Sol International Online Meeting

Late blight (LB), caused by Phytophthora infestans, and powdery mildew (PM), caused by species within the Erysiphaceae family, are the most destructive and widespread diseases in tomato (Solanum lycopersicum L.). The control of these pathogens extensively relies on fungicide application, leading to economic losses and environment impact. The disabling of susceptibility (S) genes, which facilitate pathogen infection, may provide a broad-spectrum and durable type of resistance. To this purpose, the CRISPR/Cas9 gene editing represents the most suitable and fast strategy. We selected the Powdery Mildew Resistant 4 (PMR4) gene, whose disabling has been shown to confer PM resistance in Arabidopsis and in the tomato variety ‘Moneymaker’. In order to verify whether the effect of disabling S genes is background-dependent, we knocked-out the PMR4 gene in two widely cultivated varieties, ‘San Marzano’ and ‘Oxheart’. CRISPR mutants were generated by using 4 sgRNAs targeting the FKS1dom1 and Glucan-synthase domain of the PMR4 protein in order to maximize editing efficiency. In all, 87 tomato pmr4 transformants were obtained (T0 generation) of which 70 in ‘San Marzano’ and 17 in ‘Oxheart’. Twelve of ‘San Marzano’ mutants were tested and resulted positive for the presence of Cas9 gene. The Sanger analysis performed on target editing regions revealed 3 edited plants with large deletions (~2kb) and 9 showing a prevalence of 7bp deletions. In the next future, mutants will be test with PM and LB to quantify the level of resistance.

Ruiling Li

1 Department of Agricultural, Forest and Food Science, University of Turin, Italy 2 Plant Breeding, Wageningen University & research, Droevendaalsesteeg 1, 6708PB Wageningen, The Netherlands

CRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy in polyploid crops. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, multiplex constructs comprising tandemly arrayed gRNAs are difficult to assemble, this hampering more widespread use. Here we present a comprehensive upgrade of the popular cloning platform GoldenBraid (GB), in which, on top of its classical multigene cloning software, we integrate new assembly tools for two-dimensions gRNA multiplexing with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the gRNA unspaced approaches, respectively. As functional validation, we show, among others, the assembly of up to 17 tandemly-arrayed gRNAs constructs against a subset of the Squamosa-Promoter Binding Protein-Like (SPL) and FT gene family in tobacco. With these constructs we generated a collection of Cas9-free SPL/FT mutants harboring up to 9 biallelic mutations in a single generation, unveiling their effects in flowering time, branching and juvenility. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR activators and repressors using single and multiplexing gRNAs is also validated. With the incorporation of the new CRISPR tools and part´s collection, GB4.0 turns an unprecedentedly comprehensive open platform for genetic engineering in solanaceae.

Diego Orzaez

1 Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (IBMCP), Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain

Nicotiana benthamiana has received a great deal of attention as an expression system of recombinant proteins. Varieties of therapeutic and diagnostic proteins have been produced in N. benthamiana and several of them are currently on the market. Plants have unique advantages over other expression systems such as rapid and affordable growth conditions; plants are free from pathogen; and post-translational modification is very similar to mammalian system. Several expression vectors have been developed to enable high levels of protein production in plants. In the current study, we describe the efficient transient expression of Hepatitis E open reading frame (ORF) 2 capsid protein in N. benthamiana using two expression vectors: pEAQ-HT, based on Cowpea Mosaic Virus (CPMV), and pEff, based on potato X virus (PVX). HEV ORF2 protein was purified from plant tissue by immobilized metal-anion chromatography and gradient centrifugation. The recombinant protein was used to develop an in-house ELISA for testing anti-HEV antibodies in both human and swine sera. Furthermore, the truncated HEV ORF2 protein consisting of aa residues 110 to 610 formed nanosized virus-like particles. The immunization of mice with plant-produced HEV 110-610 protein induced high levels of HEV-specific serum antibodies.

Katerina Takova <katerina.takova@gmail.com>

1 Department of Plant Physiology and Molecular Biology, University of Plovdiv, Plovdiv, 4000, Bulgaria 2 Institute of Molecular Biology and Biotechnologies, Plovdiv, 4000, Bulgaria; toneva@plantgene.eu Center of Plant Systems Biology and Biotechnology, Plovdiv, 4000, Bulgaria 3 Center of Plant Systems Biology and Biotechnology, Plovdiv, 4000, Bulgaria

Eggplant (Solanum melongena) berry flesh turns brown after cutting and exposure to air and this affects fruit quality for both industrial applications and fresh consumption. This process is caused by the enzymatic oxidation of phenolic compounds catalysed by polyphenol oxidase enzymes (PPO). In eggplant, the PPO gene family is composed by 10 members (named ppo1-10). As highlighted by transcriptional analyses, ppo4, ppo5 and ppo6 were strongly up-regulated in the flesh after cutting. A CRISPR/Cas9 approach was applied to induce knock-out mutations on these three genes in the ‘Black Beauty’ variety, designing a single sgRNA targeting a conserved region. Following transformation with the gene editing construct, in vitro regenerated plantlets were obtained. Illumina deep sequencing of amplicons of the target sites confirmed the successful editing of ppo4, ppo5 and ppo6 with the insertion of a single nucleotide as the most frequent mutation. Editing at target loci were stably inherited in the T1 and T2 progenies, while no off-target mutations were identified on the potential off-target loci. Upon cutting, edited T1 and T2 eggplant fruits showed a reduction of the typical brown coloration caused by phenolic oxidation. This study is the first example of CRISPR/Cas9 editing in eggplant, representing an alluring option for the introduction of specific traits through a targeted biotechnological approach.

Alex Maioli

1 DISAFA, Plant Genetics and Breeding, University of Torino, Grugliasco (TO), Italy; 2 Instituto de Biología Molecular y Celular de Plantas (IBMCP) CSIC-UPV, Ingeniero Fausto Elio/n, 46022 Valencia, Spain; 3 Instituto de Conservación y Mejora de la Agrodiversidad Valenciana, Universitat Politècnica de València, Camino de Vera 14, 46022 Valencia, Spain

The impact of fruit morphology on pepper composition for health-promoting compounds was investigated. For that purpose, a collection of pepper (Capsicum spp.) accessions from Ecuador, one of the hotspots in Capsicum’s origin, was analyzed for ascorbic acid, polyphenols, capsaicinoids, and prevention of cholesterol oxidation. Plant and fruit phenomics were assessed with conventional descriptors and the Tomato Analyzer digital tool. Significant differences among accessions and species revealed a large diversity within the collection. The conventional descriptors related to the magnitude of plants and fruits, as well as digital attributes under the categories of size, shape index, and latitudinal section, mostly explained the variance among Capsicum groups. On the other hand, C. frutescens displayed the highest levels of capsaicinoids, whereas the polyphenols shortly varied among the five domesticated species. C. pubescens exhibited the lowest content of ascorbic acid. Correlation test revealed that phytochemical components were negatively correlated with the morphometric fruit attributes, suggesting that huge fruits contained lower amounts of nutraceutical compounds. Multivariate analysis showed that parameters related to fruit size, shape, and nutraceutical composition primarily contribute to the arrangement of pepper accessions, suggesting that those traits have been subjected to higher selection pressures imposed by humans.

Cristina Silvar

University of Coruna

Tomato’s red fruit lycopene content is controlled, among other factors, by the decrease in Lycopene Beta Cyclase (CYC-B) gene expression. Spontaneous mutations like old gold (og) and old gold crimsom (ogc) generate non-functional CYC-B enzymes. These variants are used in breeding to increase red color. Since these mutations originated in genotypes carrying the sp mutation and CYB-B and SP genes are linked on chromosome 6, og and ogc mutants have, in consequence, determinate growth. The goal of this work is to generate novel mutations in the CYC-B gene directly into indeterminate growth tomato lines using CRIPR/Cas9. Tomato elite breeding lines ‘LT16’ and ‘LT46’, parents of a modern hybrid cultivar, were transformed with a construct containing Cas9 and specific sgRNAs expression cassettes, targeting the coding sequence of CYC-B. Transgenic regenerated plants (resistant to kanamycin) were screened for Cas9-induced mutations in the CYC-B locus using High-resolution fragment analysis and Sanger sequencing. Mutant phenotypes similar to old gold and old gold crimson phenotypes were observed in flowers and fruits of T0 plants. The progeny of one edited line was screened for plants with different combination of the mutant alleles and for T-DNA free plants. Novel CYC-B alleles were characterized by Sanger sequencing. Selected plants will be used to evaluate a new version of the indeterminate hybrid cultivar, free of T-DNA and with different allele variants of the CYC-B gene.

Ana Arruabarrena <aarruabarrena@inia.org.uy>

1 Instituto Nacional de Investigación Agropecuaria (INIA), Estación Experimental INIA Salto Grande, Uruguay. 2 Universidad de la República, Facultad de Ciencias, Uruguay.

Chlorogenic acid (CGA) and its isomers are the most abundant polyphenols in many plant species such as potato, tomato, eggplant, tobacco, coffee and apple. Due to their biological properties they are important compounds for plant protection against biotic and abiotic stresses. Their biosynthesis could occur by several different pathways, but considerable evidence suggests that the most active pathway is mediated by Hydroxycinnamoyl CoA Quinate Transferase (HQT). HQT converts caffeoyl-CoA and quinic acid to CGA and in some Solanaceous species its role has been demonstrated; in tomato the silencing of SlHQT resulted in a significant reduction of CGA and the overexpression of SlHQT led to an accumulation of CGA. To study the SlHQT contribution in CGA biosynthesis in tomato further, we designed a CRISPR/Cas9 construct to edit the SlHQT gene. Here we present the characterization of hqt mutants and the evaluation of the effect of ko mutations on phenylpropanoid metabolism in normal conditions and under abiotic stress. In addition, the hqt mutants enabled us to study the histolocalization of caffeoylquinic acids (CQAs) in tomato leaf and to understand their involvement in the photoprotection against UV light.

Fabio D'Orso <fabio.dorso@crea.gov.it>

1 CREA Research Centre for Genomics and Bioinformatics, Via Ardeatina 546, Rome, Italy 2 Metabolic Biology Department, John Innes Centre, Norwich NR4 7UH, United Kingdom

Approximately 30% of people in the United States report using Vitamin D supplements, increasing to 49% of men and 59% of women over 60. Vitamin D is needed for good skeletal health but has also been connected to improved cancer outcomes, better cardiovascular health, and good mental health. Sources of vitamin D include cod liver oil, dairy products, wild mushrooms, and UVB. There is no plant source of dietary vitamin D. Members of genus Solanum contain 7-dehydrocholesterol (7-DHC). When exposed to UVB, this begins the production of vitamin D. 7-DHC serves as the substrate of 7-Dehydrocholesterol Reductase-2 (7-DR-2) in tomato, which produces cholesterol that is used to produce 𝝰-tomatine. If 7-DR-2 is knocked out it will cause an increase in the amount of 7-DHC. Here we report our pilot research aiming to increase Vitamin D production in tomato by altering its cholesterol pathway. Two sites were selected using CrisprP 2.0 based on the lowest likelihood to produce off-target effects. The sequences to produce sgRNAs were cloned into pDIRECT_21C. This carries AtCas9 driven by the 35S promoter, sites to receive RNA sequences driven by CmYLCV promoter, bacterial kanamycin resistance, and plant hygromycin B. Two tomato lines were chosen for gene editing, MTX-851 because it was bred specifically for Minnesota’s growing conditions, and M82 because it established to be transformable by Agrobacterium. Preliminary work has shown that MTX-851 is able to regenerate through tissue culture and at least one line of M82 has been regenerated after Agrobacterium mediated transformation.

Vincenzo Averello

University of Minnesota

Despite recent advances in long-read sequencing technologies, plant genome assembly remains challenging. In particular, modern assemblies often fail to accurately reconstruct genomic repeats that are pervasive in plant genomes. Large initiatives such as the Vertebrate Genomes Project and the Human Telomere-To-Telomere (T2T) consortium have established best practices and industry standards for assembling animal genomes, but it remains unclear how these approaches translate to plant genomes. Here, we use PacBio Circular Consensus accurate long-reads (CCS), Oxford Nanopore ultra-long reads, and chromatin conformation capture (Hi-C) to establish two platinum-level Solanaceae genome assemblies. We first demonstrate the efficacy of this approach on the widely used tomato reference genotype M82, for which we report substantial improvements in accuracy, contiguity, and completeness compared to previous long-read assemblies. We then apply this approach to establish a new reference genome for the orphan crop Physalis grisea (groundcherry). This highly repetitive 1.3 Gb diploid genome also resulted in an ultra-accurate and complete chromosome-scale assembly using our innovative combination of sequencing data types and bioinformatic approaches. Using specific examples from these assemblies, we highlight new methods and best practices for plant genome assembly and scaffolding. We also discuss potential applications in pan and “personalized” genomics for tomato, as well as implications for cross-species functional studies for orphan crops and wild nightshades.

Michael Alonge

1. Department of Computer Science, Johns Hopkins University, Baltimore, MD; 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; 3. Plant Breeding and Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY; 4. Boyce Thompson Institute, Ithaca, NY; 5. Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; 6. Department of Biology, Johns Hopkins University, Baltimore, MD

The increasing availability of phylogenomic data is revealing incongruent topologies in gene trees between and even within genomes. Some of these incongruences indicate hard polytomies that are due to underlying biological processes may remain impossible to resolve. Here we focus on the large and economically important genus Solanum which with 1,230 accepted species, is one of the ten largest flowering plant genera. To provide an evolutionary framework for the large research community working on the genus, we build a new supermatrix phylogeny comprising 60% of all accepted species (740 spp.), based on two nuclear (ITS and WAXY) and seven plastid markers. To assess whether the recovered topology is robust, we explore phylogenomic analyses of a full plastome alignment of 141 species and nuclear genome dataset, composed of a target-capture dataset of 39 species based on the Angiosperm353 probes and supplemented from data extracted from published genomes and transcriptomes. Both datasets contain species from all the major recognized lineages within the genus, and we explore how various taxon sampling, missing data, phylogenetic analyses, and data partitions influence the topology of the recovered species tree. A single strongly supported topology is recovered from the different plastome phylogenomic analyses, which is incongruent with the topology recovered from the supermatrix phylogeny. We quantify gene and site concordance factors to identify where the discordance occurs and discuss the biological significance of the various hypotheses. Our findings highlight how such topological uncertainty should be incorporated in downstream macro-evolutionary and ecological analyses for non-bifurcating evolution.

Edeline Gagnon

Tropical Diversity Group, Royal Botanical Garden of Edinburgh

The Petunieae clade of Solanaceae is a culturally and economically important clade of plants with extensive floral diversity. Some of the genera, such as Petunia and Calibrachoa, are widely popular as ornamentals, due in part to their dramatic variation in floral coloration. Petunieae are native to Southern North America, Central America, South America, and the Caribbean where they grow in a diverse array of habitats including high and low deserts, tropical rain forests, coastal plains, tropical highlands, and sub-tropical wetlands. In addition to its floral diversity, the clade exhibits an assortment of other fascinating phenotypic shifts, such as transitions among life forms (trees, shrubs, herbs, and cushions). Currently, the internal relationships within Petunieae are poorly-resolved as is the placement of the clade relative to the rest of the Solanaceae. This poses a challenge to understanding biogeographic and phenotypic evolution in the clade. Here we present a phylotranscriptomic analysis spanning 70 of the approximately 145 extant species, representing one of the largest and most comprehensive studies of its kind. By characterizing phylogenetic relationships within Petunieae and combining RNA-seq data with measures of flower color we are laying the groundwork to probe the molecular mechanisms underlying the phenotypic diversification of Petunieae species over time and space.

Lucas C. Wheeler

1 University of Colorado-Boulder, Department of Ecology and Evolutionary Biology, 2 Cambridge University, Department of Genetics, 3 Natural History Museum (UK), 4 CONICET, IMBIV, 4 Universidade Federal do Rio Grande do Sul, Department of Botany

The placement of extinct taxa on phylogenies relies on scoring informative morphological characters, which can be difficult given the fragmentary nature of the fossil record. Nonetheless, advances in imaging and visualization, such as X-ray micro-Computed Tomography (μ-CT), have made it possible to non-destructively score fine-scale structural variation in fossils. Although detailed studies of Solanaceae fossils remain few, the family exhibits wide variation in seed and fruit traits to use in estimating fossil placement from morphological data. Indeed, a recent application of μ-CT to Solanaceae fossils revealed that Cantisolanum daturoides, formerly considered to be the oldest seed fossil in the family, is instead a commelinid monocot. Therefore, we reviewed most of the previously published seed fossils of Solanaceae in addition to more than 20 findings currently not published. Carpological nightshade collections by Dorofeev, Mai, and Nikitin were studied at European and Russian museums. All fossilized seeds were analyzed using stereomicroscopy and 18 were μ-CT scanned to evaluate pyritization and infer internal preservation. A high-resolution Zeiss Versa 520 μ-CT system, which incorporates phase contrast imaging, was used for scanning. We scored 13 traits, including two continuous, and performed a cluster analysis to compare these fossil seeds to extant Solanaceae (sampling at least one species per genus). This preliminary analysis allowed us to determine which morphological traits are informative and to assign putative placements of the fossils into a phylogenetic tree. These results lay the foundation for a new total evidence dating analysis for the entire family.

Rocio Deanna <rociodeanna@gmail.com>

1 Department of Ecology and Evolutionary Biology, University of Colorado at Boulder, U.S.A.; 2 Natural History Museum at London, U.K.; 3 Instituto Multidisciplinario de Biología Vegetal, IMBIV (CONICET-UNC), Argentina; 4 Komarov Botanical Institute of the Russian Academy of Sciences, Russia; 5 Museum für Naturkunde, Leibniz Institute for Evolution and Biodiversity Science, Germany.

Recently divergent species challenge taxonomists since species may share genetic and morphological features. Delimiting species is further complicated if they hybridize. The Petunia genus hybridizes in natural and artificial conditions. P. interior and P. inflata are genetically and morphologically similar, occur in sympatry, and share pollinators. We tested if they hybridize in their contact zone or if the morphological and genetic sharing is due to their recent speciation. We used genome-wide polymorphisms to search for hybrids in Snapclust and we assessed population structure in FastStructure. A generalized linear mixed modeling tested whether distance, environment, or elevation better explained the genetic divergence between species. We did not find any evidence of hybridization. Population structure resulted in three clusters: one containing four P. interior collection sites; another with five collection sites of P. inflata and one misidentified P. interior population; and another with two collection sites of P. inflata. The model that best explained the genetic divergence among populations combined geographic distances, environment variables, and elevation. Even though they seem to have all prerequisites, these species do not hybridize, suggesting some reproductive barriers. These species inhabit a transitional area between two biomes, thus geographic distances, environmental variables, and elevation are somewhat correlated, and all play roles in species divergence. These species’ ancestors lived at elevations lower than 500m, but current distribution includes sites up to 900m in elevation. Our results suggest that P. interior expanded its distribution to higher elevations, whereas P. inflata still occupies the ancestral area at lower elevations.

Pedro Pezzi

1Department of Genetics, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; 2Department of Biosciences, Universita degli Studi di Milano, Milan, Italy

The genus Petunia (Solanaceae) is endemic to southern South America with 15 wild species, which have diversified recently under ecological interactions and climate changes. Petunia altiplana grows in highland grasslands in southern Brazil, and the Pelotas river interrupts its distribution. A phylogeographic analysis involving this species showed that the riverbanks constitute reciprocal monophyletic groups, suggesting that the Pelotas river could be a phylogeographic barrier. The course of rivers explains the variability and genetic structure in different species of animals and plants, mainly in species from the Amazon Basin, but few studies address this topic in southern Brazil. Here, we used classic phylogeographic methods based on plastid intergenic spacers and microsatellites to investigate the Pelotas river's role as a barrier to gene flow among the P. altiplana populations and understand the species diversity and structure. The plastid and microsatellite results differed regarding the population structuring. The first dataset showed some differentiation between the north and south riversides, whereas the microsatellites showed low genetic structuring. Such results suggest the river acted as an ancient phylogeographic barrier, but this effect is no longer observed as contemporary gene flow was observed.

Luana Soares

Universidade Federal do Rio Grande do Sul, Laboratory of Molecular Evolution, Department of Genetics.

For conservation and sustainable use of species diversity, it is essential to characterize the environments where they develop. The objective of this research was to identify the edaphoclimatic diversity of the accessions sites of 12 species of wild tomatoes (Solanum sect. Lycopersicon) and 4 species of phylogenetically related groups (Solanum sect. Juglandifolia and sect. Lycopersicoides) using ecological descriptors of representative specimens in Latin America. Using 5,013 representative accessions and an Environmental Information System composed of 21 climatic variables (19 bioclimatic variables from WorldClim, annual evapotranspiration and altitude), and 10 edaphic variables (pH, CEC, SB, Salinity, Sodicity, Percentage of lime, clay and sand, Bulk density, TEB) in raster format with spatial resolution of 900 m, ecological descriptors (mean, maximum and minimum values) were identified through the use of geographic information systems. Subsequently, the importance and association of the variables in the geographic distribution (Principal Component Analysis) was determined, as well as the similarity of the species due to their distribution as a function of the climatic variables (Cluster Analysis). The results provided current information on the climatic conditions and the environmental breadth of the distribution of the populations of 12 species of wild tomatoes and 4 species of phylogenetically related groups.

Gabriela Ramírez-Ojeda <gabramirezo@gmail.com>

1 Universidad Autónoma Chapingo, México; 2 Facultad Ciencias Agrícolas, Universidad del Cuyo, Argentina. 3 Departamento de Agronomía, Centro Universitario de Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, México. 4 Centro Interdisciplinario de Investigación para el Desarrollo Regional Unidad Oaxaca, Instituto Politécnico Nacional, México.

Capsicum pubescens is a well-known hot chile from South American Andean highlands, nowadays cultivated from NW Argentina to Central America. The species is known only as a cultigen and not in wild form. The origin of C. pubescens has been puzzling while the knowledge of its evolutionary history and genetic diversity is scarce. A couple of wild Andean species have been proposed as its closest allies and/or putative ancestors, with which C. pubescens can interbreed. However, crossing experiments and DNA data analyses have produced some contradictory results, being C. pubescens resolved into an isolated clade.The RAD-sequencing approach was thus followed to analyze C. pubescens origin and affinities, diversification and genetic structure by using genome-wide data. The phylogenetic affinities of C. pubescens were strongly resolved, being a sister species to those considered its closest allies, but it is ascertained that none extant Capsicum species would be its wild ancestor. Within C. pubescens, two major genetic groups can be recognized: 1) individuals from Bolivia and Argentina and, 2) individuals from Peru, Ecuador and Central America, including Mexico. Based on phylogenetic reconstructions, C. pubescens area of origin could be located in the central-western Bolivian highlands. This would be in concordance with the wide morphological variation of the species in that region (i.e. flowers, fruits, pubescence, etc.). Overall, the current results allow deepening the knowledge of the affinities and distribution of the genetic diversity of the locoto chile, offering new ideas, supported by genome-wide data, about its origin and expansion.

Carolina Carrizo García

Multidisciplinary Institute of Vegetal Biology, Córdoba, Argentina & Department of Botany and Biodiversity Research, University of Vienna, Vienna, Austria

China is the world’s leading country for potato production but potato is not native to China. To gain insights into the genetic diversity of potato germplasm various studies have been performed but no study has been reported for potato landraces in China. To improve the available genepool for future potato breeding programs, a diverse population containing 292 genotypes (including foreign elite lines, local landraces and cultivars) was developed and genotyped using 30 SSR markers covering the entire potato genome. A total of 174 alleles were detected with an average of 5.5 alleles per locus. The model-based structure analysis discriminated the population into two main sub-groups, which can be further subdivided into seven groups based on collection sites. One sub-group (P1) revealed less genetic diversity than other (P2) and contained a higher number of commercial cultivars possibly indicating a slight reduction in diversity due to selection in breeding programs. The P2 sub-group showed a wider range of genetic diversity with more new and unique alleles attained from wild relatives. The potato landraces, clustered in sub-population P1 may be derived from historical population imported from ancient European and International Potato Center genotypes while sub-population P2 may be derived from modern populations from International Potato Center and European genotypes. It is proposed that in the first step, the potato genotypes were introduced from Europe to China, domesticated as landraces, and then hybridized for modern cultivars.

MuhammadAbdulRehman Rashid

1 Industrial Crops Research Institute, Yunnan Academy of Agricultural Sciences, Kunming, China; 2 Department of Bioinformatics and Biotechnology, Government College University Faisalabad, Pakistan; 3 Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China; 4 The James Hutton Institute, Invergowrie, United Kingdom; 5 Scientific Observing and Experimental Station of Potato and Rapeseed in Yunnan-Guizhou Plateau, Ministry of Agriculture, Kunming, China

The extensive trait variation across the Physalis genus is ideal to study how plant defensive strategies are shaped by a variety of factors, including herbivory, lifespan, and reproduction. Using phylogenetic comparative methods, I examined the interacting influences of plant life history (annual/perennial), mating system (self-compatible/self-incompatible), and species growth rates on both constitutive and induced anti-herbivore resistance. Contrary to long-standing assumptions in the field of plant defense, I found no evidence for a trade-off between constitutive resistance and inducibility. Instead, species growth rates and life history traits interacted to determine plant resistance. These results indicate that constitutive resistance and inducibility are not alternative strategies in Physalis and therefore can be shaped by different selective pressures.

Deidra J. Jacobsen

1 Indiana University; 2 Current address: University of Utah, School of Biological Sciences

Eggplant (Solanum melongena L.) is a member of the large Solanaceae family domesticated in India and China and is cultivated worldwide for the high nutritional value of its berries that correlates with beneficial effects as disease prevention (Mennella et al., 2012; Gürbüz et al., 2018). Eggplant is difficult to manage in terms of seed quality. Dormancy, poor seed germination rate, and low uniformity have been documented in several accessions of S. melongena and its crop wild relatives (Demir et al., 2005; Taab et al., 2009; Ranil et al., 2015). Low uniformity and low germination percentage severely impact breeding programs, with a huge economic impact on seed industries that need to increase product performance while optimizing the use of economic resources. Seed priming technology is used to enhance germination and provide faster emergence, uniform stands, and stress tolerant plants (Paparella et al. 2015). With seed priming, a low-cost and pre-sowing technique, imbibition is carried out under controlled conditions in order to delay water up-take and expand the pre-germinative metabolism, the crucial phase of seed germination, boosting antioxidant and DNA repair mechanisms (Paparella et al. 2015). In the frame of WAKE-APT project, we explore the molecular dynamics of pre-germinative metabolism in hydro-primed eggplant seeds in order to assess the response to priming, select reliable molecular hallmarks (expression patterns of antioxidant/DNA repair genes and ROS profiles) useful to discriminate between high- and low- quality lots and define improved protocols to ameliorate seed quality.

Chiara Forti

University of Study of Pavia; CREA-GB; CREA-CI

The complexity within the genus Solanum has been of great taxonomic concern over the years. Resolving this problem has led to a detailed investigation into the morphological study of the genus as it is a primary line of evidence employed in plant taxonomy. Diagnostic qualitative and quantitative characters viz. fruit colour, level of hairiness, presence of appendages, flower colour, ratio of sepals to petals, leaf length/width, fruit length/diameter differentiated the species. Appendages were observed in S. torvum. The species have globous fruit shape except for S. aethiopicum and S. melongena with oval and oblong shapes respectively. High level of similarity exists across variants of S. gilo and S. macrocarpon but can be distinguished by their fruit colour while the number of stamen is diagnostic for S. macrocarpon variants. All the species have an erect growth form except for S. americanum which is decumbent. Corymb inflorescence was observed in S. indicum. Simple umbel inflorescence in S. nigrum and S. americanum with their fruit colour being dull black and shining black respectively. These valuable diagnostic characters were explored in this study and used to construct dendrogram and key illustrations which further aided the identification and delimitation of the species.

Keywords: Solanum, Morphology, Inflorescence, Appendages, Qualitative, Quantitative, Dendrogram

Cornelius Onyedikachi Nichodemus <cornel4nic@gmail.com>

University of Port Harcourt, Department of Plant Science and Biotechnology

Cape gooseberry (Physalis peruviana L.) is a berry-bearing Solanaceae species, native to the Andean region with enormous potential for biomedical research and commercial purposes. Despite its importance, the species still lacks a more comprehensive platform for Single Nucleotide Polymorphisms (SNPs) discovery. Here we evaluated a total of 176 accessions representing wild, weedy, and commercial cultivars as well as related taxa from the Colombian germplasm bank using GBS. A robust Genotyping-By-Sequencing (GBS) pipeline platform was examined to provide accurate discovery of SNPs. A total of 7,425 SNPs, derived from P. peruviana common tags (unique 64 bp sequences shared between selected species), were identified using the GBS parameters mnLCov of 0.5 and a mnScov of 0.7. Within P. peruviana, five subpopulations with a high genetic diversity and allele fixation (HE: 0.35 to 0.36 and FIS: -0.11 to -0.01, respectively) were detected. Conversely, low genetic differentiation (FST: 0.01 to 0.05) was also observed, indicating a high gene flow among subpopulations. The results of this study provide a comprehensive insight into the genetic diversity and population structure of a relatively large cape gooseberry repository in Colombia.

Felix E. Enciso-Rodríguez <fenciso@agrosavia.co>

Centro de investigación Tibaitatá, Corporación Colombiana de Investigación Agropecuaria – Agrosavia. Mosquera, Cundinamarca, Colombia

Capsicum chacoense is native to South America distributed in Bolivia, Argentina and Paraguay, and highly appreciated as food condiment by local communities in northwestern Argentina. Fruits of “ají del monte” or “uchuquita” are harvested in their natural habitats and commercialized regionally. Traditional harvesting consists in the extraction of productive branches or the whole plant, which is the main reason for the degradation and possible loss of this valuable plant genetic resource. This concern drives a more comprehensive study about the potential for use of this wild pepper species and its conservation status. In 2020 the recovery of germplasm began in 14 locations in the Central valley of Catamarca, where “uchuquita” forms small populations associated with trees, shrubs and cactaceous species of the Monte plant community (Prosopis spp., Sarconphalus mistol, Celtis spp., Geoffroea decorticans, Parkinsonia praecox, Larrea spp., Pachycereus pringlei, Cereus forbesii). In each population an ecological characterization was made and fruits of at least 10 plants were collected. Part of the recovered seeds are maintained in a germplasm bank, while others were selected to produce plants for cultivation in field trials. Preliminary results show a good adaptation of the plants to agronomical management. Phenotypic characterization includes 20 quantitative and qualitative variables comprising the seedling, vegetative and reproductive stages, as well as fruit pungency due to capsycinoid alkaloids, since preliminary studies have shown variability in populations. An integrative study of Capsicum chacoense, including cultural traditions, allows germplasm conservation strategies and promotes its use in horticultural production and breeding programmes.

Francisco MurúaCarrizo

1. Agronomy Faculty, National University of Catamarca, Catamarca, Argentina 2. National Institute of Agriculture Research, La Consulta, Mendoza, Argentina 3. Agronomy Faculty, National University of Cuyo and CCT CONICET Mendoza, Argentina

Nicotiana benthamiana is an important research tool and a model plant, but its floral volatiles and protective insecticides have received little examination. While the originally desert-dwelling LAB isolate produces smaller flowers with structures favouring self-fertilisation, the wild QLD isolate, from a milder environment, produces flowers that favour insect pollination. Nicotiana plants produce secondary metabolites capable of attracting pollinators and repelling herbivores. Analysing the composition of floral headspace volatiles by SIFT-MS, we observed that QLD flowers emit high levels of the pollinator attractant benzyl alcohol during the night. However, the overnight emission of benzyl alcohol from LAB flowers is very low. Comparing the composition of flower and leaf tissues by LC-HESI-MS, identified differences in the production Hydroxygeranyllinallol Diterpenoid Glycosides and pyridine alkaloids, which are allelochemicals involved in plant defence and herbivory. Nicotianosides (III & IV) accumulated to higher levels in the flowers and leaves of LAB than in QLD and the reverse was found for two other diterpene glycoside forms. Most strikingly, nornicotine accumulated to levels ~50 and ~300-fold higher in QLD than in LAB flowers and leaves, respectively. RNA-seq confirmed that CYP82E4, responsible for the conversion of nicotine to nornicotine, is significantly higher in QLD. Analysis of CYP82E4 promoter identified a transcription factor (bZIP53) binding site in QLD which is mutated in LAB. This suggests a scenario in which LAB, by not producing pollinator attractants, requires less protection against herbivorous insects whereas QLD by producing floral volatiles needs to produce a potent insecticide to protect itself from being eaten.

Buddhini Ranawaka

1 Centre for Agriculture and Bioeconomy, Queensland University of Technology, Brisbane, Australia; 2 Italian National Agency for New Technologies, Energy, and Sustainable Economic Development, Casaccia Research Centre, 00123, Rome, Italy

The tribe Hyoscyameae (Solanaceae) tribe comprises eight genera with two centers of diversity: the Mediterranean-Turanian region (Atropa and Archihyoscyamus) and the Tibetan Plateau area (Przewalskia, Anisodus, and Atropanthe). The genus Scopolia includes species with disjunct distribution in eastern Asia and in the Mediterranean basin. Previous studies based on morphological or molecular characters were not able to resolve the phylogenetic affiliations of the tribe. Here, we assembled the complete chloroplast genome of Scopolia carniolica, the second species of the genus to be described. Using publicly available chloroplast genomes we performed genomic comparisons and examined the phylogenetic relationships for seven of the eight genera of Hyoscyameae. Gene content was identical across the tribe with small differences in genome length. The phylogeny of the tribe was highly resolved, with strong support for relationships among genera. The major advancements included strongly-supported phylogenetic affiliations of Atropanthe, Anisodus, and Hyoscyamus. We also studied the Hyoscyameae phylogeny using a nuclear marker, the internal transcribed spacer 2 (ITS2). Interestingly, even though the ITS2 tree was poorly resolved in general; it recovered all genera as monophyletic, except for Scopolia, which was paraphyletic in respect to Physochlaina. Finally, we re-interpreted the mitochondrial cox1 intron evolution. Two independent, intrafamilial, horizontal acquisitions were inferred, one in the ancestor of Przewalskia, Physochlaina and S. japonica, and another in Hyoscyamus, with no intron losses.

Carolina Gandini, Verónica Ibáñez

1 IBAM, Universidad Nacional de Cuyo, CONICET, Facultad de Ciencias Agrarias, Almirante Brown 500, M5528AHB, Chacras de Coria, Argentina. 2 Facultad de Ciencias Exactas y Naturales, Padre Jorge Contreras 1300, Universidad Nacional de Cuyo, M5502JMA, Mendoza, Argentina. 3 Faculty of Science and Engineering, Manchester Metropolitan University, Manchester, M1 5GD, UK. * Co-first authors.

Solanum malmeanum is a wild potato from Argentina, Brazil, Paraguay, and Uruguay. Crop wild relatives are unique reservoirs of potential traits for plant breeding. High tuber dry matter content (TDMC) confers better culinary quality, longer storage and shelf life, being an index of commercial tuber quality for potato breeders. The objective of this study was to evaluate TDMC of 11 S. malmeanum accessions (BGB015, BGB017, BGB021, BGB080, BGB081, BGB084, BGB443, BGB446, BGB447, BGB448, BGB471) compared to two clones of S. tuberosum (BRSANA, C1750-15-95) from the Embrapa Clima Temperado Potato Genebank, Pelotas, Brazil. Plants were cultivated at greenhouse in Spring 2020. Tubers were oven dried until reach constant weight at a temperature of 60ºC. TDMC [%TDMC = (dry weight/fresh weight) X 100] was calculated and the means of triplicate samples were analyzed in RStudio (v. 1.3.1093) and grouped in clusters using Scott-Knott multiple comparison (p<0.05). TDMC ranged from 21.36% to 51.74% (CV = 14.99%) and accessions were arranged in three groups. Group A comprised S. malmeanum accessions with higher TDMC: BGB021 (51.74%), BGB017 (50.48%) and BGB015 (45.45%). Group B comprised S. malmeanum accessions with lower TDMC: BGB471 (42.46%), BGB446 (40.45%), BGB443 (40.33%), BGB080 (38%), BGB084 (37.9%), BGB447 (37.46%), BGB448 (36.91%) and BGB081 (33.93%). Group C comprised the two clones of S. tuberosum, C1750-15-95 (23.20%) and BRSANA (21.36%) which bear standard TDMC for potato cultivars. S. malmeanum accessions contains much higher TDMC than minimum recommended levels (>20%) and are promising sources for this trait for potato breeding.

Rodrigo Nicolao <rodnicolao@gmail.com>

Programa de Pós-graduação em Agronomia, Universidade Federal de Pelotas, RS, Brazil; Embrapa Clima Temperado, Pelotas, RS, Brazil.

SQUAMOSA promoter binding-like proteins are known to play key roles in growth and developmental processes, including leaf development, plant architecture and juvenile-to-adult and vegetative-to-reproductive phase transitions. In this study, we report the identification of 54 putative SPL genes in Nicotiana tabaccum cv. K326 and 40 candidate SPL genes in N. benthamiana lab strain. The SPL genes of the two species were classified into 8 phylogenetic groups according to the SPL classification in Arabidopsis. The exon-intron gene structure and SBP domains were highly conserved between homeologues and ortologues. 32 of the 40 NbSPL genes and 28 of the NtSPL genes are targets of miR156. These results serve as basis for candidate gene selection for a knock-out experiment using CRISPR/Cas9 in N. tabacum that revealed some clues of the biological functions of SPL13 in branching and juvenility and of SPL9 and SPL15 in flowering delay. All together our results present a comparison of the SPL gene family in two allotetraploid species, N. benthamiana and N. tabacum, that provide the basis for elucidation of the biological role of each SPL.

Marta Vazquez-Vilar

1Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), C/ Ingeniero Fausto Elio s/n 46022 Valencia, Spain. 2University of Milan | UNIMI · Department of Bioscience, 3ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Rome, Italy. 4Centre for Tropical Crops and Biocommodities, QUT, Brisbane, QLD, Australia.

Male gametogenesis in plants occurs within the anther in a synchronized manner with the development of the anther itself. During this process, the tapetum that surrounds the developing reproductive cells has a central role acting as a nutritive tissue. Therefore, mutations in genes that affect this tissue result in male sterility. We have previously observed that male sterility triggers parthenocarpic fruit development in tomato plants (1, 2). Undoubtedly, seedless fruit production is an appealing trait for many fleshy fruit crops. To get insight into this process we generated male sterile tomato plants using CRISPR/Cas9 technology. We targeted candidate tomato genes based in protein homology to previously characterized Arabidopsis tapetum-specific genes. We have generated several loss-of-function mutations that showed male-sterile flowers and produced seedless fruits. Detailed characterization of these plants and the targeted genes is providing relevant information on the role of tapetum in pollen formation. Besides, they will help to decipher the molecular mechanisms that drive parthenocarpic fruit growth in male-sterile tomato plants, which remain largely unknown.

This work was supported by Spanish grant: RTI2018-094280-B-100.

1. Rojas-Gracia et al. 2017. The parthenocarpic hydra mutant reveals a new function for a SPOROCYTELESS-like gene in the control of fruit set in tomato. New Phytologist 214 (3): 1198-1212.

2. Medina et al 2013. Early anther ablation triggers parthenocarpic fruit development in tomato. Plant Biotecnology Journal 11 (6): 770-779.

Blanca Salazar-Sarasua <blasasa@upvnet.upv.es>

Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), Valencia, Spain

Tomato fruits have a diverse range of shapes including round, pear shaped, and very elongated. Members of the Ovate Family Protein (OFPs) regulate organ shape in a number of plant species, and in tomato, mutations in ovate and ofp20 result in elongation of the proximal end of the ovary and pear-shaped fruits. OFPs interact with TON1 Recruiting Motif (TRM) proteins and mutations in these genes have also been found to regulate organ shape in tomato and other plants. However, the molecular mechanisms regulating shape remain unclear. OFPs and TRMs are expressed throughout tomato ovary and fruit development and have been implicated in cell division and cell shape. We found that the ovate mutant has reduced cell divisions in the floral meristem and during ovary initiation resulting in a narrower carpel early in development. Double mutants with ovate and ofp20 showed a further reduction in cell division resulting in an even narrower carpel. However, while the trm5 mutant did not alter cell division frequencies, trm5 did alter cell shape early in development resulting in smaller and flatter cells. Additionally, OFP-TRM interactions result in dynamic localization of these proteins between the cytosol and microtubules in N. benthamiana. We hypothesize that OFP and TRM protein families localize to microtubules to regulate cell expansion and division throughout floral meristem and ovary development and that members may have redundant functions to ensure robust organ shape throughout plant development. This work is supported by the grant 2017-67013-26199, and the Postdoctoral Fellowship 2018-08033, from the USDA, NIFA.

Ashley Snouffer

1Center for Applied Genetic Technologies, University of Georgia, Athens, GA, USA; 2 Institute of Plant Breeding Genetics and Genomics, University of Georgia, Athens, GA, USA

Ovate Family Proteins (OFPs) interact with TONNEAU1-recruiting motif family of proteins (TRMs) at the M8 motif to control fruit shape and organ morphology in tomato and other plant species. We hypothesize that there are members of the OFP and TRM protein families that are part of a protein network that interacts with microtubules to coordinate multicellular growth and organ patterning in plants. We sought to investigate the role of OFPs and TRMs in organ initiation from tomato tissue culture given that the ubiquitous OFP-TRM interactions regulate organ morphology. This system allows us to study the cellular functions of these proteins in a system that is more amendable to manipulation than the developing flower or fruit, while still assessing protein function in living and dividing cells. The first organ initiation occurs as the callus transitions to producing shoots. This transition requires the activation of genes needed to coordinate cell division/expansion and may be regulated by certain OFPs and/or TRMs. We will 1) Investigate shooting morphogenesis in readily available mutants of OFPs and TRMs 2) Perform RNA-seq to compare the callus and shooting stages to confirm which OFPs and TRMs are active and required for organogenesis. To further investigate the functional role of the M8 motif in TRMs interaction with OFPs, we will characterize CRISPR generated in-frame mutations within the M8 domain of TRM17/20a, TRM26a, and TRM30/34a. Furthering our understanding of the cellular functions and interactions between OFPs and TRMs will broaden our understanding of plant organ morphogenesis and shape regulation.

Natacha Namphengsone

1. Institute of Plant Breeding Genetics, & Genomics, University of Georgia, Athens, USA; 2. Center for Applied Genetic Technologies, University of Georgia, Athens, GA, USA

Starch is a storage carbohydrate synthesised by plants during photosynthesis and accounts for approximately 50% of the world’s daily calorie intake. In addition to human consumption, starch is also extracted from crops for industrial applications and used as feedstock for first-generation bioethanol. In higher plants, starch is the major storage carbohydrate and accumulates in chloroplasts of leaves in light and is degraded/remobilised in the dark. Leaf or transitory starch is primarily synthesised in photosynthetic tissues during the day and undergo nocturnal breakdown, supplying the plant with sugars required for its metabolism. The synthesis and degradation of transitory starch in green leaves is therefore tightly linked to diurnal changes in the light environment. Previous studies have demonstrated that starch accumulation and degradation are finely controlled to synchronise with variations in growth conditions, including light. Analyses of a phytochrome A mutant (phyA) of tomato altered in red-light responses show alterations in photosynthetic activity and leaf starch metabolism. In comparison to its isogenic wild type, phyA shows significant changes in photosynthetic activity under photosynthetically stimulating light conditions. Analyses of transitory starch content indicate lower levels of starch in light coupled with reduced starch degradation in dark in phyA. Concomitant decrease in plant growth and shoot biomass production is also observed for phyA compared to similarly aged wild type. The functional significance of phytochrome A in regulating these responses in tomato is highlighted.

Eros V Kharshiing <eros.kharshiing@gmail.com>

St. Edmund's College, Meghalaya 793003, India

Pepper (Capsicum spp.) exhibits a vast natural variation in fruit shape that is utilized for breeding of diverse fruit types. QTL mapping studies allowed the identification of several QTLs but to-date no genes underlying fruit shape QTLs have been conclusively identified and their function verified in pepper. In this study we mapped a major QTL in chromosome 10 in a cross of elongated and round-fruited parents. Combination of RNA-seq, bulked segregant analysis (BSA) and fine mapping using fixed recombinant progenies allowed to define a 5 Mbp region that contains the QTL as well as CaOFP20, the ortholog of SlOFP20 that controls fruit shape in tomato. To prove the function of CaOFP20, we knocked down its activity by VIGS in the background of a round-fruited parent. Silenced plants exhibited elongated fruits and reduced expression of CaOFP20 in the ovary, fruit pericarp and the placenta, implicating the function of the gene in controlling fruit elongation. To further test whether the expression pattern of CaOFP20 is associated with natural variation in fruit shape, we selected 30 C. annuum accessions representing elongated and round fruits from the G2PSOL core collection. qRT-PCR of CaOFP20 indicated a strong negative correlation between the expression level of the gene and degree of fruit elongation. Histological analyses of ovary wall and fruit pericarp of QTL-NILs indicated that the QTL exerts its effect on fruit shape by modulating multiple cellular mechanisms including cell size, cell number and cell shape.

Yelena Borovsky

Institute of Plant Sciences, The Volcani Center, Agricultural Research Organization, Rishon LeZion, Israel

The timing of flowering and inflorescence architecture are critical for the reproductive success of tomato, but the gene regulatory networks underlying these traits are still hardly explored. Here we show that the tomato FRUITFULL-like (FUL-like) genes FUL2 and MADS-BOX PROTEIN 20 (MBP20) induce flowering and repress inflorescence branching. FUL1 fulfils a less prominent role and appears to depend on FUL2 and MBP20 for its upregulation in the inflorescence meristem. Our results demonstrate that MBP10, the fourth tomato FUL-like gene, has probably lost its function. The tomato FUL-like proteins cannot homodimerize, but heterodimerize with JOINTLESS (J), SlMBP21 (J2), ENHANCER OF JOINTLESS (EJ2/MADS1) and the SOC1-homolog TOMATO MADS-box gene 3 (TM3), which are co-expressed during inflorescence meristem maturation. Transcriptome analysis revealed various interesting downstream targets, including five repressors of cytokinin signaling, which are all upregulated during the vegetative-to-reproductive transition in ful1/ful2/mbp10/mbp20 mutants. Our research reveals that the four tomato FUL-like genes have diverged functions, but together regulate the important developmental processes flowering time, inflorescence architecture and fruit development.

XiaobingJiang

Wageningen University & Research, Laboratory of Molecular Biology

Understanding the complex process of aging is one of the plant breeders’ major goal to reach. Indeed, senescence timing and progression affects plant fitness, harvest time, product quality and post-harvest conservation. Senescence involves several cytological and biochemical changes that rely on numerous transcription factors, phytohormone-responsive elements and senescence-associated genes, interconnected and tightly regulated. For instance, senescence in leaves is characterized by chlorophyll breakdown, nutrient reallocation and cell wall dismantling. Aging leaves are not supposed to perform photosynthesis anymore, so plastidial and cellular structures are disrupted, and, at the same time, macromolecules accumulated before are dismantled and brought to sink organs. Among all, a group of plant-specific transcription factors known to regulate these aspects is the NAC superfamily (NAM No Apical Meristem, ATAF1/2 and CUC Cup-Shaped Cotyledon). They mediate reactions to biotic and abiotic stresses, they are involved in secondary cell-wall deposition and they regulate fruit ripening and leaf senescence. In this work we have identified a new tomato NAC transcription factor, called HEB, whose downregulation delays the onset of senescence, as demonstrated by the improved photosynthetic performance, the altered transcription of senescence-associated genes and the reduced chlorophyll dismantle. Plants where HEB has been silenced display a stay-green phenotype.

Sara Forlani <sara.forlani@unimi.it>

1Department of Biosciences, Università degli Studi di Milano, Via Celoria 26, 20133 Milano.

Light controls several aspects of plant development through a complex signalling cascade. Several B-box domain containing proteins (BBX) were identified as regulators of Arabidopsis thaliana seedling photomorphogenesis. However, the knowledge about the role of this protein family in other physiological processes and species remains scarce. To fill this gap, here BBX protein encoding genes in tomato genome were characterised. The robust phylogeny obtained revealed how the domain diversity in this protein family evolved in Viridiplantae and allowed the precise identification of 31 tomato SlBBX proteins. The mRNA profiling in different organs revealed that SlBBX genes are regulated by light and their transcripts accumulation is directly affected by the chloroplast maturation status in both vegetative and fruit tissues. As tomato fruits develops, three SlBBXs were found to be upregulated in the early stages, controlled by the proper chloroplast differentiation and by the PHYTOCHROME (PHY)-dependent light perception. Upon ripening, other three SlBBXs were transcriptionally induced by RIPENING INHIBITOR master transcriptional factor, as well as by PHY-mediated signalling and proper plastid biogenesis. Altogether, the results obtained revealed a conserved role of SlBBX gene family in the light signalling cascade and identified putative members affecting tomato fruit development and ripening.

Zoom Password: QUR8MD

BrunoSilvestre Lira <bslbsl@usp.br>

Universidade de São Paulo; Instituto de Biociências; Departamento de Botânica

MEETING PASSWORD: 2yWHiV Membrane contact sites are (MCS) are microdomains in which two membranes are very close (∼30 nm) but do not fuse. MCS are essential for non-vesicular transport of lipids which is mediated by several families of proteins containing lipid transport domains, like the synaptotagmin-like mitochondrial lipid-binding (SMP) domain. The study of these proteins has increased due to their important role in plant-environment interactions. The most studied SMP containing protein in Arabidopsis SYT1 which is involved in tolerance to multiple abiotic stresses. However, little is known about the localization and function of other SMP proteins in plants. Arabidopsis SMP sequences were used to identify their orthologues in S. lycopersicum, allowing the identification of 16 genes encoding putative SMP domains (see poster Percio et al.). Interestingly, we have found that some SMP proteins are present exclusively in plants as they do not have orthologues in yeast and human. Here, we report the subcellular localization of these new identified proteins from S. lycopersicum and A. thaliana using transient expression in N. benthamiana, confocal microscopy and a set of organelle markers. Overall, we have found two proteins that localized in ER-Golgi, one protein localized in chloroplast-ER and several proteins being part of ER-PM MCS. We are now investigating the role of some of these proteins using biochemical, genetic and cell biology approaches. This work is supported by grants from: Ministerio de Ciencia, Innovación y Universidades (grant PGC2018-098789-B-I00), Ministerio de Economía, Industria y Competitividad (grant RYC-2013-12699), UMA-FEDER (grant UMA18-FEDERJA-154) and Ministerio de Ciencia e Innovación BIO2017-82609-R.

CarolinaHuercanoRubens <carolinahuercano@uma.es>

1. Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Universidad de Málaga, Campus de Teatinos, 29071 Málaga, Spain.

Zoom Password: Fb3XUS The non-vesicular transfer of lipids between different organelle membranes is essential for their proper functioning, and it relies on the formation of membrane-contact sites (MCS). These are discrete regions where two membranes are close but not fused (10-30 nm). One of the best described MCS is stablished between the endoplasmic reticulum and the plasma membrane (ER-PM), and is well known for stress tolerance, as well as other physiological processes. The formation of those MCS is dependent of proteins which promote the apposition of the membranes and mediate the lipid transport. There are more MCS in plants aside ER-PM, such as ER-Golgi or ER-chloroplast contact sites, but which proteins are involved as well as their role in tethering and lipid transport remains elusive. The synaptotagmin-like mitochondrial-lipid-binding (SMP) domain, which is contained in proteins as SYT1 to SYT6, among others, is a domain involved in glycerolipid transport in the MCS. We have analysed the SMP-containing proteins in silico in Arabidopsis thaliana and Solanum lycopersicum, including their expression, protein domains, and phosphorylation sites. The amino acid sequence and domain analyses have allowed us to stablish groups among them. In this communication we describe the results obtained that led us to the identification of uncharacterized proteins containing SMP domains as a first step to identify their function in lipid transport. This work is supported by grants from: Ministerio de Ciencia, Innovación y Universidades (grant PGC2018-098789-B-I00), Ministerio de Economía, Industria y Competitividad (grant RYC-2013-12699), UMA-FEDER (grant UMA18-FEDERJA-154) and Ministerio de Ciencia e Innovación BIO2017-82609-R.

Francisco Percio <franciscopercio@uma.es>

Biochemistry and Molecular Biology-Plant Biotechnology Laboratory, University of Málaga, Málaga, Spain

The moment at which a plant starts flowering is very important for its reproductive success and therefore tightly regulated by many genes. Few master regulators control this process by integrating internal and environmental signals. We focus on four close homologs of the MADS-domain transcription factor SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) in tomato. These homologs show different protein-protein interactions and are thus likely to have subfunctionalized during evolution. During floral transition, STM3 is most highly expressed of these homologs. We identified two evolutionary conserved regions with putative transcription factor binding sites in the STM3 promoter, that may be responsible for its high activity. We are developing in vitro approaches to identify upstream regulators of SOC1 homologs to further unravel the tomato flowering network.

Iris Zahn <iris.zahn@wur.nl>

Plant Developmental Systems, Wageningen University & Research, The Netherlands

This study describes the metabolomic effects of four tomato fruit mutations on the cv San Marzano (SM), a worldwide famous Italian traditional tomato variety. Three single mutant lines affecting the content of all pigments (hp-1, hp-2, pd) and a double mutant (Aft_atv), which presents a typically black skin for the accumulation of anthocyanins were obtained. A deep metabolomic analysis of non-polar (NP), polar (P) and volatile (VOC) metabolite fractions in fruits revealed the differences between the mutated lines and SM recurrent variety. Regarding the NP compounds, many carotenoids increased in the fruit of hp lines, as reported in previous studies, in addition to a higher content of β-carotene detected in the Aft_atv. Furthermore, pd resulted in enriched levels of two xanthophylls (All-trans-neoxanthin and Luteoxanthin) but, simultaneously, the levels of α-and β-/γ-tochopherols decreased. Looking at the metabolites in the P fraction, some sugars (Sorbitol and Sucrose) decreased in pd, as well as Fructose/Glucose in hp-1 and Aft_atv. Conversely, many organic acids increased specifically in the Aft_atv line. Most P changes occurred in the phenylpropanoid class, with considerable increases of many metabolites mainly in the case of Aft_atv, followed by pd and hp-2 lines. Finally, the flavour/aroma volatiles were positively modified in all the mutants which showed increased levels in many Benzenoids, Lipids and Phenylalanine derivative VOCs, which are associated to sweeter taste and better aroma. In conclusion, the identified differences in the mutated lines might become new specialities of the traditional SM variety, which could bring some novel desirable nutraceutical and organoleptic properties.

Gabriella Dono

Research Centre for Cereal and Industrial Crops, CREA, SS 673, km 25.200, 71122 Foggia, Italy; gabriella.dono@crea.gov.it (G.D.)

Tomato crops are attacked by diverse pathogens resulting in enormous losses of agricultural yield. Upon sensing the pathogens, plants mount various defence responses. In addition to well-studied responses such as a Reactive Oxigen Species burst and strong up-regulation of Ethylene, Salycilic Acid, Jasmonic Acid, and other phytohormones, the Secondary metabolites (SM), are expected to be involved in the quantitative resistance of different plants to certain pathogens. Leaf blight caused by species of genus Alternaria is a devastating disease in tomatoes and only very few cultivars show strong resistance against it. A large number of studies exist, investigating the role of SM in tomato. However, these focus predominantly of fruit and flavour-related compounds, SM after infection with Alternaria remain unknown. We applied metabolomics based on UPLC-QTOF mass spectrometry in combination with multivariate data analysis to compare the metabolomic profiling in Solanum lycopersicum after treatments with A. alternata, A. solani and chitin (a general elicitor of anti-fungal plant defence responses). We find large overlaps between generic (chitin-triggered) anti-fungal defence responses and Alternaria-triggered responses, but also unique responses observed for each of the treatments and differences between the time points.

Lina Muñoz

(1) Chair of Phytophatology, Tehcnical University of Munich, Germany. (2) Bavarian Center for Biomolecular Mass Spectrometry, Technical University of Munich, Germany. (3) Chair of food Chemistry and Molecular Sensory Science. Technical University of Munich, Germany.

The biosynthesis of volatile organic compounds (VOCs) related to tomato aroma and flavor is an important quality criterion and has been related to ripening stages of the fruit. An effective inhibitor of the action of ethylene, 1-methylcyclopropene (1-MCP) was used to study the relationship between volatile biosynthesis and fruit ripening in cherry tomatoes. The fruit was treated with 1-MCP vapors at room temperature at stages E3 and E5 for 15 days after harvest. 1-MCP prevented the climacteric increase of volatile production and therefore avoided the production of ethylene and the increase in the respiration rate. Untreated fruits developed typical climacteric changes and characteristic VOCs production. Apocarotenoids were the VOCs most affected by the presence of 1-MCP at stage 5. The above demonstrates the inhibitory effect of 1-MCP on the action of ethylene irreversibly in ripening stages in cherry tomato fruits marketed in the country and it relates its strong potential to be used as a tool in the post-harvest handling of this climacteric fruit.

Lina Londoño

1 Research Group GICTA. University of Caldas, Manizales, Colombia 2 Max-Planck-Institute of Molecular Plant Physiology, D-14476 Potsdam-Golm, Germany. 3 Research group INDETSA. Catholic University of Manizales, Manizales, Colombia. 4 Research Group GIPPA. University of Caldas, Manizales, Colombia 5 Research Group MICROBIOTEC. University Libre de Pereira, Pereira, Colombia

Hypertension is a primary risk factor for cardiovascular disease, the latter causing 7.5 million deaths every year. The ɣ-aminobutyric acid (GABA) is a non-proteinogenic amino acid that has shown effective in lowering the blood pressure of hypertensive patients. Intake of GABA through the daily diet may help reduce their symptoms and might be an effective way to prevent hypertension. Tomato is one of the most produced vegetables worldwide and daily consumed in a plethora of recipes and, a part of many beneficial compounds, it also contains high levels of GABA. However, GABA levels are higher in mature tomato green fruits and rapidly decrease in ripe fruits due to two main mechanisms in the GABA pathway. In fact, GABA is synthesized from glutamate by glutamate decarboxylase (GAD) and reversibly converted to succinic semialdehyde by GABA transaminase (GABA-T). A high-GABA content Micro-Tom line was developed deleting the autoinhibitory domain of SlGAD gene by introducing a stop codon through the CRISPR/Cas9 approach (Nonaka et al., 2017). However, the same would not suitable to edit SlGABA-T gene since it would lead to severe dwarfism and affected vegetative and reproductive growth (Koike et al., 2013). Our study aims at further increasing the GABA content in tomato applying an alternative CRISPR/Cas9 approach by targeting the promoter region of SlGABA-T. In that way, using multiple target gRNAs, we expect to produce novel cis-regulatory alleles that may provide a continuum of variation in SlGABA-T gene expression and consequently a better balance between GABA accumulation and plant development.

Pietro Gramazio

1) Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan; 2) Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, Japan

Gene- or genome duplication are powerful mechanisms that can have profound evolutionary consequences. Subsequent to genetic duplication events, sub- or neofunctionalization can lead to a species’ adaptation to new ecological niches or to the fine tuning of its physiological responses. Plants use phytochromes to sense light and temperature changes in their environment and to respond accordingly with adjustments to their development. In most higher plant species phytochromes exist as a small gene family. In solanaceous plants, such as tomato, five phytochrome genes are present, of which the red-light responsive PHYB gene most recently underwent gene duplication into PHYB1 and PHYB2. We used genome-wide transcriptional network analysis to probe the genomes of mutants of PHYB1 and PHYB2 and compared them to gene networks in the wild type. We found that both genes have subfunctionalized since duplication with PHYB1 acting as a major integrator of light and auxin responses, and PHYB1 and PHYB2 antagonistically regulating aspects of photosynthesis.

Andreas Madlung <amadlung@pugetsound.edu>

*University of Puget Sound, Tacoma, WA, USA

Phytochrome-mediated light and temperature perception has been shown to be a major regulator of fruit development. Furthermore, chromatin remodelling via DNA demethylation has been described as a crucial mechanism behind the fruit ripening process; however, the molecular basis underlying the triggering of this epigenetic modification remains largely unknown. Here, through integrative analyses of the methylome, siRNAome and transcriptome of tomato fruits from phyA and phyB1B2 null mutants, we report that PHYB1 and PHYB2 control genome-wide DNA methylation during fruit development. The experimental evidence indicates that PHYB1B2 signal transduction is mediated by the coordinated expression of DNA methylases/demethylases, histone-modifying enzymes and chromatin remodelling factors, resulting in the transcriptional regulation of photosynthetic and ripening-associated genes. This new level of understanding provides insights into the orchestration of epigenetic mechanisms in response to environmental cues affecting agronomical traits.

Ricardo Bianchetti

¹Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brasil. ²Instituto Andino Patagónico de Tecnologias Biológicas y Geoambientales - CCT-CONICET, Patagonia Norte, Bariloche, Argentina. ³Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel. ⁴Instituto de Agrobiotecnología y Biología Molecular (IABIMO), CICVyA, INTA-CONICET, Argentina. ⁵Cátedra de Genética, Facultad de Agronomía, Universidad de Buenos Aires, Buenos Aires, Argentina.

Phytophthora infestans is a pathogenic oomycete that causes late blight, the most devastating and costly potato disease. Breeders rely on the availability of resistance (R) genes to P. infestans (Rpi) to develop new resistant varieties. Such Rpi receptors trigger the plant defence response upon the recognition of cognate avirulence (Avr) effector proteins of the RXLR family. In this study, we characterised two allelic variants (Rpi-chc1.1 and Rpi-chc1.2) of the late blight Rpi-chc1 gene, from the resistant wild potato relative S. chacoense. Both alleles encode a Coiled-Coil (CC) – Nucleotide-Binding (NB) – Leucine-Rich Repeat (LRR) proteins, being the first functional representatives of the CNL16 family. An allele mining approach led to the identification of 15 Rpi-chc1-like homologs, classified into four main clades. These alleles have distinct patterns of RXLR effector recognition. While Rpi-chc1.1 and other alleles from clade A recognise PexRD12 (Avr-chc1.1), Rpi-chc1.2 and other members from clade B recognise PexRD31 (Avr-chc1.2), both belonging to the PexRD12/31 effector superfamily. We hypothesise that Rpi-chc1.2 is an ancient R gene that has been overcome in the recent past by reduction of PexRD31 expression. Rpi-chc1.1 may have evolved more recently since it is still active and provides resistance to a broad spectrum of P. infestans isolates. Additionally, we identified the LRR subdomain of both Rpi-chc1.1 and Rpi-chc1.2 to be responsible for the difference in effector recognition. With this study, we expand the knowledge on the molecular plant-pathogen interaction contributing to a better R gene exploitation and consequently to a better control of the late blight disease.

Daniel MoninoLopez

Wageningen University & Research, The Netherlands

On account of changing climatic conditions, heat stress has instigated reduction in total yield by 50% of major cereal and vegetable crops at worldwide scenario. It is imperative to understand the plant developmental modulation occurring at heat sensitive reproductive stages to combat the environmental perturbations and simultaneously respond fast to adapt and survive. The study attempts to unveil the molecular mechanism of miRNA-mediated regulation in heat sensitive critical stages of flower development using contrasting tomato cultivars in response to heat stress (HS). To achieve this feat, evaluation of different tomato cultivars with known HS tolerance/sensitivity was performed using various physiological, biochemical and yield related parameters that identified CLN1621L (CLN) and CA4 as most heat-tolerant and sensitive cultivars, respectively. To account the HS sensitivity at inflorescence stage, a comparative analysis of the pollen viability and germination at different bud lengths was performed. The microtome sections highlighted the male meiotic stages as highly HS sensitive in both cultivars. The sensitive cultivar exhibited poor pollen germination in all post-meiotic stages of bud development as well. The cumulative analysis of in-house generated heat-responsive inflorescence miRNome and literature search highlighted the 21 known differentially expressing miRNAs between the two contrasting cultivars. The expression profiling of miR319 and miR169 showed differential expression at mature and precursor level and were inversely related to their targets under HS in inflorescence tissues. Transgenic tomato plants overexpressing miR169 showed better performance with more number of flower and fruit retention in overexpression lines than wild type in HS.

Sarita Jha <sarita_bt2005@yahoo.co.in>

National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-110067,India.

The ‘de penjar’ tomato (Solanum lycopersicum) is a group of local varieties from the Spanish Mediterranean region carrying the alc mutation, which provides long shelf-life. Their evolution under low input management practices has led to the selection of resilient genotypes to adverse conditions. Here we present the first evaluation of agronomic performance and nutritional fruit composition of a collection of 44 varieties of ‘de penjar’ tomato under two N levels, provided by doses of manure equivalent to 162 kg N/ha in the control and 49 kg N/ha in the low N treatment. Traits analysed included yield, fruit weight, N, fructose, glucose, citric and malic acid contents, and sugars:acids ratio. Main tomato bioactive compounds, vitamin C, lycopene and β-carotene, were also evaluated. No significant effect of N supply was found for yield and most fruit compounds, except for fructose and glucose contents, which on average were reduced by 11.5% and 15.8%, respectively, under low N conditions. Significant genotype effect was observed for all the traits studied, revealing the wide variability in the collection. Fruit weight and lycopene content showed the highest relative range of variation (more than 4-fold) under both N treatments. In addition, lycopene and β-carotene presented a strongly significant interaction genotype × N input. These results revealed the potential of ‘de penjar’ varieties as a genetic resource in breeding for low N inputs and improving the organoleptic and nutritional tomato fruit quality.

Elena Rosa-Martínez

Instituto de Conservación y Mejora de la Agrodiversidad Valenciana, Universitat Politècnica de València, Camino de Vera 14, 46022 Valencia

Salinity is an increasing problem for the decreasing arable land surface. In coastal or irrigated areas, soil salinity is particularly increasing and can reach critical levels higher than tolerated by current cultivation. Novel cultivation practices including the use of resilient plant varieties and biostimulants might help to keep these areas accessible for food production. The salt stress response of above soil tissue is well documented, however, it is the root tissue which is exposed first to salt stress conditions either from the onset of its development or when increasing salt concentrations occur throughout cultivation. Can changes in the root transcriptome be linked to the root metabolome and phenotype? Transcriptome data analysis from an RNAseq experiment with salt and biostimulant treated samples harvested at two timepoints already revealed interesting candidates. In brief, we used a principle component analysis (PCA) to reduce the dimensionality of the data. The PCA was combined with an over-representation analysis (ORA) based on Mercator functional annotations (Mercator bins). This identified e.g. a transporter regulated upon salt stress. Moreover, we found over-represented Mercator bins from secondary metabolism and two candidates from the same cellular process when differentiating samples treated with a biostimulant by the PCA. These transcriptome analyses will serve as one building block for deriving gene and metabolite networks as well as mechanistic models to characterize resilient varieties and beneficial biostimulant treatment in order to allow cultivation at currently unfavourable conditions.

Andrea Schrader

1 Laboratory of Plant Physiology, Plant Sciences Group, Wageningen University & Research, Wageningen, Netherlands; 2 Institute for Biology I, Institute for Botany and Molecular Genetics (IBMG), RWTH Aachen University, Aachen, Germany; 3 Plant Physiology Unit, Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy; 4 Green Has Italia S.p.A, Canale, Italy; 5 Institute of Bio‐ and Geosciences, IBG‐4: Bioinformatics, Forschungszentrum Jülich, Jülich, Germany; 6 Institute for Biological Data Science, Heinrich Heine University, Düsseldorf, Germany

Bacterial canker of tomato is one of the most important bacterial diseases of this crop causing important economic losses worldwide. The disease is caused by the actinomycete Clavibacter michiganensis subsp. michiganensis (Cmm). There are no effective control strategies and only few preventive methods work efficiently. Currently, some wild tomato species such as Solanum arcanum and Solanum habrochaites are tolerant to Cmm. The study of these wild species represents an important approach for identifying a natural resistance source that can be used in commercial cultivars for controlling bacterial canker. In order to gain insight related to potentially tolerance genes, we contrasted the transcriptomes of the resistant wild tomato species S. arcanum LA2157 and the susceptible species S. lycopersicum cv. Ailsa Craig. We accomplished three different strategies: mapping to the reference genome SL3.0, Semi de novo transcriptome assembly, and de novo transcriptome assembly. Our results suggest that both tomato species exhibit a high amount of differentially expressed genes from 0 hours to 24 hours after the Cmm challenge, and the more contrasting results were obtained at 8h, particularly in the wild species S. arcanum LA2157, in which several functional groups related to defense response were enriched. Global transcriptional changes seem to be conserved between both species, suggesting that resistance in S. arcanum is associated with an upstream regulatory mechanism that probably diverged during the domestication process. The future work relies on the functional validation of the potentially tolerance-related genes to in order to overexpress the candidate genes in commercial cultivars of tomato.

Leonardo Isaac Pereyra Bistraín

1. División de Biología Molecular, IPICYT, S.L.P.; 2. Centro Nacional de Supercómputo, IPICYT, S.L.P.; 3. Escuela Nacional de Estudios Superiores, ENES UNAM, Gto.

Temperature affects plant growth and productivity, influencing both the vegetative and reproductive phase. The negative effect of high temperature is a major problem because of predicted increase of earth surface in the coming years. The level of proline in anthers plays important roles in acquiring heat tolerance in tomato, although proline transport may seriously be impaired by heat stress. Here, we engineered tomato cv Microtom (MT) plants with the endogenous LePROT1 proline transporter gene under the control of the conditional HSP18.2 promoter from Arabidopsis. MT plants and two T 3 progenies were grown in optimal conditions; half of the plants were subjected to repeated heat stress (38°C, 3 h/d for 2 weeks). An in vitro experiment was also carried out to subject seedlings to an acute heat treatment. Whereas MT plants under chronic heat stress showed a decrease in anther proline content, T 3 plants had significantly more proline than either the stressed control and the unstressed transformants. This pattern was paralleled by estimation of viable pollen. At phenotypic level, transformed plants under stress performed better than controls for inflorescence length, number of flowers, fruit set and Brix value. In vitro grown T3 seedlings after heat shock showed better root growth than the controls. In the epigeal part of seedling, the proline level was higher in trasformed plants than in controls irrespectively of the treatment. Total expression levels of LePROT1 were higher in trasformed plants at 48 h after heat shock. In conclusion, our data show that proline transport engineering represents a promising tool to endow tomato plants with thermotolerance.

Andrea Mazzucato

University of Tuscia, Viterbo (Italy), Department of Agriculture and Forest Sciences (DAFNE)

Potato Spindle Tuber Viroid (PSTVd) is the smallest pathogenic RNA that infects various plants species. PSTVd pathogenicity is a complex phenomenon mediated by the specific features of the viroid molecule, host species- specific genomic traits and viroid-host interactions in a dynamic environment. Two major classes of small non-coding RNAs (21-24 nt), namely micro RNAs (miRNA) and small interfering RNAs (siRNA) have been identified to mediate post-transcriptional RNA silencing as an anti-viroid defense mechanism. Viroid- responsive miRNAs, and their potential role as regulatory determinants in viroid pathogenicity, were studied in two Bulgarian pepper cultivars: Djulunska shipka (DS) and Kurtovska kapia (KK). The viroid infection was performed with PSTVd KF 440-2 and small RNA populations were analyzed at 28 days post- inoculation. Several conserved miRNAs (miR408, miR397, miR398, miR482) display cultivar-specific expression in response to PSTVd infection, as shown by sRNA sequencing and validated by RT-qPCR. The expression dynamics of miR397a-5p and its target Laccase-like 4, evaluated by RT-qPCR at 14, 28, and 43 dpi, revealed a cultivar- specific response. Acknowledgments: This work was supported by National Science Fund Grant DN06-6

Nikol Hadjieva

1 University of Plovdiv, Dept. of Plant Physiology and Molecular Biology, 24 Tsar Assen Str. 4000 Plovdiv, Bulgaria 2 Medical University of Varna “Prof. Dr. P. Stoyanov”, Department of Biochemistry, Molecular Medicine and Nutrigenomics, 84B “Tzar Osvoboditel” Blvd., 9002 Varna, Bulgaria

MIR169 family is an evolutionarily conserved miRNA family in plants known to target Nuclear Factor-YA (NF-YA) transcripts. A systematic in-depth analysis of MIR169 family in tomato (Solanum lycopersicum) by us annotated eighteen MIR169 precursors, these include new loci for MIR169a, b and d, as well as three novel mature isoforms namely, MIR169f/g/h (Rao et al., 2020). This family has expanded by both tandem- and segmental-duplication events during evolution. A tandem-pair MIR169b/b-1 and MIR169b-2/h is bicistronic in nature coding for three MIR169b isoforms and a new variant miR169h that is evidently absent in the wild relatives S. pennellii and S. pimpinellifolium. Seven novel miR169 targets picked from degradome data were confirmed by efficient target cleavage in presence of specific MIR169 precursors as well as increased target abundance upon miR169 chelation by short-tandem-target-mimic (STTM) construct in transient assays. Expression analysis of various MIR169s under different abiotic stresses showed significant increase in response to heat stress. Yeast-one-hybrid screens and co-expression in transient assays showed that seven tomato HSFs recognize five MIR169 promoters whereas, silencing HSFs using virus induced gene silencing (VIGS) reduced miR169 expression. Functional analysis using Sly-NF-YA-VIGS tomato plants and Arabidopsis plants overexpressing At-MIR169 or At-nf-ya mutants showed a link with increased heat tolerance in both plants. In contrast, Arabidopsis plants overexpressing At-NF-YA, or those expressing a non-cleavable At-NF-YA form as well as plants inhibited for At-miRNA169 regulation are more sensitive to HS, highlighting NF-YA acts as a negative regulator of heat tolerance. We propose a retroactive control of HSFs by the miR169:NF-YA node.

SOMBIR RAO

1 National Institute of Plant Genome Research, Aruna Asaf Ali Marg, P.O. Box No. 10531, New Delhi - 110 067

The tomato- and Arabidopsis-powdery mildew interactions have become well-characterized plant systems to study the interactions between plants and obligate biotrophs. Nevertheless, many questions still remain to be answered. Previously, knock-down-based methods, such as RNAi or Virus-induced gene silencing (VIGS) have been used to identify and characterize resistance and susceptibility genes. However, residual expression of the genes targeted through these methods may affect their clear characterization. This becomes particularly problematic when studying susceptibility (S) and resistance (R) genes conferring incomplete or partial resistance. The development of CRISPR/Cas9 has allowed precise and efficient targeted mutagenesis to identify and functionally characterize the resistance genes that plants use to defend themselves against powdery mildews. We have made use of this technology to verify a few R and S genes for their contribution to (in)complete resistance and susceptibility to PMs, respectively. This technology has provided us with a better resolution for our studies, allowing us to further understand the interaction between tomato or Arabidopsis and powdery mildews.

Miguel Santillán Martínez

Wageningen University & Research

Cape gooseberry (Physalis peruviana L.) is a valuable fruit-bearing species from the Solanaceae family, recognized as a source of pharmacological compounds with anti-inflammatory, antibacterial and antitumor activities. This fruit is economically important in countries such as Colombia where stands out as the second most important export fruit. However, one of the major constraints for cape gooseberry production in Colombia is the vascular wilt caused by the fungal pathogen Fusarium oxysporum f. sp physali (Foph). In this study, we validated key genes underlying Foph recognition and cape gooseberry defense responses. Candidate genes were identified and selected from 1,653 differentially expressed genes (DEGs) derived from RNA-Seq analysis and from a previous genome wide association study (GWAS) of this plant-pathogen interaction. The RT-qPCR validation showed candidate genes highly expressed earlier during infection in a susceptible genotype, while three others were overexpressed later, mostly in the tolerant genotype. These genes are mainly involved in pathogen recognition and a subsequent signaling pathways mediated by hormones such as ethylene and salicylic acid. This study provided the first insight to uncover and understand the molecular mechanisms from the P. peruviana - Foph pathosystem. The candidate genes validated allow a better understanding of the defense response in this orphan crop at the molecular level, and can be applied for the development of molecular markers that could facilitate the identification of tolerant/susceptible genotypes for use in breeding schemes.

Gina Garzon <ggarzon@agrosavia.co>

1 Corporación Colombiana de Investigación Agropecuaria (Agrosavia), Centro de Investigación Tibaitatá, Mosquera, Cundinamarca, Colombia. 2 Department of Bioscience, University of Milan, Milan, Lombardy, Italy.

Plant genomes encode for hundreds of miRNAs which are involved in a variety of functions like growth, metabolism, development and adaptive response to biotic and abiotic stresses. In the present study, 78 small RNA datasets from a pair of tomato cultivars exhibiting contrasting response to heat stress (HS) have been analysed to identify miRNAs which are differentially expressed in different tissues and under heat stress conditions. This led to the identification of 923 unique and 1153 precursor miRNAs whose hairpin structures fulfil all defined criteria. Comparative expression analysis between HS tolerant and sensitive cultivar under varied HS regimens showed conserved to cultivar-specific HS response. In the sensitive cultivar, 108 miRNAs were downregulated in heat stress and 97 were upregulated, while in the tolerant cultivar, 55 miRNAs were downregulated and 182 were upregulated. This indicates important roles for miRNAs in heat stress tolerance in tomato. Further, the targets of all miRNAs were identified using the degradome analysis of 10 previously published degadome datasets of tomato. Based on current miRNA annotation guidelines, 15 putative novel miRNAs were selected and their precursors were confirmed by cloning followed by sequencing. Tissue-specific expression analysis revealed that two of these miRNAs were inflorescence-specific, seven were highly expressed in tomato fruits while others were leaf-specific. This data will serve as an important resource for the identification of key miRNAs and their target genes which may be involved in fruit formation or ripening pathways, flower development and tolerance to heat stress.

Apoorva Gupta

National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India

Global climate change is increasing the temperatures that crops face during their life cycle, having negative effects in yield. Understanding the genetic control of plant responses to heat is a prerequisite for developing cultivars with increased resilience. The current work reports the identification of Quantitative Trait Loci (QTL) involved in reproductive traits (flower (FLN) and fruit number (FRN) per truss, percentage of fruit set (FRS) and pollen viability (PV)) under heat stress in 168 Recombinant Inbred Line (RIL) and 52 Introgression Line (IL) populations derived from the Solanum lycopersicum var. “MoneyMaker” and S. pimpinellifolium accession TO-937 cross. Plants were cultivated under increased temperature regimen conditions: T1 (25_C day/21_C night), T2 (30_C day/25_C night) and T3 (35_C day/30_C night). The effects of the heat stress was important at T3 for FLN, whereas the effects were evident already at T2 for FRN and FRS. A total of 20 QTLs involved in reproductive traits at different temperatures were identified by multi-environmental QTL analysis. Furthermore, two ILs with introgression at chromosomes 1 and 12 showed consistent heat tolerance in replicated trials. These results represent a catalogue of QTLs and pre-breeding materials that could be used as starting point to decipher the genetic control of the genetic response of reproductive traits at different temperatures and pave the road to develop new cultivars adapted to climate change.

MaríaJose Gonzalo

1Instituto de Biología Molecular y Celular de Plantas (IBMCP) UPV-CSIC, Valencia, Spain; 2Centro de Experiencias de Cajamar, Paiporta, Spain; 3Enza Zaden España S.L. Almería, Spain; 4National Taiwan University, Taipe. Taiwan;

Capsicum annuum is one of the most cultivated Solanaceae globally, and in open field water stresses affect its fruit quality as well as fruit setting, size and ultimately yield. Following RNA-seq transcriptome analyses of a breeding line of sweet pepper (CCu07), subjected to acute water stress followed by recovery (rewatering) at three stages of plant development, we identified stage-specific and common core sets of DEGs. Among them, 2 NAC TFs: CaNAC072 and CaNAC104 were always up regulated after water stress and down regulated after recovery. Following their transient expression in Nicotiana benthamiana leaves, the two TFs were localized in the nucleus. The VIGS of CaNAC104 did not affect the pepper plant tolerance to water stress, suggesting that the stress-responsive protein CaNAC104 is a component of a complex network activated by abiotic stresses, but it does not play per se a key role in influencing the response to drought stress. Unexpectedly, VIGS of CaNAC072 improved pepper plants tolerance to water stress, as pepper plants did not display a wilting phenotype after 7 days of no watering. CaNAC072 is the orthologous of the Arabidopsis gene ANAC072, a drought responsive gene activated following water stress and conferring water stress tolerance. We generated CaNAC072 transgenic Arabidopsis plants of three genetic backgrounds, two of which carry a mutation in ANAC072 and thus show lower tolerance to water stress. However, we did not detect any recovery of function. This suggests that the interactions between a NAC protein and the cis-acting elements of target genes may vary in relation to the plant species. Taken together our results emphasize the complex role of CaNAC104 and CaNAC072 as transcriptional switches in the response of sweet pepper to water stress and highlight that further studies are needed for better clarifying their functional role.

Sergio Lanteri <sergio.lanteri@unito.it>

1. Department of Agricultural, Forest and Food Science (DISAFA) –University of Torino, Italy - 2. Plant Sciences & Natural Products, Institute of Biology Leiden, Leiden University, The Netherland - 3 Max Planck Institute of Molecular Plant Physiology, Postdam-7 Golm, Germany

Nitrogen Use Efficiency (NUE) improvement in high N demanding crops such as tomato (Solanum lycopersicum L.) has become a crucial approach for sustainable agriculture. Genotypic difference represents the basis for NUE improvement together with the understanding of both physiological and molecular mechanisms developed by genotypes differing in their NUE to cope low-NO3-. In the present study, two NUE contrasting tomato genotypes, Regina Ostuni (RO) and UC82, N-efficient and inefficient genotypes, respectively, were compared for differential expression patterns of some NO3- metabolism related genes (nitrate transport, assimilation, remobilization and storage/sequestration), in both roots and shoots, under limiting and non-limiting NO3-supply. Our results revealed no significant differences in nitrate and nitrite reductase expression levels (SlNR and SlNIR, respectively) between NO3- treatments in RO shoot, while they appeared significantly down-regulated in UC82 under limiting NO3- supply. However, SlNR transcript level was significantly higher in RO shoot compared to UC82 in NO3- limiting supply. In addition, the potential involvement of NO3- long-distance transporter genes in root (SlNRT1.5 and SlNRT1.8), remobilization and flux in shoot (SlNRT1.7 and SlNRT2.7) in RO efficiency was highlighted. Besides, the chloride channel protein (SlCLCa) expression, mediating NO3- storage in cell tonoplasts, revealed a higher NO3- storage/sequestration in both UC82 tissues. Taken together, our results suggested that the strategy of the efficient tomato genotype is mainly determined by plant ability to modulate long-distance N transport, assimilation, remobilization and sequestration genes (SlNRT1.5, SlNRT1.7, SlNRT2.7, SlNR and SlCLCa). Finally, transcriptome analyses are being performed to identify candidate genes to improve NUE in tomato.

Meriem Miyassa Aci

Università Mediterranea di Reggio Calabria

Bacterial canker of tomato caused by the vascular pathogen Clavibacter michiganensis (Cm), is considered to be one of the most important bacterial diseases of cultivated tomato. Due to the lack of effective control measures against it, Cm is regulated as a quarantine or regulated non quarantine pest in multiple countries. No resistance (R) genes against the pathogen have been identified. Furthermore, a limited number of QTLs from wild accessions, with a complex genetic background have been shown to be involved in tolerance to Cm. As a result, there are no Cm resistant tomato cultivars. We decided to investigate the potential of using impaired susceptibility (S) genes as an alternative. Arabidopsis Wall Are Thin 1 (WAT1) encodes for a plant-specific protein that is involved in auxin transportation and secondary cell wall biosynthesis. Studies have shown that mutation of WAT1 is linked to enhanced resistance of Arabidopsis to a broad range of vascular pathogens. In our study, we identified and impaired the tomato ortholog of Arabidopsis WAT1. SlWAT1 silenced transgenic tomato lines were challenged with four genetically diverse Cm strains with different aggressiveness. SlWAT1 silenced plants showed reduced susceptibility to all tested strains, without severe adverse pleiotropic effects observed. To further study the effect of a full knock-out, a mutant slwat1 line was generated through CRISPR/Cas9. Full knock-out of the gene led to loss-of-susceptibility to Cm, accompanied with severe pleiotropic defects. To our knowledge, this is the first time that impairment of a single gene has resulted in reduced susceptibility to Cm.

Eleni Koseoglou

1Plant Breeding, Wageningen University & Research, Wageningen, The Netherlands; 2Biointeractions and Plant Health, Plant Research International, Wageningen, The Netherlands

Salinity which is spreading throughout the world is one of the most important abiotic stress factors limiting the agricultural productivity worldwide. The Na+/H+ antiporter (NHX) genes play a considerable role during plant growth and development in tolerance to salt stress. The objective of this study is to identify NHX genes using version SL4.0 and version ITAG4.0 of tomato genome. Seven putative NHX genes distributed on chromosome 1, 4, 6, and 10 were identified. While SlNHX1, -2, -3, -4 and -6 genes were localized on Vacuole, SlNHX7 and SlNHX8 were localized on plasma membrane and Plasma Membrane-Vacuole, respectively. SlNHX1, -2, 3-, -4, -6 proteins have amiloride binding site [FFIYLLPPI] which is located in transmembrane helix domain-3 and is known an inhibitor of Na+/H+ exchanger activity and characteristic feature of Vacuole class NHX proteins. SlNHX4, SlNHX7 and SlNHX8 hypothetically interact with CBL-interacting protein kinases (CIPK) based on Protein-protein interaction prediction analysis. The High-throughput gene expression data analysis was retrieved from TomExpress database and Tomato Expression Atlas project. Whereas SlNHX3 gene only expressed in unopened flower, SlNHX2, -4 and SlNHX1, -6 genes showed the highest expression levels in root and mature green fruit stage, respectively. The tissue specific gene expression levels treated with salt shock stress at different salt concentrations will be performed using qRT-PCR after plants reach at targeted development stage.

Erman Cavusoglu and Ugur Sari

Department of Agricultural Biotechnology, Faculty of Agriculture, Çanakkale Onsekiz Mart University

Drought and heat stress are reported to be major abiotic stresses limiting crop yield worldwide. In particular, tomato (Solanum lycopersicum), is regarded as a heat sensitive crop, although this sensitivity varies among genotypes. We investigated physiological responses of two tomato genotypes (E42 and LA3120) that were cultivated in the greenhouses under control, heat, drought and combined stress and treated or not with a plant-based biostimulant named CycoFlow (Agriges). The application of the biostimulant stimulated growth (plants up to 11.86% taller under combined stress) and number of leaf (up to 29.89% under combined stress). In E42, plants treated with the biostimulant under drought showed the same net photosynthetic rate (PN) level as the non-treated and non-stressed plants. Moreover, in LA3120 treated plants under drought, PN levels were higher compared to non-treated plants. The Fv/Fm values in the two genotypes were affected by drought, but a higher value was observed in the treated plants of E42 compared to non-treated plants. Also, treated plants under heat stress showed a higher stomatal conductance and transpiration rate compared to non-treated plants. Interestingly, a higher content of chlorophylls was also detected in both genotypes treated with the biostimulant compared to the non-treated plants under combined stress. Altogether, the effect of the biostimulant depended on the stress applied. The outcomes of this work will be important for the selection of genotypes tolerant to abiotic stresses and will help select the management practise to be used to improve plant performances and final yields.

Maria Manuela Rigano

1 Department of Agricultural Sciences, University of Naples Federico II; 2Department of Food Science, Aarhus University, Aarhus, Denmark

Combination of abiotic stresses has negative effect on the plant growth and productivity. Plant microRNAs control entangled gene regulatory networks and have been implicated in different stress responses. The core miRNome was identified in combined heat and drought stress in tomato leaf tissue. We identified miRNAs that showed up-regulation at both mature and precursor level in individual heat, dehydration and combined stress like miR172. MIR172 family is an evolutionarily conserved miRNA family, however in-depth analysis of MIR172 family in tomato is lacking. We validate four new loci of MIR172 in addition to the four loci reported at miRBase. We also did comparative study for identification of miRNA family members loci in the closest wild relative S. pimpinellifolium and in distant wild relative S. pennellii. Expression profiles of MIR172: target pair suggest MIR172 family as pervasive regulator of various abiotic stresses and developmental pathways. This regulation of miR172 was also shown by their promoter activity confirmed by promoter MIR172:GUS-reporter assays. With the degradome data, we identify Plastidic ATP/ADP-transporter, Beta-glucosidase 01, heat shock protein 90, Sec14p-like phosphatidylinositol transfer family protein as novel targets of miR172. We validate some of them using transient assays in Nicotiana benthamiana and chelation of miR172 by short-tandem-target-mimic construct. We show different processivity of miR172 precursors coding for same mature miRNA as well as also target cleavage efficiencies by miR172:AP2a pair. The present study will help in understanding the molecular studies on the miRNA172 involved in the regulatory networks involved in individual and combined stress response in tomato.

Chandni Bansal

National Institute of Plant Genome Research, Aruna Asaf Ali Marg New Delhi-110067 INDIA

Tomato is one of the most important vegetable crops with high economic value. Improving tomato fruit taste and prolonging shelf-life is the key for success in the market. Postharvest practice, specifically low-temperature storage and early harvest are used to extend tomato shelf life, but often reduce fruit quality. It has been reported that DNA methylation is important for regulating fruit ripening through gene silencing. However, the influence of postharvest handling on DNA methylation in relation to fruit quality is still unclear. This work aimed to clarify these issues by analyzing DNA methylation using methyl-sensitive amplification polymorphism (MSAP) and semi-quantitative transcriptional analysis of marker genes, and, assessing fruit quality biomarkers. The preliminary data shown that DNA methylation was influenced more by postharvest handling than ripening stage, however, fruit quality was influenced mainly by ripening. The other part of work is ongoing: applying whole genome bisulfite sequencing (WGBS), transcriptional analysis (RNA-Seq) to connect transcriptome and methylome changes due to postharvest. Incorporating all of these data is expected to broaden our finding and advance the interpretation of tomato fruit ripening regulatory mechanisms.

Jiaqi Zhou <jiqzhou@ucdavis.edu>

1Department of Plant Sciences, UC Davis, CA, USA; 2Departamento de Produccion Vegetal, Universidad de Concepcion, Region del BioBio, Chile

Agriculture will have to feed an increasing world population, using a decreasing arable land surface. Salinity is an increasing problem, in particular in coastal or irrigated areas. Due to climate change, these traditionally fertile areas are suffering from increased soil salinity, which has reached concentrations higher than tolerated by current cultivation practices. In the near future these areas will no longer be suitable for cultivating food unless we adopt novel production practices, including the use of novel resilient plant varieties. For plants to be resilient to abiotic stresses such as salinity and drought, the root system is of vital importance. Roots are the primary organs that adapt their architecture and physiology to drought and salt stress. Their performance is key to the ability of the whole plant to take up nutrients and water. However, we have limited knowledge of how the root functions and this translates into a limited capability to control plant resilience to abiotic stress. Novel developments in biostimulants show that it is possible to affect root functioning and resilience towards abiotic stress such as high-salinity. However, despite their potential for agriculture, there is very limited knowledge on the mechanisms through which biostimulants act. In this study we use genome-wide association mapping and CRISPR/Cas9-mediated forward genetics to understand the mechanism by which biostimulants contribute to tomato resilience under salinity stress conditions. The link between changes in root architecture and salt stress resilience will be discussed together with its importance in understanding salt tolerance in tomato.

Rumyana Karlova

1 Laboratory of Plant Physiology, Plant Sciences Group, Wageningen University & Research, Wageningen, Netherlands 2 Green Has Italia S.p.A, Canale, Italy 3 Plant Physiology Unit, Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy 4Institute for Biology I, Institute for Botany and Molecular Genetics (IBMG), RWTH Aachen University, Aachen, Germany 5 INRA, UR1115, Plantes et Systèmes de culture Horticoles, CS40509, 84914 Avignon Cedex 9, France 6 Hazera 7 Institute of Bio‐ and Geosciences, IBG‐4: Plant Sciences, Forschungszentrum Jülich, Jülich, Germany 8 Institute for Biological Data Science, Heinrich Heine University, Düsseldorf, Germany

Among the strategies developed to overcome abiotic and biotic stresses, grafting was demonstrated to significantly enhance plant tolorance. Nevertheless, gaps of knowledge exist in our understanding of the mechanisms by which rootstocks mediate scion-response to, e.g., salinity and consequently in developing enhanced graft-combinations. We explored the natural variation existing in rootstock mediated response to salinity in tomato. Tomato (Solanum lycopersicum) cv M82 grafted onto 254 different tomato rootstocks, including wild and commercial races. was grown in a nethouse in semi-randomized block design and treated with saline water to a final concentration of 200 mM NaCl. Following a 34 d period, leaf tissue was collected for GC-MS based metabolite profiling and oxidative stress (MDA) estimation. In addition, morphological traits were measured at harvest. Results showed that grafting resulted in phenotypic diversification of M82. When compared with self-grafted M82 under saline condition, the highest CV was measured for MDA content and the lowest CV for fruit weight to biomass ratio. Metabolite profiling based PCA separated between “best” and “worst” combinations (determined from fruit weight and MDA analysis). Highly responsive metabolites included malate, citrate, fumarate, and Asp. Fruit weight-related traits associated with changes in several metabolites, such as glycerol-3-phosphate, threonate, shikimate, and erythronate.

Chao Song <chaosong0506@qq.com>

Ben Gurion University of the Negev

Cultivation of hydroponically grown Solanaceae and Cucurbitaceae crops is currently facing problems due to the occurrence of a root disorder called ‘hairy roots’. This disease is characterized by an extensively expanded root system, suppressed water-uptake, defective fruit development and eventually withering of the plant. The causal agents of this disease are gram-negative rhizogenic Agrobacterium biovar 1 strains harbouring a root-inducing (Ri) plasmid. To date, the molecular mechanisms behind the development of hairy roots on infected plants remain largely unknown. Protein-protein interactions are essential in the communication between a plant and a microbe and eventually determine successful mounting of the plant’s defense response or emergence of the disease. In our research, we are using an innovative, high-throughput interactomics approach to map the key events in the development of hairy root disease. More specifically, we make use of TurboID-mediated proximity labeling coupled to LC-MS/MS, which enables the identification of direct and indirect protein interactors in planta. The rationale for our interest in this topic is that hairy root cultures are proven attractive and cost-effective options for the mass production of high-value plant metabolites and can be grown in contained conditions. Hence, besides filling longstanding gaps in our fundamental understanding of this plant-pathogen interaction, we anticipate that this research line will also yield novel insights into the regulation of plant metabolism and means to increase its exploitation. Eventually, this project may also generate novel targets for tomato pest resistance engineering and breeding.

Lore Gryffroy <logry@psb.vib-ugent.be>

1. Ghent University, Department of Plant Biotechnology and Bioinformatics, Ghent, Belgium; 2. VIB Center for Plant Systems Biology, Ghent, Belgium; 3. Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium

Plant cells undergo extensive reprogramming of molecular events to combat stress conditions like heat stress (HS). One such adaptive strategy is post-transcriptional regulation by alternative-splicing (AS), which can tremendously increase the transcriptome and proteome. Experimental validation heat stress transcription factor (Hsf) AS-forms in tomato highlighted that AS-variants express either differentially or specifically under different stress condition or developmental stage. In-depth analysis of Hsf18 identified five splice variants (Hsf18-V3, Hsf18-V6, Hsf18-V7, Hsf18-V17 and Hsf18-V23) of which only Hsf18-V3, Hsf18-V6 and Hsf18-V7 have coding potential which was confirmed by Western blotting using an Hsf18 genomic context:GFP construct and GFP antibody. These variants have an intact DNA binding domain and like the full length Hsf18 are nuclear localized indicating their probable role as transcription factors (TF). Interestingly, Hsf18 is also regulated post-transcriptionally by microRNA (miR-sm-1) under HS while the variants lack the target site and thus bypass target cleavage. Furthermore, a HS responsive long non-coding RNA (lncRNA) is transcribed from the complementary strand of Hsf18 locus. Yeast one-hybrid assays confirmed that Hsf18 and variants bind to the lncRNA promoter. Further experiments confirmed that Hsf18 negatively regulates lncRNA while variants regulate its expression positively. Moreover, the expression of full-length Hsf18, its variants and the lncRNA was either non-differential or not detected in other abiotic stresses like cold, salt and drought suggesting that this module is regulating thermotolerance in tomato. The precise mechanistic regulation in this complex module involving miRNA, TF and lncRNA and role of AS in Hsfs in HS adaptation is being investigated.

JaishriRubina Das

National Institute of Plant Genome Research, New Delhi, India

Blossom end rot (BER) is one of the most devastating physiological disorders in tomato and other vegetables resulting in significant crop losses as high as 70%. Due to changes in weather patterns of excessive rain alternating with prolonged droughts, the disorder is becoming more widespread. To decipher the genetic basis of BER, an F2 population derived from a cross between BER-resistant accession BGV007900 and BER-susceptible accession BGV007936 was evaluated. The QTL-seq approach was employed to map the loci associated with BER. We identified two QTLs for BER at the bottom of chromosomes 3 and 11. Association of identified QTL(s) with the trait was confirmed in 17S28 F2 population using KASP and dCAPs markers. Confirmation and further fine-mapping were done using progeny tests of selected F2 recombinant plants and F3:4 plants. QTL interval on ch03 was narrowed down from 11.2 Mbp to 1.28 Mbp at 95% confidence intervals. Moreover, QTL interval on ch11 was narrowed down from 1.98 Mbp to 792 kb at 95% confidence intervals. To further fine-map the QTL on ch11, 192 recombinant plants will be selected by screening 1728 F4:5. Similarly, 4032 F5:6 seedlings will be screened to select 192 recombinants for fine-mapping on ch03. Ultimately, BER loci on ch03 and ch11 will be further fine-mapped and subsequent expression analysis will be implemented to identify genes that will hopefully yield molecular and developmental insights into the causes of BER. Supported by USDA AFRI grant 2020-67013-30912.

Yasin Topcu

Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Athens, Georgia, United States of America 2Department of Horticulture, University of Georgia, Athens, Georgia, United States of America

Agriculture is the economic sector that utilizes most of the freshwater resources available globally. Salinization of arable lands is a major concern due to the increasing limitations of water supply and the substantial use of continuous irrigation systems worldwide. Salinity stress has a significant impact on crop development and yield, which limits cultivation in marginal lands and agricultural use of saline water. A glycophyte crop, such as the cultivated tomato (Solanum lycopersicum), requires high amounts of water while being highly sensitive to soil salinity. Introgression of salinity resistance into tomato cultivars via conventional breeding remains challenging due to the highly polygenic nature behind abiotic traits. We aimed to domesticate tomato de novo as a high-salinity resistant crop by harnessing the feature of its wild relative, S. cheesmaniae. Based on published data, the accession LA0421 was chosen as the genetic baseline for editing genes involved in domestication traits (plant architecture, flowering, yield, fruit size, and nutritional value) due to its lower yield penalty when exposed to saline conditions. We are using a multiplex CRISPR/Cas9 strategy to produce mutated alleles for the domestication-related genes BIF, cycB, J2, EJ2, MULTI, SP, SP5G, and FW11.3 in order to create a novel, halophyte tomato harboring an inherent high-salinity resistance along with desirable cultivation traits. This proof-of-concept research aims to engineer crops using a speedy reverse breeding approach that could potentially use (at least partially) seawater hydroponics or saline soils for food production.

Estefania Tavares-Flores

1.West Virginia University, 2.Federal University of Lavras, 3.University of Sao Paulo, 4.ICAR-Sugarcane Breeding Institute.

Grafting is a vegetative propagation method widely applied in horticulture to join the top part of a plant (scion) onto the base of the stem and roots of another plant (rootstock), in order to combine positive traits from the two different plants. However, the molecular mechanisms implicated in the rootstock-scion interaction remains largely unknown. Epigenetic mechanisms play a relevant role in plant distance signalling, thus we studied the genome-wide changes induced by DNA methylation in eggplant (Solanum melongena), one of the most commonly grafted Solanaceous crops. Plants of an eggplant double haploid line (DH) derived from the commercial hybrid ‘Ecavi’ were hetero-grafted onto two commercial rootstocks: (i) Solanum torvum, which provides resistance to bacterial, Fusarium and Verticillium wilts and to root-knot nematodes, and (ii) ‘Emperador RZ’, a tomato hybrid resistant to Verticillium. Self-grafted ‘Ecavi’ plants were used as control. Hetero-grafted plants showed a significant increase in vigour compared to the self-grafted plants. We generated the first eggplant methylome (coverage 10X), which displayed 90% methylation in CG, 83% in CHG and 16% in CHH contexts, and found out that their vigour was associated with a genome-wide hypomethylation in CHH context. Following RNA-seq analyses, we also investigated the effect of DNA methylation on transcriptional activity of genes and transposable elements (TEs) in hetero-grafted and self-grafted plants. A down-regulation of younger and potentially more active LTR retrotransposons (LTR-TEs) correlated with CHH hypomethylation, suggesting that graft-induced epigenetic modifications are associated with both physiological and molecular phenotypes in grafted plants.

cinzia comino

¹ DISAFA, Agricultural, Forest and Food Sciences, Plant Genetics & Breeding, University of Torino, Italy. ² The Sainsbury Laboratory, University of Cambridge, Cambridge, UK. ³ COMAV, Institute for Conservation & Improvement of Valentian Agrodiversity, Polytechnic University of Valencia, Spain. ⁴ School of Biosciences, University of Birmingham, Birmingham, UK

Tomato is a paradigmatic case of crop domestication: a widely cultivated and consumed vegetable but with reduced genetic diversity and therefore highly vulnerable to emerging diseases and climate change. Fortunately tomato is rich in genetic resources and information to overcome those difficulties, and a coalition of those scientists and breeding experts who have generated a large amount of this information has been organized under an effective management structure of HARNESSTOM to overcome those threats. By capitalizing on the large effort done recently in several EU-funded projects to connecting phenotypes/genotypes in a large number of accessions from different germplasm banks and academia HARNESSTOM will first collect, centralize and normalize this wealth of information in a way that is easily searchable and displayed in a user-friendly manner adapted to different type of users. Second, HARNESSTOM will develop four pre-breeding programs addressing the major challenges of the field: 1) introducing DRG against the major emerging diseases, 2) improving tomato tolerance to climate change, 3) improving quality, and 4) increasing resilience in traditional European tomato by participatory breeding. An additional goal is to increase speed and efficiency in pre-breeding, which is needed to be able to respond to the emerging challenges in a timely and effective manner. Joint leadership of both academia and industry in each of the work packages and the participation of two NGOs representing different stakeholders guarantees the results of the project will have an impact in industry and society innovation.

Antonio Granell

IBMCP (CSIC-UPV) Valencia, Spain

Tomato fruit weight is a quantitative trait resulting from selections that are governed by multiple loci or QTL. Few QTLs have been cloned and confirmed in addition to other candidate loci that have been mapped. Among the cloned QTL are fw2.2, fw3.2 and fw11.3 underlying CNR, SlKLUH and CSR respectively. To identify new QTL controlling fruit weight, an F2 population developed from a cross between PI406890 with smaller fruit and BGV008041 with larger fruit was evaluated. Both accessions are fixed for known fruit weight alleles. The QTL-seq BSA approach was applied to map the loci associated with fruit weight. We identified two QTLs affecting fruit weight on the bottom of chromosomes 3 and 8. The fw3.3 QTL was confirmed in an F3 population through progeny testing of 8 F2 families to a region of 3.7Mb. This QTL was further mapped using a population of 157 recombinant plants and Inclusive Composite Interval Mapping to 1.7Mb. An additional 270 recombinants plants led to the fine mapping of the locus to a 614.5 kb region. Similarly, 145 recombinant plants confirmed the fw8.1 QTL to a 4.2 Mb region. We aim to further fine map the loci on ch3 and ch8 to a few candidate genes and investigate the effect of these genes on fruit weight in tomato.

This study has been funded by the National Science Foundation IOS 1564366.

Manoj Sapkota

1Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Athens, GA, 30602, USA 2Center for Applied Genetic Technologies, Genetics & Genomics, University of Georgia, Athens, GA 30602, USA 3College of Horticulture, South China Agricultural University, Guangzhou, 510642, China *presenting/corresponding author

Eggplant fruit shape is an important quantitative agronomic and economic trait and using suitable mapping population like a set of introgression lines (ILs) could contribute to elucidate its complex genetic. To achieve this challenging objective, a set of 16 eggplant ILs, each harboring a single marker-defined chromosomal segment from the wild eggplant relative S. incanum in the genetic background of S. melongena, was assessed for fruit shape in two environments (open field and screenhouse). Fruits of the two parents, their interspecific hybrid and ILs were phenotyped using the phenomics tool Tomato Analyzer. Many morphological differences were found among parents and the hybrid for many fruit shape traits. Significant differences were also found between ILs and the recipient parent, suggesting the existence of a relevant effect of the introgressions, even in presence of small wild donor fragments. A total of 41 stable QTLs spread over ten chromosomes were detected. Of these, twenty QTLs co-localized with other previously reported in intraspecific and interspecific eggplant populations, while twenty-one QTLs, including Blockiness, Homogeneity, Asymmetry and Internal Eccentricity traits, were novel. In addition, variations associated to genes belonging to SUN, OVATE and YABBY families described in tomato were reported in the ILs potentially underlying genes for the QTLs identified. Eleven SUN and YABBY genes were proposed as potential candidate controlling fruit shape variations in eggplant. Our results provide novel and highly relevant insights on the genetic of fruit shape in eggplant.

Giulio Mangino

1COMAV, Univeristat Politècnica de València, 46022 València, Spain; 2Meridiem Seeds S.L., 30700 Torre-Pacheco, Spain; 3Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, 305-8572 Tsukuba, Japan

Lycopene content in tomato fruit is largely under genetic control and varies greatly among genotypes. Little is known about the molecular mechanisms regulating fruit lycopene content in high-lycopene tomatoes. In the present study, 42 potential high-lycopene tomato cultigens with different genetic backgrounds were collected worldwide. High performance liquid chromatography (HPLC) analysis was used to quantify fruit carotenoid (lycopene, phytofluene, prolycopene, and beta-carotene) contents at four fruit developmental stages (i.e., breaker, orange, pink, and ripe) of each cultigen. Real-time RT-PCR was used to quantify the relative expression levels of all the 25 pathway genes individually at the breaker and ripe stages. In these cultigens, we found i) a general trend of strong expression of upstream genes prior to lycopene biosynthesis and weak expression of most downstream genes at both stages; ii) significant higher expression in 7 upstream genes at the breaker or both stages than in the negative control Moneymaker, resulting in significantly higher fruit lycopene content; and iii) significant higher expression in 8 downstream genes at the breaker or both stages than in the positive control the wild tomato, leading to significantly lower fruit lycopene content . This was the first systematic investigation of the role of the complete carotenoid biosynthesis pathway genes in fruit lycopene content across many high-lycopene cultigens, which will enable tomato breeding and gene editing for improved fruit lycopene content.

James Duduit

1Department of Horticultural Science, North Carolina State University, Raleigh, NC, 27607, USA 2Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Prov.le Lecce-Monteroni, 73100 Lecce, Italy 3Department of Plant Sciences, University of California, Davis, CA, 95616, USA 4Department of Horticultural Science, Plants for Human Health Institute, North Carolina State University, North Carolina Research Campus, 600 Laureate Way, Kannapolis, NC 28081, USA

The GRAS genes are plant-specific transcriptional factors that play critical roles in processes of plant growth and development. However, their role in fruit ripening is not fully understood. Recent genome-wide analysis of the GRAS family showed that several genes were differentially expressed during fruit development, ripening, or biotic and abiotic stress conditions. Gene expression data of tomato (Solanum lycopersicum) suggested that the gene 𝘚𝘭𝘎𝘙𝘈𝘚10 might be involved in fruit ripening. In this study, constructs using CRISPR/Cas9 technology were generated for tomato targeted mutations. Plants were transformed and T₁ progeny was obtained. All T₁ plants were screened for the absence (due to genetic segregation) of both Cas9 and NptII (kanamycin resistance) transgenes and 33% of the plants were T-DNA free. Sequence analysis revealed different mutant alleles, from 1 bp deletions to 5 bp deletions or 1 bp insertion. Moreover, no off-targets was detected in the transformed plants. Ripening processes did not seem to be influenced by the mutation of 𝘚𝘭𝘎𝘙𝘈𝘚10; however, seeds´ number was affected in some mutated lines. Expression of the gene is increased during flower anthesis in the wild type and the knockout of this gene may be interfering with flower fertilization and seed formation. However, further analysis is necessary to elucidate the role of the 𝘚𝘭𝘎𝘙𝘈𝘚10 in these processes. For that, T₂ of the lines with a reduced number of seeds are being generated and seed number and fruit development parameters will be evaluated.

Flávio Soares <fjmsoares@fc.ul.pt>

1. BioISI - Biosystems and Integrative Sciences Institute, Faculty of Sciences, University of Lisbon, Campo Grande, 1749-016 Lisboa, Portugal 2. Dept. of Plant Genomics and Biotechnology, IBMCP (CSIC-UPV), Ingeniero Fausto Elio s/n Valencia, Spain 3. UMR1287 EGFV, INRAE, Bordeaux Sciences Agro, Bordeaux University, ISVV, 33140 Villenave d'Ornon, France

Heat stress is considered the main uncontrollable factor affecting potato yield. This study was conducted aiming to evaluate the response of potato genotypes to high temperature. The potato genotypes were cultivated in growth chambers, in a 12-hour photoperiod, under two temperature conditions: control (14-27°C) and supra-optimal temperature (24-34°C). Initially, a study of the effect of supra-optimal temperature was carried out in the early stages of tuberization. Later, physiological and morpho-agronomic analyses were used to carry out non-invasive selection of potato genotypes tolerant to exposure to supra-optimal temperatures. Finally, 18 potato genotypes were characterized for heat tolerance based on these physiological and morpho-agronomic characters. As a result, it was observed that temperature did not influence induction to the tuber initiation phase. However, it prolonged the period of plant emergence, reduced the number of plants that reached the emergence stage, as well as reduced yield and quality of the tubers. Changes in the parameters of gas exchange and chlorophyll fluorescence allowed to distinguish genotypes with different levels of heat tolerance. In this way, a methodology for a fast and non-destructive selection of heat tolerant potato germplasm was established. The supra-optimal temperature caused changes in morpho-agronomic and physiological traits. The genotypes with the best responses in the supra-optimal condition were the same ones identified as superior in the control condition. The results obtained in this work will contribute to the definition of future studies to develop a potato germplasm more tolerant to the adverse effects of global warming.

Caroline Castro

1 Embrapa Clima Temperado; 2 Embrapa Algodão

As part of the UC Davis Student Collaborative Organic Plant Breeding Education (SCOPE) project, a student-led pepper breeding program specific for California organic growth environments has been conducted since 2016. Graduate and undergraduate students are trained to develop goals, select parents, evaluate and select progeny to varietal release. Thus far 6 graduate and 15 undergraduate students have been trained. After consultation with the organic industry, the following breeding goals were developed: 1) enlarged cavity (Jalapeño Popper) for jalapeño pepper, and 2) sunscald tolerance for bell pepper. Collaborating with local organic growers, open pollinated/inbred selection was done in organic environments through multiple years across different locations. A few elite lines show better performance than controls on targeted traits and yield. In addition to field selection, outreach activities (field day, taste test, chef interview, etc.) also provide meaningful information.

Mengyuan Xiao

1 Plant Breeding Center, University of California, Davis, CA, United States; 2 Agricultural Sustainability Institute, University of California, Davis, CA, United States; 3 Organic Seed Alliance, United States

While complete polyploid plant genome assembly is still a challenge (Kyriakidou et al., 2018; Kyriakidou, Anglin, et al., 2020), much can be learned from copy number variants (CNVs) in genome data. CNVs are believed to be the reason for many complex phenotypic variations in plants. In this project, the genomes of six diploid (S. stenotomum subsp. stenotomum, S. phureja, S. xajanhuiri, S. bukasovii, S. stenotomum subsp. goniocalyx), two triploid (S. juzepczukii, S. chauca), three tetraploid (S. tuberosum subsp. andigena; S. tuberosum subsp. tuberosum) and a pentaploid (S. curtilobum) potato landraces were sequenced, and together with two wild, diploid, publicly available genomes (S. commersonii and S. chacoense) were used to explore structural variation compared to two potato reference genomes; the cultivated DM1-3 and the wild S. chacoense M6. The results shows CNV clusters containing genes involved in metabolic processes, disease resistance and stress tolerance that can be used as potential biomarkers for potato breeding in order to overcome the susceptibility of the modern European cultivars to biotic and abiotic stresses (Kyriakidou et al., 2020b). Kyriakidou et al., (2018) Current strategies of polyploid plant genome sequence assembly Frontiers in Plant Science ov 21 Kyriakidou et al., (2020a) Genome assembly of six polyploid genomes. Nature Scientific Data 7:88 Kyriakidou et al., (2020b) Structural genome analysis in potato taxa. Theoretical and Applied Genetics (TAG) 133, 951–966

Maria Kyriakidou

1 Department of Plant Science, McGill University, Montreal, Canada; 2 Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, Fredericton, Canada; 3 International Potato Center, Lima, Peru

Tomato crop production is seriously limited by extreme high temperatures during summer time. Developing tomato genotypes tolerant to high temperature may be a valuable strategy to cope with climate changes. However, heat stress tolerance (HT) it a very complex trait to dissect. In this work, a tomato genome selection (GS) model was build up to increase the rate of genetic gain for yield under heat stress conditions. To reach our goal, a F4 segregating population, deriving from a tomato variety, previously selected for yield performance under heat stress, has been characterized for nine phenotypic traits (eg yield, fruit number, SSC). A PCA analysis based on phenotyping scoring allowed us to select the best and worst performers. One-hundred F4 individual have been genotyped by sequencing (GBS) to build up a prediction model. Several statistical methodologies were tested to predict the breeding value (GEBV) of candidates for the next round of selection. A robust and accurate rrBLUP regression method was developed and adopted for further analysis. After fitting the prediction model in a training population, we measured its accuracy using a test sample for which the phenotype was available. For complex trait such yield (controlled by many genes with small effect) we found a correspondence between predicted and real values up to 70 %. The model has been also applied on genotypic data of F5 individuals and validated on F6 phenotyped lines. A new breeding paradigm for optimizing tomato improvement procedures is beginning to emerge.

Maria Ercolano

University of Naples Federico II, Department of Agricultural Science, Naples Italy

Pepper is a good source of health-related metabolites such as flavonoids. QTL analysis of a segregating F2 population, derived from a cross between C.annuum Long Sweet with high levels of flavonoids and C.annuum AC2212 with low flavonoid levels, revealed a strong QTL for flavonoids on pepper chromosome 5. Data mining of the QTL region led to the identification of a candidate gene, CaMYB12-like, with high homology to SlMYB12 known to regulate flavonoids in tomatoes. Virus-induced gene silencing of CaMYB12-like in pepper fruit led to a significant decrease in the accumulation of flavonols, flavones and chalcones and downregulation of structural genes of the flavonoid pathway. Furthermore, transgenic overexpression of CaMYB12-like could partially complement the low-flavonoid phenotype of a tomato SlMYB12 mutant (y) and led to increased levels of chalcones and flavonols in transgenic fruits. Our results indicate that the CaMYB12-like transcription factor plays an essential role in regulating the flavonoid pathway in pepper fruits.

Yi Wu

1. Plant Breeding, Wageningen University and Research, Wageningen, Netherlands; 2. Agricultural Research Organization (ARO), Bet Dagan, Israel

When people think about potato and its wild relatives (Solanum sect. Petota), Brazil is certainly not the first place which comes to mind. Most cultivated and wild potatoes available worldwide in genebanks have their roots in Andean highland tropical or temperate adjacent lowland areas. The Brazilian biota has a distinct evolutionary and biogeographic history and lies under diverse environmental conditions in eastern and central South America. Thus, the Brazilian germplasm is a distinct set of genetic resources to search for novel traits in potato breeding to face a constantly changing and challenging world. The potato breeding program at Embrapa Clima Temperado started in 1946. Since 1986, wild potatoes were sampled and are currently deposited in the Herbarium ECT and the Potato Genebank. These facilities are the most representative collections of exsiccates and accessions of wild potatoes from Brazil, representing around three hundred records of S. chacoense, S. commersonii, and S. malmeanum. Accessions are continuously documented, characterized morphologically and genetically, evaluated agronomically, and phenotyped under stress conditions. Currently, six registered cultivars developed by Embrapa have at least one wild progenitor in their genealogies. Despite the efforts, we still believe our germplasm is a neglected treasure chest. To unlock the vast potential of Brazilian wild potatoes, we are increasing our efforts and collaborations within Brazil and neighbouring countries to boost sampling, characterization, and evaluation under stress conditions. The most promising genotypes are being evaluated for crossability and introgression to move them into pre-breeding initiatives and enhance their future incorporation into breeding pipelines.

Gustavo Heiden

1 Embrapa Clima Temperado, Brazil. 2 Embrapa Algodão, Brazil. 3 Programa de Pós-graduação em Agronomia, UFPel, Brazil. 4 UFPel, Brazil. 5 Programa de Pós-graduação em Fisiologia Vegetal, UFPel, Brazil. 6 Jardim Botânico do Rio de Janeiro, Brazil. 7 UFJF, Brazil. 8 UDELAR, Uruguay.

The hybrids in tomato (Solanum lycopersicum L.) are mostly cultivated due to their disease resistance, productivity, and fruit quality. The existence of heterosis has been studied in tomato for many years. However, the influence of the parental order in the cross over the hybrid’s performance, i.e. the reciprocal effect, has not been explored. This work aimed to detect heterosis and reciprocal effects in agronomic traits and metabolites. The plant material included 20 hybrids obtained by crossing two American heirlooms: Purple Pear and Green Zebra, and three RILs obtained from the Tomato Breeding Program at Facultad de Cs. Agrarias, UNR: Querubín, Gema, and ToUNR17 following a full diallelic mating design. We measured 12 agronomic traits and 28 metabolites. A PCA analysis, including the means of all traits and genotypes, showed that the hybrid’s performance was enlarged compared to the phenotypic variation of parental genotypes. Heterosis and reciprocal effect were confirmed for each cross and trait using ANOVA. There was heterosis when the hybrid had a better performance than the best parental genotype (P<0.05). The reciprocal effect occurred when, changing the parental order in the cross, the reciprocal hybrids were phenotypically different (P<0.05). We detected 12.4% of heterosis and 21% of reciprocal effect for all possible cases. Both phenomena were more frequent in metabolites. These findings highlight how important is the male and female parental genotype election in a hybrid cross, as well as the possibility of exploiting heterosis, mainly for metabolites, in the generation of new hybrids.

Agustina P. Fortuny

1 Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET – UNR, Ocampo y Esmeralda, CCT Rosario (S2000EZP), Santa Fe, Argentina; 2 Cátedra de Genética, Facultad de Ciencias Agrarias UNR, Campo Experimental Villarino S/N, (S2125ZAA) Zavalla, Santa Fe, Argentina; 3 Instituto de Investigaciones en Ciencias Agrarias de Rosario (IICAR), CONICET – UNR, Campo Experimental Villarino S/N, (S2125ZAA) Zavalla, Santa Fe, Argentina; 4 Departamento de Química Biológica, Facultad de Ciencias Bioquímicas y Farmacéuticas UNR, Suipacha 531, (S2002LRK) Rosario, Santa Fe, Argentina.

Two half-sibs were developed crossing tomato accessions of contrasting fruit colour, size and shape, from La Consulta INTA Germplasm Bank, Mendoza, Argentina. Parentals, F1 and F2 generations were evaluated through 13 morphometrical and quality fruit traits, and 18 carotenoid and chlorophyll pigments content. Segregant frequencies for pericarp colour suggest three genes with complementation and epistatic effects. Red fruits shows higher licopene and carotenes contents, and chlorophylls and pheophytins for brown fruits. Allele contributions and dominance ratios shows partial, complete and overdominance effects for different traits, and also differential correlations between them. A total of 77 transgressive F2 individuals were selected for its fruit attributes, and families were derived by selffing until F5 under a genealogical selection scheme focussed on round fruits, thick pericarp and high soluble solids content. F5 individuals were pooled by their fruit colour in red, pink, orange, dark and light yellow, brown, purple, and green groups. Fruit selection was stabilized at around 25 g weight, height-width ratio of 1.0, soluble solids rise up from 5.6-6.5 ºBrix in F1 to 8.8 ºBrix in F5, and pericarp width increased up to 27%. Broad sense heritability values of F5 colour-groups rise down with respect to F2 for size, shape and color. Each F5 colour-group shows different patterns of heritability reduction, retaining considerable variability for pigments, soluble solids content and pericarp thickness. A better understanding of the genetic components of tomato fruit attributes, and associations between agronomic traits and quality, allowed selecting individuals of outstanding characteristics for differential and gourmet markets.

Pablo Asprelli

1Agronomy Faculty, National University of Cuyo. 2La Consulta Experimental Station, National Institute of Agricultural Research. 3CCT CONICET Mendoza, Argentina.

Fruit weight has been one of the most agronomically important traits in tomatoes (Solanum lycopersicum). Volatiles, on the other hand, is an emerging trait of interest that substantially affects tomato flavor and consumer preference. The highly quantitative nature of fruit weight and the complex volatile networks make the genetic characterization of these traits challenging for plant breeders. Bulk segregant analysis (BSA) coupled with next-generation sequencing technology has become a powerful tool for quick identification of quantitative trait locus (QTL). The goal of this study is to utilize this QTL-seq method and to discover novel fruit weight and volatile QTLs by mining the allele diversity from our germplasm collection. Several F2 populations were created by crossing plants among wild, semi-domesticated and domesticated tomatoes. Current F2 bulks pooled from the phenotypic extremes for fruit weight, locule number (indirectly affect fruit weight and size) and volatiles (isovaleronitrile, 3-pentanone and benzaldehyde) can reveal QTL candidate regions after sequencing. Progeny testing and fine mapping with future selfing generations will allow us to narrow the locus to a selection of gene candidates. Gene editing tools such as CRISPR/Cas system will help us validate the causal factor. Similar pipeline will be applied to more F2 populations with segregating fruit weight and volatile profiles. Overall, this study can provide us more insights into the genetics behind tomato fruit traits and facilitate tomato breeding.

Qian Feng

1. Institute Of Plant Breeding, Genetics, And Genomics, University of Georgia, Athens, United States; 2. Center for Applied Genetic Technology, University of Georgia, Athens, United States; 3. Horticultural Sciences, University of Florida, Gainesville, United States.

With a global production of ~50 Mt, eggplant (Solanum melongena L.) represents the third most important crop of the Solanaceae family after potato and tomato, and is an important component of our daily diet. A population of 164 F6 Recombinant Inbred Lines (RILs), derived from two eggplant lines differing with respect to several key agronomic traits, ‘305E40’ and ‘67/3’, was grown to the commercial maturation stage. The fruits were harvested, separated into peel and flesh, and subjected to untargeted LC-MS analysis. A number of metabolites belonging to the glycoalkaloid, anthocyanin and polyamine classes, showed a differential accumulation in the two parental lines and F1 hybrid. Following the metabolic profiling of the RILs and by using a recently developed high-resolution map constituted of 7,249 SNPs, we identified several metabolomic quantitative trait loci (mQTLs) associated to the accumulation of metabolites. Each of the metabolic traits proved to be controlled from one to three QTLs, and for most of the traits one major mQTL (PVE≥10%) was spotted. Data on mQTL mappig and sequence analysis as well as dominance-recessivity relationships of measured compounds in the parental lines and F1 hybrid suggest the possible molecular nature of the mutations underlying the mQTLs.

Maria Sulli <maria.sulli@enea.it>

1Italian National Agency for New Technologies, Energy, and Sustainable Economic Development, Casaccia Research Centre, 00123, Rome, Italy; 2DISAFA–Plant genetics and breeding—University of Turin, 10095 Grugliasco (TO), Italy; 3CREA, Research Centre for Genomics and Bioinformatics, 26836 Montanaso Lombardo (LO), Italy.

During tomato domestication, the fruit size increased from the blueberry-sized wild tomato (SP) to cherry-sized semi-domesticated tomato (SLC) which gave rise to the large modern cultivated tomato (SLL). To date, only five fruit weight genes have been identified and the alleles leading to higher fruit weight were identified in modern tomatoes. We focus on the germplasm of SP and SLC for novel fruit weight gene discoveries and potential crop improvement. Two SLC accessions (BGV006768 and BGV007931) carry wild-type alleles of all the known fruit weight genes; yet their fruit weight differs 10-fold. We aim to identify the genetic causes of such fruit weight difference using F2 population developed from these accessions. A major fruit weight QTL (fw2.3) at the bottom of chromosome 2 was identified by bulk-segregant analysis and fine-mapped to an interval of about 100 kb near the known fruit weight gene FW2.2. Phylogeny based on SNP information at fw2.3 interval among 167 diverse tomato accessions suggests fw2.3 is not present in modern tomatoes, because the fw2.3 alleles in both BGV006768 and BGV007931 are distinct from the cultivated allele of FW2.2. Differential expression of two candidate genes between F3 progenies carrying contrasting fw2.3 genotypes narrowed down the search for candidate genes further. In addition, the fruit weight data from multiple bi-parental F2 populations derived from SLC x SLC or SLC x SP crosses were utilized to narrow down the causal variants and hopefully will lead to the identification of the causal variant in the time scale of tomato domestication.

lei zhang

1. Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Athens, GA 30602

The chlorophyll fluorescence parameter Fv/Fm reflects the maximum quantum efficiency of photosystem II (PSII) and is widely used for early stress detection. We have evaluated three S. commersonii wild potato accessions (BGB003, BGB008, BGB027) from Embrapa Clima Temperado Potato Genebank to search for phenotypic expression of genetic variation for tolerance to high temperatures. Fv/Fm were measured after 16, 28 and 43 days. We investigated the performance (high and low Fv/Fm value) in terms of photosynthetic traits under control (C) and heat stress (HS) (14/27 OC and 24/34OC day/night temperature respectively in growth chambers). The highest Fv/Fm value was observed in BGB027 followed by BGB003 in stress conditions with high Fv/Fm value accompanied by higher stomatal conductance (gs). For BGB003, high Fv/Fm was accompanied by higher total chlorophyll (CC), net photosynthetic rate (PN) and transpiration rate (E). Stomatal conductance (gs) in stress environment and photochemical quantum yield of photosystem II (Yll) in control environment was positively correlated to Fv/Fm. Genotype and environment interaction was found nonsignificant but the genotypic effect was significant which shows variation is genotypic. BGB027 with high Fv/Fm maintained significantly higher total photochemical quantum yield of photosystem II (Yll), than BGB003 accompanied by higher Stomatal conductance (gs) net photosynthetic rate (Pn), and transpiration rate (E). The evaluated genotypes may serve as sources for further studies to understand the physiological mechanisms underlying the genetic variability in heat sensitivity of photosynthesis in S. commersonii.

Ikram Bashir <ikram.pbg@gmail.com>

Programa de Pós-graduação em Agronomia, Universidade Federal de Pelotas, RS; Brazil3Embrapa Clima Temperado, Pelotas, RS, Brazil

Tomato aroma is determined by the interaction of volatile compounds (VOCs) released by the fruit with nose receptors. These compounds are both derived from primary metabolites (fatty acids and branched-chain amino acids) and secondary metabolites (phenylalanine-derived compounds and carotenoids). Aim of our researches is to collect, compare, integrate and summarize the available literature, selecting 16 scientific papers and supplemental data focused on QTLs associated with tomato aroma. Here we report our finding regarding fatty acids-derived compounds.

VOCs originated from the degradation of linolenic and linoleic acid are the most abundant in tomato fruit and provide a note of freshly cut grass to the aroma bouquet. Data collection identified a total of 108 QTLs over 28 QTLs Genomic Regions (QGRs), defined by different authors. 83 QTLS were within a 5Mb window and were merged into 15 QGRs.

Our findings provide an overview of the known flavour QTLs associated with lipid VOCs biosynthesis, providing a tool that can make it possible to (i) pinpoint the best donors for specific traits, (ii) narrow down QTL regions by combining information from different populations, and (iii) pinpoint potential candidate genes.

Matteo Martina <matteo.martina@unito.it>

1. DISAFA - Plant Genetics and Breeding, University of Torino, Italy (IT); 2. Plant Breeding, Wageningen University & Research, The Netherlands (NL)

Tomato aroma is determined by the interaction of volatile compounds (VOCs) released by the fruit with nose receptors. These compounds are both derived from primary metabolites (fatty acids and branched- chain amino acids) and secondary metabolites (phenylalanine-derived compounds and carotenoids). Aim of our researches is to collect, compare, integrate and summarize the available literature, selecting 16 scientific papers and supplemental data focused on QTLs associated with tomato aroma.

Here we report our finding regarding amino acids-derived compounds Two classes of amino acids-derived compounds can be identified, according to their origin: i) BCAA VOCs; ii) phenylalanine-derived VOCs. BCAAs derived compounds are highly volatile compounds with a low molecular weight, some of which are considered important in the development of tomato aroma. Data collection identified a total of 129 QTLs over 26 QTLs Genomic Regions (QGRs), defined by different authors. 112 QTLS were within a 5Mb window and were merged into 16 QGRs. The impact of phenylalanine-derived VOCs on tomato flavor appears not so easy to predict, since some studies show positive while others show negative effects of these compounds on consumer perception. Data collection identified a total of 81 QTLs over 25 QTLs Genomic Regions (QGRs), defined by different authors. 57 QTLS were within a 5Mb window and are merged into 13 QGRs.

Our findings provide a tool that can make it possible to (i) select donors for specific traits, (ii) narrow down QTL regions by combining information from different populations, and (iii) pinpoint potential candidate genes.

Matteo Martina <matteo.martina@unito.it>

1. DISAFA - Plant Genetics and Breeding, University of Torino, Italy (IT); 2. Plant Breeding, Wageningen University & Research, The Netherlands (NL)

In a previous report, transcriptome of S. pimpinellifolium LA0722 was aligned as S. lycopersicum cv. Heinz 1706 as a reference. In this report, we have realigned LA0722 transcriptome using as a reference the S, pimpinellifolium LA4791 genome, recently available to the scientific community. The objective was to compare results of both transcriptome analyses, one made on a same species reference genome and the other, on a related species genome. In both cases, the green mature transcriptome was used to normalize the data. Differences in the number of over-expressed genes and under-expressed genes were detected when comparing both analyses, the smallest values corresponding to alignment of LA0722 transcriptome against the same species LA4701 genome (7024 and 6676 differentially expressed genes in LA0722 between breaker and green mature stages and between red ripe and green matures stages, respectively, vs. 7313 and 6929 in the same conditions when using cv. Heinz 1706 as the reference genome). Our results pointed out the importance of using an appropriate reference for alignment of new transcriptomes.

GuillermoPratta <gpratta@unr.edu.ar>

1 CIFASIS (CONICET/UNR), 2 IICAR/Facultad de Ciencias Agrarias (CONICET/UNR)

Plastid phylogeny along with nuclear phylogeny is proven useful in studying hybridization and introgression in potato. In addition, different potato plastome types have been demonstrated to affect agronomic traits. This study reveals that while a panel of thirteen assembled and annotated potato plastomes from different potato taxa are highly similar in gene order and content, a great number of SNPs and InDels exist between them. Five different potato plastomes types (C, S, A, W, W2) were identified in the thirteen plastomes. It is interesting to observe that the two accessions of S. tuberosum ssp. andigena, ADG1 and ADG2, have different plastome types as do two accessions of S. bukasovii (BUK1 and BUK2). A conserved 48 bp deletion is observed only in the S-type plastome, which can be used as a marker for identifying S-type plastomes. The phylogenetic analysis shows that they cluster according to their plastome types. Comparing plastome phylogeny with the nuclear phylogeny of the panel reveals some congruency, with considerable differences, suggesting introgression and hybridization between potato species. The S. bukasovii (BUK1, but not BUK2), a wild potato species, and S. curtilobum (CUR) grouped with the diploid landraces. This was not observed in the nuclear phylogeny, and marks the importance of plastome analyses for a more complete understanding of potato genetic resources.

Sai Reddy Achakkagari

1Department of Plant Science, McGill University, Montreal, Canada; 2Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, Fredericton, Canada; 3International Potato Center, Lima, Peru