Unraveling the genetic basis of quantitative resistance to diseases in tomato: a meta-QTL analysis and mining of transcript profiles.

KEY MESSAGE: Whole-genome QTL mining and meta-analysis in tomato for resistance to bacterial and fungal diseases identified 73 meta-QTL regions with significantly refined/reduced confidence intervals. Tomato production is affected by a range of biotic stressors, causing yield losses and quality reductions. While sources of genetic resistance to many tomato diseases have been identified and characterized, stability of the resistance genes or quantitative trait loci (QTLs) across the resources has not been determined. Here, we examined 491 QTLs previously reported for resistance to tomato diseases in 40 independent studies and 54 unique mapping populations. We identified 29 meta-QTLs (MQTLs) for resistance to bacterial pathogens and 44 MQTLs for resistance to fungal pathogens, and were able to reduce the average confidence interval (CI) of the QTLs by 4.1-fold and 6.7-fold, respectively, compared to the average CI of the original QTLs. The corresponding physical length of the CIs of MQTLs ranged from 56 kb to 6.37 Mb, with a median of 921 kb, of which 27% had a CI lower than 500 kb and 53% had a CI lower than 1 Mb. Comparison of defense responses between tomato and Arabidopsis highlighted 73 orthologous genes in the MQTL regions, which were putatively determined to be involved in defense against bacterial and fungal diseases. Intriguingly, multiple genes were identified in some MQTL regions that are implicated in plant defense responses, including PR-P2, NDR1, PDF1.2, Pip1, SNI1, PTI5, NSL1, DND1, CAD1, SlACO, DAD1, SlPAL, Ph-3, EDS5/SID1, CHI-B/PR-3, Ph-5, ETR1, WRKY29, and WRKY25. Further, we identified a number of candidate resistance genes in the MQTL regions that can be useful for both marker/gene-assisted breeding as well as cloning and genetic transformation.

Induced Plant Defenses Against Herbivory in Cultivated and Wild Tomato.

Crop domestication and selective breeding have altered plant defense mechanisms, influencing insect-plant interactions. A reduction in plant resistance/tolerance against herbivory is generally expected in domesticated species, however, limited efforts have been made to compare inducibility of plant defenses between wild and domesticated genotypes. In the present study, the inducibility of several plant defense mechanisms (e.g. defensive chemicals, trichomes, plant volatiles) were investigated, and the performance and preference of the herbivore Helicoverpa zea were measured in three different tomato genotypes; a) wild tomato, Solanum pimpinellifolium L. (accession LA 2093), b) cherry tomato, S. lycopersicum L. var. cerasiforme (accession Matts Wild Cherry), and c) cultivated tomato, S. lycopersicum L. var. Better Boy). Enhanced inducibility of defensive chemicals, trichomes, and plant volatiles in the cultivated tomato, and a higher level of constitutive plant resistance against herbivory in the wild genotype was observed. When comparing the responses of damaged vs. undamaged leaves, the percent reduction in larval growth was higher on damaged leaves from cultivated tomato, suggesting a higher induced resistance compared to other two genotypes. While all tomato genotypes exhibited increased volatile organic compound (VOCs) emissions in response to herbivory, the cultivated variety responded with generally higher levels of VOCs. Differences in VOC patterns may have influenced the ovipositional preferences, as H. zea female moths significantly preferred laying eggs on the cultivated versus the wild tomato genotypes. Selection of traits during domestication and selective breeding could alter allocation of resources, where plants selected for higher yield performance would allocate resources to defense only when attacked.

HS-SPME-GC-MS Analyses of Volatiles in Plant Populations-Quantitating Compound × Individual Matrix Effects.

Headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography⁻mass spectrometry (GC-MS) is widely employed for volatile analyses of plants, including mapping populations used in plant breeding research. Studies often employ a single internal surrogate standard, even when multiple analytes are measured, with the assumption that any relative changes in matrix effects among individuals would be similar for all compounds, i.e., matrix effects do not show Compound × Individual interactions. We tested this assumption using individuals from two plant populations: an interspecific grape (Vitis spp.) mapping population (n = 140) and a tomato (Solanum spp.) recombinant inbred line (RIL) population (n = 148). Individual plants from the two populations were spiked with a cocktail of internal standards (n = 6, 9, respectively) prior to HS-SPME-GC-MS. Variation in the relative responses of internal standards indicated that Compound × Individual interactions exist but were different between the two populations. For the grape population, relative responses among pairs of internal standards varied considerably among individuals, with a maximum of 249% relative standard deviation (RSD) for the pair of [U13C]hexanal and [U13C]hexanol. However, in the tomato population, relative responses of internal standard pairs varied much less, with pairwise RSDs ranging from 8% to 56%. The approach described in this paper could be used to evaluate the suitability of using surrogate standards for HS-SPME-GC-MS studies in other plant populations.

Detached-Leaflet Evaluation of Tomato Germplasm for Late Blight Resistance and Its Correspondence to Field and Greenhouse Screenings.

Breeding for disease resistance requires efficient techniques for screening large plant populations. Late blight (LB), caused by the oomycete Phytophthora infestans, is one of the most devastating diseases of tomato (Solanum lycopersicum) worldwide, and there is a great interest in developing cultivars with resistance to this pathogen. Screening for LB resistance is commonly conducted under field or greenhouse conditions using whole plants. In a previous study, we demonstrated correspondence between field and greenhouse screening of tomato for LB resistance. Here, we report the use of a detached-leaflet assay for such screening. Seventy-two genotypes from two tomato species, varying in degree of resistance and susceptibility to LB, were evaluated in two replicated experiments for response to LB in a detached-leaflet assay, and the results were compared with those previously obtained from field and greenhouse screening of the same genotypes. There were significant (P < 0.001) positive correlations between replications (average r = 0.75) and experiments (average r = 0.72), suggesting that the detached-leaflet experiments were consistent. Further, there were significant (P < 0.001) positive correlations between responses in the detached-leaflet assay and those from field (r = 0.82) and greenhouse screenings (r = 0.84), suggesting reliability of the detached-leaflet assay. The results indicate the utility of the detached-leaflet assay for evaluating tomato for LB resistance, which may facilitate screening of large breeding populations.

Validation and fine mapping of lyc12.1, a QTL for increased tomato fruit lycopene content.

Lycopene content is a key component of tomato (Solanum lycopersicum L.) fruit quality, and is a focus of many tomato-breeding programs. Two QTLs for increased fruit lycopene content, inherited from a high-lycopene S. pimpinellifolium accession, were previously detected on tomato chromosomes 7 and 12 using a S. lycopersicum × S. pimpinellifolium RIL population, and were identified as potential targets for marker-assisted selection and positional cloning. To validate the phenotypic effect of these two QTLs, a BC2 population was developed from a cross between a select RIL and the S. lycopersicum recurrent parent. The BC2 population was field-grown and evaluated for fruit lycopene content using HPLC. Statistical analyses revealed that while lyc7.1 did not significantly increase lycopene content in the heterozygous condition, individuals harboring lyc12.1 in the heterozygous condition contained 70.3 % higher lycopene than the recurrent parent. To eliminate the potential pleiotropic effect of fruit size and minimize the physical size of the lyc12.1 introgression, a marker-assisted backcross program was undertaken and produced a BC3S1 NIL population (n = 1,500) segregating for lyc12.1. Lycopene contents from lyc12.1 homozygous and heterozygous recombinants in this population were measured and lyc12.1 was localized to a 1.5 cM region. Furthermore, we determined that lyc12.1 was delimited to a ~1.5 Mb sequence of tomato chromosome 12, and provided some insight into potential candidate genes in the region. The derived sub-NILs will be useful for transferring of lyc12.1 to other tomato genetic backgrounds and for further fine-mapping and cloning of the QTL.

Physiological genetic variation in tomato fruit chilling tolerance during postharvest storage.

Storage at low temperatures is a common practice to prolong postharvest life of fruit and vegetables with a minimal negative impact on human/environmental health. Storage at low temperatures, however, can be restricted due to produce susceptibility to non-freezing chilling temperatures, when injuries such as physiological disorders and decays may result in unmarketable produce. We have investigated tomato fruit response to postharvest chilling stress in a recombinant inbred line (RIL) population developed from a cross between a chilling-sensitive cultivated tomato (Solanum lycopersicum L.) breeding line and a chilling-tolerant inbred accession of the tomato wild species S. pimpinellifolium L. Screening of the fruit of 148 RILs under cold storage (1.5°C) indicated presence of significant variations in chilling tolerance, manifested by varying degrees of fruit injury. Two extremely contrasting groups of RILs were identified, chilling-tolerant and chilling-sensitive RILs. The RILs in the two groups were further investigated under chilling stress conditions, and several physiological parameters, including weight loss, chlorophyll fluorescence parameters Fv/Fm, and Performance Index (PI), were determined to be efficient markers for identifying response to chilling stress in postharvest fruit. The Fv/Fm values reflected the physiological damages endured by the fruit after cold storage, and PI was a sensitive marker for early changes in photosystem II function. These two parameters were early indicators of chilling response before occurrence of visible chilling injuries. Antioxidant activities and ascorbic acid content were significantly higher in the chilling-tolerant than the chilling-sensitive lines. Further, the expression of C-repeat/DREB binding factors (CBFs) genes swiftly changed within 1-hr of fruit exposure to the chilling temperature, and the SlCBF1 transcript level was generally higher in the chilling-tolerant than chilling-sensitive lines after 2-hr exposure to the low temperature. This research demonstrates the presence of potential genetic variation in fruit chilling tolerance in the tomato RIL population. Further investigation of the RIL population is underway to better understand the genetic, physiological, and biochemical mechanisms involved in postharvest fruit chilling tolerance in tomato.

Identification of late blight resistance quantitative trait loci in Solanum pimpinellifolium accession PI 270441.

Late blight (LB), caused by the oomycete Phytophthora infestans, is one of the most destructive diseases of the cultivated tomato (Solanum lycopersicum L.) and potato (Solanum tuberosum L.) worldwide. Genetic changes in the pathogen have resulted in the emergence of new genotypes, overcoming formerly effective fungicides or host resistance genes. We previously reported the identification of a LB-resistant accession (PI 270441) of the wild tomato species S. pimpinellifolium L. and the high heritability of its resistance. In the present study, an F2 population (n = 1,209), derived from a cross between PI 270441 and a LB-susceptible tomato breeding line (Fla. 8059), was screened for response to LB infection. Extreme resistant (n = 44) and susceptible (n = 39) F2 individuals were selected and used in a trait-based marker analysis (TBA; a.k.a selective genotyping) to identify and map quantitative trait loci (QTLs) conferring LB resistance. Reduced representation libraries (RRLs) of Fla. 8059 and PI 270441 were constructed, sequenced, and mapped to the tomato genome. A total of 13,054 single-nucleotide polymorphisms (SNPs) were identified, of which, 200 were used to construct a genetic linkage map and locate QTLs. Four LB resistance QTLs were identified on chromosomes 1, 10, and 11 of PI 270441. The markers associated with these QTLs can be used to transfer LB resistance from PI 270441 into new tomato cultivars and to develop near-isogenic lines for fine mapping of the QTL.

A new genetic linkage map of tomato based on a Solanum lycopersicum x S. pimpinellifolium RIL population displaying locations of candidate pathogen response genes.

The narrow genetic base of the cultivated tomato, Solanum lycopersicum L., necessitates introgression of new variation from related species. Wild tomato species represent a rich source of useful genes and traits. Exploitation of genetic variation within wild species can be facilitated by the use of molecular markers and genetic maps. Recently we identified an accession (LA2093) within the red-fruited wild tomato species Solanum pimpinellifolium L. with exceptionally desirable characteristics, including disease resistance, abiotic stress tolerance, and high fruit lycopene content. To facilitate genetic characterization of such traits and their exploitation in tomato crop improvement, we developed a new recombinant inbred line (RIL) population from a cross between LA2093 and an advanced tomato breeding line (NCEBR-1). Furthermore, we constructed a medium-density molecular linkage map of this population using 294 polymorphic markers, including standard RFLPs, EST sequences (used as RFLP probes), CAPS, and SSRs. The map spanned 1091 cM of the tomato genome with an average marker spacing of 3.7 cM. A majority of the EST sequences, which were mainly chosen based on the putative role of their unigenes in disease resistance, defense-related response, or fruit quality, were mapped onto the tomato chromosomes for the first time. Co-localizations of relevant EST sequences with known disease resistance genes in tomato were also examined. This map will facilitate identification, genetic exploitation, and positional cloning of important genes or quantitative trait loci in LA2093. It also will allow the elucidation of the molecular mechanism(s) underlying important traits segregating in the RIL population. The map may further facilitate characterization and exploitation of genetic variation in other S. pimpinellifolium accessions as well as in modern cultivars of tomato.

Sequencing-Based Bin Map Construction of a Tomato Mapping Population, Facilitating High-Resolution Quantitative Trait Loci Detection.

Genotyping-by-sequencing (GBS) was employed to construct a highly saturated genetic linkage map of a tomato ( L.) recombinant inbred line (RIL) population, derived from a cross between cultivar NC EBR-1 and the wild tomato L. accession LA2093. A pipeline was developed to convert single nucleotide polymorphism (SNP) data into genomic bins, which could be used for fine mapping of quantitative trait loci (QTL) and identification of candidate genes. The pipeline, implemented in a python script named SNPbinner, adopts a hidden Markov model approach for calculation of recombination breakpoints followed by genomic bins construction. The total length of the newly developed high-resolution genetic map was 1.2-fold larger than previously estimated based on restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)-based markers. The map was used to verify and refine QTL previously identified for two fruit quality traits in the RIL population, fruit weight (FW) and fruit lycopene content (LYC). Two well-described FW QTL ( and ) were localized precisely at their known underlying causative genes, and the QTL intervals were decreased by two- to tenfold. A major QTL for LYC content () was verified at high resolution and its underlying causative gene was determined to be ζ (). The RIL population, the high resolution genetic map, and the easy-to-use genotyping pipeline, SNPbinner, are made publicly available.

The tomato pan-genome uncovers new genes and a rare allele regulating fruit flavor.

Modern tomatoes have narrow genetic diversity limiting their improvement potential. We present a tomato pan-genome constructed using genome sequences of 725 phylogenetically and geographically representative accessions, revealing 4,873 genes absent from the reference genome. Presence/absence variation analyses reveal substantial gene loss and intense negative selection of genes and promoters during tomato domestication and improvement. Lost or negatively selected genes are enriched for important traits, especially disease resistance. We identify a rare allele in the TomLoxC promoter selected against during domestication. Quantitative trait locus mapping and analysis of transgenic plants reveal a role for TomLoxC in apocarotenoid production, which contributes to desirable tomato flavor. In orange-stage fruit, accessions harboring both the rare and common TomLoxC alleles (heterozygotes) have higher TomLoxC expression than those homozygous for either and are resurgent in modern tomatoes. The tomato pan-genome adds depth and completeness to the reference genome, and is useful for future biological discovery and breeding.

Identification and Mapping of Late Blight Resistance Quantitative Trait Loci in Tomato Accession PI 163245.

Late blight (LB), caused by the oomycete (Mont.) de Bary, is one of the most devastating diseases of tomato ( L.) and potato ( tuberosum L. worldwide. The importance of LB on tomato has increased due to the occurrence of aggressive and fungicide-resistant clonal lineages of . Consequently, identification and characterization of new sources of genetic resistance to LB has become a priority in tomato breeding. Previously, we reported accession PI 163245 as a promising source of highly heritable LB resistance for tomato breeding. The purpose of this study was to identify and map quantitative trait loci (QTLs) associated with LB resistance in this accession using a trait-based marker analysis (a.k.a. selective genotyping). An F mapping population ( = 560) derived from a cross between a LB-susceptible tomato breeding line (Fla. 8059) and PI 163245 was screened for LB resistance, and the most resistant ( = 39) and susceptible ( = 35) individuals were selected for genotyping. Sequencing and comparison of the reduced representation libraries (RRLs) derived from genomic DNA of the two parents resulted in the identification of 33,541 putative single nucleotide polymorphism (SNP) markers, of which, 233 genome-wide markers were used to genotype the 74 selected F individuals. The marker analysis resulted in the identification of four LB resistance QTLs conferred by PI 163245, located on chromosomes 2, 3, 10, and 11. Research is underway to develop near-isogenic lines (NILs) for fine mapping the QTLs and develop tomato breeding lines with LB resistance introduced from PI 163245.

A Solanum lycopersicum x Solanum pimpinellifolium linkage map of tomato displaying genomic locations of R-genes, RGAs, and candidate resistance/defense-response ESTs.

We have identified an accession (LA2093) within the tomato wild species Solanum pimpinellifolium with many desirable characteristics, including biotic and abiotic stress tolerance and good fruit quality. To utilize the full genetic potential of LA2093 in tomato breeding, we have developed a linkage map based on an F(2) population of a cross between LA2093 and a tomato breeding line, using 115 RFLP, 94 EST, and 41 RGA markers. The map spanned 1002.4 cM of the 12 tomato chromosomes with an average marker distance of 4.0 cM. The length of the map and linear order of the markers were in good agreement with the published maps of tomato. The ESTs were chosen based on their sequence similarities with known resistance or defense-response genes, signal-transduction factors, transcriptional regulators, and genes encoding pathogenesis-related proteins. Locations of several ESTs and RGAs coincided with locations of several known tomato resistance genes and quantitative resistance loci (QRLs), suggesting that candidate-gene approach may be effective in identifying and mapping new R genes. This map will be useful for marker-assisted exploitation of desirable traits in LA2093 and other S. pimpinellifolium accessions, and possibly for utilization of genetic variation within S. lycopersicum.

Genome mapping and molecular breeding of tomato.

The cultivated tomato, Lycopersicon esculentum, is the second most consumed vegetable worldwide and a well-studied crop species in terms of genetics, genomics, and breeding. It is one of the earliest crop plants for which a genetic linkage map was constructed, and currently there are several molecular maps based on crosses between the cultivated and various wild species of tomato. The high-density molecular map, developed based on an L. esculentum x L. pennellii cross, includes more than 2200 markers with an average marker distance of less than 1 cM and an average of 750 kbp per cM. Different types of molecular markers such as RFLPs, AFLPs, SSRs, CAPS, RGAs, ESTs, and COSs have been developed and mapped onto the 12 tomato chromosomes. Markers have been used extensively for identification and mapping of genes and QTLs for many biologically and agriculturally important traits and occasionally for germplasm screening, fingerprinting, and marker-assisted breeding. The utility of MAS in tomato breeding has been restricted largely due to limited marker polymorphism within the cultivated species and economical reasons. Also, when used, MAS has been employed mainly for improving simply-inherited traits and not much for improving complex traits. The latter has been due to unavailability of reliable PCR-based markers and problems with linkage drag. Efforts are being made to develop high-throughput markers with greater resolution, including SNPs. The expanding tomato EST database, which currently includes approximately 214 000 sequences, the new microarray DNA chips, and the ongoing sequencing project are expected to aid development of more practical markers. Several BAC libraries have been developed that facilitate map-based cloning of genes and QTLs. Sequencing of the euchromatic portions of the tomato genome is paving the way for comparative and functional analysis of important genes and QTLs.

Common QTL affect the rate of tomato seed germination under different stress and nonstress conditions.

The purpose of this study was to determine whether the rates of tomato seed germination under different stress and nonstress conditions were under common genetic controls by examining quantitative trait loci (QTL) affecting such traits. Seeds of BC(1) progeny of a cross between a slow-germinating tomato breeding line and a rapid-germinating tomato wild accession were evaluated for germination under nonstress as well as cold, salt, and drought stress conditions. In each treatment, the most rapidly-germinating seeds were selected, grown to maturity, and subjected to molecular marker analysis. A selective genotyping approach detected between 6 and 9 QTL affecting germination rate under each of the four conditions, with a total of 14 QTL identified. Ten QTL affected germination rate under 2 or 3 conditions, which were considered germination-related common QTL. Four QTL affected germination rate only in one treatment, which were considered germination-related, condition-specific QTL . The results indicated that mostly the same QTL affected seed germination under different stress and nonstress conditions, supporting a previous suggestion that similar physiological mechanisms contribute to rapid seed germination under different conditions. Marker-assisted selection for the common QTL may result in progeny with rapid seed germinability under different conditions.