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Solanaceae: Solanum), Usando Regiones Barcode Tipificación molecular y separación de especies de plantas del subgénero Leptostemonum (Solanaceae: Solanum), usando regiones barcode. Isabel Cristina Cadavid Sánchez Universidad Nacional de Colombia Facultad de Ciencias, Escuela de Biociencias Medellín, Colombia 2013 Tipificación molecular y separación de especies de plantas del subgénero Leptostemonum (Solanaceae: Solanum), usando regiones barcode. Isabel Cristina Cadavid Sánchez Tesis o trabajo de investigación presentada(o) como requisito parcial para optar al título de: Magister en Ciencias Biotecnología Director (a): Sandra Inés Uribe Soto. M.Sc,phD. Línea de Investigación: Taxonomía molecular Grupo de Investigación: Sistemática Molecular Facultad de Ciencias, Departamento Biociencias Grupo de Investigación en Sistemática Molecular Universidad Nacional de Colombia Medellín, Colombia 2013 Dedicatoria Este trabajo de tesis está dedicado a mis padres, Arnulfo Cadavid, mi angel de la guarda que me ayuda desde el cielo y Alba Nelly Sánchez, mi luz y amiga, ellos me apoyaron emocional, espiritual y económicamente, permitiéndome cumplir esta meta. A mi novio, Andres Marcelo Jímenez que a través de su apoyo incondicional y paciencia hizo mas fácil alcanzar este logro. Más que el brillo de la victoria, nos conmueve la entereza ante la adversidad Octavio Paz Agradecimientos A dios por iluminarme a través de los seres que me acompañan. A mis amigos por alegrarme los días. A la Universidad Nacional de Colombia, sede Medellín, al Grupo de Investigación en Sistemática Molecular y a COLCIENCIAS por la financiación del estudio. A la Sandra Inés Uribe Soto, directora del grupo de investigación por la dirección de la tesis. A Carlos Eduardo Giraldo, integrante del grupo de investigación, por la asesoría prestada. A Jorge Mario Vélez del Herbario de MEDEL de la Universidad Nacional de Colombia, por la colaboración en la identificación del material vegetal. Resumen y Abstract VII Resumen Solanum es el género más grande y diverso de plantas de la familia Solanaceae, con aproximadamente 1400 especies. Entre sus especies se encuentran muchas de importancia económica por su uso y explotación como alimento y aplicaciones en farmacología, además de las relaciones ecológicas y evolutivas que establecen con numerosas especies de insectos para los cuales son plantas nutricias. En el presente estudio se realizó una tipificación molecular de 17 especies de Solanum, subgénero Leptostemonum, mediante secuenciación de fragmentos de las regiones matK, trnH-psbA e ITS. Se estandarizaron las condiciones para la extracción y amplificación de los fragmentos y se evaluó su utilidad para la separación de las especies en comparación con previa determinación morfológica realizada por el especialista. Los resultados demostraron que ITS tiene el mayor número de sitios variables, diversidad nucleotídica, y las tasas más altas de discriminación (88,9%) seguido por trnH-pbsA y matK; sin embargo su amplificación produce resultados menos satisfactorios que las regiones del cloroplasto. Entre las posibles combinaciones de loci con matK + trnH-psbA +ITS, ITS + trnH-psbA e ITS + matK se obtuvo el mismo poder de discriminación (88,9%), pero debido a las ventajas de trabajar con una región codificante se recomienda usar ITS + matK para identificar especies de plantas del subgénero Leptostemonum. Palabras clave: matK, trnH-psbA, ITS, plantas, DNA barcode Abstract Genus Solanum is the largest and most diverse of family Solanaceae, with about 1400 species. Many species are economically important for its use and exploitation for food and pharmacology applications, in addition to the ecological and evolutionary relationships established with numerous species of insects for which are food plants. In this study it was performed molecular typing of species of the genus Solanum subgenus Leptostemonum using sequences of matK, trnH-psbA and ITS. Conditions for extraction and amplification of the fragments were standardized and it evaluated the utility of these for separating species compared to previous morphological determination by the specialist. Results demonstrated that ITS have the most variable sites, greater nucleotide divergences, and the highest species discrimination rates (88.9%). It was followed by trnH-psbA and matK (each 77%). However, its amplification produces less satisfactory results than the chloroplastic regions. Combinations of loci with matK+trnH-psbA+ITS, ITS+trnH-psbA and ITS + matK showed the same species discrimination rates (88.9%) but because of the advantages of working with a coding region, we recommended using ITS + matK to identify plant species of subgenus Leptostemonum. Keywords: matK, trnH-psbA, ITS, plants, DNA barcode Contenido IX Tabla de contenido Pág. Resumen ......................................................................................................................................... VII Lista de figuras ................................................................................................................................. X Lista de tablas ................................................................................................................................ XV Introducción .................................................................................................................................... 17 1. Marco teórico ........................................................................................................................... 19 1.1 La familia Solanaceae ....................................................................................... 19 1.1.1 Importancia ..................................................................................................... 19 1.2 El género Solanum ........................................................................................... 19 1.2.1 Taxonomía ..................................................................................................... 20 1.2.2 Características morfológicas .............................................................................. 20 1.2.3 Investigación ................................................................................................... 21 1.3 Subgénero Leptostemonum ................................................................................ 21 1.3.1 Importancia y estudios realizados ...................................................................... 22 1.3.2 Taxonomía ...................................................................................................... 22 1.4 Iniciativa “DNA barcoding” en plantas ............................................................... 23 1.4.1 Resultados de “DNA barcoding” en plantas en comparación con animales .............. 24 1.4.2 Proyecto DNA Barcoding en plantas de Colombia................................................ 25 1.5 Regiones “barcode” incluidas en el presente estudio ............................................ 26 1.5.1 Espaciador intergénico trnH-psbA ...................................................................... 26 1.5.2 Maturase K-matK ............................................................................................. 27 1.6 Objetivo general ............................................................................................... 31 1.7 Objetivos específicos ........................................................................................ 31 2. Metodología ............................................................................................................................. 32 2.1 Especímenes .................................................................................................... 32 2.2 Extracción de ADN .......................................................................................... 32 2.3 Obtención y edición de las secuencias de ADN .................................................... 32 2.4 Análisis de las secuencias .................................................................................. 33 3. Resultados ................................................................................................................................ 36 3.1 Colecta de ejemplares ....................................................................................... 36 3.2 Obtención y análisis de secuencias ..................................................................... 36 3.2.1 Análisis del poder de discriminación de especies .................................................. 38 4. Discusión .................................................................................................................................. 44 5. Conclusiones y recomendaciones ........................................................................................... 50 Anexo A: Comparación de dos métodos de extracción de ADN a partir de plantas del género Solanum, subgénero Leptostemonum. .......................................................................................... 165 6. Bibliografía ............................................................................................................................ 176 Contenido X Lista de figuras Pág. Figura 1. Principales clados de Solanum. …………….……………………………………..…. 52 Figura 2. a. Ejemplo de hoja compuesta b. Ejemplo de hoja simple c. ejemplo de hoja espinosa. d. Flor de Solanum inelegans e. Flor de Solanum macbridei f. Flor de Solanum peruvianum. g. Fruto de Solanum mammosum h. Fruto de Solanum pseudocapsicum i. Fruto de Solanum scabrum…… 53 Figura 3. a. Solanum jamaicense b. Solanum
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