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[EN] A collection of introgression lines (ILs) of Solanum lycopersicoides Dunal in the genetic background of cultivated tomato (S. lycopersicum L.) is a valuable tool for tomato breeding. Efficient management of the collection requires the use of molecular markers. The objective of this work was to identify polymerase chain reaction (PCR)-based markers that were polymorphic between the parents of the ILs, namely ‘VF36’ and ‘LA2951’. In total, 81 primer pairs were tested on genomic DNA from both parents. Genomic DNA of the inter-specific hybrid between the two parents, from which the IL collection had been derived, was also tested. The markers used were either cleaved amplified polymorphic sequence (CAPS) markers or were derived from a set of conserved orthologous genes. In both cases, the markers had been mapped in tomato and described in the SOL Genomics Network. Forty-seven of the markers tested produced a single PCR product in ‘VF36’ and ‘LA2951’. Eleven markers revealed polymorphisms as differences in band-sizes between the two parents. At least one restriction enzyme that generated polymorphism was identified in 29 of the remaining 36 markers. Among other applications, some of these markers have been used to identify plants carrying a target DNA fragment among segregating generations, or to delimit the length of an introgressed sequence, This research was financed by the Ministerio de Educacion y Ciencia and Ministerio de Ciencia e Innovacion, Madrid, Spain (Projects Nos. AGL2008-05114 and AGL2011-30083).

The Genebank of the Institute for the Conservation and Improvement of the Agrodiversity (COMAV) has in its collection 198 accessions of cucumber (Cucumis sativus L.) of Spanish origin, taking charge of preserving and providing their seeds. All the accessions were registered as traditional varieties or landraces, thus the collection of the COMAV represents 5% of the cucumber traditional varieties included in GENESYS.The purpose of the germplasm collections is the preservation of the genetic diversity to make it available for breeders, researchers and other users. Rationalization, that is, reduction of the size collection, is an alternative to cut costs of maintenance. With rationalization the efficiency in management and use of the collection increases; as a consequence, a correct preservation is guaranteed and at least part of the plant materials are available to users. This thesis is included in a project with the objective of the rationalization of the cucumber Spanish collection of the COMAV's Genebank. To achieve this objective, a morphological characterization of the cucumber collection, using fruit and plant descriptors, was carried out. Subsequently, a subset of the accessions were selected to be analyzed with molecular markers, concretely with SSRs (Simple sequence repeats). The morphological characterization was carried out for 206 cucumber accessions, 195 from Spain (178 held at the COMAV, 17 provided by the 'Vegetable Genebank of Zaragoza', BGHZ). Five plants per accession were characterized, with 17 qualitative and 9 quantitative descriptors, eight of them referred to plant traits and 18 related to the fruit. Fruit descriptors were evaluated in at least 25 fruits per accession. The accessions were classified in five groups: 'White', 'Short', 'French', 'Long' and 'Very long'. Principal component analysis showed that, with few exceptions, the accessions were grouped according to their phenotypic similarity. Variability found within each of the groups displayed the potential of these plant materials in breeding programmes for different traits. Maintenance of this collection is of great interest, since variability held by these accessions is not conserved in other European collections, and constitutes a source of genes for cucumber breeding. A representative subset of the accessions evaluated by morphological traits was selected for the molecular characterization. Concretely, molecular diversity of 131 Spanish accessions was evaluated with 23 SSRs. Eighteen of these SSRs were polymorphic; the mean number of alleles, mean observed heterozygosity and mean polymorphic information content were 3.2, 0.065 and 0.229, respectively. Around 60% of the alleles showed a frequency higher than 0.05, and only one allele in the SSR31399 showed a frequency lower than 0.01. In addition, three accession-specific alleles were found. A cluster analysis did not show any relation with morphological types or geographical area. Therefore, these results demonstrated that molecular diversity of the cucumber did not resemble its phenotypic variability. Finally, this study provides information for the rationalization of germplasm.Results from both studies allowed the rationalization of the cucumber Spanish collection of the COMAV. Selection of the accessions was carried out with a combined strategy, considering phenotypic traits, origin and molecular data. The selection included 47 accessions, six of the 'French' type, 15 of the 'Long' type and 24 of the 'Short' type. Moreover, the unique accessions molecularly analyzed belonging to 'Very long' and 'White' types were also selected. The analysis of the set of selected accessions confirmed that it conserved the morphological and molecular variability found in the complete collection., El Banco de Germoplasma del Instituto de Conservación y Mejora de la Agrodiversidad Valenciana (COMAV) mantiene en su colección 198 entradas de variedades tradicionales de pepino (Cucumis sativus L.) de origen español, haciéndose cargo de la conservación y cesión de sus semillas. La colección de pepino del COMAV representa el 5% de las variedades tradicionales de pepino que figuran en GENESYS. El objeto de las colecciones de germoplasma es conservar la diversidad genética y permitir el acceso a esta a mejoradores, investigadores y otros usuarios. Como alternativa para reducir los costes de mantenimiento se plantea su racionalización, es decir, la reducción del tamaño. De esta forma, se incrementa la eficiencia en el manejo y uso de las mismas, se garantiza una correcta conservación y al menos parte de los materiales se hacen accesibles a los usuarios. Esta tesis se enmarca en un proyecto cuyo objetivo es la racionalización de la colección de pepino español del Banco de Germoplasma del COMAV. Para ello se planteó en primer lugar una caracterización por caracteres de planta y fruto de la colección de entradas de pepino español, para a continuación seleccionar un subconjunto de las mismas para su análisis con marcadores moleculares de tipo microsatélite (Simple sequence repeats, SSR). La caracterización morfológica se llevó a cabo empleando 206 entradas de pepino, 195 de ellas de origen español (178 mantenidas en el COMAV y 17 proporcionadas por el Banco de Germoplasma de Hortícolas de Zaragoza, BGHZ). Se caracterizaron 5 plantas por entrada, empleando 17 descriptores cualitativos y nueve cuantitativos, ocho de ellos de planta y 18 de fruto. Los caracteres de fruto se evaluaron en al menos 25 frutos por entrada. Las entradas analizadas se clasificaron en cinco tipos según las características de fruto: "Blanco", "Corto", "Francés", "Largo" y "Muy largo". El análisis de componentes principales permitió comprobar que, con pocas excepciones, las entradas se agrupaban según su similitud fenotípica. En cualquier caso, la variabilidad observada dentro de cada uno de los grupos puso de manifiesto el potencial de los materiales evaluados para la mejora de distintos atributos. El mantenimiento de la colección resulta de especial interés, dado que la variabilidad que contiene no se encuentra conservada en otras colecciones europeas y constituye una fuente de genes de interés para la mejora del pepino. Un subconjunto representativo de las entradas evaluadas por características morfológicas se seleccionó para la caracterización molecular. En concreto, se evaluó la diversidad genética de 131 entradas españolas, empleando 23 marcadores SSRs. Dieciocho de los SSRs fueron polimórficos en la colección: los valores medios para el número de alelos, la heterocigosidad observada y el contenido en información polimórfica fueron de 3,2, 0,065 y 0,229, respectivamente. Aproximadamente el 60% de los alelos mostraron una frecuencia superior a 0,05, mientras que solo uno de los alelos para el marcador SSR31399 mostró una frecuencia inferior a 0,01. Se identificaron tres alelos específicos de entradas. En el análisis de agrupamientos las entradas no se agruparon según el tipo ni según el área geográfica. Estos resultados demostraron que la diversidad molecular de la colección de pepino no refleja la variabilidad fenotípica. Los resultados de ambas caracterizaciones han permitido llevar a cabo la racionalización de la colección de pepino español del COMAV. Para la selección de las entradas se ha llevado a cabo una estrategia combinada, considerando caracteres fenotípicos, origen y datos moleculares. La selección incluyó 47 entradas, seis de tipo "Francés", 15 del tipo "Largo" y 24 del tipo "Corto". Además, se seleccionaron las únicas entradas de tipo "Muy largo" y "Blanco" caracterizadas molecularmente. Se confirmó que el conjunto de entradas seleccionadas conservaban la variabilidad m, El Banc de Germoplasma de l'Institut de Conservació i Millora de l'Agrodiversitat Valenciana (COMAV) manté en la seua col·lecció 198 entrades de varietats de cogombre (Cucumis sativus L.) d'origen espanyol, fent-se càrrec de la conservació i cessió de les seues llavors. La col·lecció del COMAV representa el 5% de les varietats de cogombre que figuren en GENESYS.L'objectiu de les col·leccions de germoplasma és conservar la diversitat genètica i permetre l'accés a aquesta a milloradors, investigadors i altres usuaris. Com a alternativa per a reduir els costos de manteniment, es planteja la seua racionalització, és a dir, la reducció de la grandària. D'aquesta forma, augmenta l'eficiència en el maneig i ús de les mateixes, es garantix una correcta conservació i al menys part dels materials es fan accessibles als usuaris. Aquesta tesi s'emmarca en un projecte que té com a objectiu la racionalització de la col·lecció de cogombre espanyol del Banc de Germoplasma del COMAV. Per a aconseguir aquest objectiu, es va plantejar en primer lloc una caracterització per caràcters de planta i fruit de la col·lecció d'entrades de cogombre espanyol, per a continuació seleccionar un subconjunt de les mateixes per al seu anàlisi mitjançant marcadors moleculars de tipus microsatèl·lit (Simple sequence repeats, SSR). La caracterització morfològica es va dur a terme utilitzant 206 entrades de cogombre, 195 d'elles d'origen espanyol (178 mantingudes al COMAV, 17 proporcionades pel Banc de Germoplasma d'Hortícoles de Saragossa, BGHZ). Es van caracteritzar 5 plantes per entrada, emprant 17 descriptors qualitatius i nou quantitatius, huit d'ells de planta i 18 de fruit. Els caràcters de fruit s'avaluaren en al menys 25 fruits per entrada. Les entrades analitzades es classificaren en cinc tipus segons les característiques de fruit: "Blanc", "Curt", "Francés", "Llarg" i "Molt llarg". L'anàlisi de components principals va permetre comprovar que, amb poques excepcions, les entrades s'agrupaven segons la seua similitud fenotípica. En tot cas, la variabilitat observada dins de cadascun dels grups va posar de manifest el potencial dels materials avaluats per a la millora de diversos atributs. El manteniment de la col·lecció resulta d'especial interés, atés que la variabilitat que conté no es troba conservada en altres col·leccions europees i constitueix una font de gens d'interés per a la millora del cogombre.Un subconjunt representatiu de les entrades avaluades per característiques morfològiques es va seleccionar per a la caracterització molecular. En concret, es va avaluar la diversitat genètica de 131 entrades espanyoles, emprant 23 marcadors SSRs. Díhuit dels SSRs van ser polimòrfics en la col·lecció: els valors mitjans per al nombre d'al·lels, la heterocigositat observada i el contingut en informació polimòrfica van ser 3,2, 0,065 i 0,229, respectivament. Aproximadament el 60% dels al·lels van mostrar una freqüència superior a 0,05, mentre que sols un dels al·lels per al marcador SSR31399 va mostrar una freqüència inferior a 0,01. Es van identificar tres al·lels específics d'entrades. En l'anàlisi d'agrupaments les entrades no s'agruparen segons el tipus ni segons l'àrea geogràfica. Aquests resultats demostraren que la diversitat molecular de la col·lecció de cogombre no reflectix la variabilitat fenotípica.Els resultats d'ambdues caracteritzacions han permés dur a terme la racionalització de la col·lecció de cogombre espanyol del COMAV. Per a la sel·lecció de les entrades s'ha emprat una estratègia combinada, considerant caràcters fenotípics, d'origen i dades moleculars. La selecció va incloure 47 entrades, sis de tipus "Francés", 15 del tipus "Llarg" i 24 del tipus "Curt". A més, es van seleccionar les úniques entrades del tipus "Molt llarg" i "Blanc" caracteritzades molecularment. Es va confirmar que el conjunt d'entrades seleccionades conservaven la variabilitat, Valcarcel Germes, JV. (2017). Racionalización de la colección de pepino (Cucumis sativus L.) del banco de germoplasma del COMAV [Tesis doctoral no publicada]. Universitat Politècnica de València. doi:10.4995/Thesis/10251/86216., TESIS

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