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Inga Edulis Mart CZECH UNIVERSITY OF LIFE SCIENCES PRAGUE FACULTY OF TROPICAL AGRISCIENCES Genetic variation and structure of agroforestry useful trees, Inga edulis Mart. and I. ingoides (Rich.) Willd., (Fabaceae) in Amazonian Peru. Dissertation thesis Faculty of Tropical AgriSciences Department of Crop Sciences and Agroforestry Study programme: Agriculture in Tropics and Subtropics Author: Ing. Alexandr Rollo Supervisor: doc. Ing. Bohdan Lojka, Ph.D. Co-supervisor: prof. Mgr. Bohumil Mandák, Ph.D. Prague, May 2019 i DISSERTATION THESIS TOPIC DISSERTATION THESIS TOPIC Author of thesis: Ing. Alexandr Rollo Study programme: Agricultural Specialization Field of study: Tropical and Subtropical Agriculture Thesis supervisor: doc. Ing. Bohdan Lojka, Ph.D. Supervising department: Department of Crop Sciences and Agroforestry Language of a thesis: English Thesis title: Genetic variation and structure of agroforestry useful trees, Inga edulis Mart. and I. ingoides (Rich.) Willd., (Leguminosae: Mimosoideae) in Amazonian Peru Objectives of thesis: i. To evaluate genetic variation and structure of widely cultivated neotropical tree species I. edulis in geographically different anthropogenic and natural populations from Amazonian Peru. ii. To evaluate genetic variation and structure of potentionaly useful neotropical tree species I. ingoides in geographically different natural populations from Amazonian Peru. iii. To check for putative introgression between both mentioned species. Methodology: The sampling will be carried out in geographically different natural forest sites and urbanized areas in Amazonian Peru. The two Inga species are going to be identified according to morphological aspects detailed in Pennington TD, (1997). The samples will be randomly selected from sexually mature trees. Voucher specimens are going to be archived in the Regional Herbarium of Ucayali IVITA-Pucallpa, Peru. Total DNA extraction from a 20 mm by 10 mm section of silica- dried young leaf material will be performed using the Invitek, Invisorb® Spin Plant Mini Kit according to the manufacture’s instructions. Four microsatellite markers are going to be used, one (Pel5) previously developed by Daynandan et al. (1997) for Pithecellobium elegans Ducke, and three (Inga03, Inga08 and Inga33) by Hollingsworth et al. (2005) for I. edulis. Loci are going to be amplified individually in 10 µl reaction containing: 20 ng of template DNA, 5 µM of forward and reverse primer, 50 µM of dNTPs, 2 mM of MgCl2, 2 µl 5x GoTaq Flexi Buffer (Promega) and 1.0 U of GoTaq® Flexi DNA Polymerase (Promega). Amplifications were undertaken in Biometra® T1 Thermocycler using the following profile: 95 °C for 2 min; 95°C for 15 s, 55°C (Inga03) and 59°C (Inga08, Inga33 and Pel5) for 30 s, 72 °C for 30 s, 30 cycles; 72 °C for 15 min. Completed reactions will be loaded onto an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Forest City, CA, USA) and run according to the manufacture’s protocol. Allele sizes will be determined using the ROX500 internal size standard and GeneMarker® v2.4 software. Genetic variation and structure will be determined using analysis of molecular variance and Bayesian analysis of population structure, according to the objectives of the study. The proposed extent of the 80-120 pages thesis: Keywords: biodiversity conservation, domestication, edible fruits, Inga, introgression, microsatellite markers, shade trees ii DISSERTATION THESIS TOPIC Recommended information sources: 1. Dayanandan S, Bawa K, Kesseli R (1997) Conservation of microsatellites among tropical trees (leguminosae). Am J Botany 84:1658-1663 2. Hollingsworth PM, Dawson IK, Goodall-Copestake WP, Richardson JE, Weber JC, Sotelo Montes C, Pennington RT (2005) Do farmers reduce genetic diversity when they domesticate tropical trees? A case study from amazonia. Mol Ecol 14:497-501 3. Koptur S (1984) Outcrossing and pollinator limitation of fruit-set: Breeding systems of neotropical inga trees (fabaceae: Mimosoideae). Evolution 38:1130-1143 4. Nei M (1987) Molecular evolutionary genetics. Columbia University Press, New York 5. Oliveira PJC, Asner GP, Knapp DE, Almeyda A, Galván-Gildemeister R, Keene S, Raybin RF, Smith RC (2007) Land-use allocation protects the peruvian amazon. Science 317:1233-1236. doi:10.1126/science.1146324 6. Peakall R, Smouse PE (2012) Genalex 6.5: Genetic analysis in excel. Population genetic software for teaching and research—an update. Bioinformatics 28:2537-2539. doi:10.1093/bioinformatics/bts460 7. Pennington TD (1997) The genus inga: Botany. Kew: Royal Botanic Gardens, London 8. Richardson JE, Pennington RT, Pennington TD, Hollingsworth PM (2001) Rapid diversification of a species-rich genus of neotropical rain forest trees. Science 293:2242-2245. doi:10.1126/science.1061421 9. Rousset F (2008) Genepop'007: A complete re-implementation of the genepop software for windows and linux. Mol Eco Resour 8:103-106. doi:doi:10.1111/j.1471-8286.2007.01931.x 10. Weir BS, Cockerham CC (1984) Estimating f-statistics for the analysis of population structure. Evolution 38:1358-1370 Expected date: 2017/18 (SS) - FTA - State Doctoral Examinations Advisor of thesis: doc. Mgr., Bohumil Mandák, Ph.D. Electronically approved: 29. 3. 2016 Electronically approved: 6. 2. 2017 doc. Ing. Bohdan Lojka, Ph.D. doc. Ing. Bohdan Lojka, Ph.D. Head of department Chairperson of Departmental Board Electronically approved: 28. 3. 2018 doc. Ing. Jan Banout, Ph.D. Dean . iii DECLARATION OF AUTHORSHIP DECLARATION OF AUTHORSHIP I, Alexandr Rollo, hereby declare that this thesis entitled Genetic variation and structure of agroforestry useful trees, Inga edulis Mart. and I. ingoides (Rich.) Willd., (Fabaceae) in Amazonian Peru, submitted in partial fullfilment of the requirements for the degree of Ph.D. at the Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, and the work presented in it is entirely my own work. Information derived from the published or unpublished work has been acknowledged in the text and a list of references is given. Prague, May 2019 Ing. Alexandr Rollo iv ACKNOWLEDGEMENT ACKNOWLEDGEMENT First and foremost I wish to express my deep and sincere gratitude to my supervisor, doc. Ing. Bohdan Lojka, Ph.D., head of Department of Crop Sciences and Agroforestry, Faculty of Tropical AgriSciences (FTA), and my cosupervisor, prof. Mgr. Bohumil Mandák, Ph.D., Department of Ecology, Faculty of Environmental Sciences, both from Czech University of Life Sciences Prague (CULS) for their constructive comments, and for their support throughout this work. During this work I have collaborated with many colleagues for whom I have great regard, and I wish to extend my warmest thanks to all those who have helped me with this work. Adjunct Professor, Ms.C. Maria Margarida Chagas de Ataíde Ribeiro Ph.D., population geneticist and professor at the College of Agriculture of Castelo Branco, Polytecnic Institute of Castelo Branco, Portugal, Rita Maria Lorenco Da Costa, Ph.D., molecular geneticist at Laboratory of Molecular Biology, National Institute Of Biological Resources Oeiras, Portugal. My very deep gratitude belongs to Zoyla Mirella Clavo Peralta, director of Regional Herbarium of Ucayali in Pucallpa - IVITA, National University of San Marcos Lima, Peru, who provided her expertise in plant identification. My thanks are also due to doc. Dr. Ing. Pavel Vejl, head of Department of Genetics and Breeding, Faculty of Agrobiology, Food and Natural Resources (CULS), who gave me very valuable advices. Moreover, my fieldwork in Peru would not have been possible without the local inhabitants and farmers who opened their doors and let me work on their land. I gratefully acknowledge the folowing institutions which helped me realized the field work in Peru: National University of Ucayali, Pucallpa (UNU), National University of Agriculture of La Selva, Tingo María (UNAS). My deep gratitude belongs also to The National Service of Natural Areas Protected by Steate (SERNANP), Peruvian Ministery of Environment, which allowed me to enter to the Peruvian Protected Natural Area (ANP). I am also indebted to the donors, who made the study possible through their financial and technical support: Ministry of Foreign Affairs of the Czech Republic through the Czech Republic Development Aid Project entitled “Sustainable use of natural resources in Peruvian Amazon Project” No. 23/MZe/B/07-10; Ministry of Education, Youth and Sport through the scholarship on (UNU), Peru; The Academy of Science of The Czech Republic (AVCR) and The National Council for Science, Technology and Technological Innovation (CONCYTEC), Peru, through the binational Project entitled “Morphological and genetic diversity of indigenous tropical trees in the Amazon – model study of Inga edulis Mart. in Peruvian Amazon”; Foundation “Nadace Nadání Josefa, Marie a Zdeňky Hlávkových”, Czech Republic; European Union ERASMUS Training Program and to Mobility Grant of FTA (CULS). v DEDICATION to my family and all Amazon people. ...když jsme se vraceli cestou domů, tak jsme viděli dřevorubce, jak porážej strom a ten strom plakal… František Sahula, 1990 vi LIST OF ABREVIATIONS LIST OF ABREVIATIONS A Average number of alleles per locus AR Allelic richness AMOVA Analysis of Molecular Variance ANOVA Analysis of Variance ANP Protected Natural Area ARc I. edulis cultivated population Antonio Raimondi ATc I. edulis cultivated population Atalaya AVCR The Academy of Science of The Czech Republic bp base pair b.p. before present BRc I. edulis cultivated population Bretaña c cultivated CICFOR
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