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ESTUDIO DE DIVERSIDAD GENÉTICA EN ACCESIONES BOLIVIANAS DE CHIRIMOYA ( Annona Cherimola Miller) MEDIANTE MARCADORES MOLECULARES MICROSATÉLITES (SSR)

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ESTUDIO DE DIVERSIDAD GENÉTICA EN ACCESIONES BOLIVIANAS DE CHIRIMOYA ( Annona Cherimola Miller) MEDIANTE MARCADORES MOLECULARES MICROSATÉLITES (SSR) UNIVERSIDAD MAYOR DE SAN ANDRÉS FACULTAD DE AGRONOMÍA CARRERA DE INGENIERÍA AGRONÓMICA TESIS DE GRADO ESTUDIO DE DIVERSIDAD GENÉTICA EN ACCESIONES BOLIVIANAS DE CHIRIMOYA ( Annona cherimola Miller) MEDIANTE MARCADORES MOLECULARES MICROSATÉLITES (SSR) FIDEL HERNAN CORTEZ ALVAREZ La Paz – Bolivia 2010 UNIVERSIDAD MAYOR DE SAN ANDRÉS FACULTAD DE AGRONOMÍA CARRERA DE INGENIERÍA AGRONÓMICA ESTUDIO DE DIVERSIDAD GENÉTICA EN ACCESIONES BOLIVIANAS DE CHIRIMOYA ( Annona cherimola Miller) MEDIANTE MARCADORES MOLECULARES MICROSATÉLITES (SSR) Tesis de Grado presentado como requisito parcial para optar el Título de Ingeniero Agrónomo FIDEL HERNAN CORTEZ ALVAREZ Tutor: Ing. Ph. D. Jorge Antonio Rojas Beltrán …………………………… Asesor: Ing. Ph. D. Alejandro Bonifacio Flores …………………………… Tribunal Examinador: Ing. Ph. D. David Cruz Choque …………………………… Lic. Ph. D. Alberto Figueroa Soliz …………………………… Ing. M. Sc. Félix Mamani Reynoso …………………………… Aprobada Presidente Tribunal Examinador: …………………………… Ing. M. Sc. Ramiro Augusto Mendoza Morales VICEDECANO - FACULTAD DE AGRONOMÍA 2010 ÍNDICE GENERAL Índice de contenido............................................................................................... i Índice de cuadros................................................................................................... iv Índice de figuras..................................................................................................... v Dedicatoria.............................................................................................................. vii Agradecimientos .................................................................................................... viii Resumen ................................................................................................................ ix ÍNDICE DE CONTENIDO CAPÍTULO I 1. INTRODUCCIÓN ................................................................................................. 1 1.1. Objetivos......................................................................................................... 2 1.1.1. Objetivo General .......................................................................................... 2 1.1.2. Objetivos Específicos................................................................................... 2 CAPÍTULO II 2. REVISION BIBLIOGRÁFICA............................................................................... 3 2.1. Antecedentes del cultivo, origen y distribución ............................................... 3 2.2. Usos e Importancia ......................................................................................... 4 2.3. Descripción botánica y ubicación taxonómica................................................. 5 2.3.1. Descripción morfológica ............................................................................ 5 2.3.2. Ubicación taxonómica................................................................................ 7 2.4. Características ecológicas y agronómicas...................................................... 8 2.5. Biodiversidad y Agrobiodiversidad .................................................................. 9 2.5.1. Diversidad genética ................................................................................... 9 2.5.2. Recursos fitogenéticos .............................................................................. 10 2.5.3. Colección núcleo ....................................................................................... 12 2.6. Herramientas para evaluar la diversidad genética .......................................... 13 2.7. Marcadores moleculares................................................................................. 13 2.7.1. Aplicaciones y ventajas de los marcadores moleculares........................... 14 2.7.2. Clasificación de los marcadores moleculares............................................ 15 2.7.3. Selección de un marcador molecular adecuado........................................ 16 2.7.4. Microsatélites............................................................................................. 17 2.7.4.1. Aplicaciones, ventajas y limitaciones de los microsatélites .................. 18 2.7.4.2. Huellas genéticas................................................................................. 18 2.7.5. Marcadores moleculares en chirimoya ...................................................... 19 i 2.8. Técnicas para el análisis con marcadores moleculares.................................. 20 2.8.1. Colección y preservación de tejido vegetal ............................................. 20 2.8.2. Molido de la muestra............................................................................... 20 2.8.3. Extracción de ADN.................................................................................. 20 2.8.4. Electroforesis de ADN en geles de agarosa............................................ 21 2.8.5. Amplificación de secuencias microsatélites ............................................ 22 2.8.6. Separación de productos amplificados.................................................... 22 2.9. Análisis estadístico ......................................................................................... 23 2.9.1. Índices de variabilidad genética................................................................. 23 2.9.2. Análisis de estructura poblacional ............................................................. 24 2.9.2.1. Coeficientes de similitud....................................................................... 24 2.9.2.2. Análisis de conglomerados .................................................................. 25 2.9.2.3. Análisis de coordenadas principales .................................................... 25 2.10. Herramientas para el análisis geoespacial de datos..................................... 26 CAPÍTULO III 3. LOCALIZACIÓN .................................................................................................. 27 3.1. Ubicación Geográfica...................................................................................... 27 CAPÍTULO IV 4. MATERIALES Y MÉTODOS................................................................................ 27 4.1. Materiales ....................................................................................................... 27 4.1.1. Material Vegetal......................................................................................... 27 4.1.2. Material de Colecta.................................................................................... 27 4.1.3. Material de Laboratorio.............................................................................. 27 4.1.4. Material de Gabinete ................................................................................. 28 4.2. Métodos .......................................................................................................... 28 4.2.1. Número de muestras analizadas ............................................................... 29 4.2.2. Cobertura del estudio ................................................................................ 30 4.2.3. Esquema general de trabajo...................................................................... 31 4.2.4. Colecta del material vegetal ...................................................................... 32 4.2.5. Molido y conservación de muestras........................................................... 32 4.2.6. Extracción de ADN .................................................................................... 32 4.2.7. Cuantificación de ADN............................................................................... 34 4.2.8. Amplificación y separación de microsatélites ........................................... 34 4.2.8.1. Amplificación por reacción en cadena de la polimerasa (PCR)............ 34 ii 4.2.8.2. Electroforesis capilar............................................................................ 35 4.2.9. Procesamiento y análisis de datos ............................................................ 36 4.2.9.1. Índices de variabilidad genética ........................................................... 36 4.2.9.2. Cálculos para el análisis de estructura poblacional.............................. 37 CAPÍTULO V 5. RESULTADOS Y DISCUSIÓN ............................................................................ 38 5.1. Colecta de material vegetal............................................................................. 38 5.2. Molido y conservación de muestras................................................................ 38 5.3. Extracción de ADN.......................................................................................... 39 5.4. Cuantificación de ADN.................................................................................... 40 5.5. Número y frecuencias de alelos encontrados ................................................. 40 5.5.1. Número promedio de alelos por locus microsatélite................................ 42 5.5.2. Heterocigosidad (He) e Índice de contenido polimórfico (PIC)................ 43 5.6. Análisis de conglomerados ............................................................................. 44 5.6.1. Análisis de conglomerados de las chirimoyas bolivianas........................ 47 5.6.2. Análisis por grupos de individuos: plantaciones antiguas y recientes .... 49 5.7. Análisis de coordenadas principales............................................................... 56 5.7.1. Análisis por grupos de individuos: plantaciones
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