Major Impacts of Widespread Structural Variation on Gene Expression and Crop Improvement in Tomato.

Structural variants (SVs) underlie important crop improvement and domestication traits. However, resolving the extent, diversity, and quantitative impact of SVs has been challenging. We used long-read nanopore sequencing to capture 238,490 SVs in 100 diverse tomato lines. This panSV genome, along with 14 new reference assemblies, revealed large-scale intermixing of diverse genotypes, as well as thousands of SVs intersecting genes and cis-regulatory regions. Hundreds of SV-gene pairs exhibit subtle and significant expression changes, which could broadly influence quantitative trait variation. By combining quantitative genetics with genome editing, we show how multiple SVs that changed gene dosage and expression levels modified fruit flavor, size, and production. In the last example, higher order epistasis among four SVs affecting three related transcription factors allowed introduction of an important harvesting trait in modern tomato. Our findings highlight the underexplored role of SVs in genotype-to-phenotype relationships and their widespread importance and utility in crop improvement.

Loss of function of a DMR6 ortholog in tomato confers broad-spectrum disease resistance.

Plant diseases are among the major causes of crop yield losses around the world. To confer disease resistance, conventional breeding relies on the deployment of single resistance (R) genes. However, this strategy has been easily overcome by constantly evolving pathogens. Disabling susceptibility (S) genes is a promising alternative to R genes in breeding programs, as it usually offers durable and broad-spectrum disease resistance. In Arabidopsis, the S gene DMR6 (AtDMR6) encodes an enzyme identified as a susceptibility factor to bacterial and oomycete pathogens. Here, we present a model-to-crop translational work in which we characterize two AtDMR6 orthologs in tomato, SlDMR6-1 and SlDMR6-2. We show that SlDMR6-1, but not SlDMR6-2, is up-regulated by pathogen infection. In agreement, Sldmr6-1 mutants display enhanced resistance against different classes of pathogens, such as bacteria, oomycete, and fungi. Notably, disease resistance correlates with increased salicylic acid (SA) levels and transcriptional activation of immune responses. Furthermore, we demonstrate that SlDMR6-1 and SlDMR6-2 display SA-5 hydroxylase activity, thus contributing to the elucidation of the enzymatic function of DMR6. We then propose that SlDMR6 duplication in tomato resulted in subsequent subfunctionalization, in which SlDMR6-2 specialized in balancing SA levels in flowers/fruits, while SlDMR6-1 conserved the ability to fine-tune SA levels during pathogen infection of the plant vegetative tissues. Overall, this work not only corroborates a mechanism underlying SA homeostasis in plants, but also presents a promising strategy for engineering broad-spectrum and durable disease resistance in crops.

Eye tracking: empirical foundations for a minimal reporting guideline.

In this paper, we present a review of how the various aspects of any study using an eye tracker (such as the instrument, methodology, environment, participant, etc.) affect the quality of the recorded eye-tracking data and the obtained eye-movement and gaze measures. We take this review to represent the empirical foundation for reporting guidelines of any study involving an eye tracker. We compare this empirical foundation to five existing reporting guidelines and to a database of 207 published eye-tracking studies. We find that reporting guidelines vary substantially and do not match with actual reporting practices. We end by deriving a minimal, flexible reporting guideline based on empirical research (Section "An empirically based minimal reporting guideline").

Identification and Mapping of bs8, a Novel Locus Conferring Resistance to Bacterial Spot Caused by Xanthomonas gardneri.

Although cultivars possessing recessive resistance alleles provide effective control of bacterial spot of pepper (Capsicum annuum), the deployed resistance gene, bs5, is ineffective against Xanthomonas gardneri, one of the pathogenic species. Resistance against X. gardneri was identified in C. annuum accession PI 163192, and this study sought to characterize this novel resistance and to map the resistance gene(s) to the pepper genome. We crossed PI 163192 with the susceptible cultivar Early Calwonder (ECW) to develop resistant near-isogenic lines (NILs) of ECW, designated ECW80R. The novel resistance in ECW80R was determined to be quantitative, recessively inherited, and non-hypersensitive-response causing, and inhibits lesion expansion and chlorosis. Presence of the resistance in NILs decreased the in planta bacterial population by ninefold compared with ECW. Bulked segregant analysis of resistant and susceptible individuals from an F2 population using whole genome single nucleotide polymorphisms identified a major resistance locus within an approximate 6-Mbp interval on the subtelomeric region of chromosome 11. We developed markers spanning this region and used these to genotype backcross F2 populations, which further delimited the resistance locus within a 2.3-Mbp interval. The novel resistance locus has been designated bs8. ECW80R and the linked markers developed in this study should prove useful for breeders seeking to advance this resistance into commercially relevant germplasm and for pyramiding bs8 with other resistance alleles such as bs5 and bs6. The allele bs8 will help prolong the durability of bacterial spot resistance in pepper and improve resistance to multiple species of Xanthomonas.

Interaction Studies Between Meloidogyne Javanica and Fusarium Oxysporum f. Sp. lycopersici (Fol) Race 3 on Different Isolines of Tomato Cv. Tasti Lee.

The Mi gene in tomato confers resistance to Meloidogyne javanica, M. incognita, and M. arenaria, the most common tropical root-knot nematode (RKN) species found in Florida. Fusarium wilt (Fol) is another major problem in Florida tomatoes which may interact with RKN and cause more plant damage. To study the interactions between RKN, Fusarium, and Mi in tomato, two greenhouse experiments were conducted. Both experiments used different isolines (with and without I-3 and Mi genes) of the tomato cultivar Tasti Lee®. In the first experiment, all four isolines were subjected to two levels of RKN (~10,000 eggs/pot and no eggs) and two levels of Fol (1000 cc soil with 1,000 cfu/g at planting and no Fol), both applied at planting. In the second experiment, the two isolines without I-3 were exposed to the same two levels of RKN as described above and three levels of Fol (50 ml Fol with 1×106 cfu/m at planting, at 10 DAT, and no Fol). Fol reduced root-knot infection and reproduction when both Fol and RKN were inoculated at planting but not when Fol was inoculated 10 days later. Plant damage from Fol was exacerbated in the presence of RKN, especially when both pathogens were present at planting. Isolines with I-3 grew better in Fol-inoculated soil but had no effect when Fol and RKN were both present. Isolines with Mi gene reduced RKN infection and reproduction but did not affect plant damage caused by Fol. In summary, while RKN reproduction was reduced in the presence of Fol, the overall plant damage was more severe when both pathogens were present.

Characterization and elimination of linkage-drag associated with Fusarium wilt race 3 resistance genes.

KEY MESSAGE: Reducing the size of the I-3 introgression resulted in eliminating linkage-drag contributing to increased sensitivity to bacterial spot and reduced fruit size. The I-7 gene was determined to have no effect on bacterial spot or fruit size, and germplasm is now available with both the reduced I-3 introgression and I-7. Tomato (Solanum lycopersicum) production is increasingly threatened by Fusarium wilt race 3 (Fol3) caused by the soilborne fungus, Fusarium oxysporum f. sp. lycopersici. Although host resistance based on the I-3 gene is the most effective management strategy, I-3 is associated with detrimental traits including reduced fruit size and increased bacterial spot sensitivity. Previous research demonstrated the association with bacterial spot is not due to the I-3 gene, itself, and we hypothesize that reducing the size of the I-3 introgression will remedy this association. Cultivars with I-7, an additional Fol3 resistance gene, are available but are not widely used commercially, and it is unclear whether I-7 also has negative horticultural associations. To characterize the effect of I-3 on fruit size, segregating populations were developed and evaluated, revealing that the large I-3 introgression decreased fruit size by approximately 21%. We reduced the I-3 introgression from 5 to 140 kb through successive recombinant screening and crossing efforts. The reduced I-3 introgression and I-7 were then separately backcrossed into elite Florida breeding lines and evaluated for effects on bacterial spot sensitivity and fruit size across multiple seasons. The reduced I-3 introgression resulted in significantly less bacterial spot and larger fruit size than the large introgression, and it had no effect on these horticultural characteristics compared with Fol3 susceptibility. I-7 was also found to have no effect on these traits compared to Fol3 susceptibility. Together, these efforts support the development of superior Fol3-resistant cultivars and more durable resistance against this pathogen.

Ty-6, a major begomovirus resistance gene on chromosome 10, is effective against Tomato yellow leaf curl virus and Tomato mottle virus.

KEY MESSAGE: Ty-6 is a major resistance gene on chromosome 10 of tomato that provides resistance against monopartite and bipartite begomoviruses and complements resistance conferred by the known Ty-3 and ty-5 genes. Resistance to monopartite and bipartite begomoviruses is an important breeding objective for cultivated tomato. Several begomovirus resistance genes have been introgressed from related Solanum species and are available for breeding purposes. In the present study, we mapped an additional locus, Ty-6, to chromosome 10 of tomato. Ty-6 is effective against both monopartite Tomato yellow leaf curl virus (TYLCV) and bipartite Tomato mottle virus (ToMoV). Gene action is incomplete dominance, with an intermediate resistance response when Ty-6 is heterozygous. Analysis of populations segregating for Ty-6 along with Ty-3 or ty-5 indicates that the highest level of resistance against TYLCV is attained when Ty-6 is combined with an additional resistance allele. Our results also demonstrate that ty-5 is ineffective against ToMoV. Although multiple SNPs linked to Ty-6 were identified and can be used for breeding purposes, none of these were consistently polymorphic between Ty-6 and ty-6 breeding lines. Further research is underway to generate resequencing data for several Ty-6 inbred lines for the discovery of additional sequence polymorphisms that can be used for fine mapping and characterizing the Ty-6 locus.

Linkage between the I-3 gene for resistance to Fusarium wilt race 3 and increased sensitivity to bacterial spot in tomato.

KEY MESSAGE: The negative association between the I - 3 gene and increased sensitivity to bacterial spot is due to linkage drag (not pleiotropy) and may be remedied by reducing the introgression size. Fusarium wilt is one of the most serious diseases of tomato (Solanum lycopersicum L.) throughout the world. There are three races of the pathogen (races 1, 2 and 3), and the deployment of three single, dominant resistance genes corresponding to each of these has been the primary means of controlling the disease. The I-3 gene was introgressed from S. pennellii and confers resistance to race 3. Although I-3 provides effective control, it is negatively associated with several horticultural traits, including increased sensitivity to bacterial spot disease (Xanthomonas spp.). To test the hypothesis that this association is due to linkage with unfavorable alleles rather than to pleiotropy, we used a map-based approach to develop a collection of recombinant inbred lines varying for portions of I-3 introgression. Progeny of recombinants were evaluated for bacterial spot severity in the field for three seasons, and disease severities were compared between I-3 introgression haplotypes for each recombinant. Results indicated that increased sensitivity to bacterial spot is not associated with the I-3 gene, but rather with an upstream region of the introgression. A survey of public and private inbred lines and hybrids indicates that the majority of modern I-3 germplasm contains a similarly sized introgression for which the negative association with bacterial spot likely persists. In light of this, it is expected that the development and utilization of a reduced I-3 introgression will significantly improve breeding efforts for resistance to Fusarium wilt race 3.

Oculomotor atypicalities in Developmental Coordination Disorder.

Children with Developmental Coordination Disorder (DCD) fail to acquire adequate motor skill, yet surprisingly little is known about the oculomotor system in DCD. Successful completion of motor tasks is supported by accurate visual feedback. The purpose of this study was to determine whether any oculomotor differences can distinguish between children with and without a motor impairment. Using eye tracking technology, visual fixation, smooth pursuit, and pro- and anti-saccade performance were assessed in 77 children that formed three groups: children with DCD (aged 7-10), chronologically age (CA) matched peers, and a motor-match (MM) group (aged 4-7). Pursuit gain and response preparation in the pro- and anti-saccade tasks were comparable across groups. Compared to age controls, children with DCD had deficits in maintaining engagement in the fixation and pursuit tasks, and made more anti-saccade errors. The two typically developing groups performed similarly, except on the fast speed smooth pursuit and antisaccade tasks, where the CA group outperformed the younger MM group. The findings suggest that children with DCD have problems with saccadic inhibition and maintaining attention on a visual target. Developmental patterns were evident in the typically developing groups, suggesting that the pursuit system and cognitive control develop with age. This study adds to the literature by being the first to systematically identify specific oculomotor differences between children with and without a motor impairment. Further examination of oculomotor control may help to identify underlying processes contributing to DCD. A video abstract of this article can be viewed at: https://youtu.be/NinXa2KlB4M. [Correction added on 27 January 2017, after first online publication: The video abstract link was added.].

Transgenic Expression of EFR and Bs2 Genes for Field Management of Bacterial Wilt and Bacterial Spot of Tomato.

Field trials were conducted at two locations in Florida to evaluate transgenic tomato expressing the ELONGATION FACTOR TU RECEPTOR (EFR) gene from Arabidopsis thaliana, the Bs2 gene from pepper, or both Bs2 and EFR (Bs2/EFR) for managing bacterial wilt caused by Ralstonia solanacearum and bacterial spot caused by Xanthomonas perforans. Expression of EFR or Bs2/EFR in the susceptible genotype Fla. 8000 significantly reduced bacterial wilt incidence (50 to 100%) and increased total yield (57 to 114%) relative to lines expressing only Bs2 or the nontransformed Fla. 8000 control, although the marketable yield was not significantly affected. Following harvest, surviving symptomatic and nonsymptomatic plants were assessed for colonization by R. solanacearum. There were no significant differences in the population at the lower stem. Interestingly, in the middle stem, no bacteria could be recovered from EFR or Bs2/EFR lines but viable bacterial populations were recovered from Bs2 and nontransformed control lines at 102 to 105 CFU/g of stem tissue. In growth-chamber experiments, the EFR transgenic tomato lines were found to be effective against seven different R. solanacearum strains isolated from the southeastern United States, indicating utility across the southeastern United States. In all of the bacterial spot trials, EFR and Bs2/EFR lines had significantly reduced disease severity (22 to 98%) compared with the Fla. 8000 control. The marketable and total yield of Bs2/EFR were significantly higher (43 to 170%) than Fla. 8000 control in three of four field trials. These results demonstrate for the first time the potential of using the EFR gene for field management of bacterial wilt and bacterial spot diseases of tomato.

MutS HOMOLOG1-derived epigenetic breeding potential in tomato.

Evidence is compelling in support of a naturally occurring epigenetic influence on phenotype expression in land plants, although discerning the epigenetic contribution is difficult. Agriculturally important attributes like heterosis, inbreeding depression, phenotypic plasticity, and environmental stress response are thought to have significant epigenetic components, but unequivocal demonstration of this is often infeasible. Here, we investigate gene silencing of a single nuclear gene, MutS HOMOLOG1 (MSH1), in the tomato (Solanum lycopersicum) 'Rutgers' to effect developmental reprogramming of the plant. The condition is heritable in subsequent generations independent of the MSH1-RNA interference transgene. Crossing these transgene-null, developmentally altered plants to the isogenic cv Rutgers wild type results in progeny lines that show enhanced, heritable growth vigor under both greenhouse and field conditions. This boosted vigor appears to be graft transmissible and is partially reversed by treatment with the methylation inhibitor 5-azacytidine, implying the influence of mobile, epigenetic factors and DNA methylation changes. These data provide compelling evidence for the feasibility of epigenetic breeding in a crop plant.

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.

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

BACKGROUND: Decades 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. RESULTS: Here 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. CONCLUSIONS: The 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.

The influence of hallucination proneness and social threat on time perception.

INTRODUCTION: Individuals with schizophrenia frequently report disturbances in time perception, but the precise nature of such deficits and their relation to specific symptoms of the disorder is unclear. We sought to determine the relationship between hallucination proneness and time perception in healthy individuals, and whether this relationship is moderated by hypervigilance to threat-related stimuli. METHODS: 206 participants completed the Revised Launay-Slade Hallucination Scale (LSHS-R) and a time reproduction task in which, on each trial, participants viewed a face (happy, angry, neutral, or fearful) for between 1 and 5 s and then reproduced the time period with a spacebar press. RESULTS: High LSHS-R scores were associated with longer time estimates, but only during exposure to angry faces. A factor analysis of LSHS-R scores identified a factor comprising items related to reality monitoring, and this factor was most associated with the longer time estimates. CONCLUSIONS: During exposure to potential threat in the environment, duration estimates increase with hallucination proneness. The experience of feeling exposed to threat for longer may serve to maintain a state of hypervigilance which has been shown previously to be associated with positive symptoms of schizophrenia.

Idiosyncratic and dose-dependent epistasis drives variation in tomato fruit size.

Epistasis between genes is traditionally studied with mutations that eliminate protein activity, but most natural genetic variation is in cis-regulatory DNA and influences gene expression and function quantitatively. In this study, we used natural and engineered cis-regulatory alleles in a plant stem-cell circuit to systematically evaluate epistatic relationships controlling tomato fruit size. Combining a promoter allelic series with two other loci, we collected over 30,000 phenotypic data points from 46 genotypes to quantify how allele strength transforms epistasis. We revealed a saturating dose-dependent relationship but also allele-specific idiosyncratic interactions, including between alleles driving a step change in fruit size during domestication. Our approach and findings expose an underexplored dimension of epistasis, in which cis-regulatory allelic diversity within gene regulatory networks elicits nonlinear, unpredictable interactions that shape phenotypes.

Mapping of the bs5 and bs6 non-race-specific recessive resistances against bacterial spot of pepper.

Bacterial spot caused by Xanthomonas euvesicatoria is a major disease of pepper (Capsicum annuum L.) in warm and humid production environments. Use of genetically resistant cultivars is an effective approach to manage bacterial spot. Two recessive resistance genes, bs5 and bs6, confer non-race-specific resistance against bacterial spot. The objective of our study was to map these two loci in the pepper genome. We used a genotyping-by-sequencing approach to initially map the position of the two resistances. Segregating populations for bs5 and bs6 were developed by crossing susceptible Early CalWonder (ECW) with near-isogenic lines ECW50R (bs5 introgression) or ECW60R (bs6 introgression). Following fine-mapping, bs5 was delimited to a ~535 Kbp interval on chromosome 3, and bs6 to a ~666 Kbp interval in chromosome 6. We identified 14 and 8 candidate resistance genes for bs5 and bs6, respectively, based on predicted protein coding polymorphisms between ECW and the corresponding resistant parent. This research enhances marker-assisted selection of bs5 and bs6 in breeding programs and is a crucial step towards elucidating the molecular mechanisms underlying the resistances.

Redesigning the tomato fruit shape for mechanized production.

Crop breeding for mechanized harvesting has driven modern agriculture. In tomato, machine harvesting for industrial processing varieties became the norm in the 1970s. However, fresh-market varieties whose fruits are suitable for mechanical harvesting are difficult to breed because of associated reduction in flavour and nutritional qualities. Here we report the cloning and functional characterization of fs8.1, which controls the elongated fruit shape and crush resistance of machine-harvestable processing tomatoes. FS8.1 encodes a non-canonical GT-2 factor that activates the expression of cell-cycle inhibitor genes through the formation of a transcriptional module with the canonical GT-2 factor SlGT-16. The fs8.1 mutation results in a lower inhibitory effect on the cell proliferation of the ovary wall, leading to elongated fruits with enhanced compression resistance. Our study provides a potential route for introducing the beneficial allele into fresh-market tomatoes without reducing quality, thereby facilitating mechanical harvesting.

Chromosomal rearrangements between tomato and Solanum chilense hamper mapping and breeding of the TYLCV resistance gene Ty-1.

Tomato yellow leaf curl disease, a devastating disease of Solanum lycopersicum (tomato), is caused by a complex of begomoviruses generally referred to as Tomato yellow leaf curl virus (TYLCV). Almost all breeding for TYLCV resistance has been based on the introgression of the Ty-1 resistance locus derived from Solanum chilense LA1969. Knowledge about the exact location of Ty-1 on tomato chromosome 6 will help in understanding the genomic organization of the Ty-1 locus. In this study, we analyze the chromosomal rearrangement and recombination behavior of the chromosomal region where Ty-1 is introgressed. Nineteen markers on tomato chromosome 6 were used in F(2) populations obtained from two commercial hybrids, and showed the presence of a large introgression in both. Fluorescence in situ hybridization (FISH) analysis revealed two chromosomal rearrangements between S. lycopersicum and S. chilense LA1969 in the Ty-1 introgression. Furthermore, a large-scale recombinant screening in the two F(2) populations was performed, and 30 recombinants in the Ty-1 introgression were identified. All recombination events were located on the long arm beyond the inversions, showing that recombination in the inverted region was absent. Disease tests on progenies of informative recombinants with TYLCV mapped Ty-1 to the long arm between markers MSc05732-4 and MSc05732-14, an interval overlapping with the reported Ty-3 region, which led to the indication that Ty-1 and Ty-3 may be allelic. With this study we prove that FISH can be used as a diagnostic tool to aid in the accurate mapping of genes that were introgressed from wild species into cultivated tomato.