Validación de marcadores microsatélites mediante análisis de fusión de alta resolución (HRM) para la caracterización genética de Pitavia punctata (Ruiz & Pav.) Mol.
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Date
2026
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Universidad de Concepción
Abstract
El análisis de fusión de alta resolución se ha consolidado como una herramienta eficiente para la genotipificación de marcadores microsatélites en diversos sistemas biológicos. En este contexto, el estudio tuvo como objetivo validar el uso de esta metodología para la discriminación de genotipos basada en microsatélites, utilizando como modelo la especie endémica y amenazada Pitavia punctata (Ruiz&Pav.) Mol. A partir de una base de datos previa de microsatélites, se seleccionó un conjunto de marcadores candidatos que fueron evaluados mediante reacción en cadena de la polimerasa en tiempo real y análisis de fusión. Dos marcadores polimórficos fueron validados, destacando uno de ellos por su alto poder discriminante. Este marcador permitió identificar múltiples genotipos en individuos provenientes de cuatro poblaciones naturales, evidenciando variación genética y diferencias en la distribución de genotipos entre localidades. La concordancia entre los genotipos obtenidos mediante análisis de fusión y la variación alélica determinada mediante análisis de fragmentos fluorescentes confirmó que las diferencias observadas en los perfiles de fusión reflejaron variación genética real, asociada tanto a cambios en la longitud del microsatélite como a variaciones en regiones flanqueantes. Aunque el número de marcadores polimórficos fue limitado, los resultados demostraron que esta aproximación constituye una estrategia eficaz y costo-efectiva para la validación y genotipificación de microsatélites en especies no modelo, proporcionando una base metodológica para estudios futuros en genética de la conservación.
High-resolution melting analysis has emerged as an efficient tool for genotyping microsatellite markers across diverse biological systems. In this context, the present study aimed to validate the use of this approach for microsatellite-based genotype discrimination, using the endangered endemic species Pitavia punctata (Ruiz&Pav.) Mol. as a model. A set of candidate microsatellite markers was selected from a previously generated database and evaluated using real-time polymerase chain reaction followed by melting analysis. Two polymorphic markers were successfully validated, with one showing a high discriminatory power. This marker enabled the identification of multiple genotypes among individuals from four natural populations, revealing genetic variation and differences in genotype distribution among localities. The concordance between genotypes obtained through melting analysis and allelic variation determined by fluorescent fragment analysis confirmed that the observed differences in melting profiles reflected true genetic variation, associated with both changes in microsatellite length and sequence variation in flanking regions. Although the number of polymorphic markers was limited, the results demonstrated that this approach constitutes an effective and cost-efficient strategy for microsatellite validation and genotyping in non-model species, providing a methodological basis for future studies in conservation genetics.
High-resolution melting analysis has emerged as an efficient tool for genotyping microsatellite markers across diverse biological systems. In this context, the present study aimed to validate the use of this approach for microsatellite-based genotype discrimination, using the endangered endemic species Pitavia punctata (Ruiz&Pav.) Mol. as a model. A set of candidate microsatellite markers was selected from a previously generated database and evaluated using real-time polymerase chain reaction followed by melting analysis. Two polymorphic markers were successfully validated, with one showing a high discriminatory power. This marker enabled the identification of multiple genotypes among individuals from four natural populations, revealing genetic variation and differences in genotype distribution among localities. The concordance between genotypes obtained through melting analysis and allelic variation determined by fluorescent fragment analysis confirmed that the observed differences in melting profiles reflected true genetic variation, associated with both changes in microsatellite length and sequence variation in flanking regions. Although the number of polymorphic markers was limited, the results demonstrated that this approach constitutes an effective and cost-efficient strategy for microsatellite validation and genotyping in non-model species, providing a methodological basis for future studies in conservation genetics.
Description
Tesis presentada para optar al título de Ingeniero/a en Biotecnología Vegetal.
Keywords
Microsatélites (Genética), Plantas endémicas, Genotipo, Conservación de recursos naturales