Caracterización molecular del germoplasma de ñame colombiano utilizando "Selective Amplificaron of Microsatellite Polymorphic Loci" (SAMPL) en condiciones radioactivas

Molecular Characterization of Colombian Yam Germoplasm by "Selective Amplificaron of M i c rósate l lite Polymorphic Loci" (SAMPL)

En Colombia el æame (Dioscorea spp.) ha sido un cultivo básico tradicional a pequeña y mediana escala de cultivadores de la Costa Atlántica para abastecer el consumo propio y el mercado local, convirtiéndose en una gran fuente de empleo durante muchos años, sin embargo el ñame es uno de los cultivos más susceptibles a enfermedades (vg. la antracnosis y virus). En la actualidad el cultivo ha ido recuperándose progresivamente de una drástica reducción en área, como en producción, permitiendo aparentes resultados benéficos para los productores. Es imprescindible para el futuro del cultivo, que los productores cuenten con variedades seleccio­nadas para incrementar la sostenibilidad y rentabilidad del cultivo. Sin embargo esas variedades no están disponibles en la naturaleza, sino que tienen que ser mejoradas. El mejoramiento del ñame es difícil y presupone un conocimiento de la diversidad genetica del germoplasma de Dioscorea en Colombia, la cual no se conoce todavía. Por estas razones nosotros caracterizamos las variedades de ñame colombiano utilizadas en campo, así como las silvestres con una técnica de biología molecular: Selective Amplification Microsatellite Polimorphic Loci (SAMPL). Se detectaron 160 bandas identificables, (de las cuales 56 fueron monomórficas, 35% del total, con un rango de 2.8 bandas polimórficas por análisis). Con la información anterior se construyó una matriz binaria que fue transformada en un dendograma utilizando el algoritmo UPGMA. La diversidad de la colección determinada con esta técnica fue de 0.0471 con una media de similaridad de 0.9529. Palabras clave: Dioscorea spp, microsatélites, DNA

In Colombia, yam (Dioscoreu spp.) has been a basic and traditional crop for small-and medium-scale farmers. It has been an important source of family employment in the Colombian Atlantic Coast for the past several centuries. However, yam is the most fragüe crop regarding market issues and disease susceptibility (e.g. to anthracnose and viruses). At present, the crop is under progressive recovery from a previous drastic reduction in área and production, and apparently allows high profits. Is imperative for the future of the crop, that the rural farmers are supplied with selected varieties, thereby increasing their sustainability and economic yield. However, such varieties are not available, but have to be bred. Breeding of yam in turn is difficult, and presupposes knowledge about the genetic diversity in the breeding germplasm, which is not known. For these reasons, we characterized the Colombian yam varieties used in the fields as well as wild relatives with a molecular marker technique: SAMPL (Selective Amplification of Microsatellite Polymorphic Loci). Positions of 160 unequivocally scorable bands (of which 56 were monomorphic, 35% of total, with an average of 2.8 polymorphic bands per assay) were transformed into a binary character matrix and analyzed. The resulting similarity matrix was transformed into a dendrogram with the UPGMA algorithm. The diversity of the collections with this technique was 0.0471 with a mean similarity of 0.9529. Key Words: Dioscorea spp, microsatellites, DNA

	Rev. Colomb. Biotecnol. Vol. VIII N° 2 Diciembre 2006 60-66
	Molecular Characterization of Colombian Yam Germoplasm by "Selective Amplificaron of M i c rósate l lite Polymorphic Loci" (SAMPL) Caracterización molecular del germoplasma de ñame colombiano utilizando "Selective Amplificaron of Microsatellite Polymorphic Loci" (SAMPL) en condiciones radioactivas Silvia Bustamante R.'", Gustavo Buitrago H' ABSTRACT In Colombia, yam (Dioscoreu spp.) has been a basic and traditional crop for small-and medium-scale farmers. It has been an important source of family employment in the Colombian Atlantic Coast for the past several centuries. However, yam is the most fragüe crop regarding market issues and disease susceptibility (e.g. to anthracnose and viruses). At present, the crop is under progressive recovery from a previous drastic reduction in área and production, and apparently allows high profits. Is imperative for the future of the crop, that the rural farmers are supplied with selected varieties, thereby increasing their sustainability and economic yield. However, such varieties are not available, but have to be bred. Breeding of yam in turn is difficult, and presupposes knowledge about the genetic diversity in the breeding germplasm, which is not known. For these reasons, we characterized the Colombian yam varieties used in the fields as well as wild relatives with a molecular marker technique: SAMPL (Selective Amplification of Microsatellite Polymorphic Loci). Positions of 160 unequivocally scorable bands (of which 56 were monomorphic, 35% of total, with an average of 2.8 polymorphic bands per assay) were transformed into a binary character matrix and analyzed. The resulting similarity matrix was transformed into a dendrogram with the UPGMA algorithm. The diversity of the collections with this technique was 0.0471 with a mean similarity of 0.9529. Key Words: Dioscorea spp, microsatellites, DNA RESUMEN En Colombia el æame (Dioscorea spp.) ha sido un cultivo bÆsico tradicional a pequeæa y mediana escala de cultivadores de la Costa AtlÆntica para abastecer el consumo propio y el mercado local, convirtiØndose en una gran fuente de empleo durante muchos aæos, sin embargo el æame es uno de los cultivos mÆs susceptibles a enfermedades (vg. la antracnosis y virus). En la actualidad el cultivo ha ido recuperÆndose progresivamente de una drástica reducción en área, como en producción, permitiendo aparentes resultados benéficos para los productores. Es imprescindible para el futuro del cultivo, que los productores cuenten con variedades seleccio­nadas para incrementar la sostenibilidad y rentabilidad del cultivo. Sin embargo esas variedades no estÆn disponibles en la naturaleza, sino que tienen que ser mejoradas. El mejoramiento del ñame es difícil y presupone un conocimiento de la diversidad genØtica del germoplasma de Dioscorea en Colombia, la cual no se conoce todavía. Por estas razones nosotros caracterizamos las variedades de ñame colombiano utilizadas en campo, así como las silvestres con una técnica de biología molecular: Selective Amplification Microsatellite Polimorphic Loci (SAMPL). Se detectaron 160 bandas identificables, (de las cuales 56 fueron monomórficas, 35% del total, con un rango de 2.8 bandas polimórficas por análisis). Con la información anterior se construyó una matriz binaria que fue transformada en un dendograma utilizando el algoritmo UPGMA. La diversidad de la colección determinada con esta tØcnica fue de 0.0471 con una media de similaridad de 0.9529. Palabras clave: Dioscorea spp, microsatélites, DNA Recibido: octubre 06 de 2006 Aceptado: noviembre 14 de 2006
	* Instituto de Biotecnología. Universidad Nacional de Colombia. Bogotá - Colombia. ** Universidad de Frankfurt - Biozentrum Pflanzliche Molekularbiologie Marie-Curie-Str. 9/N200 D- 60439 Frankfurt amMain.
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	MOLECULAR CHARACTERIZATION OF COLOMBIAN YAM GERMOPLASM
	INTRODUCTION Yam (Dioscorea spp) belongs to the family Dioscoreaceae, order Dioscoreales. It is monocotyledonous and constitutes an important staple food crop for millions of people in the humid and subhumid tropics. Tubers provide an important food resource for people from these regions. In Co­lombia, yam (Dioscorea spp.) has been a basic and traditional crop for small- and medium-scale farmers from the Atlantic Coast (Córdoba, Sucre, Cesar, At­lántico and Bolívar departments). Formerly itwas a self-supporting crop for local markets only, but more recently became an export product. In 1989, pro-duction has been drastically affected by a massive infection of Colletotríchum gloeosporioides, the causative agent of the foliar disease anthracnose (Álvarez, 1991). In subsequent years, smallholders dramatically reduced the acreage for yams, replacing it with other crops such as cassava and plantains. Knowledge of germplasm diversity and relationships among élite materials has a significant impact on the improvement of crop plants generally (e.g. Witcombe, et ál., 2001), and of yam in particular for example the works done in Asian and Africans Dioscorea bulbifera, Guinea yam Dioscorea rotundata-D.cayenensis(Ramser, etál., 1996,1997 respectively) and Jamaican yam germplams (Asemota, et ál., 1996 ), were, using different mole­cular techniques was possible detected genomic variation for phylogeny and taxonomy studies as well as obtained information about de genome of these collection. Efficient breeding of yam in Colombia can only be achieved if its genetic diversity is known and promising varieties can be selected. This, however, is not the case. For these reasons, we characterized a comprehensive collection of the presently grown Colombian yam varieties with a molecular marker techniques: selective amplification of microsatellite polymorphic loci (SAMPL; Witsenboer, et ál. 1997). MATERIALS AND METHODS Plant material Different yam cultivars were collected from fields at different locations at the Caribbean Colombian Coast (Durangoand Padilla, 1998). The samples from África were generously donated by the Internatio-nal Institute of Tropical Agriculture (UTA Ibadan,	Nigeria). Dioscorea sansibarensis and D. vittata were provided by in the yam collection of Frankfurt University (table 1). DNA isolation DNA was ¡soled according to the procedure of Asemota (1991) with some modifications. For example, dithiotreithol (DDT) was employed for the reduction of quiñones instead of p-mercaptoethanol. SAMPL analysis The SAMPL technique makes use of compound microsatellite primers to reveal genetic polymorphisms (Morgante and Vogel, 1994). It com­bines the high multiplex ratio typical for amplification fragment length polymorphisms (AFLPs; Vos et ál. 1995) with the high level of variability typical for SSRs. The protocol was based on the procedures reported by Morgante and Vogel (1994) and Witsenboer et ál. (1997). Sequences of primers and adapters are given in Table 2. All PCR steps were performed in a Perkin Elmer 9700 thermocycler. Initial restriction enzyme digestions were performed in final volumes of 25 uL containing 200 ng of DNA témplate, 10 units of EcoRI (MBI Fermentas), 33 mM Tris-acetate (pH 7.9), 10 mM magnesium acétate, 66 mM potassium acétate, and 0.1 mg/ mL BSA. After three hours at 37 °C, 10 uL of a ligation mix containing 5.5 ng of EcoRI adapter, 1 mM ATP, 33 mM Tris-acetate (pH 7.9), 10 mM magnesium acétate, 66 mM potassium acétate, 0.1 mg/mL BSA and 2 U T4 DNA ligase (MBI Fermentas) were added. After overnight incubation at 37 °C, ligation-restriction reactions were diluted 1:4 with bi-distilled sterile water. Pre-amplifications were performed in 20 uL reactions containing 5 uL of diluted ligation products, 75 mM Tris-HCl (pH 9), 20 mM (NH4)2SO4, 0.01 % Tween-20, 1.5 mM MgCI2, 200 uM of dNTPs, 0.5 uM EcoRI adapter-specific primer, and 0.5 U Taq polymerase (Biotherm, Gene Craft). The PCR program consisted of an initial denaturation step at 94 °C (120 s), 34 cycles at 94 °C (30 s), 50 °C (30 s), and 72 °C (60 s). After pre-amplification, 3 uL aliquots were separated in 1% agarose gels and stained with ethidium bromide. The remaining samples were diluted 1: 9 and stored at -20 °C until use. Compound microsatellite primers (see table 2) were end-labelled with [y^P]-
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	Table 1. Origin of plant material
	Species c.v Origin Species c.v Origin D.alata 9805003 Córdoba D.alata Da11 África (UTA) 9503004 Sucre Da12 África (UTA) 9503005 Sucre Da13 África (UTA) 9503006 Sucre Da14 África (UTA) 9503007 Córdoba Da15 África (UTA) 9503008 Córdoba Da16 África (UTA) 9504009 Córdoba Da17 África (UTA) 9506016 Córdoba Da18 África (UTA) 9506019 Córdoba Da19 África (UTA) 9506020 Córdoba D.rotundata 9603041 Córdoba 9506021 Córdoba 9605060 Sucre 9502022 Córdoba 9804075 Córdoba 9506024 Bolívar 9804078 Córdoba 9506028 Atlántico 9804080 Córdoba 9602034 Córdoba 9804084 Córdoba 9602035 Córdoba 9804085 Córdoba 9603037 Córdoba 9404087 Córdoba 9603040 Córdoba 9804092 Córdoba 9603042 Córdoba 9903093 Córdoba 9603043 Córdoba 9903094 Córdoba 9701043 Córdoba Dr22 África (UTA) 9605044 Córdoba Dr24 África (UTA) 9605045 Bolívar Dr30 África (UTA) 9605047 Bolívar Dr32 África (UTA) 9605049 Bolívar Dr33 África (UTA) 9605052 Magdalena Dr34 África (UTA) 9605053 Magdalena Dr35 África (UTA) 9605054 Magdalena Dr36 África (UTA) 9505059 Sucre Dr37 África (UTA) 9605061 Bolívar Dr38 África (UTA) 9605063 Córdoba Dr39 África (UTA) 9605064 Antioquia Car 1 Córdoba 9605065 Córdoba D.trífida 9805001 Córdoba 9505066 Antioquia D.bulbífera 9507030 Córdoba 9605070 Córdoba D.vittata Frankfurt University collection 9701071 Córdoba D.sansibarensis Frankfurt University collection
	* c.v : cultivar number or ñame of clone Note: UTA, International Institute for Tropical Agricultura, Ibadan, Nigeria
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	MOLECULAR CHARACTERIZATION OF COLOMBIAN YAM GERMOPLASM
	ATP and polynucleotide kinase (MBI Fermentas) using standard procedures (Sambrook, et ál. 2000). Selective amplification was performed in 10 uL reactions containing 2 uL of diluted pre-amplified fragments, 0.5 uM labelled microsatellite primer, 5 uM EcoRI adapter-specific primer, 400 uM of dNTPs, 75 mM Tris-HCl (pH 9), 20 mM (NH4)2SO4, 0.01% Tween-20, 1.5 mM MgCl2, and 0.5 U Taq polymerase (Biotherm, Gene Craft). The PCR program consisted of an initial denaturation step at 94°C(120s), 11 cyclesat94°C(30s),65°C(30 s) [with a touch-down of -0.7 °C each cycle], and 72 °C (60 s), followed by 25 cycles at 94 °C (30 s), 56 °C (30 s), and 72 °C (60 s). Final extensión was at 72 °C (120 s). Products were separated on 4% sequencing gels and autoradiographed (Sambrook, et ál., 2000). The compound microsatellite primers used for the selective amplification were designed and provided by Dr. Peter Winter (unpublished data). Additionally, a newly designed adapter-primer ("Eco RI short") was employed in combination with TA-rich selective primers (Palacios, et ál. 2002).	Data analysis Unequivocally scorable SAMPL bands were transformed into a binary character matrix, with 1 for the presence, and 0 for the absence of a band at a particular position. Pairwise distances were directly computed with the STATISTICA sofware package (1984-1999 Stat Soft. Inc) using the percent disagreement. The final dendrogram was constructed using UPGMA cluster algorithm (Dhillon and Ishiki, 1999). RESULTS AND DISCUSSIONS Genomic DNAs from 36 Colombian and 9 African D. alata accessions, 12 Colombian and 11 African D. rotundata accessions, one accession each of D. trífida, D. bu l bífera, D. vittata and D. sansibarensis, respectively, were analyzed by SAMPL with a set of 5 compound microsatellite primers and a modified EcoRI adapter primer (Pala­cios, et ál., 2002) (table 2). Distinct polymorphic
	Table 2. Primers sequences
	SAMPL Adapters Sequence 5' - 3' Eco R1 1 CTCGTAGACTGCGTACC AATTGGTACGCAGTC Eco RI primer* CCATGCGTCAG Compound microsatellites primers P-SAMPL-34 TAATAATAACAACA P-SAMPL-35 TATATATATAGAAGA P-SAMPL-36 GAAGAAGAATATAT P-SAMPL-37 GAAGAAGAACACAC P-SAMPL-38 GAAGAAGAAGTGTG
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	Figure 1. SAMPL analysis of the whole D. alata and D. rotundata collections using SAMPL primer38. M: Molecular weigh marker 25pb, MBI Fermentas, 1- 5: D. trífida, D. bulbifera, D. vittata, D. sansibarensis. Car1, 6 - 26: D. rotundata, 26 - 71: D. alata.
	banding patterns were obtained with all primers, as exemplified by a subset of samples and SAMPL pri­mer 38 (figure. 1). Positions of 160 unequivocally scorable bands (of which 56 were monomorphic, 35% of total, with an average of 2.8 polymorphic bands per assay) were transformed into a binary character matrix(1 forpresence, Oforabsence)andanalyzed. The resulting similarity matrix was transformed into a dendrogram with the UPGMA algorithm. The diversity of the collections with this technique was 0.0471 with a mean similarity of 0.9529. The obtained SAMPL banding patterns clearly reflect the lack of genetic diversity observed within and between sampling sites. Basically the clusters observed obey to species separation. African accessions are found in cióse similarity with	Colombian individuáis, sincethewayof propagation applied by the farmers is in principle clonal, some accessions are still identical with the African "very likely" ancesters (Coursey and Martin, 1970; Coursey, 1976a, 1976b). On the other hand, somaclonal variation, which is the only source of variability, shows also to be rather low. Within the same species, form hundred-sixty scored characters, only four polymorphic bands were observed among individuáis form separated clusters. The same type of results were observed with DAF markers (Bustamante, et ál., 2003) were was evident the low genetic variability. Is important to develop varieties carrying as many different genes for resistance as possible, in order to provide stable resistance against a broad spectrum of the pathogens. Therefore, is imperative to extend the
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	MOLECULAR CHARACTERIZATION OF COLOMBIAN YAM GERMOPLASM
	yam germplasms in Colombia by plant breeding or introducing new varieties. ACKNOWLEDGEMENTS Research of the authors was supported by grants from GTZ (grant 95.2072.7-001.00) and BMZ (89.7860.3-01.130). S. B acknowledges a fellowship of UNESCO (Paris, France), Stiftung zur Fórderung der internationaler wissenschaftlicher Bezierungen (Frankfurt am Main, Germany), and PBA Fundation and Córdoba University for Colombian yam Collection. The authors also acknowledge Diego Riaño (Institu­to de Biotecnología, Universidad Nacional de Colombia BogotÆ, Colombia) and Carlos Molina (Plant Molecular Biology, Biocentre, University of Frankfurt, Frankfurt am Main / Germany) for their contributions. BIBLIOGRAFÍA Álvarez, A. 1991. Control de la antracnosis en æame. Instituto Colombiano Agropecuario (ICA). El Carmen de Bolívar. Documento de trabajo. Pág. 15. Asemota, H.N. 1991. A fast, simple, and efficient mini-scale method for the preparation of DNA from tissues of yam (Dioscorea spp.). Plant Mol Biol Rep. 13: 214-218. Asemota, H.N.; Ramser, J.; Lopéz-Peralta, C; Weising, K.; Kahl, G. (1996). Genetic variation and cultivar identification of Jamaican yam germplasm by random amplified polymorphic DNA analysis. Euphytica. 92: 341-351. Bustamante, S.; Guzmán, M.; Buitrago, G. 2001. Molecular characterization of some species and varieties of yam present on the Colombian Atlantic Coast. Rev Col Bio. 2: 38-43. Bustamante, S.; Guzmán, M.; Buitrago, G. 2003. Molecular characterization of Colombian yam germplasm by DNA amplification fingerprinting DAR Rev Col Bio. 5: 57-63. Coursey D.G; Martin, R.W. 1970. The past and future of yams as crop plants. In: Proceedings Second Symposium of Tropical Root Crops, Hawai i. 1: 87-90; 99-101. Coursey, D.G. 1976a. Yams Dioscorea spp. (Dioscoraceae). In: Evolution of Crop Plants. N.W. Simmonds, ed. Longmans, London an New York. Pp. 70-74. Coursey, D.G. 1976b. The origins and domestication of yams in África. In: Origins of African Plant	Domestication. J.R. Harían; J.M.J. de Wet and A.B.L. Stemler, eds. Mouton, The Hague. pp. 383-403. Durango, R.; Padilla A. 1998. Caracterización morfológica de un banco de germoplasma de ñame Dioscorea spp recolectado en la costa norte colombiana. Trabajo de grado Universi­dad de Córdoba, Montería, Colombia. Dhillon, N.P.S.; Ishiki, K. 1999. Genomic variation and genetic relationship in Ipomoea spp. Plant Breed. 118: 161-165. Morgante, M.; Vogel, J. 1994. Compound microsatellite primers for the detection of genetic polymorphisms. US Patent Application. 08/326456. Nei, M.; Li, W.H. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA. 76: 5269-5273. Paglia, G; Morgante, M. 1998. PCR-based multiplex DNA fingerprinting techniques for the analysis of conifer genomes. Mol Breed. 4: 173-177. Palacios, G.; Bustamante, S.; Molina, C.; Winter, P; Kahl, G. 2002. Electrophoretic identification of new genomic profiles with a modified SAMPL technique based on AT/AAT polymorphic repeats. Electrophoresis. 23: 3341-3345. Ramser, J.; López-Peralta, C; Wetzel, R.; Weising, K.; Kahl, G. 1996. Genomic variation and relationships in aerial yam (Dioscorea bulbifera L) detected by ramdom amplified polymorphic DNA. Genome. 39: 17-25. Ramser, J.; Weising, K.; Lopez-Peralta, C; Terhalle, W.; Terauchi, R.; Kahl, G. 1997. Molecular marker based taxonomy and phylogeny of Gui­nea yam (Dioscorea rotundata - D. cayenensis). Genome. 40: 903-915. Ramser, J.; Weising, K.; Chikaleke, V.; Kahl, G. 1997. Increased informativeness of RAPD analysis by detection of microsatellite motifs. BioTechniques. 23: 285-290. Sambrook, J.; Fritsch, E.F.; Maniatis, T. 2000. Mo­lecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Vivek, B.S.; Simón, PW. 1999. Linkage relationships among molecular markers and storage root traits of carrot (Daucus carota L ssp. sativus). Theor Appl Genet. 99: 58-64.
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	Vos, P.; Hogers, R.; Bleeker, M.; Reijans, M.; Van                production systems in Nepal and India. de Lee, T.; Hornes, M.; Frijters, A.; Pot, J.;                Euphytica. 122: 575-588. Peleman.J.; Kuiper, M.¡ Zabeau, M. 1995. Witsenboer, H.; Vogel, J.; Michelmore, R.W. 1997. AFLP: a new technique for DNA fingerprinting.                identificaron, genetic localization, and allelic Nucí Acids Res. 21: 4407-4414.                                     d¡vers¡ty of selectively amp|¡f¡ed polymorphic loci Witcombe, J.R.; Joshi, K.D.; Rana, R.B.; Virk, D.S.                ¡n lettuce and wNd relatives (Lactuca spp.). 2001. Increasing genetic diversity by                Genome. 40: 923-936. participatory varietal selection in high potential
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