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Tesis De Posgrado Abel, María Cecilia Tesis de Posgrado Estudio de la variabilidad y la diferenciación genética por medio de técnica de Isoenzimas y RAPD en poblaciones naturales de especies de pejerreyes de la República Argentina, género Odontesthes (Atherinopsidae, Atherinopsinae, Sorgentinini) Abel, María Cecilia 2002 Tesis presentada para obtener el grado de Doctor en Ciencias Biológicas de la Universidad de Buenos Aires Este documento forma parte de la colección de tesis doctorales y de maestría de la Biblioteca Central Dr. Luis Federico Leloir, disponible en digital.bl.fcen.uba.ar. Su utilización debe ser acompañada por la cita bibliográfica con reconocimiento de la fuente. This document is part of the doctoral theses collection of the Central Library Dr. Luis Federico Leloir, available in digital.bl.fcen.uba.ar. It should be used accompanied by the corresponding citation acknowledging the source. Cita tipo APA: Abel, María Cecilia. (2002). Estudio de la variabilidad y la diferenciación genética por medio de técnica de Isoenzimas y RAPD en poblaciones naturales de especies de pejerreyes de la República Argentina, género Odontesthes (Atherinopsidae, Atherinopsinae, Sorgentinini). Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. http://digital.bl.fcen.uba.ar/Download/Tesis/Tesis_3507_Abel.pdf Cita tipo Chicago: Abel, María Cecilia. "Estudio de la variabilidad y la diferenciación genética por medio de técnica de Isoenzimas y RAPD en poblaciones naturales de especies de pejerreyes de la República Argentina, género Odontesthes (Atherinopsidae, Atherinopsinae, Sorgentinini)". Tesis de Doctor. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. 2002. http://digital.bl.fcen.uba.ar/Download/Tesis/Tesis_3507_Abel.pdf Dirección: Biblioteca Central Dr. Luis F. Leloir, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Contacto: [email protected] Intendente Güiraldes 2160 - C1428EGA - Tel. (++54 +11) 4789-9293 UNIVERSIDAD DE BUENOS AIRES FACULTAD DE CIENCIAS EXACTAS Y NATURALES "Estudio de la variabilidad y la diferenciación genética por medio de técnicas de Isoeniimas y RAPD en poblaciones naturales de especies de pejerreyes (h la Qe!¿blica Argentina, género Odontesthes (Atherinops.dae, Atherinopsinae, Sorgentinini)." Autor: María Cecilia Abel Directora: Dra. María Cristina Maggese Directora Asistente: Dra. Graciela García Trabajo de Tesis para optar al grado de Doctor en Ciencias Biológicas Laboratorio de Embriología Animal Departamento de Ciencias Biológicas ry ¿(z-J CW xy“ 'ín 2002 DEDICATORIA A mi esposo Francisco, A mi hija María Constanza y A mis Padres Enseñarás a volar Pero no volarán tu vuelo Enseñarás a soñar pero no soñaran tu sueño. Enseñaras a vivirpero no vivirán tu vida Pero sabrás que cada vezque ellos vuelen, piensen, sueñen, canten, vivan... Estará la semilla del camino Enseñaa’oy aprendido Madre Teresa de Calcuta AGRADECIMIENTOS Quiero agradecer en este espacio a las personas que me han ayudado desde lo afectivo y/o desde su conocimiento a que logre este importante objetivo. .A la Dra. María Cristina Maggese, por su apoyo y por haberme brindado la posibilidad de trabajar en su laboratorio. A la Dra. Graciela García, quien me recibió en su laboratorio de Genética Evolutiva de la Facultad de Ciencias, Montevideo, me enseñó a trabajar en ADN (técnicas de extracción, PCR y RAPD), por su constante dirección en el tema y por su ayuda en lo profesional y en lo personal. A la Dra. Beatriz Saidman, quien me facilitó el aprendizaje de las técnicas de la electroforesis de isoenzimas. Al Dr. Juan Vilardi, por su constante asesoramiento y guía en el tratamiento estadístico de los resultados isoenzimáticos y de RAPD y por responder siempre a mis dudas con excelente disposición. A los sistemáticos Dra. Mirta García y Dr. Atila Gosztonyi por la determinación taxonómica de las especies estudiadas. Al Dr. Enrique Marschofi‘ por modificar parámetros de programas Fortran, imprescindibles para los análisis de RAPD. A la Dra. Graciela Guerrero y Dra. Paula Vissio, por haberme ayudado siempre incondicionalmente, por su aliento y estímulo. A la Dra. Fabiana Lonostro por contribuir a la presentación de tesis y estar siempre bien predispuesta en perder parte de su tiempo en ayudarme. A los Dres. Mario Ravaglia, Dante Paz, Gustavo Somoza, Pablo Carmanchai, Pablo Pica y Victor Cussac, quienes intervinieron en las campañas de muestreo de pejerreyes. A mis compañeras de laboratorio que alguna vez incursionaron en la Genética, Lic. Graciela Rey Vázquez y Natalia Moncaut, por escucharme, alentarme y compartir la vida de todos los días. Al Lic. Fernando Meijide, por ayudarme a recuperar archivos perdidos en la Computadora, solucionar cualquier otro inconveniente con la PC y sus criteriosas observaciones. A las chicas de Genética, Laura Ferreyra, Graciela Calgagno, Amalia Scataglini, Verónica, Andrea Alberti y Cecilia Bessega, por soportar mis constantes consultas. A Pablo Rebagliati por ayudarme con las figuras, por sus consejos, por estar siempre de buen ánimo y predisposición. A Martín Ansaldo por prestanne el espectrofotómetro. A Alejandra Volpedo por alentarme y guiarme como encarar la redacción del manuscrito. Quiero agradecer especialmente a mi esposo Francisco por comprenderme, por apoyarme y alentarme constantemente, quien no solo me acompañó en varias campañas de recolección de material sino que también, arregló fuentes de poder y cubas electroforéticas, por compartir la alegn'a del éxito y la angustia cuando no salen los experimentos. A mi tesoro, Man’a Constanza por soportar mis ausencias, mi cansancio... por ser la luz y fuerza de mi vida. También quiero agradecer especialmente a mis Padres, Elina y Oscar, por compartir la alegria del éxito y la angustia cuando no salen los experimentos, por estar siempre presentes en los momentos más dificiles colaborando constantemente con todas sus energías frente a cualquier situación. También quiero agradecer: A mi suegra Isabel, que aunque ya no esta con nosotros siempre me alentó y le hubiera gustado verme llegar a la meta. A mi hermano Carlitos, a mis cuñadas Carmencita e Isa, por el apoyo y afecto que siempre me demuestran. Este trabajo fue realizado gracias a los subsidios otorgados por la O.E.A., Universidad de Buenos Aires, Agencia Nacional de Promoción Científica y Tecnológica otorgados a la Dra. María Cristina Maggese (CONICET-PIA 0222/92, UBA-EX208., FONICYT-CONICET­ PICT N" 0183, UBA-EX141 o TW-4l, CONICET-P1P N° 4558/96, CONICET-PIP N° 0539/98, UBA-EX N° 217) INDICE DEDICATORIA r AGRADECIMIENTO fl mmm: ¡v nFchrm vu ABSTRACT Y GLOSARIO Y ABREVIATIÍRAS Ym INTRODUCCION l. Campo de ‘ r " I II. Requerimientos para programas de Conservación 4 [IL El género " ' ' 6 lII.l "' ' 6 III.2 l" " 7 III. 3 Escenarios biogeografioos y relaciones " U 10 III. 4 Historia L" o U "' 14 IIl. 4. l. Historia biogeográfica de especies de Escamas pequeñas (SS\ 14 III. 4. 2 Historia biogeográfica de especies de Escamas grandes (l S\ 16 III. 5 Caracteristicas Biológicas, Ecológicas e Importancia " ' ' IR IV. Empleo de Marcadores Moleculares para la resolución de problemas Taxonómicos Evolutivos 70 IV. l. Marcadores Bioquímioos y ‘ ' ' ' 'm IV. 2. Electroforesis de ' ' n IV. 2. l. Fundamento de la "¿mica 71 IV. 2. 2. Aportes de las isoenzimas a estudios evolutivos, taxonómicos y poblacionales..............24 IV. 3. La Técnica de RAPD 76 IV. 3. l. Fundamento de la "¿mir-2 76 IV. 3. 2. Empleo de la técnica de RAPD en estudios poblacionales, taxonómicos y v ' " 11 IV. 3. 3. Ventajas y desventajas del uso de marcadores RAPD en estudios poblacionales y - ' " 11 IV. 4. Estimación de la Variación Genética en poblaciones naturales 11 IV. S. Análisis de la Estructura Genética de las poblaciones mediante marcadores de isoenzimas y RAPD 14 IV. 6. MecanismosEvolutivos relacionadoscon la estructura genética de las poblaciones............ “.36 IV.6.l.“' " 16 IV. 6. 2. Deriva genetica 17 IV. 6. 3. El flujo gónim 17 OBJETIVOS 10 MATERIALES Y METODOS I. Muestreo 40 I.l Especies y poblaciones estudiadas mediante las técnicas de Isoenzimas y RAPD..............................40 1.2 Determinación taxonómica de las especies de f‘ J ‘ " 44 Il. Técnica de Electroforesis de ' ' 44 Il.l Sistemas Isocnzimáticos L " ’ 44 lI.2 Preparación de geles 45 II.3 Armado de geles 46 II.4 Buffer Corrida y de Geles 46 lI.5 Preparaciónde las“Mm” 46 II.6 Siembra del Material en los geles 47 II.7 Conida "' ’ ' ' 47 II.8 Revelado, fijación y preservación de los geles 47 II.9 Número de Clasificación y designación de las ' 48 II.10TincionesIsoenzimáticas ’ 4° Il. ll Determinación de los probables loci y alelns <7 IlLMétodos Estadísticos a partir de los datos de ' <7 Ill.l Cálculo de las frecuencias alélims SR III.2 Estimación de la variabilidad gpnérim sn III.3 Análisis de la Estructura P L' ' ' <0 III.4 Análisis de la Diferenciación Genética entre r ' ' ' m III.4.l Divergencia Genética y Flujo génim m IlI.4.2 Análisisjerarquizado de la distribución de la variación genética m III.5 Análisis del grado de Similitud genética entre poblaciones y/o especies't' 64 III.6 Análisis de las relaciones entre distancia genéticas y distancia o «a IV. Técnica de RAPn ¿a lV.l Extracción de ADN Total 66 IV.2 Estimación de la concentración de ADN 63 IV.3 Reacción en cadena de la Polimerasa (PCR\ m I't' J IV.4 Visualización de Fragmentos ." r 70 IV.4.l Preparación y armado de los geles 70 IV.4.2. Limpieza de gelec 71 IV.4.3. Colocación de los geles en cuba de mrn'da 71 IV.4.4. Siembra del material en los ¿”dec 71 IV.4.5. Corrida ' 72 IV.4.6. Revelado, fijación y preservación de los geles 72 IV.5 Soluciones empleadas en las técnicas de RAPD 73 V. Métodos Estadísticos a partir de los datos RAP“ 76 V.l Analisis de la divergencia genetica 77 V. l.l Distancias ‘ ' L 77 V. 1.2 F51-y Flujo gónirn 73 V.1.3 Análisis jerarquime de la distribución de la variabilidad genética 78 V.2 Correlación entre distancia genéticas y o U "' 79 V.3 Analisis Fenótim 79 V.3.l Analisis Fenético de distancias ‘ ' l 79 V.3.2 Analisis Fenético de índices FSTobtenidos por AMOVA 9m V.4 Correlación entre diferenciación genética obtenidas por RAPD y por Isozimas..............
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