Durham E-Theses Structural and molecular analyses of heterostyly in Linum tenue (Linaceae) FOROOZANI, ALIREZA How to cite: FOROOZANI, ALIREZA (2018) Structural and molecular analyses of heterostyly in Linum tenue (Linaceae), Durham theses, Durham University. Available at Durham E-Theses Online: http://etheses.dur.ac.uk/13304/ Use policy The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that: • a full bibliographic reference is made to the original source • a link is made to the metadata record in Durham E-Theses • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders. Please consult the full Durham E-Theses policy for further details. Academic Support Oce, Durham University, University Oce, Old Elvet, Durham DH1 3HP e-mail: [email protected] Tel: +44 0191 334 6107 http://etheses.dur.ac.uk 2 Structural and molecular analyses of heterostyly in Linum tenue (Linaceae) Linum tenue. Image by Stuart Brooker and Alireza Foroozani By Alireza Foroozani Department of Biosciences Durham University December 2018 Submitted for the degree of Doctor of Philosophy Declaration The material contained within this thesis has not previously been submitted for a degree at Durham University or any other university. The research reported within this thesis has been conducted by the author unless indicated otherwise. Alireza Foroozani December 2018 Ó The copyright of this thesis rests with the author. No quotation from it should be published without the author's prior written consent and information derived from it should be acknowledged. i Acknowledgements I would first like to deeply thank my primary supervisor, Dr. Adrian Brennan, for his continued guidance, patience and support throughout the entire project. Adrian, there has never been a time where I felt unable to come to you for help. You have given me the opportunity and the freedom for me to take my work in whatever direction I saw fit. I hope that we can remain friends and collaborators into the future. I would like to give special thanks to my secondary supervisor Prof. Marc Knight and all the members of Lab 19 throughout the years for their friendships and the memories we’ve shared. I would like to pay my gratitude to our collaborators: Dr. Juan Orroyo at Universidad de Sevilla, for his guidance and recommendations on the fieldwork; and to Dr. Rocio Perrez- Barrales at the University of Portsmouth, for sharing her data and knowledge of the complexity of Linum ecology. I would also like to thank the members of the Durham Ecology and Durham Molecular Ecology groups, whose friendships have been among the most important of my time in Durham. Finally, I owe a great personal thanks to Albert Lahat, whose mastery of Python was an invaluable help, and without his teaching and advice I would not have developed a lot of the skills necessary to conduct the work in these pages. David Dolan, for his bioinformatics guidance and for teaching me some of the most important lessons in computing! And last but not least Tom Batstone, for his many late-night discussions on bioinformatics and words of encouragement throughout the years. Dedication I dedicate this thesis to my mother, for her love and sacrifices. ii General Abstract Flowering plant mating systems are as varied as they are complex, and are generally considered the leading force behind angiosperm diversity and evolution. Establishing the genetic and molecular mechanisms behind phenotypic traits is important to understand how they have developed, evolved and spread across populations and taxa. Until recently, our understanding of the genetic basis of heterostyly, whereby reciprocal polymorphisms in the relative positioning of stigmas and anthers in certain angiosperm species, has been largely unknown; though has since been shown to be determined by the multiallelic S- locus. Using the study species of Linum tenue (Linaceae), this thesis investigates the ecology, trait variance and developmental progression of heterostyly in the flowers of L. tenue, making inferences on the specific control of heterostyly in this species and general speculative theories for its evolution in this taxa. Using a transcriptomic dataset derived from short-read Illumina sequence data, this thesis presents an automated method for reassembling consensus unigene sequences for the creation of a high-quality refernce transcriptome from mRNA data, providing a useful tool for a challenging aspect of gene expression studies in non-model organisms. This method is exemplified through the creation of a transcriptomic reference sequence of L. tenue vegetative and floral tissues, and through the de novo assembly of the Arabidopsis thaliana transcriptome. Next, global and differential expression analyses are used to investigate patterns in expression between polymorphic L. tenue flowers to discover candidate and proto-candidate loci determining the heterostyly syndrome and study differences in expression behaviour between the two morphotypes. This thesis provides evidence that heterostyly in L. tenue is pleiotropically controlled, and that the non-recombining nature of the S-locus can result in interesting patterns of morph-specific expression. iii Table of contents Declaration ___________________________________________________________ i Acknowledgements _____________________________________________________ ii Dedication ____________________________________________________________ ii General Abstract ______________________________________________________ iii Table of contents ______________________________________________________ iv List of abbreviations and acronyms _______________________________________ vii 1.0 General Introduction _____________________________________________ 1-29 1.1 Introduction to angiosperm reproductive biology __________________________ 2 1.1.1 Angiosperm diversity ........................................................................................................ 2 1.1.2 Angiosperm mating systems ............................................................................................. 3 1.1.3 The dimensions of plant mating strategies ........................................................................ 4 1.1.4 Promoting outbreeding ..................................................................................................... 6 1.1.5 Evolutionary transitions to self-compatibility................................................................... 11 1.2 Introduction to heterostyly ___________________________________________ 13 1.2.1 Reciprocal herkogamy: the Darwinian hypothesis ............................................................ 13 1.2.2 The ecological function: the modern perspective ............................................................ 15 1.2.3 The genetic basis (in Primula – the ‘model’ heterostylous system) ................................... 16 1.2.4 Genetic studies in other heterostylous systems ............................................................... 20 1.2.5 The origins of heterostyly ................................................................................................ 22 1.3 Introduction to Linum _______________________________________________ 23 1.3.1 The Linaceae ................................................................................................................... 23 1.3.2 The genetics of heterostyly in Linum ............................................................................... 24 1.4 Linum tenue: the study species ________________________________________ 25 1.4.1 Linum tenue: the biology, bauplan and phylogeny ........................................................... 25 1.4.2 Initial crossing experiments ............................................................................................. 27 1.4.3 Experimental design........................................................................................................ 28 1.5 Project aims _______________________________________________________ 28 2.0 The developmental and functional control of distyly in Linum tenue (Linaceae) __________________________________________ 30-61 2.0.1 Preamble ........................................................................................................................ 31 2.0.2 Abstract .......................................................................................................................... 31 2.1 Introduction _______________________________________________________ 33 2.2 Materials and Methods ______________________________________________ 39 2.2.1 Pollinator observations ................................................................................................... 39 2.2.2 Plant material ................................................................................................................. 39 2.2.3 Floral organ measures ..................................................................................................... 40 2.2.4 Analysis of floral organ length and herkogamy in open flowers ........................................ 41 2.2.5 Analysis of morph frequency and adaptive inaccuracy ..................................................... 42 2.2.6 Analysis of filament and style cell length ......................................................................... 43 iv 2.3