellifolium, were detected and are potentially utilizable as diagnostic markers. Phylogenetic analyses confirmed that S. pimpinellifolium is the closest ancestor of all cultivated tomatoes. Local accessions all clustered together and were strictly related with other cultivated tomatoes (S. lycopersicum group). Noteworthy, S. lycopersicum var. cerasiforme resulted in a mixture of both cultivated and wild tomato genotypes since one of the two analyzed accessions clustered with cultivated tomato, whereas the other with S. pimpinellifolium. Overall, our results revealed a very reduced cytoplasmic variability in cultivated tomatoes and suggest the occurrence of a cytoplasmic bottleneck during their domestication

ddRAD sequencing-based genotyping for population structure analysis in cultivated tomato provides new insights into the genomic diversity of Mediterranean 'da serbo' type long shelf-life germplasm

[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 <5kb. We then investigated SNPs showing contrasted minor frequency allele (MAF) in 'da serbo' materials, resulting in the identification of high frequencies in this germplasm of several mutations in genes related to stress tolerance and fruit maturation such as CTR1 and JAR1. Finally, a mini-core collection of 58 accessions encompassing most of the diversity was selected for further exploitation of key traits. Our findings suggest the presence of a genetic footprint of the 'da serbo' germplasm selected in the Mediterranean basin. 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Large scale phenotyping and molecular analysis in a germplasm collection of rocket salad (Eruca vesicaria) reveal a differentiation of the gene pool by geographical origin

[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.Guijarro-Real, C.; Navarro, A.; Esposito, S.; Festa, G.; Macellaro, R.; Di Cesare, C.; Fita, A.... (2020). Large scale phenotyping and molecular analysis in a germplasm collection of rocket salad (Eruca vesicaria) reveal a differentiation of the gene pool by geographical origin. 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Genotypic and environmental effects on morpho-physiological and agronomic performances of a tomato diversity panel in relation to nitrogen and water stress under organic farming

[EN] The agricultural scenario of the upcoming decades will face major challenges for the increased and sustainable agricultural production and the optimization of the efficiency of water and fertilizer inputs. Considering the current and foreseen water scarcity in several marginal and arid areas and the need for a more sustainable farming production, the selection and development of cultivars suitable to grow under low-input conditions is an urgent need. In this study, we assayed 42 tomato genotypes for thirty-two morphophysiological and agronomic traits related to plant, fruit, and root characteristics under standard (control) and no-nitrogen fertilization or water deficit (30% of the amount given to non-stressed trials) treatments in two sites (environments), which corresponded to organic farms located in Italy and Spain. A broad range of variation was found for all traits, with significant differences between the applied treatments and the cultivation sites. Dissection of genotypic (0), environmental (E), and treatment (T) factors revealed that the three main factors were highly significant for many traits, although G was the main source of variation in most cases. G x E interactions were also important, while G x T and E x T were less relevant. Only fruit weight and blossom end rot were highly significant for the triple interaction (G x E x T). Reduction of water supply significantly increased the soluble solid content in both locations, whereas both nitrogen and water stress led to a general decrease in fruit weight and total yield. Despite so, several accessions exhibited better performances than the control when cultivated under stress. Among the accessions evaluated, hybrids were promising in terms of yield performance, while overall landraces and heirlooms exhibited a better quality. This suggests the possibility of exploiting both the variation within ancient varieties and the heterosis for yield of hybrids to select and breed new varieties with better adaptation to organic farming conditions, both under optimal and suboptimal conditions. The results shed light on the strategies to develop novel varieties for organic farming, giving hints into the management of inputs to adopt for a more sustainable tomato cultivation.This work has been funded by the European Union's Horizon 2020 Research and Innovation Programme under grant agreement no. 774244 (Breeding for resilient, efficient and sustainable organic vegetable production; BRESOV).Tripodi, P.; Figás-Moreno, MDR.; Leteo, F.; Soler Aleixandre, S.; Díez Niclós, MJTDJ.; Campanelli, G.; Cardi, T.... (2022). Genotypic and environmental effects on morpho-physiological and agronomic performances of a tomato diversity panel in relation to nitrogen and water stress under organic farming. Frontiers in Plant Science. 13:1-19. https://doi.org/10.3389/fpls.2022.9365961191

The complete plastome sequences of eleven Capsicum genotypes: Insights into DNA variation and molecular evolution

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