Comparative structural profiling of trichome specialized metabolites in tomato (Solanum lycopersicum) and S. habrochaites: acylsugar profiles revealed by UHPLC/MS and NMR

Many plants accumulate large quantities of specialized metabolites in secretory glandular trichomes (SGTs), which are specialized epidermal cells. In the genus Solanum, SGTs store a diverse collection of glucose and sucrose esters. Profiling of extracts from two accessions (LA1777 and LA1392) of Solanum habrochaites using ultra-high performance liquid chromatography–mass spectrometry (UHPLC/MS) revealed wide acylsugar diversity, with up to 11 isomers annotated for each individual elemental formula. These isomers arise from differences in ester chain lengths and their positions of substitution or branching. Since fragment ion masses were not sufficient to distinguish all isomers, 24 acylsucroses were purified from S. habrochaites accessions and cultivated tomato (Solanum lycopersicum M82) and characterized using NMR spectroscopy. Two-dimensional NMR spectra yielded assignments of positions of substitution of specific acyl groups, and locations of branching. The range of substitution was wider than reported earlier, and in contrast to previous reports, tetra- and penta-acylsucroses were substituted at position 2 with acyl groups other than acetate. Because UHPLC/MS fails to yield sufficient information about structure diversity, and quantitative NMR of acylsugar mixtures is confounded by structural redundancy, the strategic combination of NMR and UHPLC/MS provides a powerful approach for profiling a class of metabolites with great structural diversity across genotypes.     

Differential and Synergistic Functionality of Acylsugars in Suppressing Oviposition by Insect Herbivores

Acylsugars are secondary metabolites exuded from type IV glandular trichomes that provide broad-spectrum insect suppression for Solanum pennellii Correll, a wild relative of cultivated tomato. Acylsugars produced by different S. pennellii accessions vary by sugar moieties (glucose or sucrose) and fatty acid side chains (lengths and branching patterns). Our objective was to determine which acylsugar compositions more effectively suppressed oviposition of the whitefly Bemisia tabaci (Gennadius) (Middle East—Asia Minor 1 Group), tobacco thrips, Frankliniella fusca (Hinds), and western flower thrips, Frankliniella occidentalis (Pergande). We extracted and characterized acylsugars from four S. pennellii accessions with different compositions, as well as from an acylsugar-producing tomato breeding line. We also fractionated the acylsugars of one S. pennellii accession to examine the effects of its components. Effects of acylsugars on oviposition were evaluated by administering a range of doses to oviposition sites of adult whiteflies and thrips in non-choice and choice bioassays, respectively. The acylsugars from S. pennellii accessions and the tomato breeding line demonstrated differential functionality in their ability to alter the distribution of whitefly oviposition and suppress oviposition on acylsugar treated substrates. Tobacco thrips were sensitive to all compositions while western flower thrips and whiteflies were more sensitive to acylsugars from a subset of S. pennellii accessions. It follows that acylsugars could thus mediate plant-enemy interactions in such a way as to affect evolution of host specialization, resistance specificity, and potentially host differentiation or local adaptation. The acylsugars from S. pennellii LA1376 were separated by polarity into two fractions that differed sharply for their sugar moieties and fatty acid side chains. These fractions had different efficacies, with neither having activity approaching that of the original exudate. When these two fractions were recombined, the effect on both whiteflies and thrips exceeded the sum of the two fractions’ effects, and was similar to that of the original exudate. These results suggest that increasing diversity of components within a mixture may increase suppression through synergistic interactions. This study demonstrates the potential for composition-specific deployment of acylsugars for herbivore oviposition suppression, either through in planta production by tomato lines, or as biocides applied by a foliar spray.     

Striking Natural Diversity in Glandular Trichome Acylsugar Composition Is Shaped by Variation at the Acyltransferase2 Locus in the Wild Tomato Solanum habrochaites

Acylsugars are polyesters of short- to medium-length acyl chains on sucrose or glucose backbones that are produced in secretory glandular trichomes of many solanaceous plants, including cultivated tomato (Solanum lycopersicum). Despite their roles in biotic stress adaptation and their wide taxonomic distribution, there is relatively little information about the diversity of these compounds and the genes responsible for their biosynthesis. In this study, acylsugar diversity was assessed for 80 accessions of the wild tomato species Solanum habrochaites from throughout the Andes Mountains. Trichome metabolites were analyzed by liquid chromatography-time of flight-mass spectrometry, revealing the presence of at least 34 structurally diverse acylsucroses and two acylglucoses. Distinct phenotypic classes were discovered that varied based on the presence of glucose or sucrose, the numbers and lengths of acyl chains, and the relative total amounts of acylsugars. The presence or absence of an acetyl chain on the acylsucrose hexose ring caused clustering of the accessions into two main groups. Analysis of the Acyltransferase2 gene (the apparent ortholog of Solyc01g105580) revealed differences in enzyme activity and gene expression correlated with polymorphism in S. habrochaites accessions that varied in acylsucrose acetylation. These results are consistent with the hypothesis that glandular trichome acylsugar acetylation is under selective pressure in some populations of S. habrochaites and that the gene mutates to inactivity in the absence of selection.Trichomes are specialized epidermal cells that protrude from the surface of a variety of plant tissues. They are thought to protect against environmental stresses such as herbivory (Kang et al., 2010a; Weinhold and Baldwin, 2011), loss of water through transpiration, and UV irradiation (Zhou et al., 2007). In particular, secreting glandular trichomes (SGTs) serve as “chemical factories” where specialized metabolites are produced, stored, or volatized (Wagner, 1991; Schilmiller et al., 2008, 2010a). In addition, SGTs produce and secrete proteins on the plant surface for insect protection (Yu et al., 1992; Thipyapong et al., 1997) and pathogen defense (Shepherd et al., 2005). SGTs also contribute to the taste and smell of plants by releasing volatile metabolites. For example, the distinctive aromas of many Mediterranean herbs of the Lamiaceae (mint family) derive from SGTs (Schilmiller et al., 2008), and compounds from the glands of hops (Humulus lupulus in the Cannabaceae) contribute to beer flavor and aroma (Wang et al., 2008). Furthermore, a number of SGT-borne metabolites are commercially valuable, especially for pharmaceutical purposes. For example, artemisinin, a widely used antimalarial, is a sesquiterpene lactone from the trichomes of Artemisia annua (Liu et al., 2011). In addition to their value in foods and medicines, trichomes provide excellent models for analyzing biosynthetic enzymes and pathways (Schilmiller et al., 2008, 2009, 2012b; Bohlmann and Gershenzon, 2009; Sallaud et al., 2009).Plants in the genus Solanum include important crop species such as potato (Solanum tuberosum), eggplant (Solanum melongena), and tomato (Solanum lycopersicum). Previous studies reported that SGTs of cultivated tomato and its wild relatives accumulate high levels of exudates containing a variety of specialized metabolites, for example flavonoids, alkaloids, and terpenoids (Wagner, 1991; Schilmiller et al., 2008, 2010a; McDowell et al., 2011). Cultivated tomato and its wild relatives have morphologically and chemically diverse trichomes. For example, Luckwill (1943) defined seven morphologically distinguishable types of trichomes in plants of this genus, including four glandular types (types 1, 4, 6, and 7; Supplemental Fig. S1; for more Solanum spp. trichome images, see Kang et al., 2010a, 2010b). The presence of specific types of trichomes and their densities vary across species and even within a single plant according to tissue types, developmental stages, and environmental conditions (Werker, 2000; Li et al., 2004). These morphologically distinct SGTs vary in the amounts and types of metabolites that they produce, accumulate, and/or secrete (Werker, 2000). For example, S. lycopersicum M82 leaf type 6 SGTs accumulate the sesquiterpenes β-caryophyllene and α-humulene, while the glands on the stem lack these metabolites (Schilmiller et al., 2010b). There are also species- and accession-specific differences in SGT metabolite profiles. For instance, methylketones accumulate in type 6 glands of a subset of Solanum habrochaites accessions (Fridman et al., 2005; Yu et al., 2010). Similarly, acylglucoses are highly abundant in type 4 glands of Solanum pennellii LA0716, while acylsucroses predominate in S. lycopersicum and S. habrochaites (Shapiro et al., 1994; McDowell et al., 2011). The chemical and morphological diversity of trichomes in different Solanum species and accessions makes the genus an attractive target for the identification of diverse trichome-borne metabolites and the major biosynthetic pathways responsible for their synthesis operating in each trichome type.The value of the comparative metabolomics approach in trichomes was recently demonstrated in studies of Solanum spp. trichome monoterpene and sesquiterpene biosynthesis (Bohlmann and Gershenzon, 2009; Sallaud et al., 2009; Schilmiller et al., 2009). It was discovered that S. lycopersicum SGTs synthesize monoterpenes from the cis-prenyldiphosphate intermediate neryldiphosphate (Sallaud et al., 2009; Schilmiller et al., 2009). This is contrary to the previous paradigm, where the trans-prenyldiphosphate geranyldiphosphate was considered the universal intermediate for monoterpene biosynthesis. An analogous example of biosynthetic innovation was reported for SGTs of S. habrochaites LA1777 (Sallaud et al., 2009), shown to produce sesquiterpenes in the plastid using the all-cis-prenyldiphosphate substrate Z,Z-farnesyldiphosphate. This is counter to the commonly described cytosolic sesquiterpene pathway, which uses the all-trans-sesquiterpene synthase substrate E,E-farnesyldiphosphate. Furthermore, a recent study demonstrated chemical diversity of trichome terpenes in geographically distinct S. habrochaites accessions associated with the evolution of terpene synthases, revealing how the plasticity of biosynthetic enzymes contributes to chemical complexity and diversity (Gonzales-Vigil et al., 2012). These observations suggest that trichome specialized metabolism is evolutionarily plastic, perhaps due to selective pressure from insects or other environmental stress agents.Acylsugars are sticky exudates made in SGTs that are thought to physically or chemically improve plant defense (Mirnezhad et al., 2010; Weinhold and Baldwin, 2011). Results from the literature indicate strong acylsugar diversity in various Solanum spp. trichomes (Schilmiller et al., 2010a, 2010b; McDowell et al., 2011). Acylsugars are categorized as either Suc or Glc esters based on the type of sugar core (Fig. 1), and they also have varying numbers and lengths of acyl chains decorating the sugar moiety. In particular, S. pennellii accumulates enormous amounts of acylsugars, up to 20% of leaf dry weight (Fobes et al., 1985). In addition, previously published data showed that total acylsugars in geographically distinct S. pennellii accessions vary in quantity, the proportion of Suc or Glc backbones, and the overall types of fatty acid esters (FAs) on the sugars (Shapiro et al., 1994). However, this study did not identify specific acylsugar types.Open in a separate windowFigure 1.Structural classes of acylsugars in Solanum species. A, Schematic structure of an acylglucose. The structure shown depicts a Glc triester composed of Glc and three acyl chains with various numbers of carbons represented as R. B, Schematic structure of an acylsucrose. The proposed structure shows a Suc tetraester with three acyl chains on the Glc ring and one on the Fru ring. If the sugar moiety is decorated with three, four, or five acyl chains, it is referred to as a Suc triester, tetraester, or pentaester, respectively. The positions of the acyl chains are currently unknown, with the exception of the most abundant acylsugar in cultivated tomato (M82) that was structurally characterized by NMR (Schilmiller et al., 2010a). In addition, a few acylsugars were isolated and reported from S. habrochaites and other species by King et al. (1990, 1993). Note the changes in nomenclature since these papers were published: Lycopersicum typicum LA1777 is now called S. habrochaites LA1777, and Lycopersicum hirsutum has been changed to S. habrochaites.To explore the detailed acylsugar chemotypes within accessions of one species, we focused on 80 accessions collected throughout the geographical range of S. habrochaites in Peru and Ecuador (Supplemental Table S1). We describe differences in sugar backbone as well as numbers and lengths of acyl chains, including the presence or absence of an acetyl group, which we found to be a major difference in accessions from the southern and northern Andes Mountains. The recent identification of the acyltransferase2 enzyme (SlAT2; encoded by Solyc01g105580), involved in acylsucrose biosynthesis in S. lycopersicum (Schilmiller et al., 2012a), permitted a test of the hypothesis that differences in expression or activity of this enzyme play an important role in the chemical diversity observed. The results extend previous evidence that Solanum spp. SGT chemistry is highly dynamic (Gonzales-Vigil et al., 2012) and show that the AT2 gene is surprisingly diverse across populations of S. habrochaites.     

Migration through a Major Andean Ecogeographic Disruption as a Driver of Genetic and Phenotypic Diversity in a Wild Tomato Species

Evolutionary dynamics at the population level play a central role in creating the diversity of life on our planet. In this study, we sought to understand the origins of such population-level variation in mating systems and defensive acylsugar chemistry in Solanum habrochaites—a wild tomato species found in diverse Andean habitats in Ecuador and Peru. Using Restriction-site-Associated-DNA-Sequencing (RAD-seq) of 50 S. habrochaites accessions, we identified eight population clusters generated via isolation and hybridization dynamics of 4–6 ancestral populations. Detailed characterization of mating systems of these clusters revealed emergence of multiple self-compatible (SC) groups from progenitor self-incompatible populations in the northern part of the species range. Emergence of these SC groups was also associated with fixation of deleterious alleles inactivating acylsugar acetylation. The Amotape-Huancabamba Zone—a geographical landmark in the Andes with high endemism and isolated microhabitats—was identified as a major driver of differentiation in the northern species range, whereas large geographical distances contributed to population structure and evolution of a novel SC group in the central and southern parts of the range, where the species was also inferred to have originated. Findings presented here highlight the role of the diverse ecogeography of Peru and Ecuador in generating population differentiation, and enhance our understanding of the microevolutionary processes that create biological diversity.     

Evolution of TPS20-related terpene synthases influences chemical diversity in the glandular trichomes of the wild tomato relative Solanum habrochaites

A systematic screen of volatile terpene production in the glandular trichomes of 79 accessions of Solanum habrochaites was conducted and revealed the presence of 21 mono‐ and sesquiterpenes that exhibit a range of qualitative and quantitative variation. Hierarchical clustering identified distinct terpene phenotypic modules with shared patterns of terpene accumulation across accessions. Several terpene modules could be assigned to previously identified terpene synthase (TPS) activities that included members of the TPS‐e/f subfamily that utilize the unusual cis‐prenyl diphosphate substrates neryl diphosphate and 2z,6z‐farnesyl diphosphate. DNA sequencing and in vitro enzyme activity analysis of TPS‐e/f members from S. habrochaites identified three previously unassigned enzyme activities that utilize these cisoid substrates. These produce either the monoterpenes α‐pinene and limonene, or the sesquiterpene 7‐epizingiberene, with the in vitro analyses that recapitulated the trichome chemistry found in planta. Comparison of the distribution of S. habrochaites accessions with terpene content revealed a strong preference for the presence of particular TPS20 alleles at distinct geographic locations. This study reveals that the unusually high intra‐specific variation of volatile terpene synthesis in glandular trichomes of S. habrochaites is due at least in part to evolution at the TPS20 locus.     

Quantitative trait loci regulating sugar moiety of acylsugars in tomato

Acylsugars are broad-spectrum insect resistance sugar esters produced at very high levels by some accessions of the wild tomato, Solanum pennellii. Transferring acylsugar production from S. pennellii LA716 to cultivated tomato through traditional breeding developed the benchmark acylsugar breeding line CU071026. The base moiety of acylsugars (sucrose vs. glucose) can vary among S. pennellii accessions. Additionally the accession S. pennellii LA716 produces almost exclusively acylglucoses, but the breeding line CU071026 derived from S. pennellii LA716 produces exclusively acylsucroses. This study uses a BC1F1 and a BC1F2 population derived from the cross CU071026 × (CU071026 × S. pennellii LA716) to identify and confirm the action of three quantitative trait loci (QTL) on chromosomes 3, 4, and 11. The QTL on chromosomes 3 and 11 are both required for acylglucose production, while addition of the chromosome 4 QTL affects the level of acylglucose produced in the presence of the QTL on chromosomes 3 and 11. A three-way interaction between these acylglucose QTL was confirmed with a post hoc ANOVA. Identification of these three QTL provides a blueprint for breeding to shift acylsucrose production to acylglucose production in tomato breeding lines. The implications of these QTL and two additional QTL affecting total acylsugar level in the BC1F2 are discussed.     

Identification of quantitative trait loci associated with acylsugar accumulation using intraspecific populations of the wild tomato, Lycopersicon pennellii

 Lycopersicon pennellii LA716, a wild relative of tomato, is resistant to a number of insect pests due to the accumulation of acylsugars exuded from type IV trichomes. These acylsugars are a class of compounds including both acylglucoses and acylsucroses. Intraspecific populations between L. pennellii LA716 and L. pennellii LA1912, the latter an accession that assorts for low-level acylsugar accumulation, were created to study the inheritance of type IV trichome density, acylsugar accumulation levels, percentage of acylsugars that are acylglucoses, and leaf area. The F₂ population was subsequently used to determine genomic regions associated with these traits. The relative proportion of acylglucoses and acylsucroses was found to be largely controlled by a single locus near TG549 on chromosome 3. One locus on chromosome 10 showed significant associations with acylsugar levels. In addition, 1 locus on chromosome 4 showed significant associations with leaf area. Ten additional loci showed modest associations with one or more of the traits examined, 5 of which have been previously reported. Received: 13 March 1997 / Accepted: 19 September 1997     

Combination of QTL affecting acylsugar chemistry reveals additive and epistatic genetic interactions to increase acylsugar profile diversity

The tomato breeding line, CU071026, was bred to accumulate high levels of the insect control compounds called acylsugars, which are exuded from glandular trichomes. The acylsugars of CU071026 exhibit a characteristic profile of acylsugar composition and constituent fatty acid acyl groups that is distinct from that of the progenitor wild tomato, Solanum pennellii LA716. A prior study reported the transfer of three QTL (FA2, FA7, and FA8), from S. pennellii LA716, that are associated with changes in acylsugar chemistry into CU071026 and demonstrated that the resulting lines, each of which possesses one of these QTL, displayed a unique acylsugar and fatty acid profile distinct from that characteristic of the acylsugars of CU071026 and each other. The current study utilized marker-assisted backcrossing to combine pairs of two of these QTL or all three of these QTL. This created a new set of lines, which allowed evaluation of the combinatory effects of FA2QTL, FA7QTL, and FA8QTL, on acylsugar level and acylsugar and fatty acid profile and diversity. Analysis of the new tomato lines revealed that these QTL interacted additively and epistatically to alter acylsugar level and chemistry, increasing the diversity of fatty acid constituents and/or acylsugar chemotypes present in the exudates of some of the lines. Extensive characterization of the lines for acylsugar level, through a spectrophotometric invertase assay, and acylsugar chemistry, through gas and liquid chromatography-mass spectrometry, allowed association of the QTL interactions with aspects of acylsugar chemotype. The evaluated fatty acids and acylsugars accumulated by the set of lines generally displayed high heritability and minimal environmental effect, which is discussed. The QTL interactions that govern a more diverse acylsugar and fatty acid profile provide valuable information for the generation of tomato lines with improved acylsugar efficacy against pests of tomato.     

Combination of Acylglucose QTL reveals additive and epistatic genetic interactions and impacts insect oviposition and virus infection

Acylsugars are important insect defense compounds produced at high levels by glandular trichomes of the wild tomato, Solanum pennellii. Marker-assisted selection was used to select for plants containing the three AGQTL named AG3QTL, AG4QTL, and AG11QTL from self-pollinated populations derived from an interspecific backcross population of CU071026 x (CU071026 x S. pennellii LA716). High acylglucose-accumulating lines were selected from these populations that possess these three AGQTL and the fewest number of extraneous S. pennellii LA716 introgressions. Incorporation of these three acylglucose QTL in the presence of the five standard S. pennellii introgressions of CU071026 altered acylsugar level and sugar moiety, demonstrating epistatic interactions between the acylglucose QTL on both of these traits. Comparison of the lines generated from the two breeding techniques indicated the three acylglucose QTL are essential but not necessarily sufficient for the production of elevated levels of acylglucose acylsugars. Fine-mapping of AG3QTL, AG4QTL, and AG11QTL resulted in less than 1 Mbp intervals for the locations of AG4QTL and AG11QTL; proposals of the causal genes underlying these acylglucose QTL are discussed. Characterization of the fatty acid profile of lines selected out of the interspecific backcross populations revealed an increase in the proportion of acylsugar n-C10 fatty acid acyl chains, possibly governed by one or more of the three acylglucose QTL. Characterization of the acylsugar profile of acylglucose lines selected from the interspecific backcross populations also demonstrated interactions among the acylglucose QTL to further modulate the diversity of acylsugars accumulated. Evaluation of an acylglucose line and controls against the tomato insect pest Frankliniella occidentalis demonstrated that levels of resistance differed among these lines and that the acylsugars accumulated by the acylglucose line were effective at reducing both F. occidentalis oviposition and incidence of Tomato spotted wilt orthotospovirus. However, of some of the acylglucose lines and hybrids tested against Spodoptera exigua did not indicate differences for larval weight gain and survival.     

Introgression of acylsugar chemistry QTL modifies the composition and structure of acylsugars produced by high-accumulating tomato lines

Acylsugars are important insect defense compounds produced at high levels by glandular trichomes of the wild tomato, Solanum pennellii. The ability to produce acylsugars at elevated levels was bred into the tomato line CU071026. This study utilized a marker-assisted backcross approach to individually introgress into CU071026 and to fine map the three quantitative trait loci (QTL) fatty acid 5 (FA5QTL), fatty acid 7 (FA7QTL), and fatty acid 8 (FA8QTL), which were previously associated with changes in acylsugar chemistry. Additional breeding with and fine mapping the previously introgressed QTL, fatty acid 2 (FA2QTL), was also conducted. The effect of these four QTL on acylsugar quality and quantity in the presence of the five introgressions of CU071026 was evaluated. Incorporation of the QTL altered acylsugar chemotype by modulating the length, orientation, and/or relative proportion of fatty acid acyl groups. The resulting quantities of acylsugar produced in most of the new lines were similar to that of CU071026; however, introgression of FA5QTL reduced acylsugar levels. The acylsugar lines containing each QTL were characterized for acylsugar level, trichome abundance, and acylsugar chemistry through gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. The novel acylsugar chemotype lines created can contribute to elucidation of the mechanism of insect resistance mediated by acylsugars and help with identification of yet-unknown genes contributing to acylsugar synthesis and diversity.     

Profiling,isolation and structure elucidation of specialized acylsucrose metabolites accumulating in trichomes of <Emphasis Type="Italic">Petunia</Emphasis> species

Introduction

Acylsugar specialized metabolites function as defenses against insect herbivores, and are the most abundant specialized metabolites produced in Solanaceous trichomes. Metabolite profiling provides the foundation for determining the genetic basis of specialized metabolism and its evolution.

Objectives

To profile and identify acylsugar specialized metabolites in three Petunia species: P. axillaris, P. integrifolia and P. exserta.

Methods

Metabolites were profiled using ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOF MS). Metabolites were purified using solid phase extraction and HPLC, and structures were established using NMR spectroscopy.

Results

Twenty-eight distinct acylsucrose formulas, representing a sampling of more than 100 different detected chemical forms, were purified from three Petunia species and structures have been proposed based on one- and two-dimensional NMR data. 15 of the 28 purified acylsugars were sucrose pentaesters that possess a malonyl group on the fructose ring. These malonate esters can be readily distinguished from other acylsugars based on distinct masses of pseudomolecular ions and fragment ions generated using multiplexed collision-induced dissociation. Chemical diversity of acylsugars was observed between Petunia species, particularly with respect to the lengths of acyl chains and specific acylation positions.

Conclusions

These findings suggest substrate selectivity of various acyltransferases in Petunia species.

     

QTL analysis of pest resistance in the wild tomato Lycopersicon pennellii: QTLs controlling acylsugar level and composition

Some accessions of Lycopersicon pennellii, a wild relative of the tomato Lycopersicon esculentum, are resistant to a number of important pests of cultivated tomato due to the accumulation of acylsugars, which constitute 90% of the exudate of type-IV trichomes in L. pennellii LA716. An interspecific F₂ population, created by the cross L. esculentum x L. pennellii LA 716, was surveyed for acylsugar accumulation and subjected to RFLP/QTL analysis to determine the genomic regions associated with the accumulation of acylglucoses, acylsucroses, and total acylsugars, as well as with acylglucoses as a percentage of total acylsugars (mole percent acylglucoses). Data were analyzed using MAPMAKER/QTL with and without a log₁₀ transformation. A threshold value of 2.4 (default value for MAPMAKER/QTL) was used, as well as 95% empirically derived threshold values. Five genomic regions, two on chromosome 2 and one each on chromosomes 3, 4 and 11, were detected as being associated with one or more aspects of acylsugar production. The L. esculentum allele is partially dominant to the L. pennellii allele in the regions on chromosomes 2 and 11, but the L. pennellii allele is dominant in the region on chromosome 3. Throughout this study, we report the comparative effects of analytical methodology on the identification of acylsugar QTLs. Similarities between our results and published results for the genus Solanum are also discussed.R. W. Doerge · S.-C. Liu · J. P. Kuai contributed equally to the paper, and we ordered randomly     

Complex Epistasis for Dobzhansky–Muller Hybrid Incompatibility in Solanum

We examined the prevalence of interactions between pairs of short chromosomal regions from one species (Solanum habrochaites) co-introgressed into a heterospecific genetic background (Solanum lycopersicum). Of 105 double introgression line (DIL) families generated from a complete diallele combination of 15 chromosomal segments, 39 (~38%) showed evidence for complex epistasis in the form of genotypic and/or allelic marker transmission distortion in DIL F₂ populations.     

Coevolution and Hierarchical Interactions of Tomato mosaic virus and the Resistance Gene Tm-1

During antagonistic coevolution between viruses and their hosts, viruses have a major advantage by evolving more rapidly. Nevertheless, viruses and their hosts coexist and have coevolved, although the processes remain largely unknown. We previously identified Tm-1 that confers resistance to Tomato mosaic virus (ToMV), and revealed that it encodes a protein that binds ToMV replication proteins and inhibits RNA replication. Tm-1 was introgressed from a wild tomato species Solanum habrochaites into the cultivated tomato species Solanum lycopersicum. In this study, we analyzed Tm-1 alleles in S. habrochaites. Although most part of this gene was under purifying selection, a cluster of nonsynonymous substitutions in a small region important for inhibitory activity was identified, suggesting that the region is under positive selection. We then examined the resistance of S. habrochaites plants to ToMV. Approximately 60% of 149 individuals from 24 accessions were resistant to ToMV, while the others accumulated detectable levels of coat protein after inoculation. Unexpectedly, many S. habrochaites plants were observed in which even multiplication of the Tm-1-resistance-breaking ToMV mutant LT1 was inhibited. An amino acid change in the positively selected region of the Tm-1 protein was responsible for the inhibition of LT1 multiplication. This amino acid change allowed Tm-1 to bind LT1 replication proteins without losing the ability to bind replication proteins of wild-type ToMV. The antiviral spectra and biochemical properties suggest that Tm-1 has evolved by changing the strengths of its inhibitory activity rather than diversifying the recognition spectra. In the LT1-resistant S. habrochaites plants inoculated with LT1, mutant viruses emerged whose multiplication was not inhibited by the Tm-1 allele that confers resistance to LT1. However, the resistance-breaking mutants were less competitive than the parental strains in the absence of Tm-1. Based on these results, we discuss possible coevolutionary processes of ToMV and Tm-1.     

Quantitative trait loci increasing acylsugars in tomato breeding lines and their impacts on silverleaf whiteflies

Solanum pennellii LA716, a wild relative of tomato, produces acylsugars, an insect resistance compound with activity against many tomato insect pests. Breeding of cultivated tomato using S. pennellii LA716 as a donor parent has led to the development of the elite acylsugar-producing tomato breeding line CU071026. CU071026 contains five introgressed S. pennellii genomic regions, and produces acylsugars at moderate levels that are effective against insect pests. A BC1F1 population was created by crossing the F1 CU071026?×?S. pennellii LA716 with CU071026 as the recurrent parent; this BC1F1 population was used to identify additional regions of the S. pennellii genome important for further improvement of acylsugar production. This population was genotyped with 94 markers in the segregating regions and phenotyped for level of acylsugar production. Using QTLNetwork 2.1 for the detection of quantitative trait loci (QTL) and epistatic interactions, this study identified five QTL for total acylsugar level. Additionally, two epistatic interactions between QTL were found to control significant levels of total acylsugar production. Two of the QTL identified were further evaluated in silverleaf whitefly (Bemisia tabaci) field cage trials using acylsugar breeding lines that differ for the presence/absence of these QTL. While high levels of silverleaf whitefly resistance were observed in all acylsugar breeding lines, lines containing the additional QTL on either chromosomes 6 or 10 had increased levels of total acylsugar production and reduced incidence of whitefly. Acylsugar lines containing the chromosome 6 QTL also had increased density of the type IV glandular trichomes which produce and exude acylsugars.     

Effects of altitude of origin on trichome-mediated anti-herbivore resistance in wild Andean potatoes

The diversity and abundance of herbivores often decreases with increasing altitude. Plants are expected to respond to reduced herbivore pressure by allocating fewer resources to anti-herbivore resistance at higher altitudes. This study uses a greenhouse experiment and parallel bioassay to examine population variability in trichome-mediated defenses across a range of accessions of Solanum berthaultii Hawkes and S. tarijense Hawkes that originated from different altitudes. S. berthaultii bears two types of glandular trichomes, type A and type B, whereas S. tarijense has type A trichomes only, and hairs resembling type B trichomes that are eglandular. Both type A and type B glandular trichomes on S. berthaultii deterred ovipositing female Phthorimaea operculella (Zell.) (Lepidoptera: Gelechiidae). The density of type A, but not type B trichomes decreased with increasing altitude of origin in S. berthaultii populations. The ratio of type A to type B trichomes on the abaxial surface of S. berthaultii negatively affected oviposition and was inversely related to altitude of origin. In S. tarijense, type A trichomes deterred, but eglandular leaf hairs stimulated oviposition. Consequently, the ratio of type A trichomes to eglandular leaf hairs was negatively related to the number of eggs laid. The total numbers of trichomes per leaf generally increased with increasing altitude of origin in S. tarijense whereas the ratio of type A trichomes to eglandular hairs declined. In the oviposition bioassay, we found no direct relationship between resistance and altitude of origin, which may have been due to differences in leaf area at the time of the bioassay. Nevertheless, the results suggest that populations of both plant species that originated from higher altitudes were generally more susceptible to ovipositing P. operculella.     

Screening Genetic Resources of Capsicum Peppers in Their Primary Center of Diversity in Bolivia and Peru

For most crops, like Capsicum, their diversity remains under-researched for traits of interest for food, nutrition and other purposes. A small investment in screening this diversity for a wide range of traits is likely to reveal many traditional varieties with distinguished values. One objective of this study was to demonstrate, with Capsicum as model crop, the application of indicators of phenotypic and geographic diversity as effective criteria for selecting promising genebank accessions for multiple uses from crop centers of diversity. A second objective was to evaluate the expression of biochemical and agromorphological properties of the selected Capsicum accessions in different conditions. Four steps were involved: 1) Develop the necessary diversity by expanding genebank collections in Bolivia and Peru; 2) Establish representative subsets of ~100 accessions for biochemical screening of Capsicum fruits; 3) Select promising accessions for different uses after screening; and 4) Examine how these promising accessions express biochemical and agromorphological properties when grown in different environmental conditions. The Peruvian Capsicum collection now contains 712 accessions encompassing all five domesticated species (C. annuum, C. chinense, C. frutescens, C. baccatum, and C. pubescens). The collection in Bolivia now contains 487 accessions, representing all five domesticates plus four wild taxa (C. baccatum var. baccatum, C. caballeroi, C. cardenasii, and C. eximium). Following the biochemical screening, 44 Bolivian and 39 Peruvian accessions were selected as promising, representing wide variation in levels of antioxidant capacity, capsaicinoids, fat, flavonoids, polyphenols, quercetins, tocopherols, and color. In Peru, 23 promising accessions performed well in different environments, while each of the promising Bolivian accessions only performed well in a certain environment. Differences in Capsicum diversity and local contexts led to distinct outcomes in each country. In Peru, mild landraces with high values in health-related attributes were of interest to entrepreneurs. In Bolivia, wild Capsicum have high commercial demand.