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Saltillo, Coahuila a 25 De Agosto De 2020 Programa De Maestría En Saltillo, Coahuila a 25 de agosto de 2020 Programa de Maestría en Ciencias en Agroplasticultura Coordinación de Posgrado P R E S E N T E Por este conducto nos permitimos informar a esta coordinación que, el documento de tesis preparado por ANTONNY URIEL VILLEGAS CHAVEZ titulado "Estudio de la diversidad genética de plantassilvestres de Guayule ( Parthenium argentatum Gray) mediante marcadores ITS” el cual fue presentado el día de hoy 25 de agosto de 20 20, ha sido modificado de acuerdo a las observaciones, comentarios y sugerencias, realizadas por el Comité Evaluador asignado. Por tal motivo, avalamos que el documento corresponde a la versión final del documento de tesis. Atentamente, SINODALES ________ ___________________________ __________________________________ Dra. Ileana Vera Reyes Dra. Mona Kassem Presidente Secretario ______________________________ Dr. Gerardo Hernandez Vera Vocal Vo. Bo. de los Asesores __________ ___ _ _ __________ _________ _______________ __________ _________ __ Dra. Ana Margarita Rodríguez Hernández Dr. Marco Antonio Castillo Campohermoso CENTRO DE INVESTIGACIÓN EN QUÍMICA APLICADA Programa de Maestría en Ciencias en Agroplasticultura TESIS Estudio de la diversidad genética de plantas silvestres de Guayule (Parthenium argentatum Gray) mediante marcadores ITS Presentada por: ANTONNY URIEL VILLEGAS CHAVEZ Para obtener el grado de: Maestro en Ciencias en Agroplasticultura Asesorada por: Dra. Ana Margarita Rodríguez Hernández Dr. Marco Antonio Castillo Campohermoso SINODALES ________________________ _____________________ Dra. Ileana Vera Reyes Dra. Mona Kassem Presidente Secretario _______________________________ Dr. Gerardo Hernández Vera Vocal Saltillo, Coahuila Agosto, 2020 CENTRO DE INVESTIGACIÓN EN QUÍMICA APLICADA Programa de Maestría en Ciencias en Agroplasticultura TESIS Estudio de la diversidad genética de plantas silvestres de Guayule (Parthenium argentatum Gray) mediante marcadores ITS Presentada por: ANTONNY URIEL VILLEGAS CHAVEZ Para obtener el grado de: Maestro en Ciencias en Agroplasticultura Asesorado por: Dra. Ana Margarita Rodríguez Hernández Dr. Marco Antonio Castillo Campohermoso Saltillo, Coahuila Agosto, 2020 Fe de erratas Por medio de la presente se hace constar que en los documentos donde aparece el título del documento de tesis de ANTONNY URIEL VILLEGAS CHAVEZ debe decir: “ Estudio de la diversidad genética de plantas silvestres de Guayule ( Parthenium argentatum Gray) mediante marcadores ITS” , y no "Estudio de la diversidad genética de plantassilvestres de Guayule ( Parthenium argentatum Gray) mediante marcadores ITS” Atentamente, Dr. Antonio Cárdenas Flores Coordinador de Posgrado en Agroplas ticultura Saltillo, Coahuila a 02 de septiembre de 2020 AGRADECIMIENTOS Al Consejo Nacional de Ciencia y Tecnología (CONACYT), por brindarme el apoyo económico durante la maestría; lo que me permitió cumplir con una meta más en el ámbito académico. Al Centro de Investigación en Química Aplicada (CIQA) que me abrió las puertas y me brindó las herramientas para finalizar mis estudios. A mis asesores la Dra. Ana Margarita Rodríguez Hernández y Dr. Marco Antonio Castillo Campohermoso por el apoyo y la confianza brindada a lo largo de este proyecto. A mis sinodales Dra. Ileana Vera Reyes, Dr. Mona Kassem y Dr. Gerardo Hernández Vera por las valiosas aportaciones recibidas para la mejora de este documento. Al la Universidad de Guadalajara por abrirme sus puertas lo que permitió llevar a cabo gran parte de este proyecto con un gran e invaluable apoyo del Dr. Gerardo. A mi familia que siempre me alentó y jamás permitió que me rindiera. A los amigos que conocí durante la maestría que permitieron que este proceso fuera más ameno. A Naza, gracias por abrirme las puertas de tu casa y corazón, gracias por todo a ti y tu hermosa familia que me abrazo como un integrante más de ella. A mi madre, gracias a ti por todo, gracia por siempre ser y estar sin importar que, este logro también es tuyo por estar a mi lado y nunca soltarme. TE AMO. “Nada en la vida es para ser temido, es sólo para ser comprendido. Ahora es el momento de entender más, de modo que podamos temer menos” Marie Curie II Resumen El guayule (Parthenium argentatum) es una especie perenne nativa del desierto chihuahuense de México, que se utiliza como materia prima para la producción de hule natural (HN), látex y productos bioenergéticos. Sin embargo, la falta de recursos de germoplasma bien caracterizados y con alta productividad (concentración de hule mayor al 17% en peso), aunado a la poca información de la diversidad genética de P. argentatum que permitan un avance en la obtención de variedades altamente productivas, se ha convertido en una limitante que impide el desarrollo de la industria del hule de guayule en México. Por lo tanto, en la presente investigación, se estudió la diversidad genética de 160 ejemplares silvestres de P. argentatum, pertenecientes a cuarenta poblaciones individuales, de 5 estados (Nuevo León, Coahuila, Zacatecas, Durango y San Luis Potosí) usando el marcador del espacio interno transcrito (ITS). Se amplifico la región ITS1-ITS2 obteniéndose secuencias de una longitud de 649 pb, con una composición nucleotídica de A: 24.1 %; G: 26.5 %; C 25.8 % y T: 23.5 %. Esta región ITS1-ITS2, presentó 152 sitios polifórmicos, 27 haplotipos únicos con una alta diversidad (Hd: 0.738 ± 0.013), una diversidad nucleotídica (Pi) de 0.0027 ± 0.0094 y una distancia genética de 0.0092. La variación genética encontrada dentro de cada población (81.19 %) fue mayor que la encontrada entre las poblaciones de cada estado (16.55 %) y la encontrada entre estados (2.26 %) según mostraron los resultados del análisis de varianza molecular (AMOVA). Este resultado nos indica que el flujo de genes de las poblaciones silvestres dentro del desierto chihuahuense es continuo ya que no hubo una correlación obvia entre la distancia genética y la distancia geográfica. Se encontraron poblaciones importantes a conservar de acuerdo con la información genética silvestre encontrada como lo son las poblaciones SL6 y SL9 en San Luis Potosí, Z5 en Zacatecas y NL2 en el estado de Nuevo León. III Índice de contenido I. ANTECEDENTES .............................................................................................................................. 1 I.1 El Guayule ......................................................................................................................... 1 I.2 Importancia económica del hule natural ......................................................................... 2 I.3 Industrias co-ubicadas e importancia industrial del hule de guayule........................... 5 I.4 Diversidad genética en la explotación comercial de cultivos ........................................ 7 I.5. Diversidad genética del guayule ................................................................................... 12 I.6. Marcadores moleculares para la diversidad genética ................................................. 13 I.7 El enfoque filogenético en estudios de diversidad y estructura genética .................. 17 I.8 Análisis bioinformáticos. ............................................................................................... 18 I.8.1 Alineamiento de secuencias............................................................................................... 18 I.8.2 Inferencia filogenética. ......................................................................................................... 20 I.8.2.1 Inferencia filogenética basada en máxima verosimilitud (ML)............................... 21 I.8.2.2 Inferencia filogenética basada en métodos bayesianos (IB). ................................. 21 I.8.3 Modelos de evolución de ADN ........................................................................................... 22 II. HIPÓTESIS ........................................................................................................................................ 25 III. OBJETIVOS .................................................................................................................................. 26 III.1 Objetivo general ............................................................................................................ 26 III.2 Objetivos específicos ................................................................................................... 26 IV. MATERIALES Y MÉTODOS ...................................................................................................... 27 IV.1 Colecta de material vegetal.......................................................................................... 27 IV.2 Extracción de ADN ....................................................................................................... 28 IV.3 Diseño de cebadores.................................................................................................... 29 IV.4 Amplificación de las regiones ITS1-ITS2 en ADN ribosómico nuclear ..................... 29 IV.5 Purificación y cuantificación de productos de PCR .................................................. 30 IV.6 Secuenciación de fragmentos amplificados .............................................................. 30 IV.7 Análisis bioinformáticos .............................................................................................. 31 IV.7.1. Estadísticas descriptivas de las secuencias y estimación de índices de diversidad genética. ......................................................................................................................
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