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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

Flowers are the defining feature of angiosperms, and function as indispensable organs for sexual reproduction. Flower colour typically plays an important role in attracting pollinators, and can show considerable variation, even between closely related species. For example, domesticated tomato (S. lycopersicum) has orange/yellow flowers, while the wild relative S. chilense (accession LA2405) has bright yellow flowers. In this study, the mechanism of flower colour formation in these two species was compared by evaluating the accumulation of carotenoids, assessing the expression genes related to carotenoid biosynthetic pathways and observing chromoplast ultrastructure. In S. chilense petals, genes associated with the lutein branch of the carotenoid biosynthetic pathway, phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), lycopene β-cyclase (LCY-B), β-ring hydroxylase (CRTR-B) and e-ring hydroxylase (CRTR-E), were highly expressed, and this was correlated with high levels of lutein accumulation. In contrast, PDS, ZDS and CYC-B from the neoxanthin biosynthetic branch were highly expressed in S. lycopersicum anthers, leading to increased β-carotene accumulation and hence an orange/yellow colour. Changes in the size, amount and electron density of plastoglobules in chromoplasts provided further evidence of carotenoid accumulation and flower colour formation. Taken together, these results reveal the biochemical basis of differences in carotenoid pigment accumulation and colour between petals and anthers in tomato.

The wild tomato species Solanum chilense and S. peruvianum are a valuable non-model system for studying plant adaptation since they grow in diverse environments facing many abiotic constraints. Here we investigate the sequence evolution of regulatory regions of drought and cold responsive genes and their expression regulation. The coding regions of these genes were previously shown to exhibit signatures of positive selection. Expression profiles and sequence evolution of regulatory regions of members of the Asr (ABA/water stress/ripening induced) gene family and the dehydrin gene pLC30-15 were analyzed in wild tomato populations from contrasting environments. For S. chilense, we found that Asr4 and pLC30-15 appear to respond much faster to drought conditions in accessions from very dry environments than accessions from more mesic locations. Sequence analysis suggests that the promoter of Asr2 and the downstream region of pLC30-15 are under positive selection in some local populations of S. chilense. By investigating gene expression differences at the population level we provide further support of our previous conclusions that Asr2, Asr4, and pLC30-15 are promising candidates for functional studies of adaptation. Our analysis also demonstrates the power of the candidate gene approach in evolutionary biology research and highlights the importance of wild Solanum species as a genetic resource for their cultivated relatives.

[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.

Simultaneous exposition to low temperature and high light radiation cause photoinhibition of photosynthetic apparatus, affecting the productivity and geographical distribution of agricultural crops. In several Solanaceous species, tolerance to low temperature stress in combination with high light has been associated with some stimulation in non-photochemical quenching (NPQ), which involved reorganization in light-harvesting complex (LHC) proteins. To study photosynthetic performance in Solanum lycopersicum L. and S. chilense (Dunal) Reiche, and to investigate transcriptional regulation of genes encoding LHC proteins and their involvement in the NPQ, plants of both species were exposed to low temperature (4 °C) and high light radiation (1300 µmol m -2 s -1 ). Lipid peroxidation, photochemical efficiency, and changes in xanthophyll cycle pigments were measured. The results presented here indicate that S. chilense showed higher tolerance to photoinhibition than S. lycopersicum under low-temperature and high light conditions, increasing light-energy consumption in photochemical processes by increasing photosynthetic capacity as indicated by photochemical quenching (qP) and relative electron transport rate (ETR) parameters. The contribution of light-harvesting chlorophyll a/b binding (LHC) protein was not related to dissipate excess excitation energy as heat (NPQ), but rather with the antioxidant function attributable to zeaxanthin as indicated by the amount of peroxidized lipids in S. chilense. We suggest that the differential expression of Lhca1 transcripts, with zeaxanthin binding sites could contribute to the greater tolerance of S. chilense to photoxidative stress.

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).

Isoprenoid compounds synthesised in the plastids are involved in plant response to water deficit. The functionality of the biosynthetic pathway of these compounds under drought stress has been analysed at the physiological and molecular levels in two related species of tomato (Solanum chilense and Solanum lycopersicum) that differ in their tolerance to abiotic challenge. Expression analysis of the genes encoding enzymes of these pathways (DXS, IPI, GGPPS, PSY1, NCED and HPT1) in plants at different RWC values shows significant differences for only GGPPS and HPT1, with higher expression in the tolerant S. chilense. Chlorophyll, carotenoids, α-tocopherol and ABA content was also determined in both species under different drought conditions. In agreement with HPT1 transcriptional activity, higher α-tocopherol content was observed in S. chilense than in S. lycopersicum, which correlates with a lower degree of lipoperoxidation in the former species. These results suggest that, in addition to lower stomatal conductance, α-tocopherol biosynthesis is part of the adaptation mechanisms of S. chilense to adverse environmental conditions.

Se determinó la respuesta de resistencia de 10 patrones de tomate a una población avirulenta de Meloidogyne javanica en maceta. Los ensayos se realizaron en primavera, cuando las temperaturas permitían la expresión fenotípica de la resistencia del gen Mi-1 (28˚C), en verano sometidos a altas temperaturas y en campo, exponiéndolos a altas densidades poblacionales del nematodo. A temperaturas inferiores a 28˚C los patrones mostraron gran variabilidad en la respuesta de resistencia que osciló entre alta y moderadamente resistente (PG-76, Gladiator, MKT-410; Brigeor, 42851, 43965, Big Power y He-man), hasta susceptible (Beaufort, Maxifort). Por encima de 28˚C, sólo dos patrones (PG-76 y He-man) inhibieron la reproducción del nematodo. Frente a distintas poblaciones de M. arenaria, M. incognita y M. javanica, el patrón PG-76, fue altamente resistente a todas las poblaciones, Brigeor osciló entre altamente resistente y moderadamente resistente, mientras que Beaufort y Maxifort mostraron menor resistencia o fueron totalmente susceptibles; además ésta varió en función de la población analizada. Se caracterizó molecularmente el locus Mi-1 en los patrones híbridos y cultivares de tomate estudiados. Se emplearon los marcadores moleculares PM3, PMi y Mi23, específicos para la caracterización del locus Mi en patrones híbridos de tomate (S. lycopersicum × S. habrochaites; S. lycopersicum × S. chilense), mediante PCR. También se realizaron análisis bioinformáticos con marcadores específicos (Mint-up/do, C172, C2S4, IMO-F1/R1, y VIGS) para determinar la presencia del gen Mi-1.2 en dichos patrones. Los resultados mostraron que los marcadores PMi y Mi23 amplifican homólogos del gen Mi-1 en S. chilense, S. habrochaites y S. peruvianum y también en S. lycopersicum (marcador Mi23). El marcador PM3 amplificó el gen Mi-1.2 en Beaufort y Maxifort (S. lycopersicum × S. habrochaites) pero no fue efectivo para los híbridos de S. chilense. El marcador molecular PM3, no pudo determinar la expresión de del gen Mi-1.2 en Beaufort y Maxifort por hallarse fuera de la secuencia codificadora (CDS) del gen. Análisis bioinformáticos indicaron que ningún marcador específico diseñado para el gen Mi-1.2, podía este gen de otros homólogos presentes en S. lypcopersicum y S. peruvianum. El nuevo marcador Pau-Do en combinación con el primer C2S4, amplificaron un fragmento de 1.494 pb en la CDS del gen Mi-1.2 en raíces y hojas de Beaufort y Maxifort. La durabilidad de la resistencia del gen Mi-1 después del cultivo reiterado de patrones de tomate se determinó en ensayos de campo durante tres años consecutivos, empleando PG-76 y Brigeor. El patrón PG-76 fue muy resistente después del 1er ciclo de cultivo, pero mostró resistencia intermedia y suscetibilidad al finalizar el 2o y el 3er año de cultivo, respectivamente. El patrón Brigeor y el cultivar de tomate resistente Monika (control) mantuvieron un nivel de resistencia intermedio al final del 3er cultivo, aunque ensayos posteriores confirmaron la aparición de virulencia. Los resultados mostraron que el cultivo reiterado de patrones de tomate resistentes seleccionó rápidamente aislados virulentos de M. javanica. El fenotipo virulento de estas poblaciones se analizó molecularmente con el marcador MVC, diseñado para distinguir las poblaciones virulentas seleccionadas de Meloidogyne de los aislados naturalmente virulentos. Se analizaron dos poblaciones japonesas seleccionadas de M. incognita y M. javanica, tres poblaciones españolas virulentas seleccionadas, una población naturalmente virulenta y una avirulenta (todas M. javanica). Las muestras de ADN se obtuvieron de individuos juveniles o de hembras adultas y se incluyeron muestras de agua sin nematodos (5 µm filtrada) procedentes del drenaje de una maceta con una planta infectada por una población virulenta japonesa. El marcador MVC amplificó ADN en las muestras de agua pero no en las que sólo contenían ADN de nematodos. Las secuencias de ADN mostraron una estrecha correlación con diversas proteínas de especies de betaproteobacterias. Los experimentos revelaron que el marcador de MVC no está relacionado con un gen de virulencia del nematodo (avr) sino con betaproteobacterias. Finalmente, se estudió la existencia de homólogos del gen Mi en las especies de tomate silvestre Solanum chilense, S. habrochaites, S. peruvianum y S. huaylasense. La respuesta de resistencia de la variedad LA-1358 de S. huaylasense varió en función de la especie del nematodo estudiada: fue resistente frente a M. arenaria y susceptible frente a M. javanica. La reproducción de M. incognita fue muy variable y no difirió de la reproducción alcanzada en los dos cultivares empleados como controles., The response of 10 Mi-1 tomato rootstocks to a Mi-avirulent population of M. javanica was determined in pot tests conducted in a greenhouse in spring when temperatures remained below the Mi-1 functionality resistance threshold (28 ˚C), and in summer when daily temperatures exceeded the Mi-1 expression threshold. Rootstocks were also evaluated in the field exposing them to high population densities of the nematode. Results on infectivity and reproduction below 28 ˚C indicated a wide variability in the resistance response of the rootstocks ranging from highly or intermediate resistance (PG-76, Gladiator, MKT-410; Brigeor, 42851, 43965, Big Power and He-man) to fully susceptible (Beaufort and Maxifort). At high temperature conditions, only PG-76 and He-man inhibited the reproduction of M. javanica. Rootstocks PG-76, Brigeor, Beaufort and Maxifort were challenged to different populations of M. arenaria, M. incognita and M. javanica. Rootstock PG-76 was highly resistant to all the populations tested, whereas the response of Brigeor ranged from highly to moderate resistance; the resistance response of rootstocks Beaufort and Maxifort varied according to the population tested. Molecular characterization of the resistance phenotype was performed for all the tomato hybrid rootstocks and cultivars tested. The markers PM3, PMi, Mi23, for the characterization of the Mi-locus of hybrid tomato rootstocks (S. lycopersicum × S. habrochaites and S. lycopersicum × S. chilense) were used for PCR reactions. In silico analyses were done with specific markers for the Mi-1.2 gene (Mint-up/do, C1/2, C2S4, IMO-F1/R1, and VIGS). Markers PMi and Mi23 were polymorphic for the Mi-1 locus in wild Solanum species (S. chilense, S. habrochaites, and S. peruvianum) and for S. lycopersicum (marker Mi23). Marker PM3 detected the Mi-1.2 gene in S. lycopersicum × S. habrochaites hybrid rootstocks, but not in the S. chilense hybrids. As marker PM3 is located outside the coding sequence of the Mi-1.2 gene, expression of this homolog could not be determined in Beaufort and Maxifort. In silico results indicated that none of the available markers for the Mi-1.2 gene could distinguish this homolog from the other Mi-homologs from S. lypcopersicum and S. peruvianum species. A new marker Pau-Do, in combination with C2S4, was designed to amplify in CDS of the Mi-1.2 gene. Amplification with these primers of cDNA from Beaufort and Maxifort indicated that the Mi-1.2 gene was expressed in both rootstocks, despite their susceptible phenotypic response to some Meloidogyne populations. The durability of the Mi-1 gene after repeated cultivation of resistant tomato rootstocks (PG-76 and Brigeor) was determined through field trials during three consecutive years. Rootstock PG-76 responded as highly resistant after the first cropping cycle, although it became fully susceptible after the second and the third cropping cycles. Rootstock Brigeor and the resistant tomato cultivar Monika (control), retained intermediate resistance levels at the end of the third year. Bioassays confirmed that selection of virulence occurred more rapidly in plots with rootstock PG-76 followed by Brigeor and the resistant tomato cultivar Monika. The virulent phenotype of the selected M. javanica populations in the field experiments was determined with MVC molecular marker, designed to distinguish selected from naturally virulent populations of Meloidogyne spp. The populations analyzed included two Japanese selected virulent populations, and the three virulent populations selected in the field trials, and one naturally virulent population and one avirulent population from Spanish. DNA samples were obtained from individual juveniles (J2) or adult females from all the selected virulent populations. Experiments included water samples free of nematodes (5-µm filtered), obtained from the draining-water of a plant infected by a Japanese selected virulent population. Amplification of DNA only occurred in samples of filtered water, but not in those containing only nematode genetic material. Sequencing and BLAST of the DNA fragments amplified by the MVC molecular marker, established a strong correlation of the amplified bands with proteins from betaproteobacteria species Overall, these results showed that the MVC marker is not related to a nematode virulence gene (avr) but to betaproteobacteria. New root-knot nematode resistant Mi-homologs were searched in accessions of the wild Solanum species. The S. huaylasense accession LA-1358 reduced reproduction of a population of M. arenaria to similar levels than the resistant tomato cultivar Anairis. Nevertheless, the resistance response of S. huaylasense accession LA-1358 was also nematode-species specific.

The plant group Solanum section Lycopersicon (the clade containing the domesticated tomato and its wild relatives) is ideal for integrating genomic tools and approaches into ecological and evolutionary research. Wild species within Lycopersicon span broad morphological, physiological, life history, mating system, and biochemical variation, and are separated by substantial, but incomplete postmating reproductive barriers, making this an ideal system for genetic analyses of these traits. This ecological and evolutionary diversity is matched by many logistical advantages, including extensive historical occurrence records for all species in the group, publicly available germplasm for hundreds of known wild accessions, demonstrated experimental tractability, and extensive genetic, genomic, and functional tools and information from the tomato research community. Here I introduce the numerous advantages of this system for Ecological and Evolutionary Functional Genomics (EEFG), and outline several ecological and evolutionary phenotypes and questions that can be fruitfully tackled in this system. These include biotic and abiotic adaptation, reproductive trait evolution, and the genetic basis of speciation. With the modest enhancement of some research strengths, this system is poised to join the best of our currently available model EEFG systems.

We present a multilocus sequencing study to assess patterns of polymorphism and divergence in the closely related wild tomato species, Solanum peruvianum and S. chilense (Solanum section Lycopersicon, Solanaceae). The data set comprises seven mapped nuclear loci (≈9.3 kb of analyzed sequence across loci) and four local population samples per species that cover much of the species' range (between 80 and 88 sequenced alleles across both species). We employ the analytical framework of divergence population genetics (DPG) in evaluating the utility of the “isolation” model of speciation to explain observed patterns of polymorphism and divergence. Whereas the isolation model is not rejected by goodness-of-fit criteria established via coalescent simulations, patterns of intragenic linkage disequilibrium provide evidence for postdivergence gene flow at two of the seven loci. These results suggest that speciation occurred under residual gene flow, implying that natural selection is one of the evolutionary forces driving the divergence of these tomato species. This inference is fully consistent with their recent divergence, conservatively estimated to be ≤0.55 million years. We discuss possible biases in the demographic parameter estimates due to the current restriction of DPG algorithms to panmictic species.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Transmission of Potato yellow mosaic virus (PYMV) (bipartite begomovirus) to tomato by 50 Bemisia tabaci biotype B individuals was observed when the acquisition access period (AAP) was at least 3 h and the inoculation access period (IAP) at least 30 min. The transmission efficiency increased with the access period to reach 92% transmission after a 48-h IAP and 48-h AAP. The transmission efficiency decreased when whiteflies were fed on PYMV non-host plant between AAP and IAP. According to these results, we inoculated nine Solanum accessions with 50 whiteflies (48-h APP and 48-h IAP) to assess their resistance level. Four of these accessions with various levels of resistance were graft inoculated with PYMV. Although none of the accessions were immune, we observed a high level of resistance to PYMV in Solanum pimpinellifolium LA2187-5 (no symptoms after vector or graft inoculation) and in Solanum chilense LA1969 (no symptoms after vector inoculation and one plant with symptoms after graft inoculation). Inheritance of LA2187-5 resistance, investigated in F1 and F2 populations, appeared to be recessive. Fewer plants were infected by PYMV in S. pimpinellifolium LA1478 after vector inoculation than after graft inoculation. We hypothesised that this was because of vector resistance, which could also be effective against other begomoviruses.

We employed a multilocus approach to examine the effects of population subdivision and natural selection on DNA polymorphism in 2 closely related wild tomato species (Solanum peruvianum and Solanum chilense), using sequence data for 8 nuclear loci from populations across much of the species' range. Both species exhibit substantial levels of nucleotide variation. The species-wide level of silent nucleotide diversity is 18% higher in S. peruvianum (pi(sil) approximately 2.50%) than in S. chilense (pi(sil) approximately 2.12%). One of the loci deviates from neutral expectations, showing a clinal pattern of nucleotide diversity and haplotype structure in S. chilense. This geographic pattern of variation is suggestive of an incomplete (ongoing) selective sweep, but neutral explanations cannot be entirely dismissed. Both wild tomato species exhibit moderate levels of population differentiation (average F(ST) approximately 0.20). Interestingly, the pooled samples (across different demes) exhibit more negative Tajima's D and Fu and Li's D values; this marked excess of low-frequency polymorphism can only be explained by population (or range) expansion and is unlikely to be due to population structure per se. We thus propose that population structure and population/range expansion are among the most important evolutionary forces shaping patterns of nucleotide diversity within and among demes in these wild tomatoes. Patterns of population differentiation may also be impacted by soil seed banks and historical associations mediated by climatic cycles. Intragenic linkage disequilibrium (LD) decays very rapidly with physical distance, suggesting high recombination rates and effective population sizes in both species. The rapid decline of LD seems very promising for future association studies with the purpose of mapping functional variation in wild tomatoes.

The differential response of 29 genotypes of tomato and wild tomato relatives (Solanum section Lycopersicon species) to cucumber mosaic virus strain Fny (CMV-Fny), alone or in combination with three different satellite RNA (satRNA) variants, allowed the identification of four disease phenotype patterns, each including plants that developed very severe symptoms (leaf malformations, top stunting and lethal necrosis) and plants that remained asymptomatic. No resistance or tolerance to CMV-Fny was observed, whilst individual host genotypes displayed latent infection upon inoculation with one (CMV-Fny/Tfn-satRNA, phenotype patterns 1 and 4), two (CMV-Fny/Tfn-satRNA and CMV-Fny/TTS-satRNA, phenotype pattern 2) or all three (the former two plus CMV-Fny/77-satRNA, phenotype pattern 3) CMV/satRNA combinations. RNA gel-blot analyses showed that latent infection generally correlated with a strong downregulation of CMV RNA accumulation levels. Introgression lines derived from a cross between Solanum habrochaites LA1777, which displayed disease phenotype pattern 2, and Solanum lycopersicum were screened for tolerance to the stunting phenotype induced by CMV-Fny/TTS-satRNA, and only one line, carrying an introgression on chromosome 6, was identified as being partially tolerant. Solanum chilense LA1932×S. lycopersicum back-cross introgression lines were screened for tolerance to lethal necrosis induced by CMV-Fny/77-satRNA (phenotype pattern 3); the tolerant phenotype was observed in 33 % of plants of the BC1F2 progeny and 1F3 progeny. Thus, potentially useful sources of tolerance to CMV/satRNA-induced diseases were identified, although the tolerant phenotypes appeared to be controlled by complex quantitative trait loci.

The effect of saline stress (NaCl, 40, 80 and 160 mmol L−1 of NaCl) on growth, plant water status and leaf antioxidant enzyme activities was investigated in a commercial cultivar of cherry tomato (Solanum lycopersicum var. cerasiforme L.) and in a wild-related species collected in a salt-affected area of North Chile (Solanum chilense Dun.). Salt stress was applied in a nutrient solution at the vegetative stage during 40 days. The highest NaCl concentration reduced shoot relative growth, fresh and dry weight and leaf area in the cultivated S. lycopersicum but had less impact on S. chilense. Both species were able to efficiently perform osmotic adjustment but S. chilense also exhibited an increase in leaf succulence. The oxidative stress estimated through malondialdehyde quantification was always higher in the cultivated S. lycopersicum, both in the absence and in the presence of salt. Total superoxide dismutase activity (EC 1.15.1.1) increased in response to the highest dose of NaCl in S. chilense but remained constant in S. lycopersicum. Salinity induced an increase in ascorbate peroxidase (EC 1.11.1.11) in S. chilense but reduced it in S. lycopersicum. It is concluded that S. chilense displays efficient strategies to cope with high NaCl doses and that management of the oxidative status is a key mechanism allowing this species to tolerate salinity.

Population genetics theory predicts that strong selection for rare, beneficial mutations or against frequent, deleterious mutations reduces polymorphism at linked neutral (or weakly selected) sites. The reduction of genetic variation is expected to be more severe when recombination rates are lower. In outbreeding species, low recombination rates are usually confined to certain chromosomal regions, such as centromeres and telomeres. In contrast, in predominantly selfing species, the rarity of double heterozygotes leads to a reduced effective recombination rate in the whole genome. We investigated the effects of restricted recombination on DNA polymorphism in these two cases, analyzing five Lycopersicon species with contrasting mating systems: L. chilense, L. hirsutum, L. peruvianum, L. chmielewskii, and L. pimpinellifolium, of which only the first three species have self-incompatibility alleles. In each species, we determined DNA sequence variation of five single-copy genes located in chromosomal regions with either high or low recombination rate. We found that the mating system has a highly significant effect on the level of polymorphism, whereas recombination has only a weak influence. The effect of recombination on levels of polymorphism in Lycopersicon is much weaker than in other well-studied species, including Drosophila. To explain these observations, we discuss a number of hypotheses, invoking selection, recombination, and demographic factors associated with the mating system. We also provide evidence that L. peruvianum, showing a level of polymorphism (almost 3%) that is comparable to the level of divergence in the whole genus, is the ancestral species from which the other species of the genus Lycopersicon have originated relatively recently.

Wild tomatoes are a valuable source of disease resistance germplasm for tomato (Solanum lycopersicum) breeders. Many species are known to possess a certain degree of resistance against certain pathogens; however, evolution of resistance traits is yet poorly understood. For some species, likeSolanum chilense, both differences in habitat and within species genetic diversity are very large. Here we aim to investigate the occurrence of spatially heterogeneous coevolutionary pressures between populations ofS. chilense. We investigate the phenotypic differences in disease resistance withinS. chilenseagainst three common tomato pathogens (Alternaria solani,Phytophthora infestansand aFusarium sp.) and confirm high degrees of variability in resistance properties between selected populations. Using generalised linear mixed models, we show that disease resistance does not follow the known demographic patterns of the species. Models with up to five available climatic and geographic variables are required to best describe resistance differences, confirming the complexity of factors involved in local resistance variation. We confirm that withinS. chilense, resistance properties against various pathogens show a mosaic pattern and do not follow environmental patterns, indicating the strength of local pathogen pressures. Our study can form the basis for further investigations of the genetic traits involved.

The wild tomato species Solanum chilense is divided into geographically and genetically distinct populations that show signs of defense gene selection and differential phenotypes when challenged with several phytopathogens, including the oomycete causal agent of late blight Phytophthora infestans. To better understand the phenotypic diversity of this disease resistance in S. chilense and to assess the effect of plant genotype versus pathogen isolate, respectively, we evaluated infection frequency in a systematic approach and with large sample sizes. We studied 85 genetically distinct individuals representing nine geographically separated populations of S. chilense. This showed that differences in quantitative resistance can be observed between but also within populations at the level of individual plants. Our data also did not reveal complete immunity in any of the genotypes. We further evaluated the resistance of a subset of the plants against P. infestans isolates with diverse virulence properties. This confirmed that the relative differences in resistance phenotypes between individuals were mainly determined by the plant genotype under consideration with modest effects of pathogen isolate used in the study. Thus, our report suggests that the observed quantitative resistance against P. infestans in natural populations of a wild tomato species S. chilense is the result of basal defense responses that depend on the host genotype and are pathogen isolate-unspecific.

[EN] Tomato (Solanum lycopersicum) is a major crop plant and a model system for fruit development. Solanum is one of the largest angiosperm genera(1) and includes annual and perennial plants from diverse habitats. Here we present a high-quality genome sequence of domesticated tomato, a draft sequence of its closest wild relative, Solanum pimpinellifolium(2), and compare them to each other and to the potato genome (Solanum tuberosum). The two tomato genomes show only 0.6% nucleotide divergence and signs of recent admixture, but show more than 8% divergence from potato, with nine large and several smaller inversions. In contrast to Arabidopsis, but similar to soybean, tomato and potato small RNAs map predominantly to gene-rich chromosomal regions, including gene promoters. The Solanum lineage has experienced two consecutive genome triplications: one that is ancient and shared with rosids, and a more recent one. These triplications set the stage for the neofunctionalization of genes controlling fruit characteristics, such as colour and fleshiness., This work was supported by: Argentina: INTA and CONICET. Belgium: Flemish Institute for Biotechnology and Ghent University. China: The State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences; Ministry of Science and Technology (2006AA10A116, 2004CB720405, 2006CB101907, 2007DFB30080) Ministry of Agriculture ('948' Program: 2007-Z5); National Natural Science Foundation (36171319); Postdoctoral Science Foundation (20070420446). European Union: FP6 Integrated Project EU-SOL PL 016214. France: Institute National de la Recherche Agronomique and Agence Nationale de la Recherche. Germany: the Max Planck Society. India: Department of Biotechnology, Government of India; Indian Council of Agricultural Research. Italy: Ministry of Research (FIRB-SOL, FIRB-Parallelomics, ItaLyco and GenoPOM projects); Ministry of Agriculture (Agronanotech and Biomassval projects); FILAS foundation; ENEA; CNR-ENEA project L. 191/2009. Japan: Kazusa DNA Research Institute Foundation and National Institute of Vegetable and Tea Science. Korea: KRIBB Basic Research Fund and Crop Functional Genomics Research Center (CFGC), MEST. Netherlands: Centre for BioSystemsGenomics, Netherlands Organization for Scientific Research. Spain: Fundacion Genoma Espana; Cajamar; FEPEX; Fundacion Seneca; ICIA; IFAPA; Fundacion Manrique de Lara; Instituto Nacional de Bioinformatica. UK: BBSRC grant BB/C509731/1; DEFRA; SEERAD. USA: NSF (DBI-0116076; DBI-0421634; DBI-0606595; IOS-0923312; DBI-0820612; DBI-0605659; DEB-0316614; DBI 0849896 and MCB 1021718); USDA (2007-02773 and 2007-35300-19739); USDA-ARS. We acknowledge the Potato Genome Sequencing Consortium for sharing data before publication; potato RNA-Seq data was provided by C. R. Buell from the NSF-funded Potato Genome Sequence and Annotation project; tomato RNA-Seq data by the USDA-funded SolCAP project, N. Sinha and J. Maloof; the Amplicon Express team for BAC pooling services; construction of the Whole Genome Profiling (WGP) physical map was supported by EnzaZaden, RijkZwaan, Vilmorin&Cie, and Takii & Co. Keygene N. V. owns patents and patent applications covering its AFLP and Whole Genome Profiling technologies; AFLP and Keygene are registered trademarks of Keygene N. V.