Exploitation of Solanum chilense and Solanum peruvianum in tomato breeding for resistance to Tomato yellow leaf curl disease Olga Julián Rodríguez Dirigido por: Ana María Pérez de Castro a M José Díez Niclós Valencia, Noviembre 2013 Agradecimientos Por fin llegó la recta final…el final de un largo camino, un camino lleno de aprendizaje, el cual creo que merece esta reflexión. Llegado este momento me planteo, ¿qué ha sido esta tesis para mí? ¿qué he aprendido de todo esto? Primero de todo quiero decir que para mí esta tesis es algo que va más allá de un título o de una serie de publicaciones. A lo largo de este doctorado he aprendido muchas cosas, algunas de carácter científico, pero también cosas acerca de la vida. Personalmente creo que el doctorado, entre otras cosas, me ha enseñado a ser más fuerte, a que las cosas en la vida cuestan y que al final todo esfuerzo tiene su recompensa. Y es que cuando las cosas no salen como uno quiere, cuando los experimentos fallan, cuando ves que el tiempo se te echa encima, etc. al final uno aprende a “caerse”, y aprender a “caerse” significa aprender a levantarse y seguir adelante. Te “caes” una y mil veces, hasta que un día te encuentras con tu tesis en la mano, miras hacia atrás y piensas: “Por fin lo conseguí”. Otra de las cosas que he aprendido a lo largo del doctorado ha sido el gran valor del trabajo en equipo. Al fin y al cabo, una tesis no es solo el trabajo de una sola persona, sino que es el esfuerzo de muchos lo que la hace posible. Por ello, quiero expresar mi más sincero agradecimiento a todas y a cada una de las personas que, de una manera u otra, han puesto su granito de arena en este proyecto: A la institución del COMAV, por haberme dado la posibilidad de realizar este doctorado. De manera muy especial, a mis directoras de tesis, Ana María Pérez y Mª José Díez, por darme la oportunidad de trabajar en su equipo, por todas las enseñanzas recibidas a lo largo de estos años y por su apoyo, tanto dentro como fuera de la tesis. A la Dra. Yuling Bai y a todo su equipo, por colaborar con nuestro grupo y hacer posibles mis dos estancias en la Universidad de Wageningen. A Itziar Di Lolli y Marina Ros, por el apoyo técnico y también por los momentos de risas y la compañía durante estos años. A mis compañeros Javier Herráiz y Santiago Vilanova, por sus enseñanzas y apoyo científico en este proyecto. A los estudiantes Juan Lizarzaburu, Silvia Corella y Enric Simón, que con sus proyectos de fin de carrera y fin de máster han hecho una valiosa contribución a esta tesis. A Myluska Caro y a Nuria García, por su apoyo en el laboratorio y por los momentos inolvidables en Wageningen. Y como no, a todos mis compañeros de batalla Carolina, Patri, Juan Pablo, Nuri, Carlos, Julia, Carolina, Vero, Alba, Carles, Alvaro, etc. con los que he compartido tantas horas, almuerzos, charlas e inestimables consejos. Finalmente, quiero dar las gracias a las dos personas que han hecho de mi la persona que soy hoy: MIS PADRES A ellos les quiero dedicar esta tesis de manera muy especial: “Mamá, no tengo palabras para agradecerte todo lo que has hecho y sigues haciendo por mí. Gracias por tu apoyo, tu paciencia, tus consejos, tus ánimos, tu compañía, tu cariño,…y un largo etcétera que nunca acabaría. Sin ti nada de esto hubiera sido posible.” “Papá, aunque ya no te tengo conmigo, no hay un solo día que no piense en ti. Siempre me decías lo orgulloso que estabas y lo mucho que creías en mí. Mil gracias, porque ese es el apoyo que he necesitado para llegar hasta donde estoy hoy. Aunque tú no estés, la fuerza de tus palabras y tu cariño siguen conmigo.” INDEX Abstract 1 English version 2 Versión en Español 4 Versió en Valencià 6 Introduction 9 1. Tomato taxonomy and related species 10 2. Exploitation of wild species in tomato breeding 13 2.1. Needs and breeding goals 13 2.2. Use of wild species in breeding 15 3. Prebreed populations 18 3.1. Types and description 18 3.2. Prebreed populations developed with wild tomato relatives 21 4. Tomato yellow leaf curl disease (TYLCD) 23 4.1. History 23 4.2. Virus biology 24 4.3. Symptoms 25 4.4. Transmission 25 4.5. Host range 27 4.6. TYLCD in Spain 29 4.7. Methods for controlling the disease 29 4.8. Breeding for resistance 30 References 39 Objectives 58 Genetic control and mapping of Solanum chilense LA1932, LA1960 and LA1971-derived resistance to Tomato yellow leaf curl disease 60 Abstract 61 Introduction 61 Material and Methods 63 Results 67 Discussion 75 References 79 Supplementary Material 87 Fine mapping of Tomato yellow leaf curl virus resistance genes derived from Solanum chilense LA1932, LA1960 and LA1971 to the Ty-1/Ty-3 region 95 Abstract 96 Introduction 96 Material and Methods 99 Results 102 Discussion 107 References 110 Supplementary Material 114 Initial development of a set of introgression lines from Solanum peruvianum PI 126944 into tomato: Exploitation of resistance to viruses 117 Abstract 118 Introduction 118 Material and Methods 120 Results 126 Discussion 134 References 138 Supplementary Material 142 General Discussion 152 Identification of new resistance genes in S. chilense: Determination of genetic control and mapping 153 Use of resistance derived from S. peruvianum PI 126944: Construction of a set of ILs 157 Future perspectives 161 References 162 Conclusions 167 ABSTRACT English version 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 2 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.