UNIVERSITÀ DEGLI STUDI DI SASSARI CORSO DI DOTTORATO DI RICERCA Scienze Agrarie Curriculum Produttività delle Piante coltivate Ciclo XXIX Morphological, chemical and genetic characterization of Citrus monstruosa, an endemism of Sardinia Dott.ssa Grazia Viglietti Coordinatore del Corso Prof. Antonello Cannas Referente di Curriculum Prof.ssa Giovanna Attene Docente Guida Dott.ssa Grazia Maria Scarpa Tutor Dott. François Luro Anno accademico 2015- 2016 Acknowledgments We would like to thank the president of the INRA Centre and also Yann Froelicher, AGAP Unit Director, in Corsica, France, for the period spent in their laboratories. For experiments and analysis thanks to UMR 1390 INRA-AGAP, San Giuliano and, in particular, my tutor François Luro. Franck Curk for his assistance and support and all INRA laboratory technicians: Gilles Constantino, Elodie Marchi and Isabelle Tur. We also want to thank University of Corse, CNRS UMR 6134 SPE, "chemistry and biomass" team, Ajaccio, in particular prof. Félix Tomi and Dr. Sylvain Sutour for the essential oil analysis. We also would like to thank the Department of Agronomy, food, natural resources, animals and environment (DAFNAE) in Padova for the period spent in their laboratories and, specially, prof. Gianni Barcaccia and dr. Giulio Galla for their support and their help about work made with them. Finally, we wish to acknowledge the collaboration of dr. Giovanna Becca and dr. Pietro Paolo Urgeghe for analysis performed by them. Grazia Viglietti - Morphological, chemical and genetic characterization of Citrus monstruosa, an endemism of Sardinia Tesi di dottorato in Scienze agrarie. Curriculum: Produttività delle piante coltivate Ciclo XXIX Università degli Studi di Sassari Anno Accademico 2015 - 2016 Index AIM AND ORGANIZATION OF THE WORK................................................................................................ 1 GENERAL INTRODUCTION ....................................................................................................................... 2 Origin and diffusion of citrus ............................................................................................................... 2 Citrus in Italy: history for a cultural identity.................................................................................... 4 Citrus in Sardinia: in search of the past ........................................................................................... 5 Pompia ............................................................................................................................................. 9 Taxonomy of citrus and genetic relationship among taxa ................................................................ 12 The citrus: 6 botanical genera ....................................................................................................... 14 Genetic origin of the most important cultivated species .............................................................. 17 Citrus essential oils and their properties ........................................................................................... 22 Essential oil composition ............................................................................................................... 22 Uses and virtues ............................................................................................................................ 23 Extraction techniques .................................................................................................................... 25 Essential oil characterization ......................................................................................................... 25 Evolution of the molecular tools for identifying the species ............................................................ 27 References ......................................................................................................................................... 32 CHAPTER I .............................................................................................................................................. 44 Karyological analysis and DNA barcoding of Pompia, a first step towards the identification of its relatives. ............................................................................................................................................ 44 Introduction ................................................................................................................................... 44 Materials and Methods ................................................................................................................. 46 Results and discussion ................................................................................................................... 51 Conclusions .................................................................................................................................... 63 References ..................................................................................................................................... 64 Supplemental material .................................................................................................................. 67 CHAPTER II ............................................................................................................................................. 88 Genetic characterization of Pompia based on SSR and InDel molecular markers ............................ 88 Introduction ................................................................................................................................... 88 Materials and Methods ................................................................................................................. 89 Results and Discussion .................................................................................................................. 94 Conclusions .................................................................................................................................... 97 References ................................................................................................................................... 100 Supplemental material ................................................................................................................ 102 Grazia Viglietti - Morphological, chemical and genetic characterization of Citrus monstruosa, an endemism of Sardinia Tesi di dottorato in Scienze agrarie. Curriculum: Produttività delle piante coltivate Ciclo XXIX Università degli Studi di Sassari Anno Accademico 2015 - 2016 CHAPTER III .......................................................................................................................................... 104 Analysis of Pompia phenotype and composition of the essential oil ............................................. 104 Introduction ................................................................................................................................. 104 Materials and Methods ............................................................................................................... 105 Results and discussion ................................................................................................................. 109 Conclusions .................................................................................................................................. 128 References ................................................................................................................................... 131 GENERAL CONCLUSIONS ..................................................................................................................... 133 Grazia Viglietti - Morphological, chemical and genetic characterization of Citrus monstruosa, an endemism of Sardinia Tesi di dottorato in Scienze agrarie. Curriculum: Produttività delle piante coltivate Ciclo XXIX Università degli Studi di Sassari Anno Accademico 2015 - 2016 AIM AND ORGANIZATION OF THE WORK Characterize an unknown species means to protect and enhance its existence, especially if it is closely linked to the territory and its traditions. Sardinia has a rich plant heritage, probably due to its geographical isolation. This biodiversity is threatened by agriculture standardized resulting in loss of local landraces. The main purpose of this work is the characterization of a species of unknown origin in Sardinia, Pompia, through the study of its morphological, chemical and genetic characteristics. The Chapter 1 is introduced the data of a first genetic analysis approach, performed with DNA barcoding technique to identify which of the species of the genus Citrus are more similar to Pompia. It is also investigated the ploidy level through chromosomal counts, karyotype and flow cytometry. In Chapter 2 it was decided to make more detailed genetic analysis using SSR and InDel molecular markers with the aim of identifying which of the Citrus species and varieties are Pompia's parents and if there is genetic variability inside Sardinian specimens. Finally, Chapter 3 contains the Pompia phenotype and its essential oil composition in leaves and fruits, comparatively with its ancestors and lemon, a same interspecific hybrid origin, to estimate the inheritance of parental characters. In second step, the variability of Pompia phenotype was investigated by the sampling of different orchards and locations in Sardinia. The present work has been corrected by a english, mother tongue,