Candidate genes and quantitative trait loci affecting fruit ascorbic acid content in three tomato populations

Fresh fruit and vegetables are a major source of ascorbic acid (vitamin C), an important antioxidant for the human diet and also for plants. Ascorbic acid content in fruit exhibits a quantitative inheritance. Quantitative trait loci (QTL) for ascorbic acid content have been mapped in three tomato populations derived from crosses between cultivated tomato varieties (Solanum lycopersicum accessions) and three related wild species or subspecies. The first population consists of a set of introgression lines derived from Solanum pennellii, each containing a unique fragment of the wild species genome. The second population is an advanced backcross population derived from a cross between a cultivated tomato and a Solanum habrochaites (formerly Lycopersicum hirsutum) accession. The third population is a recombinant inbred line population derived from the cross between a cherry tomato line and a large fruited line. Common regions controlling ascorbic acid content have been identified on chromosomes 2, 8, 9, 10, and 12. In general, the wild alleles increased ascorbic acid content, but some improvement could also be provided by S. lycopersicum. Most QTLs appeared relatively stable over years and in different environments. Mapping of candidate genes involved in the metabolism of ascorbic acid has revealed a few colocations between genes and QTLs, notably in the case of a monodehydroascorbate reductase gene and a QTL present in two of the populations on chromosome 9 (bin 9-D), and a previously mapped GDP-mannose epimerase and a QTL on chromosome 9 (bin 9-J).     

Solanum pennellii backcross inbred lines (BILs) link small genomic bins with tomato traits

We present a resource for fine mapping of traits derived from the wild tomato species Solanum pennellii (LA0716). The population of backcross inbred lines (BILs) is composed of 446 lines derived after a few generations of backcrosses of the wild species with cultivated tomato (cultivar M82; LA3475), followed by more than seven generations of self‐pollination. The BILs were genotyped using the 10K SOL‐CAP single nucleotide polymorphism (SNP) ‐Chip, and 3700 polymorphic markers were used to map recombination break points relative to the physical map of Solanum lycopersicum. The BILs carry, on average, 2.7 introgressions per line, with a mean introgression length of 11.7 Mbp. Whereas the classic 76 introgression lines (ILs) partitioned the genome into 106 mapping bins, the BILs generated 633 bins, thereby enhancing the mapping resolution of traits derived from the wild species. We demonstrate the power of the BILs for rapid fine mapping of simple and complex traits derived from the wild tomato species.     

Combining ecophysiological modelling and quantitative trait locus analysis to identify key elementary processes underlying tomato fruit sugar concentration

A mechanistic model predicting the accumulation of tomato fruit sugars was developed in order (i) to dissect the relative influence of three underlying processes: assimilate supply (S), metabolic transformation of sugars into other compounds (M), and dilution by water uptake (D); and (ii) to estimate the genetic variability of S, M, and D. The latter was estimated in a population of 20 introgression lines derived from the introgression of a wild tomato species (Solanum chmielewskii) into S. lycopersicum, grown under two contrasted fruit load conditions. Low load systematically decreased D in the whole population, while S and M were targets of genotype × fruit load interactions. The sugar concentration positively correlated to S and D when the variation was due to genetic introgressions, while it positively correlated to S and M when the variation was due to changes in fruit load. Co-localizations between quantitative trait loci (QTLs) for sugar concentration and QTLs for S, M, and D allowed hypotheses to be proposed on the processes putatively involved at the QTLs. Among the five QTLs for sugar concentration, four co-localized with QTLs for S, M, and D with similar allele effects. Moreover, the processes underlying QTLs for sugar accumulation changed according to the fruit load condition. Finally, for some genotypes, the processes underlying sugar concentration compensated in such a way that they did not modify the sugar concentration. By uncoupling genetic from physiological relationships between processes, these results provide new insights into further understanding of tomato fruit sugar accumulation.     

Yield quantitative trait loci from wild tomato are predominately expressed by the shoot

Plant yield is the integrated outcome of processes taking place above and below ground. To explore genetic, environmental and developmental aspects of fruit yield in tomato, we phenotyped an introgression line (IL) population derived from a cross between the cultivated tomato (Solanum lycopersicum) and a wild species (Solanum pennellii). Both homozygous and heterozygous ILs were grown in irrigated and non-irrigated fields and evaluated for six yield components. Thirteen lines displayed transgressive segregation that increased agronomic yield consistently over 2?years and defined at least 11 independent yield-improving QTL. To determine if these QTL were expressed in the shoots or the roots of the plants, we conducted field trials of reciprocally grafted ILs; out of 13 lines with an effect on yield, 10 QTL were active in the shoot and only IL8-3 showed a consistent root effect. To further examine this unusual case, we evaluated the metabolic profiles of fruits from both the homo- and heterozygous lines for IL8-3 and compared these to those obtained from the fruit of their equivalent genotypes in the root effect population. We observed that several of these metabolic QTL, like the yield QTL, were root determined; however, further studies will be required to delineate the exact mechanism mediating this effect in this specific line. The results presented here suggest that genetic variation for root traits, in comparison to that present in the shoot, represents only a minor component in the determination of tomato fruit yield.     

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.     

The fruit cuticles of wild tomato species exhibit architectural and chemical diversity, providing a new model for studying the evolution of cuticle function

The cuticle covers the aerial epidermis of land plants and plays a primary role in water regulation and protection from external stresses. Remarkable species diversity in the structure and composition of its components, cutin and wax, have been catalogued, but few functional or genetic correlations have emerged. Tomato (Solanum lycopersicum) is part of a complex of closely related wild species endemic to the northern Andes and the Galapagos Islands (Solanum Sect. Lycopersicon). Although sharing an ancestor <7 million years ago, these species are found in diverse environments and are subject to unique selective pressures. Furthermore, they are genetically tractable, since they can be crossed with S. lycopersicum, which has a sequenced genome. With the aim of evaluating the relationships between evolution, structure and function of the cuticle, we characterized the morphological and chemical diversity of fruit cuticles of seven species from Solanum Sect. Lycopersicon. Striking differences in cuticular architecture and quantities of cutin and waxes were observed, with the wax coverage of wild species exceeding that of S. lycopersicum by up to seven fold. Wax composition varied in the occurrence of wax esters and triterpenoid isomers. Using a Solanum habrochaites introgression line population, we mapped triterpenoid differences to a genomic region that includes two S. lycopersicum triterpene synthases. Based on known metabolic pathways for acyl wax compounds, hypotheses are discussed to explain the appearance of wax esters with atypical chain lengths. These results establish a model system for understanding the ecological and evolutionary functional genomics of plant cuticles.     

Metabolite analysis for the comparison of irrigated and non-irrigated field grown tomato of varying genotype

Every year the consequences of water deficit on crop yield and quality are profound. The observation that many wild species relatives of cultivated crops display a greater stress tolerance and the fact that the cultivated species generally display only a fraction of the allelic diversity available within the tomato clade suggest that crossing of wild species with elite cultivars could improve the stress physiology of modern crops. To assess this from the basis of chemical composition we applied an established GC-MS based metabolite profiling method to fruits from irrigated and non-irrigated tomato plants either of the cultivated tomato (Solanum lycopersicum) or of its hybrid with its wild species relative (Solanum pennellii). Results are discussed in terms of both the metabolic response to drought stress and the potential of utilizing exotic germplasm as a means to improve agronomically important characteristics of crop species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A reevaluation of the key factors that influence tomato fruit softening and integrity

The softening of fleshy fruits, such as tomato (Solanum lycopersicum), during ripening is generally reported to result principally from disassembly of the primary cell wall and middle lamella. However, unsuccessful attempts to prolong fruit firmness by suppressing the expression of a range of wall-modifying proteins in transgenic tomato fruits do not support such a simple model. 'Delayed Fruit Deterioration' (DFD) is a previously unreported tomato cultivar that provides a unique opportunity to assess the contribution of wall metabolism to fruit firmness, since DFD fruits exhibit minimal softening but undergo otherwise normal ripening, unlike all known nonsoftening tomato mutants reported to date. Wall disassembly, reduced intercellular adhesion, and the expression of genes associated with wall degradation were similar in DFD fruit and those of the normally softening 'Ailsa Craig'. However, ripening DFD fruit showed minimal transpirational water loss and substantially elevated cellular turgor. This allowed an evaluation of the relative contribution and timing of wall disassembly and water loss to fruit softening, which suggested that both processes have a critical influence. Biochemical and biomechanical analyses identified several unusual features of DFD cuticles and the data indicate that, as with wall metabolism, changes in cuticle composition and architecture are an integral and regulated part of the ripening program. A model is proposed in which the cuticle affects the softening of intact tomato fruit both directly, by providing a physical support, and indirectly, by regulating water status.     

Temporally extended gene expression of the ADP-Glc pyrophosphorylase large subunit (AgpL1) leads to increased enzyme activity in developing tomato fruit

Tomato plants (Solanum lycopersicum) harboring the allele for the AGPase large subunit (AgpL1) derived from the wild species Solanum habrochaites (AgpL1 ^H ) are characterized by higher AGPase activity and increased starch content in the immature fruit, as well as higher soluble solids in the mature fruit following the breakdown of the transient starch, as compared to fruits from plants harboring the cultivated tomato allele (AgpL1 ^E ). Comparisons of AGPase subunit gene expression and protein levels during fruit development indicate that the increase in AGPase activity correlates with a prolonged expression of the AgpL1 gene in the AgpL1 ^H high starch line, leading to an extended presence of the L1 protein. The S1 (small subunit) protein also remained for an extended period of fruit development in the AgpL1 ^H fruit, linked to the presence of the L1 protein. There were no discernible differences between the kinetic characteristics of the partially purified AGPase-L1^E and AGPase-L1^H enzymes. The results indicate that the increased activity of AGPase in the AgpL1 ^H tomatoes is due to the extended expression of the regulatory L1 and to the subsequent stability of the heterotetramer in the presence of the L1 protein, implying a role for the large subunit not only in the allosteric control of AGPase activity but also in the stability of the AGPase L1–S1 heterotetramer. The introgression line of S. lycopersicum containing the wild species AgpL1 ^H allele is a novel example of transgressive heterosis in which the hybrid multimeric enzyme shows higher activity due to a modulated temporal expression of one of the subunits.     

Trait discovery and editing in tomato

Tomato (Solanum lycopersicum), which is used for both processing and fresh markets, is a major crop species that is the top ranked vegetable produced over the world. Tomato is also a model species for research in genetics, fruit development and disease resistance. Genetic resources available in public repositories comprise the 12 wild related species and thousands of landraces, modern cultivars and mutants. In addition, high quality genome sequences are available for cultivated tomato and for several wild relatives, hundreds of accessions have been sequenced, and databases gathering sequence data together with genetic and phenotypic data are accessible to the tomato community. Major breeding goals are productivity, resistance to biotic and abiotic stresses, and fruit sensorial and nutritional quality. New traits, including resistance to various biotic and abiotic stresses and root architecture, are increasingly being studied. Several major mutations and quantitative trait loci (QTLs) underlying traits of interest in tomato have been uncovered to date and, thanks to new populations and advances in sequencing technologies, the pace of trait discovery has considerably accelerated. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing (GE) already proved its remarkable efficiency in tomato for engineering favorable alleles and for creating new genetic diversity by gene disruption, gene replacement, and precise base editing. Here, we provide insight into the major tomato traits and underlying causal genetic variations discovered so far and review the existing genetic resources and most recent strategies for trait discovery in tomato. Furthermore, we explore the opportunities offered by CRISPR/Cas9 and their exploitation for trait editing in tomato.     

Tomato GDSL1 Is Required for Cutin Deposition in the Fruit Cuticle

The plant cuticle consists of cutin, a polyester of glycerol, hydroxyl, and epoxy fatty acids, covered and filled by waxes. While the biosynthesis of cutin building blocks is well documented, the mechanisms underlining their extracellular deposition remain unknown. Among the proteins extracted from dewaxed tomato (Solanum lycopersicum) peels, we identified GDSL1, a member of the GDSL esterase/acylhydrolase family of plant proteins. GDSL1 is strongly expressed in the epidermis of growing fruit. In GDSL1-silenced tomato lines, we observed a significant reduction in fruit cuticle thickness and a decrease in cutin monomer content proportional to the level of GDSL1 silencing. A significant decrease of wax load was observed only for cuticles of the severely silenced transgenic line. Fourier transform infrared (FTIR) analysis of isolated cutins revealed a reduction in cutin density in silenced lines. Indeed, FTIR-attenuated total reflectance spectroscopy and atomic force microscopy imaging showed that drastic GDSL1 silencing leads to a reduction in ester bond cross-links and to the appearance of nanopores in tomato cutins. Furthermore, immunolabeling experiments attested that GDSL1 is essentially entrapped in the cuticle proper and cuticle layer. These results suggest that GDSL1 is specifically involved in the extracellular deposition of the cutin polyester in the tomato fruit cuticle.     

Mode of inheritance of primary metabolic traits in tomato

To evaluate components of fruit metabolic composition, we have previously metabolically phenotyped tomato (Solanum lycopersicum) introgression lines containing segmental substitutions of wild species chromosome in the genetic background of a cultivated variety. Here, we studied the hereditability of the fruit metabolome by analyzing an additional year's harvest and evaluating the metabolite profiles of lines heterozygous for the introgression (ILHs), allowing the evaluation of putative quantitative trait locus (QTL) mode of inheritance. These studies revealed that most of the metabolic QTL (174 of 332) were dominantly inherited, with relatively high proportions of additively (61 of 332) or recessively (80 of 332) inherited QTL and a negligible number displaying the characteristics of overdominant inheritance. Comparison of the mode of inheritance of QTL revealed that several metabolite pairs displayed a similar mode of inheritance of QTL at the same chromosomal loci. Evaluation of the association between morphological and metabolic traits in the ILHs revealed that this correlation was far less prominent, due to a reduced variance in the harvest index within this population. These data are discussed in the context of genomics-assisted breeding for crop improvement, with particular focus on the exploitation of wide biodiversity.     

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.     

SUN regulates vegetative and reproductive organ shape by changing cell division patterns

One of the major genes controlling the elongated fruit shape of tomato (Solanum lycopersicum) is SUN. In this study, we explored the roles of SUN in vegetative and reproductive development using near isogenic lines (NILs) that differ at the sun locus, and SUN overexpressors in both the wild species LA1589 (Solanum pimpinellifolium) and the cultivar Sun1642 background. Our results demonstrate that SUN controls tomato shape through redistribution of mass that is mediated by increased cell division in the longitudinal and decreased cell division in the transverse direction of the fruit. The expression of SUN is positively correlated with slender phenotypes in cotyledon, leaflet, and floral organs, an elongated ovary, and negatively correlated with seed weight. Overexpression of SUN leads to more extreme phenotypes than those shown in the NILs and include thinner leaf rachises and stems, twisted leaf rachises, increased serrations of the leaflets, and dramatically increased elongation at the proximal end of the ovary and fruit. In situ hybridizations of the NILs showed that SUN is expressed throughout the ovary and young fruit, particularly in the vascular tissues and placenta surface, and in the ovules and developing seed. The phenotypic effects resulting from high expression of SUN suggest that the gene is involved in several plant developmental processes.