MS – thesis June 2015 A biogeographic analysis of the species Fragaria vesca using microsatellite markers on a worldwide plant collection Hrannar Smári Hilmarsson Faculty of Land and Animal Resources MS – thesis June 2015 A biogeographic analysis of the species Fragaria vesca using microsatellite markers on a worldwide plant collection Hrannar Smári Hilmarsson 90 ECTS thesis submitted in partial fulfillment of a Magister Scientiarum degree in Agricultural Sciences Academic advisors: Dr. Jón Hallsteinn Hallsson & Dr. Timo Hytönen Agricultural University of Iceland Faculty of Land and Animal Resources Clarification of contribution I hereby declare that the work of plant collection and propagation, DNA isolation, analysis of molecular data, and writing of this thesis is my work under the supervision of my advisors, Dr. Jón Hallsteinn Hallsson and Dr. Timo Hytönen. _______________________________ Hrannar Smári Hilmarsson i Abstract The woodland strawberry, Fragaria vesca L., has the widest distribution of its genus. It can be found across North-America, all over Europe and into Asia. F. vesca has become an important research model benefitting from its small genome as well as both easy propogation and maintainance. A vast collection and research on natural variation is imperative to the advancement of research in the field of the Rosa (Rosaceae) plant family, the third most economically important of all plant families. Another importat reason for assessing the genetic population structure of the F. vesca is its vast distribution and the effect of climate change through the ages, which could shed light on the dispersal route of the species during and after the last glacial maximum. The aims of this study were to collect individuals of F. vesca representing the entire geographic distribution, assess their genetic diversity and to shed light on the origin of the Icelandic F. vesca population. In this study, a collection of 445 plants were gathered from all corners of the geographical distribution. Of those, 300 plants were analysed using 66 of the 68 microsatellite markers tested. Their mean allele number per locus was 3.94 and the mean polymorphic information content value was 0.13. Ten markers were monomorphic and 13 biallelic. The collection of plants was analysed using Beyasian clustering analysis software BAPS and STRUCTURE. The two methods resulted in contrasting number of clusters for the data. The only corresponding results were the great divergence between the American population and the European. Also it was prominent in both methods that the Icelandic population was a separate cluster in the European populations. The two genetic distance methods used showed that the Icelandic population was closest genetically to the set of cultivars used in this study. Furthermore, the results revealed no population structure in Iceland suggesting recent dispersal and refuting any suggestions of glacial survival. ii Ágrip Villijarðarberið, Fragaria vesca L., er útbreiddasta tegundin af sinni ættkvísl. Hún finnst um Norður-Ameríku, um alla Evrópu og í Asíu. F. vesca er orðin mikilvæg rannsóknar lífvera, vegna smæðar erfðamengi síns sem og vegna þess hversu auðveld hún er til ræktunar og viðhalds. Stór plöntusöfn og rannsóknir á náttúrulegum breytileika eru bráðnauðsynleg til framsækni í rannsóknum á rósafjölskyldunni (Rosaceae), sem er sú þriðja efnahagslega mikilvægasta plöntufjölskyldan í heiminum. Önnur mikilvæg ástæða fyrir því að meta stofna villijarðarberja á heimsvísu er hin mikla útbreiðsla og áhrif loftslagsbreytinga fyrri alda á uppruna og útbreiðslumynstur tegundarinnar, fyrir og eftir síðasta ísaldarskeið. Markmið þessarar rannsóknar voru að safna F. vesca plöntum sem gætu staðið fyrir heimsútbreiðslu þeirra, meta breytileika þeirra og varpa ljósa á uppruna íslenska villijarðarberjastofnsins. Í þessari rannsókn var safnað 445 plöntum frá öllum heimshornum. Af þeim voru 300 greind með 66 af 68 örtunglum sem voru prófuð. Meðal samsætu fjöldi þeirra var 3.94 og meðal upplýsinga gildi breytileikans (PIC) 0.13. Tíu örtungl sýndu eina samsætu í öllu safninu og 13 gáfu aðeins tvær samsætur. Plöntusafnið var greint með Beyasian tölfræði forritunum BAPS og STRUCTURE. Aðferðirnar tvær sýndu mismunandi fjölda hópa fyrir gögnin. Einu samstæðurnar vöru þær að Evrópski stofninn er mjög frábrugðin þeim Ameríska. Einnig sýndu niðurstöðurnar að íslenski stofninn er sérstæður frá Evrópskum stofnum. Báðar aðferðirnar sem notaðar voru til að meta erfðafjarlægðir gáfu til kynna að þau yrki sem notuð voru í rannsókninni eru skyldust íslenska stofninum. Einnig greindust engir undirhópar tengdir landfræðilegum uppruna íslensku sýnanna og því hægt að geta sér til um að F. vesca hafi nýlega borist til lands. Engin merki þess greindust í þessari rannsókn, að F. vesca hafi lifað af síðasta ísaldarskeið. iii Acknowledgements I would like to thank my advisors Dr. Jón Hallsteinn Hallsson and Dr. Timo Hytönen. I would also like to thank Dr. Sachiko Isobe at the Kazusa DNA research institute for the microsatellite analysis, Dr. Kim Hummer at the USDA National Clonal Germ plasm Repository who supplied plant material, as well as all other collectors who contributed plant material to the collection. My family receives my thanks for support and patience. The Agricultural Productivity Fund and the Memorial Fund of Hjörtur and Ragnheiður is gratefully acknowledged for financial support. iv Table of Contents Clarification of contribution .................................................................................................i Abstract................................................................................................................................ii Ágrip...................................................................................................................................iii Acknowledgements ............................................................................................................iv List of tables .....................................................................................................................viii List of figures .....................................................................................................................ix List of abbreviations .........................................................................................................xiii 1. Introduction .................................................................................................................1 1.1. Strawberries (Fragaria)...............................................................................1 1.1.1 Strawberry history and cultivation ..........................................................1 1.1.2 Taxonomy of the Fragaria genus ...........................................................2 1.1.3 Populations of the Fragaria vesca species and subspecies.....................3 1.1.4 Geographic distribution of the Fragaria vesca.......................................3 1.1.5 Fragaria vesca as a model organism ......................................................5 1.2. Molecular genetics of wild crop relatives....................................................5 1.2.1 Molecular markers and the Fragaria vesca genome...............................5 1.2.2 Natural allelic variation...........................................................................6 1.2.3 Introgression of variation from crop wild relatives.................................7 1.2.4 Biodiversity and conservation...............................................................10 1.3. Phytogeography .........................................................................................11 1.3.1 Floral history of Iceland and the North Atlantic ...................................11 1.3.2 Genetics in phytogeography..................................................................14 2. Aims of study ............................................................................................................17 3. Materials and methods...............................................................................................18 3.1. Sample collection.......................................................................................18 3.1.1 Plant propagation ..................................................................................18 3.2. Homogenization and DNA extraction .......................................................20 3.2.1 Tissue sampling and homogenization ...................................................20 3.2.2 Extraction of DNA ................................................................................21 3.3. Microsatellite amplification, analysis and fragment detection ..................21 3.4. Analysis of microsatellite data...................................................................25 3.4.1 Investigated populations........................................................................25 3.4.2 Summary statistic calculations of microsatellite data ...........................27 3.4.3 Clustering analysis ................................................................................28 v 3.4.4 Plotting distance matrices onto a map...................................................30 4. Results .......................................................................................................................31 4.1. Statistics of microsatellite data ..................................................................31 4.2. World population structure ........................................................................35