Solanum pennellii (LA5240) backcross inbred lines (BILs) for high resolution mapping in tomato.

Wild species are an invaluable source of new traits for crop improvement. Over the years, the tomato community bred cultivated lines that carry introgressions from different species of the tomato tribe to facilitate trait discovery and mapping. The next phase in such projects is to find the genes that drive the identified phenotypes. This can be achieved by genotyping a few thousand individuals resulting in fine mapping that can potentially identify the causative gene. To couple trait discovery and fine mapping, we are presenting large, recombination-rich, Backcross Inbred Line (BIL) populations involving an unexplored accession of the wild, green-fruited species Solanum pennellii (LA5240; the 'Lost' Accession) with two modern tomato inbreds: LEA, determinate, and TOP, indeterminate. The LEA and TOP BILs are in BC2F6-8 generation and include 1400 and 500 lines, respectively. The BILs were genotyped with 5000 SPET markers, showing that in the euchromatic regions there was one recombinant every 17-18 Kb while in the heterochromatin a recombinant every 600-700 Kb (TOP and LEA respectively). To gain perspective on the topography of recombination we compared five independent members of the Self-pruning gene family with their respective neighboring genes; based on PCR markers, in all cases we found recombinants. Further mapping analysis of two known morphological mutations that segregated in the BILs (self-pruning and hairless) showed that the maximal delimited intervals were 73 Kb and 210 Kb, respectively, and included the known causative genes. The 'Lost'_BILs provide a solid framework to study traits derived from a drought-tolerant wild tomato.

Diversity and genetic architecture of agro-morphological traits in a core collection of European traditional tomato.

European traditional tomato varieties have been selected by farmers given their consistent performance and adaptation to local growing conditions. Here we developed a multipurpose core collection, comprising 226 accessions representative of the genotypic, phenotypic, and geographical diversity present in European traditional tomatoes, to investigate the basis of their phenotypic variation, gene×environment interactions, and stability for 33 agro-morphological traits. Comparison of the traditional varieties with a modern reference panel revealed that some traditional varieties displayed excellent agronomic performance and high trait stability, as good as or better than that of their modern counterparts. We conducted genome-wide association and genome-wide environment interaction studies and detected 141 quantitative trait loci (QTLs). Out of those, 47 QTLs were associated with the phenotype mean (meanQTLs), 41 with stability (stbQTLs), and 53 QTL-by-environment interactions (QTIs). Most QTLs displayed additive gene actions, with the exception of stbQTLs, which were mostly recessive and overdominant QTLs. Both common and specific loci controlled the phenotype mean and stability variation in traditional tomato; however, a larger proportion of specific QTLs was observed, indicating that the stability gene regulatory model is the predominant one. Developmental genes tended to map close to meanQTLs, while genes involved in stress response, hormone metabolism, and signalling were found within regions affecting stability. A total of 137 marker-trait associations for phenotypic means and stability were novel, and therefore our study enhances the understanding of the genetic basis of valuable agronomic traits and opens up a new avenue for an exploitation of the allelic diversity available within European traditional tomato germplasm.

Multi-year field trials provide a massive repository of trait data on a highly diverse population of tomato and uncover novel determinants of tomato productivity.

Tomato (Solanum lycopersicum) is a prominent fruit with rich genetic resources for crop improvement. By using a phenotype-guided screen of over 7900 tomato accessions from around the world, we identified new associations for complex traits such as fruit weight and total soluble solids (Brix). Here, we present the phenotypic data from several years of trials. To illustrate the power of this dataset we use two case studies. First, evaluation of color revealed allelic variation in phytoene synthase 1 that resulted in differently colored or even bicolored fruit. Secondly, in view of the negative relationship between fruit weight and Brix, we pre-selected a subset of the collection that includes high and low Brix values in each category of fruit size. Genome-wide association analysis allowed us to detect novel loci associated with total soluble solid content and fruit weight. In addition, we developed eight F2 biparental intraspecific populations. Furthermore, by taking a phenotype-guided approach we were able to isolate individuals with high Brix values that were not compromised in terms of yield. In addition, the demonstration of novel results despite the high number of previous genome-wide association studies of these traits in tomato suggests that adoption of a phenotype-guided pre-selection of germplasm may represent a useful strategy for finding target genes for breeding.

Epistatic QTLs for yield heterosis in tomato.

Controlled population development and genome-wide association studies have proven powerful in uncovering genes and alleles underlying complex traits. An underexplored dimension of such studies is the phenotypic contribution of nonadditive interactions between quantitative trait loci (QTLs). Capturing of such epistasis in a genome-wide manner requires very large populations to represent replicated combinations of loci whose interactions determine phenotypic outcomes. Here, we dissect epistasis using a densely genotyped population of 1,400 backcross inbred lines (BILs) between a modern processing tomato inbred (Solanum lycopersicum) and the Lost Accession (LA5240) of a distant, green-fruited, drought-tolerant wild species, Solanum pennellii. The homozygous BILs, each harboring an average of 11 introgressions and their hybrids with the recurrent parents, were phenotyped for tomato yield components. Population-wide mean yield of the BILs was less than 50% of that of their hybrids (BILHs). All the homozygous introgressions across the genome reduced yield relative to recurrent parent, while several QTLs of the BILHs independently improved productivity. Analysis of two QTL scans showed 61 cases of less-than-additive interactions and 19 cases of more-than-additive interactions. Strikingly, a single epistatic interaction involving S. pennellii QTLs on chromosomes 1 and 7, that independently did not affect yield, increased fruit yield by 20 to 50% in the double introgression hybrid grown in irrigated and dry fields over a period of 4 y. Our work demonstrates the power of large, interspecific controlled population development to uncover hidden QTL phenotypes and how rare epistatic interactions can improve crop productivity via heterosis.

Atlas of phenotypic, genotypic and geographical diversity present in the European traditional tomato.

The Mediterranean basin countries are considered secondary centres of tomato diversification. However, information on phenotypic and allelic variation of local tomato materials is still limited. Here we report on the evaluation of the largest traditional tomato collection, which includes 1499 accessions from Southern Europe. Analyses of 70 traits revealed a broad range of phenotypic variability with different distributions among countries, with the culinary end use within each country being the main driver of tomato diversification. Furthermore, eight main tomato types (phenoclusters) were defined by integrating phenotypic data, country of origin, and end use. Genome-wide association study (GWAS) meta-analyses identified associations in 211 loci, 159 of which were novel. The multidimensional integration of phenoclusters and the GWAS meta-analysis identified the molecular signatures for each traditional tomato type and indicated that signatures originated from differential combinations of loci, which in some cases converged in the same tomato phenotype. Our results provide a roadmap for studying and exploiting this untapped tomato diversity.

European traditional tomatoes galore: a result of farmers' selection of a few diversity-rich loci.

A comprehensive collection of 1254 tomato accessions, corresponding to European traditional and modern varieties, early domesticated varieties, and wild relatives, was analyzed by genotyping by sequencing. A continuous genetic gradient between the traditional and modern varieties was observed. European traditional tomatoes displayed very low genetic diversity, with only 298 polymorphic loci (95% threshold) out of 64 943 total variants. European traditional tomatoes could be classified into several genetic groups. Two main clusters consisting of Spanish and Italian accessions showed higher genetic diversity than the remaining varieties, suggesting that these regions might be independent secondary centers of diversity with a different history. Other varieties seem to be the result of a more recent complex pattern of migrations and hybridizations among the European regions. Several polymorphic loci were associated in a genome-wide association study with fruit morphological traits in the European traditional collection. The corresponding alleles were found to contribute to the distinctive phenotypic characteristic of the genetic varietal groups. The few highly polymorphic loci associated with morphological traits in an otherwise a low-diversity population suggests a history of balancing selection, in which tomato farmers likely maintained the morphological variation by inadvertently applying a high selective pressure within different varietal types.

The potential of dynamic physiological traits in young tomato plants to predict field-yield performance.

To address the challenge of predicting tomato yields in the field, we used whole-plant functional phenotyping to evaluate water relations under well-irrigated and drought conditions. The genotypes tested are known to exhibit variability in their yields in wet and dry fields. The examined lines included two lines with recessive mutations that affect carotenoid biosynthesis, zeta z2083 and tangerine t3406, both isogenic to the processing tomato variety M82. The two mutant lines were reciprocally grafted onto M82, and multiple physiological characteristics were measured continuously, before, during and after drought treatment in the greenhouse. A comparative analysis of greenhouse and field yields showed that the whole-canopy stomatal conductance (gsc) in the morning and cumulative transpiration (CT) were strongly correlated with field measurements of total yield (TY: r2 = 0.9 and 0.77, respectively) and plant vegetative weight (PW: r2 = 0.6 and 0.94, respectively). Furthermore, the minimum CT during drought and the rate of recovery when irrigation was resumed were both found to predict resilience.

The GORKY glycoalkaloid transporter is indispensable for preventing tomato bitterness.

Fruit taste is determined by sugars, acids and in some species, bitter chemicals. Attraction of seed-dispersing organisms in nature and breeding for consumer preferences requires reduced fruit bitterness. A key metabolic shift during ripening prevents tomato fruit bitterness by eliminating α-tomatine, a renowned defence-associated Solanum alkaloid. Here, we combined fine mapping with information from 150 resequenced genomes and genotyping a 650-tomato core collection to identify nine bitter-tasting accessions including the 'high tomatine' Peruvian landraces reported in the literature. These 'bitter' accessions contain a deletion in GORKY, a nitrate/peptide family transporter mediating α-tomatine subcellular localization during fruit ripening. GORKY exports α-tomatine and its derivatives from the vacuole to the cytosol and this facilitates the conversion of the entire α-tomatine pool to non-bitter forms, rendering the fruit palatable. Hence, GORKY activity was a notable innovation in the process of tomato fruit domestication and breeding.

Genes for Yield Stability in Tomatoes.

Breeding plant varieties with adaptation to unstable environments requires some knowledge about the genetic control of yield stability. To further this goal, a meta-analysis of 12 years of field harvest data of 76 Solanum pennellii introgression lines (ILs) is conducted. Five quantitative trait loci (QTL) affecting yield stability are mapped; IL10-2-2 is unique as this introgression improved yield stability without affecting mean yield both in the historic data and in four years of field validations. Another dimension of the stability question is which genes when perturbed affect yield stability. For this the authors tested in the field 48 morphological mutants and found one 'canalization' mutant (canal-1) with a consistent effect of reducing the stability of a bouquet of traits including leaf variegation, plant size and yield. canal-1 mapped to a DNAJ chaperone gene (Solyc01g108200) whose homologues in C. elegans regulate phenotypic canalization. Additional alleles of canal-1 are generated using CRISPR/CAS9 and the resulting seedlings have uniform variegation suggesting that only specific changes in canal-1 can lead to unstable variegation and yield instability. The identification of IL10-2-2 demonstrates the value of historical phenotypic data for discovering genes for stability. It is also shown that a green-fruited wild species is a source of QTL to improve tomato yield stability.

Transpiration from Tomato Fruit Occurs Primarily via Trichome-Associated Transcuticular Polar Pores.

Nonstomatal water loss by transpiration through the hydrophobic cuticle is ubiquitous in land plants, but the pathways along which this occurs have not been identified. Tomato (Solanum lycopersicum) provides an excellent system in which to study this phenomenon, as its fruit are astomatous and a major target for desiccation resistance to enhance shelf life. We screened a tomato core collection of 398 accessions from around the world and selected seven cultivars that collectively exhibited the lowest and highest degrees of transpirational water loss for a more detailed study. The transpirational differences between these lines reflected the permeances of their isolated cuticles, but this did not correlate with various measures of cuticle abundance or composition. Rather, we found that fruit cuticle permeance has a strong dependence on the abundance of microscopic polar pores. We further observed that these transcuticular pores are associated with trichomes and are exposed when the trichomes are dislodged, revealing a previously unreported link between fruit trichome density and transpirational water loss. During postharvest storage, limited self-sealing of the pores was detected for certain cultivars, in contrast with the stem scar, which healed relatively rapidly. The abundance of trichome-associated pores, together with their self-sealing capacity, presents a promising target for breeding or engineering efforts to reduce fruit transpirational water loss.

Analysis of wild tomato introgression lines elucidates the genetic basis of transcriptome and metabolome variation underlying fruit traits and pathogen response.

Wild tomato species represent a rich gene pool for numerous desirable traits lost during domestication. Here, we exploited an introgression population representing wild desert-adapted species and a domesticated cultivar to establish the genetic basis of gene expression and chemical variation accompanying the transfer of wild-species-associated fruit traits. Transcriptome and metabolome analysis of 580 lines coupled to pathogen sensitivity assays resulted in the identification of genomic loci associated with levels of hundreds of transcripts and metabolites. These associations occurred in hotspots representing coordinated perturbation of metabolic pathways and ripening-related processes. Here, we identify components of the Solanum alkaloid pathway, as well as genes and metabolites involved in pathogen defense and linking fungal resistance with changes in the fruit ripening regulatory network. Our results outline a framework for understanding metabolism and pathogen resistance during tomato fruit ripening and provide insights into key fruit quality traits.

Quantitative trait loci analysis of seed-specialized metabolites reveals seed-specific flavonols and differential regulation of glycoalkaloid content in tomato.

Given the potential health benefits (and adverse effects), of polyphenolic and steroidal glycoalkaloids in the diet there is a growing interest in fully elucidating the genetic control of their levels in foodstuffs. Here we carried out profiling of the specialized metabolites in the seeds of the Solanum pennellii introgression lines identifying 338 putative metabolite quantitative trait loci (mQTL) for flavonoids, steroidal glycoalkaloids and further specialized metabolites. Two putative mQTL for flavonols and one for steroidal glycoalkaloids were cross-validated by evaluation of the metabolite content of recombinants harboring smaller introgression in the corresponding QTL interval or by analysis of lines from an independently derived backcross inbred line population. The steroidal glycoalkaloid mQTL was localized to a chromosomal region spanning 14 genes, including a previously defined steroidal glycoalkaloid gene cluster. The flavonoid mQTL was further validated via the use of transient and stable overexpression of the Solyc12g098600 and Solyc12g096870 genes, which encode seed-specific uridine 5'-diphosphate-glycosyltransferases. The results are discussed in the context of our understanding of the accumulation of polyphenols and steroidal glycoalkaloids, and how this knowledge may be incorporated into breeding strategies aimed at improving nutritional aspects of plants as well as in fortifying them against abiotic stress.

Development of zeaxanthin-rich tomato fruit through genetic manipulations of carotenoid biosynthesis.

The oxygenated carotenoid zeaxanthin provides numerous benefits to human health due to its antioxidant properties. Especially it is linked to protecting, together with the xanthophyll lutein, the retina in the human eye by filtering harmful blue light thus delaying the progression of age-related macular degeneration (AMD), the most prevalent cause of blindness in developed countries. Despite its high nutritional value, zeaxanthin is less available than other substantial carotenoids in our diet. To solve this shortage, we chose to develop a new food source that would contain a high concentration of natural zeaxanthin. Tomato (Solanum lycopersicum L.) was selected as the target plant since it is the second largest vegetable crop grown worldwide and its fruit characteristically synthesizes and accumulates a high concentration of carotenoids. We employed two genetic approaches in order to enhance zeaxanthin biosynthesis in tomato fruit: a transgenic metabolic engineering and classical genetic breeding. A nontransgenic tomato line, named 'Xantomato', was generated whose fruit accumulated zeaxanthin at a concentration of 39 µg/g fresh weight (or 577 µg/g dry weight), which comprised ca. 50% of total fruit carotenoids compared to zero in the wild type. This is the highest concentration of zeaxanthin reached in a primary crop. Xantomato can potentially increase zeaxanthin availability in the human diet and serve as raw material for industrial applications.

Pathways to defense metabolites and evading fruit bitterness in genus Solanum evolved through 2-oxoglutarate-dependent dioxygenases.

The genus Solanum comprises three food crops (potato, tomato, and eggplant), which are consumed on daily basis worldwide and also producers of notorious anti-nutritional steroidal glycoalkaloids (SGAs). Hydroxylated SGAs (i.e. leptinines) serve as precursors for leptines that act as defenses against Colorado Potato Beetle (Leptinotarsa decemlineata Say), an important pest of potato worldwide. However, SGA hydroxylating enzymes remain unknown. Here, we discover that 2-OXOGLUTARATE-DEPENDENT-DIOXYGENASE (2-ODD) enzymes catalyze SGA-hydroxylation across various Solanum species. In contrast to cultivated potato, Solanum chacoense, a widespread wild potato species, has evolved a 2-ODD enzyme leading to the formation of leptinines. Furthermore, we find a related 2-ODD in tomato that catalyzes the hydroxylation of the bitter α-tomatine to hydroxytomatine, the first committed step in the chemical shift towards downstream ripening-associated non-bitter SGAs (e.g. esculeoside A). This 2-ODD enzyme prevents bitterness in ripe tomato fruit consumed today which otherwise would remain unpleasant in taste and more toxic.

A Solanum neorickii introgression population providing a powerful complement to the extensively characterized Solanum pennellii population.

We present a complementary resource for trait fine-mapping in tomato to those based on the intra-specific cross between cultivated tomato and the wild tomato species Solanum pennellii, which have been extensively used for quantitative genetics in tomato over the last 20 years. The current population of backcross inbred lines (BILs) is composed of 107 lines derived after three backcrosses of progeny of the wild species Solanum neorickii (LA2133) and cultivated tomato (cultivar TA209) and is freely available to the scientific community. These S. neorickii BILs were genotyped using the 10K SolCAP single nucleotide polymorphism chip, and 3111 polymorphic markers were used to map recombination break points relative to the physical map of Solanum lycopersicum. The BILs harbor on average 4.3 introgressions per line, with a mean introgression length of 34.7 Mbp, allowing partitioning of the genome into 340 bins and thereby facilitating rapid trait mapping. We demonstrate the power of using this resource in comparison with archival data from the S. pennellii resources by carrying out metabolic quantitative trait locus analysis following gas chromatography-mass spectrometry on fruits harvested from the S. neorickii BILs. The metabolic candidate genes phenylalanine ammonia-lyase and cystathionine gamma-lyase were then tested and validated in F2 populations and via agroinfiltration-based overexpression in order to exemplify the fidelity of this method in identifying the genes that drive tomato metabolic phenotypes.

Quantitative Trait Loci Analysis Identifies a Prominent Gene Involved in the Production of Fatty Acid-Derived Flavor Volatiles in Tomato.

To gain insight into the genetic regulation of lipid metabolism in tomato, we conducted metabolic trait loci (mQTL) analysis following the lipidomic profiling of fruit pericarp and leaf tissue of the Solanum pennellii introgression lines (IL). To enhance mapping resolution for selected fruit-specific mQTL, we profiled the lipids in a subset of independently derived S. pennellii backcross inbred lines, as well as in a near-isogenic sub-IL population. We identified a putative lecithin:cholesterol acyltransferase that controls the levels of several lipids, and two members of the class III lipase family, LIP1 and LIP2, that were associated with decreased levels of diacylglycerols (DAGs) and triacylglycerols (TAGs). Lipases of this class cleave fatty acids from the glycerol backbone of acylglycerols. The released fatty acids serve as precursors of flavor volatiles. We show that LIP1 expression correlates with fatty acid-derived volatile levels. We further confirm the function of LIP1 in TAG and DAG breakdown and volatile synthesis using transgenic plants. Taken together, our study extensively characterized the genetic architecture of lipophilic compounds in tomato and demonstrated at molecular level that release of free fatty acids from the glycerol backbone can have a major impact on downstream volatile synthesis.

Canalization of Tomato Fruit Metabolism.

To explore the genetic robustness (canalization) of metabolism, we examined the levels of fruit metabolites in multiple harvests of a tomato introgression line (IL) population. The IL partitions the whole genome of the wild species Solanum pennellii in the background of the cultivated tomato (Solanum lycopersicum). We identified several metabolite quantitative trait loci that reduce variability for both primary and secondary metabolites, which we named canalization metabolite quantitative trait loci (cmQTL). We validated nine cmQTL using an independent population of backcross inbred lines, derived from the same parents, which allows increased resolution in mapping the QTL previously identified in the ILs. These cmQTL showed little overlap with QTL for the metabolite levels themselves. Moreover, the intervals they mapped to harbored few metabolism-associated genes, suggesting that the canalization of metabolism is largely controlled by regulatory genes.

De Novo Assembly of a New Solanum pennellii Accession Using Nanopore Sequencing.

Updates in nanopore technology have made it possible to obtain gigabases of sequence data. Prior to this, nanopore sequencing technology was mainly used to analyze microbial samples. Here, we describe the generation of a comprehensive nanopore sequencing data set with a median read length of 11,979 bp for a self-compatible accession of the wild tomato species Solanum pennellii We describe the assembly of its genome to a contig N50 of 2.5 MB. The assembly pipeline comprised initial read correction with Canu and assembly with SMARTdenovo. The resulting raw nanopore-based de novo genome is structurally highly similar to that of the reference S. pennellii LA716 accession but has a high error rate and was rich in homopolymer deletions. After polishing the assembly with Illumina reads, we obtained an error rate of <0.02% when assessed versus the same Illumina data. We obtained a gene completeness of 96.53%, slightly surpassing that of the reference S. pennellii Taken together, our data indicate that such long read sequencing data can be used to affordably sequence and assemble gigabase-sized plant genomes.

Bimodality of stable and plastic traits in plants.

KEY MESSAGE: We discovered an unexpected mode of bimodal distribution of stable and plastic traits, which was consistent for homologous traits of 32 varieties of seven species both in well-irrigated fields and dry conditions. We challenged archived genetic mapping data for 36 fruit, seed, flower and yield traits in tomato and found an unexpected bimodal distribution in one measure of trait variability, the mean coefficient of variation, with some traits being consistently more variable than others. To determine the degree of conservation of this distribution among higher plants, we compared 18 homologous phenotypes, including yield and seed production, across different crop species grown in a common 'crop garden' experiment. The set included 32 varieties of tomato, eggplant, pepper, melon, watermelon, sunflower and maize. Estimates of canalization were obtained using a 'canalization replication' experimental design that generated multiple estimates of the coefficient of variation of traits, as well as their reaction norms in optimal and water-stressed field plots. A common pattern of bimodal distribution of stable and plastic traits was observed for all the varieties and for a wild weed (Solanum nigrum). We propose that canalization profiles of traits in a variety of taxa were ancestrally selected to maximize adaptation and reproductive success.

Bypassing Negative Epistasis on Yield in Tomato Imposed by a Domestication Gene.

Selection for inflorescence architecture with improved flower production and yield is common to many domesticated crops. However, tomato inflorescences resemble wild ancestors, and breeders avoided excessive branching because of low fertility. We found branched variants carry mutations in two related transcription factors that were selected independently. One founder mutation enlarged the leaf-like organs on fruits and was selected as fruit size increased during domestication. The other mutation eliminated the flower abscission zone, providing "jointless" fruit stems that reduced fruit dropping and facilitated mechanical harvesting. Stacking both beneficial traits caused undesirable branching and sterility due to epistasis, which breeders overcame with suppressors. However, this suppression restricted the opportunity for productivity gains from weak branching. Exploiting natural and engineered alleles for multiple family members, we achieved a continuum of inflorescence complexity that allowed breeding of higher-yielding hybrids. Characterizing and neutralizing similar cases of negative epistasis could improve productivity in many agricultural organisms. VIDEO ABSTRACT.