Wild Tomato Species Research Articles

Analysis of wild-species introgressions in tomato inbreds uncovers ancestral origins.

BackgroundDecades of intensive tomato breeding using wild-species germplasm have resulted in the genomes of domesticated germplasm (Solanum lycopersicum) being intertwined with introgressions from their wild relatives. Comparative analysis of genomes among cultivated tomatoes and wild species that have contributed genetic variation can help identify desirable genes, such as those conferring disease resistance. The ability to identify introgression position, borders, and contents can reveal ancestral origins and facilitate harnessing of wild variation in crop breeding.ResultsHere we present the whole-genome sequences of two tomato inbreds, Gh13 and BTI-87, both carrying the begomovirus resistance locus Ty-3 introgressed from wild tomato species. Introgressions of different sizes on chromosome 6 of Gh13 and BTI-87, both corresponding to the Ty-3 region, were identified as from a source close to the wild species S. chilense. Other introgressions were identified throughout the genomes of the inbreds and showed major differences in the breeding pedigrees of the two lines. Interestingly, additional large introgressions from the close tomato relative S. pimpinellifolium were identified in both lines. Some of the polymorphic regions were attributed to introgressions in the reference Heinz 1706 genome, indicating wild genome sequences in the reference tomato genome.ConclusionsThe methods developed in this work can be used to delineate genome introgressions, and subsequently contribute to development of molecular markers to aid phenotypic selection, fine mapping and discovery of candidate genes for important phenotypes, and for identification of novel variation for tomato improvement. These universal methods can easily be applied to other crop plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0287-2) contains supplementary material, which is available to authorized users.

Quantitative genetic analysis indicates natural selection on leaf phenotypes across wild tomato species (Solanum sect. Lycopersicon; Solanaceae).

Adaptive evolution requires both raw genetic material and an accessible path of high fitness from one fitness peak to another. In this study, we used an introgression line (IL) population to map quantitative trait loci (QTL) for leaf traits thought to be associated with adaptation to precipitation in wild tomatoes (Solanum sect. Lycopersicon; Solanaceae). A QTL sign test showed that several traits likely evolved under directional natural selection. Leaf traits correlated across species do not share a common genetic basis, consistent with a scenario in which selection maintains trait covariation unconstrained by pleiotropy or linkage disequilibrium. Two large effect QTL for stomatal distribution colocalized with key genes in the stomatal development pathway, suggesting promising candidates for the molecular bases of adaptation in these species. Furthermore, macroevolutionary transitions between vastly different stomatal distributions may not be constrained when such large-effect mutations are available. Finally, genetic correlations between stomatal traits measured in this study and data on carbon isotope discrimination from the same ILs support a functional hypothesis that the distribution of stomata affects the resistance to CO2 diffusion inside the leaf, a trait implicated in climatic adaptation in wild tomatoes. Along with evidence from previous comparative and experimental studies, this analysis indicates that leaf traits are an important component of climatic niche adaptation in wild tomatoes and demonstrates that some trait transitions between species could have involved few, large-effect genetic changes, allowing rapid responses to new environmental conditions.

The genome of the stress-tolerant wild tomato species Solanum pennellii.

Solanum pennellii is a wild tomato species endemic to Andean regions in South America, where it has evolved to thrive in arid habitats. Because of its extreme stress tolerance and unusual morphology, it is an important donor of germplasm for the cultivated tomato Solanum lycopersicum. Introgression lines (ILs) in which large genomic regions of S. lycopersicum are replaced with the corresponding segments from S. pennellii can show remarkably superior agronomic performance. Here we describe a high-quality genome assembly of the parents of the IL population. By anchoring the S. pennellii genome to the genetic map, we define candidate genes for stress tolerance and provide evidence that transposable elements had a role in the evolution of these traits. Our work paves a path toward further tomato improvement and for deciphering the mechanisms underlying the myriad other agronomic traits that can be improved with S. pennellii germplasm.

Model-assisted analysis of sugar metabolism throughout tomato fruit development reveals enzyme and carrier properties in relation to vacuole expansion.

A kinetic model combining enzyme activity measurements and subcellular compartmentation was parameterized to fit the sucrose, hexose, and glucose-6-P contents of pericarp throughout tomato (Solanum lycopersicum) fruit development. The model was further validated using independent data obtained from domesticated and wild tomato species and on transgenic lines. A hierarchical clustering analysis of the calculated fluxes and enzyme capacities together revealed stage-dependent features. Cell division was characterized by a high sucrolytic activity of the vacuole, whereas sucrose cleavage during expansion was sustained by both sucrose synthase and neutral invertase, associated with minimal futile cycling. Most importantly, a tight correlation between flux rate and enzyme capacity was found for fructokinase and PPi-dependent phosphofructokinase during cell division and for sucrose synthase, UDP-glucopyrophosphorylase, and phosphoglucomutase during expansion, thus suggesting an adaptation of enzyme abundance to metabolic needs. In contrast, for most enzymes, flux rates varied irrespectively of enzyme capacities, and most enzymes functioned at <5% of their maximal catalytic capacity. One of the major findings with the model was the high accumulation of soluble sugars within the vacuole together with organic acids, thus enabling the osmotic-driven vacuole expansion that was found during cell division.

Using bioinformatic tools to identify new Methylketone Synthase 2 (MKS2) genes in the currant tomato Solanum pimpinellifolium

2-Methylketone is the organic compound containing a ketone functionality on the second carbon. This group of compounds has been known for their widespread applications in plant protection, fragrance and flavor industry, and biofuel production. Previously, two cDNAs encoding two proteins necessary for methylketone biosynthesis in the wild tomato species Solanum habrochaites subsp. glabratum were identified and designated methylketone synthase 1 (ShMKS1) and methylketone synthase 2 (ShMKS2). In searching of new MKS2 variants that are capable of using substrates of different chain lengths or degree of unsaturation, we used ShMKS2 as a query sequence for TBLASTN searches against the currant tomato (Solanum pimpinellifolium) genome. With the support of other bioinformatics tools for gene prediction, we identified three homologous genes of ShMKS2 on four contigs (contig 3697822, 6568413, 6704221, and 6708991) found in the genomic database of Solanum pimpinellifolium. Three genes encoded proteins with more than 65% identity to previously reported ShMKS2 and more than 98% identity to SlMKS2s; we named these genes as SppMKS2-1, SppMKS2-2, and SppMKS2-3. All three of these Solanum pimpinellifolium MKS2 genes had five exons and four introns (whose positions are conserved in comparison with the exon and intron positions in the homologous S. lycopersicum genes). The protein sequences of the MKS2 homologs from three Solanum species were compared and a phylogenetic tree was constructed.

A single locus confers tolerance to continuous light and allows substantial yield increase in tomato.

An important constraint for plant biomass production is the natural day length. Artificial light allows for longer photoperiods, but tomato plants develop a detrimental leaf injury when grown under continuous light--a still poorly understood phenomenon discovered in the 1920s. Here, we report a dominant locus on chromosome 7 of wild tomato species that confers continuous light tolerance. Genetic evidence, RNAseq data, silencing experiments and sequence analysis all point to the type III light harvesting chlorophyll a/b binding protein 13 (CAB-13) gene as a major factor responsible for the tolerance. In Arabidopsis thaliana, this protein is thought to have a regulatory role balancing light harvesting by photosystems I and II. Introgressing the tolerance into modern tomato hybrid lines, results in up to 20% yield increase, showing that limitations for crop productivity, caused by the adaptation of plants to the terrestrial 24-h day/night cycle, can be overcome.

Analysis of natural and induced variation in tomato glandular trichome flavonoids identifies a gene not present in the reference genome.

Flavonoids are ubiquitous plant aromatic specialized metabolites found in a variety of cell types and organs. Methylated flavonoids are detected in secreting glandular trichomes of various Solanum species, including the cultivated tomato (Solanum lycopersicum). Inspection of the sequenced S. lycopersicum Heinz 1706 reference genome revealed a close homolog of Solanum habrochaites MOMT1 3'/5' myricetin O-methyltransferase gene, but this gene (Solyc06g083450) is missing the first exon, raising the question of whether cultivated tomato has a distinct 3' or 3'/5' O-methyltransferase. A combination of mining genome and cDNA sequences from wild tomato species and S. lycopersicum cultivar M82 led to the identification of Sl-MOMT4 as a 3' O-methyltransferase. In parallel, three independent ethyl methanesulfonate mutants in the S. lycopersicum cultivar M82 background were identified as having reduced amounts of di- and trimethylated myricetins and increased monomethylated myricetin. Consistent with the hypothesis that Sl-MOMT4 is a 3' O-methyltransferase gene, all three myricetin methylation defective mutants were found to have defects in MOMT4 sequence, transcript accumulation, or 3'-O-methyltransferase enzyme activity. Surprisingly, no MOMT4 sequence is found in the Heinz 1706 reference genome sequence, and this cultivar accumulates 3-methyl myricetin and is deficient in 3'-methyl myricetins, demonstrating variation in this gene among cultivated tomato varieties.

Exploration of Three Solanum Species for Improvement of Antioxidant Traits in Tomato

Wild tomato species have been widely used for improvement of tomato disease resistance but have not been extensively explored for health-related traits. In this work, three interspecific populations derived from backcrosses between cultivated tomato and Solanum pimpinellifolium (LA1589), S. habrochaites (LA1223), and S. peruvianum (LA2172) were analyzed for water-soluble antioxidant activity, phenolic content, vitamin C content, and basic agronomic traits including fruit weight, shape, and color. The wild species accessions significantly exceeded S. lycopersicum for all three antioxidant traits with only one exception: vitamin C content in S. habrochaites LA1223. Several populations and traits showed transgressive segregation indicating that the backcross populations contained individuals with allele combinations that allowed antioxidant activity/content to exceed that of both parents. The S. habrochaites LA1223 population provided the best starting material for improvement of water-soluble antioxidant activity and phenolics content with 20% and 15% of the population, respectively, significantly exceeding the parental values for these traits. Moreover, the S. habrochaites population contained individuals that had nearly 2-fold more water-soluble antioxidant activity and phenolic content than cultivated tomato. The S. peruvianum LA2172 population was best for improvement of vitamin C content with 3-fold variation for the trait and individuals, which had twice as much vitamin C as cultivated tomato.

Characterization and mapping of a QTL derived from Solanum habrochaites associated with elevated rutin content (quercetin-3-rutinoside) in tomato

Consumption of flavonoids found in fruits and vegetables is linked to beneficial health outcomes. Tomato is among the most widely consumed and economically important vegetables worldwide and improvement of the nutrient content could lead to significant health benefits. Rutin, (quercetin-3-rutinoside), the main flavonol in tomato fruit, is confined to the peel. Rutin synthesis is limited by low expression of chalcone isomerase, the enzyme catalyzing the conversion of naringenin chalcone to naringenin quercetin. The wild tomato species Solanum habrochaites is a major source of new alleles to improve cultivated tomato (Solanum lycopersicum). AVRDC—The World Vegetable Center identified introgression line (IL) LA3984 containing a segment of S. habrochaites on chromosome 5 expressing high levels of rutin in full red ripe fruit. An AVRDC high rutin tomato line evaluated for 2 years and two seasons in Taiwan produced mean rutin content about four- to five-fold greater than the mean of all entries and about 11–12 times higher than the commercial fresh market and processing tomato cultivars. The quantitative trait locus (QTL) conditioning high rutin content was mapped to a 0.42 Mb segment on chromosome 5 flanked by markers c2_At3g55120/TaqI and ch05-4.883/ApaLI. Marker c2_Atg55120 overlaps with the chalcone-flavonone isomerase gene Solyc05g10320, and a second chalcone-flavonone isomerase gene is located 3,000 bp upstream from c2_At3g551220. Results of this project will facilitate breeding of high flavonoid tomato lines.

Response of accessions within tomato wild species, Solanum pimpinellifolium to late blight

AbstractLate blight (LB), caused by the oomycete Phytophthora infestans, is one of the most devastating diseases of the cultivated tomato (Solanum lycopersicum) worldwide. Most commercial cultivars of tomato are susceptible to LB. Previously, three major LB resistance genes (Ph‐1, Ph‐2, Ph‐3) were identified and incorporated into a few commercial cultivars of tomato. Reduced effectiveness and potential breakdown of the resistance genes has necessitated identification, characterization and utilization of new sources of resistance. We evaluated the response of 67 accessions of the wild tomato species, S. pimpinellifolium to LB, under multiple field and greenhouse (GH) conditions and compared them with six control genotypes. Sixteen accessions were identified with strong LB resistance in both field and GH experiments. However, 12 accessions exhibited resistance similar to a control line which was homozygous for Ph‐2 + Ph‐3. Genotyping accessions with molecular markers for Ph‐2 and Ph‐3 were not conclusive, indicating that resistance in these accessions could be due to these or other resistance genes. Strong correlations were observed between field and GH disease response and between foliar and stem infection.

Genetic structure of the four wild tomato species in the Solanum peruvianum s.l. species complex.

The most diverse wild tomato species Solanum peruvianum sensu lato (s.l.) has been reclassified into four separate species: Solanum peruvianum sensu stricto (s.s.), Solanum corneliomuelleri, Solanum huaylasense, and Solanum arcanum. However, reproductive barriers among the species are incomplete and this can lead to discrepancies regarding genetic identity of germplasm. We used genotyping by sequencing (GBS) of S. peruvianum s.l., Solanum neorickii, and Solanum chmielewskii to develop tens of thousands of mapped single nucleotide polymorphisms (SNPs) to analyze genetic relationships within and among species. The data set was condensed to 14,043 SNPs with no missing data across 46 sampled plants. Origins of accessions were mapped using geographical information systems (GIS). Isolation by distance, pairwise genetic distances, and number of clusters were estimated using population genetics approaches. Isolation by distance was strongly supported, especially between interspecific pairs. Eriopersicon (S. peruvianum s.s., S. corneliomuelleri, S. huaylasense) and Arcanum (S. arcanum, S. neorickii, S. chmielewskii) species groups were genetically distinct, except for S. huaylasense which showed 50% membership proportions in each group. Solanum peruvianum and S. corneliomuelleri were not significantly differentiated from each other. Many thousands of SNP markers were identified that could potentially be used to distinguish pairs of species, including S. peruvianum versus S. corneliomuelleri, if they are verified on larger numbers of samples. Diagnostic markers will be valuable for delimiting morphologically similar and interfertile species in germplasm management. Approximately 12% of the SNPs rejected a genome-wide test of selective neutrality based on differentiation among species of S. peruvianum s.l. These are candidates for more comprehensive studies of microevolutionary processes within this species complex.

Comparison of regeneration potentials in tissue cultures of primitive and cultivated tomato species (Lycopersicon sp.)

Regeneration capacities of two tomato cultivars: Potentat and Rutgers, and of three accessions of wild tomato species: &lt;em&gt;Lycopersicon peruvianum&lt;/em&gt; PI 128650, &lt;em&gt;L. peruvianum var. dentatum&lt;/em&gt; PI 128655 and &lt;em&gt;L. glandulosum&lt;/em&gt; were studied using an universal medium suitable for regeneration of those plants from leaf pieces in tissue culture. Fragments of leaf blades were taken from plants raised in greenhouse conditions and placed on a modified MS medium containing 0.3 mg/l IAA and 3.0 mg/l BAP solidified with 1% agar. The explants were transferred every 4-5 weeks on fresh medium of the same composition. It was shown that all the three primitive tomato species revealed much higher multiplication coefficients than the two cultivars. Appropriate values were: 11 - for &lt;em&gt;L. glandulosum&lt;/em&gt;, 8 - for &lt;em&gt;L. peruvianum&lt;/em&gt;, 7 - for &lt;em&gt;L. peruvianum var. dentatum&lt;/em&gt;, 4 - for &lt;em&gt;L. esculentum&lt;/em&gt; cv. Potentat and 2 - cv. Rutgers. Completely regenerated plants were obtained from all the tested species, but organogenesis occurred almost two weeks earlier in wild tomatoes than in the culitivated varieties of &lt;em&gt;L. esculentum&lt;/em&gt;.

Sequence Evolution and Expression Regulation of Stress-Responsive Genes in Natural Populations of Wild Tomato

The wild tomato species Solanum chilense and S. peruvianum are a valuable non-model system for studying plant adaptation since they grow in diverse environments facing many abiotic constraints. Here we investigate the sequence evolution of regulatory regions of drought and cold responsive genes and their expression regulation. The coding regions of these genes were previously shown to exhibit signatures of positive selection. Expression profiles and sequence evolution of regulatory regions of members of the Asr (ABA/water stress/ripening induced) gene family and the dehydrin gene pLC30-15 were analyzed in wild tomato populations from contrasting environments. For S. chilense, we found that Asr4 and pLC30-15 appear to respond much faster to drought conditions in accessions from very dry environments than accessions from more mesic locations. Sequence analysis suggests that the promoter of Asr2 and the downstream region of pLC30-15 are under positive selection in some local populations of S. chilense. By investigating gene expression differences at the population level we provide further support of our previous conclusions that Asr2, Asr4, and pLC30-15 are promising candidates for functional studies of adaptation. Our analysis also demonstrates the power of the candidate gene approach in evolutionary biology research and highlights the importance of wild Solanum species as a genetic resource for their cultivated relatives.

Chilling‐induced water stress: Variation in shoot turgor maintenance among wild tomato species from diverse habitats

Cultivated tomato, Solanum lycopersicum, suffers chilling induced wilting because water movement through its roots decreases with declining soil temperatures. Certain wild tomato species exhibit resistance to chilling-induced wilting, but the extent of this chilling tolerance in wild tomatoes is not known. • We measured shoot wilting during root chilling in wild Solanum accessions from habitats differing in elevation, temperature, and precipitation. We also measured shoot wilting during root chilling in introgression lines (ILs) with chromosome 9 segments collinear to the shoot turgor maintenance QTL stm9 region from chilling-tolerant S. habrochaites, chilling and drought-tolerant S. lycopersicoides, or drought-tolerant S. pennellii. • Wild tomato species, which experience chilling temperatures (<10°C) in their native habitat, maintain shoot turgor under root chilling. Among accessions of S. lycopersicum var. cerasiforme, a typically chilling sensitive species, shoot turgor maintenance during root chilling was correlated with the precipitation of the native habitat. By contrast, S. pennellii, a species that is typically drought adapted, did not maintain turgor under root chilling. Grafted plants with roots containing S. habrochaites and S. lycopersicoides introgressions improved shoot turgor maintenance under root chilling. • Resistance to chilling-induced water stress is an important adaptation to chilling temperatures in wild tomatoes. There is some overlap in adaptation to drought and chilling stress in some tomato species. Root-based resistance to chilling-induced water stress in wild tomatoes may involve orthologous gene(s).

Why to account for finite sites in population genetic studies and how to do this with Jaatha 2.0

With the advent of next-generation sequencing technologies, large data sets of several thousand loci from multiple conspecific individuals are available. Such data sets should make it possible to obtain accurate estimates of population genetic parameters, even for complex models of population history. In the analyses of large data sets, it is difficult to consider finite-sites mutation models (FSMs). Here, we use extensive simulations to demonstrate that the inclusion of FSMs is necessary to avoid severe biases in the estimation of the population mutation rate θ, population divergence times, and migration rates. We present a new version of Jaatha, an efficient composite-likelihood method for estimating demographic parameters from population genetic data and evaluate the usefulness of Jaatha in two biological examples. For the first application, we infer the speciation process of two wild tomato species, Solanum chilense and Solanum peruvianum. In our second application example, we demonstrate that Jaatha is readily applicable to NGS data by analyzing genome-wide data from two southern European populations of Arabidopsis thaliana. Jaatha is now freely available as an R package from the Comprehensive R Archive Network (CRAN).

Steroidal glycoalkaloid profiling and structures of glycoalkaloids in wild tomato fruit

Steroidal glycoalkaloids (SGAs) constitute one of the main groups of secondary metabolites in tomato fruit. However, the detailed composition of SGAs other than α-tomatine, dehydrotomatine and esculeoside A, remains unclear. Comparative SGA profiling was performed in eight tomato accessions, including wild tomato species by HPLC-Fourier transform ion cyclotron resonance mass spectrometry (HPLC-FTICR/MS). On the basis of molecular formulae obtained from accurate m/z and fragmentation patterns by multistage MS/ MS (MS(n)), 123 glycoalkaloids in total were screened. Detailed MS(n) analysis showed that the observed structural diversity was derived from various chemical modifications, such as glycosylation, acetylation, hydroxylation and isomerization. Total SGA content in each tomato accession was in the range of 121-1986 nmol/gfr.wt. Furthermore, the compositional variety of SGA structures was distinctive in some tomato accessions. While most tomato accessions were basically categorized as α-tomatine-rich or esculeoside A-rich group, other specific SGAs also accumulated at high levels in wild tomato. Here, five such SGAs were isolated and their structures were determined by NMR spectroscopic analysis, indicating three of them were presumably synthesized during α-tomatine metabolism.

The expanded tomato fruit volatile landscape

The present review aims to synthesize our present knowledge about the mechanisms implied in the biosynthesis of volatile compounds in the ripe tomato fruit, which have a key role in tomato flavour. The difficulties in identifiying not only genes or genomic regions but also individual target compounds for plant breeding are addressed. Ample variability in the levels of almost any volatile compound exists, not only in the populations derived from interspecific crosses but also in heirloom varieties and even in commercial hybrids. Quantitative trait loci (QTLs) for all tomato aroma volatiles have been identified in collections derived from both intraspecific and interspecific crosses with different wild tomato species and they (i) fail to co-localize with structural genes in the volatile biosynthetic pathways and (ii) reveal very little coincidence in the genomic regions characterized, indicating that there is ample opportunity to reinforce the levels of the volatiles of interest. Some of the identified genes may be useful as markers or as biotechnological tools to enhance tomato aroma. Current knowledge about the major volatile biosynthetic pathways in the fruit is summarized. Finally, and based on recent reports, it is stressed that conjugation to other metabolites such as sugars seems to play a key role in the modulation of volatile release, at least in some metabolic pathways.

A quantitative genetic basis for leaf morphology in a set of precisely defined tomato introgression lines.

Introgression lines (ILs), in which genetic material from wild tomato species is introgressed into a domesticated background, have been used extensively in tomato (Solanum lycopersicum) improvement. Here, we genotype an IL population derived from the wild desert tomato Solanum pennellii at ultrahigh density, providing the exact gene content harbored by each line. To take advantage of this information, we determine IL phenotypes for a suite of vegetative traits, ranging from leaf complexity, shape, and size to cellular traits, such as stomatal density and epidermal cell phenotypes. Elliptical Fourier descriptors on leaflet outlines provide a global analysis of highly heritable, intricate aspects of leaf morphology. We also demonstrate constraints between leaflet size and leaf complexity, pavement cell size, and stomatal density and show independent segregation of traits previously assumed to be genetically coregulated. Meta-analysis of previously measured traits in the ILs shows an unexpected relationship between leaf morphology and fruit sugar levels, which RNA-Seq data suggest may be attributable to genetically coregulated changes in fruit morphology or the impact of leaf shape on photosynthesis. Together, our results both improve upon the utility of an important genetic resource and attest to a complex, genetic basis for differences in leaf morphology between natural populations.

The Tomato Yellow Leaf Curl Virus resistance genes Ty-1 and Ty-3 are allelic and code for DFDGD-class RNA-dependent RNA polymerases.

Tomato Yellow Leaf Curl Virus Disease incited by Tomato yellow leaf curl virus (TYLCV) causes huge losses in tomato production worldwide and is caused by different related begomovirus species. Breeding for TYLCV resistance has been based on the introgression of multiple resistance genes originating from several wild tomato species. In this study we have fine-mapped the widely used Solanum chilense–derived Ty-1 and Ty-3 genes by screening nearly 12,000 plants for recombination events and generating recombinant inbred lines. Multiple molecular markers were developed and used in combination with disease tests to fine-map the genes to a small genomic region (approximately 70 kb). Using a Tobacco Rattle Virus–Virus Induced Gene Silencing approach, the resistance gene was identified. It is shown that Ty-1 and Ty-3 are allelic and that they code for a RNA–dependent RNA polymerase (RDR) belonging to the RDRγ type, which has an atypical DFDGD motif in the catalytic domain. In contrast to the RDRα type, characterized by a catalytic DLDGD motif, no clear function has yet been described for the RDRγ type, and thus the Ty-1/Ty-3 gene unveils a completely new class of resistance gene. Although speculative, the resistance mechanism of Ty-1/Ty-3 and its specificity towards TYLCV are discussed in light of the function of the related RDRα class in the amplification of the RNAi response in plants and transcriptional silencing of geminiviruses in plants.

Developmental onset of reproductive barriers and associated proteome changes in stigma/styles of Solanum pennellii.

Although self-incompatibility (SI) in plants has been studied extensively, far less is known about interspecific reproductive barriers. One interspecific barrier, known as unilateral incongruity or incompatibility (UI), occurs when species display unidirectional compatibility in interspecific crosses. In the wild tomato species Solanum pennellii, both SI and self-compatible (SC) populations express UI when crossed with domesticated tomato, offering a useful model system to dissect the molecular mechanisms involved in reproductive barriers. In this study, the timing of reproductive barrier establishment during pistil development was determined in SI and SC accessions of S. pennellii using a semi-in vivo system to track pollen-tube growth in developing styles. Both SI and UI barriers were absent in styles 5 days prior to flower opening, but were established by 2 days before flower opening, with partial barriers detected during a transition period 3–4 days before flower opening. The developmental expression dynamics of known SI factors, S-RNases and HT proteins, was also examined. The accumulation of HT-A protein coincided temporally and spatially with UI barriers in developing pistils. Proteomic analysis of stigma/styles from key developmental stages showed a switch in protein profiles from cell-division-associated proteins in immature stigma/styles to a set of proteins in mature stigma/styles that included S-RNases, HT-A protein and proteins associated with cell-wall loosening and defense responses, which could be involved in pollen–pistil interactions. Other prominent proteins in mature stigma/styles were those involved in lipid metabolism, consistent with the accumulation of lipid-rich material during pistil maturation.