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[EN] In previous studies, the FGB lab have shown that when a fragment from Chromosome 6 of Solanum chilense (which contains Ty1-3 resistance genes) is introgressed into tomato (Solanum lycopersicum), the fruit volatile profile is affected. Our working hypothesis is that a S. chilense FAD gene located next to the Ty gene is also introgressed -causing a higher expression of FAD, which is translated into a higher synthesis in fruit of volatiles produced from double-bond Fatty Acids through the Lipoxygenase Pathway. The objective of this thesis is to analyse further the effect of this S. chilense introgression by 1) determining if the change in volatiles production is restricted to tomato fruit or affects also leaves, 2) defining the differences at the genomic level between the FAD gene from tomato and S. chilense, and 3) performing a literature review to check on the extent linkage drag has on tomato breeding. We first analysed the VOCs present leaves of Solanum lycopersicum cv. Muchamiel and De la Pera traditional varieties, that either contain or not the Ty resistance gene introgression from S. chilense. We performed a SPME-HeadSpace Gas Chromatography-Mass Spectrometry analysis in order to capture, separate and quantify the volatiles present in our samples. We conducted a Targeted Metabolic Analysis to analyse possible differences in 15 specific compounds and an Untargeted Analysis to analyse all chromatograms¿ data. Results indicate no changes in the Fatty Acids-derived volatiles in leaves, indicating that the effect of the introgression could be restricted to fruit. Moreover, we performed a molecular analysis of the FAD gene sequence in both tomato and S. chilense, concluding that there is a 700bp-long repetitive element present in the promoter of the FAD gene in tomato which is absent in S. chilense species. In order to assess which alterations in the promotor sequence cause the FAD gene to present a higher expression in S. chilense than in tomato, we performed a bioinformatic analysis using the PLACE database and the informatic programs Nsite-PL and NsiteM-PL from the RegSite Database. While this repetitive element disrupts a couple of regulatory domains of the promoter, additional reasons for downregulation could be the hypermethylation of the promoter region in tomato. As this FAD gene is also required for the synthesis of Jasmonate phytohormones, aliquots of our samples are ready for future hormone analysis, since these are important for induction of defense systems in the plant. In order to assess this aspect, our samples were punctured with the zoophytophagous mirid bug Nesidiocoris tenuis and will then be compared with the control samples. Literature review identified a number of cases in tomato breeding where introgression of disease resistance genes has an effect on other traits of the plant due to linkage drag. A review of the most cultivated tomato F1 hybrids indicated that most of them carry more than three introgressions from wild relative species and are, therefore, likely to be affected regarding other traits, either beneficially or not, which ultimately affects consumer liking., [CA] S’ha observat en estudis realitzats anteriorment al laboratorio de FGB de l’IBMCP que, quan s’introgressa un fragment del Cromosoma 6 de l’espècie silvestre Solanum chilense (el qual conté els gens de resistència Ty1-3) en el genoma de la tomaca (Solanum lycopersicum), a més de conferir resistència, s’altera el perfil de compostos volàtils en el fruit. La nostra hipòtesi actual és que un gen FAD de S. Chilense que es localitza pròxim al gen de resistència Ty també s’introgressa amb aquest últim -generant una major expressió de FAD, i provocant finalment una major síntesi en fruit de volàtils produïts a partir d’Àcids Grassos de doble enllaç, a través de la Ruta de la Lipoxigenasa. L’objectiu d¿aquest treball és analitzar amb més profunditat l’efecte que té aquesta introgressió de S. Chilense en tomaca; primer, determinant si el canvi en la producció de volàtils està limitat al fruit o també afecta a les fulles; segon, indicant les diferències que hi ha a nivell genòmic entre el gen FAD en tomaca i en S. chilense; i, tercer, realitzant una revisió bibliogràfica per a determinar fins a quin punt l’esdeveniment de linkage drag afecta al cultiu de la tomaca. En primer lloc, vam analitzar els compostos volàtils (COVs) de fulles de Solanum lycopersicum de les varietats tradicionals Muchamiel i De la Pera, les quals presentaven o no la introgressió del gen Ty de S. chilense. Vam realitzar una anàlisi metabolòmica mitjançant el mètode de Microextracció en Fase Sòlida-Espai de cap-Cromatografia de Gases-Espectrometria de Masses per a capturar, separar i identificar els volàtils presents en les nostres mostres. Vam dur a terme una Anàlisi Metabòlica Dirigida per a determinar les possibles diferències en 15 compostos específics i una Anàlisi No dirigida per a extraure tota la informació dels cromatogrames. Els resultats mostren que no hi ha canvis en els volàtils procedents d’Àcids Grassos en les fulles de tomaca, indicant que els efectes de la introgressió es limiten únicament al fruit. A més a més, vam realitzar una anàlisi molecular de la seqüència del gen FAD en tomaca i en S. chilense, confirmant que hi ha un element repetitiu de 700 pb localitzat en el promotor d’aquest gen en tomaca i no en S. chilense. Amb l’objectiu d’avaluar quins canvis en la seqüència del promotor podrien estar implicats en que aquest gen presente una major expressió en S. chilense que en tomaca, vam portar a terme una anàlisi bioinformàtica utilitzant la base de dades PLACE i els programes Nsite-PL i NsiteM-PL de la base de dades RegSite. Vam concloure que aquest element repetitiu no sols es troba en la seqüència interrompent dos dominis de regulació del promotor, sinó que proposem a més altres motius que expliquen aquesta regulació negativa de FAD en tomaca, com podria ser la hipermetilació de la regió del promotor. Com aquest gen FAD també participa en la síntesi de jasmonat i aquesta hormona és important per a estimular els sistemas de defensa de la planta en resposta a la interacción amb diferents agents biòtics, hem preparat alíquotes de les nostres mostres per a realitzar una anàlisi d’hormones en el futur. Per a avaluar aquesta possibilitat, s’analitzaran mostres de fulles apicals les quals havien sigut picades per l’heteròpter zoofitòfag Nesidiocoris tenuis i es compararan amb les mostres control. Vam realitzar una revisió bibliogràfica sobre l’efecte de creuaments inter-específics en tomaca i vam identificar diversos casos en els que la introgressió de gens de resistència a malalties afectava també altres característiques de la planta, a causa de l’esdeveniment de linkage drag. D’altra banda, una anàlisi bibliogràfica de les F1 híbrides de tomaca més cultivades ens va demostrar que la majoria d’aquestes espècies posseeixen més de tres introgressions procedents d’espècies silvestres, i, per tant, és molt probable que també presenten altres.

Among viral diseases affecting cultivated tomato, Tomato yellow leaf curl disease (TYLCD) is one of the most devastating. This disease is caused by a complex of viruses of which Tomato yellow leaf curl virus (TYLCV) is regarded as the most important species. Current control strategies to fight viral diseases in tomato are mainly based on genetic resistance derived from wild relatives. In the present thesis, resistance derived from S. chilense and S. peruvianum has been exploited in breeding for resistance to TYLCD. In a previous study, TYLCV-resistant breeding lines derived from LA1932, LA1960 and LA1971 S. chilense accessions were developed. Therefore, the first objective of this thesis was to study the genetic control of the resistance derived from these accessions. With this aim, response to viral infection was assayed in segregating generations derived from the aforementioned resistant lines. The results obtained were compatible with a monogenic control of resistance. Resistance levels were higher in LA1960- and LA1971-derived F2 generations, as shown by slighter symptoms in the resistant plants and a higher number of asymptomatic plants compared with the results obtained in the LA1932-derived F2 generation. It is noteworthy that the level of resistance present in our materials is comparable to or even higher than the levels found in tomato lines homozygous for Ty-1. The response in plants heterozygous for the resistance gene was comparable to the response in homozygous plants for all three sources employed. This implies that the resistance genes derived from all three sources seem to be almost completely dominant. This effect was stronger for LA1971-derived resistance. The results were similar when comparing viral accumulation, as was expected, since a positive correlation was found in these families between viral accumulation and symptom scores. This has important implications in breeding, since the resistance will be used mostly for hybrid development. Our second objective was to map the loci associated with the major resistance genes identified. A total of 263 markers were screened, 94 of them being polymorphic between both species. Recombinant analysis allowed the resistance loci to be localized on chromosome 6, in a marker interval of 25 cM. This interval includes the Ty-1/Ty-3 region, where two S. chilense-derived TYLCD resistance loci were previously mapped. In order to test if the resistance genes identified in our populations were allelic to Ty-1 and Ty-3, further fine mapping was carried out. A total of 13 additional molecular markers distributed on chromosome 6 allowed 66 recombinants to be identified, and the resistance region to be shortened to a marker interval of approximately 950 kb, which overlaps with the Ty-1/Ty-3 region described previously by other authors. Therefore, the results obtained indicate that closely linked genes or alleles of the same gene govern TYLCV resistance in several S. chilense accessions. The third objective of the present thesis was to start the construction of a set of introgression lines (ILs) derived from Solanum peruvianum accession PI 126944 into the cultivated tomato genetic background. Once this collection of ILs is developed, it will represent a powerful tool for exploiting the resistance to different pathogens found in this particular accession in addition to other possible characters of interest. The starting plant material consisted of several segregating generations that were derived from two interspecific hybrids previously obtained by our group. Many crosses and embryo rescue were required to obtain subsequent generations due to the high sexual incompatibility that exists between tomato and PI 126944. Several mature fruits from the most advanced generations produced a few viable seeds, although embryo rescue was also employed to obtain progeny. As only a few plants were obtained by direct backcrossing, additional crosses were made in order to increase the number of descendants. A high degree of incompatibility was also found in crosses between sib plants. A total of 263 molecular markers were tested in some generations, 105 being polymorphic between tomato and PI 126944. Available generations were genotyped with these polymorphic markers in order to determine which alleles of S. peruvianum were already introgressed. On average, 79, 78 and 84 % of the S. peruvianum genome was represented in the pseudo-F2, pseudo-F4 and pseudo-F5 generations, respectively, for the markers analyzed. A reduction in the S. peruvianum genome was observed in more advanced generations, such as BC1 (56 %), pseudo-F2-BC1 (60 %) and pseudo-F3-BC1 (70 %). A greater reduction was observed in the pseudo-F3-BC2 generation (33 %). As a consequence of the reduction in the S. peruvianum genome, a loss of incompatibility was observed in some cases. The S. peruvianum genome was almost completely represented among the different plants of the most advanced generations. An evaluation for resistance to TYLCD and Tomato spotted wilt virus (TSWV) was carried out in some of the advanced generations, some of which were resistant to one or both viruses. In conclusion, we have conducted a successful and deeper exploitation of two wild species with proved resistance to TYLCD, S. chilense and S. peruvianum, identifying and fine mapping new genes of resistance., Julián Rodríguez, O. (2014). Exploitation of Solanum chilense and Solanum peruvianum in tomato breeding for resistance to Tomato yellow leaf curl disease [Tesis doctoral no publicada]. Universitat Politècnica de València. doi:10.4995/Thesis/10251/36867., Alfresco

[ES] La enfermedad del rizado amarillo del tomate (TYLCD) produce importantes pérdidas de producción en los cultivos de tomate (Solanum lycopersicum L.) en todas las zonas cálidas y templadas del mundo. La especie viral más importante causante de la enfermedad es Tomato yellow leaf curl virus (TYLCV). Se han desarrollado materiales resistentes hasta el momento con altos niveles de resistencia. S. chilense se ha citado como la especie con mayores niveles de resistencia a esta enfermedad. En concreto, la mayor parte de materiales comerciales resistentes incorporan el gen Ty-1, derivado de la entrada LA 1969 de esta especie. En cualquier caso, los materiales comerciales disponibles no suponen una solución definitiva, ya que en condiciones de elevada presión de inóculo o tras infecciones tempranas muestran síntomas, por lo que numerosos grupos de investigación continúan trabajando en la búsqueda de fuentes de resistencia. Se ha descrito resistencia en otras entradas de S. chilense, tales como LA1932, LA1960 y LA1971. En el Instituto Universitario de Conservación y Mejora de la Agrodiversidad Valenciana se dispone de líneas fijadas para la resistencia derivada de estas entradas, así como de generaciones segregantes derivadas de estas líneas. Se ha citado un control monogénico dominante para la resistencia derivada de estas entradas. Este trabajo se engloba dentro de un proyecto que tiene como objetivo identificar los loci responsables de la resistencia derivada de estas entradas y mapearlos finamente. En concreto, los objetivos de este trabajo fueron los siguientes. En primer lugar se pretendía identificar marcadores moleculares polimórficos entre tomate y las entradas de S. chilense utilizadas, para, a continuación utilizar estos marcadores para la localización de las regiones de S. chilense introgresadas en las líneas fijadas para la resistencia. Por último, se pretendía analizar la asociación entre las introgresiones de S. chilense identificadas y la resistencia a TYLCD en las generaciones segregantes disponibles. Los 18 marcadores microsatélites analizados resultaron polimórficos entre tomate y S. chilense. Se utilizaron en el genotipado estos marcadores, además de dos marcadores tipo CAPS (Cleaved Amplified Polymorphic Sequence), previamente descritos como polimórficos entre ambas especies y ligados al gen Ty-1, en el cromosoma 6. Se comprobó que únicamente la introgresión correspondiente al cromosoma 6 fue común a las cinco líneas evaluadas. En cualquier caso, todos los marcadores para los que las generaciones F1 entre las líneas y la variedad de tomate Fortuna C resultaron heterocigotas, se emplearon para el genotipado de las generaciones F2, previamente fenotipadas por su resistencia a TYLCD. Únicamente se observó asociación entre la resistencia y la presencia del alelo de S. chilense para los marcadores del cromosoma 6. En trabajos previos se había descrito la presencia de un gen de resistencia derivado de LA1932 en el cromosoma 6, el gen Ty-3. Sin embargo, es el primer estudio en el que se localizan los loci asociados a la resistencia de LA1960 y LA1971. Trabajos futuros irán encaminados a la identificación de marcadores más estrechamente ligados a los genes de resistencia, con objeto de comprobar si los loci derivados de LA1932, LA1960 y LA1971 son alélicos con los previamente descritos en el cromosoma 6, Ty-1 y Ty-3., [EN] Tomato yellow leaf curl disease (TYLCD) produces significant production losses in tomato crops (Solanum lycopersicum L.) in all warm and temperate regions of the world. The most important viral species that causes the disease is the Tomato yellow leaf curl virus (TYLCV). Resistant materials have been developed to date with high levels of resistance. S. chilense has been cited as the specie with higher levels of the resistance to this disease. Specifically, most of the commercially available resistant materials incorporate the Ty-1 gene, derived from LA 1969 accession of this species. In any case, the available commercial materials are not a definitive solution, because they show symptoms under high inoculum pressure or upon early infections. Thus many research groups are working in the search for resistance sources. Resistance has been described in other accessions of S. chilense such as LA1932, LA1960 and LA1971. In the Instituto Universitario de Conservación y Mejora de la Agrodiversidad Valenciana lines fixed for resistance derived from these accessions are available, as well as segregating generations derived from these lines. This work forms part of a project that aims to identify the loci responsible of the resistance derived from these accessions and to fine map them. Specifically, the objectives of this study were the following. The first purpose was to identify polymorphic molecular markers between tomato and S. chilense accessions used, to subsequently use these markers for the localization of the introgressed regions of S. chilense in fixed resistant lines. Finally, the objective was to analyze the association between the identified introgressions of S. chilense and TYLCD resistance detected in the segregating generations available. The 18 microsatellites markers analyzed were polymorphic between tomato and S. chilense. These markers and two CAPS markers (Cleaved Amplified Polymorphic Sequence), previously described as polymorphic between both species and linked to the Ty-1 gene on chromosome 6 were used for the genotyping. Only the introgression for the chromosome 6 was common to all five lines evaluated. In any case, all the markers for which the F1 generations derived from the cross between the lines and the tomato variety Fortuna C were heterozygous, were used for genotyping of the F2 generation, previously phenotyped for its resistance to TYLCD. Association was only observed between the resistance and the presence of S. chilense allele for the markers of chromosome 6. In previous works the presence of the resistant Ty-3 gene derived from LA1932 on chromosome 6 was described. However, it is the first study in which the resistance loci from LA1971 and LA1960 are mapped. Future works will be aimed at the identification of markers most tightly linked to the resistance genes, in order to verify if the loci derived from LA1932, LA1960 and LA1971 are allelic with the previously described Ty-1 and Ty-3 genes on chromosome 6.

La enfermedad del rizado Amarillo del tomate (TYLCD) causa importantes pérdidas económicas en los cultivos de tomate (Solanum lycopersicum L.) de la mayor parte de áreas tropicales y subtropicales del mundo. La especie viral más importante causante de TYLCD en el mundo es Tomato yellow leaf curl virus (TYLCV).