DEPARTMENT OF AGRICULTURAL SCIENCESFACULTY OF AGRICULTURE AND FORESTRYDOCTORAL PROGRAMME IN PLANT SCIENCESUNIVERSITY OF HELSINKI

Genetic and Environmental Control of Flowering in Wild and Cultivated Strawberries

ELLI KOSKELA

dissertationes schola doctoralis scientiae circumiectalis, alimentariae, biologicae. universitatis helsinkiensis 16/2016

16/2016

Helsinki 2016 ISSN 2342-5423 ISBN 978-951-51-2335-0

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DISSERTATIONES SCHOLA DOCTORALIS SCIENTIAE CIRCUMIECTALIS, ALIMENTARIAE, BIOLOGIAE 16/2016

GENETIC AND ENVIRONMENTAL CONTROL OFFLOWERING IN WILD AND CULTIVATED STRAWBERRIES

Elli Koskela

Doctoral School in Environmental, Food and Biological Sciences (YEB)Doctoral Programme in Plant Sciences (DPPS)

Department of Agricultural SciencesFaculty of Agriculture and ForestryUniversity of Helsinki

ACADEMIC DISSERTATION

To be presented, with the permission of the Faculty of Agricultural Sciences of theUniversity of Helsinki, for public examination in lecture room B3, Viikki B building,Latokartanonkaari 7, on the 2nd of September 2016, at 12 noon.

Helsinki 2016

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Supervisors: Assistant professor Timo HytönenDepartment of Agricultural SciencesUniversity of Helsinki, Finland

Professor Paula ElomaaDepartment of Agricultural SciencesUniversity of Helsinki, Finland

Follow-up group: Professor Ykä HelariuttaInstitute of BiotechnologyUniversity of Helsinki, Finland

Professor Alan SchulmanLUKE/Institute of BiotechnologyUniversity of Helsinki, Finland

Reviewers: Professor Outi SavolainenBiocenter OuluUniversity of Oulu

Professor Alan SchulmanLUKE/Institute of BiotechnologyUniversity of Helsinki, Finland

Opponent: Professor Richard ImminkPlant Research InternationalWageningen University, Netherlands

Custos: Professor Teemu TeeriDepartment of Agricultural SciencesUniversity of Helsinki, Finland

ISSN 2342-5423 (print)ISSN 2342-5431 (online)ISBN 978-951-51-2335-0 (print)ISBN 978-951-51-2336-7 (pdf)https://ethesis.helsinki.fi/Hansaprint 2016

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ACKNOWLEDGEMENTS

My scientific career with flowers started as an intern at a fruit and berry researchcentre near Wageningen (Netherlands) in the beginning of the 2000’s. Back then, Ihad a very motivating supervisor, Henk Kemp, whom I can thank for giving me ayearn to understand physiological processes in plants in more detail.

At the end of my Master’s degree around 2009 I had the honor to meet anothermotivating supervisor, doctor Dan Sargent from East Malling Research. He taughtme pretty much everything I know about genetic mapping and long lab hours.

After finishing the Master’s, the strawberry group at the Department ofAgricultural Sciences welcomed me to join their team. I would like to thank thegroup leader doctor Timo Hytönen and my supervisor professor Paula Elomaa forvaluable advice and continuous support. Also, the former group members KatiMouhu and Marja Rantanen have taught me much and more about experimentaldesign and lab methods.

This work would not have been possible without the skilled lab and greenhousetechnicians at our department. Big thanks to Marja Huovila, Eija Takala, AnuRokkanen and Marjo Kilpinen for helping me out in the lab, and to gardeners LasseKuismanen and Sanna Peltola for keeping our plants alive! Also, the greenhousetechnicians Matti Salovaara and Terttu Parkkari are to be thanked for arrangingexperimental facilities every time when needed.

I’m very greatful for all the co-authors for their input and advice. Especially ourNorwegian collaborators, Anita Sønsteby and Ola Heide are thanked for enduringso many drawbacks and hardships during the course of our projects!

My follow-up group members Ykä Helariutta and Alan Schulman have providedvaluable advice and steered my research to the right direction. I also thank Alan andOuti Savolainen for (maybe not-so-critical) pre-examination and comments.

Last but not least I thank my partner Hans Hämäläinen for putting up with myimmersion in strawberry work. He has also made the most critical comments on thelayout of this thesis. I also thank my parents, who have never stopped asking aboutmy graduation.

I acknowledge Viikki Doctoral Programme in Molecular Biosciences and RikalaHorticultural Foundation for financing this research. In addition, I have worked inprojects funded by the Academy of Finland and the Ministry of Agriculture andForestry.

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ABSTRACT

Strawberries (Fragaria sp.) belong to the large family of Rosaceae that includescommercially important crop plants such as apple, pear, peach and roses. Theeconomic impact of these species is huge and breeders around the world are strivingto keep up with consumers' demands on novelty produce. At the same time, climatechange is having an impact on the onset of flowering especially in species that aregrown in temperate climates. As flowering is a prerequisite for yield formation, it isextremely important to gain an insight on how the environmental factors, mostimportantly photoperiod and temperature, affect the timing of flowering inRosaceous species.

Although studying flowering responses directly in the cultivated species couldprovide immediate practical applications, it is often not feasible due to e.g. complexgenomics of the species, large plant size or long juvenile period. The woodlandstrawberry Fragaria vesca (L.) has arisen as a convenient model plant forstrawberries and the entire Rose family. It is a diploid species and therefore has aless complex genome than the cultivated octoploid strawberry.

The work described here begun by elucidating the molecular identity ofSEASONAL FLOWERING LOCUS (SFL), a locus controlling the switch fromseasonal to continuous flowering habit in woodland strawberry. SFL was identifiedas the woodland strawberry orthologue of TERMINAL FLOWER1 (FvTFL1) basedon its location on the strawberry genome and similarity to its Arabidopsiscounterpart, TFL1. In woodland strawberry, FvTFL1 was shown to bephotoperiodically regulated, and it was demonstrated that the continuous floweringhabit is caused by a mutation at FvTFL1. In the following experiments, alteredregulation of FvTFL1 was associated with the unique vernalisation requirement inthe artic F. vesca accession Alta-1, suggesting a previously uncharacterised functionfor a TFL1 orthologue.

The findings on FvTFL1 were extended to cultivated strawberry. It wasdemonstrated that F. × ananassa homologue of TFL1 (FaTFL1) also repressesflowering, and that differences in the regulation of FaTFL1 were associated withdifferent flowering times in strawberry cultivars. The finding that FaTFL1 is a majordeterminant in the flowering response of cultivated strawberry provides breederswith a new breeding target; producing cultivars with lowered FaTFL1 expressionlevel could expand the flowering and fruiting season of strawberries.

These results clearly demonstrate the feasibility of the model plant approach andalso highlight the importance of fundamental research. The knowledge gained onfundamental genetic pathways in model plants can be transferred to crop plants, inwhich similar genetic studies would be impossible or at least extremely complex,slow and costly to perform.

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CONTENTS

Acknowledgements ................................................................................................... iii

Abstract ..................................................................................................................... iv

Contents.......................................................................................................................v

List of original publications ...................................................................................... 6

Authorship statement .................................................................................................7

1 Introduction ...................................................................................................... 8

2 Review of the literature .................................................................................... 9

2.1 History of the cultivated strawberry ...................................................... 9

2.2 Phylogenetic relationships within Fragaria ........................................ 11

2.3 Strawberry physiology ............................................................................ 12

2.4 Pathways to flowering ............................................................................ 16

3 Aims of the study ............................................................................................ 24

4 Materials and methods................................................................................... 25

5 Results and discussion ................................................................................... 28

5.1 SFL encodes for an orthologue of TFL1 in F. vesca (I) ....................... 28

5.2 Photoperiod regulates FvTFL1 expression and flowering (I) ............ 30

5.3 Altered regulation of FvTFL1 is associated with the uniquevernalisation response in Alta-1 (II) .................................................................. 32

5.4 TFL1 is a floral repressor in F. ananassa (III) ................................. 36

6 Conclusions ..................................................................................................... 42

7 References ....................................................................................................... 43

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LIST OF ORIGINAL PUBLICATIONS

The thesis is based on the following publications, which are referred to in thetext by their Roman numerals (I–III). The publications are reprinted withpermission from their original publishers.

I Koskela EA, Mouhu K, Albani MC, Kurokura T, Rantanen M,Sargent DJ, Battey NH, Coupland G, Elomaa P and Hytönen T.2012. Mutation in TERMINAL FLOWER1 reverses thephotoperiodic requirement for flowering in the wild strawberryFragaria vesca. Plant Physiology 159: 1043–1054.

II Koskela EA, Sønsteby A, Toivainen T, Kurokura T, Heide OM,Elomaa P and Hytönen T. 2016. Altered regulation of TERMINALFLOWER1 contributes to the vernalisation response in the articwoodland strawberry accession Alta-1. Manuscript.

III Koskela EA, Sønsteby A, Flachowsky H, Heide OM, Hanke MV,Elomaa P and Hytönen T. 2016. TERMINAL FLOWER1 is abreeding target for a novel everbearing trait and tailored floweringresponses in cultivated strawberry (Fragaria × ananassa Duch.).Plant Biotechnology Journal, doi: 10.1111/pbi.12545.

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AUTHORSHIP STATEMENT

In (I), EAK carried out genetic linkage analysis for the F2 population, designedand produced the FvTFL1 vector constructs for transformation into plants, didthe transformations and analyzed the transgenic plants. RNA extraction andRT-qPCR was done by EAK, KM and MR. MR designed, produced andanalyzed the FT-RNAi transgenic plants. MCA carried out genetic linkageanalysis for the BC population. TK designed and performed the in situhybridizations. Manuscript was written by EAK, TH and KM with input fromall the authors.

In (II), the growth experiments were designed and carried out by EAK, AS, THand OMH. EAK genotyped the F2 population, extracted RNA and performedRT-qPCR analysis. TK made the crosses between Alta-1 and the other parents.TT and TH designed, carried out and analysed the GBS experiment andphenotyping of woodland strawberry accessions. Manuscript was written byEAK and TT with input from all the authors.

In (III), the growth experiments were designed and carried out by EAK, AS,TH and OMH. EAK did RNA extractions and RT-qPCR analysis. Planttransformations were done using the vectors constructed in (I) by HF andMVH, who also carried out the initial analyses of the transgenic plants. Growthexperiments for the transgenic plants were carried out by TH. Manuscript waswritten by EAK and TH with input from all the authors.

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1 INTRODUCTION

Strawberries (Fragaria sp.) belong to the large family of Rosaceae thatincludes commercially important crop plants such as apple, pear, peach androses. The economic impact of strawberry cultivation and trade is huge; in theyear 2013, the worldwide production of strawberries exceeded 7.5 millionmetric tonnes with an estimated gross value of 13.7 billion euros (FAOSTAT,2015). However, strawberry production is not without problems. Astemperature has a large effect on flowering time of strawberries, progressingclimate change is bound to have an effect on many strawberry growing areas.To meet these environmental challenges, plant breeders need tools fordeveloping new cultivars that are suitable for growing in specificenvironments. Research on genetics and genomics of strawberry could providesuch tools for more accurate selection. For instance, identification of geneswith major effects on physiological responses could provide new breedingtargets for tailored flowering time. Also, development of molecular markerslinked with specific genes could speed up the breeding process significantly.

Strawberries show a large variation in their environmental responses. Mostdiploid and octoploid strawberries are short day (SD) plants that are inducedto flower in autumn and bear flowers the following spring (Darrow, 1966).However, some accessions and cultivars are everbearers that produce newinflorescences throughout summer. This exceptional response has attracted alot of attention, as it can serve as a source for environmental adaptation.

This PhD thesis studies the genetic mechanisms controlling flowering inboth diploid and octoploid strawberries. The first article elucidates themolecular identity of the SEASONAL FLOWERING LOCUS (SFL), a majorlocus controlling the switch from SD to everbearing flowering type inwoodland strawberry, and shows that SFL encodes for an F. vesca orthologueof the floral repressor TFL1. The second article presents a previouslyuncharacterised role for a TFL1 orthologue in the unique vernalisationresponse observed in an arctic diploid strawberry accession Alta-1. In the thirdarticle, the results obtained from studies with the diploid woodland strawberryare extended to the octoploid strawberry, and the role of FaTFL1 inenvironmentally regulated flowering in the octoploid strawberry isinvestigated.

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2 REVIEW OF THE LITERATURE

Luckily for plant breeders, diversity in the Fragaria genus is large. Fragariaincludes 27 acknowledged taxa with ploidy levels ranging from diploid todecaploid and covering a wide distribution over most of Eurasia and extendingdown to Peru in South America (Staudt, 2009; Njuguna et al., 2013). TheFragaria species with by far the widest distribution range is the diploidwoodland strawberry F. vesca, which is spread over Eurasia and northernAmerica (Staudt, 2009; Liston et al., 2014). Most diploid and tetraploidstrawberry species are native to southern Asia, which has been considered theplace of origin of the most recent common ancestor of the Fragaria species oftoday (Njuguna et al., 2013; Johnson et al., 2014). The only hexaploidstrawberry species F. moschata is native to Europe and has been cultivatedthere for centuries (Liston et al., 2014). The octoploid F. chiloensis grows on anarrow coastal strip in western North America and central and southern Chile,has been cultivated in Chile for over thousand years (Hancock et al., 1999),while the octoploid Virginian strawberry F. virginiana is native to NorthernAmerica (Darrow, 1966; Liston et al., 2014). Natural hybrids betweenstrawberry species have been repeatedly reported, even between species withdifferent ploidy levels (Bringhurst, 1990). Interspecies hybridization has alsoled to the birth of the cultivated strawberry, Fragaria × ananassa (Darrow,1966).

2.1 HISTORY OF THE CULTIVATED STRAWBERRY

From the human point of view, the cultivated strawberry Fragaria × ananassais evidently the most important species of Fragaria. As a species, thecultivated strawberry is very young; it originated as an interspecific crossbetween the octoploids F. chiloensis and F. virginiana in a French gardensome time between 1714 and 1766. The maternal F. chiloensis was brought toEurope from Chile in 1714 by a French spy, Captain Frézier (Darrow, 1966).However, all the five imported plants turned out to be female and did notproduce any fruit due to lack of pollenisers. When interplanted with F.virginiana, which had been introduced earlier from Northern America, theplants produced fruit and F. chiloensis became the major strawberry speciescultivated in Europe (Darrow, 1966; Hancock et al., 1999). As time passed,strawberry seedlings with unusually large fruit and red flesh began to appearin European gardens. In 1766, botanist Duchesne determined these seedlingsto be hybrids of F. chiloensis and F. virginiana, and the new species wasnamed as F. × ananassa (Darrow, 1966).

The origins of the everbearing trait in cultivated strawberry can be tracedback to several independent sources. The first described everbearing cultivar‘Gloede’s Seedling’ was introduced in France in 1866. Although it was freely

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runnering, and thus easy to propagate clonally, it was not commerciallysuccessful (Darrow, 1966). Other early everbearing cultivars developed inEurope are thought to originate from interspecific crosses between diploideverbearing strawberries and large-fruited octoploid SD cultivars (Darrow,1917), but it is doubtful whether the parentage is correctly reported.Everbearing cultivars with European origin met only limited success, exceptfor ‘Sans Rivale’, which was the leading everbearer grown in France in the1960’s (Darrow, 1966). The first successful everbearer of American origin is‘Pan American’ introduced in 1902. ‘Pan American’ originated from a chanceseedling or clonal mutation of the SD cultivar ‘Bismarck’, and it appears in theparentage of many modern day everbearing cultivars (Darrow, 1917; Darrow,1966). The most recent source of the everbearing trait is a native population ofF. virginiana ssp. glauca in the Wasatch Mountains in Utah (Bringhurst andVoth, 1980). This germplasm introduction has resulted in the release of manycommercially important everbearing strawberry cultivars suitable for growingin North America (Ahmadi et al., 1990).

Most strawberry cultivars are descendants of the early European cultivars(Darrow, 1966), although germplasm introductions from wild progenitorspecies have been made occasionally (Bringhurst and Voth, 1980; Hancock etal., 2002; Hancock et al., 2010). As a result of a limited number of foundingparents, modern strawberry cultivars have narrow genetic diversity. Forexample, Dale and Sjulin (1990) examined pedigree data of 134 NorthAmerican cultivars released between 1960 and 1987 and discovered that thesecultivars originated from just 17 maternal founding clones. More recently,Honjo et al. (2009) detected only three maternal sources of chloroplast DNAin a study that included 75 strawberry cultivars from around the world. Lossof diversity seems to have accelerated as a result of modern breedingprogrammes; both Gil-Ariza et al. (2009) and Horvath et al. (2011) showedthat modern American and European cultivars have much lower degree ofdiversity than old European cultivars introduced before 1930. It is thereforevital that the genetic basis of strawberry breeding programmes is widened toavoid further losses of diversity.

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2.2 PHYLOGENETIC RELATIONSHIPS WITHINFRAGARIA

As F. × ananassa is such an important crop, special interest lies in identifyingthe diploid ancestors of the octoploid species. Cytological studies (Bringhurst,1990) proposed octoploid genomic composition of AAA’A’BBB’B’, suggestingan allopolyploid history and subgenomic contribution from two diploidancestral species. The work of Bringhurst (1990) also suggested that theoctoploid genomes are highly diploidized, an observation which has been laterconfirmed in several studies (Ashley et al., 2003; Rousseau-Guetin et al.,2008).

The earliest report on Fragaria phylogeny based on molecular evidencecame from a restriction fragment length polymorphism (RFLP) study onchloroplast DNA (Harrison et al., 1997). However, the authors reported onlylimited amount of variation in their samples and were unable to ascertainphylogenetic relationships at a high resolution. Potter et al. (2000) used bothchloroplast and nuclear sequence data to study phylogenetic relationships of14 Fragaria species, and concluded that F. vesca and F. nubicola are thediploid species most closely related to the polyploid species.

The most recent efforts to examine the ancestry of octoploid strawberriesuse thousands of loci, either using targeted sequence capture (Tennessen etal., 2014) or SNP marker arrays (Sargent et al., 2016). Both groups arrived atthe conclusion that one of the octoploid subgenomes originates from an F.vesca-like ancestor and one from F. iinumae, and the two groups also agreedthat all the subgenomes display disomic inheritance. However, whereasTennessen et al. (2014) identified the two remaining subgenomes to be F.iinumae-like, Sargent et al. (2016) suggested an unidentified subgenomicancestor. Irrespective of the ancestral origins of the two subgenomes, F. vescaappears to be a close relative of the cultivated strawberry, making the use of F.vesca as a model species plausible.

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2.3 STRAWBERRY PHYSIOLOGY

Strawberry life cycle

All strawberry species share similar life histories; they are small, perennialherbs capable of both sexual reproduction via flowering and clonal growth viastolon (or runner) formation (Johnson et al., 2014). Strawberry rosette crownconsists of a thick stem with short internodes. The stem terminates in mainshoot apex, which is sheltered by leaf bases (Figure 1A). Each leaf axil isaccompanied by an axillary bud, which may either remain dormant or developinto runners, new branch crowns or inflorescences (Brown and Wareing, 1965;Darrow, 1966). Fates of the axillary meristems depend on environmentalconditions, which define the yearly growth cycles of generative and vegetativegrowth.

Figure 1. Plant structure and seasonal growth cycles in strawberry. A) A LD-grown vegetativestrawberry plant with several branch crowns. Runners have been removed. Magnificationillustrates a single axillary branch crown; B) Typical seasonal growth cycle of woodland strawberrygrown in temperate zone. Flowering occurs in late spring. Vegetative growth is continued fromyoung branch crowns that were not induced in autumn. The axillary meristems of these branchcrowns produce runners during the summer months, and new branch crowns towards autumn. Inautumn, the main shoot apical meristem and the apical meristems of the oldest axillary branchcrowns develop into inflorescence meristems, which complete their development the followingspring.

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After overwintering, plants start a period of active growth manifested byoutgrowth of leaf petioles and inflorescences (Figure 1B). Flowering occurs inspring, after which plants enter a phase of active vegetative growth,characterized by ample runner production. Runner production ceases towardsautumn and axillary buds develop into branch crowns. Crown branching ispromoted by shortening days in autumn and is essential for the perennialgrowth habit of strawberries, as the young branch crowns continue thevegetative growth of the plant. During floral induction, the apical meristem ofthe main shoot develops into inflorescence meristem, which overwinters andfinishes its development the following spring (Brown and Wareing, 1965;Darrow, 1966).

Physiological responses to photoperiod and temperature in the cultivatedstrawberry

Perhaps the most important physiological response to environmentalconditions in strawberry from the point of view of yield formation is flowerinduction, and it has been extensively studied in cultivars of F. × ananassa.Most strawberry cultivars are SD plants, or June-bearers, although alsoeverbearing cultivars (Darrow and Waldo, 1934) and photoperiod-insensitiveday-neutral cultivars (Bringhurst and Voth, 1980) have been described.Everbearing cultivars originate from old European and American cultivarsintroduced in the beginning of the 1900s (Darrow, 1966; Hancock et al., 1999),whereas day-neutral cultivars originate from an introduction of F. virginianassp. glauca into the cultivated strawberry germplasm (Bringhurst and Voth,1980).

The everbearing and day-neutral F. × ananassa cultivars have beenhistorically classified into distinctive flowering types (Durner et al., 1984),although recent studies have shown that flowering in both types is actuallypromoted by long days (LDs) (Nishiyama and Kanahama, 2002; Sønsteby andHeide, 2007). The confusion in the literature about flowering types of thecultivated strawberry is probably caused by the strong photoperiod ×temperature interaction; a cultivar may appear day-neutral at one testedtemperature, but turn out to have an obligatory photoperiod requirement atanother temperature. For clarity, the term “everbearer” is used in this work torefer to strawberry cultivars showing a perpetual flowering phenotype,irrespective of the origin of the everbearing trait. Moreover, perpetualflowering strawberries were referred to as everbearers already by Darrow(1917), and therefore the term is also historically justified.

SD cultivars are induced to flower when the daylength falls below a certaincritical limit in autumn. The critical daylength is heavily dependent ontemperature; flowering is inhibited in all photoperiods at high temperaturesof above 24C, short days promote flowering at intermediate temperaturesbetween 14 and 20C whereas cooler temperatures induce flowering

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independently of photoperiod (Heide, 1977; Durner et al., 1984; Sønsteby andNes, 1998; Manakasem and Goodwin, 2001). Cultivars have markeddifferences in their responses to photoperiod and temperature; for example,early cultivars bred for growing at high altitudes in Norway flowerphotoperiod-independently even at 18C whereas cultivars developed forMiddle European conditions require SDs of 13 hours at the same temperature(Heide, 1977).

In contrast to SD cultivars, everbearing cultivars form new inflorescencesthroughout the summer and produce more than one crop per growing season.Flowering in everbearing cultivars is controlled by an interaction ofphotoperiod and temperature; everbearers have an obligatory LD requirementat the high temperature of 27C, LDs promote flowering at intermediatetemperatures between 15 and 21C, and cool temperature of 9C delaysflowering irrespective of the photoperiod (Nishiyama and Kanahama, 2002;Sønsteby and Heide, 2007).

The number of apical meristems that can be induced to form aninflorescence is directly dependent on the number of branch crowns on theplant. Therefore, branch crown formation has a large effect on subsequentyield potential, and in some cases, the high yield potential of everbearingstrawberries has been attributed to the higher number of branch crowns perplant (Camacaro et al., 2002). Crown branching is under photoperiodiccontrol and it is promoted by SDs in SD cultivars (Konsin et al., 2001; Hytönenet al., 2004). Photoperiodic control of branch crown formation in everbearingstrawberries has been studied much less, although it appears that LDs mayadvance branch crown formation at least in some everbearing cultivars(Taylor, 2002).

Runner formation is generally promoted by LDs and high temperature inboth SD and everbearing cultivars, although there are cultivar-specificdifferences (Heide, 1977; Konsin et al., 2001; Sønsteby and Heide, 2007;Bradford et al., 2010). Moreover, many everbearing cultivars produce onlylimited numbers of runners, making their nursery propagation difficult andhindering their utility for commercial production (Camacaro et al., 2002;Bradford et al., 2010).

Physiological responses to photoperiod and temperature in the diploidstrawberry

Physiological responses of the diploid F. vesca to environmental conditionsare notably similar to the responses observed in cultivars of octoploidstrawberry. Most naturally occurring F. vesca accessions are SD plants (Heideand Sønsteby, 2007), although also everbearing flowering types have beencharacterized (Brown and Wareing, 1965). The photoperiodic response of SDaccessions is strongly dependent on temperature; flowering is inhibited underall photoperiods at temperatures above 21C, short days promote flowering at

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intermediate temperatures between 15 and 18C, whereas cooler temperaturesinduce flowering independently of photoperiod (Figure 2A; Heide andSønsteby, 2007). Flowering in the everbearing types of F. vesca is promotedby LDs and high temperature (Figure 2B; Sønsteby and Heide, 2008; Mouhuet al., 2009), similarly to the situation in cultivated strawberry.

Figure2. Typical flowering responses in seasonal flowering and everbearing F. vesca genotypes.Seasonal flowering woodland strawberry genotypes (A) flower at cool temperature nearlydaylength-independently (dark grey and light grey circles for SD and LD, respectively; the degreeof circle filling depicts the approximate percentage of flowering plants), while high temperatures(white circle) inhibit flowering. Flowering in everbearing genotypes (B) is promoted by LDs andSDs inhibit flowering especially at very high temperature. Data adapted from Heide and Sønsteby(2007), Sønsteby and Heide (2008) and Rantanen et al. (2015).

Runner formation in F. vesca is promoted by LDs and high temperature(Heide and Sønsteby, 2007), similarly to the cultivated strawberry. Manyeverbearing F. vesca accessions do not form runners at all (Brown andWareing, 1965), unless they are subjected to SDs at high temperature(Sønsteby and Heide, 2008).

An accession of the diploid F. vesca displays a physiological response notcharacterised in the cultivated strawberry, or in any other strawberry speciesstudied to date. When Heide and Sønsteby (2007) studied Norwegian F. vescapopulations, they discovered an accession in the northern Norway that couldnot be induced to flower even after a 15-week exposure to SDs at 9C. Theaccession, named Alta-1 after its place of origin, required at least 5 weeks at2C, with longer cold duration accelerating flowering. Alta-1 also flowered infield conditions, albeit two weeks later than the other Norwegian populationsincluded in the experiment (Heide and Sønsteby, 2007). These resultssuggested that Alta-1 may possess an adaptation to artic conditions,preventing premature flowering in spring.

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2.4 PATHWAYS TO FLOWERING

Although the physiology of flowering and vegetative development have beenextensively studied in strawberries, much less is known about the molecularpathways controlling these events. Molecular level studies have beenconducted mostly in model plant Arabidopsis, in which both endogenous andenvironmentally regulated flowering pathways have been identified.Endogenous flowering pathways are regulated by hormones and carbohydrateassimilates. Environmental factors that affect flowering are photoperiod andtemperature; temperature regulates flowering via ambient temperature andvernalisation pathways (Huijser and Schmid, 2011). Although Arabidopsis isonly distantly related to strawberry, several genetic components of theflowering pathways in Arabidopsis have been identified also in F. vesca(Mouhu et al., 2009), indicating that similar mechanisms control at least someof the aspects of plant development in these two species. Moreover, studies inother plant species, e.g. rice and poplar, have shown that some parts of themolecular machinery controlling flowering are conserved in even distantlyrelated species (Andrés and Coupland, 2012). Therefore, knowledge onflowering pathways and comparisons of the pathways between plant speciesare vital tools for understanding the mechanisms controlling flower inductionin F. vesca.

Photoperiodic pathway

Photoperiodism, or promotion of flowering by a specific daylength, wascharacterized already in 1920 by Garner and Allard. Although the molecularbasis of photoperiodism was starting to be deciphered from the 1990sonwards, a model for photoperiodic control of flowering was proposed alreadyin 1936. Bünning (1936) suggested that plants sense seasonal changes bycoupling the observed daylength with an intrinsic circadian rhythm. Accordingto this model, an endogenous circadian clock generates a rhythm that issensitive to light only at certain times of the day. When this light-sensitiveperiod coincides with daylight, flowering responses are promoted in LD plantsand inhibited in SD plants. Later on, this model known as external coincidencemodel was refined by Pittendrigh and Minis (1964), who proposed that lightcould also affect the circadian rhythm by entraining the circadian clock.

Work on the molecular machinery controlling photoperiodic flowering inArabidopsis has shown that the model of Bünning was quite accurate. It is nowknown that an intricate machinery consisting of dozens of genes generates asteady circadian rhythm with a period of approximately 24 hours (Bouché etal., 2016). One of the outputs of the circadian clock is the rhythmic expressionpattern of CONSTANS (CO), a CCT domain transcription factor required forthe LD-dependent promotion of flowering (Putterill et al., 1995; Sawa et al.,

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2007). In addition to transcriptional regulation, CO is regulated at the proteinlevel by different mechanisms at different times of the day. CO proteinexpressed at the end of a LD is stabilized by interaction with FLAVIN-BINDING, KELCH-REPEAT, F-BOX1 protein (FKF1; Nelson et al., 2000;Song et al., 2012), whereas morning expressed CO is degraded by the action ofphytochrome B (PHYB; Valverde et al., 2004). During darkness, CO protein isdegraded by the CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1)–SUPPRESSOR OF PHYA (SPA) ubiquitin ligase complex (Andrés andCoupland, 2012).

Perception of photoperiod takes place in leaves, and all the genes involvedin the circadian clock and regulation of CO are expressed in leaf vasculartissues (Andrés and Coupland, 2012). However, inflorescences are initiated atthe shoot apical meristem. The mobile signal linking these tissues isFLOWERING LOCUS T (FT; Koornneef et al., 1991; Corbesier et al., 2007), amember of the CETS protein family (Kardailsky et al., 1999). In LDs, the COprotein expressed in phloem upregulates FT directly by binding to itspromoter (An et al., 2004; Tiwari et al., 2010). The FT protein is activelyexported from the phloem companion cells to sieve elements (Liu et al., 2012)and is transported to the shoot apex.

In the shoot apex, FT forms a complex with the bZIP transcription factorFLOWERING LOCUS D (FD; Abe et al., 2005). The FT–FD complex acts as atranscriptional activator, whose targets include the MADS box transcriptionfactor SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1;Samach et al., 2000) and the meristem identity genes APETALA1 (AP1; Abe etal., 2005), LEAFY (LFY; Moon et al., 2005) and FRUITFUL (FUL; Teper-Bamnolker and Samach, 2005). Once meristem identity genes are expressed,the shoot apical meristem is irreversibly committed to flowering (Hempel etal., 1997).

Regulation of flower induction in the shoot apex is complex, as alsoflowering inhibitive factors play roles in flower induction. One of the floralrepressors acting in the shoot apex is TERMINAL FLOWER1 (TFL1; Shannonand Meeks-Wagner, 1991), another member of the CETS protein family thatbears high resemblance to FT (Kobayashi et al., 1999). In Arabidopsis, TFL1 isrequired for maintaining inflorescence meristem indeterminacy, andmutations at TFL1 cause early flowering and development of a terminal flower(Shannon and Meeks-Wagner, 1991). TFL1 exerts its action by preventing theexpression of AP1 and LFY in the inflorescence meristem (Liljegren et al.,1999), and the expression of TFL1 is reciprocally repressed by AP1 and LFY(Liljegren et al, 1999; Kaufmann et al., 2010). TFL1 cannot bind to DNA on itsown, and it requires an interacting partner to repress flowering. It was recentlyshown that TFL1 competes with FT for binding to FD, and these interactionsare mediated by 14-3-3 proteins (Ho and Weigel, 2014).

The components of the photoperiodic pathway are fairly conserved acrossdiverse plant species, although modifications to the regulation of the pathwaygenes do exist. In the SD plant rice, the photoperiodic flowering response is

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controlled by Heading date 3a (Hd3A) and Heading date1 (Hd1), genes thatare homologous to Arabidopsis FT and CO, respectively (Kojima et al., 2002).The rice Hd1 represses flowering in LDs by inhibiting the expression of Hd3A,causing a photoperiodic flowering response opposite to that of Arabidopsis(Hayama et al., 2003). The Hd3A protein produced in SDs interacts with 14-3-3 proteins and a rice FD homologue (OsFD1) in the shoot apex and thiscomplex activates the expression of OsMADS15, a rice AP1 homologue (Taokaet al., 2011).

The function of FT as a universal floral promoter is conserved, as the genehas been shown to promote flowering in the SD plants rice (Kojima et al.,2002) and cucurbits (Lin et al., 2007), in LD plants Arabidopsis (Kardailskyet al., 1999) and barley (Yan et al., 2006) and many perennial species,including poplar (Böhlenius et al., 2006) and apple (Tränkner et al., 2010). Itappears likely that the components of the photoperiodic pathway could beconserved in Fragaria as well, although regulation of the individualcomponents is likely to differ between the annual LD plant Arabidopsis andthe perennial SD plant F. vesca.

Vernalisation pathway

Some plants require an extended period of cold before they are able to respondto other flowering-promoting stimuli. This process is termed vernalisation andit was characterised in cereals as early as 1857 by Klippart (Chouard, 1960).Later on, vernalisation requirement has been shown to exist in many annualplant species belonging to several plant families, including Brassicaceae(Arabidopsis thaliana; Napp-Zinn, 1953), Amaranthaceae (Beta vulgaris;Chroboczek, 1934) and Solanaceae (Hyoscyamys niger; Melchers, 1936). Inall these plant species, the requirement for vernalisation changes the life cycleof the species from summer annual (completing the entire life cycle within onegrowing season) to a biennial, or winter annual form, which requires a periodof cold before being able to receive flowering-inductive stimuli.

The molecular machinery controlling the vernalisation process has beenstudied in detail only in Arabidopsis, temperate grasses and beet. In winter-annual Arabidopsis accessions, a MADS box transcription factorFLOWERING LOCUS C (FLC) represses flowering in non-vernalized plantsand is upregulated by FRIGIDA (FRI) prior to vernalisation (Michaels andAmasino, 1999). FLC delays flowering by directly repressing several genesrequired for floral promotion. These genes include FT in the leaves and SOC1and FD in the shoot apex (Hepworth et al., 2002; Searle et al., 2006). Asvernalisation proceeds, the FLC locus is epigenetically and stably silenced bytrimethylation at lysine 27 of histone 3 (Wood et al., 2006). Silencing of FLCallows for the LD-dependent upregulation of FT and SOC1, which thenpromote flowering as described for the photoperiodic pathway.

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The winter growth habit in temperate grasses is caused by interactions at threeloci, VRN1, VRN2 and VRN3. VRN1 is the grass orthologue of the meristemidentity gene AP1 (Yan et al., 2003), while VRN3 is orthologous to FT (Yan etal., 2006). VRN2 encodes for a CCT domain protein that does not have closehomologues in Arabidopsis, but is downregulated by vernalisation and SDssimilarly to FLC (Yan et al., 2004). In non-vernalised plants VRN2 delaysflowering by repressing VRN1 and VRN3. Vernalisation causesdownregulation of VRN2, allowing for the subsequent LDs to promote theexpression of VRN3, which in turn upregulates VRN1 (Yan et al., 2006). Theidentity of the repressor is not the only element different between Arabidopsisand grass vernalisation pathways. In wheat, the three genes form a regulatoryfeedback loop not characterised in Arabidopsis, as VRN1 has been shown todownregulate VRN2. Moreover, the high level of VRN1 expression in leaftissues contrasts the expression pattern of Arabidopsis AP1, which is almostexclusively expressed in flowering induced shoot apical meristems (Yan et al.,2003).

In cultivated beet, the vernalisation response is determined by interactionsof a pseudo-response regulator gene BOLTING TIME CONTROL1 (BvBTC1),and two beet homologues of FT, BvFT1 and BvFT2 (Pin et al., 2012). BvFT2has the same flowering-promoting function as Arabidopsis FT, whereas BvFT1has evolved into a floral repressor (Pin et al., 2010). In annual beet, floweringis promoted in LDs through upregulation of BvBTC1, which in turn leads torepression of BvFT1 and upregulation of the floral promoter BvFT2. Inbiennial beet, the BvBTC1 gene harbors a large 28 kb insertion in its promoterregion, making the gene non-responsive to LDs. Accessions with this insertionrequire a long period of cold to sufficiently induce BvBTC1 expression topromote flowering (Pin et al., 2012).

The vernalisation pathways of Arabidopsis, temperate cereals and beetinclude different components. However, all these pathways contain arepressor expressed at a high level prior to vernalisation, and silenced asvernalisation proceeds, and the vernalisation pathways in these three speciesinvolve genes homologous to the floral promoter FT.

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Ambient temperature pathway

Although both ambient temperature and vernalisation pathways mediateresponses to changes in temperature, they are fundamentally different. Theonset of the vernalisation response requires weeks (Chouard, 1960), whereasplants respond to changes in ambient temperature typically in a matter of days(Balasubramanian et al., 2006). The term ambient temperature refers to anon-stressful temperature range, which in Arabidopsis lies between 12 and27C (Wigge, 2013). The effects of ambient temperature on flowering differfrom species to species; in Arabidopsis, elevating the temperature by 4C from23 to 27C can accelerate flowering under SDs (Balasubramanian et al., 2006).In contrast, elevating temperature from 18C to 25C has been shown to delayflowering in Boechera stricta, a perennial relative of Arabidopsis (Andersonet al., 2011).

The molecular components of the ambient temperature pathway includeboth positive and negative regulators. FT, a positive regulator of thephotoperiodic pathway, is also a component of the ambient temperaturecontrolled flowering pathway. Regulation of FT by ambient temperatureoccurs at multiple levels. Low temperature induces changes in chromatinconformation and makes the FT promoter less accessible to transcription(Kumar and Wigge, 2010). This is caused by an increased incorporation ofhistone variant H2A.Z into nucleosomes instead of the regular H2A histones.Incorporation of H2A.Z histone makes DNA pack more tightly. At elevatedtemperature, H2A.Z is evicted from nucleosomes and the FT promoter isaccessible to transcription factors (Kumar and Wigge, 2010). One of thesetranscription factors is PHYTOCHROME INTERACTING FACTOR4 (PIF4), abHLH transcription factor that binds to the FT promoter to advance floweringat high temperatures in SDs (Kumar et al., 2012).

In LDs, high temperature triggered flowering is regulated via interactionsbetween the MADS box transcription factors SHORT VEGETATIVE PHASE(SVP) and FLOWERING LOCUS M (FLM). SVP is a floral repressor that formshomodimers and is effectively degraded at high temperature (Lee et al., 2013).FLM is subject to temperature-dependent alternative splicing; at lowtemperature, the flowering-repressive splice variant FLM-β prevails.However, at higher temperatures the splice variant FLM-δ, which is impairedin its DNA binding capacity, is more abundant (Posé et al., 2013). Both splicevariants can form heterodimers with SVP, and they compete for binding toSVP to regulate flowering in an antagonistic manner. At low temperature, theSVP-SVP and SVP-FLM-β complexes actively prevent flowering bydownregulating their target genes which include SOC1 and possibly FT. Athigher temperatures the splice variant FLM-δ inhibits binding of the proteincomplexes to DNA, thus regulating flowering in a dominant-negative manner(Lee et al., 2013; Posé et al., 2013).

Also other genes have been shown to play a role in the ambient temperatureresponse in Arabidopsis. Strasser et al. (2009) found that delayed flowering

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caused by low ambient temperature could be at least partially alleviated bymutations at EARLY FLOWERING3 (ELF3) and TFL1, and that the doublemutant elf3 tfl1 was insensitive to temperature. The authors suggested thatTFL1 represses flowering at low temperature, and that ELF3 and TFL1 act ontwo independent pathways; ELF3 is regulated through PHYB, whereas TFL1is regulated through cryptochromes (Strasser et al., 2009).

The molecular components of the ambient temperature pathway have notbeen studied extensively in other plants than Arabidopsis. However, studiesin several species such as Chinese narcissus (Narcissus tazetta var. Chinensis;Li et al., 2013), chrysanthemums (Chrysanthemum sp.; Nakano et al., 2013)and Satsuma mandarin (Citrus unshiu; Nishikawa et al., 2007) suggest theinvolvement of FT homologues in ambient temperature dependent flowering;in all these species, ambient temperature-induced flowering is associated withan increased transcription of FT-like genes.

Genetic control of flowering in Fragaria

In F. × ananassa, studies into the genetic control of flowering have yieldedsomewhat conflicting results. The early studies on the genetics of floweringsuggested that the everbearing trait is controlled by a single dominant locus(Ahmadi et al., 1990; Sugimoto et al., 2005), whereas later it was proposedthat the everbearing character is controlled by several QTL (Weebadde et al.,2008). Recently, two independent studies showed that the everbearing trait inthe cultivated strawberry is controlled by a dominant QTL located on linkagegroup 4 (LGIV), and that the same QTL controls also the runnering trait(Gaston et al., 2013; Castro et al., 2015). Moreover, Honjo et al. (2015) showedby genetic complementation that the everbearing trait is caused by the samelocus in both older everbearers with European or American origin and incultivars with F. virginiana ssp. glauca germplasm in their ancestry. Also thelocus identified by Honjo et al. (2015) is likely located on the LGIV of thecultivated strawberry.

The difficulty of studying the genetics of the everbearing trait in cultivatedstrawberry with a complex octoploid genome was recognised very early.Therefore, diploid everbearing F. vesca accessions have been used forelucidating the genetic basis of the everbearing trait as early as the 1960s. Alleverbearing accessions (historically nominated F. vesca f. semperflorens) arethought to have originated from the same natural source in the Alps, and forthat reason are often called “Alpine” strawberries in older literature (Darrow,1966). The everbearing trait in woodland strawberry has been shown to berecessive, and genetic complementation experiments have demonstrated thatthe trait is indeed caused by the same gene in the studied everbearingaccessions (Brown and Wareing, 1965). This locus was subsequentlynominated the SEASONAL FLOWERING LOCUS (SFL; Albani et al., 2004).Studies into phenotypic segregation ratios revealed that the everbearing trait

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and runnering are controlled by two independent loci (Brown and Wareing,1965), the everbearing trait located on diploid Fragaria linkage group 6(LGVI), and the runnering locus on linkage group 2 (LGII; Sargent et al.,2004). Based on different chromosomal locations, it therefore seems that thegenetic basis of the everbearing character is different in diploid and octoploidstrawberries.

The genetic nature of SFL in woodland strawberry has been subject tospeculation from the 1960s. Brown and Wareing (1965) reasoned that theperpetual flowering trait arises as a result of a mutation at SFL, which wasthought to encode for a floral inhibitor. It has been proposed that SFL couldbe a gene homologous to the vernalisation pathway gene FLC (Battey, 2000).However, no FLC homologs have been found in the Rosaceae EST databasecontaining more than 500 000 sequences (Mouhu et al., 2009).

Other candidates for SFL have included a CO-like gene and GA pathwaygenes, CO because of the photoperiodic flowering response in strawberry(Mouhu et al., 2009) and GA pathway genes because GA application inhibitsflowering in strawberries (Guttridge and Thompson, 1964). However, thegenomic location of strawberry CO does not match with that of SFL (Stewart,2007). GA pathway gene are also unlikely candidates, as no differences in theexpression of GA pathway-related genes between everbearing and SDgenotypes have been observed (Mouhu et al., 2009).

Studies in other perennial species propose that SFL may encode for a CETSgene. In hybrid poplar, it has been shown that a poplar orthologue of TFL1, agene called CENTRORADIALIS-LIKE1 (CENL1), is downregulated as aresponse to SDs (Ruonala et al., 2008), and silencing poplar CENL genesresults in early flowering (Mohamed et al., 2010). Moreover, a TFL1homologue of apple, MdTFL1, is involved in maintenance of the vegetativephase (Kotoda and Wada, 2005), and silencing MdTFL1 leads to very earlyflowering (Kotoda et al., 2006).

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The CETS family

As seen in the previous chapters, the CETS genes play important roles in plantdevelopment. Interestingly, CETS proteins share similarities with human Rafkinase inhibitor proteins (RKIPs), which have been associated with celldifferentiation and cell-cycle arrest (Yeung et al., 1999). The CETS gene familyin Arabidopsis contains genes with important roles in plant development; inaddition to the florigen FT and the floral repressor TFL1, the Arabidopsisgenome contains TWIN SISTER OF FT (TSF), ARABIDOPSISCENTRORADIALIS (ATC), BROTHER OF FT AND TFL1 (BFT) and MOTHEROF FT AND TFL1 (MFT) (Kobayashi et al., 1999). MFT is associated with seedgermination and release from seed dormancy (Footit et al., 2011), ATC andBFT are floral repressors and functionally redundant with TFL1 (Yoo et al.,2010; Huang et al., 2012) and TSF acts as a floral promoter redundantly withFT (Yamaguchi et al., 2005).

TSF shares high amino acid similarity with FT, expression of TSF ispromoted in the phloem in LDs and TSF overexpressing plants flower earlyresembling the phenotype of FT overexpressors (Michaels et al., 2005;Yamaguchi et al., 2005). Moreover, TSF responds to changes in ambienttemperature in the same manner as FT, and the gene has been shown to be amajor target of the ambient temperature regulated flowering pathway (Lee etal., 2013). However, tsf single mutants do not display a flowering time relatedphenotype under LD conditions, and it was recently shown that TSF has apoorer ability to move from phloem to the shoot apex and is less stable thanFT (Jin et al., 2015). It is thus possible that TSF has a less significant biologicalfunction than FT.

The amino acid sequence of BFT resembles FT more than TFL1, and BFTexpression is upregulated by LDs similarly to FT and TSF (Yoo et al., 2010).However, BFT overexpression results in late flowering and floral defects thatresemble those caused by ectopic TFL1 expression. Modifications in BFTexpression level also change plant architecture by altering the rate of terminalto axillary inflorescence development (Yoo et al., 2010). Therefore, BFTappears to control inflorescence meristem identity redundantly with TFL1.

ATC is named after Antirrhinum CEN, with which ATC shares highprotein-level similarity. Although ATC can functionally complement the tfl1mutation when constitutively expressed, its expression pattern in vasculartissues suggests a different biological role (Mimida et al., 2001). Indeed, it hasbeen shown that ATC mRNA is a mobile floral repressor expressed in leaves inSDs. Similarly to FT and TFL1, also the ATC protein is capable of interactingwith FD and regulating the same downstream targets (Huang et al., 2012).Long-distance transport from leaves to the shoot apex was also shown forchrysanthemum anti-florigenic FT/TFL1 family protein (CsAFT). Moreover,CsAFT expression was induced after exposure to a night-break, a treatmentthat inhibits flowering in chrysanthemums (Higutchi et al., 2013).

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3 AIMS OF THE STUDY

Gaining an insight into molecular networks regulating flowering is essentialfor developing cultivars better adapted to the changing environment. As thecultivated strawberry is genetically complex, the less complicated woodlandstrawberry was used as a model plant. Therefore, the broad objective of thisstudy was to identify genes regulating flower induction in woodlandstrawberry and study how environmental factors affect the action of thesegenes. The second objective was to utilize the genetic information from studieswith woodland strawberry and examine how the homologous genes functionin cultivated strawberry.

Specific aims for the original publications were:

I Identification and molecular characterisation of the gene residingat the SEASONAL FLOWERING LOCUS (SFL) in woodlandstrawberry

II Elucidation of the role of TERMINAL FLOWER1 in thevernalisation response of arctic woodland strawberry accessionAlta-1

III Elucidation of the role of TERMINAL FLOWER1 in the control offlowering in cultivated strawberry

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4 MATERIALS AND METHODS

Methods used in this thesis are summarised in Table 1 and described in moredetail in respective publications. Methods used by co-authors are indicated inparentheses.

Table 1. Methods used in this thesis.

Method PublicationcDNA synthesis I, II, IIICrossing populations I, (II)DNA extraction I, IIGateway™ vector constructionGenotyping-by-sequencing analysis

I(II)

Genetic transformationGenotyping

I, (III)I, II

Growth experiments I, II, IIIin situ hybridisation (I)Linkage analysis I, (I)Marker design I, IIRNA extraction I, II, IIIRT-qPCR I, II, III

Plant materials and experimental conditions

To identify the SFL via positional cloning, two cross populations wereproduced; an F2 population resulting from a cross between a seasonallyflowering F. vesca accession 'Punkaharju' (National Clonal GermplasmRepository accession PI551792, abbreviated as Punk-1) and an everbearingaccession 'Hawaii-4' (H4), and a back-cross (BC) population between aseasonal F. vesca and everbearing F. vesca f. semperflorens. The F2population consisted of 978 seedlings, whereas the BC population included2996 seedlings. The seedlings were germinated and grown in LDs at 18C andthen subjected to a SD treatment for 5 weeks. After the SD treatment,flowering (presence/absence) was scored in LDs. In addition, F. vesca, H4 andArabidopsis tfl1-2 mutant in the Landsberg erecta (Ler) background weresubjected to daylength treatments described in detail in (I).

The effects of low temperature were studied in the seasonal F. vescaaccession Punk-1 and the vernalisation requiring Alta-1 accession fromnorthern Norway. The plants were subjected to several low temperaturetreatments detailed in (II). An F2 population resulting from a cross betweenAlta-1 and 'Hawaii-4' carrying the FvTFL1 hairpin construct was produced tostudy the effect of non-functional and silenced FvTFL1 on the vernalisationresponse.

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To study the effect of TFL1 on the flowering response in cultivated strawberry,five F. × ananassa cultivars (Table 2) were subjected to daylength (SD/LD)and temperature (9–21ºC) treatments described in detail in (III).

Table 2. Plant materials used in the experiments.

Species Accession name Accessionnumber

Source

A. thaliana Landsberg erectatfl1-2

N3091 Nottingham Arabidopsis Stock Centre

F. × ananassa Alaska Pioneer PI551796 Natural Resources Institute Finland (Luke)F. × ananassa Elsanta PI551579F. × ananassa Glima naF. × ananassa Honeoye PI551588 Natural Resources Institute Finland (Luke)F. × ananassa Polka na Natural Resources Institute Finland (Luke)

F. vesca Punkaharju PI551792 NCGRF. vesca Hawaii-4 PI551721 NCGRF. vesca Alta na A. Sønsteby (NIBIO Norway)

Transgenic experiments

Plasmid vectors for plant transformation were constructed as described in (I)and transformed into plant tissues via Agrobacterium-mediatedtransformation. Transformation was done by the floral dip method (Zhang etal., 2006) for Arabidopsis, by the protocol of Oosumi et al. (2006) for F. vescaand by the protocol of Fischer et al. (2014) for F. × ananassa. In total, fourdifferent constructs were transformed into four genotypic backgrounds (Table3).

In addition to direct transformation, the FvTFL1 hairpin construct forsilencing FvTFL1 was utilized for producing the Alta-1 x 'Hawaii-4' F2population. The paternal 'Hawaii-4' parent carried the FvTFL1 hairpinconstruct to promote flowering in the F1 generation.

Table 3. The vectors used and transgenes introduced into different plant genotypicbackgrounds.

Background Vector Transgene PublicationA. thaliana Ler tfl1-2 pK7WG2D-2 (overexpression) Functional FvTFL1 IF. × ananassa 'Elsanta' pK7WIWGD2D-II (RNAi) FvTFL1 hairpin IIIF. vesca 'Hawaii-4' pK7WG2D-2 (overexpression) Functional FvTFL1 IF. vesca 'Hawaii-4' pK7WG2D-2 (overexpression) Mutated FvTFL1 IF. vesca 'Hawaii-4' pK7WIWGD2D-II (RNAi) FvTFL1 hairpin I, IIF. vesca 'Hawaii-4' pK7WIWGD2D-II (RNAi) FvFT1 hairpin IF. vesca 'Punkaharju' pK7WG2D-2 (overexpression) Functional FvTFL1 IF. vesca 'Punkaharju' pK7WG2D-2 (overexpression) Mutated FvTFL1 IF. vesca 'Punkaharju' pK7WIWGD2D-II (RNAi) FvTFL1 hairpin I

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Genotyping and mapping

Genetic mapping was used in (I) and (II) to identify genes associated withqualitative flowering responses. DNA extraction was done following theprotocol of Doyle and Doyle (1990), scaled down to fit in a 1.5 ml tube. Themarkers used for genotyping are described in (I) and (II). Linkage maps wereconstructed as described by Sargent et al. (2004).

RT-qPCR

RNA for real-time quantitative PCR (RT-qPCR) was extracted according to aprotocol described earlier (Mouhu et al., 2009). In (II) and (III), RNA sampleswere treated with rDNAse (Macherey-Nagel GmbH, Düren, Germany)according to manufacturer's recommendations. cDNA was synthesized andRT-qPCR run as described in respective publications. MSI1 was used as astable reference gene and relative expression ratios were determined followingthe ΔΔCt method (Pfaffl, 2001).

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5 RESULTS AND DISCUSSION

5.1 SFL ENCODES FOR AN ORTHOLOGUE OF TFL1 INF. VESCA (I)

Genetic mapping in F2 and BC populations indicated that the SFL was locatedwithin a narrow mapping window of 248 kb in F. vesca LGVI (I, Figures S1and S2). This genetic interval contained the F. vesca homologue of TFL1(FvTFL1). FvTFL1 was selected as the most promising candidate, becausehomologues of TFL1 have been shown to repress flowering in both annual(Arabidopsis; Bradley et al., 1997; Hanano and Goto, 2011) and perennial(Malus × domestica; Kotoda et al., 2006) species. Sequencing FvTFL1 fromthe everbearing cross parents showed that both had a 2 base pair deletion inthe first exon of the gene, causing a frameshift and resulting in a putativelynonfunctional protein product. These results were in full concordance with thedata of Iwata et al. (2012), who some time earlier showed that the everbearingtrait was associated with a disruption in the reading frame of TFL1 homologuesin both F. vesca and rose.

According to Iwata et al. (2012), the loci controlling continuous floweringin rose and strawberry are orthologous and named the loci RoKSN and FvKSN,respectively. However, the same gene had been named as FvTFL1 alreadyearlier by Mimida et al. (2012). Moreover, both the conserved synteny aroundFvTFL1 (Figure 2) and Arabidopsis TFL1 and phylogenetic relationships(Mimida et al., 2012) suggest that the genes are true orthologues, supportingconsistent nomination of FvTFL1 for the F. vesca orthologue.

Figure 3. Synteny around the TFL1 locus in woodland strawberry and Arabidopsis. A 600 kbwindow of Fragaria (top) and Arabidopsis (bottom) genomes show synteny around the regionssurrounding FvTFL1 and TFL1. Syntenous regions were visualized using GEvo in the CoGeplatform (Lyons and Freeling, 2008). Green blocks represent gene models and pink lines connecthigh-scoring sequence pairs identified by BlastZ. Orange color in the Fragaria genome representsunsequenced regions.

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If FvTFL1 was indeed a floral repressor activated under LDs, we would expectto detect FvTFL1 mRNA in the shoot apical meristems of non-induced plants.Indeed, we found FvTFL1 transcripts over the entire apical meristem of LD-grown non-induced Punk-1 plants (I, Figure 4). Moreover, FvTFL1 expressionlevel in shoot apical meristems responded to photoperiodic conditions; thegene showed gradual downregulation in SDs and was again upregulated uponreturning the plants to LDs (I, Figures 4 and 5). Photoperiodic regulation of aTFL1 orthologue has not been observed in other species studied so far; inArabidopsis, TFL1 is developmentally regulated (Bradley et al., 1997; Ratcliffeet al., 1998), and the pea PsTFL1c does not exhibit environmental ordevelopmental regulation, although the loss of PsTFL1 leads to early flowering(Foucher et al., 2003).

The role of FvTFL1 as a floral repressor was confirmed by overexpressingthe functional and mutated alleles of FvTFL1 in the everbearing H4background. Plants that overexpressed mutated and putatively non-functionalFvTFL1 flowered continuously, resembling wild type H4 plants, while theplants carrying the overexpressed functional FvTFL1 remained vegetative forat least 10 months (I; Figure 1). Moreover, silencing functional FvTFL1 byRNAi in the SD-requiring Punk-1 background removed the SD requirement,causing the LD-grown transgenic plants to flower at the same time as SD-treated wild type Punk-1 plants (I, Figure 2). These data indicate that themutated everbearing allele indeed results in a non-functional protein, and thatthe functional FvTFL1 is a floral repressor that causes the seasonal floweringhabit.

The finding that the SFL encodes for a homologue of the floral repressorTFL1 is not surprising. In the monocot maize (Zea mays), overexpression ofTFL1-like genes results in delayed flowering time (Danilevskaya et al., 2010),and in pea (Pisum sativum), the loss of PsTFL1c results in early flowering(Foucher et al., 2003). In Rosaceous fruit crops, TFL1 controls also the lengthof the juvenile phase; silencing TFL1 in pear (Pyrus communis L.) and applehas resulted in accelerated life cycles, thus speeding up breeding processes(Flachowsky et al., 2012; Freiman et al., 2012). It is possible that FvTFL1 hasa similar function in maintaining the juvenile phase, as flower inductionshown by FvAP1 upregulation occurs earlier in H4 seedlings than in Punk-1seedlings grown under inductive photoperiodic conditions (I, Figure S7).

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5.2 PHOTOPERIOD REGULATES FVTFL1 EXPRESSIONAND FLOWERING (I)

After identifying FvTFL1 as SFL, we were interested in finding out how thegene functions to regulate the photoperiodic flowering response. Therefore,expression of FvTFL1 and its putative downstream targets, the floral meristemidentity genes FvAP1 and FvFUL1 were examined in shoot apical meristems ofboth wild type and transgenic plants grown under different photoperiodicconditions. In all cases, downregulation of FvTFL1 was associated with higherlevels of FvAP1 and FvFUL1 mRNAs and with earlier flowering time (I). Thecorrelation between FvAP1 upregulation and subsequent floweringcorroborated the findings of Mouhu et al. (2009) who suggested that FvAP1could be used as a marker for floral initiation.

FT proteins have been identified as major mobile floral promoters in arange of species (Turck et al., 2008), and we were interested in elucidating therole of F. vesca orthologue of FT in photoperiodic control of flowering. The F.vesca genome harbors three FT homologues (Mimida et al., 2012). Tissue-specific expression patterns and conserved synteny with the Arabidopsisorthologue (Figure 3; I, Figure 6) suggested that F. vesca FT1 was the mostlikely orthologue of FT and was studied further. The function of FvFT1 as afloral promoter was confirmed by silencing the gene in the H4 background;flowering was delayed in FvFT1-silenced plants (I, Figure 7).

Figure 4. Synteny around the FT1 locus in woodland strawberry and Arabidopsis. A 400 kbwindow of Fragaria (top) and Arabidopsis (bottom) genomes show synteny around the regionssurrounding FvFT1 and FT1. Syntenous regions were identified using SynFind tool and visualizedusing GEvo in the CoGe platform (Lyons and Freeling, 2008). Green blocks represent gene modelsand pink lines connect high-scoring sequence pairs identified by BlastZ. Orange color in theFragaria genome marks unsequenced regions.

Expression analysis showed that FvFT1 expression correlated with floweringonly in H4, where LDs promoted FvFT1 expression and early flowering. InPunk-1, LDs also resulted in an elevated FvFT1 transcript level but noflowering occurred (I, Figure 6). These data led us to build a model accordingto which functional FvTFL1 represses flowering under LDs in the SD-requiringPunk-1 accession. In the LD accession H4, the lack of functional FvTFL1 leadsto rapid FvFT1-mediated flowering. How FvTFL1 overrides the function ofFvFT1 in Punk-1 to repress FvAP1 and FvFUL1 could be explained bycompetitive binding to protein complexes; it is possible that FvTFL1 has a

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higher binding affinity to FD than FvFT1 does. Such a mechanism has beensuggested in rose, where the presence of RoKSN weakened the interactionbetween RoFD and RoFT (Randoux et al., 2014).

The photoperiodic model for flower induction was further refined byMouhu et al., (2013), who showed that FvTFL1 is regulated via the F. vescaorthologue of SOC1 (FvSOC1), and that FvSOC1 expression in turn is affectedby FvFT1. FvSOC1 is involved in the photoperiodic regulation of bothreproductive and vegetative aspects of growth; constitutive FvSOC1expression in transgenic lines results in production of runners, whereassilencing FvSOC1 by RNAi results in inflorescence formation. The authors alsoshowed that altering FvTFL1 transcription did not notably change theexpression of FvSOC1 (Mouhu et al., 2013). These data helped to explain whythe formation of branch crowns and stolons were not affected in the transgenicFvTFL1 plants (I, Figure 3). According to the results of Mouhu et al. (2013)and (I), FvSOC1 and FvTFL1 are components of the same photoperiodicflowering pathway, with FvSOC1 controlling aspects of vegetativedevelopment, but also flowering via regulation of FvTFL1. As FvTFL1putatively acts downstream of FvSOC1, changes in FvTFL1 expression affectonly the flowering response, and not patterns of vegetative development.

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5.3 ALTERED REGULATION OF FVTFL1 ISASSOCIATED WITH THE UNIQUE VERNALISATIONRESPONSE IN ALTA-1 (II)

When comparing flowering responses of six Norwegian woodland strawberryaccessions, Heide and Sønsteby (2007) inadvertently found an accessionwhich appeared to require winter before being able to flower. This Alta-1accession would not flower in SDs or LDs at temperatures ranging from 9 to21C, and when tested under field conditions, it flowered considerably laterthan the other populations (Heide and Sønsteby, 2007). Exposing Alta-1 to 2Cfor a minimum of five weeks induced flowering in a proportion of the plants,and it was concluded that, unlike any other strawberry accession studied todate, Alta-1 possesses a unique vernalisation requirement.

Seasonal flowering cycle in altered in Alta-1 (II)

Alta-1 appears to have an altered seasonal flowering cycle (Heide andSønsteby, 2007), and therefore it was of interest to elucidate whether thecorrelation between flowering time and seasonal expression patterns offlowering-related genes would differ in Alta-1 and Punk-1 plants grown undernatural conditions. The flowering time observations on the field wereconsistent with the observations of Heide and Sønsteby (2007); Alta-1flowered considerably later and continued to produce new inflorescences forlonger than Punk-1 (II, Figure 2).

Expression analysis of FvTFL1 in shoot apex samples of field grown Alta-1and Punk-1 plants collected monthly from August to December and again inMay showed that the expression of FvTFL1 was indeed higher in Alta-1 at alltested timepoints (II, Figure 1A). Moreover, downregulation of FvTFL1 byshortening days and cooling temperature occurred at a slower rate in Alta-1than in Punk-1 (II, Figure 1A and S2). The meristem identity gene FvAP1 wasupregulated in shoot apices of Punk-1 already in October, whereas in Alta-1,upregulation of FvAP1 was not observed before May (II, Figure 1C).

The expression patterns of FvTFL1 and FvAP1 suggested that floralinduction had not taken place in field-grown Alta-1 plants in December. Toconfirm these observations, Alta-1 and Punk-1 plants were taken togreenhouse in December and their flowering was observed under LD (18C)conditions. In concordance with elevated FvAP1 expression, all Punk-1 plantsflowered uniformly after 31 days in the greenhouse, whereas only 20% of Alta-1 plants flowered after approximately 51 days (II, Table S1). The observed lateand incomplete flowering of Alta-1 together with FvAP1 expression dataconfirmed that the plants had not been flowering-induced under fieldconditions, and that the greenhouse conditions were not favorable for floralinduction.

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Alta-1 requires exceptionally low temperatures to fulfil the vernalisationrequirement (II)

The work of Rantanen et al. (2015) indicated that the photoperiod-independent flowering observed in Punk-1 at cool temperature of 10C iscorrelated with downregulation of FvTFL1. Therefore, we were interested tosee whether FvTFL1 regulation was altered in the vernalisation-requiring Alta-1. To study this, runner propagated Alta-1 plants were exposed to natural SDsat 4C for 0 to 15 weeks, followed by flowering observations in LDs at 20C.These conditions were not favorable for floral induction, as none of the plantsflowered even after 15 weeks at 4C (data not shown) and FvTFL1 was notdownregulated in the shoot apices of plants sampled at any time point (II,Figure 3).

To clarify the conditions required for fulfilling the vernalisationrequirement of Alta-1, we subjected Punk-1 and Alta-1 plants to continuousLDs, three weeks of SDs and to low fluctuating temperature (±2C) for five orten weeks. In addition, Alta-1 plants were subjected to a constant temperatureof 0C for five to ten weeks. As expected, neither accession flowered in LDs,whereas Punk-1 was induced to flower by a three week exposure to SDs, andalso by the low temperature treatments. In contrast, SDs and low temperaturetreatments induced ample runner production in Alta-1 (II, Figure S3B), whichis considered diagnostic of active vegetative growth (Konsin et al., 2001;Hytönen et al., 2004). Only an exposure to low temperature for ten weekscould induce flowering in a proportion of Alta-1 plants (II, Table 1, Table S2).The expression of FvAP1 correlated with the flowering observations; the genewas upregulated in Punk-1 exposed to SDs and low temperature, whereas noupregulation was observed in Alta-1 by the end of the low temperaturetreatments (II, Figure 3C).

Alta-1 is induced to flower by LDs and cool temperature (II)

As per definition, vernalisation results in acquired competence to flower, butit does not induce flowering per se (Chouard, 1960). Based on the resultspresented in (II), the Alta-1 accession has a true vernalisation response; theaccession requires low temperature to downregulate FvTFL1 but this processdoes not induce flowering in itself. The seasonal experiments suggested thatAlta-1 is induced to flower in spring, when the days are relatively long (14–16h) and temperature is around 10C (II, Figure 1S). This prompted us tohypothesise that Alta-1 requires LDs and cool temperature after vernalisationto induce flowering. This hypothesis was tested in an experiment where field-grown, naturally vernalised Alta-1 plants were brought to greenhouse inJanuary and grown at either 10 or 20C in LDs for five weeks.

Flowering was observed only in plants subjected to 10C (II, Figure 4C andTable 2), corroborating our hypothesis that cool temperature and LDs are

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needed to induce flowering in Alta-1. However, FvTFL1 was upregulated afterfive weeks at both 10 and 20C (II, Figure 4A). At this point, it remains unclearhow the plants exposed to 10C were induced to flower although FvTFL1 wasupregulated in the shoot apices collected after five weeks at 10C. It is possiblethat upregulation of FvTFL1 was faster at 20C than at 10C and that floralinduction at 10C occurred within a narrow time window during the first weeksin LDs at 10C.

FvTFL1 expression is required for the vernalisation response (II)

As our data suggested that FvTFL1 was involved in the vernalisation responseof Alta-1, we wanted to gain experimental confirmation to this hypothesis. Wecrossed the vernalisation requiring Alta-1 with a transgenic H4 line carrying asingle copy of the FvTFL1-RNAi construct described in (I) to obtain an F1population in which approximately half of the progeny carried the transgeneand half were non-transgenic (II; Figure S7). As H4 is homozygous fornonfunctional FvTFL1 (I), and Alta-1 is homozygous for functional FvTFL1, wedid not expect the non-transgenic F1 plants to flower. As expected, none of thenon-transgenic plants in the F1 population flowered in LDs (data now shown),whereas F1 plants carrying the FvTFL1-RNAi construct showed a reduction inFvTFL1 transcript level and flowered early in LDs, (II, Figure 5). A similarflowering pattern was observed in the F2 population produced from seeds of aGFP positive F1 plant; the plants carrying the transgene or homozygous for theH4-derived nonfunctional FvTFL1 flowered in LDs (II; Table S3). These dataconvincingly show that FvTFL1 expression is required for the vernalisationresponse.

Based on these results, it was impossible to say whether the gene causingthe vernalisation response was FvTFL1 itself, or an upstream regulator ofFvTFL1. To test for the presence of a novel FvTFL1 regulator, we subjected thenon-transgenic F2 plants to SDs for six weeks, after which flowering wasobserved in LDs. If the vernalisation response was caused by an upstreamregulator of FvTFL1, the gene would be likely to segregate in the F2 population,resulting in SD-promoted flowering phenotype in a proportion of the F2plants. However, no flowering was observed in the non-transgenic F2population, suggesting that the vernalisation response is controlled by a singledominant gene that may be FvTFL1 itself, or a gene located in the proximity ofFvTFL1 on chromosome 6. It can be speculated that the vernalisation responsein Alta-1 may be caused by a mutation at FvTFL1 promoter region. Although again-of-function mutation seems unlikely, it is not without precedent. In beet,it was recently shown that a large 28 kb mutation at the promoter of keyflowering gene BvBTC1 can cause a vernalisation response (Pin et al., 2012).The vernalisation response in beet is an adaptation to northern climate, as itappears in northern accessions of Beta vulgaris ssp. maritima, the putativeancestor of the cultivated beet (Pin et al., 2010). Within Fragaria genus, Alta-

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1 is the only known example of an accession requiring vernalisation. Wephenotyped a total of 67 northern European accessions for SD-inducedflowering, and were unable to find accessions that would not flower after anexposure to inductive conditions (II, Table S4). Moreover, genome-widegenotyping data from 78 accessions (II, Figure 7) showed that Alta-1 is moreclosely related to other accessions from the Alta region in northern Norwaythan to accessions from elsewhere in northern Europe. This suggests that themutation causing the Alta-1 phenotype is a local one, and has arisen relativelyrecently. The finding also supports the notion that vernalisation requirementhas evolved independently in individual plant lineages (e.g. Ream et al., 2012).

The photoperiodic FvFT1–FvSOC1 pathway is regulated similarly in Punk-1and Alta-1 (II)

FvFT1 and FvSOC1 are photoperiodically regulated in Punk-1, with LDsupregulating and SDs downregulating their expression (I; Mouhu et al., 2013).Similarly to Punk-1, FvFT1 expression was downregulated by SDs in Alta-1 (II,Figure S4), and we were unable to find differences in FvSOC1 expressionbetween the two accessions in a range of both natural (II, Figure 1) andcontrolled conditions (II, Figure 3). As previous experiments with transgenicplants have suggested that FvTFL1 expression is activated by FvFT1 andFvSOC1 in LDs (I; Mouhu et al., 2013), we were surprised to see upregulationof FvTFL1 in SDs in Alta-1 (II, Figure 4A and S4). These data suggested thatunknown factor(s) upregulate FvTFL1 independently of the FvFT1–FvSOC1pathway.

This suggests that the vernalisation response in Alta-1 is controlled by amechanism different from that described in the perennial Brassicaceae modelspecies Arabis alpina, in which the regulation of AaSOC1 is involved in thevernalisation response (Wang et al., 2011). In Alta-1, the vernalisationresponse is likely regulated via altered expression of FvTFL1, thus adding onemore aspect to the multitude of processes that TFL1 homologues may control.

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5.4 TFL1 IS A FLORAL REPRESSOR IN F. ANANASSA(III)

The results discussed so far provide strong evidence for the role of FvTFL1 inthe environmentally controlled flowering response in woodland strawberry(I), and show that changes in the expression patterns of FvTFL1 may evenchange the entire life history of a plant (II). However, these studies wereconducted in the diploid model plant F. vesca, in which the molecular controlof flowering could be different from that of the more economically importantcultivated strawberry F. × ananassa. Therefore, it was of interest to elucidatewhether the knowledge gained from studies on molecular control of floweringin woodland strawberry could be extended to the cultivated strawberry.

Silencing FaTFL1 leads to early flowering (III)

To study the function of TFL1 in cultivated strawberry, we silenced FaTFL1 inthe SD cultivar ‘Elsanta’ by using the same FvTFL1-RNAi hairpin construct aswas used for silencing FvTFL1 in woodland strawberry. Flowering in LD-grown primary transgenic lines F138 and F139 occurred in approximately 78and 125 days, respectively, whereas no flowering was observed in the wild type‘Elsanta’ plants grown in LDs (II, Figure S3). Although the flowering times ofthe LD-grown transgenic lines may appear late, they are actually comparableto the flowering times observed for seed-propagated everbearing cultivarsgrown under continuous flowering inducing LD conditions (Table 4; Sønstebyand Heide, 2007). It has been noted that in vitro propagated plants displaymorphological features that resemble those observed in juvenile seedpropagated plants, and that this juvenile period lasts for four to five weeks(Huxley and Cartwright, 1994). Therefore, it is possible that the relatively lateflowering observed in the transgenic lines was a juvenility effect caused by invitro propagation.

Table 4. Flowering time of everbearing F1 cultivars ‘Elan’, ‘Milan’ and ‘Tarpan, ‘and transgenic‘Elsanta’ plants with silenced TFL1-RNAi grown under continuous LD conditions. Flowering timeis expressed as days after sowing.

Genotype Days to flowering standard deviation

Elsanta TFL1-RNAi F138 78,4 5,9Elsanta TFL1-RNAi F139 125,3 18,8Elan 104,9 9,2Milan 109,3 10,7Tarpan 110,02 9,2Elsanta >150

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The effect of reduced FaTFL1 expression on the seasonal growth cycle of thecultivated strawberry was evaluated using clonally propagated runner plants.In this emulated seasonal cycle, plants of wild type ‘Elsanta’ and the transgenicline F138 were first exposed to natural SDs in an unheated greenhouse,followed by a chilling period at 6C for eight weeks. After the artificial “winter”,the transgenic line showed an early and continuously flowering phenotype,producing new inflorescences until the end of the experiment, whereas thewild type ‘Elsanta’ flowered later and produced only a few inflorescences (II,Figure 1).

Promotion of flowering by reducing TFL1 expression in strawberry is in linewith the results obtained from studies with other Rosaceous species; in pearand apple, introduction of a TFL1-RNAi construct has led to greatlyaccelerated flowering, hastened inflorescence development and shortenedjuvenile period (Freiman et al., 2012; Flachowsky et al., 2012). However, thereare also species-specific differences; development of a terminal flower istypical of pears and apples with silenced TFL1, while no differences ininflorescence development were observed in transgenic cultivated strawberrylines. Moreover, TFL1-silenced transgenic apples typically died or stoppedtheir vegetative growth after flowering, displaying a life cycle more typical ofannual than perennial plants (Flachowsky et al., 2012). An explanation for thisdifference may lie in the contrasting growth patterns of pome fruits andstrawberries. In pears and apples that follow a monopodial growth pattern(Costes et al., 2014), TFL1 may be required in the maintenance of vegetativemeristems. Strawberries grow sympodially, with the vegetative apicalmeristem having strong dominance over axillary buds located in lower nodes(Heide et al., 2013; Costes et al., 2014). When the strawberry apical meristemis converted to an inflorescence meristem, the uppermost axillary meristemcontinues vegetative growth of the crown and resumes apical dominance(Heide et al., 2013). Therefore, it may be that the fate of axillary meristems instrawberries is controlled by factors other than FvTFL1, and that silencingFvTFL1 affects only the identity of the apical meristem.

Our data shows that FaTFL1 functions as a floral repressor in the cultivatedstrawberry and silencing this gene leads to a flowering phenotype resemblingthat of everbearing cultivars, but does not affect runner development.However, the gene does not appear to cause everbearing flowering in thecultivars used in recent mapping studies, as these studies have located theeverbearing locus on F. × ananassa LGIV (Gaston et al., 2013; Castro et al.,2015; Honjo et al., 2015), and based on the conserved macrosynteny betweenF. × ananassa and F. vesca (Rousseau-Guetin et al., 2008), FaTFL1 should belocated in LGVI. These results lead Gaston et al. (2013) to state that the geneticcontrol of flowering in diploid and octoploid strawberries is different.However, given that Gaston et al. (2013) utilized poorly transferable AFLPmarkers, it is difficult to say what kind of genetic landscape their QTL regionrepresents.

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Castro et al. (2015) and Honjo et al. (2015) used transferable SSR markers formapping the everbearing trait. What is noteworthy, the two studies shared acommon marker and were able to find a linkage between the marker on LGIVand the everbearing locus in four different mapping populations.Interestingly, ongoing work in our research group suggests a QTL in LGIV ofwoodland strawberry involved in the control of flowering time (Samad et al.,manuscript in preparation), providing support for the idea that the twostrawberry species have similar genetic pathways regulating flowering time.

Irrespective of the genetic identity of the previously characterised floweringtime QTL in cultivated strawberry, our results on the role of FaTFL1 as a floralrepressor provide a new perspective for strawberry breeding. Modification ofFaTFL1 expression either by conventional means or via transgenesis enablestailoring of flowering time in strawberries without affecting vegetative growth.

Photoperiod regulates FaTFL1 expression in cultivated strawberry (III)

At intermediate temperature range, photoperiod-dependent downregulationof FvTFL1 is a prerequisite to floral induction in woodland strawberry (I), andthe gene has been shown to mediate temperature regulated floweringresponses in woodland strawberry (Rantanen et al., 2015). To investigate therole of FaTFL1 in environmental responses of the cultivated strawberry, geneexpression patterns of flowering-related genes and their correlation withflowering time were studied in five strawberry cultivars.

Photoperiodic responses at 18C were studied using the cultivars‘Honeoye’, ‘Polka’ and ‘Alaska Pioneer’. The cultivars were selected based ontheir different flowering responses; according to USDA National PlantGermplasm System, ‘Honeoye’ is an early mid-season cultivar, ‘AlaskaPioneer’ is reportedly an everbearer, whereas ‘Polka’ flowers approximatelyone week later than ‘Honeoye’ under Nordic conditions (Hytönen andRichterich, personal communication).

Photoperiodic flowering responses were investigated in the three cultivarsexposed to LDs or SDs at 18C for 6 weeks. No flowering was observed in‘Honeoye’, ‘Alaska Pioneer’ or ‘Polka’ plants grown in LDs, and in SDs‘Honeoye’ flowered nearly two weeks earlier than the other two cultivars (II,Table 1). In all cultivars, SDs downregulated FaTFL1 and upregulated themeristem identity gene FaFUL1 (II, Figure 2). Downregulation of FaTFL1 inSDs was strongest in the early-flowering cultivar ‘Honeoye’ and mildest in thelate cultivar ‘Polka’ (II, Figure 2). However, downregulation of FaTFL1occurred also in LDs in cultivars ‘Honeoye’ and ‘Alaska Pioneer’, although noflowering was observed and the meristem identity gene FaFUL1 was notexpressed in LD-grown plants (II, Figure 2). It is possible that another LD-activated repressor delayed flowering in these two cultivars. The twostrawberry CENTRORADIALIS-LIKE (CEN-L) genes could be candidates forsuch a repressor. Although the CEN-L genes have not been studied in detail in

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strawberry, research in Arabidopsis and chrysanthemum suggest a role forCEN homologs in photoperiod-dependent floral repression (Huang et al.,2012; Higutchi et al., 2013).

The discrepancy between FaTFL1 downregulation and floweringphenotype could also be explained by the results obtained by Bradford et al.(2010), who found that flowering in ‘Honeoye’ was not inhibited but onlydelayed by two weeks in LDs at 17C. It is therefore possible that LD-grown‘Honeoye’ and ‘Alaska Pioneer’ could have eventually flowered if observationshad been continued for a longer time

A similar photoperiod-independent reduction in FaTFL1 expression hasbeen observed in a Japanese SD cultivar ‘Nyoho’ (Nakano et al., 2015). Apossible explanation for these observations could be that FaTFL1 expressionin some cultivars is controlled in an age-dependent manner. High TFL1expression has been shown to correlate with juvenile phase in Lolium perenne(Jensen et al., 2001) and citrus (Pillitteri et al., 2004).

Cultivar-dependent differences in FaTFL1 regulation are correlated withflowering time (III)

Interactions of photoperiod and temperature were studied in SDs and LDs attemperatures of 9, 15 and 21C using two strawberry cultivars with differenttemperature responses. ‘Elsanta’ has been reported an obligatory SD cultivarthat is not induced to flower by cool temperatures (Sønsteby and Heide, 2006),whereas ‘Glima’ flowers photoperiod-independently at temperatures below21C (Heide, 1977). Our results corroborated the earlier findings, as ‘Elsanta’did not flower in LDs at any tested temperature, whereas ‘Glima’ floweredreadily in LDs between temperatures of 9 and 15C, and a large proportion ofthe plants were induced to flower even at 21C (II, Table 2, Figure 3).

FaTFL1 expression was generally correlated with the flowering response;in ‘Elsanta’, SDs downregulated FaTFL1 and induced flowering at all testedtemperatures. In ‘Glima’, FaTFL1 transcript levels were lower than in‘Elsanta’, and photoperiod had an effect on FaTFL1 expression only in LDs at21C, correlated with delayed flowering under these conditions (II, Figure 5).The only environmental conditions under which FaTFL1 downregulation didnot correlate with flowering were LDs at 9C in the cultivar ‘Elsanta’. It ispossible that, at such a cool temperature, FaTFL1 is a stronger floral repressorthan at higher temperatures. Cool temperature has been shown to renderArabidopsis TFL1 a more potent floral repressor (Hanano and Goto, 2011; Kimet al., 2013; Strasser et al., 2009), and a similar mechanism could be at workin the cultivated strawberry.

The observed discrepancy between FaTFL1 expression and flowering undersome of the experimental conditions could also arise from the incapability ofthe RT-qPCR primers to capture the expression of all the FaTFL1 homoeologs.As demonstrated by Tennessen et al. (2014) and Sargent et al. (2016), one of

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the subgenomes of the cultivated strawberry is F. vesca-like and one is F.iinumae-like. An examination of TFL1 sequences from the putative diploidancestors of cultivated strawberry reveals SNPs at the primer binding sites ofthe TFL1-specific RT-qPCR primers (data not shown), possibly biasing ourestimation on FaTFL1 expression. Preferential expression of one of thesubgenomes has been reported in the natural allotetraploid upland cotton(Gossypium hirsutum; Hovav et al., 2008). It may therefore be that our RT-qPCR assay was not able to capture physiologically relevant changes in theexpression of the most F. iinumae-like FaTFL1 homoeolog.

The patterns of expression of FaTFL1 were notably similar to thoseobserved in woodland strawberry; in F. vesca, the temperature-dependentflowering response is mediated by FvTFL1, which is downregulatedphotoperiod-independently below the critical limit of 13C (Rantanen et al.,2015). It could be argued that the differences in environmental responsesbetween these two cultivars arise from different critical temperature limits; in‘Glima’, the critical limit for photoperiod-independent floral induction isexceptionally high, as flowering is induced in both SDs and LDs at 21C,whereas in ‘Elsanta’ the critical limit would be exceptionally low. It is alsonoteworthy, that the highest temperature used in the experiment did not causephotoperiod-independent upregulation of FaTFL1, contrasting with thefindings of Rantanen et al. (2015), who observed that a temperature of 23Cupregulated FvTFL1 in woodland strawberry in both LDs and SDs. However,the results of Sønsteby and Heide (2006) indicate, that 27C is required forphotoperiod-independent floral inhibition in ‘Elsanta’. It therefore seemslikely that the highest temperature used in the current experiment was nothigh enough to cause photoperiod-independent upregulation of FaTFL1 in thecultivars used.

In addition to woodland strawberry, flowering is correlated withdownregulation of TFL1 in several other Rosaceous species, including apple(Hättasch et al., 2008), peach (Chen et al., 2013), Japanese apricot (Esumi etal., 2010) and rose (Iwata et al., 2012). The data presented here demonstratethat downregulation of FaTFL1 in the cultivated strawberry is generallycorrelated with floral induction, and the gene responds to changes in bothphotoperiod and temperature in a cultivar-dependent manner.

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The photoperiodic FT1–SOC1–TFL1 pathway is conserved between diploidand octoploid strawberries (III)

In woodland strawberry, the photoperiodic pathway involving theupregulation of FvFT1 and FvSOC1 is activated in leaf tissues in LDs (I; Mouhuet al., 2013). FvSOC1 in turn upregulates FvTFL1, thus inhibiting flowering inLDs (Mouhu et al., 2013). To determine whether FT1 and SOC1 homologuesshow similar expression patterns in cultivated strawberry, we studied theexpression of these genes in five cultivars grown under different photoperiodsand temperatures. In the tested cultivars, FaFT1 was upregulated in leaf tissueby LDs, as was FaSOC1 (II, Figure 2, 4 and 5). Also the diurnal expressionpattern of FaFT1 in leaf tissue (II, Figure S4) was similar to that of FvFT1 (I,Figure 6) with extremely low expression in SDs and a peak in expression in themiddle of the dark period in LDs. The results on FaFT1 expression agree withthe data of Nakano et al. (2015) and Nakajima et al. (2014), who showed thatFaFT1 is upregulated in the leaf in LDs. However, Nakano et al. (2015) werenot able to detect clear photoperiod-dependent differences in FaSOC1expression in the cultivar ‘Nyoho’. The cultivars included in our experiments(II) showed much higher expression of FaSOC1 in LDs than SDs already aftertwo weeks of photoperiodic treatments (II, Figure 2). The results of Nakano etal. (2015) are, however, in agreement with the data shown by Lei et al. (2013),who could not observe clear downregulation of FaSOC1 after two weeks in SDsin the cultivar ‘Camarosa’. It therefore appears that strawberry cultivars mayhave differences in photoperiodic regulation of FaSOC1, but it is impossible tosay how these differences correlate with flowering time without directcomparisons between cultivars.

As discussed earlier, altering FvTFL1 expression in woodland strawberrydoes not affect FvSOC1 expression or patterns of vegetative development, i.e.runner and branch crown formation. The results obtained from transgeniccultivated strawberry lines were very similar to those obtained from F. vesca;the expression of FaSOC1 in TFL1-RNAi lines of cultivated strawberry werenot notably different from the wild type (II, Figure 1), and the transgenic linesproduced approximately the same number of runners as the wild type plants(II, Figure 1). These results suggest that the role of TFL1 as a floral repressoris conserved between the diploid and octoploid strawberries and renderfurther support to the idea of using woodland strawberry as a model speciesfor studying the genetics of environmental responses in strawberries.

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6 CONCLUSIONS

The results discussed above provide a significant improvement to ourunderstanding of the genetic control of flowering in strawberries.Identification of SFL as an F. vesca orthologue of TFL1 opened a route to studythe photoperiodic control of flowering at the molecular level. However,identification of FvTFL1 as the major gene controlling flowering in thewoodland strawberry would have been much more difficult without theknowledge gained from studies in the annual model plant Arabidopsis. Theprocess of utilising information from a model species to identify genescontrolling similar physiological processes in less-well studied speciesemphasises the importance of in-depth fundamental studies in model plants.

CETS genes are involved in a multitude of processes in the plant kingdom,ranging from the control of seed germination to the control of meristemidentity and yearly growth cycles. Our results on the connection between thevernalisation response and FvTFL1 regulation in Alta-1 add yet anotherprocess that a TFL1 orthologue may control, highlighting the adaptiveimportance of the gene family. Despite the multitude of processes that TFL1seems to control, there is not much information available on the identity ofgenes regulating TFL1. Future research should therefore be aimed atelucidating the genetic components that cause the observed differentialregulation of TFL1 in strawberries.

The finding that TFL1 homologues control flowering also in the cultivatedstrawberry could open up new avenues for breeding for environmentaladaptation in this important crop. Earlier studies have located the everbearingtrait in the cultivated strawberry on the F. × ananassa LGIV, and shown thatthe same locus controls also runner production. However, FaTFL1 resides onLGVI, and changes in its expression pattern do not affect runnering in thecultivated strawberry. FaTFL1 could therefore offer a novel target for breedingfor modified flowering time, without adverse effects on runner formation.

In the model plant Arabidopsis, the photoperiodic and temperature-controlled flowering pathways are integrated at the regulation of FT and SOC1.According to the data discussed here, FT1 and SOC1 may play less pronouncedroles in the control of flowering in strawberries, and instead, TFL1 has evolvedinto the main floral pathway integrator gene. The identity of components thatcontrol the expression of TFL1 remain an active topic for further studies.

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DEPARTMENT OF AGRICULTURAL SCIENCESFACULTY OF AGRICULTURE AND FORESTRYDOCTORAL PROGRAMME IN PLANT SCIENCESUNIVERSITY OF HELSINKI

Genetic and Environmental Control of Flowering in Wild and Cultivated Strawberries

ELLI KOSKELA

dissertationes schola doctoralis scientiae circumiectalis, alimentariae, biologicae. universitatis helsinkiensis 16/2016

16/2016

Helsinki 2016 ISSN 2342-5423 ISBN 978-951-51-2335-0

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