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Potencial genético de germoplasma de tomate para la resistencia al nematodo nodulador Meloidogyne spp.
dc.contributor.advisor | Ceballos Aguirre, Nelson | |
dc.contributor.author | Padilla Hurtado, Beatriz Elena | |
dc.date.accessioned | 2022-04-17T18:32:25Z | |
dc.date.available | 2025-01-31 | |
dc.date.available | 2022-04-17T18:32:25Z | |
dc.date.issued | 2022-04-07 | |
dc.identifier.uri | https://repositorio.ucaldas.edu.co/handle/ucaldas/17557 | |
dc.description | Ilustraciones | spa |
dc.description.abstract | spa:La caracterización y evaluación de los recursos fitogenéticos de tomate están dentro de las áreas de investigación estratégica para poder responder a los retos en los sistemas agrícolas productivos actuales y futuros. El tomate es originario de la cordillera de los Andes y ocupa el primer lugar dentro de la lista de producción mundial de hortalizas de la FAO (Organización de las Naciones Unidas para la Alimentación y la Agricultura). El tomate Solanum lycopersicum L. es la única especie domesticada dentro de la sección Lycopersicon del Genero Solanum. Presenta una baja diversidad, ya que contiene menos del 5% de la variación genética de sus parientes silvestres. Por tanto, sus parientes silvestres son fuente promisoria de resistencia a patógenos y reservorio de genes para la producción sostenible de la especie cultivada. Una de las principales enfermedades del cultivo del tomate es la nodulación radical causada por Meloidogyne spp, debido a que los nematodos penetran en las raíces y migran hacia los haces vasculares, lo que resulta en la formación de nódulos, que afectan la captación de nutrientes y agua por parte de la planta; ocasionando un retraso en el desarrollo, debilitamiento generalizado, amarillamiento en las hojas más viejas y una reducción considerable en la cantidad y calidad de la producción. Se han reportado pérdidas en producción causadas por éste patógeno en el cultivo de tomate entre el 25 al 68%. Las plantas han evolucionado para protegerse del ataque de patógenos mediante mecanismos de defensa, en los cuales están involucradas las Proteínas de Resistencia (PR), mecanismo conocido como resistencia gen a gen, en la cual cada gen R de la planta corresponde a un gen Avr específico del patógeno. Estos genes R están en constante selección y diversificación para mantener el mismo ritmo de la evolución de los patógenos. En la resistencia de tomate a Meloidogyne spp, un único gen R dominante, denominado Mi-1, se encontró en la especie silvestre S. peruvianum, el cual, fue introgresado en variedades de tomate aportando el rasgo de resistencia, observado como reducción en la reproducción de las especies, M. incognita, M. arenaria, y M. javanica. Los bancos de germoplasma contienen enormes recursos sin explotar de distintos alelos de los genes R, que pueden tener una aplicación potencial en programas de mejoramiento genético. La contrastación de la respuesta en campo de los diferentes genotipos frente a la enfermedad, permiten corroborar o encontrar alternativas para el uso de los recursos genéticos, derivado posiblemente de otros mecanismos de defensa inducidos en la interacción planta-patógeno. De esta forma, la evaluación del potencial genético de germoplasma de tomate para patógenos de importancia económica como Meloidogyne spp., contribuirá al aprovechamiento de este recurso fitogenético, al seleccionar accesiones e identificar genes involucrados en la resistencia, que permitan aportar información para el diseño de estrategias de manejo integrado de la enfermedad, involucrando nuevas fuentes de resistencia, o variantes de las mismas, para efectos más durables en el campo. El uso de marcadores moleculares estrechamente relacionados con genes R y su aplicación en la selección asistida por marcadores (SAM), ha tenido éxito en programas de mejoramiento genético de tomate, especialmente para resistencia a enfermedades. El marcador codominante Mi-23, tipo SCAR (siglas en inglés Sequenced Characterized Amplified Region, o secuencia amplificada de una Región conocida) ligado al gen de resistencia a Meloidogyne spp., Mi-1, permitió identificar fenotipos resistentes (dominantes y heterocigotos) y susceptibles (recesivos). En el presente estudio se evaluaron 92 accesiones mediante la amplificación del marcador molecular codominante tipo SCAR Mi-23, amplificando la banda de resistencia en 18 accesiones, 12 en heterocigosis y 6 en homocigosis resistente. En el presente estudio mediante la evaluación de diferentes densidades de población de Meloidogyne spp. (0, 1000, 2000, 3000, 4000 y 5000 individuos/planta en cuatro accesiones silvestres de tomate y dos testigos comerciales uno resistente y uno susceptible, se determinó que, la densidad de población de Meloidogyne spp. que permitió evaluar el potencial de resistencia del germoplasma de tomate a Meloidogyne spp fue de 1000 individuos por planta, siendo ésta, la densidad de población seleccionada para la fenotipificación de la resistencia de 48 accesiones de tomate silvestres. Se realizó la clasificación de la respuesta mediante la medición de las variables: índice reproductivo del patógeno y la escala de daño ocasionada en las raíces, a los 60 días de inoculadas con Meloidogyne spp. Las accesiones que amplificaron la banda de resistencia con el marcador molecular Mi-23 y presentaron una clasificación en su fenotipo comomedianamente resistente en campo, fueron LA0111 (Solanum peruvianum) y LA2076 (Solanum lycopersicum var ceraciforme), las cuales pueden ser incluidas para el desarrollo de nuevas variedades de la especie cultivada o como portainjertos resistentes a dicho patógeno, alternativas que al ser incluidas en un programa de manejo integrado, pueden mitigar el impacto de patógenos de importancia económica como Meloidogyne spp. Por medio de la amplificación y secuenciación de fragmentos de las subunidades ribosomales 28S (LSU) y 18S (SSU1 y SSU2), se determinó que el inóculo experimental, usado para la evaluación de resistencia genética de germoplasma de tomate a Meloidogyne spp. está compuesto por las especies M. incognita y M. arenaria en una proporción de aproximadamente 92.3% y 7.7% respectivamente. La resistencia genética de tomate a Meloidogyne en genotipos comerciales, se ha basado únicamente en el gen Mi-1, sin embargo, dicha resistencia no se garantiza cuando hay altas densidades de población del patógeno, biotipos virulentos del patógeno al gen Mi-1 y cuando la temperatura del suelo cultivado es superior a 28°C, por lo tanto, en el presente estudio, se llevó a cabo la identificación in silico de tres secuencias para cada uno de los genes candidatos a Mi-9 y el Mi-3 reportados previamente mediante mapeo genético como resistentes al patógeno, por su característica de termoestabilidad a temperaturas superiores a 28°C. Los genes Mi-9 y el Mi-3, fueron encontrados en las accesiones de las especies silvestres de Solanum arcanum (LA2157) y Solanum peruvianum (LA3858) respectivamente, Adicionalmente, se logró integrar el conocimiento del mapa genético, el genoma y el transcriptoma de la accesión LA3858 de S. peruvianum, registrada como resistente a Meloidogyne spp. en temperaturas del suelo hasta 32°C y reportar 3 secuencias de genes candidatos al gen Mi-3. Lo anterior permitió ampliar las alternativas de resistencia genética y aprovechamiento de los parientes silvestres de la especie cultivada para proyectar sistemas de producción de tomate más sostenibles La identificación de secuencias de genes candidatos a genes Mi con resistencia termoestable a Melodogyne spp, como son el gen Mi-9 y el Mi-3 sumado al tradicional Mi-1 y posteriores estudios experimentales de genómica funcional o silenciamiento génico, se podrán incluir dentro de las alternativas del manejo integrado del patógeno, utilizando estrategias de pirimidización de genes que, produzcan una resistencia más durable, en diferentes condiciones de siembra del cultivo de tal modo que los sistemas de producción del tomate sean más sostenibles y amigables con el ambiente. | spa |
dc.description.abstract | eng:The characterization and evaluation of tomato plant genetic resources are within the areas of strategic research to be able to respond to the challenges in current and future productive agricultural systems. The tomato is native to the Andes region and occupies the first place in the list of world vegetable production of the FAO (Food and Agriculture Organization of the United Nations). The tomato Solanum lycopersicum L. is the only domesticated species within the Lycopersicon section of the Genus Solanum. It has a low diversity, since it contains less than 5% of the genetic variation of its wild relatives. Therefore, their wild relatives are a promising source of resistance to pathogens and a reservoir of genes for the sustainable production of the cultivated species. One of the main tomato crop diseases is root nodulation caused by Meloidogyne spp, due to the fact that nematodes penetrate the roots and migrate towards the vascular bundles, resulting in the formation of nodules, which arises from the uptake of nutrients and water by the plant; causing a delay in development, generalized weakening, yellowing in the oldest leaves and a considerable reduction in the quantity and quality of production. Losses in production caused by this pathogen have been suffered in the tomato crop between 25 to 68%. Plants have evolved to protect themselves from the attack of pathogens through defense mechanisms, in which Resistance Proteins (PR) are involved, a mechanism known as gene-to-gene resistance, in which each R gene of the plant corresponds to an Avr gene pathogen specific. These R genes are under constant selection and diversification to keep pace with pathogen evolution. In tomato resistance to Meloidogyne spp, a single dominant R gene, called Mi-1, was found in the wild species S. peruvianum, which was introgressed into tomato varieties providing the resistance trait, observed as a reduction in the reproduction of the species, M. incognita, M. arenaria, and M. javanica. Germplasm banks contain enormous untapped resources of different alleles of the R genes, which may have potential application in breeding programs. The contrasting of the response in the field of the different genotypes against the disease, allows corroborating or finding alternatives for the use of genetic resources, possibly derived from other defense mechanisms induced in the plant-pathogen interaction. In this way, the evaluation of the genetic potential of tomato germplasm for pathogens of economic importance such as Meloidogyne spp., will contribute to the use of this plant genetic resource, by selecting accessions and identifying genes involved in resistance, which will provide information for the design of integrated disease management strategies, involving new sources of resistance, or variants thereof, for more lasting effects in the field. The use of molecular markers related to R genes and their application in marker-assisted selection (MAS) has been successful in tomato breeding programs, especially for disease resistance. The codominant marker Mi-23, type SCAR (acronym in English Sequenced Characterized Amplified Region, or amplified sequence of a known Region) linked to the resistance gene to Meloidogyne spp., Mi-1, allowed to identify resistant phenotypes (dominant and heterozygous) and susceptible (recessive). In the present study, 92 accessions were evaluated by amplifying the SCAR-type codominant molecular marker Mi-23, amplifying the resistance band in 18 accessions, 12 heterozygous and 6 homozygous resistant. In the present study, by evaluating different population densities of Meloidogyne spp. (0, 1,000, 2,000, 3,000, 4,000 and 5,000 individuals/plant in four wild tomato accessions and two commercial controls, one resistant and one susceptible, it was determined that the population density of Meloidogyne spp., which allowed evaluating the resistance potential of tomato germplasm to Meloidogyne spp was 1000 individuals per plant, this being the population density selected for the resistance phenotyping of 48 wild tomato accessions. The response was classified by measuring the variables: index of the pathogen and the scale of damage caused in the roots, 60 days after inoculation with Meloidogyne spp. The accessions that amplified the resistance band with the molecular marker Mi-23 and presented a classification in their phenotype as moderately resistant in the field, were LA0111 (Solanum peruvianum) and LA2076 (Solanum lycopersicum var ceraciforme), which can be included for the development of e new varieties of the cultivated species or as rootstocks resistant to said pathogen, alternatives that, when included in an integrated management program, can mitigate the impact of economically important pathogens such as Meloidogyne spp. Through the amplification and sequencing of fragments of the ribosomal subunits 28S (LSU) and 18S (SSU1 and SSU2), it was determined that the experimental inoculum, used for the evaluation of genetic resistance of tomato germplasm to Meloidogyne spp. it is composed of the species M. incognita and M. arenaria in a proportion of approximately 92.3% and 7.7%, respectively. The genetic resistance of tomato to Meloidogyne in commercial genotypes has been based solely on the Mi-1 gene, however, said resistance is not guaranteed when there are high population densities of the pathogen, virulent biotypes of the pathogen to the Mi-1 gene and when the temperature of the cultivated soil is higher than 28°C, therefore, in the present study, the in silico identification of three sequences for each of the previously reported Mi-9 and Mi-3 candidate genes was carried out by genetic mapping as resistant to the pathogen, due to its thermostability characteristic at temperatures above 28°C. The Mi-9 and Mi-3 genes were found in the accessions of the wild species of Solanum arcanum (LA2157) and Solanum peruvianum (LA3858), respectively. Additionally, it was possible to integrate the knowledge of the genetic map, the genome and the transcriptome. of the LA3858 accession of S. peruvianum, registered as resistant to Meloidogyne spp. in soil temperatures up to 32°C and report 3 candidate gene sequences to the Mi-3 gene. The foregoing allowed expanding the alternatives of genetic resistance and use of the wild relatives of the cultivated species to project more sustainable tomato production systems. The identification of candidate gene sequences for Mi genes with thermostable resistance to Melodogyne spp, such as the Mi-9 and Mi-3 genes added to the traditional Mi-1 and subsequent experimental studies of functional genomics or gene silencing, may be included within of the alternatives of the integrated management of the pathogen, using gene pyrimidization strategies that produce a more durable resistance, in different crop sowing conditions in such a way that the tomato production systems are more sustainable and friendly with the environment. | eng |
dc.description.tableofcontents | INTRODUCCIÓN / Referencias bibliográficas / CAPÍTULO I / 1. Revisión de literatura / 1.1 Importancia de los recursos fitogenéticos / 1.2 Cultivo del tomate / 1.3 Enfermedades del cultivo de tomate / 1.4 Nodulación radical causada por Meloidogyne spp / 1.5 Identificación de especies de Meloidogyne por taxonomía molecular / 1.6 Defensa de las plantas a patógenos / 1.7 Interacción planta-patógeno / 1.8 Mecanismo de infección de Meloidogyne spp. / 1.9 Mecanismos de defensa de las plantas a Meloidogyne spp. / 1.10 Resistencia genética de tomate a Meloidogyne spp / 1.11 Genes Mi de resistencia a Meloidogyne spp. / 1.12 Problemas de la resistencia genética mediada por genes Mi / 1.13 Marcadores moleculares asociados a genes Mi / 1.14 Referencias bibliográficas / CAPÍTULO II / 2. Evaluation of root-knot nematodes (Meloidogyne spp.) population density for disease resistance screening of tomato germplasm carrying the gene Mi-1 / 2.1 ABSTRACT / 2.2 INTRODUCTION / 2.3 MATERIAL AND METHODS / 2.3.1 Meloidogyne spp. Inoculum / 2.3.2 Plant material / 2.3.3 Amplification of the molecular marker SCAR Mi-23 / 2.3.4 Experimental design / 2.3.5 Variables / 2.3.6 Statistical analyses / 2.4 RESULTS AND DISCUSSION / 2.4.1 SCAR Mi-23 molecular marker amplification / 2.4.2 Phenotypic response to different population densities of Meloidogyne spp / 2.4.3 Genotype-phenotype association / 2.4.4 Agronomic variables / 2.4.5 Correlation of variables / 2.5 CONCLUSIONS / 2.6 REFERENCES / CAPÍTULO III / 3. Identificación molecular de Meloidogyne spp. en evaluación de resistencia genética en 13 germoplasma de tomate / 3.1 Resumen / 3.2 Introducción / 3.3.1 Material biológico / 3.3.2 Identificación molecular / 3.4 Resultados y análisis de secuencias / 3.5 Identificación de las especies que componen el inóculo experimental / 3.6 Análisis filogenético / 3.7 Conclusiones / 3.8 Referencias bibliográficas / CAPÍTULO IV / 4. Asociación del gen Mi-1 con la respuesta de resistencia de germoplasma de tomate a Meloidogyne spp / 4.1 Resumen / 4.2 Introducción / 4.3 Materiales y métodos / 4.3.1 Inóculo experimental de Meloidogyne spp / 4.3.2 Material vegetal / 4.3.3 Amplificación del marcador molecular Mi-23 asociado al gen Mi-1 / 4.3.4 Reacción de germoplasma de tomate a Meloidogyne spp. / 4.3.5 Diseño experimental /4.3.6 Análisis estadístico / 4.4 Resultados y discusión / 4.4.1 Amplificación del marcador molecular Mi-23 asociado al gen Mi-1 / 4.4.2 Reacción de germoplasma de tomate a Meloidogyne spp. / 4.4.3 Reacción de la interacción genotipo ambiente / 4.5 Conclusiones / 4.6 Referencias bibliográficas / CAPÍTULO V / 5. Identificación de genes de resistencia en germoplasma de tomate / Identificación de genes candidatos a Mi-9 y Mi-3 de resistencia termo-estable a Meloidogyne spp en el genoma reensamblado de Solanum arcanum y en el genoma secuenciado y ensamblado de Solanum peruvianum / 5.1 Resumen / 5.2 Introducción / 5.3 Identificación de genes candidatos al gen Mi-9 en Solanum arcanum, LA2157 / 5.3.1 Materiales y métodos / 5.3.1.1 Datos de las secuencias genómicas / 5.3.1.2 Re-ensamblaje del genoma de S. arcanum / 5.3.1.3 Integración mapa genético y genoma / 5.3.1.4 Identificación de genes candidatos al gen Mi-9 en S. arcanum / 5.3.1.5 Conversión de marcadores moleculares / 5.3.2 Resultados y discusión / 5.3.2.1 Re-ensamblaje del genoma de S. arcanum / 5.3.2.2 Análisis de sinténia / 5.3.2.3 Integración entre el mapa genético y genoma / 5.3.2.4 Identificación de genes candidatos al gen Mi-9 en S. arcanum SA-RT / 14 5.3.2.5 Conversión de marcadores moleculares / 5.4 Identificación de genes candidatos al gen Mi-3 en Solanum peruvianum, LA3858 / 5.4.1 Materiales y métodos / 5.4.1.1 Material vegetal / 5.4.1.2 Extracción de ADNg de alto peso molecular de S. peruvianum / 5.4.1.3 Secuenciación del genoma de S. peruvianum / 5.4.1.4 Ensamblaje del genoma de S. peruvianum / 5.4.1.5 Integración mapa genético y genoma / 5.4.1.6 Identificación de genes candidatos al gen Mi-3 en S. peruvianum y análisis de expresión diferencial / 5.4.2 Resultados y discusión / 5.4.2.1 Secuenciación del genoma de S. peruvianum, LA3858 / 5.4.2.2 Ensamblaje del genoma de S. peruvianum, LA3858 / 5.4.2.3 Integración entre el mapa genético y genoma / 5.4.2.4 Identificación de genes candidatos al gen Mi-3 en S. peruvianum LA3858 y análisis de expresión diferencial / 5.5 Conclusiones / 5.6 Referencias bibliográficas / CAPÍTULO VI / 6. Conclusiones y recomendaciones | spa |
dc.format.mimetype | application/pdf | spa |
dc.language.iso | eng | spa |
dc.language.iso | spa | spa |
dc.title | Potencial genético de germoplasma de tomate para la resistencia al nematodo nodulador Meloidogyne spp. | spa |
dc.type | Trabajo de grado - Doctorado | spa |
dc.contributor.researchgroup | GIPPA: Producción Agropecuaria (Categoría A1) | spa |
dc.description.degreelevel | Doctorado | spa |
dc.description.notes | No autorizó la publicación de la tesis de Doctorado ya que esta pendiente la publicación de los artículos científicos. | spa |
dc.identifier.instname | Universidad de Caldas | spa |
dc.identifier.reponame | Repositorio Institucional Universidad de Caldas | spa |
dc.identifier.repourl | https://repositorio.ucaldas.edu.co | spa |
dc.publisher.faculty | Facultad de Ciencias Agropecuarias | spa |
dc.publisher.place | Manizales | spa |
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dc.rights.accessrights | info:eu-repo/semantics/closedAccess | spa |
dc.rights.accessrights | info:eu-repo/semantics/closedAccess | spa |
dc.rights.accessrights | info:eu-repo/semantics/closedAccess | spa |
dc.rights.accessrights | info:eu-repo/semantics/closedAccess | spa |
dc.subject.proposal | Genes de resistencia | spa |
dc.subject.proposal | Resistencia genética | spa |
dc.subject.proposal | Taxonomía molecular | spa |
dc.subject.proposal | Mapa genómico | spa |
dc.subject.unesco | Biología agraria | |
dc.type.coar | http://purl.org/coar/resource_type/c_db06 | spa |
dc.type.content | Text | spa |
dc.type.driver | info:eu-repo/semantics/doctoralThesis | spa |
dc.type.version | info:eu-repo/semantics/publishedVersion | spa |
oaire.version | http://purl.org/coar/version/c_ab4af688f83e57aa | spa |
oaire.accessrights | http://purl.org/coar/access_right/c_14cb | spa |
dc.description.degreename | Doctor(a) en Ciencias Agrarias | spa |
dc.publisher.program | Doctorado en Ciencias Agrarias | spa |
dc.rights.coar | http://purl.org/coar/access_right/c_14cb | spa |