Resistance mechanisms against Bemisia tabaci in wild relatives of tomato

The silverleaf whitefly (<em>Bemisia tabaci</em>Genn.) poses a serious threat to tomato cultivation. A large part of the damage is done directly through heavy host plant colonization. Colonization has a negative impact on the plant, as the whitefly takes up nutrients from the phloem and induces phytotoxic responses, which result in irregular ripening of the fruits. However, most damage is done indirectly as the silverleaf whitefly vectors a broad range of plant pathogenic viruses. The silverleaf whitefly can successfully be controlled biologically in greenhouse cultivations, but control of the whitefly in the field is mainly based on the application of pesticides. The use of pesticides can have a negative effect on non-harmful or beneficial organisms in the field. Moreover, the effectiveness of pesticides can decline or even completely disappear through adaptation of the whitefly. An effective alternative for the use of pesticides could be the deployment of resistant cultivars. Nowadays, genetic factors responsible for whitefly resistance can be transferred faster and more efficiently into tomato cultivars through marker-assisted backcross breeding programs. Complete resistance against the whitefly is present in some crossable wild relatives of the cultivated tomato and the literature reports extensively about accessions with a high level of resistance against the whitefly. In this work, I have studied different populations that were developed by interspecific crosses between cultivated tomato and the tomato wild relatives<em>S. habrochaites</em>LYC4 and <em>S. pennellii</em>LA3791. By integrating datasets from different research disciplines, I have studied the background of whitefly resistance in these populations. Furthermore, these data were used to identify the chromosomal loci in the wild tomato relatives that harbor genes responsible for the resistance and that can be bred into cultivated tomato. The mechanisms underlying the resistance in <em>S. pennellii</em>LA3791 were studied through phenotypic resistance assays that demonstrated that survival and oviposition of the whitefly were not possible on this wild relative. Through removal of glandular cells, present on the leaf trichomes, the resistance was almost completely lost and only adult survival was still significantly different from the wild type. This result led to the hypothesis that glandular trichomes play an important role in the resistance. This was confirmed in a segregating population based on a cross between <em>S. pennellii</em>LA3791 and a susceptible cultivated tomato. Plants that lacked glandular trichomes type I and IV, had the same resistance level as the susceptible parent. Further analyses of the segregating population showed that the presence of glandular trichomes was not the only factor determining resistance, but that the composition and quantity of the metabolites in the glandular trichomes also played an important role. To gain more knowledge on the role of individual metabolites on whitefly resistance and susceptibility, we analyzed the total metabolite content of extreme phenotypes of the F₂population. Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography Time<strong>-</strong>Of<strong>-</strong>Flight Mass Spectrometry (LC-TOF-MS) were employed for the analyses of the total metabolite content. Analyses revealed that on basis of the total metabolite profiles the extreme phenotypes (susceptible versus resistant for the silverleaf whitefly) could be discriminated into two groups that were correlated with resistance or susceptibility. A number of these metabolites could be annotated, but for the majority of the components this was not possible on the basis of available literature and databases. Subsequently, I have studied the genetic basis of the phenotypic resistance parameters as well as the genetic basis of the metabolites from the GC-MS and LC-TOF-MS analyses. A genetic linkage map of the F₂mapping population was developed using DNA markers (Amplification Fragment Length Polymorphisms,AFLPs and Single Nucleotide Polymorphisms, SNPs). QTLs (Quantitative Trait Loci) were identified between the majority of the metabolites and the genetic markers (>90%) and also we found genetic linkages between whitefly resistance parameters and markers. The QTLs for metabolites and phenotypic parameters partly co-localized at the same positions on the genetic map. Several metabolite QTLs (mQTLs) co-localized with each other in so-called ‘hotspots’. Remarkably, the results of the individual phenotypic QTLs (phQTLs) for adult survival and oviposition as well as the mQTLs for the individual components did not give high explained variances (<20%), which was supported by an analysis of individual metabolite profiles, that showed a high variation in composition between F₂genotypes with an identical resistance level. On the basis of these results I hypothesized that resistance could not be explained by a specific composition of metabolites, but that multiple metabolic profiles can result in the same level of resistance in a plant. To support this hypothesis, a backcross population was developed, an F₂BC_1,by backcrossing a completely resistant F₂plant with the recurrent parent. The complete F₂BC₁population was analyzed by LC-TOF-MS to characterize the metabolite content of the progeny lines alongside resistance assays for adult survival and oviposition on these plants. Again, in this population we identified genotypes that possessed a level of resistance equal to the <em>S. pennellii</em>LA3791 donor parent. From the analyses it became clear that the complexity of the chemical profiles was reduced and that only a few components were correlated with whitefly resistance or susceptibility. A genetic linkage map with a large number of SNP markers enabled the identification of new QTLs alongside the QTLs from the previous F₂mapping that were confirmed in the F₂BC₁populations. The reduction in complexity of the chemical profile was accompanied by an increase in explained variances of both the phenotypic as well as the metabolite QTLs. The results indicate that performing phenotyping assays by scoring resistance parameters in a population along with analyzing the chemical profiles is required to identify resistance loci, which can subsequently be used in marker-assisted breeding programs. Finally I have studied an Introgression Line (IL) population, consisting of 30 lines, which each contained a different introgression of <em>S. habrochaites</em>LYC4, a whitefly-resistant wild relative of cultivated tomato. Survival and oviposition assays of the whole population revealed that there were a few lines that showed a slightly reduced susceptibility for the silverleaf whitefly. Completely resistant lines were not identified, which indicates that the resistance in this wild relative is complex and governed by the interaction of several genes at different locations on the tomato genome. Such genetic interactions, also referred to as epistatic interactions, complicate the identification of genes involved in resistance and the underlying resistance mechanisms. Therefore, I concluded that IL populations are not suitable for the elucidation of a complex trait as whitely resistance in tomato. In conclusion, this thesis demonstrates the most important aspects of susceptibility and resistance against the silverleaf whitefly in a <em>S. pennellii </em>accession and provides strong evidence for the underlying resistance mechanisms. Furthermore, we were capable of reducing the complex phenotypic and genotypic variation, which was present in the F₂population, via a backcross with the recurrent parent. This made it possible to identify three genetic loci in <em>S. pennellii </em>that play a role in whitefly resistance. A logical next step of this research would be the fine mapping of these three loci in order to enable the transfer of these loci/genes into cultivated tomato lines. By doing so, an important step towards sustainable control of the silverleaf whitefly in tomato cultivation could be made.