NGS-based genotyping, high-throughput phenotyping and genome-wide association studies laid the foundations for next-generation breeding in horticultural crops

Demographic trends and changes to climate require a more efficient use of plant genetic resources in breeding programs. Indeed, the release of high-yielding varieties has resulted in crop genetic erosion and loss of diversity. This has produced an increased susceptibility to severe stresses and a reduction of several food quality parameters. Next generation sequencing (NGS) technologies are being increasingly used to explore “gene space” and to provide high-resolution profiling of nucleotide variation within germplasm collections. On the other hand, advances in high-throughput phenotyping are bridging the genotype-to-phenotype gap in crop selection. The combination of allelic and phenotypic data points via genome-wide association studies is facilitating the discovery of genetic loci that are associated with key agronomic traits. In this review, we provide a brief overview on the latest NGS-based and phenotyping technologies and on their role to unlocking the genetic potential of vegetable crops; then, we discuss the paradigm shift that is underway in horticultural crop breeding

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

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. Moreover, we provide novel insights on LSL 'da serbo' germplasm as a promising source of alleles for tolerance to stresses.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).Esposito, S.; Cardi, T.; Campanelli, G.; Sestili, S.; Díez Niclós, MJTDJ.; Soler Aleixandre, S.; Prohens Tomás, J.... (2020). 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. Horticulture Research. 7(1):1-14. https://doi.org/10.1038/s41438-020-00353-611471Faostat 2018 http://www.fao.org/Jenkins, J. A. The origin of the cultivated tomato. Econ. Bot. 2, 379–392 (1948).Blanca, J. et al. Variation revealed by SNP genotyping and morphology provides insight into the origin of the tomato. PLoS ONE 7, e48198 (2012).Razifard, H. et al. Genomic evidence for complex domestication history of the cultivated tomato in Latin America. Mol. Biol. Evol. 37, 1118–1132 (2020).Bauchet, G. & Causse, M., Genetic diversity in tomato (Solanum lycopersicum) and its wild relatives. In Genetic Diversity in Plants (INTECH Open Access Publisher, 2012).Bai, Y. & Lindhout, P. Domestication and breeding of tomatoes: what have we gained and what can we gain in the future? Ann. Bot. 100, 1085–1094 (2007).Dwivedi, S., Goldman, I. & Ortiz, R. Pursuing the potential of heirloom cultivars to improve adaptation, nutritional, and culinary features of food crops. Agronomy 9, 441 (2019).Casañas, F., Simó, J., Casals, J. & Prohens, J. Toward an evolved concept of landrace. Front. Plant Sci. 8, 145 (2017).Klee, H. J. & Tieman, D. M. The genetics of fruit flavour preferences. Nat. Rev. Genet. 19, 347–356 (2018).Conesa, M. À., Fullana-Pericàs, M., Granell, A. & Galmés, J. Mediterranean long shelf-life landraces: an untapped genetic resource for tomato improvement. Front. Plant Sci. 10, 1651 (2020).Casals, J., Martí, R., Casañas, F. & Cebolla, J. Sugar-and-acid profile of Penjar tomatoes and its evolution during storage. Sci. Agric. 72, 314–321 (2015).Casals, J. et al. Genetic basis of long shelf life and variability into Penjar tomato. Genet. Resour. Crop Evol. 59, 219–229 (2012).van Berloo, R. et al. Diversity and linkage disequilibrium analysis within a selected set of cultivated tomatoes. Theor. Appl. Genet. 117, 89–101(2008).Robbins, M. D. et al. Mapping and linkage disequilibrium analysis with a genome-wide collection of SNPs that detect polymorphism in cultivated tomato. J. Exp. Bot. 62, 1831–1845 (2011).Sim, S. C., Robbins, M. D., Deynze, A. V., Michel, A. P. & Francis, D. M. Population structure and genetic differentiation associated with breeding history and selection in tomato (Solanum lycopersicum L.). Heredity 106, 927–935 (2011).Corrado, G., Piffanelli, P., Caramante, M., Coppola, M. & Rao, R. SNP genotyping reveals genetic diversity between cultivated landraces and contemporary varieties of tomato. BMC Genomics 14, 835 (2013).Corrado, G., Caramante, M., Piffanelli, P. & Rao, R. Genetic diversity in Italian tomato landraces: implications for the development of a core collection. Sci. Hortic. 168, 138–144 (2014).Sim, S. C. et al. High-density SNP genotyping of tomato (Solanum lycopersicum L.) reveals patterns of genetic variation due to breeding. PloS ONE 7, e45520 (2012).Sacco, A. et al. Exploring a tomato landraces collection for fruit-related traits by the aid of a high-throughput genomic platform. PLoS ONE 10, e0137139 (2015).Tranchida-Lombardo, V. et al. Genetic diversity in a collection of Italian long storage tomato landraces as revealed by SNP markers array. Plant Biosyst. 153, 288–297 (2019).Pérez de, Castro et al. Application of genomic tools in plant breeding. Curr. Genomics 13, 179–195 (2012).Peterson, B. K. et al. Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species. PLoS ONE 7, e37135 (2012).Shirasawa, K., Hirakawa, H. & Isobe, S. Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato. DNA Res. 23, 145–153 (2016).Yang, G. et al. Development of a universal and simplified ddRAD library preparation approach for SNP discovery and genotyping in angiosperm plants. Plant Met. 12, 39 (2016).Okada, Y. et al. Genome-wide association studies (GWAS) for yield and weevil resistance in sweet potato (Ipomoea batatas (L.) Lam. Plant Cell Rep. 38, 1383–1392 (2019).The Tomato Genome Consortium. The tomato gene sequence provides insights into fleshy fruit evolution. Nature 485, 635–641 (2012).Hosmani, P. S. et al. An improved de novo assembly and annotation of the tomato reference genome using single-molecule sequencing, Hi-C proximity ligation and optical maps. Preprint at https://www.biorxiv.org/content/10.1101/767764v1 (2020).Aguirre, N. et al. Optimizing ddRADseq in non-model species: a case study in Eucalyptus dunnii maiden. Agronomy 9, 484 (2019).Catchen, J., Hohenlohe, P., Bassham, S., Amores, A. & Cresko, W. Stacks: an analysis tool set for population genomics. Mol. Ecol. 22, 3124–3140 (2013).Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).Weir, B. S. Genetic Data Analysis II (Sinauer Associates Inc., Sunderland, MA, 1996).Botstein, D., White, R. L., Skolnick, M. & Davis, R. W. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32, 314–331 (1980).Alexander, D. H., Novembre, J. & Lange, K. Fast model-based estimation of ancestry in unrelated individuals. Genome Res. 19, 1655–1664 (2009).Gao, X. & Starmer, J. D. AWclust: point‐and‐click software for non‐parametric population structure analysis. BMC Bioinformatics 9, 77 (2008).Liu, K. & Muse, S. V. PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics 21, 2128–2129 (2005).RStudio Team RStudio: Integrated Development for R (RStudio, Inc., Boston, MA, 2016).De Beukelaer, H., Smýkal, P., Davenport, G. F., Fack, V. & Core Hunter, I. I. fast core subset selection based on multiple genetic diversity measures using Mixed Replica search. BMC Bioinforma. 13, 312 (2012).Schwacke, R. et al. MapMan4: a refined protein classification and annotation framework applicable to multi-omics data analysis. Mol. Plant 12, 879–892 (2019).Kong, Z. et al. Kinesin-4 functions in vesicular transport on cortical microtubules and regulates cell wall mechanics during cell elongation in plants. Mol. Plant 8, 1011–1023 (2015).Wang, X., Cai, X., Xu, C., Wang, Q. & Dai, S. Drought-responsive mechanisms in plant leaves revealed by proteomics. Int. J. Mol. Sci. 17, 1706 (2016).Xu, X., Walter, W. J., Liu, Q., Machens, I. & Nick, P. A rice class-XIV kinesin enters the nucleus in response to cold. Sci. Rep. 8, 3588 (2018).Opiyo, S. O. & Moriyama, E. N. Mining Cytochrome b561 proteins from plant genomes. Int. J. Bioinform. Res. Appl. 6, 209–221 (2010).Lee, S. et al. The small GTPase, nucleolar GTP-binding protein 1 (NOG1), has a novel role in plant innate immunity. Sci. Rep. 7, 9260 (2017).Usadel, B. et al. Extension of the visualization tool MapMan to allow statistical analysis of arrays, display of corresponding genes, and comparison with known responses. Plant Physiol. 138, 1195–1204 (2005).Sáez-Vásquez, J. & Delseny, M. Ribosome biogenesis in plants: from functional 45S ribosomal DNA organization to ribosome assembly factors. Plant Cell 31, 1945–1967 (2019).Mareri, L., Romi, M. & Cai, G. Arabinogalactan proteins: actors or spectators during abiotic and biotic stress in plants? Plant Biosyst. 153, 173–185 (2018).Zhong, S., Chang, C. in Ethylene signalling: the CTR1 protein kinase, Vol. 44 (eds Oxford, UK: Wiley-Blackwell) ch. 6 (Annual Plant Reviews, 2012).Suza, W. P. & Staswick, P. E. The role of JAR1 in jasmonoyl-L-isoleucine production in Arabidopsis wound response. Planta 227, 1221–1232 (2008).Barry, C. S. & Giovannoni, J. J. Ethylene and fruit ripening. J. Plant Growth Regul. 26, 143–159 (2007).Giovannoni, J. J. Fruit ripening mutants yield insights into ripening control. Curr. Opin. Plant Biol. 10, 283–289 (2007).Wang, R. et al. The rin, nor and Cnr spontaneous mutations inhibit tomato fruit ripening in additive and epistatic manners. Plant Sci. 294, 110436 (2020).Albrecht, E., Escobar, M. & Chetelat, R. Genetic diversity and population structure in the tomato-like nightshades Solanum lycopersicoides and S. sitiens. Ann. Bot. 105, 535–554 (2010).Xu, J. et al. Phenotypic diversity and association mapping for fruit quality traits in cultivated tomato and related species. Theor. Appl. Genet. 126, 567–581 (2013).Bauchet, G. et al. Use of modern tomato breeding germplasm for deciphering the genetic control of agronomical traits by Genome Wide Association study. Theor. Appl. Genet. 130, 875–889 (2017).Rothan, C., Diouf, I. & Causse, M. Trait discovery and editing in tomato. Plant J. 97, 73–90 (2018).Wang, T. et al. Analysis of genetic diversity and population structure in a tomato (Solanum lycopersicum L.) germplasm collection based on single nucleotide polymorphism. Genet. Mol. Res. 15, 1–12 (2016).Massaretto, I. L. et al. Recovering tomato landraces to simultaneously improve fruit yield and nutritional quality against salt stress. Front. Plant Sci. 9, 1778 (2018).Aflitos, S. et al. Exploring genetic variation in the tomato (Solanum section Lycopersicon) clade by whole-genome sequencing. Plant J. 80, 136–148 (2014).Leimu, R., Mutikainen, P. I. A., Koricheva, J. & Fischer, M. How general are positive relationships between plant population size, fitness and genetic variation? J. Ecol. 94, 942–952 (2006).Mackay, I. & Powell, W. Methods for linkage disequilibrium mapping in crops. Trends Plant Sci. 12, 57–63 (2007).Ersoz, E. S., Yu, J., Buckler, E. S. Applications of linkage disequilibrium and association mapping in Genomics-assisted crop improvement, Vol. 1 (eds. Varshney R. K., Tuberosa R.) (Dordrecht, Springer, 2008).D’Agostino, N. & Tripodi, P. NGS-based genotyping, high-throughput phenotyping and genome-wide association studies laid the foundations for next-generation breeding in horticultural crops. Diversity 9, 38 (2017).Kopeliovitch, E., Rabinowitch, H. D., Mizrahi, Y. & Kedar, N. Mode of inheritance of Alcobaca, a tomato fruit-ripening mutant. Euphytica 30, 223–225 (1981).Lobo, M., Bassett, M. J. & Hannah, L. C. Inheritance and characterization of the fruit ripening mutation in ‘alcobaca’ tomato. J. Am. Soc. Hortic. Sci. 109, 741–745 (1984).Mutschler, M., Guttieri, M., Kinzer, S., Grierson, D. & Tucker, G. Changes in ripening-related processes in tomato conditioned by the alc mutant. Theor. Appl. Genet. 76, 285–292 (1988).Conesa, M. A. et al. The postharvest tomato fruit quality of long shelf-life Mediterranean landraces is substantially influenced by irrigation regimes. Postharvest Biol. Technol. 93, 114–121 (2014).Bota, J. et al. Characterization of a landrace collection for Tomàtiga de Ramellet (Solanum lycopersicum L.) from the Balearic Islands. Genet. Resour. Crop Evol. 61, 1131–1146 (2014).Kumar, R., Tamboli, V., Sharma, R. & Sreelakshmi, Y. NAC-NOR mutations in tomato Penjar accessions attenuate multiple metabolic processes and prolong the fruit shelf life. Food Chem. 259, 234–244 (2018).Cho, Y. H. & Yoo, S. D. Novel connections and gaps in ethylene signaling from the ER membrane to the nucleus. Front. Plant Sci. 5, 733 (2015).Ju, C. et al. CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis. Proc. Natl. Acad. Sci. USA 109, 19486–19491 (2012).Achard, P. et al. Integration of plant responses to environmentally activated phytohormonal signals. Science 311, 91–94 (2006).Morello, L., Giani, S., Troina, F. & Breviario, D. Testing the IMEter on rice introns and other aspects of intron-mediated enhancement of gene expression. J. Exp. Bot. 62, 533–544 (2011).Rose, A. B., Carter, A., Korf, I. & Kojima, N. Intron sequences that stimulate gene expression in Arabidopsis. Plant Mol. Biol. 92, 337–346 (2016).Moore, S., Vrebalov, J., Payton, P. & Giovannoni, J. J. Use of genomics tools to isolate key ripening genes and analyse fruit maturation in tomato. J. Exp. Bot. 53, 2023–2030 (2002).Vrebalov, J. et al. A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus. Science 296, 343–346 (2002).Zhou, J. L., Qiu, J. & Ye, Z. H. Alteration in secondary wall deposition by overexpression of the Fragile Fiber1 kinesin-like protein in Arabidopsis. J. Integr. Plant Biol. 49, 1235–1243 (2007).Vallés, D. et al. A cysteine protease isolated from ripe fruits of Solanum granulosoleprosum (Solanaceae). Protein J. 27, 267 (2008).Guerrero, F. D., Jones, J. T. & Mullet, J. E. Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Mol. Biol. 15, 11–26 (1990).Yamaguchi-Shinozaki, K., Koizumi, M., Urao, S. & Shinozaki, K. Molecular cloning and characterization of 9 cDNAs for genes that are responsive to desiccation in Arabidopsis thaliana: sequence analysis of one cDNA clone that encodes a putative transmembrane channel protein. Plant Cell Physiol. 33, 217–224 (1992).Koizumi, M., Yamaguchi-Shinozaki, K., Tsuji, H. & Shinozaki, K. Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana. Gene 129, 175–182 (1993).Krüger, J. et al. A tomato cysteine protease required for Cf-2-dependent disease resistance and suppression of autonecrosis. Science 296, 744–747 (2002).Fan, Y., Yang, W., Yan, Q., Chen, C. & Li, J. Genome-wide identification and expression analysis of the protease inhibitor gene families in tomato. Genes 11, 1 (2020).Noctor, G. & Foyer, C. H. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. 49, 249–279 (1998).Mittler, R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405–410 (2002).Nanasato, Y., Akashi, K. & Yokota, A. Co-expression of cytochrome b561 and ascorbate oxidase in leaves of wild watermelon under drought and high light conditions. Plant Cell Physiol. 46, 1515–1524 (2005).Feeley, E. M. et al. Galectin-3 directs antimicrobial guanylate binding proteins to vacuoles furnished with bacterial secretion systems. Proc. Natl. Acad. Sci. USA 114, E1698–E1706 (2017)

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

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. Euphytica. 216(3):1-20. https://doi.org/10.1007/s10681-020-02586-xS1202163Andrey P, Maurin Y (2005) Free-D: an integrated environment for three-dimensional reconstruction from serial sections. J Neurosci Methods 145:233–244Awada L, Phillips PWB, Smyth SJ (2018) The adoption of automated phenotyping by plant breeders. Euphytica 214:148. https://doi.org/10.1007/s10681-018-2226-zBarrs HD, Weatherley PE (1962) A re-examination of the relative turgidity technique for estimating water deficits in leaves. Aust J Biol Sci 15(3):413–428Bell L, Wagstaff C (2014) Glucosinolates, myrosinase hydrolysis products, and flavonols found in rocket (Eruca sativa and Diplotaxis tenuifolia). J Agric Food Chem 62:4481–4492Bell L, Oruna Concha MJ, Wagstaff C (2015) Identification and quantification of glucosinolate and flavonol compounds in rocket salad (Eruca sativa, Eruca vesicaria and Diplotaxis tenuifolia) by LC-MS: highlighting the potential for improving nutritional value of rocket crops. Food Chem 172:852–861Bell L, Methven L, Wagstaff C (2017) The influence of phytochemical composition and resulting sensory attributes on preference for salad rocket (Eruca sativa) accessions by consumers of varying TAS2R38 diplotype. Food Chem 222:6–17Bennett RN, Mellon FA, Botting NP, Eagles J, Rosa EAS, Williamson G (2002) Identification of the major glucosinolate (4-mercaptobutyl glucosinolate) in leaves of Eruca sativa L. (salad rocket). Phytochemistry 61:25–30Bozokalfa MK, Eşiyok D, Ilbi H, Kaygisiz AT (2010) Estimates of genetic variability and association studies in quantitative plant traits of Eruca spp. landraces. Genetika 42(3):501–512Bozokalfa MK, Eşiyok D, Ilbi H, Kavak S, Kaygisiz AT (2011) Evaluation of phenotypic diversity and geographical variation of cultivated (Eruca sativa L.) and wild (Diplotaxis tenuifolia L.) rocket plant. Plant Genet Resour C 9:454–563CBI Centre for the Promotion of Imports from developing countries (2019) https://www.cbi.eu/market-information/fresh-fruit-vegetables/fresh-herbs/europe/. Accessed 21 Sept 2019Cheruiyot EK, Mumera LM, Ngetich WK, Hassanali A, Wachira F (2007) Polyhenols as potential indicators for osmotic tolerance in tea (Camellia sinensis L.). Biosci Biotechnol Biochem 71:2190–2197. https://doi.org/10.1271/bbb.70156Curtin F, Schulz P (1998) Multiple correlations and Bonferroni’s correction. Biol Psychiatr 44:775–777D’Antuono LF, Elementi S, Neri R (2008) Glucosinolates in Diplotaxis and Eruca leaves: diversity, taxonomic relations and applied aspects. Phytochemistry 69:187–199Daayf F, El Hadrami A, El-Bebany AE, Henriquez MA, Yao Z, Derksen H, El-Hadrami I, Adam LR (2012) Phenolic compounds in plant defense and pathogen counter-defense mechanisms. Rec Adv Polyphen Res 3:191–208Dalin P, Agren J, Björkman C, Huttumen P, Kärkkäinen K (2008) Leaf trichome formation and plant resistance to herbivory. In: Schaller A (ed) Induced plant resistance to herbivory. Springer, Berlin, pp 89–105Earl DA, vonHoldt BM (2012) STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv Genet Resour 4:359–361Egea-Gilabert C, Fernandez JA, Migliaro D, Martinez Sanchez JJ, Vicente MJ (2009) Genetic variability in wild vs. cultivated Eruca vesicaria populations as assessed by morphological, agronomical and molecular analyses. Sci Hortic 121:260–266Fine PVA (2015) Ecological and evolutionary drivers of geographic variation in species diversity. Annu Rev Ecol Evol Syst 46:369–392. https://doi.org/10.1146/annurev-ecolsys-112414-054102Gilardi G, Chen G, Garibaldi A, Zhiping C, Gullino ML (2007) Resistance of different rocket cultivars to wilt caused by strains of Fusarium oxysporum under artificial inoculation conditions. J Plant Pathol 89:113–117Gómez-Campo C (2003) Morphological characterization of Eruca vesicaria (Cruciferae) germplasm. Bocconea 16:615–624Guijarro-Real C, Prohens J, Rodriguez-Burruezo A, Adalid-Martínez AM, López-Gresa MP, Fita A (2019) Wild edible fool’s watercress: a potential crop with high nutraceutical properties. PeerJ 7:e6296Hall MKD, Jobling JJ, Rogers GS (2012) Some perspectives on rocket as a vegetable crop: a review. Veg Crop Res Bull 76:21–41Hanldey R, Ekbom B, Ågren J (2005) Variation in trichome density and resistance against a specialist insect herbivore in natural populations of Arabidopsis thaliana. Ecol Entomol 30:284–292Hauser MT (2014) Molecular basis of natural variation and environmental control of trichome patterning. Front Plant Sci 5:320Higdon JV, Delage B, Williams DE, Dashwood RH (2007) Cruciferous vegetables and human cancer risk: epidemiologic evidence and mechanistic basis. Pharmacol Res 55:224–236IPGRI (1999) Descriptors for rocket (Eruca spp.). International Plant Genetic Resources Institute, RomeLenzi A, Tesi R (2000) Effect of some cultural factors on nitrate accumulation in rocket [Diplotaxis tenuifolia (L.) D.C. – Eruca sativa Mill.]. Riv Agronomia 34(4):419–424Mantel NA (1967) The detection of disease clustering and a generalized regression approach. Cancer Res 27:209–220Meyer RS, Purugganan MD (2013) Evolution of crop species: genetics of domestication and diversification. Nat Rev Genet 14:840–852Ninfali P, Mea G, Giorgini S, Rocchi M, Bacchiocca M (2005) Antioxidant capacity of vegetables spices and dressings relevant to nutrition. Br J Nutr 93:257–266Padulosi S, Pignone D (1996) Rocket: a mediterranean crop for the world. Report of a workshop. IPGRI International Plant Genetic Resources Institute, Bioversity International, Rome, p 101Pane C, Sigillo L, Caputo M, Serratore G, Zaccardelli M, Tripodi P (2017) Response of rocket salad germplasm (Eruca and Diplotaxis spp.) to major pathogens causing damping-off, wilting and leaf spot diseases. Arch Phytopathol Plant Protect 50:167–177Pariyar S, Eichert T, Goldbach HE, Hunsche M, Burkhardt J (2013) The exclusion of ambient aerosols changes the water relations of sunflower (Helianthus annuus) and bean (Vicia vaba) plants. Environ Exp Bot 88:43–52Pasini F, Verardo V, Caboni MF, D’Antuono LF (2012) Determination of glucosinolates and phenolic compounds in rocket salad by HPLC-DAD−MS: evaluation of Eruca sativa Mill. and Diplotaxis tenuifolia L. genetic resources. Food Chem 133:1025–1033Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S, Rafalski A (1996) The utility of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol Breed 2:225–238Prevost A, Wilkinson MJ (1999) A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theor Appl Genet 98:107–112Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959Singleton VL, Rossi JA Jr (1965) Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Viticult 16:144–158Taiz L, Zeiger E (2006) Plant physiology, 4th edn. Sinauer Associates Inc., SunderlandTanentzap FM, Stempel A, Ryser P (2015) Reliability of leaf relative water content (RWC) measurements after storage: consequences for in situ measurements. Botany 93:535–541Taranto F, Francese G, Di Dato F, D’Alessandro A, Greco B, Onofaro Sanajà V, Pentangelo A, Mennella G, Tripodi P (2016) Leaf metabolic, genetic, and morphophysiological profiles of cultivated and wild rocket salad (Eruca and Diplotaxis spp.). J Agric Food Chem 64:5824–5836Thakur AK, Singh KH, Singh L, Nanjundan J, Khan YJ, Singh D (2018) SSR marker variations in Brassica species provide insight into the origin and evolution of Brassica amphidiploids. Hereditas 155:6. https://doi.org/10.1186/s41065-017-0041-5The Plant List (2019). http://www.theplantlist.org/tpl1.1/search?q=Eruca. Accessed 21 Sept 2019Tripodi P, Francese G, Mennella G (2017) Rocket salad: crop description, bioactive compounds and breeding perspectives. Adv Hortic Sci. https://doi.org/10.13128/ahs-21087Vavilov NI (1926) Centers of origin of cultivated plants. Bull Appl Bot Genet Plant Breed 16:1–248Wagner GJ (1991) Secreting glandular trichomes: more than just hairs. Plant Physiol 96:675–679Warwick SI, Gukel RK, Gomez-Campo C, James T (2007) Genetic variation in Eruca vesicaria (L.) Cav. Plant Genet Resour C 5:142–15

Development and application of Single Primer Enrichment Technology (SPET) SNP assay for population genomics analysis and candidate gene discovery in lettuce

Single primer enrichment technology (SPET) is a novel high-throughput genotyping method based on short-read sequencing of specific genomic regions harboring polymorphisms. SPET provides an efficient and reproducible method for genotyping target loci, overcoming the limits associated with other reduced representation library sequencing methods that are based on a random sampling of genomic loci. The possibility to sequence regions surrounding a target SNP allows the discovery of thousands of closely linked, novel SNPs. In this work, we report the design and application of the first SPET panel in lettuce, consisting of 41,547 probes spanning the whole genome and designed to target both coding (~96%) and intergenic (~4%) regions. A total of 81,531 SNPs were surveyed in 160 lettuce accessions originating from a total of 10 countries in Europe, America, and Asia and representing 10 horticultural types. Model ancestry population structure clearly separated the cultivated accessions (Lactuca sativa) from accessions of its presumed wild progenitor (L. serriola), revealing a total of six genetic subgroups that reflected a differentiation based on cultivar typology. Phylogenetic relationships and principal component analysis revealed a clustering of butterhead types and a general differentiation between germplasm originating from Western and Eastern Europe. To determine the potentiality of SPET for gene discovery, we performed genome-wide association analysis for main agricultural traits in L. sativa using six models (GLM naive, MLM, MLMM, CMLM, FarmCPU, and BLINK) to compare their strength and power for association detection. Robust associations were detected for seed color on chromosome 7 at 50 Mbp. Colocalization of association signals was found for outer leaf color and leaf anthocyanin content on chromosome 9 at 152 Mbp and on chromosome 5 at 86 Mbp. The association for bolting time was detected with the GLM, BLINK, and FarmCPU models on chromosome 7 at 164 Mbp. Associations were detected in chromosomal regions previously reported to harbor candidate genes for these traits, thus confirming the effectiveness of SPET for GWAS. Our findings illustrated the strength of SPET for discovering thousands of variable sites toward the dissection of the genomic diversity of germplasm collections, thus allowing a better characterization of lettuce collections

Genome wide association mapping for agronomic, fruit quality, and root architectural traits in tomato under organic farming conditions

[EN] Background Opportunity and challenges of the agriculture scenario of the next decades will face increasing demand for secure food through approaches able to minimize the input to cultivations. Large panels of tomato varieties represent a valuable resource of traits of interest under sustainable cultivation systems and for genome-wide association studies (GWAS). For mapping loci controlling the variation of agronomic, fruit quality, and root architecture traits, we used a heterogeneous set of 244 traditional and improved tomato accessions grown under organic field trials. Here we report comprehensive phenotyping and GWAS using over 37,300 SNPs obtained through double digest restriction-site associated DNA (dd-RADseq). Results A wide range of phenotypic diversity was observed in the studied collection, with highly significant differences encountered for most traits. A variable level of heritability was observed with values up to 69% for morphological traits while, among agronomic ones, fruit weight showed values above 80%. Genotype by environment analysis highlighted the strongest genotypic effect for aboveground traits compared to root architecture, suggesting that the hypogeal part of tomato plants has been a minor objective for breeding activities. GWAS was performed by a compressed mixed linear model leading to 59 significantly associated loci, allowing the identification of novel genes related to flower and fruit characteristics. Most genomic associations fell into the region surrounding SUN, OVATE, and MYB gene families. Six flower and fruit traits were associated with a single member of the SUN family (SLSUN31) on chromosome 11, in a region involved in the increase of fruit weight, locules number, and fruit fasciation. Furthermore, additional candidate genes for soluble solids content, fruit colour and shape were found near previously reported chromosomal regions, indicating the presence of synergic and multiple linked genes underlying the variation of these traits. Conclusions Results of this study give new hints on the genetic basis of traits in underexplored germplasm grown under organic conditions, providing a framework for the development of markers linked to candidate genes of interest to be used in genomics-assisted breeding in tomato, in particular under low-input and organic cultivation conditions.This research was supported by 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) and by 'RGV-FAO'project funded by the Italian Ministry of Agriculture, Food and Forestry. The funding bodies were not involved in the design of the study, collection, analysis, and interpretation of data, and in writing the manuscript.Tripodi, P.; Soler Aleixandre, S.; Campanelli, G.; Díez Niclós, MJTDJ.; Esposito, S.; Sestili, S.; Figás-Moreno, MDR.... (2021). Genome wide association mapping for agronomic, fruit quality, and root architectural traits in tomato under organic farming conditions. BMC Plant Biology. 21(1):1-22. https://doi.org/10.1186/s12870-021-03271-412221

Genetic and physiological analysis of tomato fruit weight and composition: influence of carbon availability on QTL detection

Throughout tomato domestication, a large increase in fruit size was associated with a loss of dry matter and sugar contents. This study aims to dissect the contributions of genetic variation and the physiological processes underlying the relationships between fruit growth and the accumulation of dry matter and sugars. Fruit quality traits and physiological parameters were measured on 20 introgression lines derived from the introgression of Solanum chmielewskii into S. lycopersicum, under high (HL, unpruned trusses) and low (LL, trusses pruned to one fruit) fruit load conditions. Inter- and intra-genotypic correlations among traits were estimated and quantitative trait loci (QTL) for size, composition, and physiological traits were mapped. LL increased almost all traits, but the response of sugar content was genotype-dependent, involving either dilution effects or differences in carbon allocation to sugars. Genotype×fruit load interactions were significant for most traits and only 30% of the QTL were stable under both fruit loads. Many QTL for fresh weight and cell or seed numbers co-localized. Eleven clusters of QTL for fresh weight and dry matter or sugar content were detected, eight with opposite allele effects and three with negative effects. Two genotypic antagonistic relationships, between fresh weight and dry matter content and between cell number and cell size, were significant only under HL; the second could be interpreted as a competition for carbohydrates among cells. The role of cuticular conductance, fruit transpiration or cracking in the relationship between fruit fresh weight and composition was also emphasized at the genetic and physiological levels

Blood ammonia levels in liver cirrhosis: a clue for the presence of portosystemic collateral veins

<p>Abstract</p> <p>Background</p> <p>Portal hypertension leads to the formation of portosystemic collateral veins in liver cirrhosis. The resulting shunting is responsible for the development of portosystemic encephalopathy. Although ammonia plays a certain role in determining portosystemic encephalopathy, the venous ammonia level has not been found to correlate with the presence or severity of this entity. So, it has become partially obsolete. Realizing the need for non-invasive markers mirroring the presence of esophageal varices in order to reduce the number of endoscopy screening, we came back to determine whether there was a correlation between blood ammonia concentrations and the detection of portosystemic collateral veins, also evaluating splenomegaly, hypersplenism (thrombocytopenia) and the severity of liver cirrhosis.</p> <p>Methods</p> <p>One hundred and fifty three consecutive patients with hepatic cirrhosis of various etiologies were recruited to participate in endoscopic and ultrasonography screening for the presence of portosystemic collaterals mostly esophageal varices, but also portal hypertensive gastropathy and large spontaneous shunts.</p> <p>Results</p> <p>Based on Child-Pugh classification, the median level of blood ammonia was 45 mcM/L in 64 patients belonging to class A, 66 mcM/L in 66 patients of class B and 108 mcM/L in 23 patients of class C respectively (p < 0.001).</p> <p>The grade of esophageal varices was concordant with venous ammonia levels (rho 0.43, p < 0.001). The best area under the curve was given by ammonia concentrations, i, e., 0.78, when comparing areas of ammonia levels, platelet count and spleen longitudinal diameter at ultrasonography. Ammonia levels predicted hepatic decompensation and ascites presence (Odds Ratio 1.018, p < 0.001).</p> <p>Conclusion</p> <p>Identifying cirrhotic patients with high blood ammonia concentrations could be clinically useful, as high levels would lead to suspicion of being in presence of collaterals, in clinical practice of esophageal varices, and pinpoint those patients requiring closer follow-up and endoscopic screening.</p