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Universidad Politécnica Salesiana Sede Quito UNIVERSIDAD POLITÉCNICA SALESIANA SEDE QUITO CARRERA: INGENIERÍA EN BIOTECNOLOGÍA DE LOS RECURSOS NATURALES Trabajo de titulación previo a la obtención del título de: INGENIERAS EN BIOTECNOLOGÍA DE LOS RECURSOS NATURALES TEMA: IDENTIFICACIÓN MOLECULAR DE HONGOS ENDÓFITOS Y MICORRÍZICOS PRESENTES EN LA RELACIÓN SIMBIÓTICA DE LA ESPECIE Caucaea pichinchae (ORCHIDACEAE) CON SU RESPECTIVO HOSPEDERO. AUTORAS: MARGARITA DE LOS ÁNGELES CAZAR IZA DANIELA BELÉN PÉREZ REYES TUTOR: MARCO FERNANDO CERNA CEVALLOS Quito, julio del 2019 Dedicatoria A Dios por ser parte de esta travesía con su sabiduría y fortaleza logré culminar el sueño de ser profesional. A mis abuelitos Blanquita y Manuelito Iza que siempre estuvieron con sus consejos oportunos brindándome seguridad en los momentos difíciles. A mis padres en especial a mi madre Nancy por darme su apoyo incondicional, inculcándome a ser una mejor persona y sobre todo por anhelar lo mejor para mi vida. A mis hermanos Fernando, Ariel por todos los momentos gratos compartidos y sus frases motivadoras permitiéndome aumentar mi fé. Margarita Cazar Al creador de todas las cosas, al que me ha dado fortaleza para continuar cada día. A mis padres Bélgica y Jorge por haberme forjado como la persona que soy, impulsándome siempre a cumplir mis sueños. A mis hermanas Fernanda, Priscila y a mi hermano Jorge que siempre han estado dispuestos a ayudarme. A mis sobrinos Brandon, Mateo y Brenda que por medio de sus alegrías me han motivado a seguir adelante. A David por brindarme su amor, apoyo incondicional y por siempre anhelar lo mejor en mi vida Daniela Pérez Agradecimientos Agradecemos a la Universidad Politécnica Salesiana, a toda la carrera de Biotecnología, a nuestros docentes por compartir sus enseñanzas y experiencias durante nuestro periodo académico permitiéndonos crecer cómo buenas profesionales. A nuestro Tutor Marco Cerna Ph.D por darnos la oportunidad de integrar el grupo de investigación NUNKUI WAKAN, brindándonos su conocimiento para el desarrollo del presente proyecto de investigación y su motivación continua. Al Ing. Alexander Hirtz por la donación de muestras y trabajo en campo, al grupo de Investigación NUNKUI WAKAN por hacer posible la ejecución, financiamiento de nuestro proyecto y a los ingenieros Elizabeth Yugsi, Byron Fuertes que forman parte del personal de los Laboratorios de Ciencias de la Vida de la carrera Ing. en Biotecnología por la colaboración oportuna. A nuestros compañeros y compañeras por los momentos compartidos durante toda la carrera y por su amistad incondicional. Índice Introducción ................................................................................................................. 1 Capítulo 1 ..................................................................................................................... 3 1. Marco conceptual .................................................................................................... 3 1.1 Familia Orchidaceae ......................................................................................... 3 1.2 Género Caucaea ............................................................................................... 4 1.2.1 Clasificación taxonómica del género Caucaea ......................................... 5 1.2.2 Descripción botánica y morfológica de Caucaea pichinchae Szlach. & Kolan. ........................................................................................................ 5 1.3 Relación simbiótica .......................................................................................... 7 1.4 Hongos endófitos .............................................................................................. 8 1.4.1 Clasificación de los hongos endófitos: ...................................................... 9 1.5 Micorrizas/Hongos micorrízicos .................................................................... 10 1.5.1 Micorrizas de orquídeas .......................................................................... 10 1.5.2 Clasificación de hongos micorrízicos ..................................................... 11 1.6 Técnicas moleculares para la identificación ................................................... 12 1.6.1 Extracción de ADN ................................................................................. 12 1.6.2 Reacción en cadena de la polimerasa ...................................................... 12 1.6.3 Secuenciación Sanger .............................................................................. 14 Capítulo 2 ................................................................................................................... 16 2. Metodología ........................................................................................................... 16 2.1 Área de estudio de la especie Caucaea pichinchae Szlach. & Kolan. ........... 16 2.2 Recolección de las muestras del género Caucaea pichinchae Szlach. & Kolan. con su respectivo hospedero ........................................................................... 16 2.3 Aislamiento de hongos provenientes de tallos y raíz ..................................... 18 2.3.1 Preparación del medio de cultivo PDA ................................................... 18 2.3.2 Protocolo de desinfección de las muestras .............................................. 18 2.3.3 Siembra de las muestras y aislamiento de hongos .................................. 19 2.4 Identificación morfológica de los subcultivos aislados de tallo y raíz ........... 19 2.5 Identificación molecular ................................................................................. 20 2.5.1 Extracción de ADN del material fúngico ................................................ 20 2.5.2 Amplificación del ADN cloroplástico y ribosómico nuclear .................. 21 2.5.3 Secuenciación .......................................................................................... 22 2.5.4 Análisis de las secuencias ....................................................................... 23 Capítulo 3 ................................................................................................................... 24 3. Resultados y discusión .......................................................................................... 24 3.1. Recolección de las muestras del género Caucaea pichinchae Szlach. & Kolan con su respectivo hospedero ........................................................................... 24 3.2. Aislamiento de hongos de tallo y raíz ............................................................ 25 3.3. Identificación morfológica de los hongos aislados ........................................ 28 3.4. Identificación molecular ................................................................................. 46 3.4.1. Amplificación del ADN cloroplástico y ribosómico nuclear .................. 46 3.4.2 Análisis de las secuencias .......................................................................... 48 3.4.3 Análisis de la biodiversidad en los puntos de recolección......................... 57 Conclusiones .............................................................................................................. 59 Recomendaciones ....................................................................................................... 60 Bibliografía ................................................................................................................ 61 Anexos: ...................................................................................................................... 72 Índice de Tablas Tabla 1 Puntos de recolección de las muestras de la especie Caucaea pichinchae Szlach. & Kolan con sus coordenadas geográficas (UTM) ............................................. 16 Tabla 2 Listado de las muestras colectadas ........................................................................ 17 Tabla 3 Preparación de la disolución para PCR con el Kit Phire Plant Direct PCR Master Mix ......................................................................................................................... 21 Tabla 4 Condiciones de temperatura para la amplificación del ADN cloroplástico y ribosómico nuclear ................................................................................................ 22 Tabla 5 Subcultivos de los hongos endófitos aislados de las muestras de tallo ............... 26 Tabla 6 Subcultivos de hongos aislados de las muestras de raíz ....................................... 27 Tabla 7 Estructuras macroscópicas y microscópicas de hongos aislados de tallos en medio PDA........................................................................................................................ 29 Tabla 8 Estructuras macroscópicas y microscópicas de hongos aislados de raíz en medio PDA........................................................................................................................ 39 Tabla 9 Secuencia del ADN cloroplástico de la especie Caucaea pichinchae Szlach & Kolan ...................................................................................................................... 49 Tabla 10 Secuencia del ADN cloroplástico de la especie Oreopanax ecuadoriensis Seem ....................................................................................................................... 50 Tabla 11 Listado de los grupos formados del árbol filogenético de las muestras de tallo ........................................................................................................................
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