Science, Irving K. Barber Faculty of (Okanagan)
Biology, Department of (Okanagan)
DSpace
UBCO
Wells, Rebecca
Master of Science - MSc
University of British Columbia
The molecular aspects of flower initiation and development have not been adequately investigated in lavender (Lavandula). In order to identify genes that control these processes, I employed RNA-Seq to obtain sequence information for transcripts originating from the vegetative shoot apical meristem (SAM) and developing inflorescence tissues of Lavandula angustifolia and Lavandula × intermedia plants, and assembled a comprehensive transcriptome of 105,294 contigs. Homology-based annotation provided gene ontology terms for the majority of transcripts, including over 100 genes homologous to those that control flower initiation and organ identity in Arabidopsis thaliana. Expression analysis revealed that most of these genes are differentially expressed during flower development. For example, LaSVP, a homolog of the floral repressor SHORT VEGETATIVE PHASE (SVP), was strongly expressed in vegetative SAM compared to developing flowers, implicating its potential involvement in flowering repression in lavender. In total, fourteen full-length lavender transcription factor homologs were identified. To investigate these genes further, I constitutively expressed each gene in transformed A. thaliana plants, evaluating the effects on flower initiation and morphology. Expression of the Lavandula genes in A. thaliana was gene- dependent. Phenotypic effects were observed for LaCC, LaFT, and LaSVP transformants. For example, constitutive expression of the LaSVP gene in A. thaliana delayed flowering and affected flower organ identity in a dosage- dependent manner. Four of the highest expressing lines produced sepals instead of petals and were sterile as they failed to develop proper seed pods. This study provides the foundation for future investigations aimed at elucidating flower initiation and development in lavender.
IDENTIFICATION, CLONING, AND FUNCTIONAL CHARACTERIZATION OF LAVANDULA FLORAL DEVELOPMENT GENES by Rebecca Wells BSc. (Honours) The University of British Columbia, 2017 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) August 2020 © Rebecca Wells 2020 i The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: Identification, cloning, and functional characterization of Lavandula floral development genes submitted by Rebecca Wells in partial fulfillment of the requirements of the degree of Master of Science in Biology Dr. Soheil Mahmoud, Biology, Irving K. Barber School of Arts and Sciences Supervisor Dr. Michael Deyholos, Biology, Irving K. Barber School of Arts and Sciences Supervisory Committee Member Dr. Louise Nelson, Biology, Irving K. Barber School of Arts and Sciences Supervisory Committee Member Dr. Hadi Mohammadi, Faculty of Applied Sciences, School of Engineering University Examiner ii Abstract The molecular aspects of flower initiation and development have not been adequately investigated in lavender (Lavandula). In order to identify genes that control these processes, I employed RNA-Seq to obtain sequence information for transcripts originating from the vegetative shoot apical meristem (SAM) and developing inflorescence tissues of Lavandula angustifolia and Lavandula × intermedia plants, and assembled a comprehensive transcriptome of 105,294 contigs. Homology-based annotation provided gene ontology terms for the majority of transcripts, including over 100 genes homologous to those that control flower initiation and organ identity in Arabidopsis thaliana. Expression analysis revealed that most of these genes are differentially expressed during flower development. For example, LaSVP, a homolog of the floral repressor SHORT VEGETATIVE PHASE (SVP), was strongly expressed in vegetative SAM compared to developing flowers, implicating its potential involvement in flowering repression in lavender. In total, fourteen full-length lavender transcription factor homologs were identified. To investigate these genes further, I constitutively expressed each gene in transformed A. thaliana plants, evaluating the effects on flower initiation and morphology. Expression of the Lavandula genes in A. thaliana was gene- dependent. Phenotypic effects were observed for LaCC, LaFT, and LaSVP transformants. For example, constitutive expression of the LaSVP gene in A. thaliana delayed flowering and affected flower organ identity in a dosage- dependent manner. Four of the highest expressing lines produced sepals instead of petals and were sterile as they failed to develop proper seed pods. This study provides the foundation for future investigations aimed at elucidating flower initiation and development in lavender. iii Lay Summary This thesis provides insight into lavender floral developemnt. Floral development has been described in other flowering plants but has yet to be studied in lavenders. Lavenders are important both economically and medicinally due to their aromatic essential oils, which are widely used in a variety of settings. As these oils are mainly produced in the flowers of these plants, it is beneficial to improve understanding of floral development. This thesis identified genes involved in flowering initiation and pattern development in lavenders and examined the functions of these genes. This was achieved through the development of a transcriptome, identification of over 100 floral development genes, and through functional characterization of a subset of these genes in A. thaliana. This research begins a new area of research and the results of this thesis provide a better understanding of floral development in lavenders, which will facilitate future studies and improvements in oil production. iv Preface Design of this project was completed by Dr. Soheil Mahmoud (Professor, UBC Okanagan). All experimentation and analyses of research data in this thesis were completed by Rebecca Wells, with the exception of some data generated for LaSVP. An honours student, Lina Bauer, was given several Lavandula genes identified in this thesis to characterize for her honours thesis. Results achieved by Lina’s LaSVP transformants have been included in this thesis in addition to the LaSVP results of the present study. Results of LaSVP transformant analysis are published in Planta (Wells et al. 2020). The heatmap and RT-PCR figures were prepared with assistance from Dr. Ayelign Adal. This thesis was written with the guidance of Dr. Soheil Mahmoud and was reviewed and approved by my supervisory committee: Dr. Soheil Mahmoud, Dr. Louise Nelson, and Dr. Michael Deyholos. Chapters 1 through 4 contain text and figures which have been published in the journal Planta. Parts of these chapters were reprinted/adapted by permission from Springer Nature: Planta 251(41): 1-11, Cloning and functional characterization of a floral repressor gene from Lavandula angustifolia, Rebecca S. Wells, Ayelign M. Adal, Lina Bauer, Elaheh Najafianashrafi and Soheil S. Mahmoud, © 2020, license 4900371164986. The publication entitled “Cloning and Functional Characterization of a Floral Repressor Gene from Lavandula angustifolia” was primarily written by Rebecca Wells and Lina Bauer, with the aid of Dr. Soheil Mahmoud and Dr. Ayelign Adal and with contributions from Elaheh Najafianashrafi in collecting data for the phylogenetic analysis. Rebecca Wells was the primary author with the duty of preparing the manuscript for submission with the aid of Dr. Soheil Mahmoud and Dr. Ayelign Adal. While completing my thesis, I also was first author on the following publication: Wells R, Truong F, Adal AM, et al (2018) Lavandula essential oils: a current review of applications in medicinal, food, and cosmetic industries of lavender. Nat Prod Commun 13:1403–1417. doi: 10.1177/1934578x1801301038 In the preparation of “Lavandula Essential Oils: A Current Review of Applications in Medicinal, Food, and Cosmetic Industries of Lavender”, Rebecca Wells and Felisha Truong were the primary authors, with contributions by Dr. Ayelign Adal and Dr. Lukman Sarker. Rebecca Wells was the primary author with the duty of preparing the manuscript for submission with the aid of Dr. Soheil Mahmoud and Dr. Ayelign Adal. v Table of Contents Abstract ........................................................................................................................................... i Lay Summary ............................................................................................................................... iii Preface ........................................................................................................................................... iv Table of Contents ...........................................................................................................................v List of Tables ............................................................................................................................... ix List of Figures ................................................................................................................................x List of Abbreviations ................................................................................................................ xiii Acknowledgements ......................................................................................................................xv Dedication .................................................................................................................................. xvi 1. Chapter 1: Introduction ............................................................................................................1 1.1 Lavenders .......................................................................................................................................... 1 1.1.1 The Genus Lavandula ................................................................................................1 1.1.2 Lavender Floral Structure ..........................................................................................2 1.1.3 Essential Oil Composition and Applications .............................................................3 1.2 Arabidopsis thaliana .............................................................................................................4 1.2.1 About Arabidopsis thaliana .......................................................................................4 1.2.2 Life Cycle and Development .....................................................................................5 1.2.3 Floral Initiation and the Transition from Vegetative to Reproductive Growth ..........6 1.3 Flowering Inductive Pathways ............................................................................................10 1.3.1 Flowering in Response to Photoperiod ....................................................................10 1.3.2 The Vernalization Pathway .......................................................................................13 1.3.3 The Autonomous Pathway ........................................................................................14 1.3.4 The Giberellin Pathway ............................................................................................14 1.4 Transcription Factors ............................................................................................................15 1.4.1 MADS-Box Transcription Factors ............................................................................16 1.5 Floral Pattern Development in Arabidopsis thaliana ...........................................................18 1.5.1 The ABC(DE) Model ................................................................................................18 1.6 Thesis Hypotheses and Objectives .......................................................................................21 1.6.1 Research Hypotheses ................................................................................................22 1.6.2 Thesis Objectives .....................................................................................................22 vi 2. Chapter 2: Materials and Methods ........................................................................................24 2.1 Transcriptome Assembly ......................................................................................................24 2.1.1. Plant Materials and Growth Conditions ...................................................................24 2.1.2. RNA Extraction and Sequencing .............................................................................24 2.1.3. De Novo Transcriptome Assembly and Annotation ................................................25 2.1.4. Differential Expression Analysis .............................................................................26 2.1.5. Candidate Selection and ORF Identification ...........................................................27 2.1.6. Primer Design ..........................................................................................................27 2.2. Cloning and Functional Characterization ...........................................................................29 2.2.1. PCR Amplification of Lavandula Floral Genes ......................................................29 2.2.2. Cloning of Lavandula angustifolia Floral Genes into the pGA482 Vector .............29 2.2.3. Bacterial Growth and Plasmid Extraction ...............................................................30 2.2.4. Restriction Digestion and Ligation ..........................................................................30 2.2.5. Heat-Shock Transformation of Competent Escherichia coli JM109 .......................31 2.2.6. Plating and Selection of Transformed Escherichia coli JM109 ..............................31 2.2.7. Preparation of Competent Agrobacterium tumefaciens ..........................................32 2.2.8. Agrobacterium tumefaciens Transformation and Selection ....................................32 2.2.9. Plant Transformation ...............................................................................................33 2.2.10. Selection of Transgenic Plants ..............................................................................35 2.2.11. Detection of Antibiotic Resistance and Floral Development Genes .....................36 2.2.12. Phenotypic Analyses ..............................................................................................37 3. Chapter 3: Results....................................................................................................................38 3.1 Literature Review and Preliminary Homolog Identification ...............................................38 3.2 RNA-Seq and Transcriptome Assembly and Analysis ........................................................38 3.3 Sequence Analysis and Phylogeny .......................................................................................52 3.4 Cloning and Transformation ................................................................................................55 3.4.1 Cloning of Candidates in the pGA482 Vector .........................................................55 3.4.2 Transformation and Selection of Transgenic Arabidopsis thaliana .........................59 3.5 Genetic Analyses of F1 Transformants ................................................................................61 3.5.1. DNA Detection of the NPT-II Gene .......................................................................61 3.5.2 cDNA Detection of Lavandula Candidate Genes .....................................................62 vii 3.6 Phenotypic Analyses of Transformants ...............................................................................65 3.6.1. LaSVP Transformant Analysis ................................................................................65 3.6.1.1. Expression of LaSVP and its Effects on Floral Transition and Morphology............................................................................................................................................67 3.6.2 LaCC Transformant Analysis ...................................................................................70 3.6.3 LaPI Transformant Analysis ....................................................................................71 3.6.4 LaAP1 Transformant Analysis .................................................................................73 3.6.5 LaCAL Transformant Analysis ................................................................................74 3.6.6 LaFT Transformant Analysis ...................................................................................75 4. Chapter 4: Discussion ..............................................................................................................77 4.1 Transcriptome Assembly and Identification of Lavandula Homologs ................................78 4.1.1. Transcriptome Assembly, Annotation, and Gene Identification .............................78 4.1.2. Sequence Analysis ...................................................................................................80 4.1.3. Expression Analysis of Lavandula Transcripts .......................................................82 4.2 Cloning and Selection of Candidates ..................................................................................83 4.2.1. Cloning in the pGA Vector and Bacterial Selection ................................................83 4.2.2. Plant Transformation and Selection .........................................................................84 4.2.3. Expression Analysis of F1 Transformants ................................................................85 4.3 Functional Characterization of Lavandula Candidates ........................................................87 4.3.1. LaSVP ......................................................................................................................89 4.3.1.1. Phylogenetic analysis of LaSVP and Other Plant Homologs ...................89 4.3.1.2 Ectopic Expression of LaSVP Alters Floral Morphology and Flowering Time ................................................................................................................................. 90 4.3.2. LaCC ........................................................................................................................92 4.3.3. LaPI..........................................................................................................................92 4.3.4. LaAP1 ......................................................................................................................94 4.3.5. LaCAL ......................................................................................................................95 4.3.6. LaFT .........................................................................................................................96 5. Chapter 5: Conclusion .............................................................................................................98 5.1. Development of a Comprehensive Lavandula Transcriptome ............................................99 5.2. Candidate Identification and Differential Expression Analysis ........................................100 viii 5.3. Cloning and Functional Characterization of Lavandula Homologs..................................102 5.4. Study Limitations ..............................................................................................................104 5.5. Summary ...........................................................................................................................106 6. Bibliography ...........................................................................................................................108 7. Appendices ..............................................................................................................................127 ix List of Tables Table 1. A. thaliana genes comprising the ABC(DE) model in Arabidopsis ..............................19 Table 2. Gene- specific primers designed for Lavandula candidates ..........................................28 Table 3. Summary of the parameters of the L. angustifolia and L. x intermedia transcriptome assemblies ......................................................................................................................................39 Table 4. Full-length, previously- reported Lavandula terpene synthase genes identified within the Lavandula transcriptome..........................................................................................................40 Table 5. Arabidopsis thaliana floral development gene homologs identified within the comprehensive Lavandula transcriptome ......................................................................................42 Table 6. Full-length Lavandula floral initiation and development genes identified within the comprehensive Lavandula transcriptome ......................................................................................51 Table 7. The percent similarity of candidate Lavandula proteins and their A. thaliana homolog based on MUSCLE alignment of proteins sequences ....................................................................52 Table 8. Percent identity matrix of the % similarity of SVP protein sequences derived from different plants ...............................................................................................................................54 Table 9. Genetic analysis of F1 transformants ..............................................................................64 Table A.1 Nanodrop Spectrometer data of extracted RNA from L. angustifolia floral and bud tissues ..........................................................................................................................................127 Table A.2 Nanodrop Spectrometer data of extracted plasmids from E. coli JM109 cells ..........127 Table A.3 Nanodrop Spectrometer data of extracted E. coli JM109 plasmids…………….…..127 Table A.4 Culture-forming units per plate of heat-shock transformed E. coli cells on LB tetracycline medium .....................................................................................................................127 Table A.5 RPKM values of floral development genes in L. angustifolia and L. x intermedia tissues based on Differential expression for RNA Seq analysis ..................................................128 Table C.1 Genes involved in floral development in A. thaliana.................................................136 Table C.2 Full-length sequences of Lavandula genes identified within the Lavandula transcriptome ...............................................................................................................................137 x List of Figures Figure 1. The ABC(DE) model and Floral Quartet Models of floral development described in A. thaliana ..........................................................................................................................................21 Figure 2. Lavandula tissue from which RNA was extracted to assemble the lavender transcriptome .................................................................................................................................25 Figure 3. Agrobacterium-mediated transformation of wild-type A. thaliana via the floral dip ...35 Figure 4. Distribution of transcript lengths in the comprehensive Lavandula transcriptome.......39 Figure 5. Classification of genes in the comprehensive Lavandula transcriptome……..…… ....41 Figure 6. Heatmap showing the relative expression levels of selected floral development genes........................................................................................................................................................50 Figure 7. Phylogenetic analysis of the LaSVP protein and other plant SVP-like proteins issue ..53 Figure 8. Multiple Sequence Alignment of protein sequences of LaSVP and other plant SVP homologs .......................................................................................................................................54 Figure 9. Detection of candidate genes with PCR using cDNA derived from L. angustifolia BD tissue ..............................................................................................................................................55 Figure 10. Antibiotic selection of transformed E. coli JM109…………………………………..56 Figure 11. Agarose gel of restriction digestion of the pGA482:: Lavandula gene constructs extracted from E. coli JM109 cells ................................................................................................57 Figure 12. Selection of transformed A. tumefaciens GV3101 with the pGA482::lavender gene constructs .......................................................................................................................................58 Figure 13. PCR amplification of lavender candidate genes from the pGA482::lavender gene constructs tissue .............................................................................................................................59 Figure 14. Representative plates of kanamycin antibiotic selection of F1 transformant seedlings ........................................................................................................................................................61 Figure 15. PCR amplification of the NPT-II gene from leaf-derived gDNA of F1 transformants using gene-specific primers……………………….………………………………………..…… 62 Figure 16. Detection of lavender candidate genes in leaf-derived cDNA from wild-type and F1 A. thaliana and transformants via PCR with gene-specific primers .................................................63 Figure 17. Effects of LaSVP overexpression on A. thaliana growth and flowering .....................66 Figure 18. Differences in floral organ structures of A. thaliana LaSVP transformants versus wild-type A. thaliana .....................................................................................................................67 xi Figure 19. Floral organ structures of A. thaliana LaSVP transformants….…….….………… ....69 Figure 20. Differences in floral organ structures of A. thaliana LaCC transformants versus wild-type A. thaliana ..............................................................................................................................70 Figure 21. Phenotype and floral organ structures of A. thaliana LaPI transformant F1 plants .....71 Figure 22. Phenotypes of the LaAP1 F1 transformants ................................................................72 Figure 23. Phenotypic comparison of LaAP1 transformant and wild-type A. thaliana ...............73 Figure 24. Phenotypes of the LaCAL transformants ....................................................................74 Figure 25. Phenotype of the LaFT transformants .........................................................................75 Figure 26. Normalization (to β-actin) of LaSVP expression and RT-PCR detection of the β-actin and LaSVP genes in wild-type A. thaliana and LaSVP transformant leaf cDNA .........................76 Figure B.1 Multiple Sequence Alignment of protein sequences of L. angustifolia Crab’s Claw (LaCC) and Arabidopsis thaliana Crab’s Claw (AtCC) ..............................................................129 Figure B.2 Multiple Sequence Alignment of protein sequences of L. angustifolia Shoot Meristemless (LaSM) and A. thaliana Shoot Meristemless (AtSM) .............................................129 Figure B.3 Multiple Sequence Alignment of protein sequences of L. angustifolia Embryonic Flower 2 (LaEMF2) and A. thaliana Embryonic Flower 2 (AtEMF2)…………………………130 Figure B.4 Multiple Sequence Alignment of protein sequences of L. angustifolia Flowering Locus T (LaFT) and A. thaliana Flowering Locus T (AtFT) .......................................................130 Figure B.5 Multiple Sequence Alignment of protein sequences of L. x intermedia Apetala2 (LiAP2) and A. thaliana Apetala2 (AtAP2)………………………………………………….….131 Figure B.6 Multiple Sequence Alignment of protein sequences of Lavandula angustifolia Cauliflower (LaCAL) and Arabidopsis thaliana Cauliflower (AtCAL)….………………….… .132 Figure B.7 Multiple Sequence Alignment of protein sequences of L. angustifolia Pistillata (LaPI) and Arabidopsis thaliana Pistillata (AtPI), which is required for petal and stamen specification .................................................................................................................................132 Figure B.8 Multiple Sequence Alignment of protein sequences of L. angustifolia Apetala1 (LaAP1) and A. thaliana Apetala1 (AtAP1)………..…………………………………….…….. 133 Figure B.9 Multiple Sequence Alignment of protein sequences of L. angustifolia Apetala 3 (LaAP3) and A. thaliana Apetala3 (AtAP3)…………….……………………………………… 133 Figure B.10 Multiple Sequence Alignment of protein sequences of L. angustifolia Suppressor of CONSTANS1 (LaSOC1) and A. thaliana Suppressor of CONSTANS1 (AtSOC1)………….......134 xii Figure B.11 Multiple Sequence Alignment of protein sequences of L. angustifolia Sepallata 3 (LaSEP3) and A. thaliana Sepallata 3 (AtSEP3)………………….…………..….. ....................134 Figure B.12 Multiple Sequence Alignment of protein sequences of L. angustifolia Agamous (LaAG) and A. thaliana Agamous (AtAG)……………………………………………………... 135 Figure B.13 Multiple Sequence Alignment of L. angustifolia Flowering Locus C (LaFLC) and A. thaliana Flowering Locus C (AtFLC)…………………………………………………….… 135 xiii List of Abbreviations BD Floral bud cDNA Complementary DNA EO Essential oil FQM Floral Quartet Model F1 Filial 1 FL Flower gDNA Genomic DNA LaAG Lavandula angustifolia Agamous LaAP1 Lavandula angustifolia Apetala 1 LaAP3 Lavandula angustifolia Apetala 3 LaARF5 Lavandula angustifolia Auxin Response Factor 5 LaCC Lavandula angustifolia Crab’s Claw LaCAL Lavandula angustifolia Cauliflower LaEMF2 Lavandula angustifolia Embryonic Flower 2 LaFLC Lavandula angustifolia Flowering Locus C LaFT Lavandula angustifolia Flowering Locus T LaSEP3 Lavandula angustifolia Sepallata 3 LaSOC1 Lavandula angustifolia Suppressor of CONSTANS1 LaSVP Lavandula angustifolia Short Vegetative Phase LiAP2 Lavandula x intermedia var. Grosso Apetala 2 LD Long day OD Optical density ORF Open reading frame PCR Polymerase chain reaction mRNA Messenger RNA RT-PCR Real-time Polymerase Chain Reaction SAM Shoot apical meristem SD Short day TF Transcription factor TPS Terpene Synthase xiv SVP Short vegetative phase VM Vegetative meristem xv Acknowledgements I would like to thank all the people who have played a role during the completion of my graduate studies. I would like to thank Dr. Soheil Mahmoud for the opportunity to work in his lab for both my honours thesis and my graduate studies and for his guidance and support throughout my undergraduate and graduate studies. I would like to thank my supervisory committee for their support, feedback, and guidance throughout my graduate studies. I would also like to thank Dr. Ayelign Adal of Dr. Mahmoud’s lab for his assistance and mentorship during my graduate studies. I would like to thank Melissa Larrabee and Elaheh Najafianashrafi for their friendship and support. I would like to express my gratitude to the faculty members at UBC Okanagan that I had the privilege of being taught by during my undergraduate and graduate studies. I would like to thank my professors for nurturing my passion for learning and academia. I would also like to thank the professors I have had the opportunity to work with during my undergraduate and graduate studies. I would like to especially thank Dr. Brendan D’Souza, Dr. Richard Plunkett, Dr. Paul Shipley, Dr. Michael Russello, Dr. Janet Kluftinger, Dr. Soheil Mahmoud, Dr. Louise Nelson, Dr. Bruce Mathieson, Dr. Michael Deyholos, and Dr. Milka Beck. I would also like to extend thanks to Barb Lucente (Biology Unit Assistant) for her continued administrative support throughout my undergraduate and graduate studies at UBCO. I would like to thank UBC Okanagan for the generous financial assistance through scholarships, awards, and bursaries throughout my undergraduate and graduate studies at UBC Okanagan, including the University Graduate Fellowship scholarships and other awards I have been granted. I would like to thank my family and friends who have supported me throughout my graduate studies. I would like to extend my sincere appreciation to Bill and Careyn Head for their endless guidance, love and support throughout my graduate studies. I would like to especially thank Kevin Head, for his continuous encouragement, generosity, patience, guidance, love and support throughout my graduate studies and who has been instrumental in the completion of my thesis. I would like to sincerely thank him for always supporting me and believing in me. I would like to express my heartfelt appreciation to him for his unwavering love and dedication and for being my biggest support throughout my graduate studies. I sincerely thank him and I dedicate this thesis to him. xvi Dedication …To Kevin… 1 Chapter 1: Introduction 1.1 Lavenders 1.1.1 The Genus Lavandula Lavenders belong to the genus Lavandula, family Lamiaceae, which includes many flowering perennial species that produce aromatic essential oils (EO) (Lis-Balchin 2002; Vakili et al. 2014). Members of the genus exhibit distribution across Madeira, the Canary Islands, Cape Verde, North Africa, the Mediterranean, South West Asia, North East Africa, the Arabian Peninsula, and India, although native to the Mediterranean region (Lis-Balchin 2002; Ziaee et al. 2015). Lavenders, as with other members of the Lamiaceae family, exhibit two-lipped flowers comprised of five fused sepals, square-shaped stems, a bilobed stigma, opposite oriented leaves, four stamens, and produce nutlet fruits (Upson and Andrews 2004). The genus comprises 39 species and three subgenera; subgenus Lavandula, which is comprised of all woody shrubs, subgenus Fabricia comprised of woody shrubs, suffruticosa shrubs, or herbaceous perennials, and subgenus Sabaudia, which is comprised of woody shrubs (Lis-Balchin 2002; Upson and Andrews 2004). The three subgenera can be further classified based on leaf and floral morphology, growth pattern as well as chromosome number (Upson and Andrews 2004). For example, members of the genus Lavandula are polyploid and thus contain a variable number of chromosomes depending on the species, with L. aristibracteata containing 18 chromosomes, L. x intermedia containing 51 chromosomes, and L. latifolia containing 36, 54, or 75 chromosomes (Upson and Andrews 2004). Recent genomic analyses have provided evidence to suggest the lavender genome is 770 Mbp – 880 Mbp in size (Urwin et al. 2007; Malli et al. 2019). In addition, members of the genus Lavandula vary with regards to their leaf and floral morphology. For example, members of the subgenus Lavandula exhibit leaves that are linear to elliptic, which are occasionally shallowly lobed (also found in the subgenus Sabaudia), whereas all species of the Lavandula genus exhibit variation in leaf number, size, extent of dissection, with some being sessile, petiolate, or even lacking leaves entirely (Upson and Andrews 2004). Lavenders are presently grown globally for their valuable ornamental flowers and essential oils (EO) in Spain, the United States, France, the UK, Portugal, Bulgaria, Australia and China (Ziaee et al. 2015). Cultivation of lavender and use of the plant dates back to ancient eras when the Greeks and Romans cultivated Lavandula stoechas for medicinal purposes and Victorian times when the EO was used in perfumes and cosmetics (Lis-Balchin 2002; Upson and 2 Andrews 2004). Lavenders can be cultivated in a variety of climates and can grow in rocky, arid, nutritionally poor soils, although there is variation in cold and frost hardiness across species (Upson and Andrews 2004). Currently, the most commonly cultivated varieties of lavenders include Lavandula angustifolia Mill (L. ang), Lavandula latifolia Medik., Lavandula stoechas, and Lavandula x intermedia Emeric ex Loisel (L. x int), which is a sterile hybrid of L. angustifolia and L. latifolia (Koulivand et al. 2013; Wells et al. 2018). Lavenders are commonly cultivated for their flowers as well as for extraction of their valuable aromatic EO, which are produced in in structures called glandular trichomes found mostly in the flowers. 1.1.2 Lavender Floral Structure The typical angiosperm flower exhibits a distinct organization of floral organs from the outermost whorl to the innermost whorl: sepals, petals, stamens, and carpels (Litt and Kramer 2010). The estimated 350,000 species of angiosperms share some floral features such as stamens and carpels; however, variation exists in organ number or appearance as well as addition of organs and variation in the presence or absence of certain organs (Litt and Kramer 2010). Like many flowering plants, lavenders exhibit the typical floral organs, although they exhibit a unique floral organization and growth pattern. An individual lavender flower is comprised of five fused sepals (the calyx), five fused petals (corolla), four stamens, and a pistil which contains a four-locular ovary arising from two fused carpels (Upson and Andrews 2004; Wells et al. 2020). Although most commonly violet, Lavandula flowers can be white, blue-violet, pink, and in some cases, black or yellow in colour (Upson and Andrews 2004). Lavenders grow in dense shrub-like bushes with flowers growing atop long, woody stems. Lavender flowers are densely packed on stem tops in structures called “spikes” and stems of lavender can exhibit varying degrees of branching (Upson and Andrews 2004). Each floral spike is comprised of numerous tiny flowers densely packed around the stem, arising from structures called “cymes”, which can each give rise to a single flower or to many flowers (Upson and Andrews 2004). These cymes give rise to flowers on each side of the stem (Upson and Andrews 2004). Below the main floral spike, some species of lavender exhibit an additional layer of flowers, referred to as the remote verticillaster (Upson and Andrews 2004). Further, ach lavender flower is considered hermaphroditic as it contains both male and female reproductive organs; however, lavenders rely on pollination via bees and other insects to maintain genetic diversity in the species (Upson and Andrews 2004). 3 Although a small amount of EO is produced in the leaves, the majority of lavender EO is produced in the flowers, making lavender flowers a valuable component of the plant. 1.1.3 Essential Oil Composition and Applications The distinct aroma of lavenders is due to their production of fragrant EO in structures within the floral tissue. The fragrant EO of lavenders are produced mainly in the flowers and to a small extent in the leaves in structures called glandular trichomes (Sarker et al. 2013). Lavandula EO are complex, biologically active EO, comprised of varying amounts of over 50 monoterpenes and sesquiterpenes, such as linalyl acetate, cineole, 1,8-cineole, linalool, borneol, cis/trans-ocimene, lavandulyl acetate, α-pinene, β-pinene camphor, β-ocimene, and terpinen-4-ol, among others (Sarker et al. 2012; Koulivand et al. 2013; Lesage-Meessen et al. 2015; Wells et al. 2018; Adal et al. 2019). Although similar constituents are found in the commonly cultivated species’ EO, the relative amount of these constituents in the EO can vary depending on the species which can also affect the aroma, chemical activity, and use of the EO of certain Lavandula species (Sarker et al. 2012). Variance in the percent composition of the constituents in the oils across species can affect the aroma, quality, chemical activity, as well as the suitability of the EO for certain commercial uses (Sarker et al. 2012). For example, L. x intermedia EO contain higher amounts of camphor compared to L. angustifolia EO (Kara and Baydar 2013). In contrast, L. angustifolia EO contain higher amounts of linalool and linalyl acetate (Lesage-Meessen et al. 2015). The differences in EO profiles account for the differences in aroma of the oils and implicate different species’ oils in different products. Additionally, EO quality, composition and yield can be affected by growth conditions, climate, cultivation technique and extraction method and, as such, different wild populations of the same species have shown distinct oil profiles due to differences in these factors (Muñoz-Bertomeu et al. 2007). EO quality is affected by the relative ratios of certain mono- and sesquiterpenes in the oils, with the most desirable constituents being linalool and linalyl acetate and these compounds produce a pleasing aroma and are abundant in L. angustifolia EO (Sarker et al. 2012). In lower quality oil, such as that of L. x intermedia, there is relatively low (compared to oils of L. angustifolia) levels of linalool and linalyl acetate, and L. x intermedia EO typically contain high amounts of camphor which provides a less pleasant and stronger odor (Landmann et al. 2007; Sarker et al. 2012). This character of L. x intermedia EO makes it well-suited for use in cleaning products, whereas L. 4 angustifolia, being low in camphor, exhibits a more delicate aroma and is thus more commonly used in perfumes and personal care products (Lesage-Meessen et al. 2015). Further, the biologically active compounds in the EO have numerous beneficial properties including insecticidal, antifungal, antibacterial, sedative, analgesic, anti-inflammatory, and antioxidant properties. Lavandula essential oils are thus widely used in a variety of commercial products, from perfumes and soaps to food, flavorings, and medicines, making lavenders commercially and medicinally important plants (Wells et al. 2018). Due to the high commercial value and diversity of applications, the world production of L. angustifolia EO is 200 tons per annum, with top L. angustifolia EO and L. x intermedia EO producing countries being Bulgaria and the UK (Lesage-Meessen et al. 2015). The world per annum production of L. x intermedia EO is approximately 1000 tons, with about 90% produced in France (Lesage-Meessen et al. 2015). In addition, lavender varieties differ in their per hectare production of EO as L. x intermedia produces approximately 100 kg/ha, while L. angustifolia produces approximately 15 kg/ha (Lesage-Meessen et al. 2015). The biosynthetic pathways of lavender EO constituents have been well-characterized, although the development of the organs in which these EO are produced, the flowers, have yet to be examined (Woronuk et al. 2010; Demissie et al. 2011). There exists variation in the amount of EO produced as well as in the quality of EO produced by the various species of commonly cultivated lavenders. 1.2 Arabidopsis thaliana 1.2.1 About Arabidopsis thaliana Arabidopsis thaliana, a member of the mustard family (Brassicaceae/Cruciferae), is a small angiosperm and a model plant used in a wide range of studies to effectively model eukaryotic systems (Meinke et al. 1998). The A. thaliana genome is comprised of only 22,000 genes encompassed on 5 chromosomes (Meinke et al. 1998). In addition, many A. thaliana genes have been studied and sequences are readily available for a wide range of genes. Arabidopsis plants are small, leafy plants which originate from Asia, Europe, and North America and produce tiny white flowers, with five petals and that produce many small seeds (Meinke et al. 1998). As such, A. thaliana is a well- studied model plant that lends well to use in a variety of studies and transformation procedures are well-documented (Meinke et al. 1998; Arabidopsis Genome 5 Initiative 2000). The most commonly utilised ecotypes include Landsberg erecta and Columbia, which lend well to studies (Meinke et al. 1998). Arabidopsis plants can grow effectively in a variety of climates and under various growth conditions, making propagation and regeneration of these plants simple and efficient in both culture and soil. For example, A. thaliana can grow under a variety of light conditions such as under long day or short day conditions and in varied nutrient conditions such as in store-bought soil mixes or growth medium in greenhouses, growth chambers, or freely in the environment (ABRC Arabidopsis Biological Resource Center 2009). Although Arabidopsis can grow in a variety of settings, growth and progression to flowering in A. thaliana is highly dependent on light conditions due to the effects of light on the plants’ internal rhythms (Searle and Coupland 2004a). Arabidopsis is photoperiod sensitive and flowers earlier under long days (LD) conditions than under short day (SD) conditions (Eriksson et al. 2006). As such, the length of the vegetative growth stage and timing of transition to the flowering stage can be altered through manipulation of the growth conditions. For example, A. thaliana are light-sensitive plants and highly responsive to alterations in light intensity as well as photoperiod. As such, the length of the vegetative growth stage and the timing of the initiation of flowering in A. thaliana can be manipulated by changing photoperiod. For example, when A. thaliana are grown in light conditions above 12 hours, flowering is initiated and early flowering can be induced in young plants when exposed to light periods above 16-hours (Searle and Coupland 2004a). Conversely, flowering initiation can be delayed when plants are exposed to photoperiods with short periods of light (less than 12 hours) and long periods of darkness (above 12 hours) (Searle and Coupland 2004a). The high dependency of A. thaliana flowering on photoperiod lends well to experimentation as flowering can be induced early or delayed by manipulation of light conditions to long-day or short-day conditions, respectively. As such, A. thaliana served as an excellent plant for the present study. 1.2.2 Life Cycle and Development Arabidopsis plants have a short life cycle, which makes them an ideal model plant and facilitates ease of use for genetic studies across generations. The entirety of the life cycle of A. thaliana can be completed in approximately six weeks, from seed germination to the maturation and seed set of the plants (Meinke et al. 1998). The life cycle of A. thaliana begins with a small 6 brown-orange seed about 0.5 mm in diameter, which requires a period of cold exposure (vernalization) before germination may occur (Meinke et al. 1998). After vernalization, light exposure is required to induce germination, at which point, the seedling produces two small true cotyledons. As seedling maturation continues, the plant’s vegetative mass increases and a rosette leaf pattern is produced (Meinke et al. 1998). After sufficient vegetative mass has been achieved, buds are produced which give rise to small white flowers at approximately 4-5 weeks of age, dependent on light conditions (Meinke et al. 1998). At maturity, each Arabidopsis flower is comprised of four floral whorls (outermost to innermost) of sepals, petals, stamens, carpel and ovules (Honma and Goto 2001; Smaczniak et al. 2012; Theißen et al. 2016). Arabidopsis flowers thus exhibit both male and female floral organs and are capable of self-pollination (Meinke et al. 1998). Arabidopsis flowers are approximately 2 mm in length and give rise to long seed pod structures called siliques, which produce the seeds and can hold over 1000 seeds at maturity (Meinke et al. 1998). The siliques house the developing seeds and, at maturity, become dry and break open, releasing the seeds. The short life cycle and the production of numerous seeds makes A. thaliana an excellent model plant to examine processes across multiple generations. In addition, floral initiation and development have been extensively studied and Arabidopsis is the model plant for describing these processes in plants. 1.2.3 Floral Initiation and the Transition from Vegetative to Reproductive Growth At some point in the angiosperm life cycle, the plant must determine the appropriate time to flower and initiate the transition from vegetative growth to reproductive growth. The appropriate timing of this stage is crucial as it is key to the plant’s reproductive success and is dependent on the integration of a variety of intricate signalling pathways and environmental cues. Plants must, therefore, possess mechanisms to sense their external environment and internal status to ensure appropriate timing of reproduction initiation (Kim and Sung 2014). This process is well-described in A. thaliana and follows a predictable sequence of events to produce the intricately organized flower. The timing of initiation of flowering can be affected by external factors such as light quality and/or quantity, photoperiod, vernalization period, nutrient availability, temperature, and water availability (Levy and Dean 1998; Boss et al. 2004). In addition, flowering can be induced by environmental stress such as crowding, or drought conditions or by endogenous signals generated within the plant, such as hormone signalling, age, 7 or sufficient increase in biomass (Levy and Dean 1998; Boss et al. 2004). The various environmental and endogenous cues sensed by the plant impact the expression of a group of floral integrator genes which impact the transition from a vegetative meristem to a reproductive meristem (Boss et al. 2004). In addition, while some pathways serve to activate the expression of certain TFs or flowering promoting genes, others serve to regulate the expression of flowering repressor proteins (Boss et al. 2004). Many plants must reach a certain level of vegetative mass to allow the plant to establish itself prior to reproductive growth, and as such, juvenile plants may be unresponsive to signals encouraging reproductive growth before sufficient vegetative mass is achieved (Levy and Dean 1998; Boss et al. 2004). During the stage of embryogenesis in flowering plants, the SAM is formed and is the meristem which will give rise to the entirety of the above-ground growth of the plant, including leaves, internodes, flowers, stems, and all lateral branches (Barton 1998; Lenhard and Laux 1999; Bowman and Eshed 2000). Initially, vegetative growth serving to increase vegetative biomass occurs at the rapidly dividing pluripotent cells of the shoot apical meristem (SAM), producing leaves in an intricate rosette pattern (Coen and Meyerowitz 1991; Yanofsky 1995; Liljegren et al. 1999). These undifferentiated cells within the SAM eventually differentiate and give rise to leaves and stems (Barton 1998). The SAM is not only a source of pluripotent stem cells which differentiate to distinct tissues, but is also the location at which the distinctive growth pattern of the stems and leaves is established (Barton 1998). Further, the SAM can be divided into 3 developmental zones: the peripheral zone containing cells which are differentiated and from which organs develop, the central zone, which contains pluripotent cells which give rise to new growth, and the rib meristem which is the site of production of the cells within the width of the stem (Barton 1998; Lenhard and Laux 1999). Cells within the SAM are pluripotent and thus have the unique capability of the potential to give rise to different tissue types as they have yet to be genetically assigned to a specific tissue (Barton 1998). As vegetative growth proceeds vertically, in the apical meristem, there exist specific bundles of cells on the sides of the meristem which are conserved to give rise to flower meristems or additional vegetative meristems (Coen and Meyerowitz 1991). This vegetative growth phase will continue until a signal is received, at which point, the SAM will convert to an inflorescence meristem which will produce an inflorescence meristem and, subsequently, a flower meristem, while suppressing leaf formation (Coen and Meyerowitz 1991; Yanofsky 1995; Koornneef et al. 1998a). Flowering is 8 bi-directional during this transition, as after the SAM has become a flowering meristem, all new primordia formed will produce flowers, but any leaf primordia formed prior to the transition will be affected by these signals and will become bracts rather than true rosette leaves (Hempel and Feldman 1994). The inflorescence meristem produces small leaves in the same rosette structure as before; however, the flower meristem produces flowers, with each meristem giving rise to a single flower (Coen and Meyerowitz 1991). Floral bud formation occurs at the edges of the floral meristem in a continuous manner, forming new buds in an indeterminate spiral pattern (Smyth et al. 1990). The inflorescence meristem will produce numerous floral meristems, whereas each floral meristem will produce only one flower with a distinct organizational pattern of the floral organs (Yanofsky 1995). The molecular mechanisms which govern this transition have been well-described in A. thaliana and flowering time is governed by multiple factors. In Arabidopsis, a variety of factors, including environmental factors, such as photoperiod and ambient temperature, influence the stage at which the plant commences flowering (Hartmann et al. 2000; Andrés et al. 2015). Photoperiod is especially important as Arabidopsis plants commence flowering in response to long photoperiods as they are long-day (LD) plants and also require vernalization prior to germination (Levy and Dean 1998). In addition, overexpression of some floral meristem identity genes can lead to an early flowering phenotype in Arabidopsis (Levy and Dean 1998). Flowering initiation requires the suppression of floral repressors or genes that promote vegetative growth, expression of genes which promote transition to a flowering meristem, and later, genes which facilitate the development of floral organs (Levy and Dean 1998; Andrés et al. 2015). In addition, genes referred to as flowering time genes are either constitutively expressed or constitutively repressed or their expression can be influenced by a variety of environment conditions (Hartmann et al. 2000). The induction of expression of floral development genes is dependent on developmental cues and changing seasonal environmental cues such as temperature, moisture, stress, and photoperiod which aid plants in determining the appropriate time to begin flowering (Hayama and Coupland 2003; Searle and Coupland 2004a). Flower initiation and development in A. thaliana thus may occur in response to external cues such as temperature, photoperiod, day length, vernalization, and light quality (Simpson and Dean 2002). For example, the protein CONSTANS (CO) is sensitive to photoperiod and when activated in response to long day length, it activates expression of downstream genes, such FLOWERING 9 LOCUS T (FT) and SUPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), which promote flowering (Koornneef et al. 1998a; Samach et al. 2000a; Wells et al. 2020). In addition, genes such as FLOWERING LOCUS C (FLC), SHORT VEGETATIVE PHASE (SVP) and EMBRYONIC FLOWER 1 and 2 (EMF 1 and EMF2) are repressors of flowering (Koornneef et al. 1998a; Hartmann et al. 2000; Samach et al. 2000a). Thus, while some genes stimulate flowering, others act as repressors The initiation of flowering thus requires the activation and suppression of certain genes in pluripotent meristematic cells to convert the plant’s vegetative meristem into a flowering meristem (Bowman and Eshed 2000; Andrés et al. 2015). For example, the genes LEAFY and APETALA1 are expressed in the flower meristem and promote flower development via suppression of shoot identity genes and flowering repressors, as well as by facilitating expression of homeotic genes required for normal flower development (Maoileidigh et al. 2014) (Maoileidigh et al. 2014). Thus, flowering time genes can be classified as repressors or activators of flowering and can be autonomously expressed or their expression can be influenced by exogenous/endogenous signals (Levy and Dean 1998). In A. thaliana , there are several types of genes involved in flowering within the floral meristem: floral meristem identity genes, floral integrators, and floral pattern development genes (Levy and Dean 1998; Theißen et al. 2016). The floral meristem identity genes include LEAFY (LFY), APETALA1 (AP1), CAULIFLOWER (CAL), and UNUSUAL FLORAL ORGANS (UFO) and serve to facilitate the transition of the meristem from a vegetative meristem to a flowering meristem by inducing certain cells in the inflorescence meristem to give rise to a floral meristem (Levy and Dean 1998; Kim et al. 2009). Floral integrator genes play a crucial role in the integration of endogenous and exogenous cues and affect the expression of floral meristem identity genes and include FT, FD, and SUPPRESSOR OF CONSTANS1 (SOC1) (Henderson and Dean 2004; Kim et al. 2009). The regulation of the expression of the floral integrator genes is dependent on external cues such as temperature, as well as internal cues, such as hormone levels (Kim et al. 2009). These genes integrate signals received to have an effect on the floral meristem identity genes based on information gathered from external and internal cues. It is of note, however, that some genes may have more than one role in flowering induction (Kim et al. 2009). The floral organ development genes require activation and function in floral pattern development by dictating which floral organ will develop in a given area of the meristem after 10 floral initiation (Levy and Dean 1998). Floral initiation is also governed by genes which are affected by signalling pathways that are acted upon by numerous endogenous and exogenous signals to either promote or inhibit flowering (Levy and Dean 1998). These pathways converge on a common set of integrator genes which activate downstream genes in response to the overlapping effects of the signalling pathways (Levy and Dean 1998; Boss et al. 2004). Thus, the initiation of flowering is commenced in response to the integration by the plant of many environmental and endogenous cues, enabling the plant to sense its environment and internal state to ensure flowering is initiated at the appropriate time (Hartmann et al. 2000; Boss et al. 2004). 1.3 Flowering Inductive Pathways In A. thaliana, there exist four main pathways which govern the initiation of flowering (Levy and Dean 1998; Kim and Sung 2014). The photoperiodic pathway and the vernalization pathways initiate flowering in response to environmental factors, with the photoperiodic pathway inducing flowering in response to long photoperiod and the vernalization pathway activating flowering in response to a period of cold exposure (vernalization) (Levy and Dean 1998). These pathways are thus responsive to environmental conditions and activate or repress flowering based on external cues (Levy and Dean 1998). In addition, two other pathways have been described- an autonomous pathway which increasingly promotes flowering as the plant matures, and the gibberellin pathway (Levy and Dean 1998; Kim and Sung 2014). 1.3.1 Flowering in Response to Photoperiod Seasonal changes in photoperiod play an important role in dictating when many plants like A. thaliana initiate flowering. Different plants flower under various light conditions. A. thaliana, for example, is a long day (LD) plant, which flowers earlier under LD conditions than under short day (SD) conditions (Searle and Coupland 2004a). Flowering time in A. thaliana is thus closely tied to photoperiod and Arabidopsis initiates flowering through the activation of certain flower development-inducing genes (Koornneef et al. 1998a; Hayama and Coupland 2003; Searle and Coupland 2004a). This photoperiodic response is governed by an internal clock which regulates the expression of flowering-related genes in response to environmental cues, allowing plants to respond to changes in photoperiod which varies on a seasonal basis (Hayama 11 and Coupland 2003; Searle and Coupland 2004a). Regulation of the rhythms in plants by this circadian clock occurs when the 24-hour circadian rhythm is generated and responds to cues, such as photoperiodic changes, by activating or repressing certain biochemical or developmental pathways, such as flowering-inducing pathways (Dunlap 1999). The transcriptional regulation and post-transcriptional regulation of certain genes is also impacted by light exposure (Hayama and Coupland 2003). The pathway senses photoperiod and relays this information to the internal signalling pathways, thus promoting flowering via the photoperiod pathway. This response to changes in light strength and length is translated into developmental changes through “flowering time genes” and their interaction with an internal circadian clock/rhythm system (Hayama and Coupland 2003). These “flowering-time genes” are governed by cues such as developmental stages, or environmental cues such as temperature, photoperiod or other seasonal changes and aid in the development of the floral meristem from which flowers arise (Becker and Theißen 2003; Searle and Coupland 2004a). It is this internal sensory system which integrates light information input and causes an effect on biochemical and signalling pathways in the plant, either promoting or inhibiting flowering (Dunlap 1999). The central system for responding to changes in photoperiod is comprised of the circadian rhythm which controls developmental pathways and the pathways which transmit the changing light signals to the internal system (Searle and Coupland 2004a). This system provides a response to environmental cues and controls the circadian rhythm and the responsive signalling pathways, allowing plants to respond to changes in photoperiod (Hayama and Coupland 2003). The internal circadian clock is responsible for producing the daily 24-hour internal rhythms and processes within the plant (Dunlap 1999). In A. thaliana and other plants, the changes in photoperiod and light quality are sensed by photoreceptors which relay the information to an internal clock mechanism (Koornneef et al. 1998a; Hayama and Coupland 2003). These photoreceptors, called phytochromes and cryptochromes, detect and transmit the light signals to the internal circadian pathway to influence internal rhythms based on light quality and quantity changes (Hayama and Coupland 2003). This circadian pathway is an internal autonomous clock, acting to create an internal 24-hour rhythm (Hayama and Coupland 2003). The clock is comprised of temperature and light signalling pathways which are input pathways, an internal oscillator which integrates these signals, and output pathways whose activation leads to a range of biological effects (Hayama and Coupland 12 2003). Thus, flowering in response to photoperiodic changes is facilitated by the action of numerous floral development genes which are activated or repressed by the oscillator in response to changes in photoperiod (Searle and Coupland 2004a). In addition, mRNA levels of these floral initiation genes can vary, with accumulations and decreases in mRNA of certain genes occurring at different times of the day (Searle and Coupland 2004a). It has also been suggested that light activates some of these flowering time proteins post-translationally (Hayama and Coupland 2003). The central pathway which translates light signals into developmental changes is comprised of the genes LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1 (TOC1), CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and mutations in any of these genes can affect the response to changing light conditions (Hayama and Coupland 2003). The phytochromes and cryptochromes sense the environmental light conditions and transmit this information to TOC1 and LHY/CCA1 (Hayama and Coupland 2003). These genes comprise the central oscillator which, in turn, regulates output pathways, such as flowering in response to photoperiod, via the activation of CONSTANS (CO) expression, which facilitates the activation of Flowering Locus T (FT) expression and floral initiation (Hayama and Coupland 2003). The gene CO facilitates initiation of flowering under long photoperiods, is highly expressed diurnally and can also become activated by exposure to light post-translationally (Robson et al. 2001; Hayama and Coupland 2003). The activation of CO in response to increased day length and favourable light conditions leads to flowering initiation through the downstream activation of additional genes such as FLOWERING LOCUS T (FT), AtP5CS2, and SOC1 (SUPPRESSOR OF CONSTANS1) (Samach et al. 2000a; Hayama and Coupland 2003; Andrés et al. 2015). Further, the Atp5CS2 gene leads to stem elongation, while the FT and SOC1 genes promote flowering through the activation of the floral meristem identity genes (Samach et al. 2000a). The floral meristem identity genes in Arabidopsis include LEAFY (LFY), APETALA1 (AP1), CAULIFLOWER (CAL), APETALA2 (AP2), and FRUITFUL (FUL), whose expression is required for the transition from a vegetative meristem to a floral meristem (Henderson and Dean 2004; Andrés et al. 2015). In addition, the FT gene interacts with the transcription factor FD to activate the expression of the genes AP1 and LFY in the meristem (Wigge et al. 2005; Andrés et al. 2015). As these genes both promote flowering and floral pattern development, the FT gene in this way mediates a response to a long daylength that subsequently initiates flowering (Andrés et 13 al. 2015). In addition, the FT gene works to activate other flowering genes such as FRUITFUL (FUL), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), LEAFY (LFY), CAULIFLOWER (CAL), APETALA3 (AP3), and APETALA1 (AP1), which are MADS-Box family transcription factors (Hayama and Coupland 2003; Mizoguchi et al. 2005; Andrés et al. 2015). Thus, the integration of external signals and the subsequent expression of certain genes together promotes the transition from a vegetative meristem to a floral meristem, induction of flowering, and floral development in A. thaliana (Wigge et al. 2005). 1.3.2 The Vernalization Pathway Another important environmental aspect which plants sense to determine the appropriate time to flower is vernalization, which is the exposure of seeds to cold temperatures for some period of time (Kim et al. 2009). Plants, especially LD flowering plants, rely on cold exposure to sense the passing of winter and the commencement of spring/summer and impending LD photoperiod required for growth, although the temperature and duration of cold exposure may vary across species (Kim et al. 2009). Further, the requirement for cold exposure before floral initiation ensures that plants do not commence flowering in the fall prior to winter, which would thwart reproductive efforts due to inability to survive cold temperatures (Kim et al. 2009). In addition, many plants requiring a vernalization period also require the LD photoperiods of spring to flower (Kim et al. 2009). Vernalization also impacts repressive pathways in A. thaliana which prevent flowering under unfavorable conditions. For example, vernalization reduces the level of transcripts of the repressor Flowering Locus C (FLC) and this repression is maintained throughout cold exposure (Sheldon et al. 2000). FLC and other related genes act as repressors of flowering through the repression of expression of the floral integrator genes, SOC1, FT, and FD. The FLC protein is a MADS-Box protein which represses flowering and is upregulated by some genes including (FRIGIDA) FRI and downregulated by other genes and vernalization (Michaels and Amasino 1999; Henderson and Dean 2004). As vernalization serves to promote flowering, the action of FLC as a repressor opposes vernalization. Once a plant has reverted to a vernalized state, this state is maintained and carried through to new growth, possibly due to epigenetic modifications (Henderson and Dean 2004). Further, overexpression of FLC can lead to delays in flowering (Michaels and Amasino 1999). Expression of the FLC gene is repressed by the vernalization 14 pathway via chromatin modifications (Kim et al. 2009). During long periods of cold exposure, transcripts of the FLC protein become near undetectable and expression of the FLC protein remains in an inactive state and is suggested to be in such a state due to stable epigenetic modifications (Michaels and Amasino 1999; Henderson and Dean 2004; Simpson 2004). Vernalization is a mitotically stable process, meaning that after cold exposure commences, the vernalization state is maintained in all cells until flowering is initiated (Boss et al. 2004). 1.3.3 The Autonomous Pathway The autonomous pathway is comprised of several proteins which promote floral initiation through the downregulation of expression of the repressor protein, FLC (Henderson and Dean 2004; Simpson 2004). Although they function in different mechanisms, the genes of the autonomous pathway all function to ultimately repress FLC (Boss et al. 2004). In this way, the autonomous pathway functions additively with the vernalization pathway to promote flowering. The autonomous pathway promotes flowering and functions independently from other flower-inducing pathways, requiring no input signals (Henderson and Dean 2004; Simpson 2004). The autonomous pathway dictates the capacity which other flowering pathways can respond to promoting cues by controlling the level of FLC mRNA (Simpson 2004). In this way, the gene of the autonomous pathway dictate how responsive the plant is to the pathways that promote flowering (Simpson 2004). Thus, the levels of mRNA of FLC are reduced by vernalization, and repressed by autonomous pathway genes, mRNA processing, and through epigenetic modifications (Henderson and Dean 2004; Simpson 2004). 1.3.4 The Gibberellin Pathway Gibberellic acid (GA) is a plant hormone which is required for the growth, development, and induction of flowering in many plants, including A. thaliana, which depends on GA signalling to induce flowering under SD conditions (Wilson et al. 1992; Gocal et al. 2001b; Moon et al. 2003a; Jung et al. 2012). The GA pathway thus promotes flowering initiation in A. thaliana and other plants (Henderson and Dean 2004). GA signalling promotes flowering through the activation of SOC1 and through the expression of the meristem identity gene, LFY, to induce flowering under SD conditions (Moon et al. 2003a; Eriksson et al. 2006). The LFY gene is a floral meristem identity gene and GA signalling leads to increased transcription from 15 the LFY promoter, increasing concentration in the SAM and facilitating development of the flower (Blázquez et al. 1998). Further, some plants transition from vegetative growth to reproductive growth in the presence of exogenous GA and a form of GA4 is seen to increase in concentration during SD at the SAM just prior to flowering (Wilson et al. 1992; Eriksson et al. 2006). Thus, multiple pathways, including the vernalization, photoperiod, gibberellin (GA), ambient-temperature pathway, and the light quality pathway transmit information and regulate expression of the integrator genes SOC1, FT, and LFY (Levy and Dean 1998; Henderson and Dean 2004; Kim and Sung 2014). The integrator genes receive this information and initiate flowering through the activation of the floral meristem identity genes AP1, AP2, FUL, CAL, LFY which convert the vegetative meristem to a floral meristem (Henderson and Dean 2004). Transition to flowering is thus dependant on a variety of endogenous and exogenous factors (Koornneef et al. 1998a; Hartmann et al. 2000). 1.4 Transcription Factors Transcription factors (TF) play an important role regulating gene expression in a variety of processes, including development, through activation or repression of transcription of mRNA from a gene’s DNA (Latchman 1993). Transcription factors are protein elements of the cell which bind to distinct locations in the promoter region on the DNA to enhance or repress the transcription of a given gene via interactions with other transcription factors and RNA polymerase (Latchman 1993). As transcription is the first step of expression of a gene, transcription factors play a crucial role in the cell (Latchman 1993). Transcription factors can be agonistic, enhancing each others’ activity, or antagonistic, interfering with each others’ activity as a given transcription factor may repress a certain gene, while another activates the same gene. In this way, the cell can produce fewer TFs than if every gene required a unique set of transcription factors. Instead, a given TF may be a repressor of one gene, but an activator of another. In addition, some transcription factors require additional protein elements, such as coactivators to effectively activate gene expression. Additionally, the levels and types of transcription factors can vary during different developmental stages of an organism as well as across different tissue types (Eeckhoute et al. 2009). For example, certain genes may be activated only during certain stages of development, while others are activated at other stages. 16 Transcription factors bind to different sites in the regulatory region of a given gene on the DNA (Latchman 1993). These sites vary in nucleotide sequence as the DNA binding domain of a given transcription factor is distinct and thus capable of recognizing a specific recognition sequence. Further, many types of TFs have been identified and can be classified based on their DNA-binding domain structure and resulting dimerization pattern (Latchman 1993). 1.4.1 MADS-Box Transcription Factors One such group of transcription factors is the MADS-Box transcription factors, which is a group of 107 TFs classified based on the presence of a highly conserved 180 base-pair DNA-binding domain called the MADS-box or MADS-domain at the amino terminus of the protein (Alvarez-Buylla et al. 2000; Pařenicová et al. 2003; Kaufmann et al. 2005). The MADS-box transcription factors are encoded by MADS-box genes and are so named based on the presence of the conserved DNA-binding domain which is named after the proteins MINICHROMOSOME MAINTENANCE 1, AGAMOUS, DEFICIENS, and SERUM RESPONSE FACTOR (Gramzow et al. 2010). The MADS-box proteins are found in a wide range of eukaryotic organisms including mammals, yeasts, insects, amphibians, and plants and play fundamental roles in important biological processes including metabolism, muscle development, regulation of oncogene expression and floral development (Shore and Sharrocks 1995; Theißen et al. 1996; Gramzow et al. 2010). In plants, the MADS-Box family of transcription factors is a developmentally important group with most of the members being genes of the floral meristem or floral identity genes which aid in specification of floral organ identity (Theißen et al. 1996; Becker and Theißen 2003). MADS-Box TFs can be classified as either Type I or Type II (Theißen et al. 1996). The type II MADS-box TFs are well-established as being implicated in regulating expression of genes involved in floral development (Pařenicová et al. 2003). MADS-Box TFs bind specifically to the CArG-box consensus sequence on DNA (CC[A/T]6GG) of genes to be regulated (Kaufmann et al. 2005). Although there are several types of MADS-domain transcription factors, MIKC-type MADS-domain transcription factors are MADS-Box transcription factors exclusively found in plants and most are involved in organ identity or floral meristem identity (Theißen et al. 1996; Kaufmann et al. 2005). The MIKC-type MADS-domain transcription factors found in plants are so named due to the domains which they exhibit, containing a highly conserved MADS-Box domain at the amino terminus, an intervening (I) 17 sequence downstream of the MADS-Box, a weakly conserved K-domain, as well as a variable C-region or C-terminus domain (Ma et al. 1991; Theißen et al. 1996; Causier et al. 2010). The main DNA-binding domain is comprised of the I domain in combination with the MADS-box domain (Causier et al. 2010). Consistent with other TFs, MADS-Box TFs affect gene expression via binding to specific sites on the DNA upstream of a given gene (Theißen et al. 1996). Most genes controlled via MADS-box genes are implicated in developmental processes in eukaryotic organisms and, more specifically, in floral development in plants (Theißen et al. 1996). The main DNA-binding domain, the MADS-Box, is a coiled coil comprised of two antiparallel, amphipathic α-helices which the DNA wraps around, allowing the helices to bind to the DNA minor groove to facilitate the TF binding (Pellegrini et al. 1995). In addition to facilitating appropriate DNA-binding, the MADS-Box of these TFs facilitates their localization to the nucleus as well as TF dimerization (Gramzow et al. 2010). In the developing flower, the Type II MADS-box TFs function not only through direct interactions with each other, forming complexes which directly regulate gene expression, but also through interactions with proteins such as chromatin remodelling proteins, among others (Smaczniak et al. 2012). These TFs form hetero- or homodimeric complexes of four TFs with other Type II MADS-box TFs through their K and I domains, whereas the C-domain is implicated in the formation of complexes with additional proteins (Causier et al. 2010). These TFs bind to CArG elements on the DNA of a given gene and promote bending of the DNA to facilitate transcription of the gene (Causier et al. 2010). The DNA binding domain binds to a sequence known as the CArG box with MADS-Box transcription factors binding in complexes of four, commonly known as “quartets” (Kaufmann et al. 2005). Although the roles of these transcription factors in floral pattern development have been described, there still remains more to discover, including the specific mechanism of action of these transcription factors and how and which genes are activated by these TFs (Smaczniak et al. 2012); However, it is known that the MADS-Box genes work through interactions with each other and other transcriptional coregulators to control the expression of these downstream genes of floral development and their expression is affected by upstream pathways (Smaczniak et al. 2012). 18 1.5 Floral Pattern Development in Arabidopsis thaliana 1.5.1 The ABC(DE) Model Although flowers are highly variable in size, number, shape, aroma, and colour, most flowers exhibit a basic arrangement of common floral organs. The model describing floral pattern development is the ABC(DE) model of floral development (originally proposed as the ABC model, but later modified to include D and E-function genes) (Coen and Meyerowitz 1991; Weigel and Meyerowitz 1994; Causier et al. 2010; Theißen et al. 2016). The basic structure of a flower is comprised of four floral “whorls” with a distinct organ present in each whorl (Causier et al. 2010). Flowers most commonly exhibit sepals in the first outermost whorl, petals in the second whorl, stamens in the third whorl and carpels (containing ovules) in the innermost, fourth whorl (Litt and Kramer 2010; Theißen et al. 2016). The ABC(DE) model describes how differential expression of homeotic floral organ identity genes (Table 1) in areas of the meristem leads to the development of the appropriate floral organ in a given whorl (Weigel and Meyerowitz 1994; Causier et al. 2010). The model was originally postulated based on floral homeotic mutants, that is mutants which fostered a mutation which caused them to exhibit disorganization of the flower with fully developed floral organs in the incorrect whorl of the flower (Schwarz-Sommer et al. 1990; Bowman et al. 1991; Coen and Meyerowitz 1991; Weigel and Meyerowitz 1994; Theißen 2001). Further, the model describes the division of the angiosperm flower into four whorls and classifies the genes responsible for floral patterning in each respective whorl (Causier et al. 2010). The model further describes how in each whorl a unique combination of A, B, C, D, and E-type homeotic genes are expressed, giving rise to a unique floral structure (Coen and Meyerowitz 1991; Causier et al. 2010). 19 Table 1. A. thaliana genes comprising the ABC(DE) model in Arabidopsis. Genes are MIKC-type MADS-box transcription factors (with the exception of AP2) functioning in floral pattern development in Arabidopsis Bowman et al. 1991; Theißen 2001; Theißen et al. 2016). Gene class Gene(s) Function Whorl A-class APETALA1 APETALA2 Sepal specification (in combination with E-class) one B-class APETALA3 PISTILLATA Petal specification (in combination with A and E-class) two C-class AGAMOUS Stamen specification (in combination with E-class) three D-class SHATTERPROOF SEEDSTICK Carpel specification (in combination with D and E-class) four E-class SEPALLATA 1-3 Specification of all floral organs all The ABC(DE) model explains that the expression of A-type genes gives rise to sepals in the first whorl, combined expression of A and B class genes produce petals in the second whorl, expression of B and C class genes together lead to stamen development in the third whorl, C-type genes facilitate carpel development in the fourth whorl, C and D- class genes give rise to ovules within the carpels, and E class genes are required in all floral whorls (Weigel and Meyerowitz 1994; Theißen et al. 2016). The A-class genes include APETALA1 (AP1) and APETALA2 (AP2), B-class genes include APETALA3 (AP3) and PISTILLATA (PI), the C-class genes are represented by AGAMOUS (AG), the class D genes include SHATTERPROOF (SHP) and SEEDSTICK (STK), and the class E genes are represented by SEPALLATA 1-3 (SEP1-3) (Table 1 & Figure 1) (Theißen et al. 2016). It is of note that AP1 and AP2 also function as floral meristem identity genes, whose expression is required for the transition from a vegetative meristem to a flowering meristem (Henderson and Dean 2004). All of the genes described by the ABC(DE) model, with the exception of AP2, encode Type II MADS-Box transcription factors which promote the development of specific floral organs in the floral whorls through regulating the expression of other floral development genes (Causier et al. 2010; Theißen et al. 2016). Loss of expression of any of these genes leads to mutant phenotypes and deviations from the classic floral pattern (Pařenicová et al. 2003; Kram et al. 2009; Takeda et al. 2011; Wils and Kaufmann 2017). Although many plants exhibit the same floral organs, variation in structure and organization is observed across the over 350, 000 angiosperm species (Litt and Kramer 2010). 20 For example, some flowers may lack certain floral organs, may exhibit floral organ fusions, or may even exhibit novel floral organs (Litt and Kramer 2010). This high structural diversity observed across flowering plants resulting in deviations from the classical ABC(DE) model can be explained by alterations in the developmental programs such as gene duplications or deletions, or changes in gene expression patterns (Litt and Kramer 2010; Theißen et al. 2016). Thus, the appropriate expression and interaction of the A, B, C, D, and E-type genes is crucial to the proper organization and development of the angiosperm flower (Figure 1). This thesis aimed to identify genes that control flower initiation and development in lavender, hypothesizing that these genes would be homologous to those that control these processes in A. thaliana and other flowering plants. A comprehensive transcriptome database corresponding to developing flowers of L. angustifolia and L. x intermedia plants was compiled and used to identify floral development-related genes. As a proof of concept, eight genes were cloned and functionally characterized through expression in A. thaliana. The results of this thesis indicate that the Lavandula genes perform a similar function to the A. thaliana homologs. Further, the database assembled in this thesis provides a valuable resource for cloning of genes that control flower development in lavenders. These genes may help improve flower timing, density (and hence EO yield) and visual appeal through plant biotechnology. 21 Figure 1. The ABC(DE) and the Floral Quartet models of floral development described in A. thaliana (Theißen et al. 2016). According to the ABC(DE) model, there exist A, B, C, D, and E-class genes which encode MADS-box transcription factors which govern floral pattern development in the different whorls of the flower. The A-class genes are APETALA1 (AP1) and APETALA2 (AP2), the B-class genes are PISTILLATA (PI) and APETALA3 (AP3), the C-class gene is AGAMOUS, the D-class genes include SHATTERPROOF and SEEDSTICK, and the E-class genes include SEPALLATA1-3 (Theißen and Saedler 2001; Theißen et al. 2016). The floral quartet model describes the mechanism in which these proteins dictate floral organ development in the floral meristem as tetrameric complexes. In whorl 1, two AP1 proteins and SEP proteins give rise to sepals. In the second whorl, the combination of one AP1 protein, one PI protein, one AP3 protein, and one SEP protein facilitates petal development. In the third whorl, one PI protein, one SEP protein, one AP3 protein, and one AG protein promote stamen identity. In the fourth whorl, carpels develop by the action of two AG proteins and two SEP proteins. Within the carpels, ovules develop via the combination of one SHP protein, one AG protein, one SEP protein, and one STK protein. These transcription factors form tetramers which bind to the promoter region of other genes to facilitate floral organ development. AP1 AP1 SEP SEP AP1 SEP PI AP3 SEP AP3 PI AG SEP SEP AG AG SEP AG SHP AG Whorl 1 Whorl 2 Whorl 3 Whorl 4 Whorl 4 Sepals Petals Stamens Carpel Ovule 22 1.6 Thesis Hypotheses and Objectives The main objectives of this thesis were to gain an understanding of the molecular mechanisms which govern floral initiation and pattern development in lavenders. 1.6.1 Research Hypotheses The hypotheses of this study were that: 1. Flower initiation and development in Lavandula are controlled by homologs of homeotic genes that control these processes in other plants including Arabidopsis thaliana. 2. Genes controlling flower initiation and development are expressed in L. angustifolia, but not in L. x intermedia plants grown under controlled light conditions in greenhouse (this hypothesis is based on the observation that under controlled light conditions, L. angustifolia plants produce flowers, but L. x intermedia plants do not). We further hypothesized that genes involved in flower initiation and development can be identified through differential gene expression analysis of greenhouse-grown L. angustifolia and L. x intermedia plants. 3. Genes involved in floral initiation and development in lavenders function similarly as in other flowering plants, and overexpression of these genes will visibly affect floral initiation and pattern development in transformed Arabidopsis plants. 1.6.2. Thesis Objectives This thesis had the following specific objectives: 1. To assemble a comprehensive transcriptome representative of L. angustifolia and L. x intermedia tissues in CLC Genomics Workbench 11. This was achieved by growing L. angustifolia and L. x intermedia plants under controlled light conditions until L. angustifolia plants flowered. RNA was extracted at different stages of flower development from L. angustifolia plants, and from the shoot apical meristem of L. x intermedia plants to assemble a comprehensive transcriptome 2. To identify genes that potentially govern floral initiation and development in lavenders and to determine if they are homologous to those described in A. thaliana and other plants. The objective was met by i) using RNA-Seq to construct transcriptomes for L. 23 angustifolia and L. x intermedia plants ii) Searching the databases for homologues of Arabidopsis thaliana genes that are known to control flower initiation and development. 3. To examine differential expression of floral initiation and development genes across different lavender tissues through RNA-Seq and differential gene expression analysis in CLC Genomics Workbench 11. This objective was met through use of the RNA-Seq and Differential Expression functions of CLC Genomics Workbench 11 to examine gene expression patterns in the comprehensive lavender transcriptome assembled from the sequences from the various tissues. 4. To functionally characterize a subset of lavender floral initiation and development-related genes through cloning and constitutive ectopic expression in Arabidopsis thaliana. This objective was met through expression of Lavandula candidate genes in stably transformed A. thaliana plants under the control of the constitutive CaMV 35S promoter. 24 Chapter 2: Materials and Methods 2.1 Transcriptome Assembly 2.1.1. Plant Materials and Growth Conditions L. angustifolia (dwarf lavender) and L. x intermedia plants were propagated from existing, mature plants growing in a greenhouse under controlled conditions of photoperiod of 16 hours light, 8 hours darkness at 25 ℃. Cuttings were obtained from the mature plants at axillary and terminal vegetative growth approximately three to four nodes below the meristem, with a total of 8 cuttings taken from L. angustifolia and 8 cuttings obtained from L. x intermedia. All leaves were trimmed from the cuttings except for the two uppermost leaves. The base of each cutting was dipped in rooting powder, planted in moistened ProMix soil, and covered with a plastic cover to maintain humidity. After approximately 1 week, covers were gradually removed. The young L. angustifolia and L. x intermedia plants were then grown in the greenhouse at 25℃ and a photoperiod of 16 hours light and 8 hours darkness. Plants were fertilized weekly with Miracle Gro fertilizer (Scott’s Canada, Canada) prepared according to manufacturer’s instructions. Young plants were transferred to larger pots, with a total of four L. x intermedia and four L. angustifolia plants transferred to growth to maturity (Wells et al. 2020). Tissue was collected from plants once sufficient vegetative mass was established, after approximately 5 months. At the time of tissue collection, the L. angustifolia plants had produced flowers, whereas the L. x intermedia plants had yet to flower. 2.1.2 RNA Extraction and Sequencing To obtain RNA from a variety of tissues, floral (FL), floral bud (BD), and shoot apical meristem (SAM) tissues were obtained from L. angustifolia and shoot apical meristem (SAM) tissues were obtained from L. x intermedia (Figure 2). L. angustifolia FL, BD and SAM tissues were obtained from one plant, and L. x intermedia SAM tissues were obtained from four plants. L. x int SAM tissues were obtained from four plants. Samples were cut and immediately flash-frozen in liquid nitrogen and stored at 80℃ (Wells et al. 2020). 25 Figure 2. Lavandula tissue from which RNA was extracted to assemble the lavender transcriptome. (a) L. angustifolia floral bud tissue (BD), (b) L. angustifolia shoot apical meristem tissue (SAM), and (c) L. angustifolia floral tissue (FL). SAM tissue was also obtained from L. x intermedia (not pictured) for transcriptome assembly, although BD and FL tissues were not available as flowering had not commenced in L. x intermedia. Arrow in (b) is indicative of location of SAM. The total RNA was extracted in triplicates for each tissue using the QIAGEN RNEasy Plant Mini Prep Kit. Frozen tissues were ground to a fine powder using a pre-chilled mortar and pestle and extractions were performed according to the manufacturer’s protocol. An amount of 5 µl of β-mercaptoethanol was added to the RLT buffer. An incubation with DNAse I was performed to remove any contaminating DNA from the sample. The Nanodrop-1000 Spectrophotometer was used to assess the integrity and concentration of the RNA. The RNA was run on a 1% agarose gel to visualize the RNA and determine RNA purity. RNA was sequenced via Illumina Sequencing at the DNA Sequencing Core Facility of the University of British Columbia (Vancouver, B.C.). To prepare for sequencing, RNA samples were diluted to a concentration of 250 ng/µl and prepared in amounts of 20 µl. Prior to sequencing at the DNA Sequencing Core Facility, the RNA quality was ensured via the Agilent 2100 Bioanalyzer to ensure only samples of sufficient quality were used for sequencing. Subsequently, following the New England Biolabs standard protocol for NEBnext Ultra II stranded mRNA, the RNA samples were utilised for illumine library preparation. Using the Illumina NextSeq 500 via Paired End (42 bp x 42 bp reads), the RNA transcripts were sequenced. Finally, using Illumina’s bcl2fastq, the transcript sequencing data was then demultiplexed and 40-43 base pair paired end reads were generated (Wells et al. 2020). 2.1.3 De Novo Transcriptome Assembly and Annotation To assemble a transcriptome, the paired end reads generated by Illumina sequencing for L. ang FL, L. ang BD, L. angustifolia SAM, and L. x intermedia SAM were input as paired reads into CLC Genomics Workbench 11.0. Using the de novo sequence assembly tool, reads for each 26 tissue type were assembled into separate transcriptomes. Reads which had large stretches of unknown sequences, as indicated by “NNNNN”, were omitted from the assemblies. The four tissue-specific assemblies were combined via de novo assembly to construct a single transcriptome, comprehensive of the L. angustifolia and L. x intermedia tissues. The De Novo assembly parameters were set as word size of 22, bubble size of 50, and a minimum contig length of 200 bp (Wells et al. 2020). To remove redundant sequences from the assembled transcriptome, the CD-HIT-EST tool with 0.98 sequence identity cut-off was used (weizhonli-lab.org/jcvi/) (Li and Godzik 2006). The transcriptome was then annotated by BLASTx against an A. thaliana protein database. To verify completeness of the transcriptome, a BLASTx analysis was performed with the transcriptome and sequences of known Lavandula terpene synthase genes to identify these genes within the transcriptome. Transcripts within the Lavandula transcriptome showing high sequence similarity to A. thaliana floral development genes were searched for within the BLASTx results. Lavandula transcripts exhibiting high homology to A. thaliana proteins of known function, as indicated by low e-value (≤ 10-10) were selected for further analysis. A low e-value was indicative of of a highly homologous match, wehereas higher e-values would indicate lower sequence homology between the given sequence and its match or matches (CLC bio a). Additionally, The BLAST2GO software in CLC Genomics Workbench 11 was then utilised for Gene Ontology mapping of the BLASTx results to provide functional analysis of the Lavandula transcripts within the transcriptome (Conesa et al. 2005). GO analyses were performed to classify transcripts into three main categories: molecular function, cellular component, or biological process and into one of many subcategories based on function (Figure 5)(Wells et al. 2020). 2.1.4 Differential Expression Analysis Differential Expression Analysis for RNA-Seq data was performed using the CLC Genomics Workbench 11.0 software (Qiagen, USA) to compare expression patterns of floral development genes across different Lavandula tissues. Expression patterns were analyzed based on RPKM (reads per kilobase mapped) and fold change expression between the tissues. Sequences with high RPKM values and large negative or large positive fold change expression values were selected for further study. 27 2.1.5 Candidate Selection and ORF Identification Candidate Lavandula floral development genes were selected based on high sequence homology to an A. thaliana floral development-related protein and by low e-value (≤ 10-10). Candidates were further selected based on length and sequence completeness and differential expression pattern in the Lavandula tissues. Each candidate transcript was then used in a BLASTx search against a previously- assembled Lavandula database to identify the most full-length sequence of the respective transcript. The open reading frame (ORF) of the Lavandula transcript was then determined by entering the transcript sequence into the NCBI Orf Finder at https://www.ncbi.nlm.nih.gov/orffinder/. The sequence and length of the ORF were compared with that of the respective A. thaliana protein and the ORF/protein sequence was determined. The corresponding cDNA sequence was then determined. If the Lavandula transcript did not generate the full-length ORF sequence, the Lavandula transcript was used in a BLASTx search against a previously- assembled Lavandula genomic DNA database to identify the full-length transcript within the genome. Once identified, the corresponding genomic sequence was entered into GeneScan (http://hollywood.mit.edu/GENSCAN.html) to identify all potential ORFs contained within the genomic sequence. The sequences of the generated ORFs were compared to the sequence of the A. thaliana protein of interest to identify the correct ORF of the homologous Lavandula protein. Once the full-length protein sequence was identified for each Lavandula candidate, the sequences were compared to the respective A. thaliana sequences to assess homology through Multiple Sequence Comparison by Log Expectation (MUSCLE) alignment (https://www.ebi.ac.uk/Tools/msa/muscle/). MUSCLE alignment was then performed to generate a percent identity value for each Lavandula A. thaliana homolog pair 2.1.6 Primer Design Primers were designed based on the full-length sequence of the open reading frame (ORF) of each Lavandula candidate gene (Table 2). Primers were constructed to include the restriction enzyme cut sites for EcoRI and KpnI at the 5’ and 3’ ends, respectively to facilitate cloning into the pGA482 (An 1986) vector for ectopic expression of the gene under the control of the constitutive CaMV promoter. The ORF sequence of each gene was entered at https://www.addgene.org/mol-bio-reference/restriction-enzymes/ to identify any restriction 28 enzyme cut sites present within the candidate gene sequences. Genes which contained either the EcoRI or KpnI restriction enzyme recognition site within their ORF were identified and primers for these genes were designed for sticky end cloning (Yamabhai 2009). The length, %GC content, melting temperature, and annealing temperature of each primer were analyzed using OligoAnalyzer 3.1 and the Integrated DNA Technologies (IDT) website (https://www.idtdna.com/calc/analyzer). Table 2. Gene-specific primers designed for Lavandula candidates. Primers were used in the PCR amplification of the respective candidate Lavandula floral development genes. Restriction enzyme cut site sequences for EcoRI and KpnI in the forward and reverse primers, respectively, facilitated cloning into the pGA482 vector. Gene Name Forward Reverse Cloned CC 5’ATCTGGCGAATTCATGGATTTGGTTCAACAA-3’ 5’-AACTTAGGTACCTCAGGCCTTAGTAATGCT-3’ CAL 5’AATATAGAATTCATGGGGAGGGGGAAGGTG-3’ 5’TATATAGGTACCTCATGCGGCAAAGCATCC-3’ SVP 5’-TCCGCAGAATTCATGGCTAGAGAGAAGATT-3’ 5’-ATTGTAGGTACCTCAGCCAGTGTAAGGAAG-3’ PI 5’-GCGCTCGAATTCATGAAAAAGGCTAAGGAG-3’ 5’-TATATAGGTACCCTAGAACCGCTCCTGCAG-3’ AP1 5’-TACGCAGAATTCATGGGTAGAGGGAAAGTG-3’ 5’-ATATAGGTACCTCATGCGGCAAAGCATCC-3’ FT 5’-TTCGCAGAATTCATGCCAAGAGATCGAGAC-3’ 5’-TATATATATAGGTACCTCACCGTCTTCTCCCGCC-3’ Not Cloned SM F1: 5’-AATTCATGCGTGTGTACAACAGTAGCACA-3’ F2: 5’-CATGCGTGTGTACAACAGTAGCACAGA-3’ 5’-ATATAGGTACCTTAGAGCAGCGCGGGCGA-3’ SEP 5’-ATTAGAATTCATGGGAAGAGGGAGAGTT-3’ R1: 5’-GTACCTTAGTTATCATGTAGCCACCCT-3’ R2: 5’-CTTAGTTATCATGTAGCCACCCTGCTA -3’ AP2 5’-TATATAGAATTCATGGAATATTCCGGCAGC-3’ 5’-TATTAGGTACCTCACCTCCAGTAGTAATG-3’ AP3 5’-TACTAGGAATTCATGGCTCGTGGGAAGATC-3’ 5’-ACTAATGGTACCTCACTCAAGCAAGGCAAA-3’ SOC1 5’-ATCGTAGAATTCATGGTGAGGGGGAAGACT-3’ 5’-TATTTAGGTACCCTATTTTTGGCACGGCCG-3’ AG 5’-CCCGTAGAATTCATGGAACAGCAAAGTGAT-3’ 5’-GCTTCCGGTACCTCAGATTAACTGAAGAGA-3’ EMF 2 F1:5’-AATTCATGCCTGGCATTCCTCTCGTG -3’ F2: 5’-CATGCCTGGCATTCCTCTCGTG-3’ 5’-TATAAAGGTACCGCTCTGTGGGTCCG-3’ FLC F1: 5’-AATTCATGGTGAGGGGGAAGGTGCAAATGAA-3’ F2: 5’-CATGGTGAGGGGGAAGGTGCAAATGAAG-3’ 5’-TATTTAGGTACC CGTCACCGCCTCCG-3’ LHY 5’-ACCGTAGAATTC ATGGATCCTTATTCTTCT-3’ 5’-TATTTAGGTACC TCATGTAGAAGCCTCTC-3’ NPT-II 5’-AGAGGCTATTCGGCTATGACTG-3’ 5’ –TCAGAAGAACTCGTCAAGAAGGC-3’ 29 2.2 Cloning and Functional Characterization 2.2.1 PCR Amplification of Lavandula Floral Genes Total RNA was extracted from frozen L. angustifolia BD and FL tissue using the QIAGEN RNEasy Plant Mini Prep Kit and according to the manufacturer’s protocol. L. angustifolia BD RNA was reverse transcribed to cDNA using the iScript cDNA Synthesis Kit (Biorad) according to the manufacturer’s protocols and using the following cycle: 25 for 5 minutes, 42 °C for 1 hour, 85 °C for 5 minutes, and a 4 °C holding temperature. The β-Actin gene was PCR amplified from the cDNA to verify cDNA quality using the following cycle: 95°C for 5 minutes, then for 30 cycles, 95 °C for 5 minutes, 58°C for 30 seconds, 72°C for 45 seconds, followed by 72°C for 5 minutes and holding at 4°C. Floral bud cDNA was used as a template to amplify the L. angustifolia candidate genes, LaCC, LaCAL, LaSVP, LaPI, LaGB, LaFT, LaAP1, and LaAP2 using the designed gene-specific primers (Table 2). Primers were centrifuged and resuspended in Tris-Hcl pH 7 to a 10 µM working stock. For sticky end primers, two primer working stocks were prepared (ie. forward primer 1 and reverse primer, forward primer 2 and reverse primer). For PCR amplification, a single Touchdown program was used to amplify all lavender candidate genes from the L. angustifolia BD cDNA. The PCR cycle was set as follows: 95 °C for 5 minutes for a single cycle, for 8 cycles: 95 °C for 30 seconds, 65 °C (-1) for 30 seconds, and 72 °C for 1 minute, followed by 33 cycles of 95 °C for 30 seconds, 57 °C for 30 seconds, 72 °C for 1 minute 30 seconds, followed by 1 cycle of 72 °C for 7 minutes with a final holding temperature of 4 °C after amplification was complete. All PCR reactions were subsequently verified on a 1% agarose gel to visualize the bands of RNA (Wells et al. 2020). 2.2.2 Cloning of Lavandula angustifolia Genes into the pGA482 Vector Each amplified L. angustifolia candidate gene was cloned into a separate pGA482 vector to place the genes under control of the cauliflower mosaic virus (CaMV) 35S promoter of the vector and to facilitate transformation of separate A. thaliana plants. To clone the L. angustifolia genes into the multiple cloning site of the pGA482 vector, the vector and the cDNA of each gene were digested with the restriction enzymes, EcoRI and KpnI. The digested vector and digested cDNA were then ligated together using DNA ligase to create the constructs, pGA482::LaCC , pGA482::LaCAL, pGA482::LaSVP, pGA482::LaPI, pGA482::LaGB, pGA482::LaFT, and pGA482::LaAP1. The constructs were then transformed into Escherichia coli JM109 cells. 30 2.2.3 Bacterial Growth and Plasmid Extraction E. coli JM109 cells were grown in Liquid LB medium containing 30 mg/mL tetracycline. Bacteria was grown overnight at 37 °C in 50 mL LB medium with 30 mg/mL tetracycline at 200 rpm. Bacterial plasmids were extracted and purified from the JM109 E. coli cells using the Plasmid DNA Miniprep Kit (BIOBASIC INC) according to the manufacturer’s protocol. Purity and quality of the extracted plasmids were verified via the Nanodrop Spectrometer 1000 (Table A.2) and via visualization on a 1% agarose gel. Bacterial plasmids were extracted from the agarose gel using the E.Z.N.A.© Gel Extraction Kit (OMEGA). Plasmid DNA concentration and purity were verified using the Nanodrop Spectrometer 100 (Table A.3). 2.2.4 Restriction Digestion and Ligation The extracted E. coli JM109 bacterial plasmids and the amplified Lavandula floral genes were digested with the restriction enzymes KnpI and EcoRI. A separate digestion reaction was prepared for each candidate gene. In a 1.5 mL tube, 5.0 µl of 10x NEB Cutsmart buffer, 3.0 µl plasmid DNA, 3 µl candidate cDNA, 2 µl high fidelity KpnI, 2 µl high fidelity EcoRI and molecular grade water (to a total reaction volume of 50 µl) were combined. Reactions were incubated in a water bath at 37 °C for two hours and reactions were stored at -20 °C. An amount of 5 µl 10x loading dye was added to each 50 µl reaction and complete restriction digestion was verified through visualization of the reactions on a 1% agarose gel with a 1 kb ladder (FroggaBio). Digested plasmid DNA and L. angustifolia cDNA were extracted from the agarose gel using the E.Z.N.A.© Gel Extraction Kit (OMEGA). Each gene’s digested cDNA was ligated into a separate pGA482 plasmid. Each reaction was prepared in a PCR tube and contained a 1:5 ratio of vector to insert. Each reaction was comprised of 200 ng of candidate cDNA, 1 µl 10x T4 DNA Ligase buffer, 1.6 µl of pGA482 vector, 1 µl of T4 DNA Ligase and molecular grade water to bring the total reaction volume to 10 µl. A control reaction was prepared with 1 µl 10x T4 DNA Ligase buffer, 1.6 µl pGA482 vector, 1 µl T4 DNA Ligase and molecular grade water to a final reaction volume of 10 µl. Reactions were centrifuged briefly and mixed by pipette. Tubes were incubated at 16 °C overnight (approx. 15 hours) in a thermocycler. The tubes were then placed back in the 31 thermocycler with the following incubation program: 23 °C for 1 hour, followed by 65 °C for 20 minutes to complete ligation. 2.2.5 Heat-Shock Transformation of Competent Escherichia coli JM109 The ligation reactions were then used in heat-shock transformation of competent Escherichia coli (E. coli) JM109 cells via the method described by Froger and Hall (2008). Briefly, pipette tips and 2 mL microfuge tubes were placed in a -80 °C freezer to pre-chill the instruments. Competent cells were thawed on ice and 10 µl of the ligation mixture (10 µl mixture per gene) was chilled in the pre-chilled microfuge tube. For each gene, the chilled 10 µl ligation mixture was added to a separate tube of thawed competent cells. Without mixing, the mixture was placed on ice for 20 minutes. The tubes were then placed in a 42 °C water bath for 1 minute to heat-shock the competent cells. Tubes were then placed on ice for 3 minutes to recover, after which, 1 mL of room temperature S.O.C medium was added to each tube to resuspend. The tubes were then placed in the rotary shaker at 37 °C for 2 hours at 200 rpm. 2.2.6 Plating and Selection of Transformed Escherichia coli JM109 To select for transformed cells, Luria Bertani (LB) tetracycline antibiotic selection plates were prepared as follows: 25 g of LB medium were combined with 1 L deionized water and 11 g agar and autoclaved for 30 minutes. Once cool, 1 mL of 30 mg/mL tetracycline was added and plates were poured. For each gene, two plates were poured, for a total of 16 plates. Selection plates were warmed at 37 °C for 30 minutes prior to plating. For the first plate, 50 µl of the ligation/competent cells mixture was spread on the plates. For the second plate, the ligation/competent cells mixture tubes were centrifuged at 11000 rpm for 2 minutes. The supernatant was then removed with a pipette and the pellet was mixed and plated on the second plate to prepare a concentrated plate. Plates were incubated overnight at 37 °C. For each candidate gene, 5 mL LB medium and 5 µl 30 mg/mL tetracycline were combined and allocated into three microfuge tubes. Three separate colonies were selected from the diluted plate of each gene and added to the microfuge tube. Tubes were shaken overnight at 37 °C at 200 rpm. Plasmids were then extracted using the Plasmid DNA Miniprep Kit (BIOBASIC INC) according to manufacturer’s protocols. Concentration of plasmid DNA was assessed using the Nanodrop Spectrophotometer and two glycerol stocks were prepared for each 32 of the 7 genes using the two most highly concentrated plasmid samples. To confirm presence of the L. angustifolia gene, the plasmids were prepared for Illumina sequencing with their gene-specific primers. In addition, a restriction digestion was performed with all constructs using EcoRI and KpnI and verified on a 1% agarose gel. 2.2.7 Preparation of Competent Agrobacterium tumefaciens Competent A. tumefaciens cells were prepared to allow for bacterial uptake of the pGA482::lavender gene constructs. A. tumefaciens cells stored at -80 °C were combined with 7 mL LB medium and 25 mg/L rifampicin. Tubes were covered in aluminum foil (as A. tumefaciens and rifampicin are light-sensitive) and shaken at 28 °C overnight. The following day, 50 mL room temperature LB medium and 25 µl of 50 mg/mL rifampicin were combined in an autoclaved flask with 500 µl of the A. tumefaciens mixture and the flask was covered in aluminum foil and incubated overnight at 28 °C at 140 rpm. The following day, the optical density (OD) was measured using the spectrophotometer to determine the phase of bacterial growth. The flask was shaken at 200 rpm at 28 °C until the OD had reached 0.6. A 20 mM Calcium Chloride (CaCl2) solution was prepared with 800 µl of 1 M CaCl2, 40 mL deionized autoclaved water and was filter-sterilized into a sterile falcon tube and placed on ice with an additional sterile falcon tube. Thirty 1.5 mL microfuge tubes were placed in the -80 °C freezer. An amount of 45 mL of cell culture was added to the pre-chilled falcon tube and centrifuged at 4 °C for 10 minutes. The supernatant was discarded, and the pellet resuspended with 5.0 mL of ice-cold CaCl2 solution. The tube was centrifuged for 5 minutes at 4000 rpm at 4 °C, the supernatant discarded, and the pellet resuspended in 3 mL ice cold CaCl2. The solution was then divided amongst 30 1.5 mL microfuge tubes in amounts of 100 µl. Microfuge tubes were individually flash-frozen in liquid nitrogen for 30 seconds and stored at -80 °C. 2.2.8 Agrobacterium tumefaciens Transformation and Selection Competent A. tumefaciens GV3101 cells were transformed with the pGA482 vectors containing the lavender genes (pGA482::lavender gene) via the freeze-thaw method (Jyothishwaran et al. 2007). Briefly, 1 tube of competent A. tumefaciens cells was thawed on ice for each gene and 2 ng of vector (pGA482::lavender gene) was added to each respective tube and tubes were incubated on ice for 5 minutes. Tubes were placed in liquid nitrogen for 5 minutes 33 and subsequently placed in a 37 °C water bath for 5 minutes. An amount of 1 mL 37 °C LB medium was added to each tube and tubes were placed on the rotary shaker at 200 rpm at 28 °C for 2.5 hours. LB antibiotic selection plates were prepared with 50 mg/mL kanamycin and 25 mg/mL rifampicin, with two plates prepared per gene. On the first plate, 100 µl of the dilute cell solution were plated and on the second plate, 100 µl of pelleted cell solution were plated. Plates were then incubated for two days at 28 °C. For each candidate gene, 3 mL LB medium and 1.5 µl 25 mg/L rifampicin and 3 µl 50 mg/L kanamycin were combined in a test tube with 2 replicates per gene. Two separate colonies were selected from the diluted plate of each gene and added to each test tube. Tubes were shaken overnight at 28 °C at 200 rpm. Plasmids were then extracted using the Plasmid DNA Miniprep Kit (BIOBASIC INC) according to manufacturer’s protocols. Glycerol stocks were prepared for each of the genes. A separate PCR reaction was then performed for each plasmid using the respective lavender gene primers to amplify each gene from its vector, including a control PCR reaction with an empty vector. Each PCR reaction included 2.5 µl 10x buffer, 0.5 µl 10 mM DNTPs, 0.5 µl Taq Polymerase, 1-1.5 µl plasmid DNA (for plasmids with concentration above 100, 1.0 µl was used and for plasmid below 100 µl, 1 µl was used), 1.5 µl 10 mM lavender gene primer, 13.5 µl molecular grade water to a total volume of 20 µl. The previous Touchdown PCR program was used to amplify the floral genes form the vector. 2.2.9 Plant Transformation To facilitate constitutive ectopic expression of the lavender genes in A. thaliana via the CaMV 35S promoter of the pGA482 vector, A. tumefaciens GV3101 containing the pGA482::Lavandula gene constructs were used. In an autoclaved Erlenmeyer flask, 200 mL room-temperature LB medium, 100 µl of 25 mg/L rifampicin, 200 µl of 50 mg/L kanamycin and 1 mL of A. tumefaciens overnight stock were combined. Flasks were covered in aluminum foil and placed on the rotary shaker at 28 °C at 200 rpm overnight until OD was 1.5-2.0. Wild-type A. thaliana var. Landsberg erecta were grown as previously mentioned until sufficient vegetative mass had been achieved and buds were apparent, but flowering had not commenced (Figure 3a). A. tumefaciens containing the pGA482::lavender gene constructs were used to transform wild-type A. thaliana via the floral dip method (Zhang et al. 2006a). Briefly, 1500 mL of infiltration medium were prepared which contained 600 µl of 200 mM 34 acetosyringone, 225 µl 0.015% Tween 80 (v/v) and 5% sucrose. For each gene, 2 falcon tubes of 50 mL overnight A. tumefaciens stock were added and centrifuged at 22 °C at 6100 rpm for 10 minutes. Supernatant was poured into a separate 200 mL beaker for each gene. An amount of 50 mL of the LB A. tumefaciens overnight stock was used to resuspend the pellet in each tube and the tubes were centrifuged again at 22 °C at 6100 rpm for 10 minutes. The pellets were resuspended with 50 mL infiltration medium which was combined with an additional 150 mL in an autoclaved beaker. Fourteen wild-type A. thaliana plants (2 per gene) were covered in aluminum foil, leaving all vegetative growth exposed, but completely covering the soil (Figure 3b). The plants were then individually inverted into the agrobacterium infiltration solution and both were placed in a desiccator for 10 minutes (figure 3c). After 10 minutes, the plants were placed on their side on a foil-lined sheet and covered to prevent light exposure. This process was repeated for all plants. Plants were left on their side in the dark for 48 hours, after which, they were uncovered and placed upright. Transformation was repeated 1 week later following the same process. For each gene, two separate transformations were performed, transforming two plants per gene and performing the transformation twice, with 4 days between transformations. A total of four plants were transformed for each gene in two transformation events, with a total of 28 plants transformed and each plant transformed twice. Transformant plants were grown simultaneously with wild-type controls until seed set and seeds were collected as available (Figure 3d). Seeds were dried and stored at 4 °C. 35 Figure 3. Agrobacterium-mediated transformation of wild-type A. thaliana via the floral dip. a) Wild-type plants were transformed at the stage of budding but prior to flowering. b) Plants were wrapped in foil to facilitate transformation. c) Vacuum infiltration in a desiccator facilitated the uptake of the A. tumefaciens containing the vector into the plant tissues. d) Seeds were collected from transformed plants at maturity when siliques had dried. 2.2.10 Selection of Transgenic Plants To select for successful transformants, transformant and wild-type seeds were grown on kanamycin antibiotic selection medium. Seeds were plated for each of the 28 plants in conjunction with wild-type controls. Wild-type seeds were grown on medium containing no antibiotic to ensure medium viability. Seeds were sterilized prior to plating through a sterilization procedure adapted from Zhang et al. (2006b). Briefly, seeds were placed in in 96% ethanol for 1 minute followed by 20 minutes in 20% bleach aqueous solution containing 1% Triton X-100. In each solution, seeds were vortexed to ensure thorough sterilization of all seed surfaces. Seeds were subsequently washed once for 10 minutes in autoclaved deionized water (with vortexing) and twice for 5 minutes to ensure thorough washing and removal of detergent, bleach, and ethanol from the seeds. Deionized water was added to the tubes to facilitate plating. Selection medium was prepared with half-strength Murashige Skoog (MS) medium containing 0.5% gellan gum and 2% sucrose and at a pH of 5.7. Kanamycin was added to the medium at a concentration of either 50 mg/L or 75 mg/L Kanamycin. Wild-type seeds were plated on medium 36 containing 50 mg/L and 75 mg/L Kanamycin as well as medium lacking Kanamycin to serve as controls. All subsequent media were prepared at 75 mg/L kanamycin to facilitate most effective selection. Selection plates were sealed with parafilm and placed in a dark 5 ℃ fridge for four days for vernalization of the seeds to facilitate germination. Plates were then transferred to a growth chamber and grown at 25 ℃ with 16 hours light and 8 hours darkness. Seeds were grown until selection was visible on plates (approximately 3-4 weeks). Positive transformants were selected based on the ability to grow on plates containing 75 mg/L Kanamycin as well as the extent of growth and root biomass. Positive transformants were carefully removed from selection plates with forceps, removing any agar attached to the roots with water, and planted in moistened soil. Additionally, wild-type control seeds germinated on medium lacking Kanamycin were transferred for growth rate comparison and phenotypic analyses. The pots containing the seedlings were covered with plastic covers to maintain humidity for two-days and then gradually removed. Plants were then grown at 23 ℃ with 8 hours light, 16 hours darkness. Seedlings were grown to maturity and phenotype was monitored throughout growth. 2.2.11 Detection of Antibiotic Resistance and Floral Development Genes from cDNA To confirm the presence of the Kanamycin resistance gene (NPT-II) and LaSVP, genomic DNA was extracted from leaf tissue using the Genomic DNA Mini Kit – Plant (Geneaid) according to the manufacturer’s protocol. DNA concentration was then determined via the NanoDrop-1000 Spectrophotometer. The NPT-II gene was amplified from the genomic DNA using gene-specific primers. Primers used for PCR amplification were as follows: NPT-II forward 5’- AGAGGCTATTCGGCTATGACTG -3’ and reverse 5’-TCAGAAGAACTCGTCAAGAAGGC-3’. For NPT-II gene amplification, the following program was used: DNA was denatured at 95°C for 5 minutes. Then for 40 cycles, 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute. Elongation occurred at 72°C for 7 minutes and the DNA was then kept at 4°C. The presence of the LaSVP gene in the transgenic plants was also confirmed through PCR amplification of the LaSVP gene from the corresponding genomic DNA. The LaSVP primer sequences were as follows: forward 5’-TCCGCAGAATTCATGGCTAGAGAGAAGATT-3’ and reverse 5’- ATTGTAGGTACCTCAGCCAGTGTAAGGAAG - 3’(Wells et al. 2020). 37 To assess the relative expression of the transgene in transformed plants, RNA was extracted from flowers using the RNeasy Mini Plant Kit (Qiagen). The resulting RNA was quantified via the NanoDrop-1000 Spectrophotometer and synthesized into cDNA using the iScript cDNA Synthesis Kit (Biorad). The following thermocycler program was used for cDNA synthesis: 25°C for 5 minutes, 42°C for 60 minutes, 85°C for 5 minutes and then held at 4°C. To semi-quantitatively measure the expression level of LaSVP in the transformed plants, the cDNA was amplified using the previously indicated primer sequence for LaSVP and the Actin forward 5’-GCGACAATGGAACTGGAATGG-3’ and reverse 5’-GGTGCCTCGGTAAGTAGAATAGG-3’ primers. Actin was amplified using the same protocol as for NPT-II (indicated above), although the cycle number was reduced to 28. The Touchdown PCR program for LaSVP consisted of the following cycle: an initial denaturation at 95 °C for 5 minutes, for 8 cycles the cDNA was kept at 95°C for 30 seconds, 65 °C (-1) for 30 seconds, and 72 °C for 1 minute and 30 seconds. The next stage consisted of 20 cycles at 95 °C for 30 seconds, 57 °C for 30 seconds, and 72°C for 1 minute and 30 seconds and the sample was then held at 4 °C. Each cDNA sample was amplified in triplicates and the PCR products were subsequently run on a 1% agarose gel. Band intensities were measured by densitometry, using the ImageJ software (https://imagej.nih.gov/ij/download.html) (Wells et al. 2020). 2.2.12 Phenotypic Analyses Transformed plants were watered and fertilized regularly with Miracle Gro fertilizer (Scott’s Canada, Canada) prepared according to manufacturer’s instructions for phenotypic comparisons between A. thaliana plants transformed with LaSVP and its counterpart wild- type. Plants were grown at 8 hours light/ 16 hours darkness, followed by 16 hours light/ 8 hours darkness at 23℃ for vegetative growth and for flowering, respectively. Plants were assessed with regards to floral morphology, seed pod morphology, and flowering time. 38 Chapter 3: Results 3.1 Literature Review and Preliminary Homolog Identification Through review of current literature describing the molecular mechanisms of flowering in A. thaliana, a subset of genes was identified which were screened for in lavenders. Preliminary BLASTx analyses were performed using a previously- assembled lavender transcriptome available in the Mahmoud lab against an annotated A. thaliana protein database. It was determined from these analyses that homologs of the previously- described A. thaliana floral initiation and developmental genes do exist in lavenders and provided preliminary evidence for the basis of this thesis. 3.2 RNA-Seq and Transcriptome Assembly and Analysis To determine if homologs of the A. thaliana floral initiation and development genes exist in lavenders, a comprehensive lavender transcriptome was assembled. Pair-end sequence data were obtained for transcripts extracted from L. angustifolia flower (FL), floral bud (BD) and shoot apical meristem (SAM) tissues, and from L. x intermedia SAM tissue using Illumina Sequencing. A separate transcriptome for each tissue was assembled using CLC Genomics Workbench 11 (Qiagen) (Table 3). The individual L. angustifolia FL, BD, and SAM transcriptomes contained 47,630, 46,786, and 39,964 contigs, respectively, and that of the L. x intermedia SAM contained 26,821 contigs (Table 3). The N50 values of the transcriptomes ranged from 500 bp from the L. x intermedia SAM to 628 bp from the L. angustifolia FL transcriptome (Table 3). The N50 represents 50% of the largest contigs within the transcriptome (CLC bio b). Most of the contigs (~ 50%) were between 200-399 bp in length, with few contigs above 2500 bp (Table 3 & Figure 4). The minimum contig length in each transcriptome was 200 bp as this was set as the minimum during assembly, while the maximum contig length varied from 6,693 bp in the L. x intermedia SAM to 12,567 bp in the L. angustifolia FL (Table 3). Transcriptome results were published in Wells et al. 2020. 39 Table 3. Summary of the parameters of the L. angustifolia and L. x intermedia transcriptome assemblies. Note that minimum contig length was set to 200 bp during assembly of all transcriptomes. FL= flower, SAM = vegetative meristem, BD = floral bud tissue. (Reprinted by permission from Springer Nature: Planta 251(41): 1-11, Cloning and functional characterization of a floral repressor gene from Lavandula angustifolia by Rebecca S. Wells, Ayelign M. Adal, Lina Bauer, Elaheh Najafianashrafi and Soheil S. Mahmoud, © 2020). Parameters L. angustifolia FL L. angustifolia BD L. angustifolia SAM L. x intermedia SAM Total paired reads 36,630,196 35,690,350 28,380,848 17,543,766 Number of contigs 47, 630 46,786 39, 964 26,821 GC content (%) 45.1 45.3 45.3 45.8 Min length (bp) 200 200 200 200 Max length (bp) 12567 9937 10146 6693 Average contig length (bp) 497 493 476 410 N50 (bp) 628 618 617 500 Total assembled bases 23,650,730 23, 051, 014 19,008,841 11,005,731 Size ranges (bp)200-399400-599600-9991000-2499> 2500Number of transcripts0100002000030000400005000060000 Figure 4. Distribution of transcript lengths in the comprehensive Lavandula transcriptome. Minimum and maximum contig lengths were 200 bp and 12,567 bp, respectively. (Reprinted by permission from Springer Nature: Planta 251(41): 1-11, Cloning and functional characterization of a floral repressor gene from Lavandula angustifolia by Rebecca S. Wells, Ayelign M. Adal, Lina Bauer, Elaheh Najafianashrafi and Soheil S. Mahmoud, © 2020). The four tissue-specific assemblies were then combined into one comprehensive transcriptome and redundant contigs were removed using CD-HIT-EST, resulting in a transcriptome with a total of 105,294 contigs (Figure 4). Most of the transcripts generated had BLASTx hits against the Arabidopsis protein database, and the transcriptome included numerous full-length sequences, including those corresponding to ten previously reported Lavandula terpene synthases (Table 4) (Landmann et al. 2007; Demissie et al. 2011, 2012; Sarker et al. 40 2013; Jullien et al. 2014; Despinasse et al. 2017; Adal et al. 2019), which are enzymes vital to lavender EO synthesis (Aprotosoaie et al. 2017). Table 4. Full-length, previously- reported Lavandula terpene synthase genes identified within the Lavandula transcriptome. A total of 10 full-length, previously- reported Lavandula terpene synthase genes were identified within the comprehensive transcriptome. Gene ontology analyses were performed using the BLAST2GO function of CLC Genomics Workbench 11. Through this analysis, annotated transcripts were classified into biological process, molecular function, and cellular component categories of GO terms (Figure 5). Of the genes which mapped to cellular components, the top GO identities were cell (nearly 50,000 sequences), organelle (> 42,500 sequences), membrane (> 30,000 sequences), and other components (Figure 5). Of the genes that were mapped to biological processes, the top GO identities were cellular process (> 42,500 sequences), metabolic process (> 37,500 sequences), response to stimulus (> 32,500 sequences), biological regulation (> 27,500 sequences), and developmental process (> 25,000 sequences) (Figure 5). In the subset of genes which mapped to molecular function, the top GO identities were binding (> 37,500 sequences), catalytic activity (> 30,000 sequences), transporter activity (nearly 5,000 sequences), and transcription regulator activity (> 3,000 sequences) (Wells et al. 2020). Accession Number Gene/protein Source Species ADQ73631.1 beta-phellandrene synthase Lavandula angustifolia ABB73045.1 (R)-linalool synthase Lavandula angustifolia ABB73044.1 limonene synthase Lavandula angustifolia ABB73046.1 trans-alpha-bergamotene synthase Lavandula angustifolia AGL98420.1 germacrene-D synthase Lavandula angustifolia AGL98418.1 cadinol synthase Lavandula angustifolia AJW68082.1 bornyl diphosphate synthase Lavandula angustifolia AFL03421.1 1,8-cineole synthase Lavandula x intermedia AGU13712.1 9-epi-caryophyllene synthase Lavandula x intermedia AGL98419.1 B-caryophyllene synthase Lavandula angustifolia 41 Figure 5. Classification of genes in the comprehensive Lavandula transcriptome. Contigs were classified based on (a) biological process, (b) molecular function, and (c), cellular component. Gene ontology studies were performed in CLC Genomics Workbench 11 to examine gene function and characterize transcripts. (Reprinted by permission from Springer Nature: Planta 251(41): 1-11, Cloning and functional characterization of a floral repressor gene from Lavandula angustifolia by Rebecca S. Wells, Ayelign M. Adal, Lina Bauer, Elaheh Najafianashrafi and Soheil S. Mahmoud, © 2020). The transcriptome contained over 100 Lavandula contigs which exhibited high sequence homology to A. thaliana floral initiation and pattern development-related genes (Wils and Kaufmann 2017) (Table S1). These genes were previously- described proteins and included both non-repressor and repressor proteins. Particularly, the transcriptome contained representative sequences corresponding to all of the A, B, C, D, and E-type MADS-Box transcription factors described in A. thaliana (Causier et al. 2010; Theißen et al. 2016). Over 100 of these floral initiation and development-related genes previously- described in A. thaliana were identified within the assembled transcriptome (Table 5). In addition full-length sequences for some of the key A. thaliana floral development genes were identified in the Lavandula transcriptome 42 including CAULIFLOWER, SHORT VEGETATIVE PHASE, PISTILLATA, FLOWERING LOCUS T, SHOOT MERISTEMLESS, APETALA1, APETALA2, APETALA3, SEPALLATA 3, LATE ELONGATED HYPOCOTYL, SUPPRESSOR OF OVEREXPRESSION OF CO1, FLOWERING LOCUS C, AGAMOUS, and EMBRYONIC FLOWER 2 (Table 6) (Wells et al. 2020). Table 5. Arabidopsis thaliana floral development gene homologs identified within the comprehensive Lavandula transcriptome. A total of 104 Lavandula genes homologous to A. thaliana floral development genes were identified within the transcriptome, including full length and partial sequences for repressor and non-repressor proteins. LaBD = L. angustifolia bud, LaFL = L. angustifolia flower, LaSAM = L. angustifolia vegetative shoot apex, and LiSAM = L. x intermedia vegetative shoot apex. A.thaliana Gene e- value LaSAM vs LiSAM LaBD vs LiSAM LaFL vs LaBD LaSAM vs LaFL Function Reference AGL-like 1.34 x 10-58 6.704 627.626 2.149 -174.384 Carpel development (Theißen et al. 2016) AGL1 4.16 x 10-40 0.000 689.089 1.068 -963.661 Floral morphogenesis (Ma et al. 1991) AGL11 2.38 x 10-71 0.000 197.384 6.858 -1770.699 Fertilization and seed development (Mizzotti et al. 2012) AGL12 2.88 0.000 8.896 6.404 -73.136 Floral transition and root proliferation (Tapia-López et al. 2008) AGL17 3.14 x 10-34 1.445 1.923 -1.921 1.455 Flowering promotion in response to increased photoperiod (Han et al. 2008) AGL21 3.24 x 10-39 1.342 2.203 1.992 -3.241 Lateral root development, seed germination (Yu et al. 2014, 2017) AGL24 3.19 x 10-11 -2.245 -290.754 0.000 153.061 Promoter of flowering, component of vernalization response (Michaels et al. 2003; Gregis 2006) AGL6 2.16 x 10-74 0.000 779.235 -1.090 -935.832 Promoter of flowering, regulator of FLC and FT genes (Ma et al. 1991; Yoo et al. 2011) AGL8 1.19 x 10-49 163.277 537.480 -7.838 2.854 Expressed in inflorescence meristem and in developing carpels (Mandel and Yanofsky 1995) ANT 2.11 x 10-130 1.192 -1.262 -59.055 88.909 Meristem establishment, organ development and growth (Horstman et al. 2014) AP1 8.72 x 10-38 0.000 1492.208 -2.113 -924.701 Sepal development, petal development (Theißen et al. 2016) 43 when combined with B-class genes. Meristem identity gene AP2 9.73 x 10-117 -2.028 1.171 -1.142 -2.064 Sepal development. Petal development when combined with B-class genes (Theißen et al. 2016) AP3 7.96 x 10-86 1.089 51.591 1.034 -48.610 Stamen development when expressed with AG (Theißen et al. 2016) ARF2 0.000 -1.410 -1.440 -1.140 1.170 Cell division, seed and organ size (Schruff et al. 2006) ARF4 5.46 x 10-163 2.220 1.650 -5.410 7.360 Male, female gametophyte development (Liu et al. 2018c) ARF5 3.55 x 10-138 1.980 1.840 -3.380 3.680 Male, female gametophyte development (Liu et al. 2018c) ARF6 0.000 1.030 -1.180 -1.050 1.290 Regulation of floral organ development (Tabata et al. 2010) ARF8 0.000 4.550 9.850 -2.220 1.040 Regulation of floral organ development (Tabata et al. 2010) BEL1 1.93 x 10-106 -4.990 -3.470 1.570 -2.240 Ovule development (Brambilla et al. 2007) BHLH10 3.87 x 10-28 6.700 1037.380 -34.250 -3.930 Flowering promotion via photoperiodic pathway (Ito et al. 2012) BLH1 1.93 x 10-106 -4.990 -3.470 1.570 -2.240 Early seedling development (Kim et al. 2013) BLH2 2.83 x 10-124 1.660 1.760 -1.620 1.530 Leaf shape establishment (Kumar et al. 2007) BLH4 7.73 x 10-104 -1.720 -5.600 1.340 2.450 Leaf shape establishment (Kumar et al. 2007) BPC1 2.40 x 10-1 -18.810 -16.380 3.050 -3.510 Aid in tissue-specific gene expression during development (Simonini et al. 2012) BPC2 1.34 x 10-93 -1.080 -1.380 -1.390 1.780 Aid in tissue-specific gene expression during development (Simonini et al. 2012) BPC3 1.67 x 10-34 -1.330 -1.400 1.120 -1.050 Aid in tissue-specific gene expression during development (Simonini et al. 2012) BPC4 1.75 x 10-96 -8.210 -8.480 1.350 -1.300 Aid in tissue-specific gene expression during development (Simonini et al. 2012) 44 BPC6 5.62 x 10-144 -1.280 -1.240 1.270 -1.300 Aid in tissue-specific gene expression during development (Simonini et al. 2012) BPE 3.01 x 10-76 2.470 4.310 1.420 -2.470 Petal growth and development (Brioudes et al. 2009) BZIP 0.000 1.410 1.990 -2.000 1.430 Flower development, defense, light and stress signalling (Dröge-Laser et al. 2018) CAL 4.35 x 10-45 10.540 187.240 -1.080 -16.190 Interaction with AP1, affects floral meristem identity (Bowman et al. 1993) COL9 2.70 x 10-103 1.360 -1.220 1.000 1.680 Flowering regulation in response to photoperiod (Cheng and Wang 2005) CC 1.43 x 10-58 12.500 500.600 6.240 -212.330 Carpel development (Bowman and Smyth 1999) CUC2 3.35 x 10-52 12.503 123.628 -2.056 -4.090 Boundary formation, organ development (Nikovics et al. 2006) CUC3 4.6 3.420 2.820 -1.440 1.770 Boundary formation, organ development (Nikovics et al. 2006) ELF3 5.24 x 10-42 -1.200 -1.290 1.140 -1.050 Flowering initiation in response to photoperiod (Zagotta et al. 1996b) ELF7 9.18 x 10-131 1.170 -1.070 1.120 1.120 Regulation of FLC expression via chromatin modification (He et al. 2004) EMF2 0.000 -1.060 1.050 -1.380 1.260 Maintains vegetative development, flowering repressor (Moon et al. 2003b) FCA 1.09 x 10-81 -2.270 -1.930 2.840 -3.320 Flowering time control, repressor (Bäurle et al. 2007) FLC 2.08 x 10-26 -4.020 -1.550 -1.570 -1.650 Flowering time gene responsive to vernalization, repressor (Hepworth et al. 2002) (Hepworth and Dean 2015) FLD 1.46 x 10-81 -1.740 1.870 1.290 -1.190 Flowering control in autonomous pathway (He et al. 2003) FPA 4.52 x 10-68 -1.090 -1.210 1.010 1.110 Floral induction, regulation of floral initiation in autonomous pathway (Schomburg et al. 2001) FT 7.55 x 10-102 401.040 2791.130 -2.710 -2.140 Flowering promotion (Teper-Bamnolker and Samach 2005) 45 FY 0.000 -1.394 -1.430 -1.258 1.301 Flowering transition regulator (Simpson et al. 2003) GI 6.48 x 10-162 -4.500 -7.580 1.440 1.180 Regulation of flowering in response to photoperiod, inducer of FT and CO (Jung et al. 2007) GNL 0.000 18.300 -1.040 1.330 -1.280 Flowering time, senescence, repressed during flowering (Richter et al. 2013) HDG1 2.22 x 10-103 1.280 1.360 -1.850 1.750 Stamen development, petal and stamen identity (Kamata et al. 2013) HDG12 0.000 2.350 1.720 -3.850 5.320 Stamen development, petal and stamen identity (Kamata et al. 2013) HDG2 1.65 x 10-141 3.140 6.520 -3.650 1.770 Stamen development, petal and stamen identity (Kamata et al. 2013) HDG5 0.000 1.240 1.400 -3.610 3.220 Stamen development, petal and stamen identity (Kamata et al. 2013) HEC1 2.06 x 10-16 12.503 90.847 -18.977 3.032 Female organ development, gynoecium development (Gremski et al. 2007) HEC2 4.90 x 10-01 -1.100 -1.170 1.800 -1.690 Female organ development, gynoecium development (Gremski et al. 2007) HEN 0.000 -1.340 -2.230 1.140 1.480 Floral organ pattern development (Chen et al. 2002) JAG 3.56 x 10-35 18.300 82.650 -1.370 -2.770 Organ growth and patterning (Schiessl et al. 2014) KAN1 4.72 x 10-59 2.800 3.760 -1.210 -2.320 Regulates lateral organ polarity (Kerstetter et al. 2001) KAN2 2.36 x 10-63 -1.580 1.200 -1.130 -1.670 Regulates lateral organ polarity (Kerstetter et al. 2001; Wu et al. 2008) KAN4 7.07 x 10-49 -1.890 -4.610 -1.200 2.960 Integument development (McAbee et al. 2006) KNAT2 4.30 x 10-71 2.700 6.200 1.030 -2.350 Carpel development (Pautot et al. 2001) LDL1 0.000 -1.670 -1.120 1.090 -1.610 Repression of seed dormancy (Zhao et al. 2015) LFY 1.05 x 10-29 0.000 62.160 -117.830 0.000 Flowering promotion, interaction with (Parcy et al. 1999) 46 homeotic floral organ genes LD 2.75 x 10-68 1.120 1.020 1.040 1.060 Promotion of floral transition (Aukerman et al. 1999) MAF5 1.00 x 10-2 -3.112 -2.421 1.261 -1.610 Flowering time protein (Ratcliffe et al. 2003) MYB21 1.13 x 10-25 0.000 275.240 28.660 -10319.740 Stamen development (Qi et al. 2015) MYB24 1.58 x 10-50 1.270 5.270 12.490 -51.510 Stamen development (Qi et al. 2015) MYC2 7.59 x 10-115 2.090 -1.430 -1.370 2.090 Stamen development, seed production (Qi et al. 2015) MYC3 2.64 x 10-51 4.080 2.940 -1.080 1.520 Stamen development, seed production (Qi et al. 2015) MYC4 1.03 x 10-103 -1.240 -1.230 -1.070 1.060 Stamen development, seed production (Qi et al. 2015) NGA1 3.00 x 10-66 2.690 5.230 -1.750 -1.100 Style development (Trigueros et al. 2009) PAN 5.44 x 10-87 3.990 1.010 -9.990 39.890 Affects floral architecture and expression of homeotic genes, regulator of AG (Maier et al. 2009) PDF2 0.000 2.120 2.400 -2.260 2.010 Embryo development (Ogawa et al. 2014) PHYB 0.000 -1.190 -1.080 1.320 -1.450 Regulation of flowering time (Hajdu et al. 2015) PI 1.64 x 10-48 1.572 223.785 -1.586 -89.107 Stamen development when expressed with AGAMOUS (Theißen et al. 2016) PIE1 0.000 -1.390 -1.430 -1.260 1.300 FLC activation, floral repression (Noh and Amasino 2003) PTL 2.63 x 10-53 12.500 131.820 -1.400 -6.400 Petal development, boundary formation between sepal and petal (Lampugnani et al. 2013) RAV1 4.13 x 10-56 1.011 -1.800 -1.154 2.119 Seed germination, seedling development (Feng et al. 2014) REV 0.000 -1.110 -1.070 2.190 -2.260 Meristem development, cell division (Talbert et al. 1995) SEP1 1.77 x 10-70 -2.249 4.910 1.374 -15.164 Expressed in all whorls, needed for expression in all whorls (Theißen 2001) SEP2 3.65 x 10-67 47.297 3606.543 1.788 -114.145 Expressed in all whorls, needed for (Theißen 2001) 47 expression in all whorls SEP3 1.09 x 10-88 24.101 4323.614 1.014 -153.090 Expressed in all whorls, needed for expression in all whorls (Theißen 2001) SHN2 2.31 x 10-33 1.940 1.510 -3.270 4.220 Expression in anthers and siliques (Shi et al. 2011) SOC1 4.07 x 10-77 -1.120 -8.920 1.040 7.730 Floral transition gene, flowering promotion (Liu et al. 2008) SPL3 7.64 x 10-34 2.680 2.960 -2.130 1.950 Transition from vegetative phase to flowering (Wu and Poethig 2006) SPL8 2.59 x 10-78 3.230 6.260 -1.470 -1.300 Pollen sac development (Unte et al. 2003) SPT 2.89 x 10-42 35.699 156.408 -1.017 -3.609 Carpel and fruit development (Groszmann et al. 2008) STM 6.80 x 10-133 -1.720 15.930 1.380 -3.430 Leaf shape establishment (Aida et al. 1999) SUP 2.69 -1.810 -1.240 2.060 -2.990 Carpel/stamen boundary conservation (Sakai et al. 2000) SVP 1.17 x 10-80 -1.390 -5.630 -4.580 18.710 Floral transition repressor, floral meristem identity specification (Gregis et al. 2013) TCP10 1.70 x 10-44 -6.210 -5.050 1.560 -1.900 Lateral organ development (Koyama et al. 2007) TCP13 3.69 x 10-44 1.750 1.040 1.180 1.440 Lateral organ development (Koyama et al. 2007) TCP15 1.32 x 10-49 2.460 3.310 -2.930 2.190 Lateral organ development (Koyama et al. 2007) TCP3 2.05 x 10-12 -1.170 -1.210 -3.350 3.500 Lateral organ development (Koyama et al. 2007) TCP4 8.67 x 10-58 -1.490 -2.370 -3.250 5.220 Lateral organ development (Koyama et al. 2007) TCP5 2.89 x 10-42 1.630 1.310 -1.350 1.700 Lateral organ development (Koyama et al. 2007) TGA10 7.95 -1.420 -1.030 -1.120 -1.220 Anther development (Murmu et al. 2010) TOE3 3.67 x 10-38 1.260 1.697 2.775 -3.702 Repressor of AG and AP2, repression of TOE3 required for pattern development (Jung et al. 2014) UFO 6.21 x 10-61 58.900 258.850 -28.650 7.740 Regulation of floral meristem and floral organ identity (Samach et al. 1999) 48 ULT 0.26 1.340 3.760 1.080 -3.000 Shoot and floral meristem size and development (Fletcher 2001) VIN3 3.76 x 10-23 -3.130 -1.660 -1.250 -1.510 Epigenetic modifications during vernalization response (Kim and Sung 2013) VRN1 7.20 x 10-19 -1.090 -1.070 -1.250 1.230 Vernalization response, repressor of FLC (Levy et al. 2002) WOX1 1.31 x 10-24 2.060 6.480 -2.670 -1.170 Petal, carpel, leaf development (Costanzo et al. 2014) WOX13 3.63 x 10-86 -1.060 -1.240 1.880 -1.600 Replum development (Romera-Branchat et al. 2013) WOX3 6.27 x 10-7 -3.030 1.160 -10.180 2.900 Sepal and petal development (Costanzo et al. 2014) WOX8 2.40 x 10-1 -1.100 1.590 -2.380 1.360 Embryo patterning, maintenance of the SAM (Costanzo et al. 2014) WOX9 3.89 x 10-20 0.000 66.260 -125.620 0.000 Embryo development, maintenance of vegetative SAM (Costanzo et al. 2014) Note: AGL-Like = Agamous-like, AGL(1,6,8,11,12,17,21,24) = Agamous-Like MADS Box Protein, ANT = Aintegumenta, AP(1-3) = Apetala, ARF(2,4,5,6,8) =Auxin Response Factor, BEL1 = Bell 1, BHLH010 = Basic Helix Loop Helix Protein 10, BLH(1,2,4) = Bel1-Like Homeodomain, BPC(1-4, 6) = Basic Pentacysteine, BPE = Big Petal P, BZIP = BZIP2 , CAL = Cauliflower, COL9 = Constans- Like 9, CRC = Crab’s Claw, CUC(2,3)= Cup-Shaped Cotyledon 2, ELF(3,7) = Early Flowering, EMF2 = Embryonic Flower 2, FCA = Flowering Control Locus A , FLC = Flowering Locus C, FLD = Flowering Locus D, FPA = FPA, FT = Flowering Locus T, FY = FY, GI = Gigantea , GNL = GNC – Like, HDG(1,2,5,12) = Homeodomain Glabrous, HEC(1,2) = Hecate, HEN = HUA Enhancer 2, JAG = Jagged, KAN(1,2,4) = Kanada, KNAT2 = Knotted – Like 2, LD = Luminidependens, LDL1 = Lysine-Specific Histone Demethylase, LFY = Leafy, MAF5 = MADS Affecting Flowering 7, MYB(21,24) = MYB, MYC = MYC, NGA1 = Ngatha 1, PAN = Periantha, PDF2 = Protodermal Factor 2, PHYB = Phytochrome B, PI =Pistillata, PIE1 = Photoperiod- Independent Early Flowering, PTL = Petal Loss, RAV1 = Related to ABI3/VP1, REV = Revoluta, SEP(1-3) = Sepallata, SHN2 = Shine 2, SOC1 = Suppressor of CONSTANS1, SPL(3,8) = Squamosa Promoter Binding Protein-Like , SPT = Spatula, STM = Shoot Meristemless, SUP = Superman, SVP = Short Vegetative Phase, TCP = TCP, TGA10 = TGA10, TCP(3,4,5,10,13,15) = named from Teosinte Branched1 (TB1), Cycloidea (CYC) Proliferating Cell Nuclear Antigen Factor (PCF1) , TOE3 = Target of Early Activation Tagged 3, UFO = Unusual Flower Organs, ULT = Ultrapetala, VIN3 = Vrenalization Insensitive 3, VRN1 Vernalization 1 , WOX(1,3,8,9,13) = Wuschel-Related Homeobox Using the Differential Expression for RNA-Seq function in CLC Genomics Workbench 11, a Differential Expression analysis was performed to examine expression patterns of the over 100 identified genes. Genes of particular interest were those that showed differential expression patterns between L. ang SAM and L. ang BD as well as L. ang SAM and L. x int SAM. Differential expression patterns were recorded for all identified A. thaliana floral development gene homologs and differential expression analysis revealed that most of the floral initiation and identity genes were differentially expressed across tissues (Figure 6). For example, the LaSVP gene was more strongly expressed in both L. angustifolia and L. x intermedia SAM tissue compared to buds and later floral stages. Expression pattern was consistent with gene function. 49 Based on the differential expression analysis, literature review of floral development gene functions, and identification of sufficiently low e- values (≤10-10) 14 full-length lavender candidate genes homologous to A. thaliana floral initiation and pattern development genes were successfully identified within the transcriptome and selected for functional characterization (Table 6). An e-value of ≤10-10 was selected as a cut off for sufficient sequence similarity. The full-length ORF sequence of the protein was determined for each of the candidates and protein length was similar between each Lavandula candidate and its respective homolog (Table 6). Candidates were further examined and a subset of 7 candidates were selected for functional characterization. 50 Figure 6. Heatmap showing the relative expression levels of selected floral development genes. Relative expression of the genes (rows) was assessed in various tissues of L. angustifolia and L. x intermedia plants. Expression values used for clustering are fold changes. LaBD = L. angustifolia bud, LaFL = L. angustifolia flower, LaSAM = L. angustifolia vegetative shoot apex, and LiSAM = L. x intermedia vegetative shoot apex. An arrow and a box show the expression patterns of the L. angustifolia SVP gene. (Reprinted by permission from Springer Nature: Planta 251(41): 1-11, Cloning and functional characterization of a floral repressor gene from Lavandula angustifolia by Rebecca S. Wells, Ayelign M. Adal, Lina Bauer, Elaheh Najafianashrafi and Soheil S. Mahmoud, © 2020). 51 Table 6. Full-length Lavandula floral initiation and development genes identified within the comprehensive Lavandula transcriptome. Genes were identified based on full-length sequence, high sequence homology to the respective A. thaliana protein homolog and gene function. Gene Lavandula protein length (aa) A. thaliana protein length (aa) e-value A. thaliana Function Reference Shoot Meristemless (LaSTM) 391 382 6.80E-133 SAM cell proliferation (Byrne et al. 2002) Crab’s Claw (LaCC) 163 181 1.43E-58 Carpel development, nectary formation, termination of floral meristem (Gross et al. 2018) Cauliflower (LaCAL) 253 255 4.35E-45 Interaction with AP1 and LFY, floral meristem identity (Bowman et al. 1993) Short Vegetative Phase (LaSVP) 230 240 1.17E-80 Floral transition repressor, meristem identity gene (Gregis 2006) Pistillata (LaPI) 187 208 1.64E-48 Stamen development, petal development (Theißen et al. 2016) Flowering Locus T (LaFT) 174 175 7.55E-102 Floral induction in response to photoperiod (Andrés et al. 2015) Apetala1 (LaAP1) 253 256 8.72 x 10-38 Sepal development Petal development when combined with B-class genes. (Theißen et al. 2016) Apetala2 (LiAP2) 433 432 9.73 x 10-117 Sepal development. Petal development when combined with B-class genes (Theißen et al. 2016) Apetala3 (LaAP3) 232 234 7.96 x 10-86 Stamen development when expressed with AGAMOUS (Theißen et al. 2016) Suppressor of CONSTANS1 (LaSOC1) 221 214 4.07 x 10-77 Integration of gibberellin and vernalization pathway during floral transition (Moon et al. 2003a) Sepallata 3 (LaSEP3) 241 251 1.09 x 10-88 Expressed in all whorls; required for development of organs in all whorls (Theißen et al. 2016) Agamous (LaAG) 247 252 1.34 x 10-58 Carpel development (Theißen et al. 2016) Embryonic Flower 2 (LaEMF2) 627 631 0.00 Repressor of flowering (Henderson and Dean 2004; Theißen et al. 2016) Flowering Locus C (LaFLC) 190 196 2.08 x 10-26 Repressor of flowering (Henderson and Dean 2004) 52 3.3 Sequence Analysis and Phylogeny To examine the percent similarity of the protein sequences of the Lavandula candidates and their respective A. thaliana homologs, MUSCLE sequence alignment with the homologous amino acid sequences was performed for each gene (Table 7, Table 8, & Figures 8, B1-B13). MUSCLE protein sequence alignment of the 14 Lavandula candidate proteins revealed that all candidates had >50% sequence similarity to their A. thaliana homolog , with LaFT being the most similar to its homolog with 78.16 % similarity, and LaFLC being the least similar to its homolog at 41.53% sequence similarity (Table 7). Further, MUSCLE alignment revealed the presence of conserved domains characteristic of the class of TFs each lavender gene belonged to. LaCC, the Lavandula homolog of the zinc finger domain TF AtCC, contained the conserved zinc finger domain and the helix loop helix domain (Figure B.1). LaSM, the homolog of AtSM, a TALE/KNOX homeobox TF, exhibited the ELK domain and the homeobox domain present in AtSM (Figure B.2). LaEMF2, the homolog of AtEMF2, exhibited the VEFS-Box and the Zinc finger domains found in AtEMF2 (Figure B.3). LiAP2, the homolog of AtAP2, exhibited the AP2/ERF2 domains found in AtAP2. In addition, LaAG, LaSVP, LaCAL, LaFLC, LaPI, LaAP1, LiAP3, LaSEP3, LaSOC1, homolog of the A. thaliana MADS-Box TFs, AAtAG, AtSVP, AtCAL, AtFLC, AtPI, AtAP1, AtAP3, AtSEP3, AtSOC1, respectively, exhibited the MADS-Box and K-Box conserved domains (Figure B.6-B.13) (Wells et al. 2020). Table 7. The percent similarity of candidate Lavandula proteins and their A. thaliana homolog based on MUSCLE alignment of protein sequences. Matrix was created by Clustal 2.1. Lavandula Protein % Identity to A. thlaliana Homolog LaAG 68.16 LaAP1 70.56 LiAP2 50.12 LaAP3 56.90 LaCAL 64.26 LaCC 66.88 LaEMF2 62.97 LaFLC 41.53 LaFT 78.16 LaPI 55.75 LaSEP3 74.17 LaSM 72.35 LaSOC1 68.22 LaSVP 67.39 To examine the relationship between LaSVP and other plant SVP-like genes, a phylogenetic tree was constructed using the amino acid sequences of these proteins (Figure 7). Although the amino acid sequence of LaSVP exhibited higher sequence similarity to EgSVP-like, BrSVP, BjSVP, and PkSVP homologs compared to AtSVP, all of these genes contained the K-Box and MADS-Box conserved domains (Figure 7). The LaSVP protein was thus found to 53 cluster most closely with the SVP homologs PkSVP (Paulowinia kawakamii) (76.21% sequence identity), EgSVP-like (Eucalyptus grandis) (69.78% sequence identity), BrSVP (Brassica rapa) (68.26% sequence identity), AtSVP (Arabidopsis thaliana) (67.39% sequence identity), CmSVP (Chrysanthemum x mortifolium) (59.46% sequence identity) and BjSVP (Brassica juncea) (67.83 % sequence identity) (Figure 8 & Table 8). Thus, LaSVP clustered most closely with PkSVP, and most distantly with CmSVP (Wells et al. 2020). Figure 7. Phylogenetic analysis of the LaSVP protein and other plant SVP-like proteins. OsMADS22, Oryza sativa; HvMADS10, Hordeum vulgare; OsMADS47, Oryza sativa, HvMADS1, Hordeum vulgare; AcSVP2, Actinidia chinensis; AeSVP3, Actinidia eriantha; StSVP, Solanum tuberosum; EsSVP-like, Epimedium sagittatum; CmSVP, Chrysanthemum × morifolium; LaSVP, Lavandula angustifolia, PkSVP, Paulownia kawakamii Actinidia chinensis, BrSVP, Brassica rapa; AtSVP, Arabidopsis thaliana; BjSVP, Brassica juncea; EgSVP-like, Eucalyptus grandis; PmSVP-like, Prunus mume; PsSVP Pisum sativum; GmSVP, Glycine max; CtSVP-like, Citrus trifoliata; AcSVP1, Actinidia chinensis; AdSVP1, Actinidia deliciosa. The scale bar indicates a divergence of 0.2 amino acid substitutions per site. The arrow indicates L. angustifolia SVP (LaSVP) studied in this thesis. (Reprinted by permission from Springer Nature: Planta 251(41): 1-11, Cloning and functional characterization of a floral repressor gene from Lavandula angustifolia by Rebecca S. Wells, Ayelign M. Adal, Lina Bauer, Elaheh Najafianashrafi and Soheil S. Mahmoud, © 2020). 54 Table 8. Percent identity matrix of the % similarity of SVP protein sequences derived from different plants. Matrix was created by Clustal2.1, where LaSVP = Lavandula angustifolia, AtSVP = Arabidopsis thaliana SVP, CmSVP = Chrysanthemum x morifolium SVP, PkSVP = Paulownia kawakamii SVP, BrSVP = Brassica rapa SVP, BjSVP = Brassica juncea SVP, and EgSVP-like = Eucalyptus grandis SVP. AtSVP and LaSVP show 67.39% sequence similarity, with highest similarity between the LaSVP and EgSVP-like protein sequences at 69.78%.(Reprinted by permission from Springer Nature: Planta 251(41): 1-11, Cloning and functional characterization of a floral repressor gene from Lavandula angustifolia by Rebecca S. Wells, Ayelign M. Adal, Lina Bauer, Elaheh Najafianashrafi and Soheil S. Mahmoud, © 2020). No. Gene 1 2 3 4 5 6 7 1 LaSVP 100.00 2 AtSVP 67.39 100.00 3 CmSVP 59.46 62.61 100.00 4 PkSVP 76.21 69.60 65.91 100.00 5 BrSVP 68.26 91.67 64.86 69.60 100.00 6 BjSVP 67.83 90.83 63.51 69.16 97.51 100.00 7 EgSVP-like 69.78 70.35 65.44 74.44 72.57 73.01 100.00 Figure 8. Multiple Sequence Alignment of protein sequences of LaSVP and other plant SVP homologs. LaSVP, AtSVP, CmSVP, PkSVP, BrSVP, BjSVP, and EgSVP-like protein sequences, where LaSVP =Lavandula angustifolia SVP, AtSVP =Arabidopsis thaliana SVP, CmSVP = Chrysanthemum x morifolium SVP, PKSVP = Paulownia kawakamii SVP, BrSVP = Brassica rapa SVP, BjSVP = Brassica juncea SVP, and EgSVP-like = Eucalyptus grandis SVP-like. The bolded portion of protein sequence indicates the sequence of two highly conserved domains of the MADS-Box transcription factors, the MADS-Box and the K-Box domains. 55 3.4 Cloning and Transformation 3.4.1. Cloning of Candidates in the pGA482 Vector Based on differential expression pattern and high sequence homology to an A. thaliana floral development protein as indicated by an e-value ≤ 10-10, a subset of eight full-length Lavandula genes was selected for cloning and functional characterization in wild-type A. thaliana. Using gene-specific primers, LaCC, LaCAL, LaSVP, LaPI, LaFT, LaAP1, and LiAP2 were successfully PCR amplified from L. angustifolia BD cDNA using gene-specific primers. The PCR reactions were visualized by 1 % agarose gel electrophoresis and bands were observed at the expected band size based on the amino acid length of each protein size at 492 bp, 762 bp, 693 bp, 564 bp, 525 bp, 762 bp, and 1302 bp for LaCC, LaCAL, LaSVP, LaPI, LaFT, LaAP1, and LiAP2, respectively (Figure 9). Figure 9. Detection of candidate genes with PCR using cDNA derived from L. angustifolia BD tissue. All 8 candidate genes, LaCC, LaCAL, LaSVP, LaPI, LaFT, LaAP1, and LiAP2, were amplified via PCR from L. angustifolia BD cDNA and were detected at the appropriate band when compared to a 1 kb ladder. All 7 of the amplified candidate genes were successfully extracted from the agarose gel, although LiAP2 extraction was not of sufficient purity and thus was excluded from the remainder of the study. The LaCC, LaCAL, LaSVP, LaPI, LaFT, and LaAP1 DNA and the pGA482 plasmid were digested with the restriction enzymes EcoRI and KpnI. All six candidate genes were successfully ligated into separate pGA482 vectors and the pGA::LaCC , pGA::LaCAL , pGA::LaSVP , pGA::LaPI , pGA::LaFT, and pGA::LaAP1 constructs were successfully prepared. The constructs were successfully transformed into competent E. coli JM109 cells and 56 colonies grew well for each candidate. Positive colonies were successfully selected for via antibiotic selection on ½ MS medium containing tetracycline antibiotic (Figure 10). For each gene construct, two plates were prepared; a 50 µl plate and a concentrated plate (Figure 10). For each construct, both the 50 µl plate and the concentrated plate exhibited colony growth, although colony growth was significantly higher on the concentrated plates (Figure 10). Control plates containing an empty pGA482 vector exhibited no growth on the 50 µl plates, but exhibited sparse growth on the concentrated plates. Figure 10. Antibiotic selection of transformed E. coli JM109. Selection was performed on Luria Bertani 30 mg/mL tetracycline selection plates. For each gene, two plates were prepared, including a 50 µl plate and concentrated plate. Two control plates were prepared which included untransformed E.coli JM109 containing the empty vector housing the tetracycline antibiotic resistance gene. All plates exhibited growth and 3 colonies were selected from each 50 µl plate. Plasmids were successfully extracted from the E. coli colonies with good purity and Illumina sequencing with gene-specific primers confirmed the presence of the correct Lavandula 57 gene insert in each respective pGA482 vector. In addition, restriction digestion of the constructs further confirmed the presence of each of the lavender gene inserts in the pGA482 vectors extracted from the E.coli colonies as bands were detected at the appropriate size for each of the eight genes on an agarose gel (Figure 11). For each gene, two bands were observed on the agarose gel, indicative of a lavender gene insert of variable size (gene-specific) and the digested vector at >10,000 bp (Figure 11). The construct for each gene was successfully digested via restriction digest with KpnI and EcoRI and the lavender genes LaCC, LaCAL, LaSVP, LaPI, LaFT, LaAP1, and LiAP2 were detected via 1% agarose gel electrophoresis at 492 bp, 762 bp, 693 bp, 564 bp, 525 bp, and 762 bp, respectively. Figure 11. Agarose gel of restriction digestion of the pGA482:: Lavandula gene constructs extracted from E. coli JM109 cells. Lanes 1-3 indicate LaCC at 492 bp, lanes 4- 5 indicate LaCAL at 762 bp, lanes 6-8 indicate LaSVP at 693 bp, and lanes 9-11 show LaPI at 564 bp. All lanes show a dark band at the top of the gel above 10,000 bp, which is indicative of the digested pGA482 vector. After validation of the presence of the respective Lavandula genes in the pGA482 vector, Agrobacterium tumefaciens GV3101 cells were transformed with the constructs via the freeze-thaw method (Jyothishwaran et al. 2007). All six constructs were successfully transformed into A. tumefaciens and positive colonies were selected for via antibiotic selection on kanamycin and rifampicin selection medium (Figure 12). For each gene construct, two plates were prepared, including a 50 µl plate and a concentrated plate (Figure 12). The 50 µl plate and the concentrated plate exhibited colony growth in all cases, although colony growth was significantly higher on the concentrated plates (Figure 12). Control plates containing an empty pGA482 vector exhibited no growth on the 50 µl plates, but exhibited sparse growth on the concentrated plates. 58 Figure 12. Representative plates of selection of transformed A. tumefaciens GV3101 with the pGA482::lavender gene constructs. Selection occurred on Luria Bertani selection plates containing 25 mg/mL rifampicin and 50 mg/mL kanamycin. For each gene, two plates were prepared, including a 50 µl plate and concentrated plate. Two control plates were prepared which included untransformed A. tumefaciens GV3101 containing the empty vector housing the antibiotic resistance genes, but without a gene insert. All plates exhibited growth and 3 colonies were isolated from each 50 µl plate for each gene. Constructs from the transformed A. tumefaciens colonies were successfully extracted and Lavandula candidate genes were PCR amplified using the extracted constructs as a template and gene-specific primers. All candidates were successfully amplified from their respective construct (Figure 13). It is of note that during the extraction of the LiAP2 plasmid from A. tumefaciens, there was insufficient sample and thus this gene was not used in the PCR reaction, nor was it further examined in the remainder of the study due to time constraints. The PCR reaction was performed to verify the successful transformation of A. tumefaciens via the freeze-thaw method. The genes LaCC, LaCAL, LaSVP, LaPI, LaFT, and LaAP1 were successfully PCR amplified from the respective extracted A. tumefaciens plasmids and visualized on a 1% agarose gel. Bands indicative of LaCC, LaCAL, LaSVP, LaPI, LaFT, and LaAP1 were observed at 492 bp, 762 bp, 693 bp, 564 bp, 525 bp, and 762 bp, respectively (Figure 13). A PCR reaction was performed with an E. coli plasmid for each gene to serve as a control as candidate gene presence had been confirmed previously (Figure 13). All E. coli plasmids again showed successful amplification of candidate genes. 59 Figure 13. PCR amplification of lavender candidate genes from the pGA482::lavender gene constructs. Constructs were extracted from transformed A. tumefaciens and were PCR amplified using gene-specific primers. a) Lanes 1-3 indicate pGA482::LaCC, lanes 5-7 show pGA482::LaCAL, lanes 10-12 indicate pGA::LaSVP, lanes 14-16 indicate pGA482::LaPI, lanes 4, 8, 13, and 17 show the respective digested E.coli JM109 plasmids for verification and lanes 9 and 18 show the 1 kB ladder. b) Lanes 1-3 show amplification from pGA482::LaAP1, lanes 6-8 show pGA482::LaFT, Lanes 4 and 9 show the respective restriction digested E. coli JM109 plasmids and lanes 5 and 10 indicate the 1 kB ladder. Lanes showing amplification from the digested E. coli JM109 plasmids show a dark band at the top of the gel above 10,000 bp, which is indicative of the pGA482 vector. 3.4.2 Transformation and Selection of Transgenic Arabidopsis thaliana To functionally characterize the lavender candidate genes, each gene was expressed in wild-type A. thaliana var. Landsberg erecta via the constitutive CaMV 35S promoter of the pGA482 vector. Transformation of A. thaliana was successfully performed via the floral dip method (Zhang et al. 2006a). For each pGA482::lavender gene construct, a total of four plants were transformed, with a total of 24 plants transformed. Plants were all successfully grown to maturity and seeds were collected from all mature plants at the point at which the siliques had dried and released seeds (Figure 14d). All plants grew well after transformation and produced normal siliques and viable seeds that germinated well. Viable seeds were successfully collected from each transformed plant. Plant transformation was first assessed through germination of F1 seeds of transformants on selection medium containing kanamycin alongside wild-type controls. Seeds were grown for approximately 3-4 weeks until selection was apparent (Figure 14). Initially, plates were prepared 60 with either 50 mg/mL kanamycin or 75 mg/mL kanamycin, but the 75 mg/mL plates proved to provide better selection and thus, 75 mg/mL kanamycin was used for all subsequent plates. While wild-type seedlings became purple or yellow and died soon after germination, some of the seeds originating from the transformed plants germinated and grew well on the selection medium (Figure 14). Seedlings from transformed plants which were able to grow on selection medium were green and exhibited increased root and leaf biomass in comparison to wild- type (Figure 14). Plates which exhibited growth of seedlings often exhibited 1-4 transformants per plate. Wild-type seeds grew well on the medium containing no antibiotic but failed to grow well on the medium containing kanamycin. Positive growth of F1 seedlings was observed for LaSVP, LaPI, LaAP1, LaCAL, LaCC and LaFT, indicating successful transformation as seedlings thus harbored the kanamycin resistance gene. For each gene, at least 10 seedlings exhibiting positive growth were identified. Seedlings growing on kanamycin selection medium were transferred to soil and were analyzed in parallel with wild-type controls. Over 40 transformant seedlings, in addition to wild-type controls, were transferred from the selection plates for genetic analyses. Although many seedlings survived to transfer, some died soon after transfer or before sufficient tissue was available. 61 Figure 14. Representative plates of kanamycin antibiotic selection of F1 transformant seedlings. Selection was performed on ½ MS 75 mg/mL kanamycin selection medium with wild-type A. thaliana controls of the same age grown under the same conditions. (a) bottom view of T2CCB1 seedlings germinated on 75 mg/mL kanamycin medium, (b) top view of T2CCB1 seedlings germinated on 75 mg/mL kanamycin selection medium. T1CCB2 transformants able to grow in the presence of kanamycin exhibited a large root biomass and broad, green leaves. (c), (d) Wild-type A. thaliana seeds. Wild-type seeds germinated but died soon after as they did not confer the antibiotic resistance gene, NPT-II. 3.5 Genetic Analyses of F1 transformants 3.5.1 DNA Detection of the NPT-II Gene Successful plant transformation was further verified by PCR amplification of the Lavandula and neomycin phosphotransferase (NPT-II) genes from leaf-derived genomic DNA (gDNA) of the F1 transformant plants using gene-specific primers for each gene. The gDNA was extracted from F1 plants with sufficient quality and purity and the gene-specific primers for NPT-II successfully amplified the NPT-II gene from the leaf gDNA of the transformants, T1SVPB1, T1SVPB4, T1SVPB5, SVP1, SVP2, SVP3, T2CALB1, T1CALA1, T1PIB1, TIPIB3, T1PIB4, 62 T1PIB5, T2PIB1, T2PIB2, T2PIA1, T2PIA2, T2AP1A3, T2AP1A4, T2AP1A8, T1FTA2. In some instances, there was insufficient leaf tissue, so cDNA only was obtained and used to amplify the NPT-II gene as in T1FTA2 and T1FTA8 (Table 9). In addition, amplification was verified through visualization of the PCR reaction for each transformant’s cDNA on a 1% agarose gel and for each transformant, the NPT-II gene was detected at the appropriate length, 693 bp (Figure 15). This result was observed for all candidates in Table 9. Figure 15. PCR amplification of the NPT-II gene from leaf-derived gDNA of F1 transformants using gene-specific primers. The NPT-II gene was successfully amplified from (1) T1AP1A3, (2) T1AP1A4, (3) T1SVPB4, (4) T1SVPB5, and (5) T1PIA1 and (6) 1 kB DNA ladder. Detection of the NPT-II gene in all candidates confirmed successful transformation with the pGA482:lavender gene constructs. 3.5.2 cDNA Detection of Lavandula Candidate Genes To confirm expression of the respective lavender gene in each transformant, flower and/or leaf RNA were extracted from each transformant plant and RNA was reverse transcribed to cDNA. cDNA was used for expression analysis as detection in gDNA would not confirm if the gene was expressed. Using the cDNA as a template, transformation was verified via PCR amplification of the respective lavender genes using gene-specific primers for each gene. The respective lavender genes were successfully amplified from the cDNA of the transformants, T1SVPB1, T1SVPB4, T1SVPB5, SVP1-3, T1CALA1, T1CALA2, T1CALB3, T1PIB3, T1PIB4, T1PIB5, T2PIB1, T2PIB2, T1PIA1, T2PIA2, T2CCB1, T2AP1A1, T2AP1A3, T2AP1A4, T2AP1A8, T1FTA2 (Table 9 & Figure 16). Successful amplification of the genes from cDNA indicated the gene was actively being expressed in these transformant tissues. In 63 some cases, there was insufficient tissue to obtain floral RNA due to altered growth. In these instances, whole plant or mixed leaf/flower RNA was obtained and analyzed. For example, transformant T2CCB1 did not produce sufficient tissue and thus cDNA derived from floral and leaf tissue was used to detect the LaCC gene in this plant. In addition, PCR amplification of the NPT-II and respective lavender gene was unsuccessful from transformants T1CALB3, T2CCB2, and T2AP1A6, although they successfully grew on selection medium (Table 9). Gene-specific primers for each Lavandula gene were also used to PCR amplify the NPT-II gene and the Lavandula genes from wild-type A. thaliana as a control. The LaSVP and LaAP1 genes were detected in the wild-type leaf cDNA and the LaSVP and LaFT genes were detected in the wild-type flower cDNA at the appropriate band weight; however, the NPT-II gene was detected only in transgenic lines and was absent in all wild-type plants. Figure 16. Detection of lavender candidate genes in leaf-derived cDNA from wild-type and F1 A. thaliana and transformants via PCR with gene-specific primers. (1) LaSVP primers amplified the A. thaliana SVP gene due to high sequence homology and nonspecific amplification in wild-type A. thaliana, (2) LaPI was not amplified from wild-type A.thaliana cDNA ,(3) LaCAL was amplified from wild-type A. thalaina cDNA (4), LaSVP was amplified from T1SVPB1 cDNA (5) LaPI was amplified from T1PIB cDNA, (6) LaCAL was amplified from T1CALA1 cDNA. 64 Table 9. Genetic analysis of F1 transformants. Results of gene detection and expression analyses of F1 transformants and wild-type A. thaliana. Transformant plants listed based on the genes detected and confirmed expression of the NPT-II and Lavandula candidate floral gene in a given tissue. T1 = seed derived from a plant from transformation 1, T2= seed derived from a plant from transformation 2, A/B = plant A or B, number at the end is indicative of the replicate, SVP1 through 3 = LaSVP transformants prepared by an honors student. Detection of genes in gDNA confirmed gene presence, while detection in cDNA confirmed active gene expression. Wild-type A. thaliana exhibited amplification of A. thaliana homologs of SVP, FT, and AP1 due to high sequence homology. Transformant Name Sample Genes Amplified T1SVPB1 Leaf gDNA Leaf RNA Flower RNA NPT-II in leaf gDNA LaSVP in flower and leaf cDNA T1SVPB4 Leaf DNA Flower RNA NPT-II in leaf gDNA LaSVP in leaf gDNA LaSVP in flower cDNA T1SVPB5 Leaf gDNA Flower RNA NPT-II in leaf gDNA LaSVP in flower cDNA SVP1 Leaf gDNA Flower RNA NPT-II in leaf gDNA LaSVP in flower cDNA SVP2 Leaf gDNA Flower RNA NPT-II in leaf gDNA LaSVP in flower cDNA SVP3 Leaf gDNA Flower RNA NPT-II in leaf gDNA LaSVP in flower cDNA T2CALB1 Leaf gDNA Flower RNA NPT-II in leaf gDNA T2CALB3 Insufficient tissue None T1CALA1 Leaf gDNA Flower RNA NPT-II in leaf gDNA LaCAL from flower cDNA T1CALA2 RNA whole plant LaCAL from plant cDNA T1CALB3 Leaf gDNA None T1PIB1 Leaf gDNA Leaf RNA NPT-II in leaf gDNA LaPI in leaf cDNA T1PIB3 Leaf gDNA Flower RNA NPT-II in leaf gDNA LaPI in floral cDNA T1PIB4 Leaf gDNA Leaf RNA NPT-II in gDNA LaPI in flower cDNA T1PIB5 Leaf gDNA Leaf RNA NPT-II in gDNA LaPI in flower cDNA T2PIB1 Leaf gDNA Leaf RNA NPT-II in leaf gDNA LaPI in leaf cDNA 65 T2PIB2 Leaf gDNA Leaf RNA NPT-II in leaf gDNA LaPI in leaf cDNA T2PIA1 Leaf gDNA Mixed leaf and flower RNA NPT-II in leaf gDNA LaPI in leaf/flower cDNA T2PIA2 Leaf gDNA Leaf RNA NPT-II in leaf gDNA LaPI in leaf cDNA T1CCA1 Insufficient tissue None T2CCB1 Mixed leaf and flower RNA LaCC in cDNA T2CCB2 Leaf RNA None T2AP1A1 Mixed leaf and flower RNA LaAP1 in cDNA T2AP1A3 Leaf gDNA NPT-II in leaf gDNA LaAP1 in leaf gDNA T2AP1A4 Leaf gDNA Leaf RNA NPT-II in leaf gDNA NPT-II in leaf cDNA LaAP1 in leaf cDNA T2AP1A6 Leaf RNA None T2AP1A8 Leaf gDNA Flower RNA NPT-II in leaf gDNA LaAP1 in flower cDNA T1FTA1 Insufficient tissue None T1FTA2 Whole plant RNA NPT-II and LaFT from plant cDNA T1FTA5 Insufficient tissue None T1FTA8 Whole plant RNA NPT-II and LaFT from plant cDNA Wild-type A. thaliana Leaf RNA Flower RNA LaSVP in leaf cDNA LAPI in leaf cDNA LaSVP in flower cDNA LaFT in flower cDNA 3.6 Phenotypic Analyses of Transformants 3.6.1. LaSVP Transformant Analysis A total of seven kanamycin resistant lines expressing the LaSVP gene and originating from independent transformation events were identified (Table 9). Compared to wild-type, plants expressing the LaSVP gene exhibited a delay in flowering (Figure 17). The LaSVP primers weakly amplified the AtSVP gene from wild-type floral cDNA in one RT-PCR and failed to amplify in another reaction due to high sequence homology between AtSVP and LaSVP in the primer flanking regions. The LaSVP transformants started bolting 5-6 days later than their wild 66 type counterparts (Figure 17). In addition, LaSVP expressers produced abnormal carpels and seed pods and failed to develop petals (Figure 18a & c). The four transgenic lines which expressed the LaSVP the strongest were SVP-1, SVP-2, T1SVPB-4, and T1SVPB-5 (Figure 18 & 19). Further, the four strongest LaSVP expressers did not produce seeds and were sterile. Some of the flowers in other transformants, exemplified by the SVP-3 line, were similar to wild type flowers, while some flowers resembled those of the SVP-1, SVP-2, T1SVPB-4, and T1SVPB-5 lines (Figure 18b & 19). Additionally, these transgenic lines developed some normal seed pods and produced viable seeds in the phenotypically normal parts of the plant, while in other parts of the plants, the seed pods were abnormal and failed to produce viable seeds (Wells et al. 2020). Figure 17. Effects of LaSVP overexpression on A. thaliana growth and flowering. The LaSVP transformant exhibited a delay in the transition to the flowering phase in comparison to wild-type A. thaliana when grown under long day conditions. (a) Wild-type (Wt) and (b) LaSVP transformant. The apparent difference in plant size (compared to WT plants) was not consistent among LaSVP transformants. (Reprinted by permission from Springer Nature: Planta 251(41): 1-11, Cloning and functional characterization of a floral repressor gene from Lavandula angustifolia by Rebecca S. Wells, Ayelign M. Adal, Lina Bauer, Elaheh Najafianashrafi and Soheil S. Mahmoud, © 2020). 67 Figure 18. Differences in floral organ structures of A. thaliana LaSVP transformants versus wild-type A. thaliana. (a) Plants exhibiting high LaSVP expression, such as SVP-2, lacked petals, produced more sepals, developed abnormal seed pods, and were sterile. (b) and (c) Plants exhibiting lower expression of the LaSVP gene, (e.g., SVP-3) exhibited normal floral development in some portions of the plant, but abnormal floral development on other stems and produced seeds. (d) Wild-type A. thaliana var. Landsberg erecta flowers and seed pods. (Reprinted by permission from Springer Nature: Planta 251(41): 1-11, Cloning and functional characterization of a floral repressor gene from Lavandula angustifolia by Rebecca S. Wells, Ayelign M. Adal, Lina Bauer, Elaheh Najafianashrafi and Soheil S. Mahmoud, © 2020). 3.6.1.1 Expression of LaSVP and its Effects on Floral Transition and Morphology Varying degrees of phenotypic abnormalities were observed across the independent transgenic lines of LaSVP transformants. To confirm LaSVP expression in transgenic plants, a fragment of the LaSVP coding sequence was amplified from floral cDNA of three representative transgenic plants by Reverse Transcriptase PCR (RT-PCR). The results demonstrated that the 68 LaSVP gene was expressed in all transgenic plants (Figure 19). RT-PCR results showed higher expression of LaSVP in SVP-1, SVP-2, and T1SVPB5 transformants than in other transgenic lines (Figure 19). The results also showed higher expression of LaSVP in T1SVPB4 (data not shown). Transformants T1SVPB1, and SVP-3 showed weak amplification of the LaSVP gene from floral cDNA (Figure 23). The A. thaliana Actin reference gene was amplified in all wild-type and transformant samples successfully. The LaSVP gene was weakly amplified from wild-type floral cDNA in one RT-PCR and failed to amplify in another reaction, although Actin was successfully amplified from wild type floral cDNA in both cases (Figure 19). In addition to varying levels of expression observed across transformants, varying levels of mutant phenotype was observed in transformants. The strength of expression of LaSVP in the transformants was associated with the intensity of the altered phenotype. In comparison to wild-type A. thaliana, LaSVP transformants exhibited a delay in flowering (Figure 17). In addition, the LaSVP expressers began bolting 5-6 days later than their counterpart wild-type plants (Figure 17). In addition, LaSVP transformants produced abnormal carpels and seed pods and failed to develop petals (Figure 18 a & c). Further, four of the strongest LaSVP expressers (SVP-1, SVP-2, T1SVPB4, and T1SVPB5) did not produce seeds and were completely sterile. Some of the flowers in other transformants, including the SVP-3 line and T1SVPB1, were similar to wild type flowers, while other flowers within the same plant resembled those of the SVP1, SVP2 and T1SVPB-4 and T1SVPB-5 lines (Figure 18 a & c, Figure 20). Thus, these plants exhibited a partially altered phenotype, with some parts of the plant being sterile and exhibiting a transgenic phenotype, and other parts of the plant resembling wild- type. These transgenic lines thus also developed some normal seed pods and produced viable seeds in the phenotypically normal parts of the plant (Wells et al. 2020). 69 Figure 19. Normalization (to β-actin) of LaSVP expression and RT-PCR detection of the β-actin and LaSVP genes in wild-type A. thaliana and LaSVP transformant floral cDNA. a) Normalized (to β-actin) expression of LaSVP in wild-type (Wt) and SVP-1, SVP-2, and SVP-3, assessed by RT-PCR. The LaSVP and β-actin genes were amplified by RT-PCR, and PCR products were resolved on an agarose (1%) gel. Band intensities were measured by densitometry, using the ImageJ software. No LaSVP amplification was observed in Wt; the apparent low expression in Wt is due densitometry background reading. b) RT-PCR detection of β-actin and the LaSVP gene in wild-type A. thaliana and SVP-1, SVP-2, and SVP-3 leaf cDNA. LaSVP was detected (~693 bp) in the transformants SVP-1, SVP-2, and SVP-3, while Actin was detected in all samples (~250 bp). SVP-2 and SVP-3 exhibited strong expression of the LaSVP gene in comparison to SVP-1. c) Normalized (to β-actin) expression of the LaSVP gene in wild-type (Wt) and two representative transformed Arabidopsis plants, T1SVPB1 and T1SVPB2 assessed by RT-PCR. The LaSVP and β-actin genes were amplified by RT-PCR, and PCR products were resolved on an agarose (1%) gel. Band intensities were measured by densitometry, using the ImageJ software. No LaSVP amplification was observed in Wt; the apparent low expression is due densitometry background reading. d) RT-PCR detection of β-actin and LaSVP in wild-type A. thaliana and T1SVP-1 and T1SVPB-5 floral cDNA. LaSVP was detected (~693 bp) in the transformants T1SVPB-1 and T1SVPB-5, while β -Actin was detected in all samples (~250 bp). T1SVPB-5 exhibited strong expression of the LaSVP gene in comparison to T1SVPB-1. ( (a) and (b) Reprinted by permission from Springer Nature: Planta 251(41): 1-11, Cloning and functional characterization of a floral repressor gene from Lavandula angustifolia by Rebecca S. Wells, Ayelign M. Adal, Lina Bauer, Elaheh Najafianashrafi and Soheil S. Mahmoud, © 2020). 70 Figure 20. Floral organ structures of A. thaliana LaSVP transformants. (a), (b), Transformant T1SVPB4, and (c) transformant T1SVPB5 lacked petals and exhibited altered carpel shape. Both T1SVPB4 and T1SVPB5 were sterile as they failed to produce viable seeds. 3.6.2 LaCC Transformant Analysis Three kanamycin resistant transformant lines which had arisen from independent transformations with pGA4882::LaCC were identified (Table 9). Several F1 seedlings grew in the presence of kanamycin but died shortly after transfer to soil. Three seedlings successfully grew in soil, although the LaCC gene was only successful amplified from one plant, T2CCB1, which differed significantly from the wild-type phenotype (Figure 21a). All LaCC transformants exhibited significantly stunted growth and a decrease in vegetative growth compared with wild-type (Figure 21). In addition, LaCC transformants exhibited leaves which curled towards the abaxial surface and had an increase in carpel abundance, but a decrease in carpel length (Figure 21). In addition, the T2CCB1 transformant exhibited a reduction in the number of cotyledons. 71 Figure 21. Differences in floral organ structures of A. thaliana LaCC transformants versus wild-type A. thaliana. (a) Transformant T2CCB1, (b) transformant T1CCA1, (c) wild-type A. thaliana var. Landsberg erecta siliques (d) transformant T2CCB2, (e) wild-type A. thaliana var. Landsberg erecta. Transformants exhibited curled leaves, increased carpel number, and a reduction in carpel length compared to wild- type. 3.6.3. LaPI Transformant Analysis A total of eight kanamycin resistant lines expressing the LaPI gene and originating from independent transformation events were identified (Table 9). All transformants exhibited expression of both the NPT-II and LaPI genes. In most cases, LaPI transformants remained phenotypically the same as wild-type plants; however, some transformants presented slight phenotypic differences (Figure 22). Transformants T1PIB1, T2PIB2, T2PIA2, and T2PIB1 exhibited a slight increase in stem and leaf growth (Figure 22 e, f, g, h). In addition, transformant T2PIA2 exhibited abnormal leaves, buds, carpels and siliques in some areas of the plant, but remained phenotypically similar to wild type in other areas of the plant (Figure 22 a). Transformants T1PIB3, T1PIB4, T1PIB5, and T1PIB1 exhibited leaves which appeared to be slightly enlarged (Figure 22, b, c, d, e). Transformant T1PIB1 exhibited both a slight increase in vegetative growth and leaf size in many leaves, although results were insignificant. Some transformants also exhibited early flowering, although many of the early flowering plants did not survive. 72 Figure 22. Phenotype and floral organ structures of A. thaliana LaPI transformant F1 plants. (a) T2PIA2, (b) T1PIB3, (c) T1PIB4 (d), T1PIB5, (e) T1PIB1, (f) T2PIB2, (g) T2PIA2, (h) T2PIB1. The LaPI transformants exhibited phenotypes similar to wild type A. thaliana, with some exceptions. T2PIA2 exhibited abnormal leaves, buds and carpels in some areas of the plant, but resembled wild type in other areas. Transformants T2PIB3, T1PIB4, T1PIB5, and T1PIB1 exhibited an apparent overall increase in leaf size. Transformants T1PIB1, T1PIB2, T1PIA2, and T2PIB1 exhibited an apparent increase in stem number and number of leaves. 73 3.6.4 LaAP1 Transformant Analysis A total of 4 plants which expressed the LaAP1 gene were identified, including T2AP1A1, T2AP1A3, T2AP1A4 and T2AP1A8. All transformants expressed both the NPT-II and the LaAP1 genes, with the exception of T2AP1A1, from which only LaAP1 was detected (Table 9). Most of the transformants exhibited a phenotype very closely resembling that of the wild- type. Although some transformants appeared to have a slight increase in bud number, this result was insignificant (Figure 23 & 24). In addition, it appeared that the LaAP1 transformants flowered at a young age, although this result was inconsistent, and it is unclear if this result was due to growth conditions. Figure 23. Phenotypes of the LaAP1 F1 transformants. (a) T2AP1A4, (b) T2AP1A1, (c) T2AP1A8, (d) T2AP1A3. LaAP1 transformants remained phenotypically similar to wild- type A. thaliana, although T2AP1A1 exhibited altered carpel structure. 74 Figure 24. Phenotypic comparison of LaAP1 transformant and wild-type A. thaliana. (a) Transformant T2AP1A8, (b) wild-type A. thaliana var. Landsberg erecta. No significant phenotypic difference was observed between LaAP1 transformant and wild type. Plants are shown at 5 weeks, 4 days of age. 3.6.5 LaCAL Transformant Analysis A total of 3 LaCAL transformants originating from 3 independent transformation events were identified through expression analysis. Over 10 seedings derived from pGA::LaCC transformants were selected from antibiotic selection plates and transferred to soil. Several transformants had insufficient tissue for testing prior to dying and were thus not tested. Of the tested transformants, T1CALA1, T2CALB1, and T1CALA1 were confirmed transgenic (Figure 25). Transformants T1CALA1, and T2CALB1, and T1CALA1 exhibited expression of the NPT-II and LaCal gene, the NPT-II gene, and LaCAL, respectively (Table 9). Of the transformants, none exhibited significant phenotypic differences compared to wild-type A. thaliana, although they all seemed to exhibit a slight reduction in vegetative growth and reduced height. 75 Figure 25. Phenotypes of the LaCAL transformants. (a) T1CALA1, (b) T2CALB1. Note that during the experiment, LaCAL transformants were referred to as LaCALF, as seen in (b). LaCAL transformants exhibited a slight reduction in height and vegetative growth compared to wild- type, although remained phenotypically very similar. 3.6.6. LaFT Transformant Analysis A total of two transformants which successfully expressed the LaFT and NPT-II genes, T1FTA2 and T1FTA8, were identified. Although over 8 transformants were selected from the antibiotic selection plates, only several transformants survived and/or had sufficient tissue for gene expression testing. Both T1FTA2 and T1FTA8 exhibited expression of NPT-II and LaFT in cDNA derived from the entire plant (Table 9). Transformants T1FTA2 and T1FTA8 both exhibited decreased vertical growth, reduced flower production, and abnormal carpels in some 76 areas of the plant when compared to wild type (Figure 26 a, b, & d). These transformants also appeared to have an overall reduction in leaf growth and flowered at an early age. In addition, transformants T1FTA1 and T1FTA5 exhibited a similar altered phenotype, although there was insufficient tissue to obtain mRNA for testing, as occurred with other LaFT F1 seedlings (Figure 26). Both T1FTA1 and T1FTA5 exhibited significantly decreased vegetative growth at 1 month of age (Figure 26 c). Both T1FTA1 and T1FTA5 exhibited a significant reduction in height and leaf biomass and had begun flowering at 1 month of age. Figure 26. Phenotype of the LaFT transformants. (a) T1FTA2, (b) T1FTA8, (c) T1FTA5, (d) wild-type A. thaliana. LaFT transformants exhibited reduced height and decreased flower production and abnormal carpels in some areas of the plants when compared to wild-type A. thaliana. Both T1FTA2 and T1FTA8 exhibited expression of NPT-II and LaFT in cDNA derived from the entire plant. Transformant T1FTA5 exhibited a transformant phenotype but had insufficient tissue for genetic testing. 77 Chapter 4: Discussion Lavenders are commercially and medicinally important plants that have been implicated for use in a variety of settings from medicinal use as an analgesic or anxiolytic to commercial use in perfumes and personal care products (Field et al. 2005; Smith et al. 2011; Soltani et al. 2013; Wells et al. 2018). Previous studies have examined the biosynthesis and biological activities of lavender EO (Landmann et al. 2007; Zuzarte et al. 2009; Woronuk et al. 2010; Demissie et al. 2011; Demissie et al. 2012; Sarker et al. 2012; Soltani et al. 2013; Lesage-Meessen et al. 2015). However, the molecular processes governing the initiation and development of flowers – the main source of essential oils in lavenders– have not been adequately investigated in this plant. Floral development has, however, been well-studied in A. thaliana, and many genes involved in floral initiation and development have been documented (Coen and Meyerowitz 1991; Theißen et al. 1996; Koornneef et al. 1998a; Levy and Dean 1998; Bowman and Eshed 2000; Hartmann et al. 2000; Samach et al. 2000a; Theißen 2001; Causier et al. 2010; Smaczniak et al. 2012; Andrés et al. 2015). The ABC(DE) model described in A. thaliana is the most well-defined model of floral development which describes the genes involved in flowering and their roles in floral pattern development (Weigel and Meyerowitz 1994; Causier et al. 2010). Additionally, many pathways governing floral initiation and repression of flowering in A. thaliana have been documented (Samach et al. 2000a; Hayama and Coupland 2003). Homologsof the A. thaliana floral development genes have been identified and studied in other plants but, to our knowledge, have yet to be formally identified in lavenders (Kang et al. 1998; Teeri et al. 2006; Bendahmane et al. 2013; Wu et al. 2017). Here, we proposed that the genes governing floral initiation and development in A. thaliana and other flowering plants are also present in the genus Lavandula. To bridge the gap in knowledge, this thesis employed a genomic approach to identify and functionally characterize genes that control flower initiation and pattern development in lavenders. We used RNA-Seq to obtain information on genes expressed during various stages of flower development in L. angustifolia and L. x intermedia plants. The information was used to develop a comprehensive Lavandula transcriptome within which Lavandula genes homologous to A. thaliana floral development genes were identified. A subset of 6 genes was successfully functionally characterized via expression in wild-type A. thaliana. The present study is the first of its kind in lavenders and has 78 thus made a significant scientific contribution and marks the start of new area of research in lavender development. 4.1 Transcriptome Assembly and Identification of Lavandula Homologs 4.1.1 Transcriptome Assembly, Annotation, and Gene Identification To examine the genes expressed at various stages of flower development, total RNA was extracted from SAM, BD, and FL tissues of mature L. x intermedia and L. angustifolia plants and was sequenced by Illumina sequencing. The L. angustifolia FL transcriptome exhibited the most contigs at 47,630 contigs, while the L. x intermedia SAM gave rise to the least amount of contigs at 26,821 (Table 3). This is likely due to the vast number of genes which are active in the flower and the floral meristem, including not only developmental genes, but genes involved in terpenoid metabolism and biosynthesis in FL tissues which are inactive in SAM tissues (Klepikova et al. 2015; Adal et al. 2019; Malli et al. 2019). RNA-Seq was then used to compile this information and assemble it into a comprehensive transcriptome of 105, 294 transcripts from a total of 118,245,160 paired end reads, representative of genes expressed in the developing tissues. CD-HIT-EST was used to remove redundant sequences which had >98% sequence similarity, ensuring that the transcriptome was comprised of unique genes and minimal duplicate transcripts. All transcriptome parameters (including N50 and transcript length distribution) were within a reasonable range when compared to previously-reported transcriptomes assembled by similar means (Duan et al. 2012; Lulin et al. 2012; Wang et al. 2012; Xu et al. 2013; Lu et al. 2014; Adal et al. 2019). Thus, the present transcriptome can be considered a reliable genomic resource for genetic studies. Using CLC Genomics Workbench 11, BLASTx analyses of the transcriptome against an annotated A. thaliana protein database assigned putative functions to the majority of transcripts by comparing the Lavandula sequences to the sequences of A. thaliana proteins with known functions. BLASTx analyses also revealed the presence of ten previously- described Lavandula terpene synthase genes within the transcriptome, which verified the comprehensiveness and quality of the transcriptome as these genes are vital for the synthesis of lavender EO constituents (Table 4) (Landmann et al. 2007; Demissie et al. 2011; Sarker et al. 2013; Jullien et al. 2014; Despinasse et al. 2017; Adal et al. 2019). 79 Annotation of the transcriptome via the BLAST2GO function of CLC Genomics Workbench 11 provided gene ontology terms for the majority of transcripts, classifying the transcripts based on cellular component, molecular function, or biological process, with few transcripts lacking annotation (Figure 5). This thorough classification of transcripts provided support for the quality and comprehensiveness of the transcriptome as the majority of transcripts could be assigned a function. The thorough gene classification was consistent with other similar RNA-Seq assembly classifications (Gao et al. 2013; Adal et al. 2019). Regarding biological process, the majority of the contigs were classified as being involved in cellular process, followed by metabolic process, response to stimulus, biological regulation and developmental process (Figure 5). These classification categories were consistent with the functions of the A. thaliana proteins to which the Lavandula transcripts matched via BLASTx analyses. As many of the identified genes within the transcriptome were development-related genes or transcription factors involved in floral initiation or pattern development, the classification of these genes functionally into response to stimulus (>32,500), biological regulation (>27,500) and developmental process (>25,000) is consistent with the corresponding A. thaliana genes’ functions in the SAM and floral meristem. With regards to molecular function, the most common category was binding (>37,000), followed by catalytic activity (>30,000) and transporter activity (approximately 5000). The abundance of transcripts classified based on binding activity is consistent with the abundance of TFs identified within the transcriptome and with the function of many TFs in protein-protein interactions and in DNA-binding to regulate gene expression in the SAM during floral initiation (Yant et al. 2010; Smaczniak et al. 2012; Theißen et al. 2016; Dinh et al. 2017). The majority of contigs matching to cellular component were classified as part of the cell (>50,000), followed by organelle component (>42,500) and membrane component (>30,000) (Fig. 4). This result is consistent with the identification of genes within the transcriptome involved in floral initiation based on the sensing and integration of endogenous and exogenous cues via regulatory pathway at the SAM to regulate flowering timing (Koornneef et al. 1998a; Boss et al. 2004; Kim et al. 2009). Gene ontology studies thus provided insight into the functions of the transcripts within the transcriptome and showed the presence of a variety of genes involved in gene regulation during floral initiation. Future studies should examine gene ontology terms of specific tissues as well as at shorter time increments to gain a more comprehensive view of these genes over time. 80 The assembled transcriptome can thus be considered a comprehensive representation of the genes expressed in Lavandula floral tissues at various developmental stages. In lieu to this, the transcriptome assembled in this study is a valuable genomic resource that can be used in future studies for the identification of additional floral initiation and development-related genes as well as genes involved in other developmental processes in lavenders. BLASTx analyses and Gene Ontology studies facilitated the identification of Lavandula candidate genes homologsto A. thaliana floral initiation and development genes within the transcriptome. The transcriptome “quality” was high as it contained full-length transcripts for numerous genes, including partial or full-length sequences for over 100 previously- described A. thaliana floral initiation and floral organ identity genes (representing all of the A, B, C, D, and E-type gene classes), and those for most of the previously reported lavender terpene synthases. Candidate genes identified within the transcriptome included homologs of previously- examined A. thaliana repressors and initiators of flowering (Table S1). Further, a total of 14 full-length lavender genes homologous to A. thaliana floral development and initiation genes were identified within the transcriptome (Table 6). From the 14 full length genes, a subset of 6 candidate genes, including LaCC, LaCAL, LaSVP, LaPI, LaFT, LaAP1, and LiAP2, were selected for functional characterization in A. thaliana. These genes were selected based on high sequence homology, full length sequence and given that these genes were homologous to genes which play vital roles in floral development in A. thaliana. For example, LaCC is the homolog of AtCC, which functions in the regulation of carpel development in A. thaliana, whereas LaAP1, the homolog of AtAP1, is vital to sepal and petal development (Bowman and Smyth 1999; Theißen et al. 2016). The presence of these genes in lavenders supported the hypothesis that there exist genes in lavenders which are homologous to genes which govern floral initiation and development in A. thaliana. 4.1.2 Sequence Analysis MUSCLE sequence alignment of the full-length Lavandula protein sequences and their respective A. thaliana homolog’s protein sequence revealed sequence similarities and differences between the pairs (Tables 7- 8, Figures 8 & B1-B13). MUSCLE alignment of the homologous pairs revealed the presence of conserved sequences respective to the TF class in each of the protein pairs. Lavandula protein sequences were similar to their respective A. thaliana 81 homologous both in amino acid sequence and protein length (Table 6). For example, the repressor protein, LaFLC, was determined to be 190 amino acids in length, while its homolog, AtFLC, is 196 amino acids in length (Table 6). This trend of similarity was observed between all homolog pairs and was an expected result as the homologous pairs contain conserved domains and are hypothesized to serve similar functions. Protein sequence similarity between the homologs ranged from 41.53-78.16%, which indicated the presence of conserved domains within the proteins, but differences in protein sequences due to being derived from different species (Table 7, Figures 8 & B1-B13). These results were consistent with homologs of the A. thaliana floral development genes described in other plants, such as homologs of PI and AG described in Oryza sativa (rice) and Lilium longiflorum (lily), respectively, which also exhibited conserved domains, but exhibited different amino acids and sequence length (Kang et al. 1998; Kyozuka et al. 2000). Many of the TFs identified in the present study, including LaAG, LaSVP, LaCAL, LaFLC, LaPI, LaAP1, LiAP3, LaSEP3, LaSOC1, are homologs of A. thaliana MADS-Box TFS, which play important roles in floral development. Alignment of these proteins with their respective homologs revealed the presence of the MADS-Box and K-Box domains characteristic to these TFs (Figures 8, B1-B13) (Shore and Sharrocks 1995; Gramzow et al. 2010). For example, alignment of genes with their A. thaliana homologs and other plant MADS-Box transcription factors revealed the presence of the conserved MADS-Box and K-box domains of the MADS-Box TFs in several of the Lavandula proteins (Figure B.1-B.12). MADS-Box genes are derived from common ancestral genes which underwent gene duplication events that caused divergence, giving rise to the plant and animal MADS-Box TFs observed today (Alvarez-Buylla et al. 2000). These gene duplication events in ancient MADS-Box genes also led to the diversification of functions of the MADS-Box genes, including the ABCDE-type TFs (Kramer and Hall 2005). It is proposed that the differences observed between the MADS-Box proteins are due to gene duplications, divergence, and changes in regulatory sequences (Force et al. 1999; Kramer and Hall 2005). Thus, although high sequence similarity between homologous MADS-Box genes is observed, sequence differences are expected between species and the MADS-Box genes identified in this study likely exhibit sequence differences outside the conserved domains for these reasons. In addition, the presence of these conserved domains provides evidence for a conserved function of these lavender proteins as MADS-Box TFs regulating the expression of 82 other genes involved in floral initiation and pattern development (Theißen 2001; Pařenicová et al. 2003; Kaufmann et al. 2005). Further, results support the hypothesis that the lavender proteins function similarly as their A. thaliana homologs Future studies should examine the 3D protein structure of these TFs and identify specific domains and interactions with other proteins. The identification of homologous genes supported the hypothesis that there exist lavender genes which are homologous to the floral initiation and pattern development genes present in A. thaliana and other plants, thus providing support for the function of the ABC(DE) model in Lavandula species. The ABC(DE) model of floral development is the most predominant and widely accepted model of floral pattern development in A. thaliana and other higher plants (Coen and Meyerowitz 1991; Kang et al. 1998; Levy and Dean 1998; Causier et al. 2010; Theißen et al. 2016). To date, numerous studies have identified homologs to these A. thaliana floral development genes in a variety of other flowering plants, including rice, wheat, and soybeans, amongst others, providing support for this model across many plants(Coen and Meyerowitz 1991; Kaufmann et al. 2005; Shitsukawa et al. 2006; Huang et al. 2014; Irish 2017; Wu et al. 2017). The identification of lavender homologs of the previously-described A. thaliana floral initiation and development genes, including the A, B, C, D, and E-class genes, provides support for the hypothesis that floral development in lavenders is similar to that in A. thaliana and other flowering plants. Further, as the ABC(DE) model is well-described in A. thaliana, homologs identified within the Lavandula transcriptome can be assigned putative functions based on the function of the respective A. thaliana homologs, thus providing insight into the genes involved in floral development in lavenders and an abundance of vital developmental genes to be examined. 4.1.3 Expression Analysis of Lavandula Transcripts To examine expression patterns of floral initiation and development genes during various stages of flower development in L. angustifolia and L. x intermedia FL, BD, and SAM tissues, the Differential Expression for RNA-Seq function of CLC Genomics was employed. Differential expression analysis revealed that many of the identified genes within the transcriptome exhibited varying levels of expression across different developmental stages (Figure 6). Some genes exhibited higher expression in the SAM than the FL or BD tissues, whereas other genes exhibited higher expression in the SAM in comparison to FL or BD tissues. Differential 83 expression across developmental stages was due to different genes being active at a given time based on their role. For example, genes which are involved in floral initiation, are more likely to be expressed in the bud or shoot apical meristem, whereas floral patterning and flower maturity genes are more likely to be active at later stages (Figure 6). The results were thus consistent with the diversity of the developmental roles of the identified genes and were consistent with other studies examining expression patterns of floral development related genes over time (Kram et al. 2009; Yant et al. 2010; Klepikova et al. 2015). For example, the gene for the repressor of flowering, FLC, was expressed in low amounts in the LaFL in comparison to the LaBD, which was consistent with studies that found levels of expression of this gene to decrease throughout floral initiation (Klepikova et al. 2015). Similarly, results showed that LaSVP, the Lavandula homolog of the A. thaliana floral repressor, AtSVP, exhibited higher expression in the LaSAM in comparison to LaFL tissues, lower expression in LaSAM versus LiSAM, lower expression in LaBD versus LiSAM, and similar expression in LaFL and LaBD tissues. This result is consistent with the role of AtSVP as a floral repressor previously-described to be active in vegetative tissues, but absent in floral tissues (Hartmann et al. 2000). Differential expression studies thus provided support for the differing roles of the identified Lavandula floral development genes and insight into their expression patterns at various stages of development. To verify expression of the selected candidates in L. angustifolia and to facilitate cloning in the pGA482 vector, all seven candidates were PCR amplified from L. angustifolia BD cDNA using gene-specific primers. All genes were successfully amplified from L. angustifolia BD cDNA, indicating that all selected candidate genes were actively expressed in L. angustifolia BD tissue (Figure 9). 4.2 Cloning and Selection of Candidates 4.2.1 Cloning in the pGA482 Vector and Bacterial Selection Due to the high protein sequence similarity between the Lavandula and A. thaliana homolog, it was proposed that the lavender genes would likely function similarly to their A. thaliana counterpart and that floral development in lavenders is governed by similar processes as in A. thaliana. To explore this hypothesis and as a proof of concept, 7 Lavandula candidate genes were functionally characterized via cloning and overexpression in wild-type A. thaliana driven by the CaMV 35S promoter of the pGA482 vector. Functional characterization of the 84 Lavandula candidate genes, LaCC, LaCAL, LaSVP, LaPI, LaFT, and LaAP1, was facilitated through cloning and expression via the pGA482 vector. Each gene was successfully ligated into a pGA482 vector via sticky end cloning, giving the constructs pGA::LaCC , pGA::LaCAL , pGA::LaSVP , pGA::LaPI , pGA::LaFT, and pGA::LaAP1, which were transformed into E.coli via heat-shock. For each construct, both the 50 µl plate and the concentrated plate exhibited colony growth, although in all cases, the concentrated plate exhibited more growth. Positive colony growth in the presence of tetracycline indicated that the cells giving rise to the colonies harbored the pGA482 vector containing the tetracycline resistance gene and the respective Lavandula gene insert. Control plates containing E. coli transformed with an empty pGA482 exhibited growth on the concentrated plates, confirming that the tetracycline resistance gene within the pGA482 vector was functional (Figure 10). All pGA482::lavender gene constructs were successfully transformed into A. tumefaciens. 4.2.2 Plant Transformation and Selection To functionally characterize the lavender candidate genes, wild-type A. thaliana var. Landsberg erecta were transformed with the pGA482::lavender gene constructs via the floral dip method (Zhang et al. 2006b). Agrobacterium-mediated plant transformation via the floral dip is a well-established protocol which has proven to be an effective means for transformation of A. thaliana in a variety of studies, as well as in other plants, such as Finger millet and Sorghum (Clough and Bent 1998; Zhang et al. 2006b; Sharma et al. 2011; De Pater et al. 2013; Huang et al. 2014; Che et al. 2018). Expression of these genes in A. thaliana enabled the study of the functions of these genes in an effective model plant. Four wild-type A. thaliana plants were transformed for each construct, for a total of 24 transformed plants. All transformed plants produced viable seeds which were grown on antibiotic selection medium to identify positive transformants. This was an effective means of selection as only seeds which had successfully taken up the pGA482::lavender gene construct harboring the kanamycin resistance gene were able to survive in the presence of antibiotic. Successful selection of transformant seedlings was observed on plates for all six genes, indicating that transformation had been successful. Initially, selection took place on plates containing either 50 mg/mL kanamycin and 75 mg/mL kanamycin, but as selection was more efficient on 75 mg/mL kanamycin, all subsequent plates had 75 mg/mL kanamycin. 85 Transformant seedlings able to grow in the presence of kanamycin had significant phenotypic differences from those which could not. Surviving seedlings exhibited green leaves and an expansive, branching root system within the medium in comparison to wild- type, which appeared yellow and failed to develop roots (Figure 14). Some plates exhibited only seedlings which died soon after germination, indicating no positive transformants. Conversely, wild-type seeds grew well on medium containing no antibiotic, thus serving as an effective control. Most seeds were able to germinate but died soon after (Figure 14). This indicated that seeds could germinate in the presence of antibiotic and use the reserves within the seed, but once the seed began drawing nutrients from the medium, the antibiotic killed the seeds lacking the NPT-II gene. Successful germination of the seeds indicated that transformation had been successful and that F1 seeds were viable. Seedlings which grew successfully in the presence of kanamycin and which were presumed to be kanamycin resistant were transferred to soil for growth and genetic analysis. For each gene, there were 5-15 seedlings transferred from plates to soil, with over 40 seedlings selected from the antibiotic plates. This result indicated that transformation success varied widely across the plants. This result is consistent with reports that transformation efficiency can be highly variable and largely dependent on the number of unopened floral buds at the time of transformation of the parental plants (Ghedira et al. 2013). Further, although numerous seedlings were transferred for each gene, some failed to survive. For example, over 14 LaAP1 seedlings were transferred from plates to soil, but few survived and/ or had sufficient tissue for testing (Table 9). Although transformation protocols were closely followed, the number of positive transformants was lower than the expected 1/100 seeds predicted by Zheng et al. (2006). This result may also be due to the use of the A. thaliana ecotype used in the present study as it has been found that A. thaliana var. Landsberg erecta exhibits lower transformation efficiency compared to other ecotypes (Zhang et al. 2006b). Future studies should examine the transformation efficiencies of other ecotypes and the effects of transformation of lavender genes into other ecotypes. 4.2.3 Expression Analysis of F1 Transformants Tissue was collected from presumed kanamycin-resistant seedlings (Table 9). The NPT-II gene was PCR amplified from gDNA extracted from transformant tissue. PCR amplification of 86 the NPT-II gene from gDNA and/or cDNA of transformants indicated that the kanamycin resistance gene was present within the transformant’s DNA and transformation had been successful. As there was a possibility that an empty vector could have been transferred to a plant, the amplification of each respective lavender candidate gene was vital. Amplification of the Lavandula genes from floral cDNA confirmed that the gene was not only present, but also was actively being expressed as mRNA. Thus, amplification of a given gene from gDNA confirmed the presence of the gene but would did not confirm expression of the gene. In some cases, such as with T2AP1A1, there was insufficient tissue for full expression analysis, but amplification of the LaAP1 gene from the cDNA provided support that the lavender gene was present and being expressed in the transformant. From this result, it could be inferred that the NPT-II gene was also present as it was transferred with the lavender gene in the vector. There were, however, some seedlings which were able to grow in the presence of kanamycin, but amplification of NPT-II and the respective lavender gene was unsuccessful. This result may have been observed due to uneven mixing of medium in some cases causing pockets lacking antibiotic within the medium, insufficient RNA quantity, or low expression of these genes in these plants below a level which was detectable. Wild-type A. thaliana plants also exhibited a low level of expression of LaSVP and LaAP1 as well as LaSVP and LaFT in the leaf cDNA and flower cDNA, respectively (Table 9). This result was likely due to the high sequence similarity between the lavender and A. thaliana gene homolog pairs in the 5’ and 3’ regions to which the primers were designed (Figures 8, B4 & B8). Similarly, LiAP2, although originating from L. x intermedia, was successfully amplified from L. angustifolia FL BD tissue due to high sequence homology in the 5’ and 3’ regions of the L. x intermedia and L. angustifolia AP2 genes. Although positive transformants were identified for all genes, identifying additional replicates in future studies would be beneficial. In the present study there were six genes that were characterized, and 24 plants transformed and, as such, many plates were prepared to screen seeds for transformants. Futures studies should focus on 1-3 genes at a time to allow for the more rapid identification of transformant seeds and increased replicates per gene. The successful PCR amplification of the lavender genes from cDNA and gDNA of F1 seedlings indicated that these genes were successfully transformed into these plants. Further, it showed that these genes could be successfully expressed in A. thaliana via the 35S CaMV 87 promoter. This method can thus be used in future studies to characterize additional lavender developmental genes in A. thaliana. Future studies should also examine the effects of the manipulation of these genes in lavenders via transformation. For example, it would be valuable to examine the effects of overexpression of certain genes responsible for petal development or to examine the effects of silencing the expression of floral repressors. Successful Agrobacterium-mediated transformation of lavenders using lavender explants hasbeen reported; however, the long generation time of lavenders poses limitations on the ability to examine the effects of transformation in short amounts of time (Nebauer et al. 2000; Mishiba et al. 2015). Thus, improving upon current lavender transformation procedures would be beneficial in many areas of study, including the study of floral development. Currently in the Mahmoud lab, such transformation procedures are being examined and optimized. 4.3 Functional Characterization of Lavandula Candidates A total of 24 F1 transformant plants were identified as indicated by growth on antibiotic selection medium and PCR amplification of NPT-II and the respective lavender gene. Although all six candidate genes were successfully expressed in wild-type A. thaliana plants, the phenotypic effects of expression of each gene varied. For example, the expression of the MADS-Box proteins LaAP1, LaCAL and LaPI did not elicit significant phenotypic effects (Figures 21 & 22). LaAP1 and LaPI are homologs of AtAP1 and AtPI, which function as MADS-Box TFs during floral development in A. thaliana (Theißen et al. 2016). In A. thaliana, these proteins function as quartets, thus binding to three other MADS-Box proteins and binding to the promoter region of a given gene (Figure 1) (Theißen et al. 2016). Although LaPI and LaAP1 were expressed in several F1 transformants, expression of these genes did not significantly impact the phenotype of these transformants. This result could be due to the nature by which these proteins function in quartets as both LaPI and LaAP1 elicit their effects on gene expression through interactions with other proteins (Theißen et al. 2016). These proteins thus rely on the ability to bind to and interact with other proteins to form a quartet, which regulates gene expression through promoter binding (Theißen et al. 2016). Each TF must thus be able to productively bind to other proteins in the quartet. For example, AtPI is involved in the regulation of petal and stamen development in A. thaliana (Theißen et al. 2016). When one AtPI protein combines with 88 one AtAP3 protein, one AtSEP protein, and one AtAP1 protein, a quartet is formed that leads to petal development in whorl 2 of the A. thaliana flower (Figure 1) (Theißen et al. 2016). In contrast, when one AtPI protein combines with one AtAP3 protein, one AtSEP protein, and one AtAG protein, the quartet binds to the promoter of genes that promote stamen development in whorl 3 of the A. thaliana (Figure 1) (Theißen et al. 2016). Thus, the ability for these proteins to effectively bind one another and a specific gene’s promoter is vital in order to elicit the desired effect on gene expression in the appropriate whorl. In the current study, expression of the Lavandula genes via the constitutive CaMV 35S promoter led to production of many copies of each protein in each A. thaliana transformant. It was hypothesized that an increase in concentration of the proteins via expression of the Lavandula orthologs in A. thaliana would lead to an increase in the processes these TFs govern. In the present study, F1 transformants of three of the Lavandula proteins, LaCC, LaSVP, and LaFT exhibited phenotypic differences from wild-type plants (Figures 18-20 & 24). In contrast, transformants expressing the MADS-Box proteins LaPI, LaAP1, and LaCAL exhibited no significant phenotypic differences in comparison to wild-type (Bowman et al. 1993; Bowman and Smyth 1999; Ferrándiz et al. 2000; Kaufmann et al. 2005) (Figures 21-23). It was hypothesized that an increase in expression of a given protein would lead to an increase in the activity that protein performs; however, this was not observed with LaPI, LaAP1, and LaCAL transformants. Thus, for these proteins an increase in protein concentration may not be associated with increase in activity. This result also may have been due to limited availability of gene promoters for the additional proteins to bind. In addition, TFs function through protein-protein interactions, so an increased concentration of one protein may not increase a given effect if the protein must interact with other proteins to elicit this effect. Additionally, the activity of the Lavandula proteins may have been limited by an incompatibility and inability to bind to available A. thaliana proteins and promoters of A. thaliana genes due to differences in the a.a. sequence between the homologou proteins. These a.a. differences may have led to incompatibilities in binding domains such that they were not sufficiently similar that they could interact with each other and form the quartets. In addition, although the concentration of LaPI, and LaAP1 proteins increased, the proteins’ effects on downstream genes did not increase as the other proteins of the quartet did not increase in concentration. In this case, there could have been a saturation effect in which the number of PI proteins increased as both AtPI and LaPI were 89 present, but the number of the other required proteins remained constant. Thus, although there were many copies of the PI protein, their effect was limited due to a limiting number of the other members of the TF quartet and a finite number of proteins to form the quartet (Deitchman et al. 2018). Further, it has been shown that A. thaliana plants which overexpress multiple MADS-Box gene exhibit a phenotypic effect which is not observed in plants in which only one MADS-Box gene is overexpressed (Krizek and Meyerowitz 1996; Honma and Goto 2001). The other proteins required for the quartet and the binding sites of the Lavandula proteins may be different than those of the A. thaliana proteins due to amino acid differences. Such differences may have led to incompatibilities in binding domains of the proteins, and thus saturation effect (Deitchman et al. 2018). Thus, an increase in concentration of some of the A. thaliana proteins via expression of the Lavandula homolog, did not have an impact in all cases. Future studies should examine the effects of overexpression of multiple Lavandula MADS-Box homologs to examine the effects of the increase in certain MADS-Box complexes, as examined in A. thaliana (Honma and Goto 2001). 4.3.1 LaSVP 4.3.1.1. Phylogenetic analysis of LaSVP and other plant homologs To examine the relationship of the LaSVP gene with other plant SVP-like genes, a phylogenetic tree was constructed using the protein sequences of plant SVP genes. Although the amino acid sequence of LaSVP showed higher sequence similarity to EgSVP-like, BrSVP, BiSVP, and PkSVP homologs AcSVP2, AcSVP3, and AdSVP3 homologs compared to AtSVP, all of these genes contained the K-Box and MADS-Box conserved domains characteristic of MADS-Box transcription factors, suggesting that they share a similar DNA-binding function as MADS-Box transcription factors (Figure 20). The MADS domain of these TFs is highly conserved, facilitating DNA binding and the K-box is a known conserved region in plant MADS-Box proteins found downstream of the MADS domain, and is an important region facilitating protein-protein interactions in these TFs (Alvarez-Buylla et al. 2000). The presence of these conserved domains in the LaSVP protein provides support for LaSVP functioning as a MADS-box TF in lavenders, homologous to other plant SVP genes. Interestingly, LaSVP was found to cluster closely with the SVP homologs, EgSVP-like and BrSVP, but was further away in relatedness to AtSVP (Fig. S2 & Table 2). This result suggests that the processes of floral 90 initiation and development in lavenders may differ from those described in A. thaliana, although this postulate requires additional investigation (Wells et al. 2020). 4.3.1.2. Ectopic Expression of LaSVP Alters Floral Morphology and Flowering Time The role of SVP as a repressor in the suppression of the transition from vegetative growth to flowering, promoting vegetative growth and altering floral morphology has been widely studied in A. thaliana (Hartmann et al. 2000; Gregis et al. 2009). Similar AtSVP homologous genes were isolated from different plant species including Poncirus trifolate, Eucalyptus grandis, Actinidia deliciosa, Chrysanthemum morifolium, and Brassica campestris. The ectopic expression of these genes in A. thaliana was observed to delay flowering time and negatively affected petal development (Brill and Watson 2004; Lee et al. 2007; Li et al. 2010; Wu et al. 2012; Gao et al. 2017). To examine the role of the LaSVP gene in floral initiation and development in lavenders, the gene was expressed in A. thaliana under the control of the constitutive ectopic CaMV 35S promoter (Wells et al. 2020). Although flower development was drastically altered in all transgenic plants, there was an apparent correlation between LaSVP expression level and flower morphology (Wells et al. 2020). To confirm LaSVP expression in transgenic plants, a fragment of the LaSVP coding sequence was amplified from flowers of three representative transgenic plants by Reverse Transcriptase PCR (RT-PCR). The results demonstrated that the LaSVP gene was expressed in all transgenic plants (Figure 20). RT-PCR results showed higher expression of LaSVP in SVP-2 than in other transgenic lines). The Actin reference gene was amplified in all samples. Further, SVP2, T1SVPB4, and T1SVPB5, which expressed the transgene stronger than the other transformants, exhibited a phenotype most drastically different than the wild-type plants, while SVP-3, SVP-4, and T1SVPB1 exhibited a milder altered phenotype in some portions of the plant (Figure 18 & 19). For example, some portions of the T1SVPB1 plant exhibited normal flowers which gave rise to normal seeds pods and produced viable seeds. In contrast, other portions of the plant exhibited the transformant phenotype, lacking petals, exhibiting additional sepals, and having wrinkled carpels failing to produce viable seeds. Thus, when affected, the flowers of the transgenic plants lacked petals, and exhibited the formation of additional sepals. When the LaSVP expression increased, the flowers lacked petals, exhibited additional sepals, and the carpels were vastly altered appearing wrinkled, wide, and failing to produce viable seeds. These results imply that the effects 91 of LaSVP on floral development may be dosage- dependent as increased expression of LaSVP coincided with a more drastic change in phenotype (Wells et al. 2020). This is consistent with studies examining the overexpression of AtSVP, which have shown that AtSVP acts as a repressor of flowering in A. thaliana in a dosage-dependent manner (Hartmann et al. 2000; Jaudal et al. 2014). Other studies in A. thaliana also indicate that altered expression of class B and C genes in the floral meristem results in homeotic conversion in floral whorls (Weigel and Meyerowitz 1994; Gregis 2006; Gregis et al. 2009; Theißen et al. 2016). However, studies on SVP mutants show that the absence of the gene does not lead to obvious morphological differences nor changes to fertility (Hartmann et al. 2000). Given this observation, the absence of expression of the SVP gene may not impact on fertility in lavenders, but the overexpression of the LaSVP gene may lead to sterility in these plants due to the absence of viable seeds. Additionally, the effects of LaSVP on floral organ structure are similar to the findings of the functions of AtSVP in A. thaliana in the control of the floral meristem and repression of B, C and E-class floral homeotic genes whose overexpression leads to morphological changes in the flower (Liu et al. 2007; Li et al. 2008; Gregis et al. 2009). In the present study, the floral morphology differed from wild-typ A. thaliana and the B, C, and E-type genes appeared to be affected; however, there was no observable effects on C-type genes as stames developed in the transformant flowers. The B, C, and E class floral homeotic genes play important roles in the organizational development of flowers, and are required for the normal development of all five floral organs (Gregis et al. 2009; Wells et al. 2020). In addition to changes in floral phenotype, flowering time was also affected by the expression of LaSVP in A. thaliana plants (Figure 17). The transgenic plants exhibited a late-flowering phenotype, compared to the wild-type counterpart. This outcome is not surprising as it has been shown that AtSVP acts as a repressor of floral initiation in A. thaliana, preventing the transition from a vegetative meristem to a flowering meristem (Gregis et al. 2013; Andrés et al. 2014). A similar result was obtained when the Chrysanthemum morfolium SVP (CmSVP) gene was expressed in A. thaliana, causing a significant delay in the onset of flowering (Gao et al. 2017). Thus, the expression of LaSVP in A. thaliana causing a delay in flowering suggests that this gene functions similarly to other plant SVP homologs. 92 4.3.2 LaCC The AtCC gene is an important gene involved in carpel development in A. thaliana and is involved in interactions with numerous proteins to regulate aspects of this process (Gross et al. 2018). The AtCC gene encodes a YABBY domain TF which acts as both an activator and a repressor (Gross et al. 2018). The LaCC TF acts as a repressor of genes which promote the floral meristem to be terminated, but acts as an activator of genes which promote development of the carpel (Gross et al. 2018). To examine the role of LaCC in lavenders, the gene was expressed in A. thaliana under the control of the constitutive ectopic CaMV 35S promoter. Although several LaCC F1 seedlings were identified on the antibiotic selection medium, only one plant that successfully expressed the LaCC gene was identified (Table 9). In several cases, the plants had insufficient tissue to extract sufficient mRNA or DNA and, in some cases, plants failed to survive. T2CCB1 (Figure 21) was the only plant which was confirmed to express LaCC. This plant exhibited curled leaves, and severely stunted growth. In addition, the morphology of the flower pattern differed from wild type in that the flowers were more dispersed along the stem rather than concentrated at the tip of the stem. This phenotype was also observed in T2CCB2 and T1CCA1, although the LaCC gene failed to be amplified from these plants. The LaCC gene is homologous to the YABBY TF AtCC (Gross et al. 2018). Previous studies have identified similar YABBY homologs in other plants, such as in maize and soybeans, which are involved in flowering processes (Strable et al. 2017; Yang et al. 2019). For example, the soybean YABBY TF, GmFILa, was altered leaf polarity and growth and increased the flowering period length when overexpressed in A. thaliana (Yang et al. 2019). Future studies should further examine the effects of overexpression of LaCC in A. thaliana as well as the effects of overexpression of AtCC and LaCC in lavenders. 4.3.3 LaPI The LaPI gene is homologous to the A. thaliana PI gene, a B-class MADS-Box TF that is vital for petal and stamen development in these plants (Figure 1) (Theißen et al. 2016). According to the ABC(DE) model, the A. thaliana PI protein forms a quartet with other MADS-Box proteins to elicit its function by combining with one AP3 protein, one SEP protein, and one AP1 protein to promote petal development in whorl 2 of the A. thaliana flower (Theißen et al. 2016). In contrast, when the PI protein combines with one AP3, one SEP, and one AG protein, 93 stamen development occurs in the third floral whorl (Figure 1) (Theißen et al. 2016). Mutations in this homeotic gene have been observed to cause sepals and carpels to develop in place of petals and stamens, respectively (Goto and Meyerowitz 1994). Homologs of the PI gene have been described in other flowering plants such as lily (Lilium longoflorum), pea (Pisum sativum), apple, Magnolia wufengensis, snapdragon (Antirrhinum majus), rice, and in the Ranunculaceae family (Kang et al. 1998; Kramer et al. 2003; Berbel et al. 2005; Tanaka et al. 2007; Wu et al. 2010; Liu et al. 2018a). To examine the role of LaPI in lavenders, the pGA482::LaPI construct was transformed into wild-type A. thaliana. A total of 8 F1 seedlings were identified which expressed both NPT-II and LaPI (Table 9). Most transformants exhibited no phenotypic differences from wild-type A. thaliana, although some transformants appeared to exhibit a slight increase in stem and leaf mass (Figure 21). One transformant, T2PIA2 exhibited abnormal carpels and siliques in some areas of the plant but remained the same as wild type in other parts of the plant (Figure 22 a). This phenotypic difference could be due to growth conditions or mutations within the plant unrelated to the expression of LaPI, as this was not observed in other transformants. It is likely that no significant phenotypic effects were observed due to the way in which the LaPI protein likely functions in a quartet as the AtPI protein does (Theißen et al. 2016). Studies have indicated that overexpression of one MADS-Box TF is insufficient to elicit a phenotypic change (Krizek and Meyerowitz 1996; Honma and Goto 2001). For example, A. thaliana overexpressing both AP3 and PI exhibited increased petal and stamen growth, whereas overexpression of one gene did not have a significant effect (Krizek and Meyerowitz 1996).In addition, within the quartet in A. thaliana, the AtPI protein forms a heterodimer with the AtAP3 protein via the proteins’ K-domains (Yang et al. 2003). In lieu of this, it is also possible that due to amino acid sequence differences in the K-domain of the LaPI protein and the AtPI protein, the LaPI proteins were unable to bind to the AtAP3 proteins to elicit an effect (Figure B.7). Future studies should examine the effects of knocking out the LaPI gene as well as the effects of concurrent overexpression of LaPI and the other MADS-Box TFs it interacts with in A. thaliana and in lavenders to fully elucidate the role this gene plays in lavenders. 94 4.3.4 LaAP1 In A. thaliana, the AP1 gene encodes a MADS-Box TF involved in the development of sepals in the outermost whorl of the A. thaliana flower (Theißen et al. 2016). Two AP1 proteins combine with two SEP proteins to form a quartet which binds to the promoter region of genes which promote sepal development in the first floral whorl (Theißen et al. 2016) (Figure 1). In addition, the AP1 protein combines with AP3, PI, and SEP in the second floral whorl to give rise to petals (Theißen et al. 2016). Genes similar to the A. thaliana AP1 gene have been described in other flowering plants such as aspen trees, grass (Lolium temulentum), poppy (Papaver somniferum and Eschscholzia californica), and bread wheat (Triticum aestivum) (Gocal et al. 2001a; Murai et al. 2003; Pabón-Mora et al. 2012; Azeez et al. 2014). Homologs of the A. thaliana AP1 gene have shown differing functions from roles in floral organ identity to effects on flowering time and growth (Gocal et al. 2001a; Murai et al. 2003; Pabón-Mora et al. 2012; Azeez et al. 2014). It was hypothesized that the lavender homolog, LaAP1, would function similarly to previously described genes and that its overexpression would impact these processes. Further, it was hypothesized that an increase in expression of PI in wild-type A. thaliana facilitated by expression of LaAP1 would enhance the effects of this gene via additional copies of the protein being present and functioning. To test this hypothesis, wild-type A. thaliana were transformed with the pGA::LaAP1 construct to constitutively and ectopically express the LaAP1 gene. The pGA::LaAP1 construct was transformed into wild-type A. thaliana. A total of 4 F1 seedlings were identified which expressed the LaAP1 gene (Table 9). It was hypothesized that an increase in expression of the AP1 gene woud result in phenotypic changes; however, this was not observed (Figure 23 & 24). Interestingly, it has been found that overexpression of A. thaliana AP1 leads to a decrease in time to flowering in tomato and citrus. Further, it was reported that overexpression of the MdMADS5 gene found in apple, a gene similar to AP1, led to phenotypic changes and early flowering (Kotoda et al. 2002). This was not observed in the present study as there was no significant phenotypic difference between LaAP1 transformants and wild-type A. thaliana (Figure 23 & 24). Although there appeared to be a slight increase in floral bud mass in some transformants, such as in T2AP1A4, this increase was not significant or consistent between transformants. In addition, although many LaAP1 transformants flowered at a young age, this was not consistent between all transformants it is unclear if this effect was due to light conditions in the growth chamber. It is possible that there were no significant phenotypic effects due to 95 incompatibility of the binding domains of the LaAP1 protein and the proteins which must interact with or due to saturation effects described previously. Further, the LaAP1 gene could function differently than that of A. thaliana AP1. Future studies should examine in-depth the effects of overexpression of LaAP1 in a large-scale transformation with only LaAP1 to determine if LaAP1 significantly impacts flowering time in these plants. 4.3.5 LaCAL In A. thaliana, the CAL gene is thought to interact similarly to AP1 and is involved in the enhancing LFY gene expression, promoting the initiation of flowering and the floral meristem identity (Bowman et al. 1993; Ferrándiz et al. 2000). The CAL gene encodes a MADS-Box TF and functions similarly to AP1 and plays a significant role in the floral transition (Bowman et al. 1993; Ferrándiz et al. 2000; Alvarez-Buylla et al. 2006; Kempin et al. 2017). Further it has been shown that the sequences of AP1 and CAL are similar and the genes are likely products of a gene duplication event as the genes also serve mostly redundant functions during floral initiation (Yanofsky 1995; Alvarez-Buylla et al. 2006). Interestingly, it has been shown that CAL cannot function in place of AP1 in A. thaliana, but, AP1 can function in place of CAL (Alvarez-Buylla et al. 2006). In addition, mutations in AP1 and CAL in combination with other MADS-Box genes have been shown to prevent flowering in A. thaliana Landsberg erecta (Ferrándiz et al. 2000). It was hypothesized that LaCAL would function similarly to AtCAL. Further, it was hypothesized that in increase in expression of CAL in wild-type A. thaliana facilitated by expression of LaCAL would enhance the effects of this gene via additional copies of the protein being present and functioning. To test this hypothesis, wild-type A. thaliana were transformed with the pGA::LaCAL construct to constitutively and ectopically express the LaCAL gene. Three F1 seedlings were identified which expressed genes via the CaMV 35S promoter of the pGA vector (Table 9). Two plants expressed the NPT-II gene, and two plants expressed LaCAL, with one plant expressing both (Table 9). Several other plants were identified, but there was insufficient tissue or plant did not amplify either the NPT-II or LaCAL genes. The LaCAL transformants did not exhibit any significant phenotypic changes (Figure 25). A few seedlings, including T1CALB3, appeared to flower at a young age, but this plant was not confirmed to express either the NPT-II or the LaCAL gene and as such, this was not a significant result. The lack of impact of overexpression of LaCAL may have been due to the redundancy of the CAL 96 and AP1 gene functions or due to an inability of the LaCAL homologto interact with the proteins which A. thaliana CAL interacts (Bowman et al. 1993; Ferrándiz et al. 2000; Alvarez-Buylla et al. 2006). Future studies should examine the effects of knockout studies of LaCAL in lavenders to examine the function of LaCAL and LaAP1 in lavenders to determine if the redundant relationship between these genes in A. thaliana is observed in Lavandula. 4.3.6 LaFT The A. thaliana FT gene is a floral pathway integrator and plays a vital role in the induction of flowering in response to photoperiod (Kobayashi et al. 1999; Simpson and Dean 2002; Mizoguchi et al. 2005; Ho and Weigel 2014; Andrés et al. 2015). In A. thaliana, FT promotes flowering in response to long days and is activated by the protein CO in the floral meristem (Koornneef et al. 1998b; Kardailsky et al. 1999; Samach et al. 2000b; Yoo et al. 2016). Further, AtFT promotes flowering through the activation of several downstream floral meristem identity genes and mutations in FT and CO cause a delay in the transition to flowering (Samach et al. 2000b; Mizoguchi et al. 2005). Homolog of the AtFT gene have been identified and described to function in flowering and development in other flowering plants such as Medicago (Medicago trunculata), Garden pea (Pisum sativum), Norway Spruce (Picea abies L. Karst.), and soybean (Glycine max L. Merr.) (Gyllenstrand et al. 2007; Hecht et al. 2011; Laurie et al. 2011; Sun et al. 2011). It was hypothesized that the Lavandula homolog of AtFT, LaFT, would function similarly in lavenders. Further, it was hypothesized that an increase in the expression of FT in wild-type A. thaliana facilitated by expression of LaFT would enhance the effects of this gene via additional copies of the protein being present and functioning. To test this hypothesis, wild-type A. thaliana were transformed with the pGA::LaFT construct to constitutively and ectopically express the LaFT gene. Over 8 LaFT transformants were identified from antibiotic selection plates and grown in soil. Of the plants transferred, there were only two (T1FTA2 and T1FTA8) from the genomic DNA of which the NPT-II and LaFT genes could be amplified (Table 9). Several transformants, including T1FTA1 and T1FTA5 had insufficient tissue to extract cDNA and mRNA. The NPT-II and LaFT genes was also amplified from the cDNA obtained from T1FTA2 and T1FTA8 transformants, indicating that these genes were indeed overexpressed in the transformed plants. (Figure 26). The transformed plants exhibited abnormal sepals, abnormal siliques which varied 97 in size and length, and abnormal flowers (Figure 26). In addition, all transformants exhibited a significant reduction in vegetative growth and leaf mass. Due to the phenotypic differences observed in T1FTA1 and T1FTA5, it is possible that these plants were transformed, but that NPT-II and LaFT were not amplified due to low quality or concentration of mRNA available due to lack of tissue. All LaFT transformants exhibited a phenotype significantly different than that of wild type, with decreased vegetative growth and an apparent reduced time to flowering. A similar reduction in height and decrease in time to flowering was found when AtFT was overexpressed in cassava (Manihot esculenta, Crantz), (Adeyemo et al. 2017). A similar decrease in time to flowering was found when AtFT was overexpressed in Eucalyptus trees and in apples, with apples also showing perpetual flowering (Malus X domestica Borkh.) (Tanaka et al. 2014; Klocko et al. 2016). Thus, FT overexpression leads to early flowering in a variety of plants. As LaFT overexpression also had significant effects on the phenotype of transformants in the present study, further analysis of the LaFT gene would be beneficial for elucidating this gene’s function in lavenders. In addition, future studies should examine the effects of overexpression of AtFT in lavenders in addition to the overexpression of LaFT in lavenders. Further, as many LaFT transformants did not survive or exhibited significantly decreased overall growth, it would be valuable to examine the effects of overexpression of LaFT in A. thaliana using a method in which mRNA and cDNA can be efficiently extracted from minimal tissue. Future studies should examine the effects of overexpression of LaFT in lavenders to determine if similar effects are observed. An early flowering phenotype in Lavandula would be beneficial as it may increase floral production early on, which could allow for increased production of lavender EO. 98 Chapter 5: Conclusion Lavenders are a commercially and medicinally important plant, with many species being cultivated globally for their valuable EO. Lavender EOs have been implicated in a variety of settings from use medicinally as an anxiolytic agent to use in perfumes and personal care products (Rastogi et al. 2001; Herrera 2004; Kasper et al. 2010; Grunebaum et al. 2011). The biological activities and diverse applications of lavender EO constituents and the identification of genes involved in the biosynthetic pathways of important oil constituents have been examined in previous studies, with numerous important genes being identified (Woronuk et al. 2010; Demissie et al. 2011; Sarker et al. 2012; Mendoza-Poudereux et al. 2014; Adal et al. 2019). Thus, significant advances have been made in elucidating the details of important pathways and enzymes involved in terpenoid biosynthesis in lavenders. Although flowers are the main site of EO in lavenders, the molecular mechanisms governing floral initiation and pattern development in these plants have not been adequately investigated. These processes have, however, been examined in other flowering plants and are best described in the model plant A. thaliana (Kang et al. 1998; Searle and Coupland 2004a; Lee et al. 2007; Litt and Kramer 2010; Hecht et al. 2011; Wu et al. 2012, 2017; Melzer and Ru 2016; Gao et al. 2017). To bridge the gap in knowledge, a transcriptomic approach was employed in this thesis to identify and functionally characterize genes that control flower initiation and pattern development in lavenders. Specifically, the objectives of this thesis were to assemble a comprehensive transcriptome database representative of various developmental stages in lavenders, to identify lavender homologs of A. thaliana floral developmental genes, and to functionally characterize a subset of these floral development related genes. The objectives of this thesis were achieved through the assembly of a comprehensive lavender transcriptome from FL, BD, and vegetative SAM tissues of L. x intermedia and L. angustifolia tissues, identification of Lavandula homologs of previously- described A. thaliana floral development genes within the transcriptome via CLC Genomics Workbench 11, and through the expression of Lavandula homologs in wild-type A. thaliana driven by the CaMV 35S promoter of the pGA482 vector. As a proof of concept, several Lavandula homologs were functionally characterized through their overexpression in wild-type A. thaliana. The method employed in this thesis demonstrates a comprehensive procedure for the identification, cloning, and characterization of lavender floral development genes which can be extended to future 99 studies to examining developmental or regulatory genes. This study is the first of its kind in lavenders and the results provide a rich genomic resource for a novel area of research in lavenders. Additionally, the results of this thesis provide valuable insight into a previously unstudied process in lavenders with the identification and characterization of key lavender floral development genes and provides the potential for the manipulation of these genes to improve EO production or yield. 5.1 Development of a Comprehensive Lavandula Transcriptome This thesis first aimed to identify genes involved in floral development by assembling a comprehensive Lavandula transcriptome representative of L. angustifolia and L. x intermedia tissues at different developmental stages to determine if gene homologs to the A. thaliana floral development genes existed in lavenders. A comprehensive literature review was conducted to identify floral initiation pathway genes and floral pattern development genes previously described in A. thaliana. Based on the presence of homologous floral initiation and pattern development genes in A. thaliana and other flowering plants, it was hypothesized that homologsof these genes would exist and function similarly in lavenders (Kang et al. 1998; Theißen 2001; Causier et al. 2010; Ito et al. 2012; Huang et al. 2014; Jaudal et al. 2014; Theißen et al. 2016). Preliminary BLASTx analysis using a previously assembled lavender transcriptome against an annotated A. thaliana protein database revealed the presence of lavender genes with high sequence similarity to many A. thaliana floral initiation and floral development genes. This result provided support for the hypothesis that these genes exist in lavenders. To discover the floral development-related genes in Lavandula, a comprehensive transcriptome database derived from flowers, buds and shoot apical meristem tissues was developed. Assembly of a comprehensive Lavandula transcriptome was then achieved by extracting total RNA from L. x intermedia vegetative SAM and L. angustifolia vegetative SAM, FL, and BD tissues and transcripts were sequenced via Illumina sequencing. Using the RNA-Seq function of CLC Genomics Workbench 11, a comprehensive lavender transcriptome database of 105,294 contigs representative of developing lavender floral tissues was assembled. The parameters of the resulting transcriptome were comparable to other plant transcriptomes and the transcriptome was validated through the identification of 10 important lavender terpene synthase genes (Duan et al. 2012; Lulin et al. 2012; Xu et al. 2013; Lu et al. 2014; Liu et al. 2018b; Adal 100 et al. 2019). This transcriptome thus provides a valuable genomic resource for future studies. Additionally, gene ontology studies classified transcripts based on biological process, molecular function, and cellular component (Figure 5). Although the transcriptome assembled in the present study was comprehensive and reliable, these parameters could be enhanced in future studies by obtaining RNA from additional lavender replicates and from developing floral tissues at smaller increments of time. These additions would allow for an increased comprehensiveness of the transcriptome and thorough representation all genes involved incrementally in floral development, including those which may be expressed transiently, or in low levels in some developmental stages. In addition, species-specific transcriptomes could be assembled to examine the interspecific differences in these genes across lavender species. The comprehensive Lavandula transcriptome constructed in this thesis provides insight into the genes involved in flowering and floral pattern development in Lavandula and provides a valuable genomic resource for future studies. Gene ontology studies provide putative functions for the majority of transcripts within the transcriptome, which may be useful in identifying genes of interest to analyze in future studies. Thus, the transcriptome assembled in this study provides a comprehensive database for the identification and functional characterization of additional important floral initiation and development related genes in lavenders. The assembly of this transcriptome has made a significant contribution to what is known about floral development in lavenders. The identification and characterization of additional genes from this transcriptome will provide further insight into floral development in lavenders as well as the potential for manipulation of the expression of these genes to alter floral development. 5.2 Candidate Identification and Differential Expression Analysis The second objective of this thesis was to identify Lavandula floral initiation and development related genes within the transcriptome and to identify candidates for functional characterization in A. thaliana. The Lavandula transcriptome was annotated via BLASTX analysis in CLC Gemonics Workbench 11 against an annotated A. thaliana protein database. These analyses resulted in the identification of Lavandula transcripts within the transcriptome showing high sequence similarity to A. thaliana floral development related genes. Specifically, over 100 lavender genes homologous to A. thaliana floral initiation and pattern development 101 genes, including repressors and non-repressor proteins, as well as the MADS-Box ABC(DE)-type genes described in A. thaliana were identified within the transcriptome (Table 5). This result supported the hypothesis that there exist floral initiation and pattern development genes in lavenders which are homologous to those described in A. thaliana and other flowering plants. The identification of over 100 Lavandula genes with high sequence homology to A. thaliana floral initiation and development genes provided excellent support for the hypothesis that lavenders develop in a similar way to other flowering plants. Differential expression analyses were performed to examine the expression patterns of the identified genes in L. angustifolia and L. x intermedia SAM, FL, and BD tissues. Results of these analysese provided insight into the expression patterns of the over 100 floral development related genes identified within the transcriptome and revealed that the expression pattern of many genes varied significantly across developmental stages (Figure 6). For example, LaSVP exhibited high expression in the LaSAM in comparison to the LaFL, but low expression in the LaBD in comparison to the LiSAM. This result was consistent with the role of LaSVP as a floral repressor (Hartmann et al. 2000; Liu et al. 2007; Wells et al. 2020). Thus, the results of the differential expression analyses in this study provided insight into the expression patterns of Lavandula floral development genes and was reflective of the variety of roles of these genes. In addition, the results demonstrate that the expression patterns of these lavender genes similarly varied across developmental stages and certain genes may be active or inactive at a given stage, depending on their role in floral development. This result is consistent with what is known about gene expression and was also consistent with other studies showing differential gene expression during the various stages floral development (Kram et al. 2009; Yant et al. 2010; Klepikova et al. 2015). The analysis of expression patterns of the identified genes provides a starting point for subsequent studies examining important floral development genes. Further analysis of these expression patterns may facilitate the identification of additional key genes, thus providing a valuable genomic resource for future studies. In addition, it would be valuable to examine expression patterns of the Lavandula floral developmental genes parallel to their homologs through short increments of time to examine expression patterns through time as well as the expression patterns at the moment of floral transition. 102 Based on homology to an A. thaliana protein sequence and differential expression pattern, 14 full-length Lavandula candidate genes were identified within the transcriptome (Table 6). The identification of lavender genes homologous to those described in A. thaliana indicated that the ABC(DE) floral pattern development model and the initiation processes described in A. thaliana may also be used to describe flowering in lavenders (Koornneef et al. 1998a; Searle and Coupland 2004a, b; Cheng and Wang 2005; Melzer and Ru 2016; Theißen et al. 2016; Irish 2017). To examine sequence similarity between the homologous proteins, MUSCLE alignment of the protein sequences was performed. Results of the analysis indicated high sequence similarity of the Lavandula proteins and each respective A. thaliana homolog and the presence of conserved domains found in other MADS-Box proteins (Alvarez-Buylla et al. 2000; Becker and Theißen 2003). In addition, there were differences in the amino acid sequences between the homologous pairs, as expected in different species. It is hypothesized that the sequence differences may lead to functional differences in these proteins. Together, these results supported the hypothesis that these genes exist and function similarly in lavenders. The identification of Lavandula floral initiation and development-related genes in this study is the first formal description of these genes in lavender and provides the first glimpse into floral initiation and pattern development in lavenders. These genes have not been formally examined in lavenders and the results of this thesis serve to bridge this gap in knowledge and begin a new area of research in lavenders. With the identification of over 100 floral development-related genes, including numerous full-length genes, this transcriptome serves as a rich genomic resource for future studies examining floral development and other developmental processes in lavenders, thus providing a starting point for many future studies. 5.3 Cloning and Functional Characterization of Lavandula Homologs Given the high sequence similarity between lavender floral development genes and those of other flowering plants, it was hypothesized that these genes would function in lavenders as they do in A. thaliana. As a proof of concept, 6 of the full-length genes were functionally characterized through overexpression in wild-type A. thaliana var. Landsberg erecta driven by the CaMV 35S promoter. It was hypothesized that given the high sequence similarity to A. thaliana floral development related genes, the lavender homologs would function similarly to the 103 A. thaliana genes. Transformation was successful for each of the lavender genes, LaPI, LaAP1, LaCC, LaFT, LaCAL, and LaSVP and F1 transformants expressing the transformed gene were identified for each gene construct. Functional characterization of these genes provided insight into the function of these genes in lavenders and provided evidence that these genes likely function similarly in lavenders as they do in A. thaliana and other plants. Ectopic expression of these genes in A. thaliana had varying phenotypic effects, with the most drastic phenotypic effects observed in LaSVP and LaFT mutants. The LaSVP gene, was shown to be a repressor of flowering in lavenders through functional annotation in Arabidopsis. As found in previous overexpression studies, this gene repressed the development of flowers and delayed time to flowering. Ectopic expression of the LaSVP gene in A. thaliana caused a marked phenotypic change, including suppression of petal formation, increased sepal development, sterility, as well as a delay in flowering in comparison to wild- type plants. The results of this study indicate that LaSVP acts as a floral repressor in a dosage-dependent manner, functioning similarly to other homologs of SVP, including AtSVP (Lis-Balchin and Hart 1999; Jaudal et al. 2014). Further, the effects of LaSVP on floral organ structure are consistent with the function of AtSVP in control in the floral meristem and its role in repression of B, C, D, and E-type floral homeotic genes, having morphological implications (Liu et al. 2007; Li et al. 2008; Gregis et al. 2009). LaFT transformants exhibited an early onset of flowering and a significant reduction in vegetative and floral growth. Transformants LaPI, LaCAL and LaAP1 did not exhibit significant phenotypic differences compared to wild type. It was hypothesized that these genes also function similarly to their A. thaliana homologs. It is possible that the lack of phenotypic effects could be due to the manner in which these genes function as MADS-Box TFs in protein complexes (Figure 1) (Theißen et al. 2016). For example, AtPI is a B-class gene which binds to the DNA in a quartet with AtSEP, AtAP3, and AtAP1 to promote petal development and with AtSEP, AtAP3, and AtAG to promote stamen development (Causier et al. 2010). It is also possible that amino acid differences in the binding domains of the Lavandula and A. thaliana impacted the ability of the proteins to interact productively. It is also possible that the increase in concentration of a given TF did not increase functions elicited by that TF as the concentrations of the other MADS-box TFs and other interacting proteins did not change. Thus, it is possible that there was no phenotypic change due to a saturation effect with there being a finite number of the other TFs available to bind with the overexpressed lavender TFs (Deitchman et al. 2018). In addition, there 104 may be differences in post-translational modifications of the proteins in A. thaliana and Lavandula. One limitation of this study thus may be that some of the MADS-Box TFS must be overexpressed in combination with other TFs in order to fully determine the effects of overexpression on floral morphology, as the quartets are limited by the TF with the lowest concentration. Several studies examining double mutants of flowering development genes to have significant effects (Pautot et al. 2001; Mizoguchi et al. 2005; Gregis 2006; Hecht et al. 2011; Zhao et al. 2015). As such, future studies should examine the effects of overexpression of multiple Lavandula floral development genes in A. thaliana. Future studies should further functionally characterize the genes examined in the present study as well as functionally characterize additional repressors and initiators of floral development identified within the Lavandula transcriptome. It would be of interest to simultaneously overexpress all member of a quartet in a given whorl to examine the effects on flowering (Figure 1). The genes identified within this thesis should also be further functionally characterized through knockout studies in A. thaliana to examine the developmental effects of the absence of expression of these genes and further establish their role in floral development in lavenders. These genes should then be examined through overexpression and knockout studies in Lavandula. Although transformation methods have been described in lavenders, the consistency and efficacy of transformation is reported to be inconsistent and the long generation time makes short-term gene expression studies difficult (Mishiba et al. 2000; Nebauer et al. 2000). Recent progress has been made in the Mahmoud lab towards the optimization of effective Lavandula transformation protocols. 5.4 Study Limitations Although these analyses provided valuable insight into the expression patterns and provided a starting point for future studies, some limitations existed. Limitations of the analyses included lack of biological replicates used for transcriptome assembly, lack of replication of the differential expression analysis, and absence of qPCR of all identified genes. Future studies should include an increased number of biological replicates from which RNA is extracted for transcriptome assembly to increase accuracy of the representation of the totality of the genes expressed in these tissues, thus increasing the strength of the transcriptome and the expression analysis results. In addition, future studies may include additional Lavandula species in the 105 transcriptome assembly to examine expression patterns across tissues in other species and examine differences in the sequences in the floral development genes across various lavender species. As there were few biological replicates used, there could exist bias in the expression patterns of the genes due to lack of replicate source tissue. An increase in the number of biological replicates would thus strengthen the transcriptome. In addition, it would be beneficial to assemble multiple transcriptomes and perform Differential Expression analyses on the various transcriptomes to provide replicates of the differential expression results, thus strengthening the results. The observation of the same or similar expression patterns of the identified genes across numerous tissues in numerous transcriptomes would provide additional support for the expression patterns elucidated in this thesis. Thus, increased biological replicates and increased replication of expression analyses would provide further support for the observed expression patterns. In addition, a large-scale qPCR analysis of all identified candidate genes could be performed to validate the results of the differential expression analyses. This large-scale qPCR was beyond the scope of this study but may be beneficial to provide further support for the expression patterns elucidated in this study. Lastly, it would be beneficial in future studies to examine additional developmental stages and identify additional genes which were not identified in this study. In addition, it would be interesting to examine the expression patterns of these genes at smaller time intervals. Future studies should further characterize the initiation pathways which govern flower initiation in lavenders. As the generation time between A. thaliana and lavenders differs greatly, it would be valuable to examine the differences in the initiation pathways in lavenders and A. thaliana and the photoperiodic response in lavenders. A further understanding of the initiation pathways and the genes involved in the photoperiodic response in lavenders would potentially lead to the manipulation of flowering time in lavender through the manipulation of expression of these genes either through overexpression or through silencing. In addition, pattern development should also be further examined in lavenders. The identification of the genes which lead to the duplications of flowers along the lavender spike and the division of the SAM are an important area of research. A further understanding of the division of the lavender SAM as well as the processes which govern the development of the lavender floral pattern may lead to the ability to manipulate the expression of genes within the SAM which govern floral pattern in lavenders. 106 5.5 Summary The objective of this thesis was to examine the previously unstudied area of floral development in lavenders. The objective was successfully achieved by the completion of three main goals. First, a comprehensive transcriptome database was assembled from RNA of lavender tissues at different developmental stages. The parameters of this transcriptome were comparable to other plant transcriptomes assembled in a similar manner. Second, BLASTx analysis of the transcriptome against an annotated A. thaliana protein database facilitated the identification of over 100 Lavandula homologs of previously- described A. thaliana floral development genes and assigned putative functions to these genes. Lastly, a subset of these Lavandula genes were functionally characterized through ectopic expression in wild-type A. thaliana. Of the characterized genes, the floral repressor, LaSVP, was identified and ectopic expression of this gene in A. thaliana caused marked phenotypic changes including suppression of petal formation, increased sepal development, sterility, and delayed flowering. In addition, LaFT transformants exhibited a significant decrease in vegetative growth and decreased time to flowering, which was consistent with other studies (Hecht et al. 2011; Azeez et al. 2014; Tanaka et al. 2014; Klocko et al. 2016). Similarities in the function of LaSVP and AtSVP and LaFT and AtFT, provides evidence that floral development in lavenders may closely resemble that of A. thaliana and that the genes described in A. thaliana likely function similarly in lavenders. The present study has made a significant contribution to research in floral development in lavenders. This study is the first of its kind and a valuable lavender genomic resource was generated. The work in this thesis and the genes identified in the present study have given rise to additional studies. Specifically, this thesis provided the basis and the genes for three undergraduate theses and the start of several additional studies in the Mahmoud lab, providing a significant contribution to research in lavenders. Several genes of the over 100 genes discovered in this study were given to undergraduate students to classify in three separate honors theses. These genes were characterized, providing further significant contributions to research in lavender floral development. Thus, the results of this study have significantly contributed to the Mahmoud lab and developmental research in lavenders. The results of this thesis mark the first formal identification and characterization of floral initiation and pattern development genes in lavenders and provide a significant contribution research in these plants. Further, the identification and characterization of the floral repressor, 107 LaSVP, has provided valuable insight into a previously- unstudied process in lavenders with the publication of these results in Planta (Wells et al. 2020). The results of this study provide evidence that flowering in lavenders is governed by similar processes as are observed in A. thaliana and other plants as the floral development genes in Lavandula function similarly to those reported in A. thaliana. In addition, the identification of over 100 floral development-related genes within the transcriptome provides a resource for future studies to functionally characterize other floral development-related lavender genes. The successful expression of these Lavandula genes in A. thaliana provides an efficient means of functional characterization of other lavender floral development related genes. The techniques utilised in this thesis may thus be used in future studies to functionally characterize other floral development genes. The results of this thesis provide compelling evidence that the floral development genes in Lavandula likely function similarly to those reported in A. thaliana and other plants. Furthermore, this study provides a rich genomic resource for future identification of other developmental genes and insight into the genes involved in flowering and floral pattern development in the genus Lavandula. 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Sample µg/ µl 260/230 230/280 1 228.9 2.30 1.89 2 238.0 2.09 1.86 Table A.3 Nanodrop Spectrometer data of extracted E. coli JM109 plasmids. Sample µg/ µl 260/230 230/280 CC 100.1 1.88 0.96 CAL 74.9 1.93 1.06 SVP 70.3 1.88 1.96 PI 92.9 1.87 0.48 GB 80.1 1.83 0.59 FT 79.8 1.88 1.85 AP1 96.7 1.92 1.54 AP2 31.2 1.93 0.32 Table A.4 Culture-forming units per plate of heat-shock transformed E. coli cells on LB tetracycline medium. Gene Dilute Plate Concentrated Plate CC 9 >100 CAL 10 >100 SVP 73 >100 PI 48 >100 GB 41 >100 FT 60 >100 AP1 61 >100 Plasmid 0 0 128 Table A.5 RPKM values of floral development genes in L. angustifolia and L. x intermedia tissues based on Differential expression for RNA Seq analysis. Gene Tissue L. x intermedia VM L. x angustifolia VM L. x angustifolia FL L. angustifolia BD CC 0.00 0.36 102.40 16.68 CAL 4.44 7.09 41.56 45.93 SVP 25.42 11.51 0.61 2.80 PI 0.68 1.23 145.75 234.96 FT 0 10.55 28.90 79.67 AP1 0 0 24.68 53.02 Not cloned: SM 2.39 16.82 73.94 54.64 AP2 7.63 1.84 0 3.05 AP3 2.62 3.16 197.12 193.87 SOC1 28.03 27.61 4.56 4.47 FLC 8.01 22.30 25.79 22.31 SEP3 0 0.99 198.45 198.96 AG 0 0.19 45.95 21.73 EMF2 10.62 16.29 13.32 17.90 129 Appendix B – Supplementary Figures Figure B.1 Multiple Sequence Alignment of protein sequences of L. angustifolia Crab’s Claw (LaCC) and Arabidopsis thaliana Crab’s Claw (AtCC). AtCC has zinc finger and helix loop helix domains and is a member of the YABBY family of transcription factors which is involved in carpel and nectary development (Bowman and Smyth 1999). Figure B.2 Multiple Sequence Alignment of protein sequences of L. angustifolia Shoot Meristemless (LaSM) and A. thaliana Shoot Meristemless (AtSM). AtSM is a TALE/KMNOX Homeobox transcription factor involved in cell proliferation in the SAM (Aida et al. 1999; Byrne et al. 2002). The bolded portions of protein sequence indicate the sequence of the ELK Domain and homeobox. 130 Figure B.3 Multiple Sequence Alignment of protein sequences of L. angustifolia Embryonic Flower 2 (LaEMF2) and A. thaliana Embryonic Flower 2 (AtEMF2). AtEMF2 is a transcription factor involved in repression of flowering in A. thaliana (Moon et al. 2003b). The Zinc finger and VEFS-Box domains are shown in bold. Figure B.4 Multiple Sequence Alignment of protein sequences of L. angustifolia Flowering Locus T (LaFT) and A. thaliana Flowering Locus T (AtFT). AtFT is a gene involved in floral initiation in response to photoperiod (Andrés et al. 2015). The FT protein acts as a phosphatidylethanolamine binding transcription factor (Wang et al. 2015). 131 Figure B.5 Multiple Sequence Alignment of protein sequences of L. x intermedia Apetala2 (LiAP2) and A. thaliana Apetala2 (AtAP2). AtAP2 is a transcription factor involved in sepal and petal development in A. thaliana (Theißen et al. 2016)s. The bolded portion of the protein sequence indicates the sequence of the two DNA-binding domains. 132 Figure B.6 Multiple Sequence Alignment of protein sequences of Lavandula angustifolia Cauliflower (LaCAL) and Arabidopsis thaliana Cauliflower (AtCAL). AtCAL is a MADS-Box TF involved in floral meristem identity specification in A. thaliana (Bowman et al. 1993). The bolded portion of protein sequence indicates the sequence of two highly conserved domains of the MADS-Box transcription factors, the MADS-Box and the K-Box domains. Figure B.7 Multiple Sequence Alignment of protein sequences of L. angustifolia Pistillata (LaPI) and Arabidopsis thaliana Pistillata (AtPI), which is required for petal and stamen specification. The bolded portion of protein sequence indicates the sequence of two highly conserved domains of the MADS-Box transcription factors, the MADS-Box and the K-Box domains (Krizek and Meyerowitz 1996; Theißen et al. 2016). 133 Figure B.8 Multiple Sequence Alignment of protein sequences of L. angustifolia Apetala1 (LaAP1) and A. thaliana Apetala1 (AtAP1). AtAP1 is a MADS-Box TF involved in sepal and petal development (Theißen et al. 2016). The bolded portion of protein sequence indicates the sequence of two highly conserved domains of the MADS-Box transcription factors, the MADS-Box and the K-Box domains. Figure B.9 Multiple Sequence Alignment of protein sequences of L. angustifolia Apetala 3 (LaAP3) and A. thaliana Apetala3 (AtAP3). AtAP3 is a MADS-Box transcription factor involved in stamen development in A. thaliana (Theißen et al. 2016). The bolded portion of protein sequence indicates the sequence of two highly conserved domains of the MADS-Box transcription factors, the MADS-Box and the K-Box domains. 134 Figure B.10 Multiple Sequence Alignment of protein sequences of L. angustifolia Suppressor of CONSTANS1 (LaSOC1) and A. thaliana Suppressor of CONSTANS1 (AtSOC1). AtSOC1 is a MADS-Box transcription factor involved in floral transition (Moon et al. 2003a). The bolded portion of protein sequence indicates the sequence of two highly conserved domains of the MADS-Box transcription factors, the MADS-Box and the K-Box domains. Figure B.11 Multiple Sequence Alignment of protein sequences of L. angustifolia Sepallata 3 (LaSEP3) and A. thaliana Sepallata 3 (AtSEP3). AtSEP3 is a MADS-Box transcription factor required for floral organ development (Theißen et al. 2016). The bolded portion of protein sequence indicates the sequence of two highly conserved domains of the MADS-Box transcription factors, the MADS-Box and the K-Box domains. 135 Figure B.12 Multiple Sequence Alignment of protein sequences of L. angustifolia Agamous (LaAG) and A. thaliana Agamous (AtAG). AtAG is a MADS-Box transcription factor involved in carpel development in A. thaliana (Theißen et al. 2016). The bolded portion of protein sequence indicates the sequence of two highly conserved domains of the MADS-Box transcription factors, the MADS-Box and K-Box domains. Figure B.13 Multiple Sequence Alignment of L. angustifolia Flowering Locus C (LaFLC) and A. thaliana Flowering Locus C (AtFLC). AtFLC is a floral repressor in A. thaliana (Henderson and Dean 2004). 136 Appendix C - Supplementary Tables Table C.1 Genes involved in floral development in A. thaliana. Genes include ABCDE-type MADS-Box and other transcription factors including, corepressors, repressors, and floral initiation genes. Gene Gene Type Function Reference ABCDE-Type Genes APETALA1 (AP1) MADS-Box A-type function gene Transcription factor Sepal development. Petal development when combined with B-class genes. Meristem identity gene (Yanofsky 1995) APETALA2 (AP2) MADS-Box A-type function gene Transcription factor Sepal development. Petal development when combined with B-class genes. (Theißen et al. 2016) APETALA3 (AP3) MADS-Box B-type function transcription factor Stamen development when combined with AGAMOUS (Theißen et al. 2016) PISTILLATA (PI) MADS-Box B-type function gene Transcription factor Stamen development when expressed with AGAMOUS (Theißen et al. 2016) AGAMOUS (AG) MADS-Box C-type function gene Transcription factor Carpel development (Theißen et al. 2016) SEEDSTICK (STK) MADS-Box D-type function gene Transcription factor Ovule identity (Theißen et al. 2016) SHATTERPROOF1 (SHP1) MADS-Box D-type function gene Transcription factor Ovule identity (Theißen et al. 2016) SEPALLATA1-3 (SEP1-3) MADS-Box E-type function gene Transcription factor Expressed in all whorls (Theißen 2001) Repressors SHORT VEGETATIVE PHASE (SVP) Repressor of flowering-time genes Represses flowering and during vegetative growth phase. (Hartmann et al. 2000) Additional Genes CLAVATA 1 Floral meristem identity gene Establishes and maintains floral meristem Maintains floral meristem size (Clark et al. 1993) CAULIFLOWER (CAL) Floral initiation gene Specification of floral meristem identity (Yanofsky 1995) LEAFY (LFY) Floral initiation gene Meristem identity gene Aids in stamen and petal identity (Yanofsky 1995) FCA Flowering time controlling gene Promotion of flowering (Page et al. 1999) CONSTANS (CO) Circadian rhythm gene Initiation of flowering Flowering time control gene (Searle and Coupland 2004a) FLOWERING LOCUS T (FT) Circadian rhythm gene Flowering time gene and initiation of flowering in response to day length. Overexpression leads to severe early flowering. Mutation leads to late flowering under long days. Activation of AP1, AP3, SOC1, FUL, CAL (Searle and Coupland 2004a; Mizoguchi et al. 2005) SUPPRESSOR OF OVEREXPRESSIOELF3N OF CO 1 (SOC1) Circadian rhythm gene Initiation of flowering in response to day length. Overexpression leads to severe early flowering, (Searle and Coupland 2004a) The early flowering 3 (ELF3) Flowering initiation in response to photoperiod Flowering initiation in response to photoperiod. Mutants cause extreme early flowering Regulation of floral identity genes and floral meristem genes (Zagotta et al. 1996a) 137 Table C.2 Full-length sequences of Lavandula genes identified within the Lavandula transcriptome. Source tissue is the tissue from which the gene was originally amplified during construction of the comprehensive Lavandula transcriptome, where VM = vegetative meristem, FL = flower, and BD = floral bud and ORF = open reading frame. Gene name Source tissue Lavandula ORF sequence Shoot Meristemless L. angustifolia SAM ATGCGTGTGTACAACAGTAGCACAGATAAAACAAACAAACAAACAGAAAACATTTTTTTTCCTGGGTTTATAAAGAGAGAGAGAGGCATATTATTAGCTGGAGTATTAGAGAAAGAGAGAGAAATGGAGCGCGGCGGTGGCGGTGGCTCGAGTAGCAATTCTTGTTTTATGTCTTTCGGAGAAAACAATAATGGGTTTTGCCCTATACTCATGATGCCTTCGATGGTCTCTTCTCACTCTAATCCGGATCCGGGAAATAACCTATTTCTCCCTCTTACTCATCAAGACATGAACCGCGGCGGCGGCGGCAGTTCGTCGGTGATGGTTGAAGATCAGAATAGCCATAACAAGAATATTACGACCCCAGGGTATTATTTTATGGAGAACAGTAATGAAGCCGGTGGTTGTTCTGCTAGGGCTAAAATCATGGCTCATCCTCACTACCACCGCCTCTTGTCAGCTTACGCCAACTGCCAAAAGGTAGGAGCGCCGCCGGAAGTAGTGGCGAAGCTGGAGGAGGCCTGCGCTTCCGTGGCGGCGATGAACCGGCAAGGCACAAGCTGCGTGGGCGAAGATCCTGCGCTGGATCAATTCATGGAGGCCTACTGTGAGATGCTGAGCAAGTACGAGCAAGAACTGTCGAAACCCTTCAAAGAAGCCATGCTTTTCCTTTCCAGAATTGAGTGCCAGTTCAAAGCCCTCACCCTCTCGCCTCCCACCTCCGCTGCTTGTAATGATGCAATGGAGACGAATGGGTCATCCGAAGAAGAGACCGATGCCAACAACAGTTTCATAGACCCTCAAGCAGAAGACAGAGAACTCAAAGGCCAGCTCCTACGCAAATACAGCGGATACTTAGGCAGCCTCAAGCAAGAATTCATGAAGAAGCGAAAGAAAGGCAAGCTGCCTAAGGAAGCTCGGCAGCAGCTGCTCGAATGGTGGAGCCGCCACTACAAATGGCCTTATCCATCAGAATCACAGAAGCTGGCGTTGGCTGAGTCGACAGGGCTTGACCAGAAGCAGATAAACAACTGGTTTATCAACCAAAGGAAGAGGCATTGGAAGCCGTCTGAGGATATGCAGTTTGTAGTGATGGATGCTGCTCATCCTCATTACTACATGGATAATGTGTTGGGTAACCCTTTTCCGATGGATATTTCGCCCGCGCTGCTCTAA Crab’s claw L. angustifolia FL ATGGATTTGGTTCAACAAGCTTCCGAACATGTTTGCTACGTCCGCTGCACCTTCTGCAACACCGTTCTCGCGGTTGGGATTCCATGCAAGAGGCTGATGGACACTCTGACAGTGAAATGTGGGCATTGCAGCAATCTCTCATTTCTTAGCACAAAACCTCAGCCTCAAGGACACTGCTATGATCATCAAACAAGCGTTCATTATCAGGCTTTCTGTAATGAGATCAAGAAGGGGCAGTCTGCATCATCTTCTTCATCCACTTCGAGCGAACCCTTGTCCCCAAAAGCTCCATTTGTGGTCAAACCTCCTGAGAAGAAGCATAGGCTACCATCTGCATACAATCGGTTTATGAAAGAAGAGATTCAGCGCATCAAGGCGGCTAATCCGGAGATACCACATAGAGAGGCTTTCAGTGCAGCTGCAAAAAATTGGGCTAGGTATATTCCAAACACCCCTCAGGCGTCTGGTTCTAGCAGCATTACTAAGGCCTGA Cauliflower L. angustifolia BD ATGGGGAGGGGGAAGGTGGAGCTGAAGAGAATCGAGAACAAGGTGAACCAGCAGGTGACTTTCTCGAAGAGGCGA138 TCGGGGCTTCTGAAGAAAGCCCATGAGATCTCTGTGTTGTGTGATGCTGAAGTTGCTTTGATTGTTTTCTCTCATAAGGGGAAGTTGTTTGAGTACTCCACTGATTCTTGCATGGATAGGATCCTTGAGAAGTATGAAAGATACTCATTTGCAGAAAGGCAGTTAATTGCTAGTGAGCCTGAGTCACCTGTTAATTGGACCCTCGAACACAGCAAACTTAAGGCTAGGATTGAGCTCTTGCAAAGAAATCATAGGCACTACATGGGCGAAGATTTGGACTCGATGAGCGTGAAAGATCTGCAGAATCTGGAGCAGCAGCTTGACACTTCGCTTAAATGCATAAGATCAAGAAAAAATCAGCTCTTGTTCGATTCGATCTCCGAGTTGCAGCAAAAGGAGAAAGCTATACAAGAGCAGAACAGCATGCTGGTAAAGAAGATCAAAGAGAAGGAGAATGAAATGGGACAAATGGGACAACAGTCACAGTGGGTGCAGCAAAACCCTAGCACCAACACAGCAGCACTTCCACCACCAGCACCACCACCTCAATATCTCATGTCATCTCAACTCCCTTGCTTAAACATAGGAGGGGGATCGTACGAGGAAGCATCCGAGGCCACAAGGAACGAGCTAGACCTCACTCTTGACTCACTCTATTCTTGCCATCTTGGATGCTTTGCCGCATGA Short Vegetative Phase L. angustifolia SAM ATGGCTAGAGAGAAGATTCAGATCAAGAAAATCGACAACGTCACCGCCCGGCAGGTCACTTTCTCCAAGAGGAGAAGAGGGCTCTTCAAGAAGGCCCAAGAACTCGCCGTTCTCTGCGACGCCGATGTGGGACTCATCATCTTCTCCTCCACCGGCAAACTCTTCGAATATGCCAGTACAAGTATGGATGATATAGTGGGAAGGCATAATTTGCACTCCAAGAACCTTAGCAAGTTGGATCAGCCATGCCTCGAGCTGCAGCTAGTGGAGGATAGCAACCACTCCAGGCTGAGCAAAGAAGTGGTTGAGAGAAGCCATCAGCTAAGGCACATGAGAGGAGAAGAACTTCACGAATTAAGTGTTGAAGAGCTGCATCATCTGGAGAAGTCTCTTGAAGTTGGACTAACTCGCGTCATAGAGAAGAAGGGAGAGAAAATCATGACCGAGATTGGTCAGCTTCAAGAAAAGGGGATGGAACTCATGGAAGAGAACAAACGACTAAGGATGCAGGTGAGAGATTTATCGAATGGGGCGAGGAGAATGGGCACAATTACTAGTGAGTCAGAGATAGTAATGTACGAAGAAGGGCAGTCGTCGGAGTCCGTCAAAAACGCCTGCAACTCCACCGGCCCGCCGCAAGACTACGACAGCTCATATACATCTCTCAAGCTCGGGCTTCCTTACACTGGCTGA Pistillata L. angustifolia FL ATGAAAAAGGCTAAGGAGATCAGCGTCTTGTGTGATGCTCAAGTCTCCGTTATCATATTCGCTAGTTCTGGGAAGATGCACGACTTCTGCAGCCCTTCTACTACGCTGGTTGACATGTTGGATCAGTACCATAAACTCTCTGGGAAGAGGCTCTGGGATGCAAAACATGAGCACTTGGAGAATGAAATCAACAGAATCAAGAAAGAGAATGACACCATGCAGATTGAGCTGAGGCACCTGAAAGGAGAAGATATATCGATTTTGAGTTACAAGGAGCTGATGGGGTTGGAGGAAGCTCTTGAGAATGGAATCACAACACTCAAAGCCAAGCAGATGGAGTTCGTCCGGATGATGAGGAAACATAATGACATGATGGAGGAGGAGAACCAGAACCTCCTATTTAAGCTGAGGCAGATGCATTTGGATCCAATGGATGACACCGTACTGGAAGCGCAAGGTGTGTATGATCATCAGGGTGTTGCTGTTGCAGACTACGAAACACACCAGATGCCGTTCGCCTTCCGTGTCCAGCCAATGCAGCCTAATCTGCAGGAGCGGTTCTAG 139 Flowering Locus T L. angustifolia BD ATGCCAAGAGATCGAGACCCTCTAGTGGTAGGTAGAGTTATAGGAGATGTTTTGGATCCATTCACAAGATCCATTGGACTGAGAGTTGTTTATGGGAACAGAGAGGTTACTAATGGTTGCGAGTTTAGGCCCTCTCAAATTGTCAACCAGCCTAGGGTTGAAGTTGGAGGGGAAGATCTTCGCACTTTCTTCACTCTGGTCATGGTGGACCCTGATGCTCCAAGCCCCAGTGATCCTAATCTTAGAGAATACTTGCACTGGCTAGTGACTGATATTCCAGCAACTACTGGAGCAACCTTTGGACAAGAGATCGTGTGTTACGAGAGTCCACGGCCTTCGATGGGGATCCACCGCCTGGTCTACATGTTGTTCCGGCAGTTGGGGCGGCAGACGGTCTACGCCCCCGGTTGGCGCCAAAACTTCAACACGAGGGACTTTGCGGAGCTCTACAACCTTGGCTCCCCGGTGGCTGCGGTGTATTATAACTGTCAGAGAGAGAGTGGAACAGGCGGGAGAAGACGGTGA Apetala1 L. angustifolia FL ATGGGTAGAGGGAAAGTGCAACTGAAGAGGATAGAGAACAAAATCAACAGACAAGTAACCTTCTCTAAGAGGAGAGTTGGCTTGTTGAAGAAAGCCCATGAGATCTCTGTGTTGTGTGATGCTGAAGTTGCTTTGATTGTTTTCTCTCATAAGGGGAAGTTGTTTGAGTACTCCACTGATTCTTGCATGGATAGGATCCTTGAGAAGTATGAAAGATACTCATTTGCAGAAAGGCAGTTAATTGCTAATGAGCCTGAGTCACCTGTTAACTGGACCATCGAACACAGCAAACTTAAGGCTAGGATTGAGCTCTTGCAAAGAAATCATAGGCACTACATGGGCGAAGATTTGGACTCGATGAGCGTGAAAGATCTGCAGAATCTGGAGCAGCAGCTTGACACTTCGCTTAAATGCATAAGATCAAGAAAAAATCAGCTCTTGTTCGATTCGATCTCCGAGTTGCAGCAAAAGGAGAAAGCTATACAAGAGGAGAACAGCATTCTGGTAAAGAAGATCAAAGAGAAGGAGAATGAAATGGGACAAATGGGACAACAGTCACAGTGGGTGCAGCAAAACCCTAGCACCAACACAGCAGCACTTCCACCACCAGCACCACCACCTCAATATCTCATGTCATCTCAACTCCCTTGCTTAAACATAGGAGGGGGATCGTACGAGGAAGCATCCGAGGCCACAAGGAACGAGCTAGACCTAACTCTTGACTCACTGTATTCTTGCCATCTTGGATGCTTTGCCGCATGA Apetala2 L. x intermedia SAM ATGGAATATTCCGGCAGCTCTAGTTCCTCCATTGTTAATGTGGAGACTTCGAGCACCGCCGGCGATGAGAACTCATGCTCCGATCATTTCGTCGGTTTCGATATTTGGAAGAGCAACTGCGATTACGAGGAGAGTGAGAAATCGAGCTGCGGTTACGTGACGAAGGAGTTTTTCCCGGGGACCGGAGGGGGGCCGACGCAGTGTTTGGATCTCTCTGAGAACCGGCATGAGCTGATGGAGCAGAGGATTATTTCTCGGGAGGAGCAGCAGCAGAAGCGGCAGCTAGTGAAGAAGAGTCGGCGGGGACCTAGGTCTCGCAGCTCTCAGTATCGCGGCGTCACATTCTATCGCCGCACCGGACGCTGGGAATCGCACATTTGGGACTGTGGTAAACAAGTTTATTTAGGGGGATTTGACACTGCACATGCTGCGGCACGGGCATACGACCGTGCTGCAATTAAATTTCGTGGACTGGATGCAGACATCAATTTTAATGTTGATGATTATTATGAAGATTTTAAGCAGATGAAGAATTTAACCAAGGAAGAATTTGTGCACATACTTCGGCGCAAGAGTACAGGATTCTCAAGGGGAAGTTCGAAGTATAGGGGGGTGACGTTGCACAAATGTGGACGGTGGGAAGCTCGGAT 140 GGGGCAGTTTCTTGGCAAGAAGTATATATATCTTGGATTGTTTGACACTGAGATAGAAGCTGCAAGGGCATATGATAAAGCTGCTATCAAGTGCAATGGAAGAGAAGCAGTTACCAATTTTGAGCCGAGTACATATGGTGAAGAGCTGAGCTCTGAGGGTGTCATTGGAGCTGGCGAGAACCACAATCTCAACCTGAATTTGAGCATTGCTCCCCCTGAAGGAGCTCATTATGAAATTCATAACATGGAGATGGGAAGCTCTCAGATGCACAGTGGCTTATGTAACCGGCCTGAAGGCGGACTAAAGAGCTCTGCTTCAGCCTCTGTGGCAGCTCAATCTTCCCATGAACATGTAAATGTATGTGAATACCCTTTTGTTTGGAGGGGAGTCGATTCTTGTCTTCCTCCCATTTATAAGGGAACAACAACAGAGAAGGTTGCGGAACTTGATTCTAATGAACATTGGACTAGAAACTCCCCGAACAATCTTCATGGGGATTATCCTTTACCATTCTTCTCTACTGCAGCATCATCAGGATTCTCTACTTCATCAGCTGGTATCCATCAGTTGCCCTTCAGCTCGAGATCCCCCAACCATTATCTTCCACCTGTCACCAATTTCAACAATGGATCTCATTACTACTGGAGGTGA Apetala3 L. angustifolia BD ATGGCTCGTGGGAAGATCCAGATCAAGAAAATAGAGAACCAAACAAATAGGCAGGTAACTTACTCCAAGAGAAGAAATGGTTTGTTCAAGAAAGCTCATGAGCTTACTGTTCTTTGTGACGCAAAGATATCTATCCTTATGATCTCCAGTACTCAGAAACTTCATGAATATATCAGCCCCACAGTCACGACAAAGCAGATTTTTGATGAATACCAGAAGACTGCTAGGGTTGATCTTTGGAGCTCCCACTACGAGAAAATGCAAGAGCACTTGAAGAAGCTGAAGGAGATTAACAGGAATCTGAGAAAGGAGATTAGGCAACGGATGGGAGAAAGCTTGAACGACCTACCGTACGACCATATGGTTAATCTCATCGAAGATATGGACAAGTCTCTCAAGTTGATTCGCGAAAAAAAGTACAAAGTCATCAGCAGCAAAATTGATACCGGCAAGAAAAAGCTGAGGAATGTTGAAGAAATACATAGAGGCCTTGTGCTTCGATTTGATGCAAAGCAGGATGATCCACACTACGGCTTAGTTGAGAATGAAGGTGATTATAATTCCGTTCTGGGATTTCCACACGGCGGGGCAAGGATCATCGCGCTTCGCCTGCCGCCTAATCACCACCCTCACCACCACCACCATGCTAGCCTTCACAGTGGAACCGGAGCCTCAGACCTCACTACATTTGCCTTGCTTGAGTGA Suppressor of CONSTANS1 L. x intermedia SAM ATGGTGAGGGGGAAGACTCAGATGAGGCGTATAGAGAACGCCACAAGCAGGCAGGTGACCTTCTCAAAGAGGAGAAATGGGCTGCTCAAGAAGGCCTTTGAGCTCTCAGTTCTTTGTGATGCTGAGGTGGCACTCATCATCTTCTCTCCTAGAGGCAAGCTCTATGAATTTGCAAGCTCAAGCATGCATGAAACGATCGAGCGCTACCAGAAGCACACGAAAGATAATCAAGCCAACAACCTTCCTCCCGAGCACAACATGCAGCACTTGAAGCATGAAGCAGCAAGCATGATGAAAAAAATCGAGCAGCTAGAACAAGCAAAGCGGAAGCTGCTTGGGGAAAGCTTAGGCTCATGCAACATGGAGGAGCTGCAGCAGCTAGAACAGCAGCTCGAGCGGAGTGTCACCACCATTCGTGCAAGGAAGATGCAAGTGTTCAAGCAGCAGATCGAGCACTTGAAGGAGAAGGAGAAAGCCCTAGCTGCTGAGAATGCAATGCTATCTGAGAAGTTTGTGCTTCAAACGCAAGGAGGGAGAGGGGAAGGAGGGTCGAAGGAGGAGAGGGCGGTGGCGCCGTCGGCGGAGATAAGTGAGGTATCGGATGTGG141 AGACGGAGTTGTTTATTGGGCCGCCTGAGACGAGGAATAAACGGCCGTGCCAAAAATAG Sepallata 3 L. angustifolia FL ATGGGAAGAGGGAGAGTTGAGCTGAAGAGGATAGAGAACAAGATCAACAGGCAAGTGACCTTTGCTAAGCGAAGGAATGGGCTTTTGAAGAAAGCTTACGAGCTTTCGGTCCTTTGTGACGCTGAGGTTGCTCTGATTATCTTCTCCAATAGAGGAAAGCTGTACGAGTTTTGCAGCAGCTCTAGCAGCATGCTCAAGACACTGGAAAGGTATCAGAAATGCAACTATGCAGCTCCAGATACCGGTGTATCGACAAGCGAGGCTCTGGAACTTAGTAGCCAGCAGGAATATTTGAAGCTAAAAGGAAGCTATGAATCCCTGCAACGCACTCAAAGGAACCTTCTTGGTGATGATCTTGGTCCTTTGAGCAGCAAGGAGCTCGAATCACTCGAGAGGCAACTCGATATGTCACTCAAGGCGATCCGATCAACACGGACTCAAGCAATGCTGGATACACTTCAAGATCTTCAGAATAAGGAGCTTGCCCTCAATGAAGCTAACAAGAGTTTGAAACATCGGTTGGAGGGGAATCAAATGAATCTATCATGGAATCAGAATGCTCATGATGTGGCCTATGGAAGACAGCAACCACACCAGCCTCAAGCTGATGGCTTTTTTCATCCTTTGGACTGTGAACCCACTCTGCAAATTGGGTACCATACTGATGCAGCAGCAGGAGGGTCTAGTGTGAATAACTACATAGCAGGGTGGCTACATGATAACTAA ARF5 L. angustifolia FL ATGGGTCTTCTCAGCATAATCCGGAAAATTAAGCGCAAAGAAAAGGAGATGCGCATTCTCATGGTGGGTCTTGATAACTCTGGAAAGACGACAATAGTGATGAAGATTAACGGTGAAGACACTAGTATAATCAGTCCTACACTTGGATTCAACATCAAAACCATGGCGTACCAAAAGTATACTTTGAATATATGGGATGTTGGAGGGCAGAAAACCATAAGGTCTTATTGGAGGAATTATTTCGAGCAAACTGATGGGATGGTCTGGGTGGTGGATAGTTCAGACCTGCGAAGGCTGGATGATTGCAAATATGAGTTAGATAATCTTCTCAAAGAGGAGAGACTATCAGGAGCATCATTGTTGATTTTTGCAAATAAGCAAGACATACAAGGTTCCCTGTCGCCTGATGAGATAGCTAGAGTGCTCAACTTAGAATCCATGGATGGAAGCAGACACTGGAAAATAGTTGGGTGTAGTGCAGTGACTGGGGAAGGCCTGCTCCAAGGCTTCGATTGGTTGGTTCAAGACATTGCCTCCAGAATTTACGTGCTTGACTAA Agamous L. angustifolia FL ATGGAACAGCAAAGTGATCGATCAAGGGAGATGTCATCAGAGAGGAAGATTGGGAGGGGGAAGATCGAGATCAAACGGATCGAGAACACGACGAACCGGCAGGTAACTTTCTGCAAAAGGCGCAATGGTTTGCTGAAGAAGGCGTATGAATTATCAGTGCTTTGTGATGCTGAGGTTGCTCTAGTTGTTTTCTCAAGCAGAGGCCGCCTCTATGAGTATGCTAACAACAGTGTTAAAGCAACCATTGAGAGGTACAAGAAAGCATCCTCAGATTCCTCAAGTAGTGGCTCTATTTCTGAAGCCAATACTCAGTATTACCAGCAGGAGTCTTCAAAACTTCGTGCACAGATCAGTAATTTGCAGAATAATAACAAAAACATGGTTGGCGAATGTTTAGGGGCTTTAAATCTGAGAGAGCTCAAGAATCTGGAAGGCAGAGTTGAAAGAGCAATTAGCAAAATCCGAGCCAAGAAGAATGAACTATTGTTTGCTGAGATCGAGTACATGCAGAAGAGGCAGGAGATTGACTTGCATCAGAACAATCAGTACCTTCGAGCAAAGATAGCTGAGAGT142 GAGAGAGAGCAGCAGCACATGAACTTGATGCCTGGTAGTTCTGATCAGTATCATGAGCTCGTGCAACCTCATCAGCCGTTCGATGATCGGGATTATCTTCAAGTCAATAGCCTCCAACCCTCAGATCAGGATTACCATAGGCACGACCAGACATCTCTTCAGTTAATCTGA Embryonic flower 2 L. angustifolia SAM ATGCCTGGCATTCCTCTCGTGGCTCGTGAGACTGCGAATTGTGTTTGCAATTGCAGTTACTCTAGAGGTGCAGACCATATGTGTCGCCAAGATCCCCGTGCGCATTTGTCCCCTGAAGAGCAAATTGCAGCTGAGGAAAGCCTATCAATTTACTGCAAGCCTGTTGAATTGTATAACATTCTTCAGCGCCGTGCTGTTAGAAATCCATCCTTTCTTCAGAGATGTTTGCGGTACAAAATACAAGCAAAGCACAAGAAAAGGATTCAAATGACAATCTCCCTACCTTCAACTGTAAATGATGAATCTCGAATTCAAAGTCTATTTCCTTTGTATATAATGTTGGCAAGACCCGTTTATAGTCCTTCAGTTGTAGAGGAATCTGCAGTTTATCGATTCAGTCGAGAATGTATATTGACTAAATGTACGGGAGATGATGCAACGAACCAAGATGAAGCAAATTTTATTCTTCCTGAGATTAATAAACTATCAGCTGAAGTCAAATCTGGCTCTCTTTCGATTTTGTTTGTTAGCTGCGCTGAATTGTTTAAGGAGCATCGGGATGAGCTGTTATTTCCTGCTAATGCTGGAGGTCACTGCTTTCTGGGTAAGATTCCAATGGAGTTACTTCATTTGTCATGGGAGAAGTCTCTGAACTTGAGTTTCGGCAAGAGAGCCGAGATGCTGTCTACTGTTGACTTACATTCCTGCTTCCTGAAGACGGGCTGTTTTGGAGAAGACAAATGTTTTTCATTTCATAGTCCTCATGCTGGAGCTATGCCTATGTCTCAACAACTGCAAGTCCGCATTGCCGCTGAAGAACTTGGCTTCAGGGAGAGATCTGCTTATGATTCGTTTGCATTCAGTGACATTCCTTCCTCTACTTTGCCTCATATTATCAGGTTGAGAACTGGAAATGTTGTCTTCAACTACAGATACTATGATAACAAGTTGCAGAGGACCGAAGTGACGGAGGACTTTGCATGCCCTTTCTGTTTGGTCAAATGTGCAAGCTTTAAGGGTTTGAGATATCATTTGCCCTCCAGTCATGACTTATTCAGCTTTGAGTTTTGGGTGACAGAGGAATTTCAAGCTGTCAATGTATCTGTCAAGACTGATACGTGGAGATCTGAGATCGTTGCGGCTGGTGTTGACCCTAAGCAACAAACATTTTTCTTTTGTTCTAAGGCACCACGACGTAGAAAAATGAAGAGTCCGCTGCAAAAATCAAAGTTTGTGCATCCTCTTGTCTTAGATTCAGAAATGCTGGGGTCAGCCAATGAGCTTAGAGAAAAGACCATTGGCGTGGAAGAGAGTGCGGACTGTGATGCATCAAGTCCCATAGTTTCATCTGCCACAGCTCATTCATATGCAGACCCTGAATGTGTCCAGTCATTACCTGGAAGCAATCTTGCACCTACAGCATTGCTACAGTTTGCAAAGACAAGGAAATTGTCAGTTGAGCGCTCTGATCCTAGGAATCGTGCACTCTTAAAGAAGCGGCAGTTCTTCCACTCTCATAGAGCACAACCCATGGCCATGGAGCAAGTCCTATCAGATCGCGATAGTGAGGATGAAGTTGATGATGATGTTGCAGATCTTGAAGACCGAAGGATGCTGGATGACTTTGTGGATGTCACAAAAGATGAGAAGCAATTGATGCATCTTTGGAACTCTTTTGTTAGGAAGCAAAGGGTACTGGCAGATGGTCATATCTCTTGGGCATGCGAGGCTTTCTCCAAATTGCACAGACAGGATCTTGTTCAGACACCTGCCTTACTTTGGTGCTGGAGATTATTTATGATCAAGCTGTGGAACCATGGCCTTCTGGACGCTCGTACGATGAACAGCTGTAACGTAATTCTCGAGCAATCGGACCCACAGAGC 143 Flowering locus C L. angustifolia FL ATGGTGAGGGGGAAGGTGCAAATGAAGAGAATTGAGAATGCATCAAGCCGACAAGTAACCTTTTCGAAGAGGCGAAATGGCCTGCTGAAGAAGGCGTACGAGCTCTCGATTCTCTGCGATGCAGAAGTGGCTTTGATCATCTTCTCTTCTAAAGGGAGACTCTATGAATTCTCAAGCTCCAACATGCAGAAGTCAATAGAGAAATATCTGGAACTTACAAAAGATCGCCATTCTAGCATTGAAGTTGAACAACGCATGCAGCATATAAAACATGAAGCAGCATTCATGGCAAAGAAGATAGAGCTATTGGAGAATTCACACCGGAAGCTTTTAGGGCATAATCTGGGATCATGCTCCATGGATGAGCTTCAAGAGATTGATAACCAGTTGGAGAGAAGCCTTAAGAACATAAGGGCCAGAAAGGTTCAAGTATTCAAGGAGGAAACAGAGAAACTAAAAGCAAAGGAGAAATTTCTCTTCGAAGAAAACCAAAGACTCAGAGAAAAGATTGGGTTGAAGCAAAAGAATGAAGCAGAAATGCATAAAGAAATTGGGAGTTGCAGCCGGAGCACGGTGAGCGCGGAGGCGGTGACG Late elongated hypocotyl L. angustifolia BD ATGGATCCTTATTCTTCTGGAGAAGACCCAGTCATCAAGACAAGAAAACCTTATACAATTACTAAGCAGCGTGAGAGATGGACTGAAGAGGAGCATAATAGGTTTCTAGAAGCTCTCAAGCTCTATGGTCGTGCTTGGCAGCGAATAGAAGAACATATAGGAACCAAGACTGCTGTGCAGATAAGAAGTCATGCACAAAAGTTTTTTACAAAGTTGGAAAAGGAGGCTATTGTTAAAGGAGTTCCAATAGGACAATCTCTTGATATCGATATCCCTCCACCACGGCCTAAAAGAAAACCAAACAATCCTTATCCTCGAAAGACAAGTGTGGGAACTCCCGCTTCCCAGGTTGAAGTGAAGGATGGACTATCAAATCCTATTTCTTCCTCATGTCAGAGTAAAGTGACCATGAATGCGGAGAGAGAACCTGAGCAATCTTGCGACTATGCGAAATTCGGAAATAGTAAATACCATGACAAGGATAATTTTTCTGAGGCTTTCACTACTCTCAAGGAAGCTTCCTGTGAATCCCCATCGTCAGCATACAAAACTGCCTCCTTATCTGATGCTCCTAGAAAATCCTGTACATTTCGGCAATTTGCCCCACTCTCTAGAGAAGCAATGACTCAAGATGTAACCACTGCAATCCATGTTACTACCGAAGCCAAAGGGATGAAGAAGTCTGATATTGAACAGCCTTTCCAGGATAATCTCACTTTGAGGGTCTCAAATGTTGGTGATCCATACCTTTTACAAGAGAAATCTGTCCATACTAGAAAAGTAGATGAGTTGAAACAGACTGAAAATAATGGAAGTGAAACTACTGCTGTGCAGGCCTCTCAGAACTATCCTCGCCATGTTCCTGTGCACATTCTTGATGGGAGCTTAGGAATGAGGGCATACAATATCTCCCCCGATGTGTCATATGCAGATTTTCGCCATATGGGTGGAGTCCATGCTCATCAAAATCTATTCATGAACCCAGCTTCATCTGCTTCTTCTGAGCATTATAGCAATGCATCAAAATCTTCTGTTCACCAGGCATTTCCTAGTTATCATCCTGTCTTCGCTCCTATCCAAAGCCAAGACGATCCCCAGTCGTGTCTGCATGTATCACCGGCGTTTTCCAGTCTAATTGTGTCTTCTCTCTCCCAAAATCCTGCAGCCTATGCCGCAGCTAGTTTTGCAGCAACTTTGTGGCCGTCTCTGAACGTGGAAGCTTCAGTTGAGCCATCTACAACTCCCAGTATGGCTGCAATTGCTGCAGCAACTGTAGCAGCAGCAACTGCATGGTGGGCAGCCCATGGCCTACTTCCTTTATGTGCTCCATATCATCCTGGCTTTACCTGTTCTCCTGCATCAGCATCTGCAGCTCCAATACCTAGCAGCCGAGGTATAACTGTTAACAATGAAAGAAGGGAGAGTCCTGAT144 CCTACCTTAGGGGGTCAGCAACTGGAACCGGAATGTTCGGAGGCTCTGCAAGAACAGCATTCAGTTTCAAAGTCACCATTGTTATCCTCATCAGATTCTGAAGAAAGTGAAGGTGCAAAAAAGAATGTTGAGTCAACCGCTCCTGAAACAACAAAAGATGAGGTTACTACGACTGAGCCGCATGATGCAGACAAGTCAACTAACAAAAAGCTTGTGGACCGTTCTTCATGTGGATCAAATACACCTTCCAGCAGTGAAGTAGAAGCTGAGGCTTTGGAAAAGGATACAAAGGAGAAAGACGAAAAACATATGAACGAGAAAGAGAAGCAAGAAGAACTTGATGCTATGCTTAATGTTAGTGATCCATTTGGTCGTCGTTGTAGAAGCACAAGCAATGCTGGTGCCGGTGACTCTTGGAAGGAAGTATCAGAGGAGGGCCGACTGGCATTTGACGCGCTCTTCTCTAGAGAAATACTACCACAGAGTTTTTCACCTCCACATAAGGGCAAGAAAACCTGTAACAAGGAAAGTGAGAAGGATGAAAGCAGAATGCAGTTAGACCTTAATGAGAAGACATGGGATACATGTTCACCCTTGATTAGTGAAACCATGAAAGAGGGAGCCCTACATACAGGACTAGAACTAGGACAAGGTAAGCTAAAAGCTCGACGGACAGGATTTAAACCTTACAAGAGATGTTCAGTGGAAGCCAAGGAGTTCAAGGTGTCAGCCAACGGCCAAGATGAAGAGAAATGTCCGAAACGATTACGCTTGGAAGGAGAGGCTTCTACATGA
Thesis/Dissertation
eng
Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Identification, cloning, and functional characterization of Lavandula floral development genes
Text