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

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

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

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

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

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

Canalization of Tomato Fruit Metabolism.

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

A New Advanced Backcross Tomato Population Enables High Resolution Leaf QTL Mapping and Gene Identification.

Quantitative Trait Loci (QTL) mapping is a powerful technique for dissecting the genetic basis of traits and species differences. Established tomato mapping populations between domesticated tomato (Solanum lycopersicum) and its more distant interfertile relatives typically follow a near isogenic line (NIL) design, such as the S. pennellii Introgression Line (IL) population, with a single wild introgression per line in an otherwise domesticated genetic background. Here, we report on a new advanced backcross QTL mapping resource for tomato, derived from a cross between the M82 tomato cultivar and S. pennellii This so-called Backcrossed Inbred Line (BIL) population is comprised of a mix of BC2 and BC3 lines, with domesticated tomato as the recurrent parent. The BIL population is complementary to the existing S. pennellii IL population, with which it shares parents. Using the BILs, we mapped traits for leaf complexity, leaflet shape, and flowering time. We demonstrate the utility of the BILs for fine-mapping QTL, particularly QTL initially mapped in the ILs, by fine-mapping several QTL to single or few candidate genes. Moreover, we confirm the value of a backcrossed population with multiple introgressions per line, such as the BILs, for epistatic QTL mapping. Our work was further enabled by the development of our own statistical inference and visualization tools, namely a heterogeneous hidden Markov model for genotyping the lines, and by using state-of-the-art sparse regression techniques for QTL mapping.

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.

Domestication selected for deceleration of the circadian clock in cultivated tomato.

The circadian clock is a critical regulator of plant physiology and development, controlling key agricultural traits in crop plants. In addition, natural variation in circadian rhythms is important for local adaptation. However, quantitative modulation of circadian rhythms due to artificial selection has not yet been reported. Here we show that the circadian clock of cultivated tomato (Solanum lycopersicum) has slowed during domestication. Allelic variation of the tomato homolog of the Arabidopsis gene EID1 is responsible for a phase delay. Notably, the genomic region harboring EID1 shows signatures of a selective sweep. We find that the EID1 allele in cultivated tomatoes enhances plant performance specifically under long day photoperiods, suggesting that humans selected slower circadian rhythms to adapt the cultivated species to the long summer days it encountered as it was moved away from the equator.

In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network.

Plant glandular secreting trichomes are epidermal protuberances that produce structurally diverse specialized metabolites, including medically important compounds. Trichomes of many plants in the nightshade family (Solanaceae) produce O-acylsugars, and in cultivated and wild tomatoes these are mixtures of aliphatic esters of sucrose and glucose of varying structures and quantities documented to contribute to insect defense. We characterized the first two enzymes of acylsucrose biosynthesis in the cultivated tomato Solanum lycopersicum. These are type I/IV trichome-expressed BAHD acyltransferases encoded by Solyc12g006330--or S. lycopersicum acylsucrose acyltransferase 1 (Sl-ASAT1)--and Solyc04g012020 (Sl-ASAT2). These enzymes were used--in concert with two previously identified BAHD acyltransferases--to reconstruct the entire cultivated tomato acylsucrose biosynthetic pathway in vitro using sucrose and acyl-CoA substrates. Comparative genomics and biochemical analysis of ASAT enzymes were combined with in vitro mutagenesis to identify amino acids that influence CoA ester substrate specificity and contribute to differences in types of acylsucroses that accumulate in cultivated and wild tomato species. This work demonstrates the feasibility of the metabolic engineering of these insecticidal metabolites in plants and microbes.

A Feedback-Insensitive Isopropylmalate Synthase Affects Acylsugar Composition in Cultivated and Wild Tomato.

Acylsugars are insecticidal specialized metabolites produced in the glandular trichomes of plants in the Solanaceae family. In the tomato clade of the Solanum genus, acylsugars consist of aliphatic acids of different chain lengths esterified to sucrose, or less frequently to glucose. Through liquid chromatography-mass spectrometry screening of introgression lines, we previously identified a region of chromosome 8 in the Solanum pennellii LA0716 genome (IL8-1/8-1-1) that causes the cultivated tomato Solanum lycopersicum to shift from producing acylsucroses with abundant 3-methylbutanoic acid acyl chains derived from leucine metabolism to 2-methylpropanoic acid acyl chains derived from valine metabolism. We describe multiple lines of evidence implicating a trichome-expressed gene from this region as playing a role in this shift. S. lycopersicum M82 SlIPMS3 (Solyc08g014230) encodes a functional end product inhibition-insensitive version of the committing enzyme of leucine biosynthesis, isopropylmalate synthase, missing the carboxyl-terminal 160 amino acids. In contrast, the S. pennellii LA0716 IPMS3 allele found in IL8-1/8-1-1 encodes a nonfunctional truncated IPMS protein. M82 transformed with an SlIPMS3 RNA interference construct exhibited an acylsugar profile similar to that of IL8-1-1, whereas the expression of SlIPMS3 in IL8-1-1 partially restored the M82 acylsugar phenotype. These IPMS3 alleles are polymorphic in 14 S. pennellii accessions spread throughout the geographical range of occurrence for this species and are associated with acylsugars containing varying amounts of 2-methylpropanoic acid and 3-methylbutanoic acid acyl chains.

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.

Plant domestication versus crop evolution: a conceptual framework for cereals and grain legumes.

'Domestication syndrome' (DS) denotes differences between domesticated plants and their wild progenitors. Crop plants are dynamic entities; hence, not all parameters distinguishing wild progenitors from cultigens resulted from domestication. In this opinion article, we refine the DS concept using agronomic, genetic, and archaeobotanical considerations by distinguishing crucial domestication traits from traits that probably evolved post-domestication in Near Eastern grain crops. We propose that only traits showing a clear domesticated-wild dimorphism represent the pristine domestication episode, whereas traits showing a phenotypic continuum between wild and domesticated gene pools mostly reflect post-domestication diversification. We propose that our approach may apply to other crop types and examine its implications for discussing the timeframe of plant domestication and for modern plant science and breeding.

Resolution by recombination: breaking up Solanum pennellii introgressions.

Quantitative trait locus (QTL) genetics retains an important role in the study of biological and agronomic processes; however, its genetic resolution is often comparatively low. Community-based strategies are thus required to address this issue. Here we detail such a strategy wherein the widely used Solanum pennellii introgression lines (ILs) in the genetic background of the cultivated tomato (Solanum lycopersicum) are broken up into molecular marker-defined sublines as a community resource for map-based cloning.